FIELD OF THE INVENTION <EOH>The present invention relates to a process for the polymerization of olefins."],["<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides a process for homopolymerization of ethylene or copolymerization of ethylene with alpha-olefins by contacting ethylene or ethylene and alpha-olefin with a catalyst composition comprising: (a) a solid catalyst precursor comprising at least one vanadium compound, at least one magnesium compound and a polymeric material; and (b) a cocatalyst comprising at least one aluminum compound.","Most preferably, the component (a) used in the catalyst composition for the process of the present invention further comprises at least one further transition metal compound and/or at least one alcohol.","As a result of the present invention polyolefin and especially polyethylenes and copolymers of ethylene and alpha-olefins are provided which have a density of about 0.88 to 0.98 g/cm 3 and weight average molecular weight of about 500 to 900 000 g/mole and molecular weight distribution range of about 2 to about 30.The products have a uniform spherical particle morphology, very low level of fines and catalyst residues, improved thermal stability, excellent optical and improved environmental stress cracking resistance (ESCR).","detailed-description description=\"Detailed Description\" end=\"lead\"?"],["FIELD OF THE INVENTION The present invention relates to a process for the polymerization of olefins.","DESCRIPTION OF THE PRIOR ART Several publications are referenced in this application.","These references describe the state of the art to which this invention pertains, and are incorporated herein by reference.","The application of vanadium based Ziegler-Natta catalysts for industrial use has been limited to vanadium trichloride and vanadium oxytrichloride as homogenous catalysts in solution polymerization for the production of ethylene-propylene copolymers.","To attain suitable activity for polymerization these catalysts require the use of halogenated organic molecules such as chloroform and trichlorofluoromethane as a promoter.","In the absence of the promoter the catalyst activity is low.","Further, the kinetic behavior of these catalysts during polymerization display high initial rates of polymerization followed by a sharp decrease with time, decay type rate-time kinetics, and as a result produce resin of poor morphology.","The application of vanadium based catalysts for ethylene polymerization, high density and linear low density polyethylene production, has been comparatively very limited.","Attempts have been made to support vanadium based catalysts on silica or magnesium chloride to produce catalysts for ethylene polymerization.","U.S. Pat.","No.","4,508,842 describes a catalyst preparation in which a complex of VCl3 and tetrahydrofuran (THF) is treated with silica, the solvent is then removed from the solid by distillation and a boron trihalide or alkylaluminum halide modifier is added to the solid.","In addition, chloroform is used as a promoter with the catalyst for ethylene polymerization.","However the productivity and stability of this catalyst system is relatively poor.","U.S. Pat.","Nos.","5,534,472 and 5,670,439 describe a silica supported vanadium catalyst prepared by the prior contacting of silica with an organomagnesium compound and a trialkylaluminum compound.","The catalysts are suitable for the production of ethylene-hexene copolymer.","However, the polymerizations are conducted with trichlorofluoromethane or dibromomethane promoters.","Further, procedures typically used for the preparation of suitable magnesium chloride and silica supports such as spray drying or re-crystallization processes are complicated and expensive.","Also, the inorganic supports remain in the product, which can affect the product properties, such as optical properties, or processing.","It is an object of the present invention to provide a process for polymerization of olefins which overcomes the drawbacks of the prior art, especially providing a process which is cheap in carrying out and which produces a polymer with improved productivity and activity without using halogenated organic molecules as a promoter.","SUMMARY OF THE INVENTION The present invention provides a process for homopolymerization of ethylene or copolymerization of ethylene with alpha-olefins by contacting ethylene or ethylene and alpha-olefin with a catalyst composition comprising: (a) a solid catalyst precursor comprising at least one vanadium compound, at least one magnesium compound and a polymeric material; and (b) a cocatalyst comprising at least one aluminum compound.","Most preferably, the component (a) used in the catalyst composition for the process of the present invention further comprises at least one further transition metal compound and/or at least one alcohol.","As a result of the present invention polyolefin and especially polyethylenes and copolymers of ethylene and alpha-olefins are provided which have a density of about 0.88 to 0.98 g/cm3 and weight average molecular weight of about 500 to 900 000 g/mole and molecular weight distribution range of about 2 to about 30.The products have a uniform spherical particle morphology, very low level of fines and catalyst residues, improved thermal stability, excellent optical and improved environmental stress cracking resistance (ESCR).","DETAILED DESCRIPTION OF THE INVENTION Further advantages and features of the present invention will become apparent by the following detailed description in connection with the accompanying drawing.","In the drawing the Figure is a polymerization kinetic rate-time profile for ethylene polymerization using the catalyst composition of the present invention described in Example 1 below and a process of the present invention described in Example 5 below.","The profile shows a rate build up followed by a stable rate-time profile resulting in product polymer of spherical morphology and uniform particle size distribution.","The ethylene homopolymers and copolymers which may be prepared by the process of the present invention can have a wide density range of about 0.88 to 0.98 g/cm3.The process of the present invention provides polyolefins, and preferably high density polyethylene and linear low density polyethylene.","The density of the polymer at a given melt index can be regulated by the amount of the alpha-olefin comonomer used.","The amount of alpha-olefin comonomer needed to achieve the same density is varied according to the type of comonomer used.","These alpha-olefins have 4 to 10 carbon atoms and include 1-butene, 1-pentene, 4-methyl-1-pentene, 1-hexene, 1-heptene, 1-octene and/or mixtures thereof.","The weight average molecular weight (Mw) of the polymers produced by the process of the present invention is in the range of about 500 to 900 000 g/mole or higher, preferably from about 10 000 to 750 000 g/mole; depending on the amount of hydrogen used, the polymerization temperature and the polymer density attained.","The homopolymers and copolymers of the present invention have a melt index (MI) range of more than 0 and up to 100, preferably between 0.3 to 50.The polymers of such wide range of MI are capable of being used in film and molding applications.","The molecular weight distribution (MWD) of the polymers produced by the process of the present invention expressed as weight average molecular weight/number average molecular weight (Mw/Mn), is in the range of about 2 to about 30.The polymers produced by the process of the present invention are granular materials, uniform and spherical particles with an average particle size of about 0.1 to 4 mm in diameter and contain a very low level of fines.","The solid catalyst precursor used in the present invention comprises a polymeric material in the form of particles having a mean particle diameter of about 5 to 1 000 microns and a pore volume of at least about 0.05 cm3/g and a pore diameter of about 20 to 10 000 angstroms, preferably from about 500 to 10 000 angstroms, and a surface area of about 0.1 to 100 m2/g, preferably from about 0.2 to 30 m2/g.","The vanadium compound used for the catalyst composition for the process of the present invention is represented by the general formulas V(OR1)nX4-n, V(R2)nX4-n, VX3 and VOX3, wherein R1 and R2 represent an alkyl group, aryl group or cycloalkyl group having 1 to 20 carbon atoms, X represents a halogen and n represents a number satisfying 0≦n≦4.Examples of R1 and R2 include alkyl groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl and the like.","Preferred examples of the above mentioned vanadium compounds are selected from the group comprising vanadium tetraethoxide, vanadium tetrapropoxide, vanadium tetrabutoxide, vanadium trichloride, vanadium tetrachloride, vanadium oxytrichloride, vanadium dichlorodiethoxide and/or the like.","Vanadium tetrachloride and/or vanadium oxytrichloride are most preferred.","The transition metal compound which may be used for the catalyst composition for the process of the present invention is represented by the general formulas Tm(OR3)nX4-n, TmOX3 and Tm(R4)nX4-n, wherein Tm represents a transition metal of Group IVB, VB, or VIB, wherein R3 and R4 represent an alkyl group, aryl group or cycloalkyl group having 1 to 20 carbon atoms, X represents a halogen atom and n represents a number satisfying 0≦n≦4.Non-limiting examples of the transition metal are titanium and vanadium, examples of R3 and R4 include alkyl groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl and the like.","Preferred examples of the above mentioned transition metal compounds are selected from the group comprising titanium trichloromethoxide, titanium dichlorodimethoxide, titanium tetramethoxide, titanium trichloroethoxide, titanium dichlorodiethoxide, titanium tetraethoxide, titanium trichloropropoxide, titanium dichlorodipropoxide, titanium chlorotripropoxide, titanium tetrapropoxide, titanium trichlorobutoxide, titanium dichlorodibutoxide, titanium tetrabutoxide, vanadium tetrachloride, vanadium tetraethoxide, vanadium tetrapropoxide, vanadium tetrabutoxide, vanadium oxytrichloride, vanadium dichlorodiethoxide and/or the like.","Titanium tetraethoxide, titanium tetrapropoxide and/or titanium tetrabutoxide are most preferred.","The alcohol compound which may be used for the catalyst composition for the process of the present invention includes compounds represented by the general formula R5OH, wherein R5 is an alkyl group, aryl group or cycloalkyl group having 1 to 20 carbon atoms.","Examples of R5 include groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, cyclohexyl, phenyl, methylphenyl, ethylphenyl and the like.","Preferred examples of the above mentioned alcohols are selected from the group comprising methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, cyclohexanol, phenol, methylphenol, ethylphenol and/or mixtures thereof.","The magnesium compound used for the catalyst composition for the process of the present invention include Grignard compounds represented by the general formula R6MgX, wherein R6 is a hydrocarbon group having 1 to 20 carbon atoms and X is a halogen atom, preferably chlorine.","Other preferred magnesium compounds are represented by the general formula R7R8Mg, wherein R7 and R8 are each a hydrocarbon group having 1 to 20 carbon atoms.","Preferred examples of the above mentioned magnesium compounds include dialkylmagnesium such as diethylmagnesium, di-n-propylmagnesium, di-isopropylmagnesium, di-n-butylmagnesium, di-isobutylmagnesium butylethylmagnesium, dihexylmagnesium, dioctylmagnesium, butyloctylmagnesium; alkylmagnesium chloride such as ethylmagnesium chloride, butylmagnesium chloride, hexylmagnesium chloride and/or mixtures thereof.","The polymer particles used for the catalyst composition for the process of the present invention have a spherical shape with a mean particle diameter of about 5 to 1 000 microns and a pore volume of at least about 0.05 cm3/g and a pore diameter of about 20 to 10 000 angstroms, preferably from about 500 to 10 000 angstroms, and a surface area of about 0.1 to 100 m2/g, preferably from about 0.2 to 30 m2/g.","Examples of the above polymeric supports used for the catalyst composition for the process of the present invention are selected from the group comprising polymer particles of polyvinylchloride, polyvinylalcohol, polyketone, hydrolyzed polyketone, ethylene-vinylalcohol copolymer, polycarbonate and the like.","Among these polymeric materials polyvinylchloride is more preferred and non-crosslinked polyvinylchloride particles are most preferred.","Polyvinylchloride having a molecular weight in the range of about 5 000 to 500 000 g/mole is most preferred.","The use of the polymer particles mentioned in the present invention, in catalyst preparation offers significant advantages over traditional olefin polymerization catalysts using supports such as silica or magnesium chloride.","In comparison to the silica supported catalyst, the polymer particles described in catalyst preparation of the present invention do not require high temperature and prolonged dehydration steps prior to their use in catalyst synthesis, thereby simplifying the synthesis process and thus reducing the overall cost of catalyst preparation.","Further, the cost of the polymeric support used in the present invention is substantially cheaper than silica or magnesium chloride supports.","In addition, the catalyst of the present invention uses significantly lower levels of catalyst components for catalyst preparation than silica or magnesium chloride supported catalysts.","Also, the process of the present invention exhibits a higher activity than processes with conventional silica or magnesium chloride supported catalysts.","According to one embodiment of the present invention, a polyvinylchloride support is used in the process.","The synthesis of the solid catalyst precursor of the present invention involves introducing the polymeric material described above into a vessel and then adding a diluent.","Suitable diluents include isopentane, hexane, cyclohexane, heptane, isooctane and pentamethylheptane.","The polymeric material is then treated with a magnesium compound described above at a temperature in the range of about 10° C. to 130° C. The ratio of magnesium compound to the polymer support may be in the range of about 0.05 to 20 mmol per gram of polymer, preferably 0.1 to 10 mmol per gram polymer.","The solvent is then vaporized using a nitrogen purge at a temperature in the range of about 20° C. to 100° C. The resulting free-flowing solid product is then slurried.","Suitable solvents for slurring include isopentane, hexane, cyclohexane, heptane, isooctane and pentamethylheptane.","The magnesium modified polymeric material is then treated with a transition metal compound and/or an alcohol described above at a temperature in the range of about 10° C. to 130° C. According to the present invention titanium tetramethoxide, titanium tetraethoxide, titanium tetrapropoxide, titanium tetrabutoxide are preferred transition metal compounds and methanol, ethanol, n-propanol, isopropanol, n-butanol are preferred alcohols.","The resulting material is then treated with a vanadium compound described above at a temperature in the range of about 10° C. to 130° C. According to the present invention vanadium tetrachloride and/or vanadium oxytrichloride are preferred vanadium compounds.","The produced solid catalyst precursor is then washed several times with a suitable solvent such as isopentane, hexane, cyclohexane, heptane, or isooctane.","The solid catalyst precursor is then dried using a nitrogen purge at a temperature in the range of 20° C. to 100° C. In the final dried solid catalyst precursor, the molar ratio of vanadium to magnesium is in the range of about 0.01 to 50.In the case where the transition metal compound is used in catalyst preparation the molar ratio of vanadium to transition metal in the catalyst precursor is in the range of about 0.01 to 50, and in the case where alcohol is used in catalyst preparation the molar ratio of vanadium to OH groups in the catalyst precursor is in the range of about 0.01 to 50.The catalyst compositions used for the process of the present invention are not subjected to halogenation treatments, for example chlorination treatments.","The thus-formed catalyst precursor of the present invention is suitably activated by aluminum compounds, also known as cocatalysts.","The activation process can be a one step in which the catalyst is fully activated in the reactor, or two steps, in which the catalyst is partially activated outside the reactor and the complete activation occurs inside the reactor.","Unlike catalyst compositions described in the prior art the catalyst compositions of the present invention do not require promoters such as chloroform, trichlorofluoromethane or dibromomethane during polymerization.","The aluminum compounds, also known as cocatalyst, used in the process of the present invention are represented by the general formula R9nAlX3-n, wherein R9 represents a hydrocarbon group having 1 to 10 carbon atoms, X represents a halogen and represents a number satisfying 0≦n≦3.Illustrative but not limiting examples include trialkylaluminums such as trimethylaluminum, triethylaluminum, tri-isobutylaluminum or tri-n-hexylaluminum; dialkylaluminum chloride such as dimethylaluminum chloride, diethylaluminum chloride.","The preferred activators of the above general formula are trimethylaluminum, triethylaluminum, tri-isobutylaluminum and tri-n-hexylaluminum.","Examples of other suialuminum compounds of the present invention are represented by the general formula R10R11Al—O—AlR12R13, wherein R10, R11, R12 and R13 are either the same or different linear, branched or cyclic alkyl group having 1 to 12 carbon atoms, such as methyl, ethyl, propyl or isobutyl.","The preferred examples are methylaluminoxane (MAO) and modified methylaluminoxane (MMAO).","Further, mixtures of alkylaluminum compounds and aluminoxane compounds described above can also be conveniently used in the present invention.","The cocatalyst in the present invention can be added to the catalyst precursor before and/or during the polymerization reaction.","The cocatalyst in the present invention can be used in an amount of about 10 to 5 000 in terms moles of aluminum in the cocatalyst to moles of transition metal in the catalyst precursor, and is preferably in the range of 20 to 3 000.The process of the present invention can be carried out in slurry, solution and gas phase conditions.","Gas phase polymerization can be carried out in stirred bed reactors and in fluidized bed reactors.","Suitable ethylene pressures are in the range of about 3 to 40 bar, more preferably 5 to 30 bar and suitable polymerization temperatures are in the range of about 30° C. to 110° C., preferably 50° C. to 95° C. In addition to polyethylene homopolymer, ethylene copolymers with alpha-olefins having 4 to 10 carbon atoms are readily prepared by the present invention.","Particular examples include ethylene/1-butene, ethylene/1-pentene, ethylene/1-heptene, ethylene/1-hexene, ethylene/1-octene and ethylene/4-methyl 1-pentene.","The density of the polymer at a given melt index can be regulated by the amount and nature of the alpha-olefin comonomer used.","Hydrogen can be very conveniently used during polymerization using the catalyst described in the present invention to regulate the molecular weight of the polymer product.","Catalysts described in the prior art display undesirable decay type polymerization rate-time profiles, however the catalysts described in the present invention display build up followed by stable polymerization rate-time profiles, as seen in the Figure, such profiles are very suitable in gas, slurry and solution phase polymerization processes and provide polymer of good morphology and high resin bulk density.","EXAMPLES The following examples are intended to be illustrative of this invention.","They are, of course, not be taken in any way limiting on the scope of this invention.","Numerous changes and modifications can be made with respect to the invention.","Test Methods The properties of the polymers produced in the following examples were determined by the following test methods: Mw, Mn, and MWD were measured at 135° C. by Gel Permeation Chromatography (GPC) using mixed mode columns and trichlorobenzene as solvent.","Melting point (Mp) was determined by Differential Scanning Calorimetry (DSC).","Example 1 Synthesis of Solid Catalyst Precursor (A) To a three-necked round bottom flask, equipped with a condenser and a magnetic stirrer, was placed 10.0 g of polyvinylchloride spheres with an average particle size of 36 microns.","The flask containing the polyvinylchloride was heated up to 70° C. using an oil bath and then evacuated at 30 mm Hg pressure for 30 minutes.","The flask and its contents were then purged with dried nitrogen and the polyvinylchloride was slurried using 30 cm3 of isopentane.","Then 1.5 cm3 of butylmagnesium chloride (2.0 molar in diethylether) was added to the slurry and the resultant mixture was stirred for 60 minutes at an oil bath temperature of 35° C., under reflux conditions.","The isopentane was then evaporated to obtain a free-flowing powder by using a nitrogen purge at 35° C. Then the magnesium-modified polyvinylchloride was slurried using 30 cm3 of isopentane and 2.0 cm3 of titanium tetraethoxide (1.0 molar in n-hexane) was added to the slurry and the resulting mixture was stirred at 35° C. for 40 minutes.","A dark green/brown colored solid was produced.","Then 8.0 cm3 of vanadium tetrachloride (1.0 molar in n-hexane) was added to contents of the flask and the resulting mixture was stirred at 35° C. for 20 minutes.","The supernatant liquid was decanted and the resulting solid product was washed by stirring with 80 cm3 of isopentane and then removing the isopentane, then washed again twice with 80 cm3 of isopentane in each wash.","Finally, the solid catalyst was dried using a nitrogen purge at 35° C. to yield a free-flowing brown colored solid product.","The solid catalyst precursor was analyzed by atomic adsorption spectroscopy and was found to contain 0.57% by weight magnesium, 0.54% by weight titanium and 1.77% by weight vanadium.","Example 2 Ethylene Polymerization An autoclave with a volume of 3 liters was purged with nitrogen at 130° C. for 30 minutes.","After cooling the autoclave to 85° C. the reactor was purged with hydrogen and then 1.5 liters of n-hexane were introduced to the reactor.","The reactor was then pressurized to 3 barg hydrogen pressure.","Then 5.0 cm3 of tri-isobutylaluminum, TIBAL (1.0 molar in n-hexane) was injected into the reactor.","Ethylene was introduced to the reactor such as to raise the reactor pressure to 15 barg.","This was followed by injection of 0.1 g of the solid catalyst precursor “A” described in Example 1 after being slurried in 20 cm3 of n-hexane solvent.","Polymerization was carried out for 1 hour; with ethylene supplied on demand to maintain the total reactor pressure at 15 barg.","390 grams of polyethylene were recovered with a catalyst productivity of 3900 gPE/g catalyst, a resin bulk density of 0.290 g/cm3, a weight average molecular weight of 94 050 g/mole, a number average molecular weight of 28 350, a molecular weight distribution of 3.32 and a melting point of 135° C. Examples 3,4 Ethylene Polymerization An autoclave with a volume of 3 liters was purged with nitrogen at 130° C. for 30 minutes.","After cooling the autoclave to 85° C. the reactor was purged with hydrogen and then 1.5 liters of n-hexane were introduced to the reactor.","The reactor was then pressurised to 3 barg hydrogen pressure.","Then, the desired quantity of triethylaluminum, TEAL (1.0 molar in n-hexane), described in Table 1 below, was injected into the reactor.","Ethylene was introduced to the reactor such as to raise the reactor pressure to 15 barg.","This was followed by injection of 0.1 g of the solid catalyst precursor “A” described in Example 1 after being slurried in 20 cm3 of n-hexane solvent.","Polymerization was carried out for 1 hour; with ethylene supplied on demand to maintain the total reactor pressure at 15 barg.","Catalyst performance and product polymer properties are described in Table 1.TABLE 1 Influence of TEAL Concentration Productivity/ Bulk TEAL/ Yield/ (g PE/g Density/ Mw/ Mn/ DSC/ Example (mmol) (g PE) catalyst) (g/cm3) (1000 g/mol) (1000) MWD (Mp/° C.) 3 4 500 5000 0.310 135 36.4 3.71 138 4 6 455 4550 0.303 109 35.2 3.10 137 Examples 5,6 Ethylene Polymerization An autoclave with a volume of 3 liters was purged with nitrogen at 130° C. for 30 minutes.","After cooling the autoclave to 85° C. the reactor was purged with hydrogen and then 1.5 liters of n-hexane were introduced to the reactor.","The reactor was then pressurised to the desired hydrogen pressure, described in Table 2 below.","Then 4.0 cm3 of triethylaluminum, TEAL (1.0 molar in n-hexane) was injected into the reactor.","Ethylene was introduced to the reactor such as to raise the reactor pressure to 15 barg.","This was followed by injection of 0.1 g of the solid catalyst precursor “A” described in Example 1 after being slurried in 20 cm3 of n-hexane solvent.","Polymerization was carried out for 1 hour; with ethylene supplied on demand to maintain the total reactor pressure at 15 barg.","Catalyst performance and product polymer properties are described in Table 2.TABLE 2 Influence of Hydrogen Pressure Hydrogen Productivity/ Bulk Pressure/ Yield/ (g PE/g Density/ Mw/ Mn/ DSC/ Example (barg) (g PE) catalyst) (g/cm3) (1000 g/mol) (1000) MWD (Mp/° C.) 5 2 490 4900 0.290 151 43.3 3.48 139 3 3 500 5000 0.310 135 36.4 3.71 13 6 4 305 3050 0.260 114 33.7 3.37 137 Example 7 Ethylene-Butene Copolymerization An autoclave with a volume of 3 liters was purged with nitrogen at 130° C. for 30 minutes.","After cooling the autoclave to 85° C. the reactor was purged with hydrogen and then 1.5 liters of n-hexane were introduced to the reactor.","Then the desired quantity of butene, described in Table 3 below, was added to the reactor.","The reactor was then pressurized to 3 barg hydrogen pressure.","Then 4.0 cm3 of triethylaluminum, TEAL (1.0 molar in n-hexane) was injected into the reactor.","Ethylene was introduced to the reactor such as to raise the reactor pressure to 15 barg.","This was followed by injection of 0.1 g of the solid catalyst precursor “A” described in Example 1 after being slurried in 20 cm3 of n-hexane solvent.","Polymerization was carried out for 1 hour; with ethylene supplied on demand to maintain the total reactor pressure at 15 barg.","Catalyst performance and product polymer properties are described in Table 3.TABLE 3 Influence of Butene Productivity/ Bulk Butene/ Yield/ (g PE/g Density/ Mw/ Mn/ DSC/ Example (cm3) (g PE) catalyst) (g/cm3) (1000 g/mol) (1000) MWD (Mp/° C.) 3 0 500 5000 0.310 135 36.4 3.71 138 7 10 502 5020 0.300 135 35.7 3.77 138 Example 8 Ethylene-Hexene Copolymerization An autoclave with a volume of 3 liters was purged with nitrogen at 130° C. for 30 minutes.","After cooling the autoclave to 85° C. the reactor was purged with hydrogen and then 1.5 liters of n-hexane were introduced to the reactor.","Then the desired quantity of hexene, described in Table 4 below, was added to the reactor.","The reactor was then pressurized to 3 barg hydrogen pressure.","Then 4.0 cm3 of triethylaluminum, TEAL (1.0 molar in n-hexane) was injected into to the reactor.","Ethylene was introduced to the reactor such as to raise the: reactor pressure to 15 barg.","This was followed by injection of 0.1 g of the solid catalyst precursor “A” described in Example 1 after being slurried in 20 cm3 of n-hexane solvent.","Polymerization was carried out for 1 hour; with ethylene supplied on demand to maintain the total reactor pressure at 15 barg.","Catalyst performance and product polymer properties are described in Table 4.TABLE 4 Influence of Hexene Productivity/ Bulk Hexene/ Yield/ (g PE/g Density/ Mw/ Mn/ DSC/ Example (cm3) (g PE) catalyst) (g/cm3) (1000 g/mol) (1000) MWD (Mp/° C.) 3 0 500 5000 0.310 135 36.4 3.71 138 8 10 476 4760 0.302 134 41.8 3.21 137 Example 9 Synthesis of Solid Catalyst Precursor (B) To a three-necked round bottom flask, equipped with a condenser and a magnetic stirrer, was placed 10.0 g of polyvinylchloride spheres with an average particle size of 36 microns.","The flask containing the polyvinylchloride was heated up to 70° C. using an oil bath and then evacuated at 30 mm Hg pressure for 30 minutes.","The flask and its contents were then purged with dried nitrogen and the polyvinylchloride was slurried using 30 cm3 of isopentane.","Then 1.5 cm3 of butylmagnesium chloride (2.0 molar in diethylether) was added to the slurry and the resultant mixture was stirred for 60 minutes at an oil bath temperature of 35° C., under reflux conditions.","The isopentane was then evaporated to obtain a free-flowing powder by using a nitrogen purge at 35° C. Then the magnesium-modified polyvinylchloride was slurried using 30 cm3 of isopentane and 2.0 cm3 of titanium tetraethoxide (1.0 molar in n-hexane) was added to the slurry and the resulting mixture was stirred at 35° C. for 40 minutes.","A dark green/brown colored solid was produced.","Then 8.0 cm3 of vanadium oxytrichloride (1.0 molar in n-hexane) was added to contents of the flask and the resulting mixture was stirred at 35° C. for 20 minutes.","The supernatant liquid was decanted and the resulting solid product was washed by stirring with 80 cm3 of isopentane and then removing the isopentane, then washed again twice with 80 cm3 of isopentane in each wash.","Finally, the solid catalyst was dried using a nitrogen purge at 35° C. to yield a free-flowing brown colored solid product.","The solid catalyst precursor was analyzed by atomic adsorption spectroscopy and was found to contain 0.74% by weight magnesium, 0.56% by weight titanium and 1.50% by weight vanadium.","Example 10 Ethylene Polymerization An autoclave with a volume of 3 liters was purged with nitrogen at 130° C. for 30 minutes.","After cooling the autoclave to 85° C. the reactor was purged with hydrogen and then 1.5 liters of n-hexane were introduced to the reactor.","The reactor was then pressurised to 3 barg hydrogen pressure.","Then, 4.0 cm3 of triethylaluminum, TEAL (1.0 molar in n-hexane) was injected into the reactor.","Ethylene was introduced to the reactor such as to raise the reactor pressure to 15 barg.","This was followed by injection of 0.1 g of the solid catalyst precursor “B” described in Example 9 after being slurried in 20 cm3 of n-hexane solvent.","Polymerization was carried out for 1 hour; with ethylene supplied on demand to maintain the total reactor pressure at 15 barg.","200 grams of polyethylene were recovered with a catalyst productivity of 2000 gPE/g catalyst, a resin bulk density of 0.260 g/cm3, a weight average molecular weight of 157 000 g/mole, a number average molecular weight of 44 950, a molecular weight distribution of 3.49 and a melting point of 138° C. The features disclosed in the foregoing description, in the claims and/or in the drawing may, both separately and in any combination thereof, be material for realising the invention in diverse forms thereof."]],"string":"[\n [\n \"Process for the polymerization of olefins\",\n \"The present invention relates to a process for homopolymerization of ethylene or copolymerization of ethylene with alpha-olefins by contacting ethylene or ethylene and alpha-olefin with a catalyst composition comprising: (a) a solid catalyst precursor comprising at least one vanadium compound, at least one magnesium compound and a polymeric material or a solid catalyst precursor comprising at least one vanadium compound, at least one further transition metal compound and/or at least one alcohol, at least one magnesium compound and a polymeric material; and (b) a cocatalyst comprising an aluminum compound.\"\n ],\n [\n \"1.A process for homopolymerization of ethylene or copolymerization of ethylene with alpha-olefins comprising contacting ethylene or ethylene and alpha-olefin with a catalyst composition comprising: (i) a solid catalyst precursor comprising at least one vanadium compound represented by the general formulas V(OR1)nX4-n, V(R2)nX4-n, VX3 and VOX3 wherein R1 and R2 represent an alkyl group, aryl group or cycloalkyl group having 1 to 20 carbon atoms, and X represents a halogen and n represents a number satisfying 0≦n≦4, at least one Grignard compound represented by the general formula R6MgX wherein R6 is a hydrocarbon group having 1 to 20 carbon atoms and X is a halogen atom or a dialkyl magnesium compound represented by the general formula R7R8Mg, wherein R7 and R8 are each a hydrocarbon group having 1 to 20 carbon atoms, and a polymeric support; and (ii) a cocatalyst comprising an aluminum compound represented by the general formula R9nAlX3-n wherein R9 represents a hydrocarbon group having 1 to 10 carbon atoms; X represents a halogen and n represents a number satisfying 0≦n≦3, or by the general formula R10R11Al—O—AlR12R13, wherein R10, R11, R12 and R13 are either the same or different linear, branched or cyclic alkyl group having 1 to 12 carbons, or is an aluminoxane.\",\n \"2.The process according to claim 1 wherein said solid catalyst precursor further comprises at least transition metal compound represented by the general formulas Tm(OR3)nX4-n, TmOX3 and Tm(R4)nX4-n, wherein Tm is titanium or vanadium, R3 and R4 represent an alkyl group, aryl group or cycloalkyl group having 1 to 20 carbon atoms, X represents a halogen atom and n represents a number satisfying 0≦n≦4 or at least one alcohol represented by the general formula R5OH, wherein R5 is an alkyl group, aryl group or cycloalkyl group having 1 to 20 carbon atoms.\",\n \"3.\",\n \"(canceled) 4.\",\n \"(canceled) 5.The process according to claim 1, wherein said vanadium compound is selected from the group comprising vanadium tetraethoxide, vanadium tetrapropoxide, vanadium tetrabutoxide, vanadium trichloride, vanadium tetrachloride, vanadium oxytrichloride, and vanadium dichlorodiethoxide.\",\n \"6.The process according to claim 5, wherein said vanadium compound comprises vanadium tetrachloride or vanadium oxytrichloride.\",\n \"7.\",\n \"(canceled) 8.The process according to claim 2, wherein the transition metal compound is selected from the group comprising titanium trichloromethoxide, titanium dichlorodimethoxide, titanium tetramethoxide, titanium trichloroethoxide, titanium dichlorodiethoxide, titanium tetraethoxide, titanium trichloropropoxide, titanium dichlorodipropoxide, titanium chlorotripropoxide, titanium tetrapropoxide, titanium trichlorobutoxide, titanium dichlorodibutoxide, titanium tetrabutoxide, vanadium tetrachloride, vanadium tetraethoxide, vanadium tetrapropoxide, vanadium tetrabutoxide, vanadium oxytrichloride, and vanadium dichlorodiethoxide.\",\n \"9.The process according to claim 8, wherein the transition metal compound comprises titanium tetraethoxide, titanium tetrapropoxide or titanium tetrabutoxide.\",\n \"10.\",\n \"(canceled) 11.The process according to claim 2, wherein the alcohol is selected from the group comprising methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, cyclohexanol, phenol, methylphenol, and ethylphenol.\",\n \"12.\",\n \"(canceled) 13.The process according to claim 5, wherein the magnesium compound comprises diethylmagnesium, di-n-propylmagnesium, di-isopropylmagnesium, di-n-butylmagnesium, di-isobutylmagnesium butylethylmagnesium, dihexylmagnesium, dioctylmagnesium, butyloctylmagnesium, ethylmagnesium chloride, butylmagnesium chloride, or hexylmagnesium chloride.\",\n \"14.The process according to claim 5, wherein the polymeric support is in the form of particles having a mean particle diameter of about 5 to 1000 microns, a pore volume of at least about 0.05 cm3/g, a pore diameter of about 20 to 10000 angstroms and a surface area of about 0.1 to 100 m2/g.\",\n \"15.The process according to claim 14, wherein said polymeric support has a pore diameter from about 500 to 10000 angstroms and a surface area from about 0.2 to 30 m2/g.\",\n \"16.The process according to claim 15, wherein the polymeric support is comprised of polyvinylchloride, polyvinylalcohol, polyketone, hydrolyzed polyketone, ethylene-vinylalcohol copolymer, or polycarbonate.\",\n \"17.(canceled).\",\n \"18.The process according to claim 15, wherein the polymeric support is comprised of polyvinylchloride having a molecular weight in the range of about 5000 to 500000 g/mole.\",\n \"19.The process according to claim 18, wherein the Grignard compound is used in the range of about 0.05 to 20 mmol per gram of polymeric support.\",\n \"20.The process according to claim 1, wherein the molar ratio of vanadium to magnesium in the catalyst precursor is in the range of about 0.01 to 50.21.The process according to claim 2, wherein the molar ratio of vanadium to transition metal in said transition compound in the catalyst precursor is of about 0.01 to 50.22.The process according to claim 2, wherein said catalyst precursor comprises an alcohol and the molar ratio of vanadium to OH groups in said alcohol in the catalyst precursor is in the range of about 0.01 to 50.23.\",\n \"(canceled) 24.The process according to claim 6, wherein the aluminum compound is trimethylaluminum, triethylaluminum, tri-isobutylaluminum or tri-n-hexylaluminum.\",\n \"25.\",\n \"(canceled) 26.The process according to claim 5, wherein the aluminum compound comprises methyl aluminoxane (MAO) or modified methyl aluminoxane (MMAO).\",\n \"27.The process according to claim 5, wherein the aluminum compound comprises a mixture of an alkylaluminum and an aluminoxane.\",\n \"28.The process according to claim 2, wherein the ratio of moles of aluminum in said cocatalyst to the moles of transition metal in said catalyst precursor is about 10 to 5000.29.\",\n \"(canceled) 30.\",\n \"(canceled) 31.\",\n \"(canceled)\"\n ],\n [\n \"<SOH> FIELD OF THE INVENTION <EOH>The present invention relates to a process for the polymerization of olefins.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides a process for homopolymerization of ethylene or copolymerization of ethylene with alpha-olefins by contacting ethylene or ethylene and alpha-olefin with a catalyst composition comprising: (a) a solid catalyst precursor comprising at least one vanadium compound, at least one magnesium compound and a polymeric material; and (b) a cocatalyst comprising at least one aluminum compound.\",\n \"Most preferably, the component (a) used in the catalyst composition for the process of the present invention further comprises at least one further transition metal compound and/or at least one alcohol.\",\n \"As a result of the present invention polyolefin and especially polyethylenes and copolymers of ethylene and alpha-olefins are provided which have a density of about 0.88 to 0.98 g/cm 3 and weight average molecular weight of about 500 to 900 000 g/mole and molecular weight distribution range of about 2 to about 30.The products have a uniform spherical particle morphology, very low level of fines and catalyst residues, improved thermal stability, excellent optical and improved environmental stress cracking resistance (ESCR).\",\n \"detailed-description description=\\\"Detailed Description\\\" end=\\\"lead\\\"?\"\n ],\n [\n \"FIELD OF THE INVENTION The present invention relates to a process for the polymerization of olefins.\",\n \"DESCRIPTION OF THE PRIOR ART Several publications are referenced in this application.\",\n \"These references describe the state of the art to which this invention pertains, and are incorporated herein by reference.\",\n \"The application of vanadium based Ziegler-Natta catalysts for industrial use has been limited to vanadium trichloride and vanadium oxytrichloride as homogenous catalysts in solution polymerization for the production of ethylene-propylene copolymers.\",\n \"To attain suitable activity for polymerization these catalysts require the use of halogenated organic molecules such as chloroform and trichlorofluoromethane as a promoter.\",\n \"In the absence of the promoter the catalyst activity is low.\",\n \"Further, the kinetic behavior of these catalysts during polymerization display high initial rates of polymerization followed by a sharp decrease with time, decay type rate-time kinetics, and as a result produce resin of poor morphology.\",\n \"The application of vanadium based catalysts for ethylene polymerization, high density and linear low density polyethylene production, has been comparatively very limited.\",\n \"Attempts have been made to support vanadium based catalysts on silica or magnesium chloride to produce catalysts for ethylene polymerization.\",\n \"U.S. Pat.\",\n \"No.\",\n \"4,508,842 describes a catalyst preparation in which a complex of VCl3 and tetrahydrofuran (THF) is treated with silica, the solvent is then removed from the solid by distillation and a boron trihalide or alkylaluminum halide modifier is added to the solid.\",\n \"In addition, chloroform is used as a promoter with the catalyst for ethylene polymerization.\",\n \"However the productivity and stability of this catalyst system is relatively poor.\",\n \"U.S. Pat.\",\n \"Nos.\",\n \"5,534,472 and 5,670,439 describe a silica supported vanadium catalyst prepared by the prior contacting of silica with an organomagnesium compound and a trialkylaluminum compound.\",\n \"The catalysts are suitable for the production of ethylene-hexene copolymer.\",\n \"However, the polymerizations are conducted with trichlorofluoromethane or dibromomethane promoters.\",\n \"Further, procedures typically used for the preparation of suitable magnesium chloride and silica supports such as spray drying or re-crystallization processes are complicated and expensive.\",\n \"Also, the inorganic supports remain in the product, which can affect the product properties, such as optical properties, or processing.\",\n \"It is an object of the present invention to provide a process for polymerization of olefins which overcomes the drawbacks of the prior art, especially providing a process which is cheap in carrying out and which produces a polymer with improved productivity and activity without using halogenated organic molecules as a promoter.\",\n \"SUMMARY OF THE INVENTION The present invention provides a process for homopolymerization of ethylene or copolymerization of ethylene with alpha-olefins by contacting ethylene or ethylene and alpha-olefin with a catalyst composition comprising: (a) a solid catalyst precursor comprising at least one vanadium compound, at least one magnesium compound and a polymeric material; and (b) a cocatalyst comprising at least one aluminum compound.\",\n \"Most preferably, the component (a) used in the catalyst composition for the process of the present invention further comprises at least one further transition metal compound and/or at least one alcohol.\",\n \"As a result of the present invention polyolefin and especially polyethylenes and copolymers of ethylene and alpha-olefins are provided which have a density of about 0.88 to 0.98 g/cm3 and weight average molecular weight of about 500 to 900 000 g/mole and molecular weight distribution range of about 2 to about 30.The products have a uniform spherical particle morphology, very low level of fines and catalyst residues, improved thermal stability, excellent optical and improved environmental stress cracking resistance (ESCR).\",\n \"DETAILED DESCRIPTION OF THE INVENTION Further advantages and features of the present invention will become apparent by the following detailed description in connection with the accompanying drawing.\",\n \"In the drawing the Figure is a polymerization kinetic rate-time profile for ethylene polymerization using the catalyst composition of the present invention described in Example 1 below and a process of the present invention described in Example 5 below.\",\n \"The profile shows a rate build up followed by a stable rate-time profile resulting in product polymer of spherical morphology and uniform particle size distribution.\",\n \"The ethylene homopolymers and copolymers which may be prepared by the process of the present invention can have a wide density range of about 0.88 to 0.98 g/cm3.The process of the present invention provides polyolefins, and preferably high density polyethylene and linear low density polyethylene.\",\n \"The density of the polymer at a given melt index can be regulated by the amount of the alpha-olefin comonomer used.\",\n \"The amount of alpha-olefin comonomer needed to achieve the same density is varied according to the type of comonomer used.\",\n \"These alpha-olefins have 4 to 10 carbon atoms and include 1-butene, 1-pentene, 4-methyl-1-pentene, 1-hexene, 1-heptene, 1-octene and/or mixtures thereof.\",\n \"The weight average molecular weight (Mw) of the polymers produced by the process of the present invention is in the range of about 500 to 900 000 g/mole or higher, preferably from about 10 000 to 750 000 g/mole; depending on the amount of hydrogen used, the polymerization temperature and the polymer density attained.\",\n \"The homopolymers and copolymers of the present invention have a melt index (MI) range of more than 0 and up to 100, preferably between 0.3 to 50.The polymers of such wide range of MI are capable of being used in film and molding applications.\",\n \"The molecular weight distribution (MWD) of the polymers produced by the process of the present invention expressed as weight average molecular weight/number average molecular weight (Mw/Mn), is in the range of about 2 to about 30.The polymers produced by the process of the present invention are granular materials, uniform and spherical particles with an average particle size of about 0.1 to 4 mm in diameter and contain a very low level of fines.\",\n \"The solid catalyst precursor used in the present invention comprises a polymeric material in the form of particles having a mean particle diameter of about 5 to 1 000 microns and a pore volume of at least about 0.05 cm3/g and a pore diameter of about 20 to 10 000 angstroms, preferably from about 500 to 10 000 angstroms, and a surface area of about 0.1 to 100 m2/g, preferably from about 0.2 to 30 m2/g.\",\n \"The vanadium compound used for the catalyst composition for the process of the present invention is represented by the general formulas V(OR1)nX4-n, V(R2)nX4-n, VX3 and VOX3, wherein R1 and R2 represent an alkyl group, aryl group or cycloalkyl group having 1 to 20 carbon atoms, X represents a halogen and n represents a number satisfying 0≦n≦4.Examples of R1 and R2 include alkyl groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl and the like.\",\n \"Preferred examples of the above mentioned vanadium compounds are selected from the group comprising vanadium tetraethoxide, vanadium tetrapropoxide, vanadium tetrabutoxide, vanadium trichloride, vanadium tetrachloride, vanadium oxytrichloride, vanadium dichlorodiethoxide and/or the like.\",\n \"Vanadium tetrachloride and/or vanadium oxytrichloride are most preferred.\",\n \"The transition metal compound which may be used for the catalyst composition for the process of the present invention is represented by the general formulas Tm(OR3)nX4-n, TmOX3 and Tm(R4)nX4-n, wherein Tm represents a transition metal of Group IVB, VB, or VIB, wherein R3 and R4 represent an alkyl group, aryl group or cycloalkyl group having 1 to 20 carbon atoms, X represents a halogen atom and n represents a number satisfying 0≦n≦4.Non-limiting examples of the transition metal are titanium and vanadium, examples of R3 and R4 include alkyl groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl and the like.\",\n \"Preferred examples of the above mentioned transition metal compounds are selected from the group comprising titanium trichloromethoxide, titanium dichlorodimethoxide, titanium tetramethoxide, titanium trichloroethoxide, titanium dichlorodiethoxide, titanium tetraethoxide, titanium trichloropropoxide, titanium dichlorodipropoxide, titanium chlorotripropoxide, titanium tetrapropoxide, titanium trichlorobutoxide, titanium dichlorodibutoxide, titanium tetrabutoxide, vanadium tetrachloride, vanadium tetraethoxide, vanadium tetrapropoxide, vanadium tetrabutoxide, vanadium oxytrichloride, vanadium dichlorodiethoxide and/or the like.\",\n \"Titanium tetraethoxide, titanium tetrapropoxide and/or titanium tetrabutoxide are most preferred.\",\n \"The alcohol compound which may be used for the catalyst composition for the process of the present invention includes compounds represented by the general formula R5OH, wherein R5 is an alkyl group, aryl group or cycloalkyl group having 1 to 20 carbon atoms.\",\n \"Examples of R5 include groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, cyclohexyl, phenyl, methylphenyl, ethylphenyl and the like.\",\n \"Preferred examples of the above mentioned alcohols are selected from the group comprising methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, cyclohexanol, phenol, methylphenol, ethylphenol and/or mixtures thereof.\",\n \"The magnesium compound used for the catalyst composition for the process of the present invention include Grignard compounds represented by the general formula R6MgX, wherein R6 is a hydrocarbon group having 1 to 20 carbon atoms and X is a halogen atom, preferably chlorine.\",\n \"Other preferred magnesium compounds are represented by the general formula R7R8Mg, wherein R7 and R8 are each a hydrocarbon group having 1 to 20 carbon atoms.\",\n \"Preferred examples of the above mentioned magnesium compounds include dialkylmagnesium such as diethylmagnesium, di-n-propylmagnesium, di-isopropylmagnesium, di-n-butylmagnesium, di-isobutylmagnesium butylethylmagnesium, dihexylmagnesium, dioctylmagnesium, butyloctylmagnesium; alkylmagnesium chloride such as ethylmagnesium chloride, butylmagnesium chloride, hexylmagnesium chloride and/or mixtures thereof.\",\n \"The polymer particles used for the catalyst composition for the process of the present invention have a spherical shape with a mean particle diameter of about 5 to 1 000 microns and a pore volume of at least about 0.05 cm3/g and a pore diameter of about 20 to 10 000 angstroms, preferably from about 500 to 10 000 angstroms, and a surface area of about 0.1 to 100 m2/g, preferably from about 0.2 to 30 m2/g.\",\n \"Examples of the above polymeric supports used for the catalyst composition for the process of the present invention are selected from the group comprising polymer particles of polyvinylchloride, polyvinylalcohol, polyketone, hydrolyzed polyketone, ethylene-vinylalcohol copolymer, polycarbonate and the like.\",\n \"Among these polymeric materials polyvinylchloride is more preferred and non-crosslinked polyvinylchloride particles are most preferred.\",\n \"Polyvinylchloride having a molecular weight in the range of about 5 000 to 500 000 g/mole is most preferred.\",\n \"The use of the polymer particles mentioned in the present invention, in catalyst preparation offers significant advantages over traditional olefin polymerization catalysts using supports such as silica or magnesium chloride.\",\n \"In comparison to the silica supported catalyst, the polymer particles described in catalyst preparation of the present invention do not require high temperature and prolonged dehydration steps prior to their use in catalyst synthesis, thereby simplifying the synthesis process and thus reducing the overall cost of catalyst preparation.\",\n \"Further, the cost of the polymeric support used in the present invention is substantially cheaper than silica or magnesium chloride supports.\",\n \"In addition, the catalyst of the present invention uses significantly lower levels of catalyst components for catalyst preparation than silica or magnesium chloride supported catalysts.\",\n \"Also, the process of the present invention exhibits a higher activity than processes with conventional silica or magnesium chloride supported catalysts.\",\n \"According to one embodiment of the present invention, a polyvinylchloride support is used in the process.\",\n \"The synthesis of the solid catalyst precursor of the present invention involves introducing the polymeric material described above into a vessel and then adding a diluent.\",\n \"Suitable diluents include isopentane, hexane, cyclohexane, heptane, isooctane and pentamethylheptane.\",\n \"The polymeric material is then treated with a magnesium compound described above at a temperature in the range of about 10° C. to 130° C. The ratio of magnesium compound to the polymer support may be in the range of about 0.05 to 20 mmol per gram of polymer, preferably 0.1 to 10 mmol per gram polymer.\",\n \"The solvent is then vaporized using a nitrogen purge at a temperature in the range of about 20° C. to 100° C. The resulting free-flowing solid product is then slurried.\",\n \"Suitable solvents for slurring include isopentane, hexane, cyclohexane, heptane, isooctane and pentamethylheptane.\",\n \"The magnesium modified polymeric material is then treated with a transition metal compound and/or an alcohol described above at a temperature in the range of about 10° C. to 130° C. According to the present invention titanium tetramethoxide, titanium tetraethoxide, titanium tetrapropoxide, titanium tetrabutoxide are preferred transition metal compounds and methanol, ethanol, n-propanol, isopropanol, n-butanol are preferred alcohols.\",\n \"The resulting material is then treated with a vanadium compound described above at a temperature in the range of about 10° C. to 130° C. According to the present invention vanadium tetrachloride and/or vanadium oxytrichloride are preferred vanadium compounds.\",\n \"The produced solid catalyst precursor is then washed several times with a suitable solvent such as isopentane, hexane, cyclohexane, heptane, or isooctane.\",\n \"The solid catalyst precursor is then dried using a nitrogen purge at a temperature in the range of 20° C. to 100° C. In the final dried solid catalyst precursor, the molar ratio of vanadium to magnesium is in the range of about 0.01 to 50.In the case where the transition metal compound is used in catalyst preparation the molar ratio of vanadium to transition metal in the catalyst precursor is in the range of about 0.01 to 50, and in the case where alcohol is used in catalyst preparation the molar ratio of vanadium to OH groups in the catalyst precursor is in the range of about 0.01 to 50.The catalyst compositions used for the process of the present invention are not subjected to halogenation treatments, for example chlorination treatments.\",\n \"The thus-formed catalyst precursor of the present invention is suitably activated by aluminum compounds, also known as cocatalysts.\",\n \"The activation process can be a one step in which the catalyst is fully activated in the reactor, or two steps, in which the catalyst is partially activated outside the reactor and the complete activation occurs inside the reactor.\",\n \"Unlike catalyst compositions described in the prior art the catalyst compositions of the present invention do not require promoters such as chloroform, trichlorofluoromethane or dibromomethane during polymerization.\",\n \"The aluminum compounds, also known as cocatalyst, used in the process of the present invention are represented by the general formula R9nAlX3-n, wherein R9 represents a hydrocarbon group having 1 to 10 carbon atoms, X represents a halogen and represents a number satisfying 0≦n≦3.Illustrative but not limiting examples include trialkylaluminums such as trimethylaluminum, triethylaluminum, tri-isobutylaluminum or tri-n-hexylaluminum; dialkylaluminum chloride such as dimethylaluminum chloride, diethylaluminum chloride.\",\n \"The preferred activators of the above general formula are trimethylaluminum, triethylaluminum, tri-isobutylaluminum and tri-n-hexylaluminum.\",\n \"Examples of other suialuminum compounds of the present invention are represented by the general formula R10R11Al—O—AlR12R13, wherein R10, R11, R12 and R13 are either the same or different linear, branched or cyclic alkyl group having 1 to 12 carbon atoms, such as methyl, ethyl, propyl or isobutyl.\",\n \"The preferred examples are methylaluminoxane (MAO) and modified methylaluminoxane (MMAO).\",\n \"Further, mixtures of alkylaluminum compounds and aluminoxane compounds described above can also be conveniently used in the present invention.\",\n \"The cocatalyst in the present invention can be added to the catalyst precursor before and/or during the polymerization reaction.\",\n \"The cocatalyst in the present invention can be used in an amount of about 10 to 5 000 in terms moles of aluminum in the cocatalyst to moles of transition metal in the catalyst precursor, and is preferably in the range of 20 to 3 000.The process of the present invention can be carried out in slurry, solution and gas phase conditions.\",\n \"Gas phase polymerization can be carried out in stirred bed reactors and in fluidized bed reactors.\",\n \"Suitable ethylene pressures are in the range of about 3 to 40 bar, more preferably 5 to 30 bar and suitable polymerization temperatures are in the range of about 30° C. to 110° C., preferably 50° C. to 95° C. In addition to polyethylene homopolymer, ethylene copolymers with alpha-olefins having 4 to 10 carbon atoms are readily prepared by the present invention.\",\n \"Particular examples include ethylene/1-butene, ethylene/1-pentene, ethylene/1-heptene, ethylene/1-hexene, ethylene/1-octene and ethylene/4-methyl 1-pentene.\",\n \"The density of the polymer at a given melt index can be regulated by the amount and nature of the alpha-olefin comonomer used.\",\n \"Hydrogen can be very conveniently used during polymerization using the catalyst described in the present invention to regulate the molecular weight of the polymer product.\",\n \"Catalysts described in the prior art display undesirable decay type polymerization rate-time profiles, however the catalysts described in the present invention display build up followed by stable polymerization rate-time profiles, as seen in the Figure, such profiles are very suitable in gas, slurry and solution phase polymerization processes and provide polymer of good morphology and high resin bulk density.\",\n \"EXAMPLES The following examples are intended to be illustrative of this invention.\",\n \"They are, of course, not be taken in any way limiting on the scope of this invention.\",\n \"Numerous changes and modifications can be made with respect to the invention.\",\n \"Test Methods The properties of the polymers produced in the following examples were determined by the following test methods: Mw, Mn, and MWD were measured at 135° C. by Gel Permeation Chromatography (GPC) using mixed mode columns and trichlorobenzene as solvent.\",\n \"Melting point (Mp) was determined by Differential Scanning Calorimetry (DSC).\",\n \"Example 1 Synthesis of Solid Catalyst Precursor (A) To a three-necked round bottom flask, equipped with a condenser and a magnetic stirrer, was placed 10.0 g of polyvinylchloride spheres with an average particle size of 36 microns.\",\n \"The flask containing the polyvinylchloride was heated up to 70° C. using an oil bath and then evacuated at 30 mm Hg pressure for 30 minutes.\",\n \"The flask and its contents were then purged with dried nitrogen and the polyvinylchloride was slurried using 30 cm3 of isopentane.\",\n \"Then 1.5 cm3 of butylmagnesium chloride (2.0 molar in diethylether) was added to the slurry and the resultant mixture was stirred for 60 minutes at an oil bath temperature of 35° C., under reflux conditions.\",\n \"The isopentane was then evaporated to obtain a free-flowing powder by using a nitrogen purge at 35° C. Then the magnesium-modified polyvinylchloride was slurried using 30 cm3 of isopentane and 2.0 cm3 of titanium tetraethoxide (1.0 molar in n-hexane) was added to the slurry and the resulting mixture was stirred at 35° C. for 40 minutes.\",\n \"A dark green/brown colored solid was produced.\",\n \"Then 8.0 cm3 of vanadium tetrachloride (1.0 molar in n-hexane) was added to contents of the flask and the resulting mixture was stirred at 35° C. for 20 minutes.\",\n \"The supernatant liquid was decanted and the resulting solid product was washed by stirring with 80 cm3 of isopentane and then removing the isopentane, then washed again twice with 80 cm3 of isopentane in each wash.\",\n \"Finally, the solid catalyst was dried using a nitrogen purge at 35° C. to yield a free-flowing brown colored solid product.\",\n \"The solid catalyst precursor was analyzed by atomic adsorption spectroscopy and was found to contain 0.57% by weight magnesium, 0.54% by weight titanium and 1.77% by weight vanadium.\",\n \"Example 2 Ethylene Polymerization An autoclave with a volume of 3 liters was purged with nitrogen at 130° C. for 30 minutes.\",\n \"After cooling the autoclave to 85° C. the reactor was purged with hydrogen and then 1.5 liters of n-hexane were introduced to the reactor.\",\n \"The reactor was then pressurized to 3 barg hydrogen pressure.\",\n \"Then 5.0 cm3 of tri-isobutylaluminum, TIBAL (1.0 molar in n-hexane) was injected into the reactor.\",\n \"Ethylene was introduced to the reactor such as to raise the reactor pressure to 15 barg.\",\n \"This was followed by injection of 0.1 g of the solid catalyst precursor “A” described in Example 1 after being slurried in 20 cm3 of n-hexane solvent.\",\n \"Polymerization was carried out for 1 hour; with ethylene supplied on demand to maintain the total reactor pressure at 15 barg.\",\n \"390 grams of polyethylene were recovered with a catalyst productivity of 3900 gPE/g catalyst, a resin bulk density of 0.290 g/cm3, a weight average molecular weight of 94 050 g/mole, a number average molecular weight of 28 350, a molecular weight distribution of 3.32 and a melting point of 135° C. Examples 3,4 Ethylene Polymerization An autoclave with a volume of 3 liters was purged with nitrogen at 130° C. for 30 minutes.\",\n \"After cooling the autoclave to 85° C. the reactor was purged with hydrogen and then 1.5 liters of n-hexane were introduced to the reactor.\",\n \"The reactor was then pressurised to 3 barg hydrogen pressure.\",\n \"Then, the desired quantity of triethylaluminum, TEAL (1.0 molar in n-hexane), described in Table 1 below, was injected into the reactor.\",\n \"Ethylene was introduced to the reactor such as to raise the reactor pressure to 15 barg.\",\n \"This was followed by injection of 0.1 g of the solid catalyst precursor “A” described in Example 1 after being slurried in 20 cm3 of n-hexane solvent.\",\n \"Polymerization was carried out for 1 hour; with ethylene supplied on demand to maintain the total reactor pressure at 15 barg.\",\n \"Catalyst performance and product polymer properties are described in Table 1.TABLE 1 Influence of TEAL Concentration Productivity/ Bulk TEAL/ Yield/ (g PE/g Density/ Mw/ Mn/ DSC/ Example (mmol) (g PE) catalyst) (g/cm3) (1000 g/mol) (1000) MWD (Mp/° C.) 3 4 500 5000 0.310 135 36.4 3.71 138 4 6 455 4550 0.303 109 35.2 3.10 137 Examples 5,6 Ethylene Polymerization An autoclave with a volume of 3 liters was purged with nitrogen at 130° C. for 30 minutes.\",\n \"After cooling the autoclave to 85° C. the reactor was purged with hydrogen and then 1.5 liters of n-hexane were introduced to the reactor.\",\n \"The reactor was then pressurised to the desired hydrogen pressure, described in Table 2 below.\",\n \"Then 4.0 cm3 of triethylaluminum, TEAL (1.0 molar in n-hexane) was injected into the reactor.\",\n \"Ethylene was introduced to the reactor such as to raise the reactor pressure to 15 barg.\",\n \"This was followed by injection of 0.1 g of the solid catalyst precursor “A” described in Example 1 after being slurried in 20 cm3 of n-hexane solvent.\",\n \"Polymerization was carried out for 1 hour; with ethylene supplied on demand to maintain the total reactor pressure at 15 barg.\",\n \"Catalyst performance and product polymer properties are described in Table 2.TABLE 2 Influence of Hydrogen Pressure Hydrogen Productivity/ Bulk Pressure/ Yield/ (g PE/g Density/ Mw/ Mn/ DSC/ Example (barg) (g PE) catalyst) (g/cm3) (1000 g/mol) (1000) MWD (Mp/° C.) 5 2 490 4900 0.290 151 43.3 3.48 139 3 3 500 5000 0.310 135 36.4 3.71 13 6 4 305 3050 0.260 114 33.7 3.37 137 Example 7 Ethylene-Butene Copolymerization An autoclave with a volume of 3 liters was purged with nitrogen at 130° C. for 30 minutes.\",\n \"After cooling the autoclave to 85° C. the reactor was purged with hydrogen and then 1.5 liters of n-hexane were introduced to the reactor.\",\n \"Then the desired quantity of butene, described in Table 3 below, was added to the reactor.\",\n \"The reactor was then pressurized to 3 barg hydrogen pressure.\",\n \"Then 4.0 cm3 of triethylaluminum, TEAL (1.0 molar in n-hexane) was injected into the reactor.\",\n \"Ethylene was introduced to the reactor such as to raise the reactor pressure to 15 barg.\",\n \"This was followed by injection of 0.1 g of the solid catalyst precursor “A” described in Example 1 after being slurried in 20 cm3 of n-hexane solvent.\",\n \"Polymerization was carried out for 1 hour; with ethylene supplied on demand to maintain the total reactor pressure at 15 barg.\",\n \"Catalyst performance and product polymer properties are described in Table 3.TABLE 3 Influence of Butene Productivity/ Bulk Butene/ Yield/ (g PE/g Density/ Mw/ Mn/ DSC/ Example (cm3) (g PE) catalyst) (g/cm3) (1000 g/mol) (1000) MWD (Mp/° C.) 3 0 500 5000 0.310 135 36.4 3.71 138 7 10 502 5020 0.300 135 35.7 3.77 138 Example 8 Ethylene-Hexene Copolymerization An autoclave with a volume of 3 liters was purged with nitrogen at 130° C. for 30 minutes.\",\n \"After cooling the autoclave to 85° C. the reactor was purged with hydrogen and then 1.5 liters of n-hexane were introduced to the reactor.\",\n \"Then the desired quantity of hexene, described in Table 4 below, was added to the reactor.\",\n \"The reactor was then pressurized to 3 barg hydrogen pressure.\",\n \"Then 4.0 cm3 of triethylaluminum, TEAL (1.0 molar in n-hexane) was injected into to the reactor.\",\n \"Ethylene was introduced to the reactor such as to raise the: reactor pressure to 15 barg.\",\n \"This was followed by injection of 0.1 g of the solid catalyst precursor “A” described in Example 1 after being slurried in 20 cm3 of n-hexane solvent.\",\n \"Polymerization was carried out for 1 hour; with ethylene supplied on demand to maintain the total reactor pressure at 15 barg.\",\n \"Catalyst performance and product polymer properties are described in Table 4.TABLE 4 Influence of Hexene Productivity/ Bulk Hexene/ Yield/ (g PE/g Density/ Mw/ Mn/ DSC/ Example (cm3) (g PE) catalyst) (g/cm3) (1000 g/mol) (1000) MWD (Mp/° C.) 3 0 500 5000 0.310 135 36.4 3.71 138 8 10 476 4760 0.302 134 41.8 3.21 137 Example 9 Synthesis of Solid Catalyst Precursor (B) To a three-necked round bottom flask, equipped with a condenser and a magnetic stirrer, was placed 10.0 g of polyvinylchloride spheres with an average particle size of 36 microns.\",\n \"The flask containing the polyvinylchloride was heated up to 70° C. using an oil bath and then evacuated at 30 mm Hg pressure for 30 minutes.\",\n \"The flask and its contents were then purged with dried nitrogen and the polyvinylchloride was slurried using 30 cm3 of isopentane.\",\n \"Then 1.5 cm3 of butylmagnesium chloride (2.0 molar in diethylether) was added to the slurry and the resultant mixture was stirred for 60 minutes at an oil bath temperature of 35° C., under reflux conditions.\",\n \"The isopentane was then evaporated to obtain a free-flowing powder by using a nitrogen purge at 35° C. Then the magnesium-modified polyvinylchloride was slurried using 30 cm3 of isopentane and 2.0 cm3 of titanium tetraethoxide (1.0 molar in n-hexane) was added to the slurry and the resulting mixture was stirred at 35° C. for 40 minutes.\",\n \"A dark green/brown colored solid was produced.\",\n \"Then 8.0 cm3 of vanadium oxytrichloride (1.0 molar in n-hexane) was added to contents of the flask and the resulting mixture was stirred at 35° C. for 20 minutes.\",\n \"The supernatant liquid was decanted and the resulting solid product was washed by stirring with 80 cm3 of isopentane and then removing the isopentane, then washed again twice with 80 cm3 of isopentane in each wash.\",\n \"Finally, the solid catalyst was dried using a nitrogen purge at 35° C. to yield a free-flowing brown colored solid product.\",\n \"The solid catalyst precursor was analyzed by atomic adsorption spectroscopy and was found to contain 0.74% by weight magnesium, 0.56% by weight titanium and 1.50% by weight vanadium.\",\n \"Example 10 Ethylene Polymerization An autoclave with a volume of 3 liters was purged with nitrogen at 130° C. for 30 minutes.\",\n \"After cooling the autoclave to 85° C. the reactor was purged with hydrogen and then 1.5 liters of n-hexane were introduced to the reactor.\",\n \"The reactor was then pressurised to 3 barg hydrogen pressure.\",\n \"Then, 4.0 cm3 of triethylaluminum, TEAL (1.0 molar in n-hexane) was injected into the reactor.\",\n \"Ethylene was introduced to the reactor such as to raise the reactor pressure to 15 barg.\",\n \"This was followed by injection of 0.1 g of the solid catalyst precursor “B” described in Example 9 after being slurried in 20 cm3 of n-hexane solvent.\",\n \"Polymerization was carried out for 1 hour; with ethylene supplied on demand to maintain the total reactor pressure at 15 barg.\",\n \"200 grams of polyethylene were recovered with a catalyst productivity of 2000 gPE/g catalyst, a resin bulk density of 0.260 g/cm3, a weight average molecular weight of 157 000 g/mole, a number average molecular weight of 44 950, a molecular weight distribution of 3.49 and a melting point of 138° C. The features disclosed in the foregoing description, in the claims and/or in the drawing may, both separately and in any combination thereof, be material for realising the invention in diverse forms thereof.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10467389"}}},{"rowIdx":364,"cells":{"text":{"kind":"list like","value":[["Modulating apparatus and method, and dsv control bit producing method","The present invention relates to a modulation apparatus and method and a DSV-control-bit generating method for suppressing an increase in circuit size of the modulation apparatus.","When an input data stream is supplied to a DSV control bit determination unit 31, the DSV control bit determination unit 31 determines a DSV control bit to be inserted into the input data stream.","Upon supplying the input data stream to the DSV control bit determination unit 31, the input data stream is simultaneously supplied to a delay processor 32.","The input data stream is delayed for a predetermined delay time and supplied to a determined-DSV-control-bit insertion unit 33.","The determined-DSV-control-bit insertion unit 33 inserts the DSV control bit determined by the DSV control bit determination unit 3.1 into a predetermined position of the input data stream supplied by the delay means and supplies the input data stream containing the DSV control bit to a modulator 34.","The modulator 34 modulates the input data stream containing the DSV control bit into a code string in accordance with a predetermined coding rule (for example, 1,7PP modulation)."],["1.A modulation apparatus for generating a channel bit string from an input bit string and generating a recording code string or a transmission code string from the channel bit string, comprising: DSV-control-bit generating means for generating a DSV control bit to be inserted into the input bit string in order to control a DSV of the recording code string or the transmission code string; timing adjusting means for adjusting transmission timing for transmitting the input bit string; DSV-control-bit-inserted bit string generating means for generating a DSV-control-bit-inserted bit string by inserting the DSV control bit generated by the DSV-control-bit generating means into a predetermined position of the input bit string whose transmission timing is adjusted by the timing adjusting means; and first modulation means for modulating the DSV-control-bit-inserted bit string generated by the DSV-control-bit-inserted bit string generating means-into the channel bit string on the basis of a conversion rule (d, k; m, n; r).","2.The modulation apparatus according to claim 1, further comprising NRZI means for performing NRZI modulation of the channel bit string generated by modulation by the first modulation means to generate the recording code string or the transmission code string.","3.The modulation apparatus according to claim 1, wherein the conversion rule states that the remainder of the number of “1s” in a predetermined block of the input bit string or the DSV-control-bit-inserted bit string divided by two equals the remainder of the number of “1s” in the corresponding block of the channel bit string divided by two.","4.The modulation apparatus according to claim 1, wherein the conversion rule states that the number of consecutive minimum run length d of the channel bit string is limited to a predetermined number or less.","5.The modulation apparatus according to claim 1, wherein the conversion rule states a variable-length code (d, k; m, n; r), where the minimum run length d=1, the maximum run length k=7, the length of basic data prior to conversion m=2, and the length of basic channel bits subsequent to conversion n=3.6.The modulation apparatus according to claim 1, wherein data of m length, which is the length of basic data, is input for a time period in which the channel bit string of n length, which is the length of the basic channel bits, is output.","7.The modulation apparatus according to claim 1, wherein the DSV-control-bit generating mean includes: first-candidate-bit-inserted bit string generating means for generating a first-candidate-bit-inserted bit string, which is a candidate for the DSV-control-bit-inserted bit string, by inserting a first candidate bit for the DSV control bit into the predetermined position of the input bit string; second-candidate-bit-inserted bit string generating means for generating a second-candidate-bit-inserted bit string, which is another candidate for the DSV-control-bit-inserted bit string, by inserting a second candidate bit for the DSV control bit into the predetermined position of the input bit string; second modulation means for modulating, on the basis of the same conversion rule as the conversion rule used by the first modulation means, the first-candidate-bit-inserted bit string generated by the first-candidate-bit-inserted bit string generating means into a first candidate channel bit string, which is a candidate for the channel bit string, and for modulating the second-candidate-bit-inserted bit string generated by the second-candidate-bit-inserted bit string generating means into a second candidate channel bit string, which is another candidate for the channel bit string; DSV calculating means for calculating a DSV of each of the first and second candidate channel bit strings generated by modulation by the second modulation means; and DSV control bit determining means for determining one of the first and second candidate bits as the DSV control bit on the basis of the DSVs calculated by the DSV calculating means.","8.The modulation apparatus according to claim 7, wherein the DSV calculating means includes: section DSV calculating means for calculating a section DSV of a current DSV control section of each of the first and second candidate channel bit strings; cumulative DSV calculating means for calculating a cumulative DSV on the basis of the determination result by the DSV control bit determining means; and adding means for calculating the DSV by adding each section DSV calculated by the section DSV calculating means and the cumulative DSV immediately before the current DSV control section, the cumulative DSV being calculated by the cumulative DSV calculating means.","9.The modulation apparatus according to claim 7, wherein the first and second modulation means each have a minimum number of registers required to perform modulation based on the coding rule.","10.The modulation apparatus according to claim 1, further comprising: first sync signal inserting means for inserting a sync pattern including a preset unique pattern into the channel bit string, wherein the DSV-control-bit generating means includes second sync signal inserting means for inserting the same sync pattern as the sync pattern inserted by the first sync signal inserting means into each of the first and second candidate channel bit strings generated by modulating the first- and second-candidate-bit-inserted bit strings generated by inserting the first and second candidate bits, respectively, into the input bit string, and wherein the DSV calculating means calculates the DSV on the basis of each of the first and second candidate channel bit strings, each of which includes the sync pattern inserted by the second sync signal inserting means.","11.The modulation apparatus according to claim 1, wherein the timing adjusting means adjusts the transmission timing by adding a delay time to the input bit string.","12.The modulation apparatus according to claim 1, wherein the timing adjusting means inserts a temporary value prior to determination of the DSV control bit into the input bit string at predetermined intervals.","13.The modulation apparatus according to claim 1, further comprising checking information generating means for calculating a final cumulative DSV of the recording code string or the transmission code string, determining whether or not the calculated final cumulative DSV is within a predetermined range, and generating checking information on the basis of the determination result, wherein the DSV-control-bit generating means generates the DSV control bit on the basis of the checking information generated by the checking information generating means.","14.The modulation apparatus according to claim 13, wherein, when it is determined that the final cumulative DSV is out of the predetermined range, the checking information generating means resets the final cumulative DSV to zero and generates an error signal serving as the checking information, and wherein the DSV-control-bit generating means internally calculates a cumulative DSV for generating the DSV control bit and, when the error signal is generated by the checking information generating means, resets the cumulative DSV to zero.","15.A modulation method for a modulation apparatus for generating a channel bit string from an input bit string and generating a recording code string or a transmission code string from the channel bit string, comprising: a DSV-control-bit generating step of generating a DSV control bit to be inserted into the input bit string in order to control a DSV of the recording code string or the transmission code string; a timing adjusting step of adjusting transmission timing for transmitting the input bit string; a DSV-control-bit-inserted bit string generating step of generating a DSV-control-bit-inserted bit string by inserting the DSV control bit generated in the DSV-control-bit generating step into a predetermined position of the input bit string whose transmission timing is adjusted in the timing adjusting step; and a modulation step of modulating the DSV-control-bit-inserted bit string generated in the DSV-control-bit-inserted bit string generating step into the channel bit string on the basis of a conversion rule (d, k; m, n; r).","16.A recording medium providing a computer-readable computer program for controlling a modulation apparatus for generating a channel bit string from an input bit string and generating a recording code string or a transmission code string from the channel bit string, the program comprising: a DSV-control-bit generating step of generating a DSV control bit to be inserted into the input bit string in order to control a DSV of the recording code string or the transmission code string; a timing adjusting step of adjusting transmission timing for transmitting the input bit string; a DSV-control-bit-inserted bit string generating step of generating a DSV-control-bit-inserted bit string by inserting the DSV control bit generated in the DSV-control-bit generating step into a predetermined position of the input bit string whose transmission timing is adjusted in the timing adjusting step; and a modulation step of modulating the DSV-control-bit-inserted bit string generated in the DSV-control-bit-inserted bit string generating step into the channel bit string on the basis of a conversion rule (d, k; m, n; r).","17.A program for causing a computer that controls a modulation apparatus for generating a channel bit string from an input bit string and generating a recording code string or a transmission code string from the channel bit string to perform a process comprising: a DSV-control-bit generating step of generating a DSV control bit to be inserted into the input bit string in order to control a DSV of the recording code string or the transmission code string; a timing adjusting step of adjusting transmission timing for transmitting the input bit string; a DSV-control-bit-inserted bit string generating step of generating a DSV-control-bit-inserted bit string by inserting the DSV control bit generated in the DSV-control-bit generating step into a predetermined position of the input bit string whose transmission timing is adjusted in the timing adjusting step; and a modulation step of modulating the DSV-control-bit-inserted bit string generated in the DSV-control-bit-inserted bit string generating step into the channel bit string on the basis of a conversion rule (d, k; m, n; r).","18.A DSV-control-bit generating method for generating a DSV control bit to be inserted into an input bit string, comprising: a first-candidate-bit-inserted bit string generating step of generating a first-candidate-bit-inserted bit string, which is a candidate for a DSV-control-bit-inserted bit string, by inserting a first candidate bit for the DSV control bit into a predetermined position of the input bit string; a second-candidate-bit-inserted bit string generating step of generating a second-candidate-bit-inserted bit string, which is another candidate for the DSV-control-bit-inserted bit string, by inserting a second candidate bit for the DSV control bit into the predetermined position of the input bit string; a modulation step of modulating, on the basis of the same conversion rule as a conversion rule applied when modulating the input bit string, the first-candidate-bit-inserted bit string generated in the first-candidate-bit-inserted bit string generating step into a first candidate channel bit string, which is a candidate for a channel bit string generated from the input bit string, and for modulating the second-candidate-bit-inserted bit string generated in the second-candidate-bit-inserted bit string generating step into a second candidate channel bit string, which is another candidate for the channel bit string; a DSV calculating step of calculating a DSV of each of the first and second candidate channel bit strings generated by modulation in the modulation step; and a DSV control bit determining step of determining one of the first and second candidate bits as the DSV control bit on the basis of the DSVs calculated in the DSV calculating step.","19.A recording medium providing a computer-readable program for generating a DSV control bit to be inserted into an input bit string, the program comprising: a first-candidate-bit-inserted bit string generating step of generating a first-candidate-bit-inserted bit string, which is a candidate for a DSV-control-bit-inserted bit string, by inserting a first candidate bit for the DSV control bit into a predetermined position of the input bit string; a second-candidate-bit-inserted bit string generating step of generating a second-candidate-bit-inserted bit string, which is another candidate for the DSV-control-bit-inserted bit string, by inserting a second candidate bit for the DSV control bit into the predetermined position of the input bit string; a modulation step of modulating, on the basis of the same conversion rule as a conversion rule applied when modulating the input bit string, the first-candidate-bit-inserted bit string generated in the first-candidate-bit-inserted bit string generating step into a first candidate channel bit string, which is a candidate for a channel bit string generated from the input bit string, and for modulating the second-candidate-bit-inserted bit string generated in the second-candidate-bit-inserted bit string generating step into a second candidate channel bit string, which is another candidate for the channel bit string; a DSV calculating step of calculating a DSV of each of the first and second candidate channel bit strings generated by modulation in the modulation step; and a DSV control bit determining step of determining one of the first and second candidate bits as the DSV control bit on the basis of the DSVs calculated in the DSV calculating step.","20.A program for generating a DSV control bit to be inserted into an input bit string, the program causing a computer to perform a process comprising: a first-candidate-bit-inserted bit string generating step of generating a first-candidate-bit-inserted bit string, which is a candidate for a DSV-control-bit-inserted bit string, by inserting a first candidate bit for the DSV control bit into a predetermined position of the input bit string; a second-candidate-bit-inserted bit string generating step of generating a second-candidate-bit-inserted bit string, which is another candidate for the DSV-control-bit-inserted bit string, by inserting a second candidate bit for the DSV control bit into the predetermined position of the input bit string; a modulation step of modulating, on the basis of the same conversion rule as a conversion rule applied when modulating the input bit string, the first-candidate-bit-inserted bit string generated in the first-candidate-bit-inserted bit string generating step into a first candidate channel bit string, which is a candidate for a channel bit string generated from the input bit string, and for modulating the second-candidate-bit-inserted bit string generated in the second-candidate-bit-inserted bit string generating step into a second candidate channel bit string, which is another candidate for the channel bit string; a DSV calculating step of calculating a DSV of each of the first and second candidate channel bit strings generated by modulation in the modulation step; and a DSV control bit determining step of determining one of the first and second candidate bits as the DSV control bit on the basis of the DSVs calculated in the DSV calculating step."],["<SOH> BACKGROUND ART <EOH>In transferring data to a predetermined transmission line or in recording data onto a recording medium, such as a magnetic disk, an optical disk, or a magneto-optical disk, the data is modulated to suit the transfer or recording.","One known modulation method is block coding.","Block coding is to divide a data stream into blocks of m×i bits (hereinafter the blocks are referred to as data words), and each data word is converted into a codeword of n×i bits in accordance with an appropriate coding rule.","When i=1, the resultant code is a fixed-length code.","When i can be of multiple values, that is, when i is selected from a range of 1 to i max (maximum i) and conversion is performed with the selected i, the resultant code is a variable-length code.","The code generated by block coding is defined as a variable-length code (d, k; m, n; r).","In the above description, i denotes the constraint length, and i max is r (maximum constraint length); d denotes the minimum number of consecutive “0s” between consecutive “1s”, e.g., the minimum run length of “0”; and k denotes the maximum number of consecutive “0s” between consecutive “1s”, e.g., the maximum run length of “0”.","In recording the code generated as described above onto an optical disk, a magneto-optical disk, or the like, such as a compact disk (CD) or mini disk (MD), the variable-length code is subjected to NRZI (Non Return to Zero Inverted) modulation in which “1” is inverted whereas “0” is not inverted, and recording is done on the basis of the NRZI-modulated variable-length code (hereinafter referred to as the recording code string).","There is a system, such as that on a magneto-optical disk in an initial ISO format having a not-so-high recording density, which records a modulated recording bit string that has not been subjected to NRZI modulation.","The minimum inversion interval of the recording code string is denoted by Tmin, and the maximum inversion interval of the recording code string is denoted by Tmax.","Higher recording density in the direction of linear velocity is realized when the minimum inversion interval Tmin is longer, that is, when the minimum run length d is greater.","On the other hand, in terms of clock reading, it is preferable to have a shorter maximum inversion interval Tmax, that is, a smaller maximum run length k. Various modulations methods have been proposed.","Specifically, modulation systems proposed or actually used in, for example, optical disks, magnetic disks, magneto-optical disks, and the like are described as follows.","For example, RLL codes (Run Length Limited Code) with the minimum run length d=2 include an EFM (Eight to Fourteen Modulation) code (may also be represented as (2, 10: 8, 17; 1)) used in CD, MD, and the like; an 8-16 code (may also be represented as (2, 10: 8, 16; 1)) used in DVD (Digital Video Disk); an RLL (2-7) (may also be represented as (2, 7; 1, 2; r)) used in PD (Phase Change Disk); and the like.","RLL codes with the minimum run length d=1 include a fixed-length RLL (1-7) (may also be represented as (1, 7; 2, 3; 1)) used in an ISO-format MO disk (Magnetic-Optical Disk); and a variable-length RLL (1-7) (may also be represented as (1, 7; 2, 3; r)) used in a disk drive for a high-density optical disk, magneto-optical disk, or the like.","A conversion table for the variable-length RLL (1-7) is as follows: TABLE 1 RLL (1, 7, 2, 3, 2) Data word Codeword i = 1 11 00x 10 010 01 10x i = 2 0011 000 00x 0010 000 010 0001 100 00x 0000 100 010 The symbol x in the conversion table corresponds to 1 when the subsequent codeword is 0 and corresponds to 0 when the subsequent codeword is 1.The maximum constraint length r is 2.The parameters of the variable-length RLL (1-7) are (1, 7; 2, 3; 2).","When the bit interval of the recording code string is denoted by T, the minimum inversion interval Tmin expressed as (d+1) is 2(=1+1)T. When the bit interval of the data stream is denoted by Tdata, the minimum inversion interval Tmin expressed as (m/n)×2 is 1.33(=(⅔)×2)Tdata.","In the above description, m/n denotes conversion at the ratio m:n. For example, ⅔ denotes conversion at 2:3 (conversion of a data word of 2×i bits into a codeword of 3×i bits).","The maximum inversion interval Tmax expressed as (k+1)T is 8(=7+1)T ((=(⅔)×8Tdata=5.33Tdata).","The detection window margin Tw is expressed as (m/n)×Tdata and is 0.67(=⅔)Tdata.","In a code string (channel bit string) generated by RLL (1-7) modulation in Table 1, 2T which is Tmin occurs most frequently, which is followed by 3T, 4T, and so forth.","The fact that edge information, such as 2T or 3T, occurs many times with a short cycle is advantageous in reading clock.","In contrast, when the recording linear density becomes higher, the minimum run length causes a problem.","Specifically, when 2T (the minimum run length) occurs consecutively, the recording waveform is easily distorted because the waveform output of 2T is smaller than the others and is influenced more easily by defocus, tangential tilt, or the like.","In recording at high linear density, recording with consecutive minimum marks is influenced more easily by disturbance, such as noise, and this may easily lead to a data read error.","In such a case in which a data read error occurs, the error often resides in shift of start-edge and end-edge of the consecutive minimum marks.","In other words, the generated bit error length becomes longer.","In order to solve this problem, the consecutive minimum run lengths are controlled to better suit high linear density.","In contrast, in recording onto a recording medium or in data transfer, code modulation based on each medium (transfer) is done.","When a modulated code contains a DC component, fluctuation or jitter may be caused in various error signals, such as for a tracking error in servo control of a disk drive.","It is preferable for the modulated code not to contain a DC component.","In order to solve this problem, DSV (Digital Sum Value) control is proposed.","DSV is the sum of bits of an NRZI-modulated (level-coded) bit string (channel bit string) in which “1” corresponds to +1 and “0” corresponds to −1.DSV serves as a reference for a DC component in the code string.","Minimizing the absolute value of DSV, that is, performing DSV control, enables suppression of a DC component in the code string.","In the code modulated according to the variable-length RLL (1-7) table shown in Table 1, no DSV control is performed.","In such a case, DSV control is performed by calculating DSV of the modulated channel bit string at a predetermined interval and inserting predetermined DSV control bits into the code string.","Basically, the DSV control bits are redundant bits.","In view of the efficiency of code conversion, the fewer the DSV control bits, the better.","It is preferable that the minimum run length d and the maximum run length k remain unchanged by the inserted DSV control bits.","Changes of (d, k) influence the reading and writing characteristics.","In order to satisfy the above requirements, DSV control must be performed in as efficiently as possible.","While the actual RLL code must satisfy the minimum run length requirement, the maximum run length requirement need not be satisfied.","There is a format that uses a pattern exceeding the maximum run length for a sync signal.","For example, although EFM plus for DVD has a maximum run length of 11T, EFM plus for DVD allows for 14T as a matter of convenience of the format.","By exceeding the maximum run length, for example, the capability of detecting a sync signal or the like is enhanced greatly.","In the RLL (1-7) format with improved conversion efficiency, it is important to “control the consecutive minimum run lengths so as to better suit the high linear density” and to “perform DSV control as efficiently as possible” in association with an increase in linear density.","Accordingly, the assignee of the present invention et al.","discloses, in Japanese Patent Application No.","10-150280, a conversion table including, as a conversion code, a basic code where d=1, k=7, m=2, and n=3; a coding rule that the remainder of the number of “1s” in each element of a data stream divided by two must be one or zero and equal the remainder of the number of “1s” in a converted channel bit string divided by two; a first replacement code for limiting the consecutive minimum run lengths d to a predetermined number or less; and a second replacement code for satisfying the run length limitation.","Specifically, when a disk drive with high linear density reads/writes an RLL code, a pattern with consecutive minimum run lengths often causes a long error.","When DSV control is performed on an RLL code, such as the RLL (1-7) code, DSV control bits need to be inserted into a code string (channel bit string) at arbitrary intervals.","As described above, since the DSV control bits are redundant bits, it is preferable to have fewer DSV control bits.","In order to maintain the minimum run length or the maximum run length, the DSV control bits of at least 2 bits or greater are necessary.","The assignee of the present invention et al.","discloses, in Japanese Patent Application No.","10-150280, an RLL code with the minimum run length d=1 (d, k; m, n) and a conversion table, which is shown in Table 2, for limiting the number of consecutive minimum run lengths and for performing complete DSV control using efficient control bits while maintaining the minimum run length and the maximum run length (hereinafter referred to as a 1,7PP table; and a code according to 1,7PP table is referred to as a 1,7PP code): TABLE 2 1, 7PP (d, k, m, n, r) = (1, 7, 2, 3, 4) Data word Codeword 11 *0* 10 001 01 010 0011 010 100 0010 010 000 0001 000 100 000011 000 100 100 000010 000 100 000 000001 010 100 100 000000 010 100 000 “110111 001 000 000 (next010) 00001000 000 100 100 100 00000000 010 100 100 100 if xx1 then *0* = 000 xx0 then *0* = 101 Termination table 00 000 0000 010 100 “110111 001 000 000 (next010) When next channel bits are ‘010’ convert ‘11 01 11’ to ‘001 000 000’ after using main table and termination table.","As an example of a modulation apparatus using the 1,7PP table, the assignee of the present invention discloses, in Japanese Patent Application No.","10-150280, a modulation apparatus 1 shown in FIG.","1 .","The modulation apparatus 1 includes a DSV control bit determination and insertion unit 11 for determining “1” or “0” serving as a DSV control bit and inserting the DSV control bit into an input data stream at arbitrary intervals; a modulator 12 for modulating the data stream containing the DSV control bits; and an NRZI unit 13 for converting the output of the modulator 12 into a recording code string.","Although not shown in the diagram, the modulation apparatus 1 includes a timing management unit for generating a timing signal, supplying the timing signal to the above components, and managing timing.","In Japanese Patent Application No.","09-342416, the assignee of the present invention et al.","discloses a specific example of another modulation apparatus, namely, a modulation apparatus 2 shown in FIG.","2 .","The modulation apparatus includes a DSV control bit insertion unit 21 for inserting “1” or “0” serving as a DSV control bit into a data stream at arbitrary intervals.","At this time, there is a data stream into which the DSV control bit “ 1 ” is inserted and another data stream into which the DSV control bit “ 0 ” is inserted.","The modulation apparatus further includes a modulator 22 for modulating the data stream containing the DSV control bits and a DSV controller 23 for NRZI-modulating the modulated code string into level data, calculating DSV of the level data, and consequently outputting a DSV-controlled recording code string.","As described above, the 1,7PP code is advantageous in solving the above problems.","In contrast, compared with a modulation apparatus using a known method or a technique for performing DSV control on the RLL (1,7) code, the configuration of the known modulation apparatus using the 1,7PP code is complicated, and the circuit size is increased.","For example, in the modulation apparatus 2 shown in FIG.","2 , the register configuration in the modulator 22 is shown in FIG.","3 .","Specifically, the modulator 22 has a modulation (1,7PP modulation) portion and a delay portion corresponding to a DSV control interval (DSV section) in one integrated unit in order to transfer data corresponding to the DSV control interval to the DSV controller 23 at a subsequent stage.","As a result, the modulator 22 needs two registers, that is, an input register 22 a (register 22 a for the data stream) and an output register 22 b (register 22 b for the channel bit string).","The number of registers required corresponds to the DSV control interval.","Two pairs of registers (registers 22 a and 22 b ) are necessary for the DSV control bit “0” and the DSV control bit “1”."],["<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.","1 is a block diagram showing an example of the configuration of a known modulation apparatus.","FIG.","2 is a block diagram showing an example of the configuration of another known modulation apparatus.","FIG.","3 is a diagram showing an example of the register configuration in a modulator of the modulation apparatus shown in FIG.","2 .","FIG.","4 is a block diagram showing an example of the configuration of a modulation apparatus according to the present invention.","FIG.","5 is a block diagram showing details of the configuration of the modulation apparatus shown in FIG.","4 .","FIG.","6 is a flowchart for describing the operation of the modulation apparatus shown in FIG.","4 .","FIG.","7 is a diagram for describing the data format at each stage of a data stream modulated by the modulation apparatus shown in FIG.","4 .","FIG.","8 is a chart for describing timing of data to be input to the modulation apparatus shown in FIG.","4 .","FIG.","9 is a diagram showing an example of the register configuration in a modulator of the modulation apparatus shown in FIG.","4 .","FIG.","10 is a block diagram showing an example of the configuration of another modulation apparatus according to the present invention.","detailed-description description=\"Detailed Description\" end=\"lead\"?"],["TECHNICAL FIELD The present invention relates to modulation apparatuses and methods and DSV-control-bit generating methods, and more particularly relates to a modulation apparatus and method and a DSV-control-bit generating method for suppressing an increase in circuit size.","BACKGROUND ART In transferring data to a predetermined transmission line or in recording data onto a recording medium, such as a magnetic disk, an optical disk, or a magneto-optical disk, the data is modulated to suit the transfer or recording.","One known modulation method is block coding.","Block coding is to divide a data stream into blocks of m×i bits (hereinafter the blocks are referred to as data words), and each data word is converted into a codeword of n×i bits in accordance with an appropriate coding rule.","When i=1, the resultant code is a fixed-length code.","When i can be of multiple values, that is, when i is selected from a range of 1 to i max (maximum i) and conversion is performed with the selected i, the resultant code is a variable-length code.","The code generated by block coding is defined as a variable-length code (d, k; m, n; r).","In the above description, i denotes the constraint length, and i max is r (maximum constraint length); d denotes the minimum number of consecutive “0s” between consecutive “1s”, e.g., the minimum run length of “0”; and k denotes the maximum number of consecutive “0s” between consecutive “1s”, e.g., the maximum run length of “0”.","In recording the code generated as described above onto an optical disk, a magneto-optical disk, or the like, such as a compact disk (CD) or mini disk (MD), the variable-length code is subjected to NRZI (Non Return to Zero Inverted) modulation in which “1” is inverted whereas “0” is not inverted, and recording is done on the basis of the NRZI-modulated variable-length code (hereinafter referred to as the recording code string).","There is a system, such as that on a magneto-optical disk in an initial ISO format having a not-so-high recording density, which records a modulated recording bit string that has not been subjected to NRZI modulation.","The minimum inversion interval of the recording code string is denoted by Tmin, and the maximum inversion interval of the recording code string is denoted by Tmax.","Higher recording density in the direction of linear velocity is realized when the minimum inversion interval Tmin is longer, that is, when the minimum run length d is greater.","On the other hand, in terms of clock reading, it is preferable to have a shorter maximum inversion interval Tmax, that is, a smaller maximum run length k. Various modulations methods have been proposed.","Specifically, modulation systems proposed or actually used in, for example, optical disks, magnetic disks, magneto-optical disks, and the like are described as follows.","For example, RLL codes (Run Length Limited Code) with the minimum run length d=2 include an EFM (Eight to Fourteen Modulation) code (may also be represented as (2, 10: 8, 17; 1)) used in CD, MD, and the like; an 8-16 code (may also be represented as (2, 10: 8, 16; 1)) used in DVD (Digital Video Disk); an RLL (2-7) (may also be represented as (2, 7; 1, 2; r)) used in PD (Phase Change Disk); and the like.","RLL codes with the minimum run length d=1 include a fixed-length RLL (1-7) (may also be represented as (1, 7; 2, 3; 1)) used in an ISO-format MO disk (Magnetic-Optical Disk); and a variable-length RLL (1-7) (may also be represented as (1, 7; 2, 3; r)) used in a disk drive for a high-density optical disk, magneto-optical disk, or the like.","A conversion table for the variable-length RLL (1-7) is as follows: TABLE 1 RLL (1, 7, 2, 3, 2) Data word Codeword i = 1 11 00x 10 010 01 10x i = 2 0011 000 00x 0010 000 010 0001 100 00x 0000 100 010 The symbol x in the conversion table corresponds to 1 when the subsequent codeword is 0 and corresponds to 0 when the subsequent codeword is 1.The maximum constraint length r is 2.The parameters of the variable-length RLL (1-7) are (1, 7; 2, 3; 2).","When the bit interval of the recording code string is denoted by T, the minimum inversion interval Tmin expressed as (d+1) is 2(=1+1)T. When the bit interval of the data stream is denoted by Tdata, the minimum inversion interval Tmin expressed as (m/n)×2 is 1.33(=(⅔)×2)Tdata.","In the above description, m/n denotes conversion at the ratio m:n. For example, ⅔ denotes conversion at 2:3 (conversion of a data word of 2×i bits into a codeword of 3×i bits).","The maximum inversion interval Tmax expressed as (k+1)T is 8(=7+1)T ((=(⅔)×8Tdata=5.33Tdata).","The detection window margin Tw is expressed as (m/n)×Tdata and is 0.67(=⅔)Tdata.","In a code string (channel bit string) generated by RLL (1-7) modulation in Table 1, 2T which is Tmin occurs most frequently, which is followed by 3T, 4T, and so forth.","The fact that edge information, such as 2T or 3T, occurs many times with a short cycle is advantageous in reading clock.","In contrast, when the recording linear density becomes higher, the minimum run length causes a problem.","Specifically, when 2T (the minimum run length) occurs consecutively, the recording waveform is easily distorted because the waveform output of 2T is smaller than the others and is influenced more easily by defocus, tangential tilt, or the like.","In recording at high linear density, recording with consecutive minimum marks is influenced more easily by disturbance, such as noise, and this may easily lead to a data read error.","In such a case in which a data read error occurs, the error often resides in shift of start-edge and end-edge of the consecutive minimum marks.","In other words, the generated bit error length becomes longer.","In order to solve this problem, the consecutive minimum run lengths are controlled to better suit high linear density.","In contrast, in recording onto a recording medium or in data transfer, code modulation based on each medium (transfer) is done.","When a modulated code contains a DC component, fluctuation or jitter may be caused in various error signals, such as for a tracking error in servo control of a disk drive.","It is preferable for the modulated code not to contain a DC component.","In order to solve this problem, DSV (Digital Sum Value) control is proposed.","DSV is the sum of bits of an NRZI-modulated (level-coded) bit string (channel bit string) in which “1” corresponds to +1 and “0” corresponds to −1.DSV serves as a reference for a DC component in the code string.","Minimizing the absolute value of DSV, that is, performing DSV control, enables suppression of a DC component in the code string.","In the code modulated according to the variable-length RLL (1-7) table shown in Table 1, no DSV control is performed.","In such a case, DSV control is performed by calculating DSV of the modulated channel bit string at a predetermined interval and inserting predetermined DSV control bits into the code string.","Basically, the DSV control bits are redundant bits.","In view of the efficiency of code conversion, the fewer the DSV control bits, the better.","It is preferable that the minimum run length d and the maximum run length k remain unchanged by the inserted DSV control bits.","Changes of (d, k) influence the reading and writing characteristics.","In order to satisfy the above requirements, DSV control must be performed in as efficiently as possible.","While the actual RLL code must satisfy the minimum run length requirement, the maximum run length requirement need not be satisfied.","There is a format that uses a pattern exceeding the maximum run length for a sync signal.","For example, although EFM plus for DVD has a maximum run length of 11T, EFM plus for DVD allows for 14T as a matter of convenience of the format.","By exceeding the maximum run length, for example, the capability of detecting a sync signal or the like is enhanced greatly.","In the RLL (1-7) format with improved conversion efficiency, it is important to “control the consecutive minimum run lengths so as to better suit the high linear density” and to “perform DSV control as efficiently as possible” in association with an increase in linear density.","Accordingly, the assignee of the present invention et al.","discloses, in Japanese Patent Application No.","10-150280, a conversion table including, as a conversion code, a basic code where d=1, k=7, m=2, and n=3; a coding rule that the remainder of the number of “1s” in each element of a data stream divided by two must be one or zero and equal the remainder of the number of “1s” in a converted channel bit string divided by two; a first replacement code for limiting the consecutive minimum run lengths d to a predetermined number or less; and a second replacement code for satisfying the run length limitation.","Specifically, when a disk drive with high linear density reads/writes an RLL code, a pattern with consecutive minimum run lengths often causes a long error.","When DSV control is performed on an RLL code, such as the RLL (1-7) code, DSV control bits need to be inserted into a code string (channel bit string) at arbitrary intervals.","As described above, since the DSV control bits are redundant bits, it is preferable to have fewer DSV control bits.","In order to maintain the minimum run length or the maximum run length, the DSV control bits of at least 2 bits or greater are necessary.","The assignee of the present invention et al.","discloses, in Japanese Patent Application No.","10-150280, an RLL code with the minimum run length d=1 (d, k; m, n) and a conversion table, which is shown in Table 2, for limiting the number of consecutive minimum run lengths and for performing complete DSV control using efficient control bits while maintaining the minimum run length and the maximum run length (hereinafter referred to as a 1,7PP table; and a code according to 1,7PP table is referred to as a 1,7PP code): TABLE 2 1, 7PP (d, k, m, n, r) = (1, 7, 2, 3, 4) Data word Codeword 11 *0* 10 001 01 010 0011 010 100 0010 010 000 0001 000 100 000011 000 100 100 000010 000 100 000 000001 010 100 100 000000 010 100 000 “110111 001 000 000 (next010) 00001000 000 100 100 100 00000000 010 100 100 100 if xx1 then *0* = 000 xx0 then *0* = 101 Termination table 00 000 0000 010 100 “110111 001 000 000 (next010) When next channel bits are ‘010’ convert ‘11 01 11’ to ‘001 000 000’ after using main table and termination table.","As an example of a modulation apparatus using the 1,7PP table, the assignee of the present invention discloses, in Japanese Patent Application No.","10-150280, a modulation apparatus 1 shown in FIG.","1.The modulation apparatus 1 includes a DSV control bit determination and insertion unit 11 for determining “1” or “0” serving as a DSV control bit and inserting the DSV control bit into an input data stream at arbitrary intervals; a modulator 12 for modulating the data stream containing the DSV control bits; and an NRZI unit 13 for converting the output of the modulator 12 into a recording code string.","Although not shown in the diagram, the modulation apparatus 1 includes a timing management unit for generating a timing signal, supplying the timing signal to the above components, and managing timing.","In Japanese Patent Application No.","09-342416, the assignee of the present invention et al.","discloses a specific example of another modulation apparatus, namely, a modulation apparatus 2 shown in FIG.","2.The modulation apparatus includes a DSV control bit insertion unit 21 for inserting “1” or “0” serving as a DSV control bit into a data stream at arbitrary intervals.","At this time, there is a data stream into which the DSV control bit “1” is inserted and another data stream into which the DSV control bit “0” is inserted.","The modulation apparatus further includes a modulator 22 for modulating the data stream containing the DSV control bits and a DSV controller 23 for NRZI-modulating the modulated code string into level data, calculating DSV of the level data, and consequently outputting a DSV-controlled recording code string.","As described above, the 1,7PP code is advantageous in solving the above problems.","In contrast, compared with a modulation apparatus using a known method or a technique for performing DSV control on the RLL (1,7) code, the configuration of the known modulation apparatus using the 1,7PP code is complicated, and the circuit size is increased.","For example, in the modulation apparatus 2 shown in FIG.","2, the register configuration in the modulator 22 is shown in FIG.","3.Specifically, the modulator 22 has a modulation (1,7PP modulation) portion and a delay portion corresponding to a DSV control interval (DSV section) in one integrated unit in order to transfer data corresponding to the DSV control interval to the DSV controller 23 at a subsequent stage.","As a result, the modulator 22 needs two registers, that is, an input register 22a (register 22a for the data stream) and an output register 22b (register 22b for the channel bit string).","The number of registers required corresponds to the DSV control interval.","Two pairs of registers (registers 22a and 22b) are necessary for the DSV control bit “0” and the DSV control bit “1”.","DISCLOSURE OF INVENTION In view of these circumstances, it is an object of the present invention to suppress an increase in circuit size of a modulation apparatus.","A modulation apparatus of the present invention includes DSV-control-bit generating means for generating a DSV control bit to be inserted into an input bit string in order to control a DSV of a recording code string or a transmission code string; timing adjusting means for adjusting transmission timing for transmitting the input bit string; DSV-control-bit-inserted bit string generating means for generating a DSV-control-bit-inserted bit string by inserting the DSV control bit generated by the DSV-control-bit generating means into a predetermined position of the input bit string whose transmission timing is adjusted by the timing adjusting means; and first modulation means for modulating the DSV-control-bit-inserted bit string generated by the DSV-control-bit-inserted bit string generating means into a channel bit string on the basis of a conversion rule (d, k; m, n; r).","The modulation apparatus may further include NRZI means for performing NRZI modulation of the channel bit string generated by modulation by the first modulation means to generate the recording code string or the transmission code string.","The conversion rule may state that the remainder of the number of “1s” in a predetermined block of the input bit string or the DSV-control-bit-inserted bit string divided by two equals the remainder of the number of “1s” in the corresponding block of the channel bit string divided by two.","The conversion rule may state that the number of consecutive minimum run length d of the channel bit string is limited to a predetermined number or less.","The conversion rule may state a variable-length code (d, k; m, n; r) where the minimum run length d=1, the maximum run length k=7, the length of basic data prior to conversion m=2, and the length of basic channel bits subsequent to conversion n=3.Data of m length, which is the length of basic data, may be input for a time period in which the channel bit string of n length, which is the length of the basic channel bits, is output.","The DSV-control-bit generating mean may include first-candidate-bit-inserted bit string generating means for generating a first-candidate-bit-inserted bit string, which is a candidate for the DSV-control-bit-inserted bit string, by inserting a first candidate bit for the DSV control bit into the predetermined position of the input bit string; second-candidate-bit-inserted bit string generating means for generating a second-candidate-bit-inserted bit string, which is another candidate for the DSV-control-bit-inserted bit string, by inserting a second candidate bit for the DSV control bit into the predetermined position of the input bit string; second modulation means for modulating, on the basis of the same conversion rule as the conversion rule used by the first modulation means, the first-candidate-bit-inserted bit string generated by the first-candidate-bit-inserted bit string generating means into a first candidate channel bit string, which is a candidate for the channel bit string, and for modulating the second-candidate-bit-inserted bit string generated by the second-candidate-bit-inserted bit string generating means into a second candidate channel bit string, which is another candidate for the channel bit string; DSV calculating means for calculating a DSV of each of the first and second candidate channel bit strings generated by modulation by the second modulation means; and DSV control bit determining means for determining one of the first and second candidate bits as the DSV control bit on the basis of the DSVs calculated by the DSV calculating means.","The DSV calculating means may include section DSV calculating means for calculating a section DSV of a current DSV control section of each of the first and second candidate channel bit strings; cumulative DSV calculating means for calculating a cumulative DSV on the basis of the determination result by the DSV control bit determining means; and adding means for calculating the DSV by adding each section DSV calculated by the section DSV calculating means and the cumulative DSV immediately before the current DSV control section, the cumulative DSV being calculated by the cumulative DSV calculating means.","The first and second modulation means each may have a minimum number of registers required to perform modulation based on the coding rule.","The modulation apparatus may further include first sync signal inserting means for inserting a sync pattern including a preset unique pattern into the channel bit string.","The DSV-control-bit generating means may include second sync signal inserting means for inserting the same sync pattern as the sync pattern inserted by the first sync signal inserting means into each of the first and second candidate channel bit strings generated by modulating the first- and second-candidate-bit-inserted bit strings generated by inserting the first and second candidate bits, respectively, into the input bit string.","The DSV calculating means may calculate the DSV on the basis of each of the first and second candidate channel bit strings, each of which includes the sync pattern inserted by the second sync signal inserting means.","The timing adjusting means may adjust the transmission timing by adding a delay time to the input bit string.","The timing adjusting means may insert a temporary value prior to determination of the DSV control bit into the input bit string at predetermined intervals.","The modulation apparatus may further include checking information generating means for calculating a final cumulative DSV of the recording code string or the transmission code string, determining whether or not the calculated final cumulative DSV is within a predetermined range, and generating checking information on the basis of the determination result.","The DSV-control-bit generating means may generate the DSV control bit on the basis of the checking information generated by the checking information generating means.","When it is determined that the final cumulative DSV is out of the predetermined range, the checking information generating means may reset the final cumulative DSV to zero and generate an error signal serving as the checking information.","The DSV-control-bit generating means may internally calculate a cumulative DSV for generating the DSV control bit and, when the error signal is generated by the checking information generating means, may reset the cumulative DSV to zero.","A modulation method of the present invention includes a DSV-control-bit generating step of generating a DSV control bit to be inserted into an input bit string in order to control a DSV of a recording code string or a transmission code string; a timing adjusting step of adjusting transmission timing for transmitting the input bit string; a DSV-control-bit-inserted bit string generating step of generating a DSV-control-bit-inserted bit string by inserting the DSV control bit generated in the DSV-control-bit generating step into a predetermined position of the input bit string whose transmission timing is adjusted in the timing adjusting step; and a modulation step of modulating the DSV-control-bit-inserted bit string generated in the DSV-control-bit-inserted bit string generating step into a channel bit string on the basis of a conversion rule (d, k; m, n; r).","A program on a recording medium of the present invention includes a DSV-control-bit generating step of generating a DSV control bit to be inserted into an input bit string in order to control a DSV of a recording code string or a transmission code string; a timing adjusting step of adjusting transmission timing for transmitting the input bit string; a DSV-control-bit-inserted bit string generating step of generating a DSV-control-bit-inserted bit string by inserting the DSV control bit generated in the DSV-control-bit generating step into a predetermined position of the input bit string whose transmission timing is adjusted in the timing adjusting step; and a modulation step of modulating the DSV-control-bit-inserted bit string generated in the DSV-control-bit-inserted bit string generating step into a channel bit string on the basis of a conversion rule (d, k; m, n; r).","A program of the present invention includes a DSV-control-bit generating step of generating a DSV control bit to be inserted into an input bit string in order to control a DSV of a recording code string or a transmission code string; a timing adjusting step of adjusting transmission timing for transmitting the input bit string; a DSV-control-bit-inserted bit string generating step of generating a DSV-control-bit-inserted bit string by inserting the DSV control bit generated in the DSV-control-bit generating step into a predetermined position of the input bit string whose transmission timing is adjusted in the timing adjusting step; and a modulation step of modulating the DSV-control-bit-inserted bit string generated in the DSV-control-bit-inserted bit string generating step into a channel bit string on the basis of a conversion rule (d, k; m, n; r).","According to a modulation apparatus and method, a recording medium, and a program of the present invention, a DSV control bit to be inserted into an input bit string is generated in order to control a DSV of a recording code string or a transmission code string.","Transmission timing for transmitting the input bit string is adjusted.","The generated DSV control bit is inserted into a predetermined position of the input bit string, whose transmission timing is adjusted, to generate a DSV-control-bit-inserted bit string.","The generated DSV-control-bit-inserted bit string is modulated into a channel bit string on the basis of a conversion rule (d, k; m, n; r).","A DSV-control-bit generating method of the present invention includes a first-candidate-bit-inserted bit string generating step of generating a first-candidate-bit-inserted bit string, which is a candidate for a DSV-control-bit-inserted bit string, by inserting a first candidate bit for a DSV control bit into a predetermined position of an input bit string; a second-candidate-bit-inserted bit string generating step of generating a second-candidate-bit-inserted bit string, which is another candidate for the DSV-control-bit-inserted bit string, by inserting a second candidate bit for the DSV control bit into the predetermined position of the input bit string; a modulation step of modulating, on the basis of the same conversion rule as a conversion rule applied when modulating the input bit string, the first-candidate-bit-inserted bit string generated in the first-candidate-bit-inserted bit string generating step into a first candidate channel bit string, which is a candidate for a channel bit string, and for modulating the second-candidate-bit-inserted bit string generated in the second-candidate-bit-inserted bit string generating step into a second candidate channel bit string, which is another candidate for the channel bit string; a DSV calculating step of calculating a DSV of each of the first and second candidate channel bit strings generated by modulation in the modulation step; and a DSV control bit determining step of determining one of the first and second candidate bits as the DSV control bit on the basis of the DSVs calculated in the DSV calculating step.","A program on a recording medium of the present invention includes a first-candidate-bit-inserted bit string generating step of generating a first-candidate-bit-inserted bit string, which is a candidate for a DSV-control-bit-inserted bit string, by inserting a first candidate bit for a DSV control bit into a predetermined position of an input bit string; a second-candidate-bit-inserted bit string generating step of generating a second-candidate-bit-inserted bit string, which is another candidate for the DSV-control-bit-inserted bit string, by inserting a second candidate bit for the DSV control bit into the predetermined position of the input bit string; a modulation step of modulating, on the basis of the same conversion rule as a conversion rule applied when modulating the input bit string, the first-candidate-bit-inserted bit string generated in the first-candidate-bit-inserted bit string generating step into a first candidate channel bit string, which is a candidate for a channel bit string, and for modulating the second-candidate-bit-inserted bit string generated in the second-candidate-bit-inserted bit string generating step into a second candidate channel bit string, which is another candidate for the channel bit string; a DSV calculating step of calculating a DSV of each of the first and second candidate channel bit strings generated by modulation in the modulation step; and a DSV control bit determining step of determining one of the first and second candidate bits as the DSV control bit on the basis of the DSVs calculated in the DSV calculating step.","A program of the present invention includes a first-candidate-bit-inserted bit string generating step of generating a first-candidate-bit-inserted bit string, which is a candidate for a DSV-control-bit-inserted bit string, by inserting a first candidate bit for a DSV control bit into a predetermined position of an input bit string; a second-candidate-bit-inserted bit string generating step of generating a second-candidate-bit-inserted bit string, which is another candidate for the DSV-control-bit-inserted bit string, by inserting a second candidate bit for the DSV control bit into the predetermined position of the input bit string; a modulation step of modulating, on the basis of the same conversion rule as a conversion rule applied when modulating the input bit string, the first-candidate-bit-inserted bit string generated in the first-candidate-bit-inserted bit string generating step into a first candidate channel bit string, which is a candidate for a channel bit string, and for modulating the second-candidate-bit-inserted bit string generated in the second-candidate-bit-inserted bit string generating step into a second candidate channel bit string, which is another candidate for the channel bit string; a DSV calculating step of calculating a DSV of each of the first and second candidate channel bit strings generated by modulation in the modulation step; and a DSV control bit determining step of determining one of the first and second candidate bits as the DSV control bit on the basis of the DSVs calculated in the DSV calculating step.","According to a DSV-control-bit generating method, a recording medium, and a program of the present invention, a first-candidate-bit-inserted bit string, which is a candidate for a DSV-control-bit-inserted bit string, is generated by inserting a first candidate bit for a DSV control bit into a predetermined position of an input bit string.","A second-candidate-bit-inserted bit string, which is another candidate for the DSV-control-bit-inserted bit string, is generated by inserting a second candidate bit for the DSV control bit into the predetermined position of the input bit string.","On the basis of the same conversion rule as a conversion rule applied when modulating the input bit string, the generated first-candidate-bit-inserted bit string is modulated into a first candidate channel bit string, which is a candidate for channel bits; and the generated second-candidate-bit-inserted bit string is modulated into a second candidate channel bit string, which is another candidate for the channel bits.","A DSV of each of the first and second candidate channel bit strings is calculated.","One of the first and second candidate bits is determined as the DSV control bit on the basis of the calculated DSVs.","BRIEF DESCRIPTION OF THE DRAWINGS FIG.","1 is a block diagram showing an example of the configuration of a known modulation apparatus.","FIG.","2 is a block diagram showing an example of the configuration of another known modulation apparatus.","FIG.","3 is a diagram showing an example of the register configuration in a modulator of the modulation apparatus shown in FIG.","2.FIG.","4 is a block diagram showing an example of the configuration of a modulation apparatus according to the present invention.","FIG.","5 is a block diagram showing details of the configuration of the modulation apparatus shown in FIG.","4.FIG.","6 is a flowchart for describing the operation of the modulation apparatus shown in FIG.","4.FIG.","7 is a diagram for describing the data format at each stage of a data stream modulated by the modulation apparatus shown in FIG.","4.FIG.","8 is a chart for describing timing of data to be input to the modulation apparatus shown in FIG.","4.FIG.","9 is a diagram showing an example of the register configuration in a modulator of the modulation apparatus shown in FIG.","4.FIG.","10 is a block diagram showing an example of the configuration of another modulation apparatus according to the present invention.","BEST MODE FOR CARRYING OUT THE INVENTION FIG.","4 shows an example of the configuration of a modulation apparatus 3 according to the present invention.","The modulation method employed by the modulation apparatus 3 is not limited.","In this example, the modulation apparatus 3 converts a data stream into, for example, a variable-length code (d, k; m, n; r)=(1, 7; 2, 3; 4).","An input unit 38 receives an input data stream supplied from the outside and supplies the input data stream to a DSV control bit determination unit 31 and a delay processor 32.The DSV control bit determination unit 31 executes a predetermined arithmetic operation to determine the DSV control bit “1” or “0” to be inserted into the supplied input data stream and supplies the determination result to a determined-DSV-control-bit insertion unit 33.The delay processor 32 delays the supplied input data stream for a predetermined delay time and supplies the delayed input data stream to the determined-DSV-control-bit insertion unit 33.Specifically, the delay processor 32 adjusts the transmission timing to supply the input data stream to the determined-DSV-control-bit insertion unit 32.The predetermined delay time is set on the basis of the timing to insert, by the determined-DSV-control-bit insertion unit 33 described below, the DSV control bit determined by the DSV control bit determination unit 31 into a predetermined position of the input data stream output by the delay processor 32.When the determined-DSV-control-bit insertion unit 33 receives, at a predetermined time, the input data stream supplied by the delay processor 32 and the DSV-control-bit determination result (the result indicating whether the DSV control bit is “0” or “1”) supplied at a predetermined time by the DSV control bit determination unit 31, the determined-DSV-control-bit insertion unit 33 inserts a DSV control bit corresponding to the received determination result into the predetermined position (hereinafter referred to as the DSV position) of the received input data stream to generate a new data stream (hereinafter referred to as a DSV-control-bit-inserted bit string in order to be distinguished from other data streams) and supplies the DSV-control-bit-inserted bit string to a modulator 34.Specifically, the predetermined delay time of the delay processor 32 is set so that a bit corresponding to the DSV position of the input data stream will be input to the determined-DSV-control-bit insertion unit 33 at the above-described predetermined time.","The modulator 34 modulates the DSV-control-bit-inserted bit string into a code string (channel bit string) in accordance with a predetermined conversion rule (e.g., 1,7PP table shown in Table 2) and supplies the code string to a sync signal insertion unit 35.The sync signal insertion unit 35 inputs a prepared sync signal to a predetermined position of the code string supplied by the modulator 34 at a predetermined time (differing from that of the determined-DSV-control-bit insertion unit 33) and supplies the resultant code string to an NRZI unit 36.The sync signal consists of a predetermined pattern containing a predetermined number of channel bits and is referred to as Frame Sync hereinafter.","The NRZI unit 36 performs NRZI modulation of the code string supplied by the sync signal insertion unit 35 to generate a recording code string and outputs the recording code string to the outside and to a cumulative DSV checking unit 37.As described above, bit-string permutation by inverting 1 and not inverting 0 of a code string is referred to as NRZI modulation.","In other words, the code string prior to being NRZI-modulated is a bit string indicating the edge position, whereas the NRZI-modulated recording code string corresponds to a bit string indicating the H/L (High/Low) level of recording data.","The cumulative DSV checking unit 37 receives the recording code string supplied by the NRZI unit 36, calculates the cumulative DSV so far (hereinafter referred to as the final cumulative DSV in order to be distinguished from other DSVs), determines whether or not the calculated final cumulative DSV is within a predetermined range, and generates checking information based on the determination result.","Specifically, when the cumulative DSV checking unit 37 determines that the final cumulative DSV is out of the predetermined range, the cumulative DSV checking unit 37 resets the final cumulative DSV to “0” or sets the final cumulative DSV to a predetermined initial value, generates an error signal serving as the checking information, and supplies the error signal to the DSV control bit determination unit 31.Specifically, the cumulative DSV checking unit 37 determines whether or not the final cumulative DSV at each moment exceeds the predetermined range (e.g., a range from −128 to +127 or a range from 0 to 255 when represented in sign and magnitude notation).","When it is determined that the final cumulative DSV exceeds the predetermined range, the error signal is supplied to a cumulative DSV calculator 55, which will be described later, in the DSV control bit determination unit 31 shown in FIG.","5.The cumulative DSV calculator 55 resets the calculated cumulative DSV so far to “0” or sets the calculated cumulative DSV so far to the predetermined initial value.","In this example, when the cumulative DSV checking unit 37 determines that the final cumulative DSV is within the predetermined range, the cumulative DSV checking unit 37 generates no checking information.","Alternatively, the cumulative DSV checking unit 37 may generate a signal corresponding to “normal” or the like serving as the checking information.","Referring to FIG.","5, the modulation apparatus 3 will be described in more detail.","Specifically, FIG.","5 shows an example of the detailed configuration of the modulation apparatus 3.In FIG.","5, the symbol “+” in the square represents a portion that processes a data stream and functions as an insertion unit or a selector.","In order to simplify the description, the symbol is simply referred to as an adder.","Referring to FIG.","5, the input data stream received by the input unit 38 is supplied to the DSV control bit determination unit 31 and the delay processor 32 at the same time.","In the DSV control bit determination unit 31, an adder 42 inserts “0” serving as a first candidate bit for the DSV control bit into the predetermined position (DSV position) of the received input data stream to generate a data stream serving as a first candidate for the DSV-control-bit-inserted bit string (hereinafter referred to as a first-candidate-bit-inserted bit string) and supplies the first-candidate-bit-inserted bit string to a 1,7PP modulator 45.The 1,7PP modulator 45 modulates the first-candidate-bit-inserted bit string into a code string (hereinafter referred to as a first candidate code string in order to be distinguished from other code strings) in accordance with, for example, the above-described 1,7PP table shown in Table 2 and supplies the first candidate code string to an adder 48.The adder 48 inserts, at a predetermined time, Frame Sync (sync signal) into a predetermined position of the first candidate code string supplied by the 1,7PP modulator 45 and supplies the resultant code string to an NRZI unit 51.As described above, for example, the 1,7PP table shown in Table 2 is used in this example.","The 1,7PP table contains a termination table for inserting Frame Sync.","The 1,7PP modulator 45 performs termination based on the termination table.","Specifically, termination is to set, on a data stream, a break point at a position immediately before a position at which Frame Sync is to be inserted and to terminate table-based conversion (modulation) at the break point.","Since the 1,7PP table has a variable-length structure, the table-based conversion has a variable end position.","The modulation apparatus 3 uses the above-described termination table if necessary to terminate the table-based conversion at an arbitrary position on the data stream in units of two.","The adder 48 inserts Frame Sync containing the predetermined number of channel bits into a position immediately after the end position of the table-based conversion at the time the table-based conversion is terminated.","As described above, Frame Sync contains a predetermined pattern distinguishable from other code strings (a unique pattern not included in conversion codes in the conversion table).","In this example, the same Frame Sync is inserted by the above-described adder 48, an adder 49, and an adder 65, which will be described later.","The NRZI unit 51 performs NRZI modulation of the first candidate code string supplied by the adder 48 to generate a recording code string (hereinafter referred to as a first candidate symbol code string in order to be distinguished from other recording code strings) and supplies the first candidate recording code string to a section DSV calculator 53.The section DSV calculator 53 calculates a DSV in a predetermined DSV section (hereinafter referred to as a section DSV in order to be distinguished from other DSVs) on the basis of the first candidate recording code string and supplies the section DSV to an adder 56.The adder 56 adds the section DSV of the first candidate recording code string and the cumulative DSV so far supplied by the cumulative DSV calculator 55 described below and supplies the sum to a comparator 58.In contrast, an adder 43 inserts “1” serving as a second candidate bit for the DSV control bit into the predetermined position (DSV position) of the received input data stream to generate a data stream serving as a second candidate for the DSV-control-bit-inserted bit string (hereinafter referred to as a second-candidate-bit-inserted bit string) and supplies the second-candidate-bit-inserted bit string to a 1,7PP modulator 46.The second-candidate-bit-inserted bit string supplied to the 1,7PP modulator 46 is modulated, as in the above-described first-candidate-bit-inserted bit string, by the 1,7PP modulator 46 into a code string (hereinafter referred to as a second candidate code string to be distinguished from other code strings).","The adder 49 inserts Frame Sync into the second candidate code string at a predetermined time (predetermined position).","An NRZI unit 52 performs NRZI modulation of the second candidate code string containing Frame Sync to generate a recording code string (hereinafter referred to as a second candidate recording code string to be distinguished from other recording code strings).","The second candidate recording code string is supplied to a section DSV calculator 54.The section DSV calculator 54 calculates a section DSV in a predetermined DSV section on the basis of the second candidate recording code string and supplies the section DSV to an adder 57.The adder 57 adds the section DSV of the second candidate recording code string and the cumulative DSV so far supplied by the cumulative DSV calculator 55 described below and supplies the sum to the comparator 58.Accordingly, the cumulative DSV of the data stream generated by inserting “0” serving as a candidate for the DSV control bit into the input data stream (the first-candidate-bit-inserted bit string) and the cumulative DSV of the data stream generated by inserting “1” serving as another candidate for the DSV control bit into the input data stream (the second-candidate-bit-inserted bit string) are supplied to the comparator 58.The comparator 58 compares the absolute values of the two cumulative DSVs, selects the data stream having the cumulative DSV whose absolute value is smaller (first- or second-candidate-bit-inserted bit string), and determines, as the DSV control bit to be actually inserted into the input data stream, the first or second candidate DSV control bit included in the selected data stream (“0” when the first-candidate-bit-inserted bit string is selected and “1” when the second-candidate-bit-inserted bit string is selected).","Specifically, the comparator 58 supplies a DSV control bit selection signal (signal indicating “1” or “0”) corresponding to the determined DSV control bit to an AND operator 62.The comparator 58 supplies the cumulative DSV of the selected data stream to the cumulative DSV calculator 55.The cumulative DSV calculator 55 receives the cumulative DSV supplied by the comparator 58 and determines the received cumulative DSV as the cumulative DSV.","When the subsequent section DSV of the first or second candidate recording code string is supplied to the adder 56 or the adder 57, the cumulative DSV calculator 55 supplies the cumulative DSV determined immediately before the supply of the section DSV to the adder 56 or the adder 57.As described above, when the final cumulative DSV corresponding to the currently output recording code string exceeds the predetermined range, the cumulative DSV checking unit 37 supplies the error signal to the cumulative DSV calculator 55.The cumulative DSV calculator 55 receives the error signal and resets the currently determined cumulative DSV to 0 or sets the currently determined cumulative DSV to the predetermined initial value.","In the delay processor 32, an adder 60 inserts “0” serving as a temporary value prior to determination of the DSV control bit into the predetermined position (DSV position) of the input data stream supplied by the input unit 38 to generate a new data stream (hereinafter referred to as a temporary-DSV-control-bit-inserted bit string to be distinguished from other data streams) and supplies the temporary-DSV-control-bit-inserted bit string to a DSV section delay shift register 61.Specifically, the temporary-DSV-control-bit-inserted bit string is the same data stream as the first-candidate-bit-inserted bit string generated by the above-described adder 42.In this example, “0” is inserted as the temporary value prior to determination of the DSV control bit.","When a combination of logic circuits in the determined-DSV-control-bit insertion unit 33 described below changes, “1” may be inserted.","A temporary-DSV-control-bit-inserted bit string in this case is the same data stream as the second-candidate-bit-inserted bit string generated by the above-described adder 43.The DSV section delay shift register 61 delays the temporary-DSV-control-bit-inserted bit string for a predetermined delay time and supplies the delayed temporary-DSV-control-bit-inserted bit string to the determined-DSV-control-bit insertion unit 33.The DSV section delay shift register 61 contains registers, the number of which corresponds to a delay of x bits corresponding to the DSV control section, and, if necessary, a delay of α bits corresponding to a circuit delay (delay corresponding to the circuit delay α shown in FIG.","5).","The order of the adder 60 and the shift register 61 of the delay processor 32 may be reversed.","Specifically, the adder 60 may insert “0” serving as the temporary value prior to determination of the DSV control bit into the input data stream delayed by the shift register 61 to generate a temporary-DSV-control-bit-inserted bit string and may supply the temporary-DSV-control-bit-inserted bit string to the determined-DSV-control-bit insertion unit 33.In the determined-DSV-control-bit insertion unit 33, the AND operator 62 performs an AND operation (logical AND) of “1” supplied at a predetermined time by a DSV control bit position gate 64 and the DSV control bit selection signal indicating “0” or “1” supplied by the above-described comparator 58 and supplies the logical operation result to an OR operator 63.The OR operator 63 performs an OR operation (logical OR) of the operation result (“1” or “0”) supplied by the AND operator 62 and predetermined bit data of the temporary-DSV-control-bit-inserted bit string supplied by the DSV section delay shift register 61 and supplies the logical operation result to the 1,7PP modulator 34.When the delay processor 32 (DSV section delay shift register 61) supplies bit data corresponding to the DSV position of the temporary-DSV-control-bit-inserted bit string (“0” inserted by the adder 60 and serving as the temporary value prior to determination of the DSV control bit) to the OR operator 63, the above-described DSV control bit position gate 64 supplies “1” to the AND operator 62 at that time.","As described above, when “1” serving as the DSV control bit selection signal is supplied to the AND operator 62 and “1” is supplied by the DSV control bit position gate 64 to the AND operator 62 (when a bit corresponding to the DSV position is supplied to the OR operator 63), the AND operator 62 supplies “1” serving as the logical operation result to the OR operator 63.Specifically, the OR operator 63 receives “1” supplied by the AND operator 62 and bit data that is supplied by the delay processor 32 and that corresponds to the DSV position, that is, “0” inserted as the temporary DSV control bit by the adder 60, performs an OR operation of the received “1” and “0”, and supplies “1” serving as the logical operation result to the 1,7PP modulator 34.In other words, when the DSV control bit determined by the DSV control bit determination unit 31 is “1”, the determined-DSV-control-bit insertion unit 33 converts “0 (temporary value prior to determination of the DSV control bit)” inserted into the DSV position by the adder 60 into “1 (DSV control bit determined by the DSV control bit determination unit 31)”.","In contrast, when the DSV control bit determined by the DSV control bit determination unit 31 is “0”, the determined-DSV-control-bit insertion unit 33 uses “0 (temporary value prior to determination of the DSV control bit)” inserted into the DSV position by the adder 60, unchanged, as the DSV control bit (performs no conversion).","Accordingly, the determined-DSV-control-bit insertion unit 33 inserts the DSV control bit determined by the DSV control bit determination unit 31 into the DSV position of the temporary-DSV-control-bit-inserted bit string (position at which the temporary value prior to determination of the DSV control bit is inserted) to generate a DSV-control-bit-inserted bit string and supplies the DSV-control-bit-inserted bit string to the 1,7PP modulator 34.Since the 1,7PP modulator 34 has the same configuration as the above-described 1,7PP modulator 45 and the 1,7PP modulator 46, and since the adder 65 of the sync signal insertion unit 35 has the same configuration as the above-described adder 48 and adder 49, descriptions of the 1,7PP modulator 34 and the adder 65 are omitted.","Since the NRZI unit 36 and the cumulative DSV checking unit 37 have already been described, descriptions of the NRZI unit 36 and the cumulative DSV checking unit 37 are omitted.","The modulator 34 in FIG.","4 and the 1,7PP modulator 34 in FIG.","5 are the same modulator.","In order to emphasize that the modulator performs 1,7PP modulation, in FIG.","5, the modulator is expressed as the 1,7PP modulator 34.With reference to the flowchart of FIG.","6, the operation of the modulation apparatus 3 will now be described.","An input data stream 71 shown in FIG.","7 is supplied to the modulation apparatus 3.In step S11, the modulation apparatus 3 receives the input data stream 71.The time at which the input data stream 71 is input is as shown in FIG.","8.Specifically, a channel bit string (code string) 74 output by the 1,7PP modulator 34 is output as a serial recording code string in synchronization with a predetermined clock 75.In other words, one codeword is output every clock cycle.","In contrast, the input data stream 71 is input in accordance with the conversion ratio m/n of the 1,7PP modulator 34.Specifically, in this example, the conversion ratio is ⅔.","The amount of data of a codeword is three, whereas the amount of data of a data word of the input data stream 71 is two.","As shown in FIG.","8, the modulation apparatus 3 receives, of the input data stream 71, predetermined two data words within two clock cycles and stops receiving the input data stream 71 for one clock cycle.","Accordingly, a mismatch in the conversion ratio between the input data and the output code is adjusted.","Referring to FIG.","7, a DSV section of the input data stream 71 has x bits.","A 1-bit DSV control bit is inserted at the end of each x-bit data.","In order to be distinguished from unit data, i.e., a data word, to be modulated by the 1,7PP modulator 34, the x-bit data is referred to as data Dk (k is an integer).","In other words, the DSV position of data Dk is immediately after the end of the data Dk.","Data D1 into which Frame Sync is inserted has a short DSV section.","Specifically, Data D1 consists of x-Fsx(⅔) bits (Fs is the number of bits of Frame Sync).","Referring back to FIG.","6, in step S12, the modulation apparatus 3 determines the DSV control bit and inserts the DSV control bit into the predetermined position of the input data stream 71 to generate a DSV-control-bit-inserted bit string 72 shown in FIG.","7.Specifically, when each data word of the input data stream 71 is supplied to the DSV control bit determination unit 31 in the order shown in FIG.","8, the DSV control bit determination unit 31 receives each data word and determines the DSV control bit to be inserted into the DSV position of x-bit data received, that is, data Dk.","At the same time, the input data stream 71 is also supplied to the delay processor 32 in the order shown in FIG.","8 and delayed for a predetermined delay time, and the delayed input data stream 71 is supplied to the determined-DSV-control-bit insertion unit 33.The determined-DSV-control-bit insertion unit 33 inserts the DSV control bit (bit “0” or “1”) of the data Dk, which is determined by the DSV control bit determination unit 31, into the DSV position of the data Dk at the time the bit at the DSV position of the data Dk is supplied by the delay processor 32 to generate the DSV-control-bit-inserted bit string 72 and supplies the DSV-control-bit-inserted bit string 72 to the 1,7PP modulator 34.In step S13, the modulation apparatus 3 performs 1,7PP modulation of the DSV-control-bit-inserted bit string 72 to generate a predetermined code string.","Specifically as described above (as shown in FIG.","8), since the input data is received in units of two data words (stopped for one clock cycle), the 1,7PP modulator 34 modulates the DSV-control-bit-inserted bit string 72 in units of two data words.","In other words, the 1,7PP modulator 34 processes the data in units of three clock cycles (three channel bits).","The timing is generated by a counter or the like (not shown).","The register configuration in the 1,7PP modulator 34 is as shown in FIG.","9.Also, the register configuration in each of the 1,7PP modulator 45 and the 1,7PP modulator 46 is as shown in FIG.","9.Specifically, an input register 81 of the 1,7PP modulator 34 (register 81 associated with the DSV-control-bit-inserted bit string 72 supplied to the 1,7PP modulator 34) and an output register 82 (register 82 associated with a code string 73 output by the 1,7PP modulator 34) have minimum number of registers required to modulate each data word in accordance with the 1,7PP table shown in Table 2.Specifically, the input register 81 is provided with a number of registers which are 12 bits.","The output register 82 is provided with a number of registers which are 18 bits.","The 1,7PP modulator 34 includes a timing control register (not shown).","The number of registers required by the 1,7PP modulator 34 is the minimum number of registers required to modulate each data word and does not depend on the DSV interval of the format (in this example, the x-bit section).","In other words, the registers required by the 1,7PP modulator 34 are provided without taking into consideration a delay corresponding to a DSV control interval portion.","When the input data word consists of two bits (constraint length i=1), the 1,7PP modulator 34 places the bits into [0, 1] of the input register 81 shown in FIG.","9.Referring to [0,1] replaced with the corresponding bits, the 1,7PP modulator 34 modulates the bits when a predetermined condition is satisfied and places the channel bits into [0, 1, 2] of the output register 82.Similarly, when the input data word consists of four bits (constraint length i=2), the 1,7PP modulator 34 refers to [0, 1, 2, 3] of the input register 81 containing the corresponding bits, modulates the bits when a predetermined condition is satisfied, and places the channel bits into [0, 1, 2, 3, 4, 5] of the output register 82.When the input data word consists of six bits (constraint length i=3), the 1,7PP modulator 34 refers to [0, 1, 2, 3, 4, 5] of the input register 81 containing the corresponding bits, modulates the bits when a predetermined condition is satisfied, and places the channel bits into [0, 1, 2, 3, 4, 5, 6, 7, 8] of the output register 82.In the case of the maximum constraint length, that is, when the input data word consists of eight bits (constraint length i=4), the 1,7PP modulator 34 refers to [0, 1, 2, 3, 4, 5, 6, 7] of the input register 81 containing the corresponding bits, modulates the bits when a predetermined condition is satisfied, and places the channel bits into [0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11] of the output register 82.When processing the Prohibit rmtr portion (110111-next_cbit:010), the 1,7PP modulator 34 refers to [0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11] of the input register 81 and, when a predetermined condition is satisfied, replaces predetermined positions of the output register 82 with the channel bits.","Accordingly, the 1,7PP modulator 34 uses the input register 81 and the output register 82 to perform 1,7PP modulation of the DSV-control-bit-inserted bit string 72 to generate a channel bit string (code string) and supplies the channel bit string (code string) to the sync signal insertion unit 35.In step S14, the modulation apparatus 3 inserts Frame Sync into a predetermined position of the code string output by the 1,7PP modulator 34 to generate the code string (channel bit string) 73 shown in FIG.","7.Specifically, when the 1,7PP modulator 34 performs the above-described termination, the sync signal insertion unit 35 inserts Frame Sync (sync signal) at the head of the immediate data Dk (data D1 in this example) to generate the code string (channel bit string) 73 shown in FIG.","7 and supplies the code string 73 to the NRZI unit 36.Subsequent to the insertion of Frame Sync into the code string 73, DSV control bits are inserted into the code string 73 at equal intervals (span 1=span 2=span 3), thus achieving appropriate DSV control.","More specifically, when the 1,7PP modulator 34 performs termination, the 1,7PP modulator 34 stops outputting data.","At the same time, the adder 65 of the sync signal insertion unit 35 changes the selector and adds Frame Sync of a predetermined length.","When Frame Sync is added, the adder 65 changes the selector (to the original state), and the 1,7PP modulator 34 resumes data output (the code string 74 is supplied to the sync signal insertion unit 65).","Although the above method is described as an example of a method of inserting Frame Sync, the method is not limited to the above-described method.","For example, the 1,7PP modulator 34 may perform termination and then supply a temporary code string of the same length as predetermined Frame Sync to the sync signal insertion unit 35, and the sync signal insertion unit 35 may replace the temporary code string with the predetermined Frame Sync.","In step S15, the modulation apparatus 3 performs NRZI modulation of the code string 73 to generate a recording code string and outputs the recording code string to the outside.","Specifically, the NRZI unit 36 performs NRZI modulation of the code string 73 supplied by the sync signal insertion unit 35 to generate a recording code string and outputs the recording code string to the outside and to the cumulative DSV checking unit 37.The cumulative DSV checking unit 37 receives the recording code string supplied by the NRZI unit 36, calculates the final cumulative DSV, and determines whether or not the calculated final cumulative DSV is within a predetermined range.","When it is determined that the final cumulative DSV is out of the predetermined range, the cumulative DSV checking unit 37 supplies the determination result to the cumulative DSV calculator 55 of the DSV control bit determination unit 31.The cumulative DSV calculator 55 resets the calculated cumulative DSV so far to 0 or sets the calculated cumulative DSV so far to a predetermined initial value.","The cumulative DSV supplied to the cumulative DSV calculator 55 of the DSV control bit determination unit 31 may be a value supplied by the comparator 58 shown in FIG.","5 or may be, for example, the final cumulative DSV calculated by the above-described cumulative DSV checking unit 37.Specifically, supplying the final cumulative DSV calculated by the cumulative DSV checking unit 37 to the cumulative DSV calculator 55 at a predetermined time enables the cumulative DSV calculator 55 to operate in a manner similar to the above-described operation.","The code string output by the NRZI unit 36 is a recording code string in this example.","In the case of transmission of the output result, the NRZI unit 36 outputs a transmission code string.","In this case, the operation of the solid-state imaging device 3 remains unchanged.","As described above, according to the modulation apparatus 3 of the present invention, as shown in FIG.","9, the number of registers (the input register 81 and the output register 82) in each of the 1,7PP modulator 34, the 1,7PP modulator 45, and the 1,7PP modulator 46 is the minimum number of registers required to perform 1,7PP modulation of each data word and does not depend on the DSV control interval since the portion that performs 1,7PP modulation has an independent structure.","On the other hand, registers in a known modulating apparatus (e.g., the modulation apparatus 2 shown in FIG.","2) must be of sufficient number corresponding to the DSV control interval, as shown in FIG.","3.In the delay processor 32, only the DSV section delay shift register 61) is required, which consists of registers, the number of which corresponds to the total of the channel bit string corresponding to the DSV control interval and the circuit delay α (only one shift register is necessary).","Accordingly, the registers required in the modulation apparatus 3 of the present invention are more compact than those in a known modulation apparatus.","As a result, the manufacturer can make circuits of the modulation apparatus 3 more compact.","A reduction in the number of registers reduces, for example, the power consumption.","In particular, when the DSV control interval is increased or the conversion table for converting a data word into a codeword becomes smaller, the advantage of using the modulation apparatus 3 becomes more apparent.","A series of the above-described processes may be executed by hardware or software.","In the latter case, for example, the modulation apparatus 4 includes a personal computer shown in FIG.","10.Referring to FIG.","10, a CPU 101 performs various processes in accordance with a program stored in a ROM 102 or a program loaded from a storage unit 108 into a RAM 103.If necessary, the RAM 103 stores data necessary for the CPU 101 to perform the various processes.","The CPU 101, the ROM 102, and the RAM 103 are interconnected with one another via a bus 104.An input/output interface 105 is connected to the bus 104.An input unit 106 including a keyboard and a mouse, an output unit 107 including a display, the storage unit 108 including a hard disk, a communication unit 109 including a modem and a terminal adapter are connected to the input/output interface 105.The communication unit 109 performs communication via a network including the Internet.","If necessary, a drive 110 is connected to the input/output interface 105.A magnetic disk 121, an optical disk 122, a magneto-optical disk 123, or a semiconductor memory 124 is appropriately placed on the drive 111, and a computer program read from the placed medium is installed into the storage unit 108 if necessary.","When a series of processes is to be performed using software, a program providing the software is installed via a network or a recording medium onto a computer included in dedicated hardware or, for example, a general personal computer capable of performing various functions by installing therein various programs.","As shown in FIG.","10, the recording medium includes a packaged medium including the magnetic disk 121 (including a floppy disk), the optical disk 122 (including a CD-ROM (Compact Disk-Read Only Memory) and DVD (Digital Versatile Disk)), the magneto-optical disk 123 (including MD (Mini-Disk)), or the semiconductor memory 124, all of which have recorded thereon the program and which are distributed, separately from the apparatus, to supply the program to the user.","Also, the recording medium includes the ROM 102 or the hard disk included in the storage unit 108, which have recorded thereon the program and which are included in advance in the apparatus to be supplied to the user.","In this specification, steps for writing the program stored in the recording medium not only include time-series processing performed in accordance with the described order but also include parallel or individual processing, which may not necessarily be performed in time series.","INDUSTRIAL APPLICABILITY As described above, according to a modulation apparatus and method and to a DSV-control-bit generating method of the present invention, an increase in circuit size of the modulation apparatus is suppressed."]],"string":"[\n [\n \"Modulating apparatus and method, and dsv control bit producing method\",\n \"The present invention relates to a modulation apparatus and method and a DSV-control-bit generating method for suppressing an increase in circuit size of the modulation apparatus.\",\n \"When an input data stream is supplied to a DSV control bit determination unit 31, the DSV control bit determination unit 31 determines a DSV control bit to be inserted into the input data stream.\",\n \"Upon supplying the input data stream to the DSV control bit determination unit 31, the input data stream is simultaneously supplied to a delay processor 32.\",\n \"The input data stream is delayed for a predetermined delay time and supplied to a determined-DSV-control-bit insertion unit 33.\",\n \"The determined-DSV-control-bit insertion unit 33 inserts the DSV control bit determined by the DSV control bit determination unit 3.1 into a predetermined position of the input data stream supplied by the delay means and supplies the input data stream containing the DSV control bit to a modulator 34.\",\n \"The modulator 34 modulates the input data stream containing the DSV control bit into a code string in accordance with a predetermined coding rule (for example, 1,7PP modulation).\"\n ],\n [\n \"1.A modulation apparatus for generating a channel bit string from an input bit string and generating a recording code string or a transmission code string from the channel bit string, comprising: DSV-control-bit generating means for generating a DSV control bit to be inserted into the input bit string in order to control a DSV of the recording code string or the transmission code string; timing adjusting means for adjusting transmission timing for transmitting the input bit string; DSV-control-bit-inserted bit string generating means for generating a DSV-control-bit-inserted bit string by inserting the DSV control bit generated by the DSV-control-bit generating means into a predetermined position of the input bit string whose transmission timing is adjusted by the timing adjusting means; and first modulation means for modulating the DSV-control-bit-inserted bit string generated by the DSV-control-bit-inserted bit string generating means-into the channel bit string on the basis of a conversion rule (d, k; m, n; r).\",\n \"2.The modulation apparatus according to claim 1, further comprising NRZI means for performing NRZI modulation of the channel bit string generated by modulation by the first modulation means to generate the recording code string or the transmission code string.\",\n \"3.The modulation apparatus according to claim 1, wherein the conversion rule states that the remainder of the number of “1s” in a predetermined block of the input bit string or the DSV-control-bit-inserted bit string divided by two equals the remainder of the number of “1s” in the corresponding block of the channel bit string divided by two.\",\n \"4.The modulation apparatus according to claim 1, wherein the conversion rule states that the number of consecutive minimum run length d of the channel bit string is limited to a predetermined number or less.\",\n \"5.The modulation apparatus according to claim 1, wherein the conversion rule states a variable-length code (d, k; m, n; r), where the minimum run length d=1, the maximum run length k=7, the length of basic data prior to conversion m=2, and the length of basic channel bits subsequent to conversion n=3.6.The modulation apparatus according to claim 1, wherein data of m length, which is the length of basic data, is input for a time period in which the channel bit string of n length, which is the length of the basic channel bits, is output.\",\n \"7.The modulation apparatus according to claim 1, wherein the DSV-control-bit generating mean includes: first-candidate-bit-inserted bit string generating means for generating a first-candidate-bit-inserted bit string, which is a candidate for the DSV-control-bit-inserted bit string, by inserting a first candidate bit for the DSV control bit into the predetermined position of the input bit string; second-candidate-bit-inserted bit string generating means for generating a second-candidate-bit-inserted bit string, which is another candidate for the DSV-control-bit-inserted bit string, by inserting a second candidate bit for the DSV control bit into the predetermined position of the input bit string; second modulation means for modulating, on the basis of the same conversion rule as the conversion rule used by the first modulation means, the first-candidate-bit-inserted bit string generated by the first-candidate-bit-inserted bit string generating means into a first candidate channel bit string, which is a candidate for the channel bit string, and for modulating the second-candidate-bit-inserted bit string generated by the second-candidate-bit-inserted bit string generating means into a second candidate channel bit string, which is another candidate for the channel bit string; DSV calculating means for calculating a DSV of each of the first and second candidate channel bit strings generated by modulation by the second modulation means; and DSV control bit determining means for determining one of the first and second candidate bits as the DSV control bit on the basis of the DSVs calculated by the DSV calculating means.\",\n \"8.The modulation apparatus according to claim 7, wherein the DSV calculating means includes: section DSV calculating means for calculating a section DSV of a current DSV control section of each of the first and second candidate channel bit strings; cumulative DSV calculating means for calculating a cumulative DSV on the basis of the determination result by the DSV control bit determining means; and adding means for calculating the DSV by adding each section DSV calculated by the section DSV calculating means and the cumulative DSV immediately before the current DSV control section, the cumulative DSV being calculated by the cumulative DSV calculating means.\",\n \"9.The modulation apparatus according to claim 7, wherein the first and second modulation means each have a minimum number of registers required to perform modulation based on the coding rule.\",\n \"10.The modulation apparatus according to claim 1, further comprising: first sync signal inserting means for inserting a sync pattern including a preset unique pattern into the channel bit string, wherein the DSV-control-bit generating means includes second sync signal inserting means for inserting the same sync pattern as the sync pattern inserted by the first sync signal inserting means into each of the first and second candidate channel bit strings generated by modulating the first- and second-candidate-bit-inserted bit strings generated by inserting the first and second candidate bits, respectively, into the input bit string, and wherein the DSV calculating means calculates the DSV on the basis of each of the first and second candidate channel bit strings, each of which includes the sync pattern inserted by the second sync signal inserting means.\",\n \"11.The modulation apparatus according to claim 1, wherein the timing adjusting means adjusts the transmission timing by adding a delay time to the input bit string.\",\n \"12.The modulation apparatus according to claim 1, wherein the timing adjusting means inserts a temporary value prior to determination of the DSV control bit into the input bit string at predetermined intervals.\",\n \"13.The modulation apparatus according to claim 1, further comprising checking information generating means for calculating a final cumulative DSV of the recording code string or the transmission code string, determining whether or not the calculated final cumulative DSV is within a predetermined range, and generating checking information on the basis of the determination result, wherein the DSV-control-bit generating means generates the DSV control bit on the basis of the checking information generated by the checking information generating means.\",\n \"14.The modulation apparatus according to claim 13, wherein, when it is determined that the final cumulative DSV is out of the predetermined range, the checking information generating means resets the final cumulative DSV to zero and generates an error signal serving as the checking information, and wherein the DSV-control-bit generating means internally calculates a cumulative DSV for generating the DSV control bit and, when the error signal is generated by the checking information generating means, resets the cumulative DSV to zero.\",\n \"15.A modulation method for a modulation apparatus for generating a channel bit string from an input bit string and generating a recording code string or a transmission code string from the channel bit string, comprising: a DSV-control-bit generating step of generating a DSV control bit to be inserted into the input bit string in order to control a DSV of the recording code string or the transmission code string; a timing adjusting step of adjusting transmission timing for transmitting the input bit string; a DSV-control-bit-inserted bit string generating step of generating a DSV-control-bit-inserted bit string by inserting the DSV control bit generated in the DSV-control-bit generating step into a predetermined position of the input bit string whose transmission timing is adjusted in the timing adjusting step; and a modulation step of modulating the DSV-control-bit-inserted bit string generated in the DSV-control-bit-inserted bit string generating step into the channel bit string on the basis of a conversion rule (d, k; m, n; r).\",\n \"16.A recording medium providing a computer-readable computer program for controlling a modulation apparatus for generating a channel bit string from an input bit string and generating a recording code string or a transmission code string from the channel bit string, the program comprising: a DSV-control-bit generating step of generating a DSV control bit to be inserted into the input bit string in order to control a DSV of the recording code string or the transmission code string; a timing adjusting step of adjusting transmission timing for transmitting the input bit string; a DSV-control-bit-inserted bit string generating step of generating a DSV-control-bit-inserted bit string by inserting the DSV control bit generated in the DSV-control-bit generating step into a predetermined position of the input bit string whose transmission timing is adjusted in the timing adjusting step; and a modulation step of modulating the DSV-control-bit-inserted bit string generated in the DSV-control-bit-inserted bit string generating step into the channel bit string on the basis of a conversion rule (d, k; m, n; r).\",\n \"17.A program for causing a computer that controls a modulation apparatus for generating a channel bit string from an input bit string and generating a recording code string or a transmission code string from the channel bit string to perform a process comprising: a DSV-control-bit generating step of generating a DSV control bit to be inserted into the input bit string in order to control a DSV of the recording code string or the transmission code string; a timing adjusting step of adjusting transmission timing for transmitting the input bit string; a DSV-control-bit-inserted bit string generating step of generating a DSV-control-bit-inserted bit string by inserting the DSV control bit generated in the DSV-control-bit generating step into a predetermined position of the input bit string whose transmission timing is adjusted in the timing adjusting step; and a modulation step of modulating the DSV-control-bit-inserted bit string generated in the DSV-control-bit-inserted bit string generating step into the channel bit string on the basis of a conversion rule (d, k; m, n; r).\",\n \"18.A DSV-control-bit generating method for generating a DSV control bit to be inserted into an input bit string, comprising: a first-candidate-bit-inserted bit string generating step of generating a first-candidate-bit-inserted bit string, which is a candidate for a DSV-control-bit-inserted bit string, by inserting a first candidate bit for the DSV control bit into a predetermined position of the input bit string; a second-candidate-bit-inserted bit string generating step of generating a second-candidate-bit-inserted bit string, which is another candidate for the DSV-control-bit-inserted bit string, by inserting a second candidate bit for the DSV control bit into the predetermined position of the input bit string; a modulation step of modulating, on the basis of the same conversion rule as a conversion rule applied when modulating the input bit string, the first-candidate-bit-inserted bit string generated in the first-candidate-bit-inserted bit string generating step into a first candidate channel bit string, which is a candidate for a channel bit string generated from the input bit string, and for modulating the second-candidate-bit-inserted bit string generated in the second-candidate-bit-inserted bit string generating step into a second candidate channel bit string, which is another candidate for the channel bit string; a DSV calculating step of calculating a DSV of each of the first and second candidate channel bit strings generated by modulation in the modulation step; and a DSV control bit determining step of determining one of the first and second candidate bits as the DSV control bit on the basis of the DSVs calculated in the DSV calculating step.\",\n \"19.A recording medium providing a computer-readable program for generating a DSV control bit to be inserted into an input bit string, the program comprising: a first-candidate-bit-inserted bit string generating step of generating a first-candidate-bit-inserted bit string, which is a candidate for a DSV-control-bit-inserted bit string, by inserting a first candidate bit for the DSV control bit into a predetermined position of the input bit string; a second-candidate-bit-inserted bit string generating step of generating a second-candidate-bit-inserted bit string, which is another candidate for the DSV-control-bit-inserted bit string, by inserting a second candidate bit for the DSV control bit into the predetermined position of the input bit string; a modulation step of modulating, on the basis of the same conversion rule as a conversion rule applied when modulating the input bit string, the first-candidate-bit-inserted bit string generated in the first-candidate-bit-inserted bit string generating step into a first candidate channel bit string, which is a candidate for a channel bit string generated from the input bit string, and for modulating the second-candidate-bit-inserted bit string generated in the second-candidate-bit-inserted bit string generating step into a second candidate channel bit string, which is another candidate for the channel bit string; a DSV calculating step of calculating a DSV of each of the first and second candidate channel bit strings generated by modulation in the modulation step; and a DSV control bit determining step of determining one of the first and second candidate bits as the DSV control bit on the basis of the DSVs calculated in the DSV calculating step.\",\n \"20.A program for generating a DSV control bit to be inserted into an input bit string, the program causing a computer to perform a process comprising: a first-candidate-bit-inserted bit string generating step of generating a first-candidate-bit-inserted bit string, which is a candidate for a DSV-control-bit-inserted bit string, by inserting a first candidate bit for the DSV control bit into a predetermined position of the input bit string; a second-candidate-bit-inserted bit string generating step of generating a second-candidate-bit-inserted bit string, which is another candidate for the DSV-control-bit-inserted bit string, by inserting a second candidate bit for the DSV control bit into the predetermined position of the input bit string; a modulation step of modulating, on the basis of the same conversion rule as a conversion rule applied when modulating the input bit string, the first-candidate-bit-inserted bit string generated in the first-candidate-bit-inserted bit string generating step into a first candidate channel bit string, which is a candidate for a channel bit string generated from the input bit string, and for modulating the second-candidate-bit-inserted bit string generated in the second-candidate-bit-inserted bit string generating step into a second candidate channel bit string, which is another candidate for the channel bit string; a DSV calculating step of calculating a DSV of each of the first and second candidate channel bit strings generated by modulation in the modulation step; and a DSV control bit determining step of determining one of the first and second candidate bits as the DSV control bit on the basis of the DSVs calculated in the DSV calculating step.\"\n ],\n [\n \"<SOH> BACKGROUND ART <EOH>In transferring data to a predetermined transmission line or in recording data onto a recording medium, such as a magnetic disk, an optical disk, or a magneto-optical disk, the data is modulated to suit the transfer or recording.\",\n \"One known modulation method is block coding.\",\n \"Block coding is to divide a data stream into blocks of m×i bits (hereinafter the blocks are referred to as data words), and each data word is converted into a codeword of n×i bits in accordance with an appropriate coding rule.\",\n \"When i=1, the resultant code is a fixed-length code.\",\n \"When i can be of multiple values, that is, when i is selected from a range of 1 to i max (maximum i) and conversion is performed with the selected i, the resultant code is a variable-length code.\",\n \"The code generated by block coding is defined as a variable-length code (d, k; m, n; r).\",\n \"In the above description, i denotes the constraint length, and i max is r (maximum constraint length); d denotes the minimum number of consecutive “0s” between consecutive “1s”, e.g., the minimum run length of “0”; and k denotes the maximum number of consecutive “0s” between consecutive “1s”, e.g., the maximum run length of “0”.\",\n \"In recording the code generated as described above onto an optical disk, a magneto-optical disk, or the like, such as a compact disk (CD) or mini disk (MD), the variable-length code is subjected to NRZI (Non Return to Zero Inverted) modulation in which “1” is inverted whereas “0” is not inverted, and recording is done on the basis of the NRZI-modulated variable-length code (hereinafter referred to as the recording code string).\",\n \"There is a system, such as that on a magneto-optical disk in an initial ISO format having a not-so-high recording density, which records a modulated recording bit string that has not been subjected to NRZI modulation.\",\n \"The minimum inversion interval of the recording code string is denoted by Tmin, and the maximum inversion interval of the recording code string is denoted by Tmax.\",\n \"Higher recording density in the direction of linear velocity is realized when the minimum inversion interval Tmin is longer, that is, when the minimum run length d is greater.\",\n \"On the other hand, in terms of clock reading, it is preferable to have a shorter maximum inversion interval Tmax, that is, a smaller maximum run length k. Various modulations methods have been proposed.\",\n \"Specifically, modulation systems proposed or actually used in, for example, optical disks, magnetic disks, magneto-optical disks, and the like are described as follows.\",\n \"For example, RLL codes (Run Length Limited Code) with the minimum run length d=2 include an EFM (Eight to Fourteen Modulation) code (may also be represented as (2, 10: 8, 17; 1)) used in CD, MD, and the like; an 8-16 code (may also be represented as (2, 10: 8, 16; 1)) used in DVD (Digital Video Disk); an RLL (2-7) (may also be represented as (2, 7; 1, 2; r)) used in PD (Phase Change Disk); and the like.\",\n \"RLL codes with the minimum run length d=1 include a fixed-length RLL (1-7) (may also be represented as (1, 7; 2, 3; 1)) used in an ISO-format MO disk (Magnetic-Optical Disk); and a variable-length RLL (1-7) (may also be represented as (1, 7; 2, 3; r)) used in a disk drive for a high-density optical disk, magneto-optical disk, or the like.\",\n \"A conversion table for the variable-length RLL (1-7) is as follows: TABLE 1 RLL (1, 7, 2, 3, 2) Data word Codeword i = 1 11 00x 10 010 01 10x i = 2 0011 000 00x 0010 000 010 0001 100 00x 0000 100 010 The symbol x in the conversion table corresponds to 1 when the subsequent codeword is 0 and corresponds to 0 when the subsequent codeword is 1.The maximum constraint length r is 2.The parameters of the variable-length RLL (1-7) are (1, 7; 2, 3; 2).\",\n \"When the bit interval of the recording code string is denoted by T, the minimum inversion interval Tmin expressed as (d+1) is 2(=1+1)T. When the bit interval of the data stream is denoted by Tdata, the minimum inversion interval Tmin expressed as (m/n)×2 is 1.33(=(⅔)×2)Tdata.\",\n \"In the above description, m/n denotes conversion at the ratio m:n. For example, ⅔ denotes conversion at 2:3 (conversion of a data word of 2×i bits into a codeword of 3×i bits).\",\n \"The maximum inversion interval Tmax expressed as (k+1)T is 8(=7+1)T ((=(⅔)×8Tdata=5.33Tdata).\",\n \"The detection window margin Tw is expressed as (m/n)×Tdata and is 0.67(=⅔)Tdata.\",\n \"In a code string (channel bit string) generated by RLL (1-7) modulation in Table 1, 2T which is Tmin occurs most frequently, which is followed by 3T, 4T, and so forth.\",\n \"The fact that edge information, such as 2T or 3T, occurs many times with a short cycle is advantageous in reading clock.\",\n \"In contrast, when the recording linear density becomes higher, the minimum run length causes a problem.\",\n \"Specifically, when 2T (the minimum run length) occurs consecutively, the recording waveform is easily distorted because the waveform output of 2T is smaller than the others and is influenced more easily by defocus, tangential tilt, or the like.\",\n \"In recording at high linear density, recording with consecutive minimum marks is influenced more easily by disturbance, such as noise, and this may easily lead to a data read error.\",\n \"In such a case in which a data read error occurs, the error often resides in shift of start-edge and end-edge of the consecutive minimum marks.\",\n \"In other words, the generated bit error length becomes longer.\",\n \"In order to solve this problem, the consecutive minimum run lengths are controlled to better suit high linear density.\",\n \"In contrast, in recording onto a recording medium or in data transfer, code modulation based on each medium (transfer) is done.\",\n \"When a modulated code contains a DC component, fluctuation or jitter may be caused in various error signals, such as for a tracking error in servo control of a disk drive.\",\n \"It is preferable for the modulated code not to contain a DC component.\",\n \"In order to solve this problem, DSV (Digital Sum Value) control is proposed.\",\n \"DSV is the sum of bits of an NRZI-modulated (level-coded) bit string (channel bit string) in which “1” corresponds to +1 and “0” corresponds to −1.DSV serves as a reference for a DC component in the code string.\",\n \"Minimizing the absolute value of DSV, that is, performing DSV control, enables suppression of a DC component in the code string.\",\n \"In the code modulated according to the variable-length RLL (1-7) table shown in Table 1, no DSV control is performed.\",\n \"In such a case, DSV control is performed by calculating DSV of the modulated channel bit string at a predetermined interval and inserting predetermined DSV control bits into the code string.\",\n \"Basically, the DSV control bits are redundant bits.\",\n \"In view of the efficiency of code conversion, the fewer the DSV control bits, the better.\",\n \"It is preferable that the minimum run length d and the maximum run length k remain unchanged by the inserted DSV control bits.\",\n \"Changes of (d, k) influence the reading and writing characteristics.\",\n \"In order to satisfy the above requirements, DSV control must be performed in as efficiently as possible.\",\n \"While the actual RLL code must satisfy the minimum run length requirement, the maximum run length requirement need not be satisfied.\",\n \"There is a format that uses a pattern exceeding the maximum run length for a sync signal.\",\n \"For example, although EFM plus for DVD has a maximum run length of 11T, EFM plus for DVD allows for 14T as a matter of convenience of the format.\",\n \"By exceeding the maximum run length, for example, the capability of detecting a sync signal or the like is enhanced greatly.\",\n \"In the RLL (1-7) format with improved conversion efficiency, it is important to “control the consecutive minimum run lengths so as to better suit the high linear density” and to “perform DSV control as efficiently as possible” in association with an increase in linear density.\",\n \"Accordingly, the assignee of the present invention et al.\",\n \"discloses, in Japanese Patent Application No.\",\n \"10-150280, a conversion table including, as a conversion code, a basic code where d=1, k=7, m=2, and n=3; a coding rule that the remainder of the number of “1s” in each element of a data stream divided by two must be one or zero and equal the remainder of the number of “1s” in a converted channel bit string divided by two; a first replacement code for limiting the consecutive minimum run lengths d to a predetermined number or less; and a second replacement code for satisfying the run length limitation.\",\n \"Specifically, when a disk drive with high linear density reads/writes an RLL code, a pattern with consecutive minimum run lengths often causes a long error.\",\n \"When DSV control is performed on an RLL code, such as the RLL (1-7) code, DSV control bits need to be inserted into a code string (channel bit string) at arbitrary intervals.\",\n \"As described above, since the DSV control bits are redundant bits, it is preferable to have fewer DSV control bits.\",\n \"In order to maintain the minimum run length or the maximum run length, the DSV control bits of at least 2 bits or greater are necessary.\",\n \"The assignee of the present invention et al.\",\n \"discloses, in Japanese Patent Application No.\",\n \"10-150280, an RLL code with the minimum run length d=1 (d, k; m, n) and a conversion table, which is shown in Table 2, for limiting the number of consecutive minimum run lengths and for performing complete DSV control using efficient control bits while maintaining the minimum run length and the maximum run length (hereinafter referred to as a 1,7PP table; and a code according to 1,7PP table is referred to as a 1,7PP code): TABLE 2 1, 7PP (d, k, m, n, r) = (1, 7, 2, 3, 4) Data word Codeword 11 *0* 10 001 01 010 0011 010 100 0010 010 000 0001 000 100 000011 000 100 100 000010 000 100 000 000001 010 100 100 000000 010 100 000 “110111 001 000 000 (next010) 00001000 000 100 100 100 00000000 010 100 100 100 if xx1 then *0* = 000 xx0 then *0* = 101 Termination table 00 000 0000 010 100 “110111 001 000 000 (next010) When next channel bits are ‘010’ convert ‘11 01 11’ to ‘001 000 000’ after using main table and termination table.\",\n \"As an example of a modulation apparatus using the 1,7PP table, the assignee of the present invention discloses, in Japanese Patent Application No.\",\n \"10-150280, a modulation apparatus 1 shown in FIG.\",\n \"1 .\",\n \"The modulation apparatus 1 includes a DSV control bit determination and insertion unit 11 for determining “1” or “0” serving as a DSV control bit and inserting the DSV control bit into an input data stream at arbitrary intervals; a modulator 12 for modulating the data stream containing the DSV control bits; and an NRZI unit 13 for converting the output of the modulator 12 into a recording code string.\",\n \"Although not shown in the diagram, the modulation apparatus 1 includes a timing management unit for generating a timing signal, supplying the timing signal to the above components, and managing timing.\",\n \"In Japanese Patent Application No.\",\n \"09-342416, the assignee of the present invention et al.\",\n \"discloses a specific example of another modulation apparatus, namely, a modulation apparatus 2 shown in FIG.\",\n \"2 .\",\n \"The modulation apparatus includes a DSV control bit insertion unit 21 for inserting “1” or “0” serving as a DSV control bit into a data stream at arbitrary intervals.\",\n \"At this time, there is a data stream into which the DSV control bit “ 1 ” is inserted and another data stream into which the DSV control bit “ 0 ” is inserted.\",\n \"The modulation apparatus further includes a modulator 22 for modulating the data stream containing the DSV control bits and a DSV controller 23 for NRZI-modulating the modulated code string into level data, calculating DSV of the level data, and consequently outputting a DSV-controlled recording code string.\",\n \"As described above, the 1,7PP code is advantageous in solving the above problems.\",\n \"In contrast, compared with a modulation apparatus using a known method or a technique for performing DSV control on the RLL (1,7) code, the configuration of the known modulation apparatus using the 1,7PP code is complicated, and the circuit size is increased.\",\n \"For example, in the modulation apparatus 2 shown in FIG.\",\n \"2 , the register configuration in the modulator 22 is shown in FIG.\",\n \"3 .\",\n \"Specifically, the modulator 22 has a modulation (1,7PP modulation) portion and a delay portion corresponding to a DSV control interval (DSV section) in one integrated unit in order to transfer data corresponding to the DSV control interval to the DSV controller 23 at a subsequent stage.\",\n \"As a result, the modulator 22 needs two registers, that is, an input register 22 a (register 22 a for the data stream) and an output register 22 b (register 22 b for the channel bit string).\",\n \"The number of registers required corresponds to the DSV control interval.\",\n \"Two pairs of registers (registers 22 a and 22 b ) are necessary for the DSV control bit “0” and the DSV control bit “1”.\"\n ],\n [\n \"<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.\",\n \"1 is a block diagram showing an example of the configuration of a known modulation apparatus.\",\n \"FIG.\",\n \"2 is a block diagram showing an example of the configuration of another known modulation apparatus.\",\n \"FIG.\",\n \"3 is a diagram showing an example of the register configuration in a modulator of the modulation apparatus shown in FIG.\",\n \"2 .\",\n \"FIG.\",\n \"4 is a block diagram showing an example of the configuration of a modulation apparatus according to the present invention.\",\n \"FIG.\",\n \"5 is a block diagram showing details of the configuration of the modulation apparatus shown in FIG.\",\n \"4 .\",\n \"FIG.\",\n \"6 is a flowchart for describing the operation of the modulation apparatus shown in FIG.\",\n \"4 .\",\n \"FIG.\",\n \"7 is a diagram for describing the data format at each stage of a data stream modulated by the modulation apparatus shown in FIG.\",\n \"4 .\",\n \"FIG.\",\n \"8 is a chart for describing timing of data to be input to the modulation apparatus shown in FIG.\",\n \"4 .\",\n \"FIG.\",\n \"9 is a diagram showing an example of the register configuration in a modulator of the modulation apparatus shown in FIG.\",\n \"4 .\",\n \"FIG.\",\n \"10 is a block diagram showing an example of the configuration of another modulation apparatus according to the present invention.\",\n \"detailed-description description=\\\"Detailed Description\\\" end=\\\"lead\\\"?\"\n ],\n [\n \"TECHNICAL FIELD The present invention relates to modulation apparatuses and methods and DSV-control-bit generating methods, and more particularly relates to a modulation apparatus and method and a DSV-control-bit generating method for suppressing an increase in circuit size.\",\n \"BACKGROUND ART In transferring data to a predetermined transmission line or in recording data onto a recording medium, such as a magnetic disk, an optical disk, or a magneto-optical disk, the data is modulated to suit the transfer or recording.\",\n \"One known modulation method is block coding.\",\n \"Block coding is to divide a data stream into blocks of m×i bits (hereinafter the blocks are referred to as data words), and each data word is converted into a codeword of n×i bits in accordance with an appropriate coding rule.\",\n \"When i=1, the resultant code is a fixed-length code.\",\n \"When i can be of multiple values, that is, when i is selected from a range of 1 to i max (maximum i) and conversion is performed with the selected i, the resultant code is a variable-length code.\",\n \"The code generated by block coding is defined as a variable-length code (d, k; m, n; r).\",\n \"In the above description, i denotes the constraint length, and i max is r (maximum constraint length); d denotes the minimum number of consecutive “0s” between consecutive “1s”, e.g., the minimum run length of “0”; and k denotes the maximum number of consecutive “0s” between consecutive “1s”, e.g., the maximum run length of “0”.\",\n \"In recording the code generated as described above onto an optical disk, a magneto-optical disk, or the like, such as a compact disk (CD) or mini disk (MD), the variable-length code is subjected to NRZI (Non Return to Zero Inverted) modulation in which “1” is inverted whereas “0” is not inverted, and recording is done on the basis of the NRZI-modulated variable-length code (hereinafter referred to as the recording code string).\",\n \"There is a system, such as that on a magneto-optical disk in an initial ISO format having a not-so-high recording density, which records a modulated recording bit string that has not been subjected to NRZI modulation.\",\n \"The minimum inversion interval of the recording code string is denoted by Tmin, and the maximum inversion interval of the recording code string is denoted by Tmax.\",\n \"Higher recording density in the direction of linear velocity is realized when the minimum inversion interval Tmin is longer, that is, when the minimum run length d is greater.\",\n \"On the other hand, in terms of clock reading, it is preferable to have a shorter maximum inversion interval Tmax, that is, a smaller maximum run length k. Various modulations methods have been proposed.\",\n \"Specifically, modulation systems proposed or actually used in, for example, optical disks, magnetic disks, magneto-optical disks, and the like are described as follows.\",\n \"For example, RLL codes (Run Length Limited Code) with the minimum run length d=2 include an EFM (Eight to Fourteen Modulation) code (may also be represented as (2, 10: 8, 17; 1)) used in CD, MD, and the like; an 8-16 code (may also be represented as (2, 10: 8, 16; 1)) used in DVD (Digital Video Disk); an RLL (2-7) (may also be represented as (2, 7; 1, 2; r)) used in PD (Phase Change Disk); and the like.\",\n \"RLL codes with the minimum run length d=1 include a fixed-length RLL (1-7) (may also be represented as (1, 7; 2, 3; 1)) used in an ISO-format MO disk (Magnetic-Optical Disk); and a variable-length RLL (1-7) (may also be represented as (1, 7; 2, 3; r)) used in a disk drive for a high-density optical disk, magneto-optical disk, or the like.\",\n \"A conversion table for the variable-length RLL (1-7) is as follows: TABLE 1 RLL (1, 7, 2, 3, 2) Data word Codeword i = 1 11 00x 10 010 01 10x i = 2 0011 000 00x 0010 000 010 0001 100 00x 0000 100 010 The symbol x in the conversion table corresponds to 1 when the subsequent codeword is 0 and corresponds to 0 when the subsequent codeword is 1.The maximum constraint length r is 2.The parameters of the variable-length RLL (1-7) are (1, 7; 2, 3; 2).\",\n \"When the bit interval of the recording code string is denoted by T, the minimum inversion interval Tmin expressed as (d+1) is 2(=1+1)T. When the bit interval of the data stream is denoted by Tdata, the minimum inversion interval Tmin expressed as (m/n)×2 is 1.33(=(⅔)×2)Tdata.\",\n \"In the above description, m/n denotes conversion at the ratio m:n. For example, ⅔ denotes conversion at 2:3 (conversion of a data word of 2×i bits into a codeword of 3×i bits).\",\n \"The maximum inversion interval Tmax expressed as (k+1)T is 8(=7+1)T ((=(⅔)×8Tdata=5.33Tdata).\",\n \"The detection window margin Tw is expressed as (m/n)×Tdata and is 0.67(=⅔)Tdata.\",\n \"In a code string (channel bit string) generated by RLL (1-7) modulation in Table 1, 2T which is Tmin occurs most frequently, which is followed by 3T, 4T, and so forth.\",\n \"The fact that edge information, such as 2T or 3T, occurs many times with a short cycle is advantageous in reading clock.\",\n \"In contrast, when the recording linear density becomes higher, the minimum run length causes a problem.\",\n \"Specifically, when 2T (the minimum run length) occurs consecutively, the recording waveform is easily distorted because the waveform output of 2T is smaller than the others and is influenced more easily by defocus, tangential tilt, or the like.\",\n \"In recording at high linear density, recording with consecutive minimum marks is influenced more easily by disturbance, such as noise, and this may easily lead to a data read error.\",\n \"In such a case in which a data read error occurs, the error often resides in shift of start-edge and end-edge of the consecutive minimum marks.\",\n \"In other words, the generated bit error length becomes longer.\",\n \"In order to solve this problem, the consecutive minimum run lengths are controlled to better suit high linear density.\",\n \"In contrast, in recording onto a recording medium or in data transfer, code modulation based on each medium (transfer) is done.\",\n \"When a modulated code contains a DC component, fluctuation or jitter may be caused in various error signals, such as for a tracking error in servo control of a disk drive.\",\n \"It is preferable for the modulated code not to contain a DC component.\",\n \"In order to solve this problem, DSV (Digital Sum Value) control is proposed.\",\n \"DSV is the sum of bits of an NRZI-modulated (level-coded) bit string (channel bit string) in which “1” corresponds to +1 and “0” corresponds to −1.DSV serves as a reference for a DC component in the code string.\",\n \"Minimizing the absolute value of DSV, that is, performing DSV control, enables suppression of a DC component in the code string.\",\n \"In the code modulated according to the variable-length RLL (1-7) table shown in Table 1, no DSV control is performed.\",\n \"In such a case, DSV control is performed by calculating DSV of the modulated channel bit string at a predetermined interval and inserting predetermined DSV control bits into the code string.\",\n \"Basically, the DSV control bits are redundant bits.\",\n \"In view of the efficiency of code conversion, the fewer the DSV control bits, the better.\",\n \"It is preferable that the minimum run length d and the maximum run length k remain unchanged by the inserted DSV control bits.\",\n \"Changes of (d, k) influence the reading and writing characteristics.\",\n \"In order to satisfy the above requirements, DSV control must be performed in as efficiently as possible.\",\n \"While the actual RLL code must satisfy the minimum run length requirement, the maximum run length requirement need not be satisfied.\",\n \"There is a format that uses a pattern exceeding the maximum run length for a sync signal.\",\n \"For example, although EFM plus for DVD has a maximum run length of 11T, EFM plus for DVD allows for 14T as a matter of convenience of the format.\",\n \"By exceeding the maximum run length, for example, the capability of detecting a sync signal or the like is enhanced greatly.\",\n \"In the RLL (1-7) format with improved conversion efficiency, it is important to “control the consecutive minimum run lengths so as to better suit the high linear density” and to “perform DSV control as efficiently as possible” in association with an increase in linear density.\",\n \"Accordingly, the assignee of the present invention et al.\",\n \"discloses, in Japanese Patent Application No.\",\n \"10-150280, a conversion table including, as a conversion code, a basic code where d=1, k=7, m=2, and n=3; a coding rule that the remainder of the number of “1s” in each element of a data stream divided by two must be one or zero and equal the remainder of the number of “1s” in a converted channel bit string divided by two; a first replacement code for limiting the consecutive minimum run lengths d to a predetermined number or less; and a second replacement code for satisfying the run length limitation.\",\n \"Specifically, when a disk drive with high linear density reads/writes an RLL code, a pattern with consecutive minimum run lengths often causes a long error.\",\n \"When DSV control is performed on an RLL code, such as the RLL (1-7) code, DSV control bits need to be inserted into a code string (channel bit string) at arbitrary intervals.\",\n \"As described above, since the DSV control bits are redundant bits, it is preferable to have fewer DSV control bits.\",\n \"In order to maintain the minimum run length or the maximum run length, the DSV control bits of at least 2 bits or greater are necessary.\",\n \"The assignee of the present invention et al.\",\n \"discloses, in Japanese Patent Application No.\",\n \"10-150280, an RLL code with the minimum run length d=1 (d, k; m, n) and a conversion table, which is shown in Table 2, for limiting the number of consecutive minimum run lengths and for performing complete DSV control using efficient control bits while maintaining the minimum run length and the maximum run length (hereinafter referred to as a 1,7PP table; and a code according to 1,7PP table is referred to as a 1,7PP code): TABLE 2 1, 7PP (d, k, m, n, r) = (1, 7, 2, 3, 4) Data word Codeword 11 *0* 10 001 01 010 0011 010 100 0010 010 000 0001 000 100 000011 000 100 100 000010 000 100 000 000001 010 100 100 000000 010 100 000 “110111 001 000 000 (next010) 00001000 000 100 100 100 00000000 010 100 100 100 if xx1 then *0* = 000 xx0 then *0* = 101 Termination table 00 000 0000 010 100 “110111 001 000 000 (next010) When next channel bits are ‘010’ convert ‘11 01 11’ to ‘001 000 000’ after using main table and termination table.\",\n \"As an example of a modulation apparatus using the 1,7PP table, the assignee of the present invention discloses, in Japanese Patent Application No.\",\n \"10-150280, a modulation apparatus 1 shown in FIG.\",\n \"1.The modulation apparatus 1 includes a DSV control bit determination and insertion unit 11 for determining “1” or “0” serving as a DSV control bit and inserting the DSV control bit into an input data stream at arbitrary intervals; a modulator 12 for modulating the data stream containing the DSV control bits; and an NRZI unit 13 for converting the output of the modulator 12 into a recording code string.\",\n \"Although not shown in the diagram, the modulation apparatus 1 includes a timing management unit for generating a timing signal, supplying the timing signal to the above components, and managing timing.\",\n \"In Japanese Patent Application No.\",\n \"09-342416, the assignee of the present invention et al.\",\n \"discloses a specific example of another modulation apparatus, namely, a modulation apparatus 2 shown in FIG.\",\n \"2.The modulation apparatus includes a DSV control bit insertion unit 21 for inserting “1” or “0” serving as a DSV control bit into a data stream at arbitrary intervals.\",\n \"At this time, there is a data stream into which the DSV control bit “1” is inserted and another data stream into which the DSV control bit “0” is inserted.\",\n \"The modulation apparatus further includes a modulator 22 for modulating the data stream containing the DSV control bits and a DSV controller 23 for NRZI-modulating the modulated code string into level data, calculating DSV of the level data, and consequently outputting a DSV-controlled recording code string.\",\n \"As described above, the 1,7PP code is advantageous in solving the above problems.\",\n \"In contrast, compared with a modulation apparatus using a known method or a technique for performing DSV control on the RLL (1,7) code, the configuration of the known modulation apparatus using the 1,7PP code is complicated, and the circuit size is increased.\",\n \"For example, in the modulation apparatus 2 shown in FIG.\",\n \"2, the register configuration in the modulator 22 is shown in FIG.\",\n \"3.Specifically, the modulator 22 has a modulation (1,7PP modulation) portion and a delay portion corresponding to a DSV control interval (DSV section) in one integrated unit in order to transfer data corresponding to the DSV control interval to the DSV controller 23 at a subsequent stage.\",\n \"As a result, the modulator 22 needs two registers, that is, an input register 22a (register 22a for the data stream) and an output register 22b (register 22b for the channel bit string).\",\n \"The number of registers required corresponds to the DSV control interval.\",\n \"Two pairs of registers (registers 22a and 22b) are necessary for the DSV control bit “0” and the DSV control bit “1”.\",\n \"DISCLOSURE OF INVENTION In view of these circumstances, it is an object of the present invention to suppress an increase in circuit size of a modulation apparatus.\",\n \"A modulation apparatus of the present invention includes DSV-control-bit generating means for generating a DSV control bit to be inserted into an input bit string in order to control a DSV of a recording code string or a transmission code string; timing adjusting means for adjusting transmission timing for transmitting the input bit string; DSV-control-bit-inserted bit string generating means for generating a DSV-control-bit-inserted bit string by inserting the DSV control bit generated by the DSV-control-bit generating means into a predetermined position of the input bit string whose transmission timing is adjusted by the timing adjusting means; and first modulation means for modulating the DSV-control-bit-inserted bit string generated by the DSV-control-bit-inserted bit string generating means into a channel bit string on the basis of a conversion rule (d, k; m, n; r).\",\n \"The modulation apparatus may further include NRZI means for performing NRZI modulation of the channel bit string generated by modulation by the first modulation means to generate the recording code string or the transmission code string.\",\n \"The conversion rule may state that the remainder of the number of “1s” in a predetermined block of the input bit string or the DSV-control-bit-inserted bit string divided by two equals the remainder of the number of “1s” in the corresponding block of the channel bit string divided by two.\",\n \"The conversion rule may state that the number of consecutive minimum run length d of the channel bit string is limited to a predetermined number or less.\",\n \"The conversion rule may state a variable-length code (d, k; m, n; r) where the minimum run length d=1, the maximum run length k=7, the length of basic data prior to conversion m=2, and the length of basic channel bits subsequent to conversion n=3.Data of m length, which is the length of basic data, may be input for a time period in which the channel bit string of n length, which is the length of the basic channel bits, is output.\",\n \"The DSV-control-bit generating mean may include first-candidate-bit-inserted bit string generating means for generating a first-candidate-bit-inserted bit string, which is a candidate for the DSV-control-bit-inserted bit string, by inserting a first candidate bit for the DSV control bit into the predetermined position of the input bit string; second-candidate-bit-inserted bit string generating means for generating a second-candidate-bit-inserted bit string, which is another candidate for the DSV-control-bit-inserted bit string, by inserting a second candidate bit for the DSV control bit into the predetermined position of the input bit string; second modulation means for modulating, on the basis of the same conversion rule as the conversion rule used by the first modulation means, the first-candidate-bit-inserted bit string generated by the first-candidate-bit-inserted bit string generating means into a first candidate channel bit string, which is a candidate for the channel bit string, and for modulating the second-candidate-bit-inserted bit string generated by the second-candidate-bit-inserted bit string generating means into a second candidate channel bit string, which is another candidate for the channel bit string; DSV calculating means for calculating a DSV of each of the first and second candidate channel bit strings generated by modulation by the second modulation means; and DSV control bit determining means for determining one of the first and second candidate bits as the DSV control bit on the basis of the DSVs calculated by the DSV calculating means.\",\n \"The DSV calculating means may include section DSV calculating means for calculating a section DSV of a current DSV control section of each of the first and second candidate channel bit strings; cumulative DSV calculating means for calculating a cumulative DSV on the basis of the determination result by the DSV control bit determining means; and adding means for calculating the DSV by adding each section DSV calculated by the section DSV calculating means and the cumulative DSV immediately before the current DSV control section, the cumulative DSV being calculated by the cumulative DSV calculating means.\",\n \"The first and second modulation means each may have a minimum number of registers required to perform modulation based on the coding rule.\",\n \"The modulation apparatus may further include first sync signal inserting means for inserting a sync pattern including a preset unique pattern into the channel bit string.\",\n \"The DSV-control-bit generating means may include second sync signal inserting means for inserting the same sync pattern as the sync pattern inserted by the first sync signal inserting means into each of the first and second candidate channel bit strings generated by modulating the first- and second-candidate-bit-inserted bit strings generated by inserting the first and second candidate bits, respectively, into the input bit string.\",\n \"The DSV calculating means may calculate the DSV on the basis of each of the first and second candidate channel bit strings, each of which includes the sync pattern inserted by the second sync signal inserting means.\",\n \"The timing adjusting means may adjust the transmission timing by adding a delay time to the input bit string.\",\n \"The timing adjusting means may insert a temporary value prior to determination of the DSV control bit into the input bit string at predetermined intervals.\",\n \"The modulation apparatus may further include checking information generating means for calculating a final cumulative DSV of the recording code string or the transmission code string, determining whether or not the calculated final cumulative DSV is within a predetermined range, and generating checking information on the basis of the determination result.\",\n \"The DSV-control-bit generating means may generate the DSV control bit on the basis of the checking information generated by the checking information generating means.\",\n \"When it is determined that the final cumulative DSV is out of the predetermined range, the checking information generating means may reset the final cumulative DSV to zero and generate an error signal serving as the checking information.\",\n \"The DSV-control-bit generating means may internally calculate a cumulative DSV for generating the DSV control bit and, when the error signal is generated by the checking information generating means, may reset the cumulative DSV to zero.\",\n \"A modulation method of the present invention includes a DSV-control-bit generating step of generating a DSV control bit to be inserted into an input bit string in order to control a DSV of a recording code string or a transmission code string; a timing adjusting step of adjusting transmission timing for transmitting the input bit string; a DSV-control-bit-inserted bit string generating step of generating a DSV-control-bit-inserted bit string by inserting the DSV control bit generated in the DSV-control-bit generating step into a predetermined position of the input bit string whose transmission timing is adjusted in the timing adjusting step; and a modulation step of modulating the DSV-control-bit-inserted bit string generated in the DSV-control-bit-inserted bit string generating step into a channel bit string on the basis of a conversion rule (d, k; m, n; r).\",\n \"A program on a recording medium of the present invention includes a DSV-control-bit generating step of generating a DSV control bit to be inserted into an input bit string in order to control a DSV of a recording code string or a transmission code string; a timing adjusting step of adjusting transmission timing for transmitting the input bit string; a DSV-control-bit-inserted bit string generating step of generating a DSV-control-bit-inserted bit string by inserting the DSV control bit generated in the DSV-control-bit generating step into a predetermined position of the input bit string whose transmission timing is adjusted in the timing adjusting step; and a modulation step of modulating the DSV-control-bit-inserted bit string generated in the DSV-control-bit-inserted bit string generating step into a channel bit string on the basis of a conversion rule (d, k; m, n; r).\",\n \"A program of the present invention includes a DSV-control-bit generating step of generating a DSV control bit to be inserted into an input bit string in order to control a DSV of a recording code string or a transmission code string; a timing adjusting step of adjusting transmission timing for transmitting the input bit string; a DSV-control-bit-inserted bit string generating step of generating a DSV-control-bit-inserted bit string by inserting the DSV control bit generated in the DSV-control-bit generating step into a predetermined position of the input bit string whose transmission timing is adjusted in the timing adjusting step; and a modulation step of modulating the DSV-control-bit-inserted bit string generated in the DSV-control-bit-inserted bit string generating step into a channel bit string on the basis of a conversion rule (d, k; m, n; r).\",\n \"According to a modulation apparatus and method, a recording medium, and a program of the present invention, a DSV control bit to be inserted into an input bit string is generated in order to control a DSV of a recording code string or a transmission code string.\",\n \"Transmission timing for transmitting the input bit string is adjusted.\",\n \"The generated DSV control bit is inserted into a predetermined position of the input bit string, whose transmission timing is adjusted, to generate a DSV-control-bit-inserted bit string.\",\n \"The generated DSV-control-bit-inserted bit string is modulated into a channel bit string on the basis of a conversion rule (d, k; m, n; r).\",\n \"A DSV-control-bit generating method of the present invention includes a first-candidate-bit-inserted bit string generating step of generating a first-candidate-bit-inserted bit string, which is a candidate for a DSV-control-bit-inserted bit string, by inserting a first candidate bit for a DSV control bit into a predetermined position of an input bit string; a second-candidate-bit-inserted bit string generating step of generating a second-candidate-bit-inserted bit string, which is another candidate for the DSV-control-bit-inserted bit string, by inserting a second candidate bit for the DSV control bit into the predetermined position of the input bit string; a modulation step of modulating, on the basis of the same conversion rule as a conversion rule applied when modulating the input bit string, the first-candidate-bit-inserted bit string generated in the first-candidate-bit-inserted bit string generating step into a first candidate channel bit string, which is a candidate for a channel bit string, and for modulating the second-candidate-bit-inserted bit string generated in the second-candidate-bit-inserted bit string generating step into a second candidate channel bit string, which is another candidate for the channel bit string; a DSV calculating step of calculating a DSV of each of the first and second candidate channel bit strings generated by modulation in the modulation step; and a DSV control bit determining step of determining one of the first and second candidate bits as the DSV control bit on the basis of the DSVs calculated in the DSV calculating step.\",\n \"A program on a recording medium of the present invention includes a first-candidate-bit-inserted bit string generating step of generating a first-candidate-bit-inserted bit string, which is a candidate for a DSV-control-bit-inserted bit string, by inserting a first candidate bit for a DSV control bit into a predetermined position of an input bit string; a second-candidate-bit-inserted bit string generating step of generating a second-candidate-bit-inserted bit string, which is another candidate for the DSV-control-bit-inserted bit string, by inserting a second candidate bit for the DSV control bit into the predetermined position of the input bit string; a modulation step of modulating, on the basis of the same conversion rule as a conversion rule applied when modulating the input bit string, the first-candidate-bit-inserted bit string generated in the first-candidate-bit-inserted bit string generating step into a first candidate channel bit string, which is a candidate for a channel bit string, and for modulating the second-candidate-bit-inserted bit string generated in the second-candidate-bit-inserted bit string generating step into a second candidate channel bit string, which is another candidate for the channel bit string; a DSV calculating step of calculating a DSV of each of the first and second candidate channel bit strings generated by modulation in the modulation step; and a DSV control bit determining step of determining one of the first and second candidate bits as the DSV control bit on the basis of the DSVs calculated in the DSV calculating step.\",\n \"A program of the present invention includes a first-candidate-bit-inserted bit string generating step of generating a first-candidate-bit-inserted bit string, which is a candidate for a DSV-control-bit-inserted bit string, by inserting a first candidate bit for a DSV control bit into a predetermined position of an input bit string; a second-candidate-bit-inserted bit string generating step of generating a second-candidate-bit-inserted bit string, which is another candidate for the DSV-control-bit-inserted bit string, by inserting a second candidate bit for the DSV control bit into the predetermined position of the input bit string; a modulation step of modulating, on the basis of the same conversion rule as a conversion rule applied when modulating the input bit string, the first-candidate-bit-inserted bit string generated in the first-candidate-bit-inserted bit string generating step into a first candidate channel bit string, which is a candidate for a channel bit string, and for modulating the second-candidate-bit-inserted bit string generated in the second-candidate-bit-inserted bit string generating step into a second candidate channel bit string, which is another candidate for the channel bit string; a DSV calculating step of calculating a DSV of each of the first and second candidate channel bit strings generated by modulation in the modulation step; and a DSV control bit determining step of determining one of the first and second candidate bits as the DSV control bit on the basis of the DSVs calculated in the DSV calculating step.\",\n \"According to a DSV-control-bit generating method, a recording medium, and a program of the present invention, a first-candidate-bit-inserted bit string, which is a candidate for a DSV-control-bit-inserted bit string, is generated by inserting a first candidate bit for a DSV control bit into a predetermined position of an input bit string.\",\n \"A second-candidate-bit-inserted bit string, which is another candidate for the DSV-control-bit-inserted bit string, is generated by inserting a second candidate bit for the DSV control bit into the predetermined position of the input bit string.\",\n \"On the basis of the same conversion rule as a conversion rule applied when modulating the input bit string, the generated first-candidate-bit-inserted bit string is modulated into a first candidate channel bit string, which is a candidate for channel bits; and the generated second-candidate-bit-inserted bit string is modulated into a second candidate channel bit string, which is another candidate for the channel bits.\",\n \"A DSV of each of the first and second candidate channel bit strings is calculated.\",\n \"One of the first and second candidate bits is determined as the DSV control bit on the basis of the calculated DSVs.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS FIG.\",\n \"1 is a block diagram showing an example of the configuration of a known modulation apparatus.\",\n \"FIG.\",\n \"2 is a block diagram showing an example of the configuration of another known modulation apparatus.\",\n \"FIG.\",\n \"3 is a diagram showing an example of the register configuration in a modulator of the modulation apparatus shown in FIG.\",\n \"2.FIG.\",\n \"4 is a block diagram showing an example of the configuration of a modulation apparatus according to the present invention.\",\n \"FIG.\",\n \"5 is a block diagram showing details of the configuration of the modulation apparatus shown in FIG.\",\n \"4.FIG.\",\n \"6 is a flowchart for describing the operation of the modulation apparatus shown in FIG.\",\n \"4.FIG.\",\n \"7 is a diagram for describing the data format at each stage of a data stream modulated by the modulation apparatus shown in FIG.\",\n \"4.FIG.\",\n \"8 is a chart for describing timing of data to be input to the modulation apparatus shown in FIG.\",\n \"4.FIG.\",\n \"9 is a diagram showing an example of the register configuration in a modulator of the modulation apparatus shown in FIG.\",\n \"4.FIG.\",\n \"10 is a block diagram showing an example of the configuration of another modulation apparatus according to the present invention.\",\n \"BEST MODE FOR CARRYING OUT THE INVENTION FIG.\",\n \"4 shows an example of the configuration of a modulation apparatus 3 according to the present invention.\",\n \"The modulation method employed by the modulation apparatus 3 is not limited.\",\n \"In this example, the modulation apparatus 3 converts a data stream into, for example, a variable-length code (d, k; m, n; r)=(1, 7; 2, 3; 4).\",\n \"An input unit 38 receives an input data stream supplied from the outside and supplies the input data stream to a DSV control bit determination unit 31 and a delay processor 32.The DSV control bit determination unit 31 executes a predetermined arithmetic operation to determine the DSV control bit “1” or “0” to be inserted into the supplied input data stream and supplies the determination result to a determined-DSV-control-bit insertion unit 33.The delay processor 32 delays the supplied input data stream for a predetermined delay time and supplies the delayed input data stream to the determined-DSV-control-bit insertion unit 33.Specifically, the delay processor 32 adjusts the transmission timing to supply the input data stream to the determined-DSV-control-bit insertion unit 32.The predetermined delay time is set on the basis of the timing to insert, by the determined-DSV-control-bit insertion unit 33 described below, the DSV control bit determined by the DSV control bit determination unit 31 into a predetermined position of the input data stream output by the delay processor 32.When the determined-DSV-control-bit insertion unit 33 receives, at a predetermined time, the input data stream supplied by the delay processor 32 and the DSV-control-bit determination result (the result indicating whether the DSV control bit is “0” or “1”) supplied at a predetermined time by the DSV control bit determination unit 31, the determined-DSV-control-bit insertion unit 33 inserts a DSV control bit corresponding to the received determination result into the predetermined position (hereinafter referred to as the DSV position) of the received input data stream to generate a new data stream (hereinafter referred to as a DSV-control-bit-inserted bit string in order to be distinguished from other data streams) and supplies the DSV-control-bit-inserted bit string to a modulator 34.Specifically, the predetermined delay time of the delay processor 32 is set so that a bit corresponding to the DSV position of the input data stream will be input to the determined-DSV-control-bit insertion unit 33 at the above-described predetermined time.\",\n \"The modulator 34 modulates the DSV-control-bit-inserted bit string into a code string (channel bit string) in accordance with a predetermined conversion rule (e.g., 1,7PP table shown in Table 2) and supplies the code string to a sync signal insertion unit 35.The sync signal insertion unit 35 inputs a prepared sync signal to a predetermined position of the code string supplied by the modulator 34 at a predetermined time (differing from that of the determined-DSV-control-bit insertion unit 33) and supplies the resultant code string to an NRZI unit 36.The sync signal consists of a predetermined pattern containing a predetermined number of channel bits and is referred to as Frame Sync hereinafter.\",\n \"The NRZI unit 36 performs NRZI modulation of the code string supplied by the sync signal insertion unit 35 to generate a recording code string and outputs the recording code string to the outside and to a cumulative DSV checking unit 37.As described above, bit-string permutation by inverting 1 and not inverting 0 of a code string is referred to as NRZI modulation.\",\n \"In other words, the code string prior to being NRZI-modulated is a bit string indicating the edge position, whereas the NRZI-modulated recording code string corresponds to a bit string indicating the H/L (High/Low) level of recording data.\",\n \"The cumulative DSV checking unit 37 receives the recording code string supplied by the NRZI unit 36, calculates the cumulative DSV so far (hereinafter referred to as the final cumulative DSV in order to be distinguished from other DSVs), determines whether or not the calculated final cumulative DSV is within a predetermined range, and generates checking information based on the determination result.\",\n \"Specifically, when the cumulative DSV checking unit 37 determines that the final cumulative DSV is out of the predetermined range, the cumulative DSV checking unit 37 resets the final cumulative DSV to “0” or sets the final cumulative DSV to a predetermined initial value, generates an error signal serving as the checking information, and supplies the error signal to the DSV control bit determination unit 31.Specifically, the cumulative DSV checking unit 37 determines whether or not the final cumulative DSV at each moment exceeds the predetermined range (e.g., a range from −128 to +127 or a range from 0 to 255 when represented in sign and magnitude notation).\",\n \"When it is determined that the final cumulative DSV exceeds the predetermined range, the error signal is supplied to a cumulative DSV calculator 55, which will be described later, in the DSV control bit determination unit 31 shown in FIG.\",\n \"5.The cumulative DSV calculator 55 resets the calculated cumulative DSV so far to “0” or sets the calculated cumulative DSV so far to the predetermined initial value.\",\n \"In this example, when the cumulative DSV checking unit 37 determines that the final cumulative DSV is within the predetermined range, the cumulative DSV checking unit 37 generates no checking information.\",\n \"Alternatively, the cumulative DSV checking unit 37 may generate a signal corresponding to “normal” or the like serving as the checking information.\",\n \"Referring to FIG.\",\n \"5, the modulation apparatus 3 will be described in more detail.\",\n \"Specifically, FIG.\",\n \"5 shows an example of the detailed configuration of the modulation apparatus 3.In FIG.\",\n \"5, the symbol “+” in the square represents a portion that processes a data stream and functions as an insertion unit or a selector.\",\n \"In order to simplify the description, the symbol is simply referred to as an adder.\",\n \"Referring to FIG.\",\n \"5, the input data stream received by the input unit 38 is supplied to the DSV control bit determination unit 31 and the delay processor 32 at the same time.\",\n \"In the DSV control bit determination unit 31, an adder 42 inserts “0” serving as a first candidate bit for the DSV control bit into the predetermined position (DSV position) of the received input data stream to generate a data stream serving as a first candidate for the DSV-control-bit-inserted bit string (hereinafter referred to as a first-candidate-bit-inserted bit string) and supplies the first-candidate-bit-inserted bit string to a 1,7PP modulator 45.The 1,7PP modulator 45 modulates the first-candidate-bit-inserted bit string into a code string (hereinafter referred to as a first candidate code string in order to be distinguished from other code strings) in accordance with, for example, the above-described 1,7PP table shown in Table 2 and supplies the first candidate code string to an adder 48.The adder 48 inserts, at a predetermined time, Frame Sync (sync signal) into a predetermined position of the first candidate code string supplied by the 1,7PP modulator 45 and supplies the resultant code string to an NRZI unit 51.As described above, for example, the 1,7PP table shown in Table 2 is used in this example.\",\n \"The 1,7PP table contains a termination table for inserting Frame Sync.\",\n \"The 1,7PP modulator 45 performs termination based on the termination table.\",\n \"Specifically, termination is to set, on a data stream, a break point at a position immediately before a position at which Frame Sync is to be inserted and to terminate table-based conversion (modulation) at the break point.\",\n \"Since the 1,7PP table has a variable-length structure, the table-based conversion has a variable end position.\",\n \"The modulation apparatus 3 uses the above-described termination table if necessary to terminate the table-based conversion at an arbitrary position on the data stream in units of two.\",\n \"The adder 48 inserts Frame Sync containing the predetermined number of channel bits into a position immediately after the end position of the table-based conversion at the time the table-based conversion is terminated.\",\n \"As described above, Frame Sync contains a predetermined pattern distinguishable from other code strings (a unique pattern not included in conversion codes in the conversion table).\",\n \"In this example, the same Frame Sync is inserted by the above-described adder 48, an adder 49, and an adder 65, which will be described later.\",\n \"The NRZI unit 51 performs NRZI modulation of the first candidate code string supplied by the adder 48 to generate a recording code string (hereinafter referred to as a first candidate symbol code string in order to be distinguished from other recording code strings) and supplies the first candidate recording code string to a section DSV calculator 53.The section DSV calculator 53 calculates a DSV in a predetermined DSV section (hereinafter referred to as a section DSV in order to be distinguished from other DSVs) on the basis of the first candidate recording code string and supplies the section DSV to an adder 56.The adder 56 adds the section DSV of the first candidate recording code string and the cumulative DSV so far supplied by the cumulative DSV calculator 55 described below and supplies the sum to a comparator 58.In contrast, an adder 43 inserts “1” serving as a second candidate bit for the DSV control bit into the predetermined position (DSV position) of the received input data stream to generate a data stream serving as a second candidate for the DSV-control-bit-inserted bit string (hereinafter referred to as a second-candidate-bit-inserted bit string) and supplies the second-candidate-bit-inserted bit string to a 1,7PP modulator 46.The second-candidate-bit-inserted bit string supplied to the 1,7PP modulator 46 is modulated, as in the above-described first-candidate-bit-inserted bit string, by the 1,7PP modulator 46 into a code string (hereinafter referred to as a second candidate code string to be distinguished from other code strings).\",\n \"The adder 49 inserts Frame Sync into the second candidate code string at a predetermined time (predetermined position).\",\n \"An NRZI unit 52 performs NRZI modulation of the second candidate code string containing Frame Sync to generate a recording code string (hereinafter referred to as a second candidate recording code string to be distinguished from other recording code strings).\",\n \"The second candidate recording code string is supplied to a section DSV calculator 54.The section DSV calculator 54 calculates a section DSV in a predetermined DSV section on the basis of the second candidate recording code string and supplies the section DSV to an adder 57.The adder 57 adds the section DSV of the second candidate recording code string and the cumulative DSV so far supplied by the cumulative DSV calculator 55 described below and supplies the sum to the comparator 58.Accordingly, the cumulative DSV of the data stream generated by inserting “0” serving as a candidate for the DSV control bit into the input data stream (the first-candidate-bit-inserted bit string) and the cumulative DSV of the data stream generated by inserting “1” serving as another candidate for the DSV control bit into the input data stream (the second-candidate-bit-inserted bit string) are supplied to the comparator 58.The comparator 58 compares the absolute values of the two cumulative DSVs, selects the data stream having the cumulative DSV whose absolute value is smaller (first- or second-candidate-bit-inserted bit string), and determines, as the DSV control bit to be actually inserted into the input data stream, the first or second candidate DSV control bit included in the selected data stream (“0” when the first-candidate-bit-inserted bit string is selected and “1” when the second-candidate-bit-inserted bit string is selected).\",\n \"Specifically, the comparator 58 supplies a DSV control bit selection signal (signal indicating “1” or “0”) corresponding to the determined DSV control bit to an AND operator 62.The comparator 58 supplies the cumulative DSV of the selected data stream to the cumulative DSV calculator 55.The cumulative DSV calculator 55 receives the cumulative DSV supplied by the comparator 58 and determines the received cumulative DSV as the cumulative DSV.\",\n \"When the subsequent section DSV of the first or second candidate recording code string is supplied to the adder 56 or the adder 57, the cumulative DSV calculator 55 supplies the cumulative DSV determined immediately before the supply of the section DSV to the adder 56 or the adder 57.As described above, when the final cumulative DSV corresponding to the currently output recording code string exceeds the predetermined range, the cumulative DSV checking unit 37 supplies the error signal to the cumulative DSV calculator 55.The cumulative DSV calculator 55 receives the error signal and resets the currently determined cumulative DSV to 0 or sets the currently determined cumulative DSV to the predetermined initial value.\",\n \"In the delay processor 32, an adder 60 inserts “0” serving as a temporary value prior to determination of the DSV control bit into the predetermined position (DSV position) of the input data stream supplied by the input unit 38 to generate a new data stream (hereinafter referred to as a temporary-DSV-control-bit-inserted bit string to be distinguished from other data streams) and supplies the temporary-DSV-control-bit-inserted bit string to a DSV section delay shift register 61.Specifically, the temporary-DSV-control-bit-inserted bit string is the same data stream as the first-candidate-bit-inserted bit string generated by the above-described adder 42.In this example, “0” is inserted as the temporary value prior to determination of the DSV control bit.\",\n \"When a combination of logic circuits in the determined-DSV-control-bit insertion unit 33 described below changes, “1” may be inserted.\",\n \"A temporary-DSV-control-bit-inserted bit string in this case is the same data stream as the second-candidate-bit-inserted bit string generated by the above-described adder 43.The DSV section delay shift register 61 delays the temporary-DSV-control-bit-inserted bit string for a predetermined delay time and supplies the delayed temporary-DSV-control-bit-inserted bit string to the determined-DSV-control-bit insertion unit 33.The DSV section delay shift register 61 contains registers, the number of which corresponds to a delay of x bits corresponding to the DSV control section, and, if necessary, a delay of α bits corresponding to a circuit delay (delay corresponding to the circuit delay α shown in FIG.\",\n \"5).\",\n \"The order of the adder 60 and the shift register 61 of the delay processor 32 may be reversed.\",\n \"Specifically, the adder 60 may insert “0” serving as the temporary value prior to determination of the DSV control bit into the input data stream delayed by the shift register 61 to generate a temporary-DSV-control-bit-inserted bit string and may supply the temporary-DSV-control-bit-inserted bit string to the determined-DSV-control-bit insertion unit 33.In the determined-DSV-control-bit insertion unit 33, the AND operator 62 performs an AND operation (logical AND) of “1” supplied at a predetermined time by a DSV control bit position gate 64 and the DSV control bit selection signal indicating “0” or “1” supplied by the above-described comparator 58 and supplies the logical operation result to an OR operator 63.The OR operator 63 performs an OR operation (logical OR) of the operation result (“1” or “0”) supplied by the AND operator 62 and predetermined bit data of the temporary-DSV-control-bit-inserted bit string supplied by the DSV section delay shift register 61 and supplies the logical operation result to the 1,7PP modulator 34.When the delay processor 32 (DSV section delay shift register 61) supplies bit data corresponding to the DSV position of the temporary-DSV-control-bit-inserted bit string (“0” inserted by the adder 60 and serving as the temporary value prior to determination of the DSV control bit) to the OR operator 63, the above-described DSV control bit position gate 64 supplies “1” to the AND operator 62 at that time.\",\n \"As described above, when “1” serving as the DSV control bit selection signal is supplied to the AND operator 62 and “1” is supplied by the DSV control bit position gate 64 to the AND operator 62 (when a bit corresponding to the DSV position is supplied to the OR operator 63), the AND operator 62 supplies “1” serving as the logical operation result to the OR operator 63.Specifically, the OR operator 63 receives “1” supplied by the AND operator 62 and bit data that is supplied by the delay processor 32 and that corresponds to the DSV position, that is, “0” inserted as the temporary DSV control bit by the adder 60, performs an OR operation of the received “1” and “0”, and supplies “1” serving as the logical operation result to the 1,7PP modulator 34.In other words, when the DSV control bit determined by the DSV control bit determination unit 31 is “1”, the determined-DSV-control-bit insertion unit 33 converts “0 (temporary value prior to determination of the DSV control bit)” inserted into the DSV position by the adder 60 into “1 (DSV control bit determined by the DSV control bit determination unit 31)”.\",\n \"In contrast, when the DSV control bit determined by the DSV control bit determination unit 31 is “0”, the determined-DSV-control-bit insertion unit 33 uses “0 (temporary value prior to determination of the DSV control bit)” inserted into the DSV position by the adder 60, unchanged, as the DSV control bit (performs no conversion).\",\n \"Accordingly, the determined-DSV-control-bit insertion unit 33 inserts the DSV control bit determined by the DSV control bit determination unit 31 into the DSV position of the temporary-DSV-control-bit-inserted bit string (position at which the temporary value prior to determination of the DSV control bit is inserted) to generate a DSV-control-bit-inserted bit string and supplies the DSV-control-bit-inserted bit string to the 1,7PP modulator 34.Since the 1,7PP modulator 34 has the same configuration as the above-described 1,7PP modulator 45 and the 1,7PP modulator 46, and since the adder 65 of the sync signal insertion unit 35 has the same configuration as the above-described adder 48 and adder 49, descriptions of the 1,7PP modulator 34 and the adder 65 are omitted.\",\n \"Since the NRZI unit 36 and the cumulative DSV checking unit 37 have already been described, descriptions of the NRZI unit 36 and the cumulative DSV checking unit 37 are omitted.\",\n \"The modulator 34 in FIG.\",\n \"4 and the 1,7PP modulator 34 in FIG.\",\n \"5 are the same modulator.\",\n \"In order to emphasize that the modulator performs 1,7PP modulation, in FIG.\",\n \"5, the modulator is expressed as the 1,7PP modulator 34.With reference to the flowchart of FIG.\",\n \"6, the operation of the modulation apparatus 3 will now be described.\",\n \"An input data stream 71 shown in FIG.\",\n \"7 is supplied to the modulation apparatus 3.In step S11, the modulation apparatus 3 receives the input data stream 71.The time at which the input data stream 71 is input is as shown in FIG.\",\n \"8.Specifically, a channel bit string (code string) 74 output by the 1,7PP modulator 34 is output as a serial recording code string in synchronization with a predetermined clock 75.In other words, one codeword is output every clock cycle.\",\n \"In contrast, the input data stream 71 is input in accordance with the conversion ratio m/n of the 1,7PP modulator 34.Specifically, in this example, the conversion ratio is ⅔.\",\n \"The amount of data of a codeword is three, whereas the amount of data of a data word of the input data stream 71 is two.\",\n \"As shown in FIG.\",\n \"8, the modulation apparatus 3 receives, of the input data stream 71, predetermined two data words within two clock cycles and stops receiving the input data stream 71 for one clock cycle.\",\n \"Accordingly, a mismatch in the conversion ratio between the input data and the output code is adjusted.\",\n \"Referring to FIG.\",\n \"7, a DSV section of the input data stream 71 has x bits.\",\n \"A 1-bit DSV control bit is inserted at the end of each x-bit data.\",\n \"In order to be distinguished from unit data, i.e., a data word, to be modulated by the 1,7PP modulator 34, the x-bit data is referred to as data Dk (k is an integer).\",\n \"In other words, the DSV position of data Dk is immediately after the end of the data Dk.\",\n \"Data D1 into which Frame Sync is inserted has a short DSV section.\",\n \"Specifically, Data D1 consists of x-Fsx(⅔) bits (Fs is the number of bits of Frame Sync).\",\n \"Referring back to FIG.\",\n \"6, in step S12, the modulation apparatus 3 determines the DSV control bit and inserts the DSV control bit into the predetermined position of the input data stream 71 to generate a DSV-control-bit-inserted bit string 72 shown in FIG.\",\n \"7.Specifically, when each data word of the input data stream 71 is supplied to the DSV control bit determination unit 31 in the order shown in FIG.\",\n \"8, the DSV control bit determination unit 31 receives each data word and determines the DSV control bit to be inserted into the DSV position of x-bit data received, that is, data Dk.\",\n \"At the same time, the input data stream 71 is also supplied to the delay processor 32 in the order shown in FIG.\",\n \"8 and delayed for a predetermined delay time, and the delayed input data stream 71 is supplied to the determined-DSV-control-bit insertion unit 33.The determined-DSV-control-bit insertion unit 33 inserts the DSV control bit (bit “0” or “1”) of the data Dk, which is determined by the DSV control bit determination unit 31, into the DSV position of the data Dk at the time the bit at the DSV position of the data Dk is supplied by the delay processor 32 to generate the DSV-control-bit-inserted bit string 72 and supplies the DSV-control-bit-inserted bit string 72 to the 1,7PP modulator 34.In step S13, the modulation apparatus 3 performs 1,7PP modulation of the DSV-control-bit-inserted bit string 72 to generate a predetermined code string.\",\n \"Specifically as described above (as shown in FIG.\",\n \"8), since the input data is received in units of two data words (stopped for one clock cycle), the 1,7PP modulator 34 modulates the DSV-control-bit-inserted bit string 72 in units of two data words.\",\n \"In other words, the 1,7PP modulator 34 processes the data in units of three clock cycles (three channel bits).\",\n \"The timing is generated by a counter or the like (not shown).\",\n \"The register configuration in the 1,7PP modulator 34 is as shown in FIG.\",\n \"9.Also, the register configuration in each of the 1,7PP modulator 45 and the 1,7PP modulator 46 is as shown in FIG.\",\n \"9.Specifically, an input register 81 of the 1,7PP modulator 34 (register 81 associated with the DSV-control-bit-inserted bit string 72 supplied to the 1,7PP modulator 34) and an output register 82 (register 82 associated with a code string 73 output by the 1,7PP modulator 34) have minimum number of registers required to modulate each data word in accordance with the 1,7PP table shown in Table 2.Specifically, the input register 81 is provided with a number of registers which are 12 bits.\",\n \"The output register 82 is provided with a number of registers which are 18 bits.\",\n \"The 1,7PP modulator 34 includes a timing control register (not shown).\",\n \"The number of registers required by the 1,7PP modulator 34 is the minimum number of registers required to modulate each data word and does not depend on the DSV interval of the format (in this example, the x-bit section).\",\n \"In other words, the registers required by the 1,7PP modulator 34 are provided without taking into consideration a delay corresponding to a DSV control interval portion.\",\n \"When the input data word consists of two bits (constraint length i=1), the 1,7PP modulator 34 places the bits into [0, 1] of the input register 81 shown in FIG.\",\n \"9.Referring to [0,1] replaced with the corresponding bits, the 1,7PP modulator 34 modulates the bits when a predetermined condition is satisfied and places the channel bits into [0, 1, 2] of the output register 82.Similarly, when the input data word consists of four bits (constraint length i=2), the 1,7PP modulator 34 refers to [0, 1, 2, 3] of the input register 81 containing the corresponding bits, modulates the bits when a predetermined condition is satisfied, and places the channel bits into [0, 1, 2, 3, 4, 5] of the output register 82.When the input data word consists of six bits (constraint length i=3), the 1,7PP modulator 34 refers to [0, 1, 2, 3, 4, 5] of the input register 81 containing the corresponding bits, modulates the bits when a predetermined condition is satisfied, and places the channel bits into [0, 1, 2, 3, 4, 5, 6, 7, 8] of the output register 82.In the case of the maximum constraint length, that is, when the input data word consists of eight bits (constraint length i=4), the 1,7PP modulator 34 refers to [0, 1, 2, 3, 4, 5, 6, 7] of the input register 81 containing the corresponding bits, modulates the bits when a predetermined condition is satisfied, and places the channel bits into [0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11] of the output register 82.When processing the Prohibit rmtr portion (110111-next_cbit:010), the 1,7PP modulator 34 refers to [0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11] of the input register 81 and, when a predetermined condition is satisfied, replaces predetermined positions of the output register 82 with the channel bits.\",\n \"Accordingly, the 1,7PP modulator 34 uses the input register 81 and the output register 82 to perform 1,7PP modulation of the DSV-control-bit-inserted bit string 72 to generate a channel bit string (code string) and supplies the channel bit string (code string) to the sync signal insertion unit 35.In step S14, the modulation apparatus 3 inserts Frame Sync into a predetermined position of the code string output by the 1,7PP modulator 34 to generate the code string (channel bit string) 73 shown in FIG.\",\n \"7.Specifically, when the 1,7PP modulator 34 performs the above-described termination, the sync signal insertion unit 35 inserts Frame Sync (sync signal) at the head of the immediate data Dk (data D1 in this example) to generate the code string (channel bit string) 73 shown in FIG.\",\n \"7 and supplies the code string 73 to the NRZI unit 36.Subsequent to the insertion of Frame Sync into the code string 73, DSV control bits are inserted into the code string 73 at equal intervals (span 1=span 2=span 3), thus achieving appropriate DSV control.\",\n \"More specifically, when the 1,7PP modulator 34 performs termination, the 1,7PP modulator 34 stops outputting data.\",\n \"At the same time, the adder 65 of the sync signal insertion unit 35 changes the selector and adds Frame Sync of a predetermined length.\",\n \"When Frame Sync is added, the adder 65 changes the selector (to the original state), and the 1,7PP modulator 34 resumes data output (the code string 74 is supplied to the sync signal insertion unit 65).\",\n \"Although the above method is described as an example of a method of inserting Frame Sync, the method is not limited to the above-described method.\",\n \"For example, the 1,7PP modulator 34 may perform termination and then supply a temporary code string of the same length as predetermined Frame Sync to the sync signal insertion unit 35, and the sync signal insertion unit 35 may replace the temporary code string with the predetermined Frame Sync.\",\n \"In step S15, the modulation apparatus 3 performs NRZI modulation of the code string 73 to generate a recording code string and outputs the recording code string to the outside.\",\n \"Specifically, the NRZI unit 36 performs NRZI modulation of the code string 73 supplied by the sync signal insertion unit 35 to generate a recording code string and outputs the recording code string to the outside and to the cumulative DSV checking unit 37.The cumulative DSV checking unit 37 receives the recording code string supplied by the NRZI unit 36, calculates the final cumulative DSV, and determines whether or not the calculated final cumulative DSV is within a predetermined range.\",\n \"When it is determined that the final cumulative DSV is out of the predetermined range, the cumulative DSV checking unit 37 supplies the determination result to the cumulative DSV calculator 55 of the DSV control bit determination unit 31.The cumulative DSV calculator 55 resets the calculated cumulative DSV so far to 0 or sets the calculated cumulative DSV so far to a predetermined initial value.\",\n \"The cumulative DSV supplied to the cumulative DSV calculator 55 of the DSV control bit determination unit 31 may be a value supplied by the comparator 58 shown in FIG.\",\n \"5 or may be, for example, the final cumulative DSV calculated by the above-described cumulative DSV checking unit 37.Specifically, supplying the final cumulative DSV calculated by the cumulative DSV checking unit 37 to the cumulative DSV calculator 55 at a predetermined time enables the cumulative DSV calculator 55 to operate in a manner similar to the above-described operation.\",\n \"The code string output by the NRZI unit 36 is a recording code string in this example.\",\n \"In the case of transmission of the output result, the NRZI unit 36 outputs a transmission code string.\",\n \"In this case, the operation of the solid-state imaging device 3 remains unchanged.\",\n \"As described above, according to the modulation apparatus 3 of the present invention, as shown in FIG.\",\n \"9, the number of registers (the input register 81 and the output register 82) in each of the 1,7PP modulator 34, the 1,7PP modulator 45, and the 1,7PP modulator 46 is the minimum number of registers required to perform 1,7PP modulation of each data word and does not depend on the DSV control interval since the portion that performs 1,7PP modulation has an independent structure.\",\n \"On the other hand, registers in a known modulating apparatus (e.g., the modulation apparatus 2 shown in FIG.\",\n \"2) must be of sufficient number corresponding to the DSV control interval, as shown in FIG.\",\n \"3.In the delay processor 32, only the DSV section delay shift register 61) is required, which consists of registers, the number of which corresponds to the total of the channel bit string corresponding to the DSV control interval and the circuit delay α (only one shift register is necessary).\",\n \"Accordingly, the registers required in the modulation apparatus 3 of the present invention are more compact than those in a known modulation apparatus.\",\n \"As a result, the manufacturer can make circuits of the modulation apparatus 3 more compact.\",\n \"A reduction in the number of registers reduces, for example, the power consumption.\",\n \"In particular, when the DSV control interval is increased or the conversion table for converting a data word into a codeword becomes smaller, the advantage of using the modulation apparatus 3 becomes more apparent.\",\n \"A series of the above-described processes may be executed by hardware or software.\",\n \"In the latter case, for example, the modulation apparatus 4 includes a personal computer shown in FIG.\",\n \"10.Referring to FIG.\",\n \"10, a CPU 101 performs various processes in accordance with a program stored in a ROM 102 or a program loaded from a storage unit 108 into a RAM 103.If necessary, the RAM 103 stores data necessary for the CPU 101 to perform the various processes.\",\n \"The CPU 101, the ROM 102, and the RAM 103 are interconnected with one another via a bus 104.An input/output interface 105 is connected to the bus 104.An input unit 106 including a keyboard and a mouse, an output unit 107 including a display, the storage unit 108 including a hard disk, a communication unit 109 including a modem and a terminal adapter are connected to the input/output interface 105.The communication unit 109 performs communication via a network including the Internet.\",\n \"If necessary, a drive 110 is connected to the input/output interface 105.A magnetic disk 121, an optical disk 122, a magneto-optical disk 123, or a semiconductor memory 124 is appropriately placed on the drive 111, and a computer program read from the placed medium is installed into the storage unit 108 if necessary.\",\n \"When a series of processes is to be performed using software, a program providing the software is installed via a network or a recording medium onto a computer included in dedicated hardware or, for example, a general personal computer capable of performing various functions by installing therein various programs.\",\n \"As shown in FIG.\",\n \"10, the recording medium includes a packaged medium including the magnetic disk 121 (including a floppy disk), the optical disk 122 (including a CD-ROM (Compact Disk-Read Only Memory) and DVD (Digital Versatile Disk)), the magneto-optical disk 123 (including MD (Mini-Disk)), or the semiconductor memory 124, all of which have recorded thereon the program and which are distributed, separately from the apparatus, to supply the program to the user.\",\n \"Also, the recording medium includes the ROM 102 or the hard disk included in the storage unit 108, which have recorded thereon the program and which are included in advance in the apparatus to be supplied to the user.\",\n \"In this specification, steps for writing the program stored in the recording medium not only include time-series processing performed in accordance with the described order but also include parallel or individual processing, which may not necessarily be performed in time series.\",\n \"INDUSTRIAL APPLICABILITY As described above, according to a modulation apparatus and method and to a DSV-control-bit generating method of the present invention, an increase in circuit size of the modulation apparatus is suppressed.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10467399"}}},{"rowIdx":365,"cells":{"text":{"kind":"list like","value":[["Systems and methods for automatic carburetor enrichment during cold start","An automatic carburetor (20) enrichment system that controls the air-fuel mixture during cold start of an engine (10) having a carburetor including a fuel bowl (22) and an induction passage (23), includes a sensor (50) that provides a signal indicative of an engine temperature at engine start, a fuel line (30) connected between the fuel bowl (22) and the induction passage, a solenoid valve (40) disposed in the fuel line, and a controller (10) that receives the signal and sets a duty cycle of the solenoid valve associated with the engine temperature to increase the air-fuel ratio of the air-fuel mixture at engine start.","The automatic carburetor enrichment system reduces cranking time during cold start, eliminates the need for driver input during cold start, prevents engine stalling without assistance from the operator during the warm up phase, provides a simpler, more cost effective and reliable carburetor enrichment, provides self-drowning protection without the use of an electronic idle switch and eliminates the risk of engine drowning when the engine is cranked with the choke ON and the ignition switches OFF."],["1.A carburetor enrichment system that controls an air-fuel ratio of an air-fuel mixture supplied to an internal combustion engine during cold start, the engine having a carburetor, that is supplied fuel from a fuel reservoir, and includes an induction passage, the carburetor enrichment system comprising: a sensor that provides a signal indicative of an engine temperature; a fuel line connected between the fuel reservoir and the induction passage; a solenoid valve disposed in the fuel line; and a controller that receives the signal and sets a duty cycle of the solenoid valve associated with the engine temperature to increase the air-fuel ratio of the air-fuel mixture.","2.A carburetor enrichment system according to claim 1, wherein the controller sets the duty cycle of the solenoid valve at one of a) an engine start time and b) a boost duration time associated with the engine temperature.","3.A carburetor enrichment system according to claim 2, wherein the engine start time is determined when the engine reaches a predetermined idle threshold speed.","4.A carburetor enrichment system according to claim 2, wherein the controller stores a plurality of engine temperatures and associated boost duration times.","5.A carburetor enrichment system according to claim 1, wherein the controller stores a plurality of engine temperatures and associated duty cycles.","6.A carburetor enrichment system according to claim 2, wherein the controller reduces the duty cycle of the solenoid valve at a predetermined rate after one of a) the engine start time and b) the boost duration time.","7.A carburetor enrichment system according to claim 6, wherein the predetermined rate is constant.","8.A carburetor enrichment system according to claim 2, wherein the duty cycle of the solenoid valve is a maximum duty cycle prior to one of a) the engine start time and b) the boost duration time.","9.A carburetor enrichment system that controls an air-fuel ratio of an air-fuel mixture supplied to an internal combustion engine during cold start the engine having a carburetor, that is supplied fuel from a fuel reservoir, and includes an induction passage, the carburetor enrichment system comprising: a sensor that provides a signal indicative of an engine temperature; a fuel line connected between the fuel reservoir and the induction passage; a solenoid valve disposed in the fuel line; and a controller that sets a duty cycle of the solenoid valve to a maximum duty cycle, receives the signal, reduces the duty cycle from the maximum duty cycle to a determined duty cycle associated with the engine temperature, and further reduces the duty cycle at a predetermined rate after one of a) engine start and b) a boost duration time associated with the engine temperature.","10.A carburetor enrichment system according to claim 9, wherein the engine start time is determined when the engine reaches a predetermined idle threshold speed.","11.A method of controlling an air-fuel ratio of an air-fuel mixture supplied to an internal combustion engine during cold start, the engine having a carburetor including an induction passage that is supplied fuel by a fuel line connected between a fuel reservoir and the induction passage, the fuel line having a solenoid valve disposed therein, the method comprising: determining an engine temperature; and setting a duty cycle of the solenoid valve associated with the engine temperature to increase the air-fuel ratio of the air-fuel mixture.","12.A method according to claim 11, wherein setting the duty cycle occurs at one of a) an engine start time and b) a boost duration time associated with the engine temperature.","13.A method according to claim 12, wherein the engine start time is determined when the engine reaches a predetermined idle threshold speed.","14.A method according to claim 12, wherein a plurality of engine temperatures are associated with a plurality of boost duration times.","15.A method according to claim 11, wherein a plurality of engine temperatures ar associated with a plurality of duty cycles.","16.A method according to claim 12, further comprising reducing the duty cycle of the solenoid valve at a predetermined rate after one of a) the engine start time and b) the boost duration time.","17.A method according to claim 16, wherein the predetermined rate is constant.","18.A method according to claim 12, wherein the duty cycle of the solenoid valve is a maximum duty cycle prior to one of a) the engine start time and b) the boost duration time.","19.A method for controlling an air-fuel ratio of an air-fuel mixture supplied to an internal combustion engine during cold start the engine having a carburetor including an induction passage that is supplied by a fuel line connected between a fuel reservoir and the induction passage, the fuel line having a solenoid valve disposed therein, the method comprising: determining an engine temperature; setting a duty cycle of the solenoid valve to a maximum duty cycle; reducing the duty cycle from the maximum duty cycle to a determined duty cycle associated with the engine temperature; and further reducing the duty cycle at a predetermined rate after one of a) engine start and b) a boost duration time associated with the engine temperature.","20.A method according to claim 19, wherein the engine start time is determined when the engine reached a predetermined idle threshold speed."],["<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention The present invention relates to systems and methods for automatic carburetor enrichment during cold start of an engine.","2.Description of the Related Art In internal combustion engines having carburetor controlled fuel supplies, for example, engines used in vehicles such as snowmobiles, personal watercraft, and all terrain vehicles, the rate of fuel flow in a fixed or variable venturi carburetor is dependent on the pressure differential between the venturi and the fuel bowl, also known as the float bowl or float chamber.","In a conventional float bowl carburetor the pressure differential is measured between the pressure in the fluid float chamber, which is normally atmospheric, and the pressure at the discharge orifice of the fuel metering system which is normally located in or adjacent the venturi in the induction passage.","For optimum combustion, the relationship between the mass air flow and the mass fuel flow delivered to the engine by the carburetor should be kept to a controllable rate.","A fixed or variable venturi may be used to provide a constant relationship between the mass air flow and the mass fuel flow.","As the air velocity in the induction passage increases, a pressure reduction, or vacuum, is created in the venturi.","The pressure reduction creates a pressure differential between the induction passage and the fuel in the float chamber causing fuel to be drawn into the induction passage at a flow rate that is proportional to the pressure differential.","The pressure reduction is mainly a function of the air velocity through the induction passage.","However, at a given velocity, the mass air flow rate is dependent on the air density which in turn is dependent on the barometric pressure and temperature.","For a given air velocity, the induction passage delivers a reduced mass air flow at higher altitudes due to the reduced barometric pressure and correspondingly reduced air density.","Operating the engine at higher altitudes thus causes the engine to be supplied with an over rich air-fuel mixture.","Conversely, for a given air velocity, the induction passage delivers an increased mass air flow at lower temperatures due to the increased air density.","Operating the engine at lower temperatures thus causes the engine to be supplied with an over lean air-fuel mixture.","U.S. Pat.","No.","5,021,198 to Bostelmann, the entire contents of which are herein incorporated by reference, discloses a carburetor with a high altitude compensator that includes a pressure splitter connected with the lower pressure of the venturi throat in the area where the fuel delivery line opens out and with the induction pressure in the area of the inlet end of the air flow passage.","The pressure splitter includes a pressure line with two chokes that are connected in series.","The fuel bowl is connected to the pressure line between the two chokes and one or both of the chokes is controlled as a function of the specific air density.","The control system of U.S. Pat.","No.","5,021,198 cannot provide an enriched air-fuel mixture during cold start When the engine is cold it is difficult to start because it is difficult to create a sufficient amount of fuel vapor in the combustion chamber because atomization and vaporization are less effective at lower temperatures.","It is therefore necessary to increase the amount of fuel in order to compensate for the lack of atomization.","It has been known to increase the amount of fuel by using a manual primer or an air pump enrichment system at the carburetor.","U.S. application Ser.","No.","08/948,064, the entire contents of which are hereby incorporated by reference, discloses an electronic compensation system for an internal combustion engine including a manifold connected to the float chamber and the venturi of each carburetor.","A barometric pressure sensor and an engine temperature sensor provide signals to an electronic control unit that controls first and second solenoids connected to the manifold.","During a cold start, the first solenoid is controlled to provide pressurized gas, provided by a pressure line in communication with the crankcase interior, from the manifold to the float chambers of the carburetors to increase the fuel flow and enrich the air-fuel mixture.","As the engine temperature increases, the electronic control unit responds to signals from the engine temperature sensor to reduce the duty cycle of the first solenoid and thus reduce the supply of pressurized gas to the float chambers until the normal engine operating temperature is reached.","The second solenoid is controlled during normal engine operation to apply the underpressure, or vacuum, from the venturis of the carburetors to the manifold to reduce the float chamber pressure to decrease the fuel flow when the air density is reduced, for example, at increased altitudes.","The electronic compensation system of U.S. application Ser.","No.","08/948,064 requires the use of an air pump when the rotation speed of the engine c corresponds to idling or higher.","While completely reliable during operation, condensation of fuel, oil, and water in the air pump decreases the reliability of the air pump.","The electronic compensation system of U.S. application Ser.","No.","08/948,064 also requires an expensive idle switch to signal the position of the throttle during idling to the electronic control unit and to limit fuel distribution to avoid flooding the engine.","In addition, the electronic compensation system of U.S. application Ser.","No.","08/948,064 requires the insertion of an impulse line from the interior of the crankcase to the air pump to activate a diaphragm of the air pump."],["<SOH> SUMMARY OF THE INVENTION <EOH>There exists a need for an engine control system that automatically provides an enriched air-fuel mixture during cold start and that does not require a manifold and an air pump for providing pressurized gas to the float chamber and an impulse line for activating the diaphragm of the air pump.","There also exists a need for an engine control system that automatically reduces the enriched air-fuel mixture as the engine operating temperature approaches a normal engine operating temperature and that does not require an idle switch for limiting the fuel distribution to the engine when in off-idle mode.","A system for automatic carburetor enrichment during cold start according to the present invention controls an air-fuel ratio of an air-fuel mixture supplied to an internal combustion engine during cold start, the engine having a carburetor and being supplied fuel from a fuel reservoir, for example a fuel bowl, and an induction passage, and includes a sensor that provides a signal indicative of an engine temperature at engine start, a fuel line connected between the fuel bowl and the induction passage, a solenoid valve disposed in the fuel line, and a controller that receives the signal and sets a duty cycle of the solenoid valve associated with the engine temperature to increase the air-fuel ratio of the air-fuel mixture at engine start A method according to the present invention for controlling the air-fuel ratio of an air fuel mixture supplied to a carburetor of an internal combustion engine during cold start, the engine having a carburetor including a fuel bowl, a fuel line between the fuel reservoir and an induction passage of the carburetor, and a solenoid valve disposed in the fuel line, includes determining a temperature of the engine, determining a duty cycle of the solenoid valve associated with the determined temperature, setting the duty cycle of the solenoid valve to the associated duty cycle at an engine start time to increase the air-fuel ratio of the air-fuel mixture at engine start."],["BACKGROUND OF THE INVENTION 1.Field of the Invention The present invention relates to systems and methods for automatic carburetor enrichment during cold start of an engine.","2.Description of the Related Art In internal combustion engines having carburetor controlled fuel supplies, for example, engines used in vehicles such as snowmobiles, personal watercraft, and all terrain vehicles, the rate of fuel flow in a fixed or variable venturi carburetor is dependent on the pressure differential between the venturi and the fuel bowl, also known as the float bowl or float chamber.","In a conventional float bowl carburetor the pressure differential is measured between the pressure in the fluid float chamber, which is normally atmospheric, and the pressure at the discharge orifice of the fuel metering system which is normally located in or adjacent the venturi in the induction passage.","For optimum combustion, the relationship between the mass air flow and the mass fuel flow delivered to the engine by the carburetor should be kept to a controllable rate.","A fixed or variable venturi may be used to provide a constant relationship between the mass air flow and the mass fuel flow.","As the air velocity in the induction passage increases, a pressure reduction, or vacuum, is created in the venturi.","The pressure reduction creates a pressure differential between the induction passage and the fuel in the float chamber causing fuel to be drawn into the induction passage at a flow rate that is proportional to the pressure differential.","The pressure reduction is mainly a function of the air velocity through the induction passage.","However, at a given velocity, the mass air flow rate is dependent on the air density which in turn is dependent on the barometric pressure and temperature.","For a given air velocity, the induction passage delivers a reduced mass air flow at higher altitudes due to the reduced barometric pressure and correspondingly reduced air density.","Operating the engine at higher altitudes thus causes the engine to be supplied with an over rich air-fuel mixture.","Conversely, for a given air velocity, the induction passage delivers an increased mass air flow at lower temperatures due to the increased air density.","Operating the engine at lower temperatures thus causes the engine to be supplied with an over lean air-fuel mixture.","U.S. Pat.","No.","5,021,198 to Bostelmann, the entire contents of which are herein incorporated by reference, discloses a carburetor with a high altitude compensator that includes a pressure splitter connected with the lower pressure of the venturi throat in the area where the fuel delivery line opens out and with the induction pressure in the area of the inlet end of the air flow passage.","The pressure splitter includes a pressure line with two chokes that are connected in series.","The fuel bowl is connected to the pressure line between the two chokes and one or both of the chokes is controlled as a function of the specific air density.","The control system of U.S. Pat.","No.","5,021,198 cannot provide an enriched air-fuel mixture during cold start When the engine is cold it is difficult to start because it is difficult to create a sufficient amount of fuel vapor in the combustion chamber because atomization and vaporization are less effective at lower temperatures.","It is therefore necessary to increase the amount of fuel in order to compensate for the lack of atomization.","It has been known to increase the amount of fuel by using a manual primer or an air pump enrichment system at the carburetor.","U.S. application Ser.","No.","08/948,064, the entire contents of which are hereby incorporated by reference, discloses an electronic compensation system for an internal combustion engine including a manifold connected to the float chamber and the venturi of each carburetor.","A barometric pressure sensor and an engine temperature sensor provide signals to an electronic control unit that controls first and second solenoids connected to the manifold.","During a cold start, the first solenoid is controlled to provide pressurized gas, provided by a pressure line in communication with the crankcase interior, from the manifold to the float chambers of the carburetors to increase the fuel flow and enrich the air-fuel mixture.","As the engine temperature increases, the electronic control unit responds to signals from the engine temperature sensor to reduce the duty cycle of the first solenoid and thus reduce the supply of pressurized gas to the float chambers until the normal engine operating temperature is reached.","The second solenoid is controlled during normal engine operation to apply the underpressure, or vacuum, from the venturis of the carburetors to the manifold to reduce the float chamber pressure to decrease the fuel flow when the air density is reduced, for example, at increased altitudes.","The electronic compensation system of U.S. application Ser.","No.","08/948,064 requires the use of an air pump when the rotation speed of the engine c corresponds to idling or higher.","While completely reliable during operation, condensation of fuel, oil, and water in the air pump decreases the reliability of the air pump.","The electronic compensation system of U.S. application Ser.","No.","08/948,064 also requires an expensive idle switch to signal the position of the throttle during idling to the electronic control unit and to limit fuel distribution to avoid flooding the engine.","In addition, the electronic compensation system of U.S. application Ser.","No.","08/948,064 requires the insertion of an impulse line from the interior of the crankcase to the air pump to activate a diaphragm of the air pump.","SUMMARY OF THE INVENTION There exists a need for an engine control system that automatically provides an enriched air-fuel mixture during cold start and that does not require a manifold and an air pump for providing pressurized gas to the float chamber and an impulse line for activating the diaphragm of the air pump.","There also exists a need for an engine control system that automatically reduces the enriched air-fuel mixture as the engine operating temperature approaches a normal engine operating temperature and that does not require an idle switch for limiting the fuel distribution to the engine when in off-idle mode.","A system for automatic carburetor enrichment during cold start according to the present invention controls an air-fuel ratio of an air-fuel mixture supplied to an internal combustion engine during cold start, the engine having a carburetor and being supplied fuel from a fuel reservoir, for example a fuel bowl, and an induction passage, and includes a sensor that provides a signal indicative of an engine temperature at engine start, a fuel line connected between the fuel bowl and the induction passage, a solenoid valve disposed in the fuel line, and a controller that receives the signal and sets a duty cycle of the solenoid valve associated with the engine temperature to increase the air-fuel ratio of the air-fuel mixture at engine start A method according to the present invention for controlling the air-fuel ratio of an air fuel mixture supplied to a carburetor of an internal combustion engine during cold start, the engine having a carburetor including a fuel bowl, a fuel line between the fuel reservoir and an induction passage of the carburetor, and a solenoid valve disposed in the fuel line, includes determining a temperature of the engine, determining a duty cycle of the solenoid valve associated with the determined temperature, setting the duty cycle of the solenoid valve to the associated duty cycle at an engine start time to increase the air-fuel ratio of the air-fuel mixture at engine start.","BRIEF DESCRIPTION OF THE DRAWINGS The present invention will further be described with reference to the accompanying drawings wherein: FIG.","1 is a schematic illustration of an exemplary automatic carburetor enrichment system according to the present invention; FIG.","2 is a graphical representation of an exemplary relationship between a solenoid duty cycle and an engine start time according to the present invention; FIG.","3 is a graphical representation of a table including duty cycles values at various engine starting temperatures; FIG.","4 is a graphical representation of a table including enrichment mode durations at various engine starting temperatures; and FIG.","5 is a graphical representation of a value of the duty cycle slope at an idle threshold speed.","DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS Various exemplary embodiments of the present invention will now be described in detail with reference to the drawings.","Referring to FIG.","1, an automatic carburetor enrichment system for an internal combustion engine 10 includes a carburetor 20 having a throttle valve 21.It should be appreciated that a carburetor having an air inlet of fixed size may also be used.","The carburetor 20 also includes a float chamber 22 configured in the usual manner to ensure a constant fuel level within the chamber 22.It should be appreciated however that fuel may be supplied to the carburetor from a fuel reservoir separate from the carburetor.","A fuel delivery line 30 extends from the float chamber 22 into the induction passage 23 of the carburetor 20.A solenoid valve 40 controls the flow of fuel from the float chamber 22 to the induction passage 23.A check valve 60 provides a balanced distribution of fuel to the cylinders of the engine 10 and prevents fuel from flowing back from the induction passage 23 to the float chamber 22 through the fuel delivery line 30.An electronic control unit 70 receives a signal indicative of the engine temperature from an engine temperature sensor 50.The engine temperature sensor 50 provides a signal indicative of the engine temperature by sensing, for example, the temperature of the engine coolant.","The electronic control unit 70 controls the duty cycle of the solenoid valve 40 based on the temperature sensed by the temperature sensor 50.The duty cycle of the solenoid valve 40 is the percentage of the opening time of the solenoid valve in relation to its fixed cycle time.","The solenoid valve 40 has a fixed frequency of, for example, 10 Hz and the electronic control unit 70 controls the duty cycle of the solenoid valve 40 to increase the amount of fuel delivered to the induction passage 23 through the fuel delivery line 30 during cold start.","The electronic control unit 70 reduces the duty cycle of the solenoid valve 40 at a constant rate until it reaches zero.","This prevents an over enrichment of fuel mixture once the engine has started, which may otherwise damage the ignition components (spark plugs) or reduce the engine performance.","Referring to FIG.","2, the duty cycle at the beginning of engine cranking is set at 100%, regardless of the engine temperature, to minimize the crank time before engine start-up.","Immediately upon engine start, defined as the electronic control unit 70 detecting an engine speed corresponding to an idle threshold speed, indicated in FIG.","5, the electronic control unit 70 reduces the duty cycle of the solenoid valve 40 to a value associated with the temperature determined by the sensor 50 that is stored in a table, such as the table shown in FIG.","3.Although the duty cycle is shown in FIG.","2 as being changed from 100% to the associated value in zero time, it should be appreciated that the change in duty cycle does require some time, albeit a very short time.","The table may be stored, for example, in a memory of the electronic control unit 70.The A/D value in FIG.","3 is an analog to digital parameter readable by the electronic control unit 70.The idle threshold speed shown in FIG.","5 is empirically determined.","The rate at which the duty cycle will decrease after engine start will be determined by dividing the duty cycle pitch by the time pitch, shown in FIG.","5.For example, the duty cycle will be reduced 1% every two seconds.","The duty cycle pitch and the time pitch are empirically determined for the vehicle and are not dependent on the temperature.","The duty cycle/motor temp table shown in FIG.","3 includes duty cycle values at four temperatures, ranging from 40° C. to −40° C. The duty cycle values at the four temperatures are empirically determined.","Although the table shown in FIG.","3 includes duty cycle values at four temperatures, it should be appreciated that the table may include duty cycle values at any number of temperatures.","If the signal from the temperature sensor 50 indicates a temperature that is not stored in the table shown in FIG.","3, the electronic control unit 70 extrapolates a duty cycle value from the table based on the signal provided by the temperature sensor 50.Referring again to FIG.","2, if the engine does not start before a predetermined boost duration time To, such as the boost duration time shown in the table of FIG.","4, the electronic control unit 70, at time To, will reduce the duty cycle of the solenoid valve 40 to that corresponding to the engine temperature at the beginning of cranking, found in FIG.","3, and continue to reduce it at the constant rate determined by the duty cycle pitch and the time pitch of FIG.","5.The boost duration time To is also empirically determined and is temperature dependent Again, assuming that the engine temperature, as determined by the signal from the temperature sensor 50, is not stored in the tables of FIGS.","3 and 4, the electronic control unit 70 extrapolates a value of the temperature and the boost duration time To.","Thereafter, regardless of the start time of the engine after the boost duration time To, the duty cycle of the solenoid valve 40 continues to decrease at the constant rate until it reaches zero.","The automatic carburetor enrichment system according to the present invention reduces cranking time during cold start and eliminates the need for driver input during cold start.","The automatic carburetor enrichment system according to the present invention also prevents engine stalling without assistance from the operator during the warm up phase and provides a simpler, more cost effective and reliable carburetor enrichment than current systems, including those that use an air pump.","The automatic carburetor enrichment system according to the present invention provides self-drowning protection without the use of an electronic idle switch and eliminates the risk of engine drowning when the engine is cranked with the choke ON and the ignition switches OFF.","Although the present invention has been described with reference to the exemplary embodiments described above, it should be appreciated that various modifications would be within the level of ordinary skill in the art.","For example, a manual choke may be included as a backup to the automatic carburetor enrichment system.","As another example, although the electronic control unit has been described as determining the engine temperature at the beginning of engine start, it should be appreciated that the electronic control unit may monitor the engine temperature a plurality of times, or even continuously, during engine start and control the solenoid valve duty cycle based on the plural signals or the continuous signal from the temperature sensor.","Additionally, although the duty cycle has been described as reduced at a constant linear rate, it should be appreciated that the duty cycle may be reduced at an exponential or logarithmic rate.","Accordingly, the scope of the protection sought by the grant of Letters Patent is defined by the Claims appended hereto."]],"string":"[\n [\n \"Systems and methods for automatic carburetor enrichment during cold start\",\n \"An automatic carburetor (20) enrichment system that controls the air-fuel mixture during cold start of an engine (10) having a carburetor including a fuel bowl (22) and an induction passage (23), includes a sensor (50) that provides a signal indicative of an engine temperature at engine start, a fuel line (30) connected between the fuel bowl (22) and the induction passage, a solenoid valve (40) disposed in the fuel line, and a controller (10) that receives the signal and sets a duty cycle of the solenoid valve associated with the engine temperature to increase the air-fuel ratio of the air-fuel mixture at engine start.\",\n \"The automatic carburetor enrichment system reduces cranking time during cold start, eliminates the need for driver input during cold start, prevents engine stalling without assistance from the operator during the warm up phase, provides a simpler, more cost effective and reliable carburetor enrichment, provides self-drowning protection without the use of an electronic idle switch and eliminates the risk of engine drowning when the engine is cranked with the choke ON and the ignition switches OFF.\"\n ],\n [\n \"1.A carburetor enrichment system that controls an air-fuel ratio of an air-fuel mixture supplied to an internal combustion engine during cold start, the engine having a carburetor, that is supplied fuel from a fuel reservoir, and includes an induction passage, the carburetor enrichment system comprising: a sensor that provides a signal indicative of an engine temperature; a fuel line connected between the fuel reservoir and the induction passage; a solenoid valve disposed in the fuel line; and a controller that receives the signal and sets a duty cycle of the solenoid valve associated with the engine temperature to increase the air-fuel ratio of the air-fuel mixture.\",\n \"2.A carburetor enrichment system according to claim 1, wherein the controller sets the duty cycle of the solenoid valve at one of a) an engine start time and b) a boost duration time associated with the engine temperature.\",\n \"3.A carburetor enrichment system according to claim 2, wherein the engine start time is determined when the engine reaches a predetermined idle threshold speed.\",\n \"4.A carburetor enrichment system according to claim 2, wherein the controller stores a plurality of engine temperatures and associated boost duration times.\",\n \"5.A carburetor enrichment system according to claim 1, wherein the controller stores a plurality of engine temperatures and associated duty cycles.\",\n \"6.A carburetor enrichment system according to claim 2, wherein the controller reduces the duty cycle of the solenoid valve at a predetermined rate after one of a) the engine start time and b) the boost duration time.\",\n \"7.A carburetor enrichment system according to claim 6, wherein the predetermined rate is constant.\",\n \"8.A carburetor enrichment system according to claim 2, wherein the duty cycle of the solenoid valve is a maximum duty cycle prior to one of a) the engine start time and b) the boost duration time.\",\n \"9.A carburetor enrichment system that controls an air-fuel ratio of an air-fuel mixture supplied to an internal combustion engine during cold start the engine having a carburetor, that is supplied fuel from a fuel reservoir, and includes an induction passage, the carburetor enrichment system comprising: a sensor that provides a signal indicative of an engine temperature; a fuel line connected between the fuel reservoir and the induction passage; a solenoid valve disposed in the fuel line; and a controller that sets a duty cycle of the solenoid valve to a maximum duty cycle, receives the signal, reduces the duty cycle from the maximum duty cycle to a determined duty cycle associated with the engine temperature, and further reduces the duty cycle at a predetermined rate after one of a) engine start and b) a boost duration time associated with the engine temperature.\",\n \"10.A carburetor enrichment system according to claim 9, wherein the engine start time is determined when the engine reaches a predetermined idle threshold speed.\",\n \"11.A method of controlling an air-fuel ratio of an air-fuel mixture supplied to an internal combustion engine during cold start, the engine having a carburetor including an induction passage that is supplied fuel by a fuel line connected between a fuel reservoir and the induction passage, the fuel line having a solenoid valve disposed therein, the method comprising: determining an engine temperature; and setting a duty cycle of the solenoid valve associated with the engine temperature to increase the air-fuel ratio of the air-fuel mixture.\",\n \"12.A method according to claim 11, wherein setting the duty cycle occurs at one of a) an engine start time and b) a boost duration time associated with the engine temperature.\",\n \"13.A method according to claim 12, wherein the engine start time is determined when the engine reaches a predetermined idle threshold speed.\",\n \"14.A method according to claim 12, wherein a plurality of engine temperatures are associated with a plurality of boost duration times.\",\n \"15.A method according to claim 11, wherein a plurality of engine temperatures ar associated with a plurality of duty cycles.\",\n \"16.A method according to claim 12, further comprising reducing the duty cycle of the solenoid valve at a predetermined rate after one of a) the engine start time and b) the boost duration time.\",\n \"17.A method according to claim 16, wherein the predetermined rate is constant.\",\n \"18.A method according to claim 12, wherein the duty cycle of the solenoid valve is a maximum duty cycle prior to one of a) the engine start time and b) the boost duration time.\",\n \"19.A method for controlling an air-fuel ratio of an air-fuel mixture supplied to an internal combustion engine during cold start the engine having a carburetor including an induction passage that is supplied by a fuel line connected between a fuel reservoir and the induction passage, the fuel line having a solenoid valve disposed therein, the method comprising: determining an engine temperature; setting a duty cycle of the solenoid valve to a maximum duty cycle; reducing the duty cycle from the maximum duty cycle to a determined duty cycle associated with the engine temperature; and further reducing the duty cycle at a predetermined rate after one of a) engine start and b) a boost duration time associated with the engine temperature.\",\n \"20.A method according to claim 19, wherein the engine start time is determined when the engine reached a predetermined idle threshold speed.\"\n ],\n [\n \"<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention The present invention relates to systems and methods for automatic carburetor enrichment during cold start of an engine.\",\n \"2.Description of the Related Art In internal combustion engines having carburetor controlled fuel supplies, for example, engines used in vehicles such as snowmobiles, personal watercraft, and all terrain vehicles, the rate of fuel flow in a fixed or variable venturi carburetor is dependent on the pressure differential between the venturi and the fuel bowl, also known as the float bowl or float chamber.\",\n \"In a conventional float bowl carburetor the pressure differential is measured between the pressure in the fluid float chamber, which is normally atmospheric, and the pressure at the discharge orifice of the fuel metering system which is normally located in or adjacent the venturi in the induction passage.\",\n \"For optimum combustion, the relationship between the mass air flow and the mass fuel flow delivered to the engine by the carburetor should be kept to a controllable rate.\",\n \"A fixed or variable venturi may be used to provide a constant relationship between the mass air flow and the mass fuel flow.\",\n \"As the air velocity in the induction passage increases, a pressure reduction, or vacuum, is created in the venturi.\",\n \"The pressure reduction creates a pressure differential between the induction passage and the fuel in the float chamber causing fuel to be drawn into the induction passage at a flow rate that is proportional to the pressure differential.\",\n \"The pressure reduction is mainly a function of the air velocity through the induction passage.\",\n \"However, at a given velocity, the mass air flow rate is dependent on the air density which in turn is dependent on the barometric pressure and temperature.\",\n \"For a given air velocity, the induction passage delivers a reduced mass air flow at higher altitudes due to the reduced barometric pressure and correspondingly reduced air density.\",\n \"Operating the engine at higher altitudes thus causes the engine to be supplied with an over rich air-fuel mixture.\",\n \"Conversely, for a given air velocity, the induction passage delivers an increased mass air flow at lower temperatures due to the increased air density.\",\n \"Operating the engine at lower temperatures thus causes the engine to be supplied with an over lean air-fuel mixture.\",\n \"U.S. Pat.\",\n \"No.\",\n \"5,021,198 to Bostelmann, the entire contents of which are herein incorporated by reference, discloses a carburetor with a high altitude compensator that includes a pressure splitter connected with the lower pressure of the venturi throat in the area where the fuel delivery line opens out and with the induction pressure in the area of the inlet end of the air flow passage.\",\n \"The pressure splitter includes a pressure line with two chokes that are connected in series.\",\n \"The fuel bowl is connected to the pressure line between the two chokes and one or both of the chokes is controlled as a function of the specific air density.\",\n \"The control system of U.S. Pat.\",\n \"No.\",\n \"5,021,198 cannot provide an enriched air-fuel mixture during cold start When the engine is cold it is difficult to start because it is difficult to create a sufficient amount of fuel vapor in the combustion chamber because atomization and vaporization are less effective at lower temperatures.\",\n \"It is therefore necessary to increase the amount of fuel in order to compensate for the lack of atomization.\",\n \"It has been known to increase the amount of fuel by using a manual primer or an air pump enrichment system at the carburetor.\",\n \"U.S. application Ser.\",\n \"No.\",\n \"08/948,064, the entire contents of which are hereby incorporated by reference, discloses an electronic compensation system for an internal combustion engine including a manifold connected to the float chamber and the venturi of each carburetor.\",\n \"A barometric pressure sensor and an engine temperature sensor provide signals to an electronic control unit that controls first and second solenoids connected to the manifold.\",\n \"During a cold start, the first solenoid is controlled to provide pressurized gas, provided by a pressure line in communication with the crankcase interior, from the manifold to the float chambers of the carburetors to increase the fuel flow and enrich the air-fuel mixture.\",\n \"As the engine temperature increases, the electronic control unit responds to signals from the engine temperature sensor to reduce the duty cycle of the first solenoid and thus reduce the supply of pressurized gas to the float chambers until the normal engine operating temperature is reached.\",\n \"The second solenoid is controlled during normal engine operation to apply the underpressure, or vacuum, from the venturis of the carburetors to the manifold to reduce the float chamber pressure to decrease the fuel flow when the air density is reduced, for example, at increased altitudes.\",\n \"The electronic compensation system of U.S. application Ser.\",\n \"No.\",\n \"08/948,064 requires the use of an air pump when the rotation speed of the engine c corresponds to idling or higher.\",\n \"While completely reliable during operation, condensation of fuel, oil, and water in the air pump decreases the reliability of the air pump.\",\n \"The electronic compensation system of U.S. application Ser.\",\n \"No.\",\n \"08/948,064 also requires an expensive idle switch to signal the position of the throttle during idling to the electronic control unit and to limit fuel distribution to avoid flooding the engine.\",\n \"In addition, the electronic compensation system of U.S. application Ser.\",\n \"No.\",\n \"08/948,064 requires the insertion of an impulse line from the interior of the crankcase to the air pump to activate a diaphragm of the air pump.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>There exists a need for an engine control system that automatically provides an enriched air-fuel mixture during cold start and that does not require a manifold and an air pump for providing pressurized gas to the float chamber and an impulse line for activating the diaphragm of the air pump.\",\n \"There also exists a need for an engine control system that automatically reduces the enriched air-fuel mixture as the engine operating temperature approaches a normal engine operating temperature and that does not require an idle switch for limiting the fuel distribution to the engine when in off-idle mode.\",\n \"A system for automatic carburetor enrichment during cold start according to the present invention controls an air-fuel ratio of an air-fuel mixture supplied to an internal combustion engine during cold start, the engine having a carburetor and being supplied fuel from a fuel reservoir, for example a fuel bowl, and an induction passage, and includes a sensor that provides a signal indicative of an engine temperature at engine start, a fuel line connected between the fuel bowl and the induction passage, a solenoid valve disposed in the fuel line, and a controller that receives the signal and sets a duty cycle of the solenoid valve associated with the engine temperature to increase the air-fuel ratio of the air-fuel mixture at engine start A method according to the present invention for controlling the air-fuel ratio of an air fuel mixture supplied to a carburetor of an internal combustion engine during cold start, the engine having a carburetor including a fuel bowl, a fuel line between the fuel reservoir and an induction passage of the carburetor, and a solenoid valve disposed in the fuel line, includes determining a temperature of the engine, determining a duty cycle of the solenoid valve associated with the determined temperature, setting the duty cycle of the solenoid valve to the associated duty cycle at an engine start time to increase the air-fuel ratio of the air-fuel mixture at engine start.\"\n ],\n [\n \"BACKGROUND OF THE INVENTION 1.Field of the Invention The present invention relates to systems and methods for automatic carburetor enrichment during cold start of an engine.\",\n \"2.Description of the Related Art In internal combustion engines having carburetor controlled fuel supplies, for example, engines used in vehicles such as snowmobiles, personal watercraft, and all terrain vehicles, the rate of fuel flow in a fixed or variable venturi carburetor is dependent on the pressure differential between the venturi and the fuel bowl, also known as the float bowl or float chamber.\",\n \"In a conventional float bowl carburetor the pressure differential is measured between the pressure in the fluid float chamber, which is normally atmospheric, and the pressure at the discharge orifice of the fuel metering system which is normally located in or adjacent the venturi in the induction passage.\",\n \"For optimum combustion, the relationship between the mass air flow and the mass fuel flow delivered to the engine by the carburetor should be kept to a controllable rate.\",\n \"A fixed or variable venturi may be used to provide a constant relationship between the mass air flow and the mass fuel flow.\",\n \"As the air velocity in the induction passage increases, a pressure reduction, or vacuum, is created in the venturi.\",\n \"The pressure reduction creates a pressure differential between the induction passage and the fuel in the float chamber causing fuel to be drawn into the induction passage at a flow rate that is proportional to the pressure differential.\",\n \"The pressure reduction is mainly a function of the air velocity through the induction passage.\",\n \"However, at a given velocity, the mass air flow rate is dependent on the air density which in turn is dependent on the barometric pressure and temperature.\",\n \"For a given air velocity, the induction passage delivers a reduced mass air flow at higher altitudes due to the reduced barometric pressure and correspondingly reduced air density.\",\n \"Operating the engine at higher altitudes thus causes the engine to be supplied with an over rich air-fuel mixture.\",\n \"Conversely, for a given air velocity, the induction passage delivers an increased mass air flow at lower temperatures due to the increased air density.\",\n \"Operating the engine at lower temperatures thus causes the engine to be supplied with an over lean air-fuel mixture.\",\n \"U.S. Pat.\",\n \"No.\",\n \"5,021,198 to Bostelmann, the entire contents of which are herein incorporated by reference, discloses a carburetor with a high altitude compensator that includes a pressure splitter connected with the lower pressure of the venturi throat in the area where the fuel delivery line opens out and with the induction pressure in the area of the inlet end of the air flow passage.\",\n \"The pressure splitter includes a pressure line with two chokes that are connected in series.\",\n \"The fuel bowl is connected to the pressure line between the two chokes and one or both of the chokes is controlled as a function of the specific air density.\",\n \"The control system of U.S. Pat.\",\n \"No.\",\n \"5,021,198 cannot provide an enriched air-fuel mixture during cold start When the engine is cold it is difficult to start because it is difficult to create a sufficient amount of fuel vapor in the combustion chamber because atomization and vaporization are less effective at lower temperatures.\",\n \"It is therefore necessary to increase the amount of fuel in order to compensate for the lack of atomization.\",\n \"It has been known to increase the amount of fuel by using a manual primer or an air pump enrichment system at the carburetor.\",\n \"U.S. application Ser.\",\n \"No.\",\n \"08/948,064, the entire contents of which are hereby incorporated by reference, discloses an electronic compensation system for an internal combustion engine including a manifold connected to the float chamber and the venturi of each carburetor.\",\n \"A barometric pressure sensor and an engine temperature sensor provide signals to an electronic control unit that controls first and second solenoids connected to the manifold.\",\n \"During a cold start, the first solenoid is controlled to provide pressurized gas, provided by a pressure line in communication with the crankcase interior, from the manifold to the float chambers of the carburetors to increase the fuel flow and enrich the air-fuel mixture.\",\n \"As the engine temperature increases, the electronic control unit responds to signals from the engine temperature sensor to reduce the duty cycle of the first solenoid and thus reduce the supply of pressurized gas to the float chambers until the normal engine operating temperature is reached.\",\n \"The second solenoid is controlled during normal engine operation to apply the underpressure, or vacuum, from the venturis of the carburetors to the manifold to reduce the float chamber pressure to decrease the fuel flow when the air density is reduced, for example, at increased altitudes.\",\n \"The electronic compensation system of U.S. application Ser.\",\n \"No.\",\n \"08/948,064 requires the use of an air pump when the rotation speed of the engine c corresponds to idling or higher.\",\n \"While completely reliable during operation, condensation of fuel, oil, and water in the air pump decreases the reliability of the air pump.\",\n \"The electronic compensation system of U.S. application Ser.\",\n \"No.\",\n \"08/948,064 also requires an expensive idle switch to signal the position of the throttle during idling to the electronic control unit and to limit fuel distribution to avoid flooding the engine.\",\n \"In addition, the electronic compensation system of U.S. application Ser.\",\n \"No.\",\n \"08/948,064 requires the insertion of an impulse line from the interior of the crankcase to the air pump to activate a diaphragm of the air pump.\",\n \"SUMMARY OF THE INVENTION There exists a need for an engine control system that automatically provides an enriched air-fuel mixture during cold start and that does not require a manifold and an air pump for providing pressurized gas to the float chamber and an impulse line for activating the diaphragm of the air pump.\",\n \"There also exists a need for an engine control system that automatically reduces the enriched air-fuel mixture as the engine operating temperature approaches a normal engine operating temperature and that does not require an idle switch for limiting the fuel distribution to the engine when in off-idle mode.\",\n \"A system for automatic carburetor enrichment during cold start according to the present invention controls an air-fuel ratio of an air-fuel mixture supplied to an internal combustion engine during cold start, the engine having a carburetor and being supplied fuel from a fuel reservoir, for example a fuel bowl, and an induction passage, and includes a sensor that provides a signal indicative of an engine temperature at engine start, a fuel line connected between the fuel bowl and the induction passage, a solenoid valve disposed in the fuel line, and a controller that receives the signal and sets a duty cycle of the solenoid valve associated with the engine temperature to increase the air-fuel ratio of the air-fuel mixture at engine start A method according to the present invention for controlling the air-fuel ratio of an air fuel mixture supplied to a carburetor of an internal combustion engine during cold start, the engine having a carburetor including a fuel bowl, a fuel line between the fuel reservoir and an induction passage of the carburetor, and a solenoid valve disposed in the fuel line, includes determining a temperature of the engine, determining a duty cycle of the solenoid valve associated with the determined temperature, setting the duty cycle of the solenoid valve to the associated duty cycle at an engine start time to increase the air-fuel ratio of the air-fuel mixture at engine start.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS The present invention will further be described with reference to the accompanying drawings wherein: FIG.\",\n \"1 is a schematic illustration of an exemplary automatic carburetor enrichment system according to the present invention; FIG.\",\n \"2 is a graphical representation of an exemplary relationship between a solenoid duty cycle and an engine start time according to the present invention; FIG.\",\n \"3 is a graphical representation of a table including duty cycles values at various engine starting temperatures; FIG.\",\n \"4 is a graphical representation of a table including enrichment mode durations at various engine starting temperatures; and FIG.\",\n \"5 is a graphical representation of a value of the duty cycle slope at an idle threshold speed.\",\n \"DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS Various exemplary embodiments of the present invention will now be described in detail with reference to the drawings.\",\n \"Referring to FIG.\",\n \"1, an automatic carburetor enrichment system for an internal combustion engine 10 includes a carburetor 20 having a throttle valve 21.It should be appreciated that a carburetor having an air inlet of fixed size may also be used.\",\n \"The carburetor 20 also includes a float chamber 22 configured in the usual manner to ensure a constant fuel level within the chamber 22.It should be appreciated however that fuel may be supplied to the carburetor from a fuel reservoir separate from the carburetor.\",\n \"A fuel delivery line 30 extends from the float chamber 22 into the induction passage 23 of the carburetor 20.A solenoid valve 40 controls the flow of fuel from the float chamber 22 to the induction passage 23.A check valve 60 provides a balanced distribution of fuel to the cylinders of the engine 10 and prevents fuel from flowing back from the induction passage 23 to the float chamber 22 through the fuel delivery line 30.An electronic control unit 70 receives a signal indicative of the engine temperature from an engine temperature sensor 50.The engine temperature sensor 50 provides a signal indicative of the engine temperature by sensing, for example, the temperature of the engine coolant.\",\n \"The electronic control unit 70 controls the duty cycle of the solenoid valve 40 based on the temperature sensed by the temperature sensor 50.The duty cycle of the solenoid valve 40 is the percentage of the opening time of the solenoid valve in relation to its fixed cycle time.\",\n \"The solenoid valve 40 has a fixed frequency of, for example, 10 Hz and the electronic control unit 70 controls the duty cycle of the solenoid valve 40 to increase the amount of fuel delivered to the induction passage 23 through the fuel delivery line 30 during cold start.\",\n \"The electronic control unit 70 reduces the duty cycle of the solenoid valve 40 at a constant rate until it reaches zero.\",\n \"This prevents an over enrichment of fuel mixture once the engine has started, which may otherwise damage the ignition components (spark plugs) or reduce the engine performance.\",\n \"Referring to FIG.\",\n \"2, the duty cycle at the beginning of engine cranking is set at 100%, regardless of the engine temperature, to minimize the crank time before engine start-up.\",\n \"Immediately upon engine start, defined as the electronic control unit 70 detecting an engine speed corresponding to an idle threshold speed, indicated in FIG.\",\n \"5, the electronic control unit 70 reduces the duty cycle of the solenoid valve 40 to a value associated with the temperature determined by the sensor 50 that is stored in a table, such as the table shown in FIG.\",\n \"3.Although the duty cycle is shown in FIG.\",\n \"2 as being changed from 100% to the associated value in zero time, it should be appreciated that the change in duty cycle does require some time, albeit a very short time.\",\n \"The table may be stored, for example, in a memory of the electronic control unit 70.The A/D value in FIG.\",\n \"3 is an analog to digital parameter readable by the electronic control unit 70.The idle threshold speed shown in FIG.\",\n \"5 is empirically determined.\",\n \"The rate at which the duty cycle will decrease after engine start will be determined by dividing the duty cycle pitch by the time pitch, shown in FIG.\",\n \"5.For example, the duty cycle will be reduced 1% every two seconds.\",\n \"The duty cycle pitch and the time pitch are empirically determined for the vehicle and are not dependent on the temperature.\",\n \"The duty cycle/motor temp table shown in FIG.\",\n \"3 includes duty cycle values at four temperatures, ranging from 40° C. to −40° C. The duty cycle values at the four temperatures are empirically determined.\",\n \"Although the table shown in FIG.\",\n \"3 includes duty cycle values at four temperatures, it should be appreciated that the table may include duty cycle values at any number of temperatures.\",\n \"If the signal from the temperature sensor 50 indicates a temperature that is not stored in the table shown in FIG.\",\n \"3, the electronic control unit 70 extrapolates a duty cycle value from the table based on the signal provided by the temperature sensor 50.Referring again to FIG.\",\n \"2, if the engine does not start before a predetermined boost duration time To, such as the boost duration time shown in the table of FIG.\",\n \"4, the electronic control unit 70, at time To, will reduce the duty cycle of the solenoid valve 40 to that corresponding to the engine temperature at the beginning of cranking, found in FIG.\",\n \"3, and continue to reduce it at the constant rate determined by the duty cycle pitch and the time pitch of FIG.\",\n \"5.The boost duration time To is also empirically determined and is temperature dependent Again, assuming that the engine temperature, as determined by the signal from the temperature sensor 50, is not stored in the tables of FIGS.\",\n \"3 and 4, the electronic control unit 70 extrapolates a value of the temperature and the boost duration time To.\",\n \"Thereafter, regardless of the start time of the engine after the boost duration time To, the duty cycle of the solenoid valve 40 continues to decrease at the constant rate until it reaches zero.\",\n \"The automatic carburetor enrichment system according to the present invention reduces cranking time during cold start and eliminates the need for driver input during cold start.\",\n \"The automatic carburetor enrichment system according to the present invention also prevents engine stalling without assistance from the operator during the warm up phase and provides a simpler, more cost effective and reliable carburetor enrichment than current systems, including those that use an air pump.\",\n \"The automatic carburetor enrichment system according to the present invention provides self-drowning protection without the use of an electronic idle switch and eliminates the risk of engine drowning when the engine is cranked with the choke ON and the ignition switches OFF.\",\n \"Although the present invention has been described with reference to the exemplary embodiments described above, it should be appreciated that various modifications would be within the level of ordinary skill in the art.\",\n \"For example, a manual choke may be included as a backup to the automatic carburetor enrichment system.\",\n \"As another example, although the electronic control unit has been described as determining the engine temperature at the beginning of engine start, it should be appreciated that the electronic control unit may monitor the engine temperature a plurality of times, or even continuously, during engine start and control the solenoid valve duty cycle based on the plural signals or the continuous signal from the temperature sensor.\",\n \"Additionally, although the duty cycle has been described as reduced at a constant linear rate, it should be appreciated that the duty cycle may be reduced at an exponential or logarithmic rate.\",\n \"Accordingly, the scope of the protection sought by the grant of Letters Patent is defined by the Claims appended hereto.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10467451"}}},{"rowIdx":366,"cells":{"text":{"kind":"list like","value":[["Sequence of the photorhabdus luminescens strain tt01 genome and uses","The present invention relates to the genomic sequence and to nucleotide sequences encoding polypeptides of Photorhabdus luminescens.","The present invention further relates to polypeptides involved in operons involved in the biosynthesis of antibiotics or toxins, as well as polypeptides with activity of the antibiotic or toxin.","Uses of the aforementioned polypeptides in pesticides, bactericides, or fungicides is provides.","In addition, the present invention provides vectors, cells, or animals containing the sequences of the present invention."],["1-56.","(canceled) 57.An isolated nucleotide sequence derived from the Photorhabdus luminescens genome, comprising a sequence selected from the group consisting of SEQ ID No.","1 to SEQ ID No.","41 and SEQ ID No.","5826 to SEQ ID No.","5834.58.An isolated nucleotide sequence derived from the Photorhabdus luminescens genome, selected from the group consisting of: a) a nucleotide sequence comprising at least 75% identity with a sequence chosen selected from the group consisting of SEQ ID No.","1 to SEQ ID No.","41 and SEQ ID No.","5826 to SEQ ID No.","5834; b) a nucleotide sequence comprising a fragment of a sequence selected from the group consisting of SEQ ID No.","1 to SEQ ID No.","41 and SEQ ID No.","5826 to SEQ ID No.","5834; c) a nucleotide sequence complementary to a nucleotide sequence as defined in a) or b); d) a nucleotide sequence of the RNA corresponding to one of the sequences as defined in a), b) or c); and e) a nucleotide sequence as defined in a), b), c) or d), which has been modified.","59.The nucleotide sequence as claimed in claim 58, wherein the nucleotide sequence is a fragment of a nucleotide sequence selected from the group consisting of SEQ ID No.","1 to SEQ ID No.","41 and SEQ ID No.","5826 to SEQ ID No.","5834, and wherein said fragment encodes a polypeptide selected from the group consisting of SEQ ID No.","42 to SEQ ID No.","3855 or a polypeptide encoded by a sequence selected from the group consisting of SEQ ID No.","5835 to SEQ ID No.","10784.60.The nucleotide sequence as claimed in claim 59, wherein: a) said nucleotide sequence encodes a polypeptide selected from the group consisting of SEQ ID No.","61, SEQ ID No.","62, SEQ ID No.","67, SEQ ID No.","171, SEQ ID No.","221, SEQ ID No.","268, SEQ ID No.","288, SEQ ID No.","380, SEQ ID No.","426, SEQ ID No.","438, SEQ ID No.","448, SEQ ID No.","453, SEQ ID No.","455, SEQ ID No.","456, SEQ ID No.","458, SEQ ID No.","501, SEQ ID No.","516, SEQ ID No.","530, SEQ ID No.","542, SEQ ID No.","551, SEQ ID No.","720, SEQ ID No.","761, SEQ ID No.","762, SEQ ID No.","814, SEQ ID No.","859, SEQ ID No.","860, SEQ ID No.","861, SEQ ID No.","862, SEQ ID No.","869, SEQ ID No.","1079, SEQ ID No.","1168, SEQ ID No.","1174, SEQ ID No.","1176, SEQ ID No.","1413, SEQ ID No.","1414, SEQ ID No.","1415, SEQ ID No.","1416, SEQ ID No.","1417, SEQ ID No.","1457, SEQ ID No.","1651, SEQ ID No.","1856, SEQ ID No.","1869, SEQ ID No.","2021, SEQ ID No.","2080, SEQ ID No.","2152, SEQ ID No.","2162, SEQ ID No.","2173, SEQ ID No.","2251, SEQ ID No.","2295, SEQ ID No.","2306, SEQ ID No.","2317, SEQ ID No.","2328, SEQ ID No.","2340, SEQ ID No.","2342, SEQ ID No.","2351, SEQ ID No.","2500, SEQ ID No.","3228, SEQ ID No.","3230, SEQ ID No.","3311, SEQ ID No.","3317, SEQ ID No.","3318, SEQ ID No.","3319, SEQ ID No.","3320, SEQ ID No.","3322, SEQ ID No.","3323, SEQ ID No.","3326, SEQ ID No.","3327, SEQ ID No.","3328, SEQ ID No.","3375, SEQ ID No.","3376, SEQ ID No.","3377, SEQ ID No.","3378, SEQ ID No.","3422, SEQ ID No.","3489, SEQ ID No.","3503, SEQ ID No.","3609, SEQ ID No.","3623, SEQ ID No.","3624, SEQ ID No.","3772, SEQ ID No.","3783, SEQ ID No.","3788 and SEQ ID No.","3794; or b) said nucleotide sequence is selected from the group consisting of SEQ ID No.","5835 to SEQ ID No.","10784.61.A nucleotide sequence, comprising a nucleotide sequence selected from the group consisting of: a) a nucleotide sequence as claimed in claim 59; b) a nucleotide sequence comprising at least 75% identity with a nucleotide sequence as claimed in claim 59; c) a complementary or RNA nucleotide sequence corresponding to a sequence as defined in a) or b); d) a nucleotide sequence of a representative fragment of a sequence as defined in a) or c); and e) a sequence as defined in a) or c), which has been modified.","62.A polypeptide encoded by a nucleotide sequence as claimed in claim 58.63.A polypeptide as claimed in claim 62, wherein: a) said polypeptide is selected from the group consisting of SEQ ID No.","61, SEQ ID No.","62, SEQ ID No.","67, SEQ ID No.","171, SEQ ID No.","221, SEQ ID No.","268, SEQ ID No.","288, SEQ ID No.","380, SEQ ID No.","426, SEQ ID No.","438, SEQ ID No.","448, SEQ ID No.","453, SEQ ID No.","455, SEQ ID No.","456, SEQ ID No.","458, SEQ ID No.","501, SEQ ID No.","516, SEQ ID No.","530, SEQ ID No.","542, SEQ ID No.","551, SEQ ID No.","720, SEQ ID No.","761, SEQ ID No.","762, SEQ ID No.","814, SEQ ID No.","859, SEQ ID No.","860, SEQ ID No.","861, SEQ ID No.","862, SEQ ID No.","869, SEQ ID No.","1079, SEQ ID No.","1168, SEQ ID No.","1174, SEQ ID No.","1176, SEQ ID No.","1413, SEQ ID No.","1414, SEQ ID No.","1415, SEQ ID No.","1416, SEQ ID No.","1417, SEQ ID No.","1457, SEQ ID No.","1651, SEQ ID No.","1856, SEQ ID No.","1869, SEQ ID No.","2021, SEQ ID No.","2080, SEQ ID No.","2152, SEQ ID No.","2162, SEQ ID No.","2173, SEQ ID No.","2251, SEQ ID No.","2295, SEQ ID No.","2306, SEQ ID No.","2317, SEQ ID No.","2328, SEQ ID No.","2340, SEQ ID No.","2342, SEQ ID No.","2351, SEQ ID No.","2500, SEQ ID No.","3228, SEQ ID No.","3230, SEQ ID No.","3311, SEQ ID No.","3317, SEQ ID No.","3318, SEQ ID No.","3319, SEQ ID No.","3320, SEQ ID No.","3322, SEQ ID No.","3323, SEQ ID No.","3326, SEQ ID No.","3327, SEQ ID No.","3328, SEQ ID No.","3375, SEQ ID No.","3376, SEQ ID No.","3377, SEQ ID No.","3378, SEQ ID No.","3422, SEQ ID No.","3489, SEQ ID No.","3503, SEQ ID No.","3609, SEQ ID No.","3623, SEQ ID No.","3624, SEQ ID No.","3772, SEQ ID No.","3783, SEQ ID No.","3788 and SEQ ID No.","3794; or b) said polypeptide is encoded by a nucleotide sequence selected from the group consisting of SEQ ID No.","5835 to SEQ ID No.","10784.64.A polypeptide, comprising a polypeptide selected from the group consisting of: a) a polypeptide as claimed in claim 62; b) a polypeptide exhibiting at least 80% identity with a polypeptide of sequence selected from the group consisting of SEQ ID No.","42 to SEQ ID No.","3855 or with a polypeptide encoded by a sequence selected from the group consisting of SEQ ID No.","5835 to SEQ ID No.","10784; c) a fragment of at least 5 amino acids of a polypeptide of sequence selected from the group consisting of SEQ ID No.","42 to SEQ ID No.","3855 or with a polypeptide encoded by a sequence selected from the group consisting of SEQ ID No.","5835 to SEQ ID No.","10784; d) a biologically active fragment of a polypeptide of sequence selected from the group consisting of SEQ ID No.","42 to SEQ ID No.","3855 or with a polypeptide encoded by a sequence selected from the group consisting of SEQ ID No.","5835 to SEQ ID No.","10784; and e) a polypeptide of sequence selected from the group consisting of SEQ ID No.","42 to SEQ ID No.","3855 or with a polypeptide encoded by a sequence selected from the group consisting of SEQ ID No.","5835 to SEQ ID No.","10784, or as defined in c) or d), which has been modified.","65.A nucleotide sequence encoding a polypeptide as claimed in claim 62.66.The nucleotide sequence as claimed in claim 58, wherein said nucleotide sequence encodes a polypeptide of P. luminescens having toxin activity or antibiotic activity, or is involved in the synthesis of toxins or antibiotics.","67.The polypeptide as claimed in claim 62, wherein said polypeptide is a polypeptide of P. luminescens having toxin activity or antibiotic activity, or is involved in the synthesis of toxins or antibiotics, or a fragment thereof.","68.A recording medium, comprising one or more nucleotide sequences as claimed in claim 57.69.The recording medium as claimed in claim 68, wherein said recording medium is selected from the group consisting of a CD-ROM, a computer disk and a computer server.","70.A method of identifying primers or probes for determining genes in strains related to P. luminescens comprising inspecting the sequences recorded on a recording medium as claimed in claim 68 and identifying a primer pair or probe corresponding to a desired nucleotide or to a nucleotide sequence encoding a polypeptide with a desired function.","71.A method of studying the genetic polymorphism of strains related to P. luminescens comprising obtaining a nucleotide sequence from a strain related to P. luminescens sequencing the nucleotide sequence comparing the nucleotide sequence to the nucleotide sequences recorded on a recording medium as claimed in claim 68.72.A method for the automatic annotation of genes originating from a genome other than P. luminescens comprising obtaining genomic DNA from a strain other than P. luminescens sequencing the genomic DNA comparing the nucleotide sequence of the genomic DNA to the nucleotide sequences recorded on a recording medium as claimed in claim 68 to assign putative function associated therewith.","73.A recording medium, comprising one or more polypeptides sequences as claimed in claim 62.74.The recording medium as claimed in claim 73, wherein said recording medium is selected from the group consisting of a CD-ROM, a computer disk and a computer server.","75.A method of identifying primers or probes for determining genes in strains related to P. luminescens comprising inspecting the sequences recorded on a recording medium as claimed in claim 73 and identifying a primer pair or probe corresponding to a desired nucleotide or to a nucleotide sequence encoding a polypeptide with a desired function.","76.A method of studying the genetic polymorphism of strains related to P. luminescens comprising obtaining a nucleotide sequence from a strain related to P. luminescens sequencing the nucleotide sequence comparing the nucleotide sequence to the nucleotide sequences encoding the polypeptide sequences recorded on a recording medium as claimed in claim 73.77.A method for the automatic annotation of genes originating from a genome other than P. luminescens comprising obtaining genomic DNA from a strain other than P. luminescens sequencing the genomic DNA comparing the nucleotide sequence of the genomic DNA to the nucleotide sequences encoding the polypeptide sequences recorded on a recording medium as claimed in claim 73 to assign putative function associated therewith 78.A primer or a probe, comprising one or more sequences as claimed in claim 57.79.The nucleotide sequence as claimed in claim 78, wherein said primer or probe is labeled with a radioactive compound or with a nonradioactive compound.","80.The nucleotide sequence as claimed in claim 78, wherein said primer or probe is immobilized on a support by a covalent or noncovalent interaction.","81.The nucleotide sequence as claimed in claim 80, wherein said support is a high density filter or a DNA chip.","82.A DNA chip or a filter, comprising one or more nucleotide sequences as claimed in claim 78.83.The DNA chip or the filter as claimed in claim 82, further comprising one or more nucleotide sequences from a cell from a source selected from the group consisting of a plant, an animal and a microorganism other than P. luminescens, wherein said nucleotide sequences are immobilized on the support of said chip.","84.The DNA chip or the filter as claimed in claim 83, wherein said cell is a cell or microorganism sensitive to a toxin or an antibiotic produced by P. luminescens, a bacterium of the genus Photorhabdus, and a variant of P. luminescens.","85.A kit for detecting and/or quantifying the expression of at least one gene of P. luminescens, comprising a DNA chip or a filter as claimed in claim 82.86.A cloning and/or expression vector, comprising a nucleotide sequence as claimed in claim 57.87.The cloning and/or expression vector as claimed in claim 86, comprising a nucleotide sequence selected from the group consisting of SEQ ID No.","3856 to SEQ ID No.","5825 and SEQ ID No.","5835 to SEQ ID No.","10784, or fragments thereof.","88.A host cell, transformed with a vector as claimed in claim 86.89.A plant or an animal, except a human, comprising a transformed cell as claimed in claim 88.90.A method for preparing a polypeptide, comprising culturing a cell transformed with a vector as claimed in claim 88 under conditions which allow the expression of said polypeptide, and recovering the resultant recombinant polypeptide.","91.A recombinant polypeptide obtained by the method as claimed in claim 90.92.A method for preparing a polypeptide as claimed in claim 62, comprising chemical synthesizing said polypeptide and isolating said polypeptide.","93.An antibody selected from the group consisting of a monoclonal antibody, a polyclonal antibody, and a chimeric antibody, or a fragment thereof, wherein said antibody specifically recognizes a polypeptide as claimed in claim 62.94.The antibody as claimed in claim 93, wherein said antibody is a labeled antibody.","95.A method for detecting and/or identifying bacteria belonging to the species P. luminescens, in a biological sample, comprising a) contacting said biological sample with an antibody as claimed in claim 93; b) detecting the formation of an antigen-antibody complex.","96.A method for detecting the expression of a gene of P. luminescens, comprising contacting a strain of P. luminescens with an antibody as claimed in claim 93, and detecting the formation of an antigen-antibody complex.","97.A kit for performing the method as claimed in claim 95, comprising the following elements: a) said antibody; and at least one of either b) reagents for constituting the medium suitable for an immunoreaction; or c) reagents for detecting the antigen-antibody complexes resulting from the immunoreaction.","98.A kit for performing the method as claimed in claim 96, comprising the following elements: a) said antibody; b) reagents for constituting the medium suitable for an immunoreaction; and c) reagents for detecting the antigen-antibody complexes resulting from the immunoreaction.","99.The polypeptide as claimed in claim 62, wherein said polypeptide is immobilized on a support 100.The polypeptide as claimed in claim 99, wherein said support a protein chip.","101.The polypeptide as claimed in claim 91, wherein said polypeptide is immobilized on a support 102.The polypeptide as claimed in claim 101, wherein said support a protein chip.","103.The antibody as claimed in claim 93, wherein said polypeptide is immobilized on a support 104.The antibody as claimed in claim 103, wherein said support a protein chip.","105.A protein chip comprising one or more polypeptide as claimed in claim 62 immobilized on the support of said chip.","106.The protein chip as claimed in claim 105, further comprising one or more polypeptides from a cell from a source selected from the group consisting of a plant, an animal and a microorganism other than P. luminescens, wherein said polypeptides are immobilized on the support of said chip.","107.The protein chip as claimed in claim 106, wherein said cell is a cell or microorganism sensitive to a toxin or an antibiotic produced by P. luminescens, a bacterium of the genus Photorhabdus, and a variant of P. luminescens.","108.A kit for detecting and/or quantifying the expression of at least one gene of P. luminescens, comprising a protein chip as claimed in claim 105.109.A protein chip comprising one or more polypeptide as claimed in claim 91 immobilized on the support of said chip.","110.The protein chip as claimed in claim 109, further comprising one or more polypeptides from a cell from a source selected from the group consisting of a plant, an animal and a microorganism other than P. luminescens, wherein said polypeptides are immobilized on the support of said chip.","111.The protein chip as claimed in claim 110, wherein said cell is a cell or microorganism sensitive to a toxin or an antibiotic produced by P. luminescens, a bacterium of the genus Photorhabdus, and a variant of P. luminescens.","112.A kit for detecting and/or quantifying the expression of at least one gene of P. luminescens, comprising a protein chip as claimed in claim 109.113.A protein chip comprising one or more antibodies as claimed in claim 93 immobilized on the support of said chip.","114.The protein chip as claimed in claim 113, further comprising one or more polypeptides from a cell from a source selected from the group consisting of a plant, an animal and a microorganism other than P. luminescens, wherein said polypeptides are immobilized on the support of said chip.","115.The protein chip as claimed in claim 114, wherein said cell is a cell or microorganism sensitive to a toxin or an antibiotic produced by P. luminescens, a bacterium of the genus Photorhabdus, and a variant of P. luminescens.","116.A kit for detecting and/or quantifying the expression of at least one gene of P. luminescens, comprising a protein chip as claimed in claim 113.117.A method for detecting and/or identifying bacteria belonging to the species P. luminescens, in a biological sample, comprising: a) isolating the DNA, or cDNA from the RNA, from the biological sample; b) specifically amplifying the DNA of bacteria belonging to the species P. luminescens using at least one primer as claimed in claim 78; c) identifying the amplification products.","118.A kit or set for detecting and/or identifying bacteria belonging to the species P. luminescens, comprising: a) a nucleotide probe and/or primer as claimed in claim 78; and at least one of b) reagents required for carrying out a hybridization reaction; or c) reagents for a DNA amplification reaction.","119.A composition comprising one or more nucleotide sequences as claimed in claim 57.120.A pharmaceutical composition comprising the composition as claimed in claim 119 and a pharmaceutically acceptable vehicle.","121.A biopesticidal composition comprising the composition as claimed in claim 119.122.A composition comprising one or more polypeptides as claimed in claim 62.123.A pharmaceutical composition comprising the composition as claimed in claim 122 and a pharmaceutically acceptable vehicle.","124.A biopesticidal composition comprising the composition as claimed in claim 122.125.A composition comprising a vector as claimed in claim 86.126.A pharmaceutical composition comprising the composition as claimed in claim 125 and a pharmaceutically acceptable vehicle.","127.A biopesticidal composition comprising the composition as claimed in claim 125.128.A composition comprising one or more antibodies as claimed in claim 93.129.A pharmaceutical composition comprising the composition as claimed in claim 128 and a pharmaceutically acceptable vehicle.","130.A biopesticidal composition comprising the composition as claimed in claim 128.131.A method of preparing a toxin or an antibiotic comprising culturing a cell as claimed in claim 88, and expressing a polypeptide involved in production of a toxin or an antibiotic.","132.A genomic library of a bacterium of the genus Photorhabdus.","133.The genomic DNA library of a bacterium of the genus Photorhabdus as claimed in claim 132, wherein said DNA library is cloned into a plasmid.","134.The genomic DNA library as claimed in claim 132, wherein said bacterium is P. luminescens or P. luminescens strain TT01.135.The genomic DNA library as claimed in claim 132, wherein said genomic DNA library is the genomic DNA library deposited with the CNCM on May 12, 2000, under accession No.","I-2478.136.A method for identifying at least one nucleotide sequence of P. luminescens not present in the genome of another species of bacterium or for identifying at least one nucleotide sequence of a genome of a bacterium of a species other than P. luminescens and not present in the P. luminescens genome, comprising: a) aligning the genomic sequences of the other bacterial species with the nucleotide sequences of P. luminescens as claimed in claim 57; and b) analyzing the data obtained by said aligning to identify and isolate said sequences only present in one or the other genome.","137.A method for identifying at least one nucleotide sequence of P. luminescens not present in the genome of another species of bacterium or for identifying at least one nucleotide sequence of a genome of a bacterium of a species other than P. luminescens and not present in the P. luminescens genome, comprising: a) aligning the genomic sequences of the other bacterial species with the genomic DNA library as claimed in claim 134; and b) analyzing the data obtained by said aligning to identify and isolate said sequences only present in one or the other genome."],["The invention relates to the genomic sequence and to nucleotide sequences encoding polypeptides of Photorhabdus luminescens, such as polypeptides involved in operons for biosynthesis of antibiotics, or of toxins, or polypeptides with activity of the toxin or antibiotic type which can be used as a pesticide, bactericide or fungicide, and also to vectors which include said sequences, and to cells or animals transformed with these vectors.","Photorhabdus luminescens is an entomopathogenic, commensal intestinal bacterium of a nematode and insect parasite.","This bacterium is both a model for studying host-parasite interactions and a bacterium which has many industrial applications because of its ability to synthesize numerous toxins (insecticides, bactericides and fungicides) and to secrete numerous enzymes.","In order to obtain an overall understanding of the genetic determinants involved in these processes, sequencing of the Photorhabdus luminescens genome was carried out.","The choice of the TT01 strain of Photorhabdus luminescens, subspecies laumondii, the sequencing of whose genome was carried out in the present invention, is very important since this strain has several advantages: its genome is stable; it can be cultured on a Petri dish; it is central in the phylogenetic tree, and therefore representative of the species; and its associated nematode is known and cultured (Heterorhabditis bacteriophora HP88, Trinidad).","The present invention thus relates to the nucleotide and polypeptide sequences of Photorhabdus luminescens strain TT01.Thus, an object of the present invention is to disclose the sequence of the genome of Photorhabdus luminescens strain TT01, contained in the genomic library prepared from the genome of this strain and deposited with the CNCM [French National Collection of Cultures and Micro-organisms] on May 12, 2000, under the number I-2478, and of all the genes and noncoding regulatory sequences contained in said genome.","Photorhabdus luminescens strain TT01 is also identified in the present application by Photorhabdus luminescens , in an interchangeable manner.","The invention also relates to novel tools for typing Photorhabdus strains.","These tools might be of the DNA “chip” type or of another type.","The novel characteristics of these typing tools will be as follows: rapidity and simplicity of use; high capacity for discriminating between strains; and possibility of providing information on the genomic content of the strain analyzed.","The present invention therefore relates to an isolated nucleotide sequence derived from the Photorhabdus luminescens genome, characterized in that it comprises a sequence chosen from the sequences SEQ ID No.","1 to SEQ ID No.","41 and the sequences SEQ ID No.","5826 to SEQ ID No.","5834.The sequences SEQ ID No.","1 to SEQ ID No.","41 represent the sequences of 41 contigs which altogether cover the genomic sequence of Photorhabdus luminescens TT01.It has been possible to reassemble these sequences SEQ ID No.","1 to SEQ ID No.","41 and to smooth them back out into 9 new contigs which altogether also cover the genomic sequence of Photorhabdus luminescens TT01.The sequences SEQ ID No.","5826 to SEQ ID No.","5834 represent the sequences of these 9 contigs.","The nucleotide sequences SEQ ID No.","1 to SEQ ID No.","41 and SEQ ID No.","5826 to SEQ ID No.","5834 were obtained by sequencing the Photorhabdus luminescens TT01 genome using the “shotgun” technique (cf.","examples).","Despite the great precision of these sequences SEQ ID No.","1 to SEQ ID No.","41 or SEQ ID No.","5826 to SEQ ID No.","5834, it is possible that these sequences do not give a 100% perfect representation, after assembly, of the nucleotide sequence of the Photorhabdus luminescens TT01 genome, and that some rare sequencing errors or indeterminations remain in these sequences.","In the present invention, the presence of an indetermination of an amino acid is denoted by “Xaa” and that of a nucleotide is denoted by “N” or “n” in the sequence listing hereinafter.","These few rare errors or indeterminations may be easily demonstrated and corrected by those skilled in the art using the whole chromosome and/or its representative fragments according to the invention, and standard methods of amplification, cloning and sequencing, it being possible for the sequences obtained to be easily compared, in particular by means of computer software, and using computer-readable media for recording the sequences according to the invention, as described, for example, below.","After correction of these possible rare errors or indeterminations, the corrected nucleotide sequence obtained would still comprise at least 97%, preferably at least 98%, 98.5%, 99% or 99.9%, identity with the genomic sequence obtained after assembly of these nucleotide sequences SEQ ID No.","1 to SEQ ID No.","41 or SEQ ID No.","5826 to SEQ ID No.","5834.The present invention also relates to an isolated nucleotide sequence derived from the Photorhabdus luminescens genome, characterized in that it is chosen from: a) a nucleotide sequence comprising at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with a sequence chosen from the sequences SEQ ID No.","1 to SEQ ID No.","41 or SEQ ID No.","5826 to SEQ ID No.","5834; b) a nucleotide sequence comprising a representative fragment of a sequence chosen from the sequences SEQ ID No.","1 to SEQ ID No.","41 or SEQ ID No.","5826 to SEQ ID No.","5834; c) a nucleotide sequence complementary to a nucleotide sequence as defined in a) or b); d) a nucleotide sequence of the RNA corresponding to one of the sequences as defined in a), b) or c); e) a nucleotide sequence as defined in a), b), c) or d), which has been modified; f) a nucleotide sequence which hybridizes, under high stringency conditions, with a sequence chosen from SEQ ID No.","1 to SEQ ID No.","41 or SEQ ID No.","5826 to SEQ ID No.","5834, and which comprises at least 20 nucleotides, preferably at least 25, 30, 50, 75, 100, 150, 200, 250, 500, 750, 1 000, 1 500, 2 000 or 2 500 nucleotides.","More particularly, a subject of the present invention is also a nucleotide sequence included in one of the sequences SEQ ID No.","1 to SEQ ID No.","41 or one of the sequences SEQ ID No.","5826 to SEQ ID No.","5834, and in that it encodes a polypeptide chosen from the polypeptides of sequence SEQ ID No.","42 to SEQ ID No.","3855 or from the polypeptides encoded by a sequence SEQ ID No.","5835 to SEQ ID No.","10784.Preferably, the polypeptides encoded by one of the sequences SEQ ID No.","5835 to SEQ ID No.","10784 are the polypeptides for which the sequence of at least 5 amino acids is obtained by taking as reading frame the first nucleotide of the sequences SEQ ID No.","5835 to SEQ ID No.","10784.Very preferably, a subject of the invention is also a nucleotide sequence, characterized in that it encodes a polypeptide whose function annotated in table I hereinafter, final column, or in table II hereinafter, penultimate column, corresponds to an activity of the toxin and/or antibiotic type, or to an operon involved in the synthesis of a toxin and/or of an antibiotic, which polypeptide is preferably chosen from: a) the polypeptides of sequences SEQ ID No.","61, SEQ ID No.","62, SEQ ID No.","67, SEQ ID No.","171, SEQ ID No.","221, SEQ ID No.","268, SEQ ID No.","288, SEQ ID No.","380, SEQ ID No.","426, SEQ ID No.","438, SEQ ID No.","448, SEQ ID No.","453, SEQ ID No.","455, SEQ ID No.","456, SEQ ID No.","458, SEQ ID No.","501, SEQ ID No.","516, SEQ ID No.","530, SEQ ID No.","542, SEQ ID No.","551, SEQ ID No.","720, SEQ ID No.","761, SEQ ID No.","762, SEQ ID No.","814, SEQ ID No.","859, SEQ ID No.","860, SEQ ID No.","861, SEQ ID No.","862, SEQ ID No.","869, SEQ ID No.","1079, SEQ ID No.","1168, SEQ ID No.","1174, SEQ ID No.","1176, SEQ ID No.","1413, SEQ ID No.","1414, SEQ ID No.","1415, SEQ ID No.","1416, SEQ ID No.","1417, SEQ ID No.","1457, SEQ ID No.","1651, SEQ ID No.","1856, SEQ ID No.","1869, SEQ ID No.","2021, SEQ ID No.","2080, SEQ ID No.","2152, SEQ ID No.","2162, SEQ ID No.","2173, SEQ ID No.","2251, SEQ ID No.","2295, SEQ ID No.","2306, SEQ ID No.","2317, SEQ ID No.","2328, SEQ ID No.","2340, SEQ ID No.","2342, SEQ ID No.","2351, SEQ ID No.","2500, SEQ ID No.","3228, SEQ ID No.","3230, SEQ ID No.","3311, SEQ ID No.","3317, SEQ ID No.","3318, SEQ ID No.","3319, SEQ ID No.","3320, SEQ ID No.","3322, SEQ ID No.","3323, SEQ ID No.","3326, SEQ ID No.","3327, SEQ ID No.","3328, SEQ ID No.","3375, SEQ ID No.","3376, SEQ ID No.","3377, SEQ ID No.","3378, SEQ ID No.","3422, SEQ ID No.","3489, SEQ ID No.","3503, SEQ ID No.","3609, SEQ ID No.","3623, SEQ ID No.","3624, SEQ ID No.","3772, SEQ ID No.","3783, SEQ ID No.","3788 and SEQ ID No.","3794; or b) the polypeptides encoded by the sequences SEQ ID No.","5835 to SEQ ID No.","10784 homologous to the sequences as defined in a) above, as indicated in the final column of table II.","These 82 polypeptides of sequence as defined in paragraph a) above, whose function is associated with an activity of the toxin or antibiotic type, or their homologous polypeptide of table II as defined in paragraph b) above, could be identified, for example, by the presence of a consensus motif associated with these functions or by the presence of sequences juxtaposing them on the genome and involved in this type of activity.","More generally, the present invention also relates to the nucleotide sequences derived from SEQ ID No.","1 to SEQ ID No.","41 or SEQ ID No.","5826 to SEQ ID No.","5834, and encoding a polypeptide of P. luminescens, such that they can be isolated from SEQ ID No.","1 to SEQ ID No.","41 or SEQ ID No.","5826 to SEQ ID No.","5834.In addition, the nucleotide sequences characterized in that they comprise a nucleotide sequence chosen from: a) a nucleotide sequence encoding a polypeptide chosen from the sequences SEQ ID No.","42 to SEQ ID No.","3855 or from the polypeptides encoded by the sequences SEQ ID No.","5835 to SEQ ID No.","10784, preferably from the 82 polypeptide sequences above selected for their function associated with an activity of the toxin or antibiotic type, or their homologous peptide as defined in table II, in the final column; b) a nucleotide sequence comprising at least 75% identity with a nucleotide sequence as defined in a), preferably at least 80%, 85%, 90%, 95%, 98% or 99% identity; c) a complementary or RNA nucleotide sequence corresponding to a sequence as defined in a) or b); d) a nucleotide sequence of a representative fragment of a sequence as defined in a) or c); and e) a sequence as defined in a) or c), which has been modified are also subjects of the invention.","The terms “nucleic acid”, “nucleic acid sequence”, “polynucleotide”, “oligonucleotide”, “polynucleotide sequence” and “nucleotide sequence”, terms which will be used indifferently in the present description, are intended to denote a precise chain of nucleotides, which may or may not be modified, making it possible to define a fragment or a region of a nucleic acid, which may or may not comprise unnatural nucleotides, and which may correspond equally to a double-stranded DNA, a single-stranded DNA and products of transcription of said DNAs.","Thus, the nucleic acid sequences according to the invention also encompass PNAs (Peptide Nucleic Acids).","It should be understood that the present invention does not concern the nucleotide sequences in their natural chromosomal environment, i.e.","in their natural state.","They are sequences which have been isolated and/or purified, i.e.","they have been taken directly or indirectly, for example by copying, their environment having been at least partially modified.","The nucleic acids obtained by chemical synthesis are thus also intended to be denoted.","For the purpose of the present invention, the term “percentage identity” between two nucleic acid or amino acid sequences is intended to denote a percentage of nucleotides or of amino acid residues which are identical between the two sequences to be compared, obtained after best alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly and over their entire length.","The term “best alignment” or “optimal alignment” is intended to denote the alignment for which the percentage identity determined as below is the highest.","Sequence comparisons between two nucleic acid or amino acid sequences are conventionally carried out by comparing these sequences after having optimally aligned them, said comparison being carried out by segment or by “window of comparison” so as to identify and compare the local regions of sequence similarity.","The optimal alignment of the sequences for the comparison can be carried out, besides manually, by means of the local homology algorithm of Smith and Waterman (1981, Ad.","App.","Math., 2:482), by means of the local homology algorithm of Neddleman and Wunsch (1970, J. Mol.","Biol., 48:443), by means of the similarity search method of Pearson and Lipman (1988, Proc.","Natl.","Acad.","Sci.","USA, 85:2444), by means of computer programs using these algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.).","In order to obtain the optimal alignment, the BLAST program is preferably used, with the BLOSUM 62 matrix.","The PAM or PAM250 matrices can also be used.","The percentage identity between two nucleic acid or amino acid sequences is determined by comparing these two sequences when optimally aligned, the nucleic acid or amino acid sequence to be compared possibly comprising additions or deletions with respect to the reference sequence, for optimal alignment between these two sequences.","The percentage identity is calculated by determining the number of identical positions for which the nucleotide or amino acid residue is identical in the two sequences, dividing this number of identical positions by the total number of positions compared, and multiplying the result obtained by 100 so as to obtain the percentage identity between these two sequences.","The expression “nucleic acid sequences exhibiting a percentage identity of at least 75%, preferably 80%, 85%, 90%, 95%, 98% or 99%, after optimal alignment, with a reference sequence” is intended to denote the nucleic acid sequences exhibiting, compared to the reference nucleic acid sequence, certain modifications such as in particular a deletion, a truncation, an extension, a chimeric fusion and/or a substitution, in particular of the point type, and the nucleic acid sequence of which exhibits at least 75%, preferably 80%, 85%, 90%, 95%, 98% or 99%, identity, after optimal alignment, with the reference nucleic acid sequence.","They are preferably sequences for which the complementary sequences are capable of hybridizing specifically with the reference sequences.","Preferably, the specific or high stringency hybridization conditions will be such that they provide at least 75%, preferably 80%, 85%, 90%, 95%, 98% or 99%, identity, after optimal alignment, between one of the two sequences and the sequence complementary thereto.","A hybridization under high stringency conditions means that the conditions of temperature and of ionic strength are chosen such that they make it possible to maintain the hybridization between two complementary DNA fragments.","By way of illustration, high stringency conditions for the hybridization step for the purposes of defining the polynucleotide fragments described above are advantageously as follows.","The DNA-DNA or DNA-RNA hybridization is carried out in two steps: (1) prehybridization at 42° C. for 3 hours in phosphate buffer (20 mM, pH 7.5) containing 5×SSC (1×SSC corresponds to a solution of 0.15M NaCl+0.015M sodium citrate), 50% of formamide, 7% of sodium dodecyl sulfate (SDS), 10× Denhardt's, 5% of dextran sulfate and 1% of salmon sperm DNA; (2) hybridization per se for 20 hours at a temperature which depends on the length of the probe (i.e.",": 42° C. for a probe>100 nucleotides in length) followed by 2 washes of 20 minutes at 20° C. in 2×SSC+2% SDS and 1 wash of 20 minutes at 20° C. in 0.1×SSC+0.1% SDS.","The final wash is carried out in 0.1×SSC+0.1% SDS for 30 minutes at 60° C. for a probe>100 nucleotides in length.","The high stringency hybridization conditions described above for a polynucleotide of defined length can be adjusted by those skilled in the art for longer or shorter oligonucleotides, according to the teaching of Sambrook et al.","(1989, Molecular cloning: a laboratory manual.","2nd Ed.","Cold Spring Harbor).","In addition, the expression “representative fragment of sequences according to the invention” is intended to denote any nucleotide fragment having at least 15 consecutive nucleotides, preferably at least 20, 25, 30, 50, 75, 100, 150, 200, 300 and 450 consecutive nucleotides, of the sequence from which it is derived.","The term “representative fragment” is intended to mean in particular a nucleic acid sequence encoding a biologically active fragment of a polypeptide, as defined later.","The term “representative fragment” is also intended to mean the intergenic sequences, and in particular the nucleotide sequences carrying the regulatory signals (promoters, terminators, or even enhancers, etc).","Among said representative fragments, preference is given to those having nucleotide sequences corresponding to open reading frames, referred to as ORF sequences, included in general between an initiation codon and a stop codon, or between two stop codons, and encoding polypeptides, preferably with at least 100 amino acids, such as, for example, without being limited thereto, the ORF sequences which will subsequently be described.","The numbering of the ORF nucleotide sequences which will subsequently be used in the present description corresponds to the numbering of the amino acid sequences of the proteins encoded by said ORFs for the sequences SEQ ID No.","42 to SEQ ID No.","3855.The numbering of the ORF nucleotide sequences SEQ ID No.","5835 to SEQ ID No.","10784 will subsequently be used in the present description for the numbering of the amino acid sequences of the proteins encoded by said ORFs SEQ ID No.","5835 to SEQ ID No.","10784.The representative fragments according to the invention can be obtained, for example, by specific amplification such as PCR or after digestion, with suitable restriction enzymes, of nucleotide sequences according to the invention, this method being described in particular in the work by Sambrook et al.","Said representative fragments can also be obtained by chemical synthesis when they are not too long, according to methods well known to those skilled in the art.","The sequences containing sequences of the invention, or representative fragments, are also intended to include the sequences which are naturally framed by sequences which exhibit at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with the sequences according to the invention.","The term “modified nucleotide sequence” is intended to mean any nucleotide sequence obtained by mutagenesis according to techniques well known to those skilled in the art, and comprising modifications compared to the normal sequences, preferably at most 10% of modified nucleotides compared to these normal sequences, for example mutations in the regulatory and/or promoter sequences for expression of the polypeptide, in particular leading to a modification of the level of expression or of the activity of said polypeptide.","The term “modified nucleotide sequence” is also intended to mean any nucleotide sequence encoding a modified polypeptide as defined below.","The representative fragments according to the invention can also be probes or primers, which can be used in methods for detecting, identifying, assaying or amplifying nucleic acid sequences.","For the purpose of the invention, a probe or primer is defined as being a single-stranded nucleic acid fragment or a denatured double-stranded fragment comprising, for example, from 12 bases to a few kb, in particular from 15 to a few hundred bases, preferably from 15 to 50 or 100 bases, and having a specificity of hybridization under given conditions so as to form a hybridization complex with a target nucleic acid.","The probes and primers according to the invention can be labeled directly or indirectly with a radioactive or nonradioactive compound using methods well known to those skilled in the art, in order to obtain a detectable and/or quantifiable signal (patent FR 78 10975 and bDNA of Chiron EP 225 807 and EP 510 085).","The unlabeled sequences of polynucleotides according to the invention can be used directly as a probe or primer.","The sequences are generally labeled to obtain sequences which can be used for many applications.","The labeling of the primers or of the probes according to the invention is carried out with radioactive elements or with nonradioactive molecules.","Among the radioactive isotopes used, mention may be made of 32P, 33P 35S, 3H or 125I.","The nonradioactive entities are selected from ligands such as biotin, avidin, streptavidin or digoxigenin, haptens, dyes, and luminescent agents such as radioluminescent, chemiluminescent, bioluminescent, fluorescent or phosphorescent agents.","The polynucleotides according to the invention can thus be used as a primer and/or a probe in methods using in particular the PCR (polymerase chain reaction) technique (Rolfs et al., 1991, Berlin: Springer-Verlag).","This technique requires choosing pairs of oligonucleotide primers framing the fragment which must be amplified.","Reference may, for example, be made to the technique described in U.S. Pat.","No.","4,683,202.The amplified fragments can be identified, for example after agarose or polyacrylamide gel electrophoresis, or after a chromatography technique such as gel filtration or ion exchange chromatography, and then sequenced.","The specificity of the amplification can be controlled using the nucleotide sequences of polynucleotides of the invention as a matrix, plasmids containing these sequences or else the derived amplification products.","The amplified nucleotide fragments can be used as reagents in hybridization reactions in order to demonstrate the presence, in a biological sample, of a target nucleic acid of sequence complementary to that of said amplified nucleotide fragments.","The invention is also directed toward the nucleic acids which can be obtained by amplification using primers according to the invention.","Other techniques for amplifying the target nucleic acid can advantageously be employed as an alternative to PCR (PCR-like), using a pair of primers of nucleotide sequences according to the invention.","The term “PCR-like” is intended to denote all the methods using direct or indirect reproductions of nucleic acid sequences, or else in which the labeling systems have been amplified; these techniques are, of course, known.","In general, they involve amplification of the DNA with a polymerase; when the sample of origin is an RNA, a reverse transcription should be carried out beforehand.","A very large number of methods currently exist for this amplification, such as, for example, the SDA (strand displacement amplification) technique (Walker et al., 1992, Nucleic Acids Res., 20:1691), the TAS (transcription-based amplification system) technique described by Kwoh et al.","(1989, Proc.","Natl.","Acad.","Sci.","USA, 86, 1173), the 3SR (self-sustained sequence replication) technique described by Guatelli et al.","(1990, Proc.","Natl.","Acad.","Sci.","USA, 87: 1874), the NASBA (nucleic acid sequence based amplification) technique described by Kievitis et al.","(1991, J. Virol.","Methods, 35, 273), the TMA (transcription mediated amplification) technique, the LCR (ligase chain reaction) technique described by Landegren et al.","(1988, Science, 241, 1077), the RCR (repair chain reaction) technique described by Segev (1992, Kessler C. Springer Verlag, Berlin, N.Y., 197-205), the CPR (cycling probe reaction) technique described by Duck et al.","(1990, Biotechniques, 9, 142) and the Q-beta-replicase amplification technique described by Miele et al.","(1983, J. Mol.","Biol., 171, 281).","Some of these techniques have since been improved.","When the target polynucleotide to be detected is an mRNA, an enzyme of the reverse transcriptase type is advantageously used, prior to carrying out an amplification reaction using the primers according to the invention, or to carrying out a method of detection using the probes of the invention, in order to obtain a cDNA from the mRNA contained in the biological sample.","The cDNA obtained will then serve as a target for the primers or the probes used in the method of amplification or of detection according to the invention.","The probe hybridization technique can be carried out in various ways (Matthews et al., 1988, Anal.","Biochem., 169, 1-25).","The most general method consists in immobilizing the nucleic acid, extracted from the cells of various tissues or from cells in culture, on a support (such as nitrocellulose, nylon or polystyrene) and in incubating, under well-defined conditions, the immobilized target nucleic acid with the probe.","After hybridization, the excess probe is removed and the hybrid molecules formed are detected by the appropriate method (measurement of the radioactivity, of the fluorescence or of the enzyme activity associated with the probe).","According to another embodiment of the nucleic acid probes according to the invention, the latter can be used as capture probes.","In this case, a probe, termed “capture probe”, is immobilized on a support and is used to capture, by specific hybridization, the target nucleic acid obtained from the biological sample to be tested, and the target nucleic acid is then detected using a second probe, termed “detection probe”, labeled with a readily detectable element.","Among the advantageous nucleic acid fragments, mention should thus in particular be made of antisense oligonucleotides, i.e.","oligonucleotides whose structure provides, by hybridization with the target sequence, inhibition of the expression of the corresponding product.","Mention should also be made of sense oligonucleotides which, by interaction with proteins involved in regulating the expression of the corresponding product, will induce either inhibition or activation of this expression.","Preferably, the probes or primers according to the invention are immobilized on a support in a covalent or noncovalent manner.","In particular, the support can be a DNA chip or a high or medium density filter, also a subject of the present invention (patents WO 97/29212, WO 98/27317, WO 97/10365 and WO 92/10588).","The term “DNA chip” or “high density filter” is intended to denote a support on which are attached DNA sequences, it being possible to pinpoint each one of them by its geographical location.","These chips or filters differ mainly by their size, the material of the support and, optionally, the number of DNA sequences which are attached thereto.","The probes or primers according to the first invention can be attached to solid supports, in particular the DNA chips, by various methods of production.","In particular, in situ synthesis can be carried out by photochemical addressing or by inkjet.","Other techniques consist in carrying out an ex situ synthesis and attaching the probes to the support of the DNA chip by mechanical or electronic addressing or by inkjet.","These various methods are well known to those skilled in the art.","A nucleotide sequence (probe or primer) according to the invention therefore makes it possible to detect and/or amplify specific nucleic acid sequences.","In particular, the detection of these said sequences is facilitated when the probe is attached to a DNA chip or to a high density filter.","The use of DNA chips or of high density filters in fact makes it possible to determine the gene expression in an organism having a genomic sequence close to P. luminescens, and to type the strain in question.","The genomic sequence of P. luminescens, supplemented by the identification of the genes of these organisms, as presented in the present invention, serves as a basis for constructing these DNA chips or filters.","The preparation of these filters or chips consists in synthesizing oligonucleotides, corresponding to the 5′ and 3′ ends of the genes or to more internal fragments, in order to amplify fragments of an appropriate length, for example of between approximately 300 and 800 bases.","These oligonucleotides are chosen using the genomic sequence and its annotations disclosed in the present invention.","The temperature for pairing these oligonucleotides at the corresponding places on the DNA should be approximately the same for each oligonucleotide.","This makes it possible to prepare DNA fragments corresponding to each gene using appropriate PCR conditions in a highly automated environment.","The amplified fragments are then immobilized on filters or supports made of glass, silicon or synthetic polymers and these media are used for the hybridization.","The availability of such filters and/or chips and of the corresponding annotated genomic sequence makes it possible to study the expression of large sets, or even all, of the genes of Photorhabdus luminescens , in particular of P. luminescens TT01, by preparing the complementary DNAs and hybridizing them to the DNA or to the oligonucleotides immobilized on the filters or the chips.","Similarly, the filters and/or the chips make it possible to study the strain or species variability by preparing the DNA of these organisms and hybridizing them to the DNA or to the oligonucleotides immobilized on the filters or the chips.","The differences between the genomic sequences of the various strains or species can greatly affect the intensity of the hybridization and, consequently, disturb the interpretation of the results.","It may therefore be necessary to have the precise sequence of the genes of the strain intended to be studied.","The method for detecting the genes described later in detail, involving determining the sequence of random fragments of a genome and organizing them according to the sequence of the genome of P. luminescens, in particular of P. luminescens TT01, disclosed in the present invention, may be very useful.","The nucleotide sequences according to the invention can be used in DNA chips to carry out mutation analysis.","This analysis is based on constituting chips capable of analyzing each base of a nucleotide sequence according to the invention.","For this purpose, use may in particular be made of the techniques of microsequencing on a DNA chip.","The mutations are detected by extension of immobilized primers which hybridize to the matrix of the analyzed sequences, just at a position adjacent to that of the mutated nucleotide sought.","A single-stranded, RNA or DNA matrix of the sequences to be analyzed will advantageously be prepared according to conventional methods, using products amplified according to PCR-type techniques.","The single-stranded DNA or RNA matrices thus obtained are then deposited onto the DNA chip, under conditions which allow their specific hybridization to the immobilized primers.","A thermostable polymerase, for example Tth or Taq DNA polymerase, specifically extends the 3′ end of the immobilized primer with a labeled nucleotide analog complementary to the nucleotide at the position of the variable site; for example, thermocycling is carried out in the presence of fluorescent dideoxyribonucleotides.","The experimental conditions will be adjusted in particular to the chips used, to the immobilized primers, to the polymerases used and to the labeling system chosen.","One advantage of microsequencing, compared to techniques based on probe hybridization, is that it makes it possible to identify all the variable nucleotides with optimal discrimination under homogeneous reaction conditions; when used on DNA chips, it allows optimal resolution and specificity for the routine and industrial detection of mutations in a multiplex.","A DNA chip or a filter can be an extremely advantageous tool for determining, detecting and/or identifying a microorganism.","Thus, the DNA chips according to the invention which also contain at least one nucleotide sequence of a microorganism other than Photorhabdus luminescens, immobilized on the support of said chip, are also preferred.","The microorganism chosen is preferably done so from the bacteria of the genus Photorhabdus (hereinafter referred to as P. luminescens-related bacteria), or the variants of Photorhabdus luminescens TT01.A DNA chip or a filter according to the invention is a very useful element of certain kits or sets for detecting and/or identifying microorganisms, in particular bacteria belonging to the species Photorhabdus luminescens , which are also the subject of the invention.","Moreover, the DNA chips or the filters according to the invention, containing probes or primers specific for Photorhabdus luminescens , are very advantageous elements of kits or sets for detecting and/or quantifying the expression of Photorhabdus luminescens genes.","Specifically, the control of gene expression is a critical point for optimizing the growth and yield of a strain, either by allowing the expression of one or more new genes, or by modifying the expression of genes already present in the cell.","The present invention provides all of the sequences naturally active in P. luminescens which allow gene expression.","It thus makes it possible to determine all the sequences expressed in P. luminescens.","It also provides a tool for pinpointing the genes whose expression follows a given pattern.","To do this, the DNA of all or some of the genes of P. luminescens can be amplified using primers according to the invention, and then attached to a support such as, for example, glass or nylon or a DNA chip, in order to construct a tool for following the expression profile of these genes.","This tool, consisting of this support containing the coding sequences, serves as a matrix of hybridization to a mixture of labeled molecules reflecting the messenger RNAs expressed in the cell (in particular the labeled probes according to the invention).","By repeating this experiment at various moments and combining all of these data using suitable processing, the expression profiles of all these genes are then obtained.","The knowledge of the sequences which follow a given regulatory scheme can also be exploited to search for, in a directed manner, for example by homology, other sequences following, overall, but in a slightly different way, the same regulatory scheme.","In addition, it is possible to isolate each control sequence present upstream of the segments acting as probes and to follow the activity thereof using a suitable means such as a reporter gene (luciferase, β-galactosidase, GFP).","These isolated sequences can then be modified and assembled by metabolic engineering with sequences of interest with a view to the optimal expression thereof.","The invention also relates to the polypeptides encoded by a nucleotide sequence according to the invention, preferably by a representative fragment of the preceding sequences, corresponding to an ORF sequence.","In particular, the polypeptides of Photorhabdus luminescens TT01 of SEQ ID No.","42 to SEQ ID No.","3855 or encoded by SEQ ID No.","5835 to SEQ ID No.","10784 are a subject of the invention.","The invention also comprises the polypeptides characterized in that they comprise a polypeptide chosen from: a) a polypeptide of sequence SEQ ID No.","42 to SEQ ID No.","3855 or encoded by a sequence SEQ ID No.","5835 to SEQ ID No.","10784; b) a polypeptide exhibiting at least 80%, preferably 85%, 90%, 95% and 98%, identity with a polypeptide according to the invention; c) a fragment of at least 5 amino acids of a polypeptide as defined in a); d) a biologically active fragment of a polypeptide as defined in a); and e) a polypeptide as defined in a), b), c) or d), which has been modified.","The nucleotide sequences encoding the polypeptides described above are also a subject of the invention.","In the present description, the terms “polypeptides”, “polypeptide sequences”, “peptides” and “proteins” are interchangeable.","The term “polypeptide” comprises any amino acid sequence making it possible to generate an antibody response.","It should be understood that the invention does not concern the polypeptides in natural form, i.e.","they are not taken in their natural environment.","On the other hand, it concerns those which it has been possible to isolate or obtain by purification from natural sources, or else those obtained by genetic recombination or by chemical synthesis, and they can then comprise unnatural amino acids as will be described below.","The expression “polypeptide exhibiting a certain percentage identity with another”, for which the expression “homologous polypeptide” will also be used, is intended to denote the polypeptides exhibiting, compared to the natural polypeptides, certain modifications, in particular a deletion, addition or substitution of at least one amino acid, a truncation, an extension, a chimeric solution and/or a mutation, or the polypeptides exhibiting post-translational modifications.","Among the homologous polypeptides, preference is given to those in which the amino acid sequence exhibits at least 80%, preferably 85%, 90%, 95%, 98% or 99%, identity, after optimal alignment, with the amino acid sequences of the polypeptides according to the invention.","In the case of a substitution, one or more consecutive or nonconsecutive amino acid(s) may be replaced with “equivalent” amino acids.","The expression “equivalent amino acids” is here aimed at denoting any amino acid capable of being substituted for one of the amino acids of the basic structure without, however, essentially modifying the biological activities of the corresponding peptides as they will be defined subsequently.","These equivalent amino acids can be determined either based on their structural homology with the amino acids for which they substitute, or based on results of comparative biological activity assays between the various polypeptides liable to be produced.","By way of example, mention is made of the substitution possibilities which can be made without this resulting in a profound modification of the biological activity of the corresponding modified polypeptide.","It is thus possible to replace leucine with valine or isoleucine, aspartic acid with glutamic acid, glutamine with asparagine, arginine with lysine, etc, it naturally being possible to envision the reverse substitutions under the same conditions.","The homologous polypeptides also correspond to the polypeptides encoded by the nucleotide sequences which exhibit a certain percentage identity with the nucleotide sequences of the invention or which are identical, as previously defined, and thus comprise, in the present definition, mutated polypeptides or polypeptides corresponding to inter- or intraspecies variations which can exist in Photorhabdus, and which correspond in particular to truncations, substitutions, deletions and/or additions of at least one amino acid residue.","It is understood that the percentage identity between two polypeptides is calculated in the same way as between two nucleic acid sequences.","Thus, the percentage identity between two polypeptides is calculated, after optimal alignment of these two sequences, on a window of maximum homology.","To define said window of maximum homology, the same algorithms as for the nucleic acid sequences can be used.","The expression “biologically active fragment of a polypeptide according to the invention” is intended to denote in particular a polypeptide fragment, as defined below, exhibiting at least one of the biological characteristics of the polypeptides according to the invention, in particular in that it is capable of exerting, in general, even a partial activity, such as, for example: an enzymatic (metabolic) activity or an activity which may be involved in the biosynthesis or the biodegradation of organic or inorganic compounds, or preferably a toxic or antibiotic activity, in particular for insects or microorganisms (bacteria or fungi), or else an activity involved in the biosynthesis of these toxins or antibiotics; such proteins with enzymatic activity may in particular be used in methods for screening and/or selecting compounds capable of modifying this activity, in particular of inhibiting it; a structural activity (cell envelope, chaperone molecule, ribosome).","Proteins corresponding especially to extramembrane proteins may in particular be used as an immunogen for producing mono- or polyclonal antibodies directed specifically against these extramembrane proteins; a transport activity (energy transport, ion transport); or an activity in protein secretion; an activity in the process of replication, amplification, preparation, transcription, translation or maturation, in particular of DNA, of RNA or of proteins.","The expression “polypeptide fragment according to the invention” is intended to denote a polypeptide comprising at least 5 amino acids, preferably 10, 15, 25, 50, 100 and 150 amino acids.","The polypeptide fragments can correspond to isolated or purified fragments naturally present in the strains of Photorhabdus, or to fragments which can be obtained by cleavage of said polypeptide with a proteolytic enzyme, such as trypsin or chymotrypsin or collagenase, or with a chemical reagent (cyanogen bromide, CNBr) or by placing said polypeptide in a very acidic environment (for example at pH=2.5).","Polypeptide fragments can also be prepared by chemical synthesis, from hosts transformed with an expression vector according to the invention which contain a nucleic acid which allows expression of said fragment and is placed under the control of the appropriate regulatory and/or expression elements.","The term “modified polypeptide” of a polypeptide according to the invention is intended to denote a polypeptide obtained by genetic recombination or by chemical synthesis as described later, which exhibits at least one modification compared to the normal sequence, and preferably at most 10% of modified amino acids compared to the normal sequence.","These modifications may in particular be made on amino acids necessary for the specificity or the effectiveness of the activity, or responsible for the structural conformation, for the charge or for the hydrophobicity of the polypeptide according to the invention.","It is thus possible to create polypeptides with equivalent, increased or decreased activity, or with equivalent, stricter or broader specificity.","Among the modified polypeptides, mention should be made of the polypeptides in which up to five amino acids can be modified, truncated at the N- or C-terminal end, or else deleted, or added.","As is indicated, the aim of the modifications to a polypeptide is in particular: to allow its use in methods of biosynthesis or of biodegradation of organic or inorganic compounds, or in the biosynthesis of toxins or of antibiotics, to allow its use in methods of replication, of amplification, of repair and of regulation of transcription, translation or maturation in particular of DNA, RNA or proteins; to allow its enhanced secretion, to modify its solubility, or the effectiveness or specificity of its activity, or else to facilitate its purification.","Chemical synthesis also has the advantage of being able to use unnatural amino acids or nonpeptide bonds.","Thus, it may be advantageous to use unnatural amino acids, for example in the D form, or amino acid analogs, in particular sulfur-containing forms.","The invention also relates to the nucleic acid or peptide sequences according to the present invention with the exception of the nucleic acid or peptide sequences described in documents WO 99/54472, WO 99/42589, WO 99/03328, WO 98/08932 and EP 0 823 215.The present invention provides the nucleotide sequence of the Photorhabdus luminescens TT01 genome in the form of 41 contigs or in the form of 9 contigs, and also some polypeptide sequences.","The nucleic acid or peptide sequences below, characterized by their function, can also be identified by their nucleotide and amino acid sequence with reference to table I. Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01 with activity of the toxin and/or antibiotic type, or involved in the synthesis of these toxins and/or antibiotics.","Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in amino acid biosynthesis.","Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the biosynthesis of cofactors, prosthetic groups and transporters.","Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a cell envelope polypeptide or a polypeptide present at the surface of Photorhabdus luminescens TT01, or one of its fragments.","Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the cellular machinery.","Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in central intermediate metabolism.","Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in energy metabolism.","Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in fatty acid and phospholipid metabolism.","Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the metabolism of nucleotides, purines, pyrimidines or nucleosides.","Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in regulatory functions.","Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the replication process.","Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the transcription process.","Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the translation process.","Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the process of protein transport and binding.","Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in adaptation to atypical conditions.","Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, [lacuna] in sensitivity to medicinal products and analogs.","Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in functions relating to transposons.","Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide specific for Photorhabdus luminescens TT01, or one of its fragments.","In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, [lacuna] activity of the toxin and/or antibiotic type, or involved in the synthesis of these toxins and/or antibiotics.","In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in amino acid biosynthesis.","In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the biosynthesis of cofactors, prosthetic groups and transporters.","In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a cell envelope polypeptide or a surface polypeptide of Photorhabdus luminescens TT01, or one of its fragments.","In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the cellular machinery.","In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in central intermediate metabolism.","In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in energy metabolism.","In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in fatty acid and phospholipid metabolism.","In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the metabolism of nucleotides, purines, pyrimidines or nucleosides.","In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in regulatory functions.","In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the replication process.","In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the transcription process.","In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the translation process.","In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the process of protein transport and binding.","In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in adaptation to atypical conditions.","In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, [lacuna] in sensitivity to medicinal products and analogs.","In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in functions relating to transposons.","In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide specific for Photorhabdus luminescens TT01, or one of its fragments.","A subject of the invention is also the operons involved in the synthesis of antibiotics and/or of toxins.","Table I provides the list of some polypeptides according to the invention, and also their location in the contigs represented by SEQ ID No.","1 to SEQ ID No.","41, and the similarities observed after comparison in the databases.","Table II provides the list of some polypeptides according to the invention, and also their location in the contigs represented by SEQ ID No.","5826 to SEQ ID No.","5834, and the similarities observed after comparison in the databases.","In table II, contigs 1 to 9 are identified by the sequences SEQ ID No.","5826 to SEQ ID No.","5834.Entirely preferably, a subject of the invention is also the polypeptides whose functions annotated in table I, final column, or whose functions annotated in table II, penultimate column, correspond to activities of the toxin and/or antibiotic type, or to polypeptides involved in the synthesis of these toxins and/or antibiotics, which polypeptides are preferably chosen from: a) the polypeptides of sequence SEQ ID No.","61, SEQ ID No.","62, SEQ ID No.","67, SEQ ID No.","171, SEQ ID No.","221, SEQ ID No.","268, SEQ ID No.","288, SEQ ID No.","380, SEQ ID No.","426, SEQ ID No.","438, SEQ ID No.","448, SEQ ID No.","453, SEQ ID No.","455, SEQ ID No.","456, SEQ ID No.","458, SEQ ID No.","501, SEQ ID No.","516, SEQ ID No.","530, SEQ ID No.","542, SEQ ID No.","551, SEQ ID No.","720, SEQ ID No.","761, SEQ ID No.","762, SEQ ID No.","814, SEQ ID No.","859, SEQ ID No.","860, SEQ ID No.","861, SEQ ID No.","862, SEQ ID No.","869, SEQ ID No.","1079, SEQ ID No.","1168, SEQ ID No.","1174, SEQ ID No.","1176, SEQ ID No.","1413, SEQ ID No.","1414, SEQ ID No.","1415, SEQ ID No.","1416, SEQ ID No.","1417, SEQ ID No.","1457, SEQ ID No.","1651, SEQ ID No.","1856, SEQ ID No.","1869, SEQ ID No.","2021, SEQ ID No.","2080, SEQ ID No.","2152, SEQ ID No.","2162, SEQ ID No.","2173, SEQ ID No.","2251, SEQ ID No.","2295, SEQ ID No.","2306, SEQ ID No.","2317, SEQ ID No.","2328, SEQ ID No.","2340, SEQ ID No.","2342, SEQ ID No.","2351, SEQ ID No.","2500, SEQ ID No.","3228, SEQ ID No.","3230, SEQ ID No.","3311, SEQ ID No.","3317, SEQ ID No.","3318, SEQ ID No.","3319, SEQ ID No.","3320, SEQ ID No.","3322, SEQ ID No.","3323, SEQ ID No.","3326, SEQ ID No.","3327, SEQ ID No.","3328, SEQ ID No.","3375, SEQ ID No.","3376, SEQ ID No.","3377, SEQ ID No.","3378, SEQ ID No.","3422, SEQ ID No.","3489, SEQ ID No.","3503, SEQ ID No.","3609, SEQ ID No.","3623, SEQ ID No.","3624, SEQ ID No.","3772, SEQ ID No.","3783, SEQ ID No.","3788 and SEQ ID No.","3794; or b) the polypeptides encoded by the sequences SEQ ID No.","5835 to SEQ ID No.","10784, homologous to the sequences as defined in a), as indicated in the final column of table II.","The subject of the present invention is also the nucleotide and/or polypeptide sequences according to the invention, characterized in that said sequences are recorded on a recording medium, the form and nature of which facilitate the reading, analysis and/or exploitation of said sequence(s).","These media may also contain other information extracted from the present invention, in particular the similarities with already known sequences, and/or information concerning the nucleotide and/or polypeptide sequences of a cell of a plant, of an animal or of a microorganism other than P. luminescens, in particular a cell or microorganism sensitive to a toxin or an antibiotic produced by P. luminescens, a bacterium of the genus Photorhabdus, or a variant of P. luminescens, in order to facilitate the comparative analysis and the exploitation of the results obtained.","Among these said recording media, preference is given in particular to computer-readable media, such as magnetic, optical, electrical or hybrid media, in particular computer disks, CD-ROMs and computer servers.","Such recording media are also a subject of the invention.","The recording media according to the invention, with the information provided, are very useful for choosing nucleotide primers or probes for determining genes in Photorhabdus luminescens TT01 or strains related to this organism.","Similarly, the use of these media for studying the genetic polymorphism of strains related to Photorhabdus luminescens TT01, in particular by determining the regions of colinearity, is very useful insofar as these media provide not only the nucleotide sequence of the Photorhabdus luminescens TT01 genome, but also the genomic organization in said sequence.","Thus, the uses of recording media according to the invention are also subjects of the invention.","The analysis of homology between various sequences is in fact advantageously performed using sequence comparison programs, such as the Blast program, or the programs of the GCG package, described above.","The invention is also directed toward the cloning and/or expression vectors which contain a nucleotide sequence according to the invention.","The vectors according to the invention preferably comprise elements which allow the expression and/or the secretion of the nucleotide sequences in a given host cell.","The vector should then comprise a promoter, translation initiation and termination signals, and also regions suitable for regulating transcription.","It must be possible for it to be maintained stably in the host cell and it may optionally contain particular signals which specify secretion of the translated protein.","These various elements are chosen and optimized by those skilled in the art as a function of the cellular host used.","To this effect, the nucleotide sequences according to the invention can be inserted into vectors which replicate autonomously in the host chosen, or may be vectors which integrate in the host chosen.","Such vectors are prepared by methods commonly used by those skilled in the art, and the resulting clones can be introduced into a suitable host by standard methods, such as lipofection, electroporation, heat shock or chemical methods.","The vectors according to the invention are, for example, vectors of plasmid or viral origin.","They are useful for transforming host cells in order to clone or express the nucleotide sequences according to the invention.","Among these vectors, preference is also given to the cloning and/or expression vectors according to the invention, characterized in that they contain a nucleotide sequence chosen from the sequences SEQ ID No.","3856 to SEQ ID No.","5825, and SEQ ID No.","5835 to SEQ ID No.","10784, or their fragment derived from the P. luminescens genome, in particular the sequences encoding the polypeptides with toxin or antibiotic activity or involved in these activities, in particular those mentioned above whose functions annotated in table I below correspond to these activities.","The invention also comprises the host cells transformed with a vector according to the invention.","The cellular host may be chosen from prokaryotic or eukaryotic systems, for example bacterial cells, but also yeast cells or animal cells, in particular mammalian cells.","Insect cells or plant cells may also be used.","The preferred host cells according to the invention are in particular prokaryotic cells, preferably bacteria belonging to the genus Photorhabdus or to the species Photorhabdus luminescens , more particularly Photorhabdus luminescens TT01.The invention also relates to the plants and animals, except humans, which comprise a transformed cell according to the invention.","The transformed cells according to the invention can be used in methods for preparing recombinant polypeptides according to the invention.","The methods for preparing a polypeptide according to the invention in recombinant form, characterized in that they use a vector and/or a cell transformed with a vector according to the invention, are themselves included in the present invention.","Preferably, a cell transformed with a vector according to the invention is cultured under conditions which allow the expression of said polypeptide, and said recombinant polypeptide is recovered.","As has been mentioned, the cellular host can be chosen from prokaryotic or eukaryotic systems.","In particular, it is possible to identify nucleotide sequences according to the invention which facilitate secretion in such a prokaryotic or eukaryotic system.","A vector according to the invention carrying such a sequence can therefore be advantageously used for producing recombinant proteins intended to be secreted.","As a result, the purification of these recombinant proteins of interest will be facilitated by the fact that they are present in the cell culture supernatant rather than inside the host cells.","The polypeptides according to the invention may also be prepared by chemical synthesis.","Such a method of preparation is also a subject of the invention.","Those skilled in the art are aware of the methods of chemical synthesis, for example the techniques using solid phases (see in particular Steward et al., 1984, Solid phase peptides synthesis, Pierce Chem.","Company, Rockford, 111, 2nd ed., (1984)) or techniques using partial solid phases, by fragment condensation or by conventional synthesis in solution.","The polypeptides obtained by chemical synthesis, and possibly comprising corresponding unnatural amino acids, are also included in the invention.","The hybrid polypeptides according to the invention are very useful for obtaining monoclonal or polyclonal antibodies capable of specifically recognizing the polypeptides according to the invention.","These specific polyclonal or monoclonal antibodies can be obtained by the standard methods well known to those skilled in the art, after immunizing a mammal using these polypeptides (or their corresponding nucleic acid) or, for example, according to the conventional method of hybridoma culture described by Köhler and Milstein (1975, Nature, 256, 495) for the monoclonal antibodies.","Such monoclonal or polyclonal antibodies, their fragments, or the chimeric antibodies, which recognize the polypeptides according to the invention, are also subjects of the invention.","The antibodies according to the invention are, for example, chimeric antibodies, humanized antibodies, or Fab or F(ab′)2 fragments.","They can also be in the form of immunoconjugates or of antibodies which are labeled in order to obtain a detectable and/or quantifiable signal.","Thus, the antibodies according to the invention can be used in a method for detecting and/or identifying bacteria belonging to the genus Photorhabdus and/or to the species Photorhabdus luminescens , in a biological sample, characterized in that it comprises the following steps: a) bringing the biological sample into contact with an antibody according to the invention; b) demonstrating the antigen-antibody complex possibly formed.","The antibodies according to the invention can also be used in order to detect expression of a gene of Photorhabdus luminescens TT01.Specifically, the presence of the expression product of a gene recognized by an antibody specific for said expression product can be detected by the presence of an antigen-antibody complex formed after the Photorhabdus luminescens strain TT01 has been brought into contact with an antibody according to the invention.","The bacterial strain used may have been “prepared”, i.e.","centrifuged, lyzed and/or placed in an appropriate reagent for constituting the medium suitable for the immunoreaction.","In particular, preference is given to a method for detecting expression in the gene, corresponding to Western blotting, which may be performed after polyacrylamide gel electrophoresis of a lysate of the bacterial strain, in the presence or absence of reducing conditions (SDS-PAGE).","After migration and separation of the proteins on the polyacrylamide gel, said proteins are transferred onto a suitable membrane (for example made of nylon) and the presence of the protein or of the polypeptide of interest is detected by bringing said membrane into contact with an antibody according to the invention.","Thus, the present invention also comprises the kits or sets for carrying out a method as described (for detecting the expression of a gene of Photorhabdus luminescens TT01, or for detecting and/or identifying bacteria belonging to the species Photorhabdus luminescens), comprising the following elements: a) a polyclonal or monoclonal antibody according to the invention; b) optionally, the reagents for constituting the medium suitable for the immunoreaction; c) optionally, the reagents for demonstrating the antigen-antibody complexes produced by the immunoreaction.","The polypeptides and the antibodies according to the invention can advantageously be immobilized on a support, in particular a protein chip.","Such a protein chip is the subject of the invention and may also contain at least one polypeptide of a microorganism other than Photorhabdus luminescens , or an antibody directed against a compound of a microorganism other than Photorhabdus luminescens .","The protein chips or high density filters containing proteins according to the invention can be constructed in the same way as the DNA chips according to the invention.","In practice, it is possible to carry out the synthesis of the polypeptides directly attached to the protein chip, or to carry out an ex situ synthesis followed by a step of attaching the synthesized polypeptide to said chip.","The latter method is preferable when the intention is to attach proteins of considerable size to the support, these proteins being advantageously prepared by genetic engineering.","However, if the intention is to attach only peptides to the support of said chip, it may be more advantageous to synthesize said peptides directly in situ.","The protein chips according to the invention can advantageously be used in kits or sets for detecting and/or identifying bacteria related to the species Photorhabdus luminescens , or to a microorganism, or more generally in kits or sets for detecting and/or identifying microorganisms.","When the polypeptides according to the invention are attached to DNA chips, the presence of antibodies in the samples tested is sought, the attachment of an antibody according to the invention to the support of said protein chip allowing identification of the protein for which said antibody is specific.","Preferably, an antibody according to the invention is attached to the support of the protein chip, and the presence of the corresponding antigen, specific for Photorhabdus luminescens , is detected.","A protein chip described above can be used to detect gene products, in order to establish an expression profile for said genes, in addition to a DNA chip according to the invention.","The protein chips according to the invention are also extremely useful for proteomic experiments which study interactions between the various proteins of a cell of a plant, of an animal, such as an insect, or of a microorganism other than P. luminescens.","Thus, the invention also comprises a protein chip according to the invention, characterized in that it also contains at least one polypeptide of a cell of a plant, of an animal or of a microorganism other than P. luminescens, immobilized on the support of said chip, preferably said cell or other microorganism is chosen from a cell or microorganism sensitive to a toxin or an antibiotic produced by P. luminescens.","In a simplified manner, representative peptides of the various proteins of an organism are attached to a support.","Said support is then brought into contact with labeled proteins and, after an optional rinsing step, interactions between said labeled proteins and the peptides attached to the protein chip are detected.","Thus, the protein chips comprising a polypeptide sequence according to the invention or an antibody according to the invention are a subject of the invention, as are the kits or sets containing them.","The present invention also covers a method for detecting and/or identifying bacteria belonging to the species Photorhabdus luminescens , in a biological sample, which uses a nucleotide sequence according to the invention.","It should be understood that, in the present invention, the term “biological sample” concerns samples taken from a living organism (in particular blood, tissues, organs or others taken from a mammal) or a sample containing biological material, i.e.","DNA or RNA.","Such a biological sample also comprises food compositions containing bacteria (for example cheeses, dairy products), but also food compositions containing yeasts (beers, breads) or others.","The term “biological sample” also concerns bacteria isolated from these samples or food compositions.","The method of detection and/or identification using the nucleotide sequences according to the invention may be diverse in nature.","A method comprising the following steps is preferred: a) optionally isolating the DNA from the biological sample to be analyzed, or obtaining a cDNA from the RNA of the biological sample; b) specifically amplifying the DNA of bacteria belonging to the species Photorhabdus luminescens using at least one primer according to the invention; c) demonstrating the amplification products.","This method is based on the specific amplification of the DNA, in particular via a chain amplification reaction.","A method comprising the following steps is also preferred: a) bringing a nucleotide probe according to the invention into contact with a biological sample, the nucleic acid contained in the biological sample having, where appropriate, previously been made accessible to hybridization, under conditions which allow hybridization of the probe to the nucleic acid of a bacterium belonging to the species Photorhabdus luminescens ; b) demonstrating the hybrid possibly formed between the nucleotide probe and the DNA of the biological sample.","Such a method should not be limited to detecting the presence of the DNA contained in the biological sample to be tested, it can also be used to detect the RNA contained in said sample.","This method encompasses in particular Southern and Northern blotting.","Another preferred method according to the invention comprises the following steps: a) bringing a nucleotide probe immobilized on a support according to the invention into contact with a biological sample, the nucleic acid of the sample having, where appropriate, previously been made accessible to hybridization, under conditions which allow hybridization of the probe to the nucleic acid of a bacterium belonging to the species Photorhabdus luminescens ; b) bringing the hybrid formed between the nucleotide probe immobilized on a support and the nucleic acid contained in the biological sample, where appropriate after removing the DNA of the biological sample which has not hybridized with the probe, into contact with a labeled nucleotide probe according to the invention; c) demonstrating the new hybrid formed in step b).","This method is advantageously used with a DNA chip according to the invention, the nucleic acid being sought hybridizing with a probe present at the surface of said chip, and being detected using a labeled probe.","This method is advantageously carried out by combining a prior step of amplifying the DNA or the complementary DNA optionally obtained by reverse transcription, using primers according to the invention.","Thus, the present invention also encompasses the kits or sets for detecting and/or identifying bacteria belonging to the species Photorhabdus luminescens , characterized in that it comprises the following elements: a) a nucleotide probe according to the invention; b) optionally, the reagents required for carrying out a hybridization reaction; c) optionally, at least one primer according to the invention and also the reagents required for a DNA amplification reaction.","Similarly, the present invention also encompasses the kits or sets for detecting and/or identifying bacteria belonging to the species Photorhabdus luminescens TT01, characterized in that it comprises the following elements: a) a nucleotide probe, termed capture probe, according to the invention; b) an oligonucleotide probe, termed detection probe, according to the invention; c) optionally, at least one primer according to the invention and also the reagents required for a DNA amplification reaction.","Finally, the kits or sets for detecting and/or identifying bacteria belonging to the species Photorhabdus luminescens , characterized in that they comprise the following elements: a) at least one primer or one probe according to the invention; b) optionally, the reagents required to carry out a DNA amplification reaction; c) optionally, a component for verifying the sequence of the amplified fragment, more particularly an oligonucleotide probe according to the invention, are also subjects of the present invention.","Preferably, said primers and/or probes and/or polypeptides and/or antibodies according to the present invention, used in the methods and/or kits or sets according to the present invention, are chosen from the primers and/or probes and/or polypeptides and/or antibodies specific for the species Photorhabdus luminescens.","Preferably, these elements are chosen from the nucleotide sequences encoding a secreted protein, from the secreted polypeptides, or from the antibodies directed against secreted polypeptides of Photorhabdus luminescens.","A subject of the present invention is also the strains of Photorhabdus luminescens TT01 containing one or more mutation(s) in a nucleotide sequence according to the invention, in particular an ORF sequence, or regulatory elements thereof (in particular promoters).","According to the invention, preference is given to the strains of Photorhabdus luminescens TT01 exhibiting one or more mutation(s) in the nucleotide sequences encoding polypeptides preferably with activity of the toxin or antibiotic type, or involved in their biosynthesis, or else, in another aspect, involved in the cellular machinery, in particular secretion, central intermediate metabolism, energy metabolism, and processes of amino acid synthesis, of transcription and of translation, and of polypeptide synthesis.","Said mutations may lead to inactivation of the gene or, in particular, when they are located in the regulatory elements of said gene, to overexpression of this gene.","According to the present invention, the strains of Photorhabdus luminescens TT01 exhibiting one or more mutation(s) may be used to validate the function of a wild-type gene of Photorhabdus luminescens .","The invention also relates to the use of a nucleotide sequence according to the invention, of a polypeptide according to the invention, of an antibody according to the invention, and/or of a cell according to the invention, for selecting an organic or inorganic compound capable of modulating, regulating, inducing or inhibiting gene expression in a plant or animal cell or in a microorganism other than P. luminescens, the resistance or sensitivity of which to at least one toxin or antibiotic produced by P. luminescens it is, for example, desired to modify.","The invention also comprises a method for selecting compounds capable of binding to a polypeptide or one of its fragments according to the invention, capable of binding to a nucleotide sequence according to the invention, or capable of recognizing an antibody according to the invention, and/or capable of modulating, regulating, inducing or inhibiting gene expression, and/or of modifying growth or cell replication of eukaryotic or prokaryotic cells, or capable of inducing, inhibiting or increasing, in an animal or plant organism, resistance or sensitivity to at least one toxin or antibiotic produced by P. luminescens, said method comprising the following steps: a) bringing said compound into contact with said polypeptide or said nucleotide sequence and/or with a transformed cell according to the invention; b) determining the ability of said compound to bind to said polypeptide or said nucleotide sequence, or to modulate, regulate, induce or inhibit gene expression, or to modulate growth or cell replication, or to induce, inhibit or increase, in an animal or plant organism, resistance or sensitivity to at least one toxin or antibiotic produced by P. luminescens.","The transformed cells according to the invention may advantageously be used as a model and may be used in methods for studying, identifying and/or selecting compounds liable to be responsible for resistance or sensitivity to at least one toxin or antibiotic produced by P. luminescens.","The compounds which may be selected can be organic compounds such as polypeptides or carbohydrates or any other organic or inorganic compounds which are already known, or new organic compounds developed using molecular modeling techniques and obtained by chemical or biochemical synthesis, these techniques being known to those skilled in the art.","The invention relates to the compounds which can be selected using a method of selection according to the invention.","The invention also relates to a composition, in particular pesticidal or pharmaceutical composition, comprising a compound chosen from the following compounds: a) a nucleotide sequence according to the invention; b) a polypeptide according to the invention; c) a vector according to the invention; d) an antibody according to the invention; and e) a compound which can be selected using a method of selection according to the invention, optionally in combination with a pharmaceutically acceptable vehicle.","The invention also relates to a pharmaceutical composition according to the invention, for preventing or treating an infection with a microorganism, such as a bacterium or a fungus, sensitive to at least one toxin or antibiotic produced by P. luminescens.","The invention also relates to a pesticidal composition, in particular against insects, bacteria and/or fungi, according to the invention, for preventing or treating plants infested with animals, such as insects, or with a microorganism, such as a bacterium or a fungus, sensitive to at least one toxin or antibiotic produced by P. luminescens.","The invention also comprises the use of a transformed cell according to the invention, for preparing a toxin or an antibiotic produced by P. luminescens.","The expression “pharmaceutically acceptable vehicle” is intended to denote a compound or a combination of compounds making up a pharmaceutical composition causing no side effects and which makes it possible, for example, to facilitate administration of the active compound, to increase its lifetime and/or its effectiveness in the organism, to increase its solubility in solution or else to improve its conservation.","These pharmaceutically acceptable vehicles are well known and will be adjusted by those skilled in the art as a function of the nature and of the method of administration of the active compound chosen.","Preferably, these pharmaceutical compounds will be administered systemically, in particular intravenously, intramuscularly, intradermally or subcutaneously, or orally.","Their methods of administration, dosages and pharmaceutical forms which are optimal can be determined according to the criteria generally taken into account in establishing a treatment suitable for a patient, such as, for example, the age or the body weight of the patient, the seriousness of his or her general condition, the tolerance to the treatment and the side effects observed.","Finally, the invention comprises the use of a composition according to the invention, for preparing a medicinal product intended for the prevention or treatment of an infection with a microorganism, such as a bacterium or a fungus, sensitive to at least one toxin or antibiotic produced by P. luminescens.","Moreover, a subject of the present invention is also a genomic DNA library of a bacterium of the genus Photorhabdus, preferably Photorhabdus luminescens , preferably the strain TT01.The genomic DNA libraries described in the present invention, in particular the BAC library deposited with the CNCM [French National Collection of Cultures and Microorganisms] on May 12, 2000, under No.","I-2478 and which covers the Photorhabdus luminescens TT01 genome.","The invention also relates to a method for identifying at least one nucleotide sequence of P. luminescens not present in the genome of another species of bacterium, in particular a pathogenic bacterium, and/or for identifying at least one nucleotide sequence of a genome of a bacterium, in particular a pathogenic bacterium, of a species other than P. luminescens and not present in the P. luminescens genome, characterized in that it comprises the following steps: a) the nucleotide sequences of P. luminescens according to the invention, or contained in a genomic library according to the invention, are aligned with the genomic sequence of the other bacterial species, or one of its fragments; and b) the data obtained with this alignment are processed in order to isolate and identify said sequence(s) only present in one or the other genome.","Such a method, also called “subtractive genomic method”, can be used here to identify a sequence responsible for the pathogenicity of a bacterium, such as a gram-negative bacterium, for which the P. luminescens bacterium, which is not pathogenic, can serve as a model for comparison.","The present invention thus relates to the methods for isolating a polynucleotide of interest present in a strain of Photorhabdus and absent from another strain or species, which use at least one DNA library based, for example, on a plasmid pcDNA2.1 containing the Photorhabdus genome.","The method according to the invention for isolating a polynucleotide of interest can comprise the following steps: a) isolating at least one polynucleotide contained in a clone of the library of DNA of Photorhabdus origin; b) isolating: at least one genomic polynucleotide or cDNA of a bacterium, said bacterium belonging to a strain or species different from the Photorhabdus strain used to construct the DNA library of step a) or, alternatively, at least one polynucleotide contained in a clone of a DNA library prepared from the genome of a bacterium belonging to a strain or species different from the Photorhabdus strain used to construct the DNA library of step a), and hybridizing the polynucleotide of step a) to the polynucleotide of step b); c) selecting the polynucleotides of step a) which have not formed a hybridization complex with the polynucleotides of step b); d) characterizing the selected polynucleotide.","The polynucleotide of step a) can be prepared by digesting at least one recombinant clone with a suitable restriction enzyme and, optionally, amplifying the polynucleotide insert which results therefrom.","Thus, the method of the invention allows those skilled in the art to carry out comparative genomic studies between a bacterium of the genus Photorhabdus and between, for example, a pathogenic strain or species.","In particular, it is possible to study and determine the regions of polymorphism between said strains.","The present invention also relates to the use of the nucleic acid sequences or of the polypeptides according to the invention: for preparing biopesticides, in particular entomotoxins, antibiotics, antifungal agents or cytotoxins, for secreting proteins, as virulence factors, for control via quorum-sensing, for identifying targets for human diseases for which Photorhabdus luminescens is a model (in particular the plague or whooping cough), and for identifying targets against pathogenic Gram-negative bacteria using the subtractive genomic method (such as, for example, by comparison with E. coli or other pathogenic gram-negative bacteria).","The present invention also relates to the use of the polypeptides according to the present invention, for screening compounds capable of modulating the activity of these polypeptides, in particular the polypeptides with enzymatic activity.","Other characteristics and advantages of the invention appear in the following examples: EXAMPLES Example 1 Materials and Methods The strategy for sequencing the genome of Photorhabdus luminescens strain TT01 is based on random (shotgun) sequencing.","The first step of this study consisted in cloning the genomic DNA of the Photorhabdus luminescens bacterium into various vectors (plasmids and BACs).","Photorhabdus luminescens genomic DNA libraries used.","Three genomic DNA libraries were prepared: I)—A genomic DNA library in a high copy number bacterial vector (pcDNA2.1, Invitrogen).","Average insert size 1.5 kb.","II)—A genomic DNA library in a low copy number bacterial vector (pSYX34).","Average insert size 10 kb.","III)—A genomic DNA library in a BAC vector (pBeloBAC11, California Institute of Technology).","Average insert size 50 to 100 kb.","Two batches of DNA of the TT01 strain of the Photorhabdus luminescens bacterium were extracted on Oct. 30, 1998 and Apr.","14, 1999.I) Establishing a Genomic DNA Library in the Bacterial Vector pcDNA2.1 (Invitrogen) A) Preparation of the Vector: pcDNA2.1 We prepared the plasmid pcDNA2.1 by carrying out two midipreps (QIAGEN KIT) in parallel according to the conditions recommended by the manufacturer.","The vector pcDNA2.1 was digested with the BstX1 restriction enzyme.","B) Preparation of the Chromosomal DNA of Photorhabdus luminescens Strain TT01 and Ligation into the Vector pcDNA2.1 1) Dissolving of the DNA A dry pellet of genomic DNA of the strain TT01, prepared on Oct. 30, 1998, was taken up in 200 μl of 10:1 TE, and then dissolved for 30 minutes at 65° C. Its concentration was estimated at 0.15 μg/μl.","2) Nebulization of the DNA 50 μl of genomic DNA strain TT01 in an amount of H2O sufficient for 2 ml were nebulized for 45 sec at a pressure of 1 bar of nitrogen, and centrifuged for 2 min at 600 rpm in order to recover the entire volume.","3) Precipitation of the DNA This DNA was then precipitated with sodium acetate (2 ml of DNA+0.2 ml of 3M Na acetate, pH 5.2,+5 ml of absolute ethanol; 2 h at −20° C.), centrifuged for 30 min at 14 000 rpm and at 4° C., and then redissolved in 100 μl of water.","4) Analysis of the DNA 4 μl were loaded onto a 1% agarose TBE gel.","DNA fragments of the expected size of 500 bp to 3 kb were visualized.","5) Repair of the DNA In order to blunt end the DNA fragments, repair was carried out using T4 DNA polymerase.","The following are mixed in 2 separate tubes: 48 μl of DNA from step 3), 100 μl of H2O, 20 μl of 5× ligation buffer, 2 μl of dNTP mix (10 mM), 5 μl of T4 DNA polymerase (Boehringer).","Incubation is carried out for 25 min at ambient temperature and the reaction is then stopped by heating for 15 min at 75° C. 6) Precipitation of the DNA This DNA was then precipitated overnight at −20° C. with sodium acetate (1/10 volume of Na acetate and 2.5 volume of absolute ethanol) and centrifuged, and the DNA pellet obtained was then air-dried and redissolved in 30 μl of water.","7) Ligation of the DNA This DNA was ligated overnight at 16° C. with the Bstx A+Bstx B linkers (Invitrogen) in the presence of 2 μl of ligase.","8) Preparation of the Inserts After migration of the DNA ligated to the linkers in a 1% agarose TAE gel, at 70 volts, the region of interest (between 1 and 3 Kb) was cut into four fragments.","9) Purification of the Inserts The agarose fragments containing the regions of interest were purified using Geneclean (BIO101) according to the conditions recommended by the manufacturer.","An aliquot was loaded onto an agarose minigel in order to validate the quality of the purification.","10) Ligation of the Inserts into the Vector pcDNA2.1 (Invitrogen) 4 μl of DNA (insert after the Geneclean purification step, step 9), 2 μl of plasmid pcDNA2.1 (after the BstX1 digestion step and then Geneclean purification), 2 μl of ligation buffer (10×), add 2 μl of ligase, 10 μl H2O (total volume: 20 μl).","The mixture is incubated overnight at 16° C. 11) Transformation of Ultracompetent XL2 Blue Cells (Stratagene) The genomic DNA library obtained in step 10 was integrated into ultracompetent XL2 Blue cells (Stratagene) according to the conditions recommended by the manufacturer.","Analysis of the inserts of 24 clones by digestion with the PvuII restriction enzyme and by sequencing of the ends was carried out and gave satisfactory results.","II) Establishing a Genomic DNA Library in the Bacterial Vector pSYX34 The PARTIAL FILL-IN technique, developed in the laboratory, allowed us to construct a library of genomic DNA of the TT01 strain of the bacterium Photorhabdus luminescens , cloned into the plasmid pSYX34 (average insert size 10 Kb).","Note: in this case, digestion of the vector pSYX34 with the Sal I restriction enzyme frees the following ends: 5′TCGAC-G-5′ The PARTIAL FILL-IN was carried out in the presence of dCTP and dTTP deoxynucleotides.","5′TCGAC-CTG-5′ A) Preparation of the Vector: pSYX34 1) Production of the Vector Plasmid pSYX34 was prepared by carrying out two midipreps, QIAGEN KIT, in parallel according to the conditions recommended by the manufacturer.","2) Digestion of the Plasmid pSYX34 with the Sal I Restriction Enzyme The final volume will be 100 μl: 20 μl pSYX34, 10 μl of buffer H, 66 μl of H2O, 4 μl Sal I.","The mixture is incubated for 2 h at 37° C. An aliquot and also a molecular weight marker were loaded onto an agarose minigel in order to validate the quality of the purification.","3) Chloroform Extraction Chloroform extraction makes it possible to stop the enzyme digestion and to remove any traces of protein.","After digestion, 95 μl are recovered, 105 μl of 10 mM TE are added, 200 μl of chloroform are added.","The mixture is vortexed and centrifuged for 1 min at 1 000 rpm.","The aqueous phase (upper phase) is recovered.","4) Precipitation with Sodium Acetate 1/10 volume of sodium acetate (3M, pH: 5.2), i.e.","20 μl, are added, followed by 2.5 volumes of clean absolute ethanol, i.e.","500 μl (bottle kept at −20° C.).","The mixture is left at −20° C. for at least 1 hour (it is possible to leave it at −20° C. overnight).","The mixture is centrifuged at 4° C. (cold room) for 30 min at 14 000 rpm.","The supernatant is removed using the vacuum pump.","400 μl of 70% ethanol are added and the mixture is centrifuged at 4° C. for 5 min at 14 000 rpm.","The supernatant is removed using the vacuum pump and the pellet is left to dry for approximately one hour on the bench.","The pellet is resuspended in 20 μl of 10 mM TE (1/10).","5) Partial Fill-in Final volume of the reaction: 50 μl; 20 μl of pSYX34 digested with Sal I 5 μl of synthesis buffer, 2.5 μl of 1 mM nucleotide (C-T) mix, 20.5 μl of water, 2 μl of Klenow (2 U/μl).","The mixture is left for 30 min at ambient temperature.","Verification is carried out on a 1% agarose minigel, followed by freezing at −20° C. Mixture of C-T Nucleotides: Mix: 2 μl of T nucleotides (100 mM), 2 μl of C nucleotides (100 mM), 16 μl of 10 mM TRIS, pH=7.5.The nucleotides are thus diluted to 1/10, for a concentration of 10 mM.","A second dilution to 1/10 in 10 mM TRIS buffer will be carried out so as to have a concentration of 1 mM.","In addition, during the reaction, the nucleotides are diluted to 1/20 (2.5 μl in 50 μl), thus obtaining a final concentration of nucleotides of 50 μM.","6) Purification of the Vector pSYX34: Preparative Gel Prepare a 1% agarose TAE gel, large wells will be provided for the samples.","Take 30 μl of pGB2 after the partial fill-in (20 μl will therefore remain in the freezer), add 6 μl of loading solution.","In parallel, load the molecular weight marker in the following proportions: 5 μl H2O, 5 μl of the 1 Kb DNA marker, 2 μl of the loading solution.","Allow to migrate at 70 volts.","Cut the gel on the marker side using a scalpel and locate, by taking a photo and using a ruler, the region of interest; in our case, the band corresponding to 4 Kb will be taken.","Each sample will be cut in half.","The weight of each sample will indicate its approximate volume.","There will therefore be four samples to purify by the Geneclean II technique.","7) Purification of the Vector pSYX34 Take up in 3 volumes of sodium iodide solution (1 ml for a sample of 0.2 g), place at 49° C., shaking every 2 minutes, until the agarose has completely dissolved.","Add 8 μl of microbeads: GLASSMILK R, leave at ambient temperature for 5-10 min, the DNA attaches to the microbeads.","Centrifuge for 2 min at 14 000 rpm, remove the supernatant by suction.","Wash 3 times with the New Wash solution (600 μl/eppendorf, vortex, centrifuge for a few seconds at 10 000 rpm); this solution was prepared and is kept at −20° C. Resuspend the pellet in 10 μl of H2O, vortex, leave for 5 min at 49° C. This allows the DNA to detach from the microbeads.","Centrifuge for 2 min at 14 000 rpm.","Recover the supernatants in new eppendorfs (No.","1 and No.","2, etc) Add 10 μl of H2O again to the pellets, so as to be sure to recover everything, vortex, leave for 2 min at 49° C. Centrifuge for 2 min at 14 000 rpm.","Recover the supernatants in the preceding eppendorfs (No.","1 and No.","2, etc).","There are approximately 20 μl of supernatant, centrifuge for 2 min at 14 000 rpm.","Transfer the supernatants into new eppendorfs, so as to be sure that there are no longer any microbeads.","Load 1 μl of each preparation (+5 μl of H2O+2 μL of loading solution) onto a 1% agarose minigel, allow to migrate at 80 volts for 2 hours.","The most concentrated samples will be kept, they will then be frozen at −20° C. B) Preparation of the Chromosomal DNA of Photorhabdus luminescens Strain TT01 1) Dissolving of the DNA A dry pellet of genomic DNA of the strain TT01 was taken up in 200 μl of 10:1 TE and then dissolved for 30 min at 65° C. Its concentration was estimated at 0.15 μg/μl.","2) Partial Digestion of the Genomic DNA with the Sau3A Restriction Enzyme 15 μl of DNA were digested with 2, 1, 0.5, 0.25, 0.125, 0.0625 or 0.03125 units of Sau3A restriction enzyme for 1 h at 37° C. 3) Chloroform Extraction Chloroform extraction makes it possible to stop the enzyme digestion and to remove any traces of protein.","After digestion, 100 μl of chromosomal DNA are recovered.","100 μl of 10 mM TE are added, 200 μl of chloroform are added.","The mixture is vortexed and centrifuged for 1 min at 1 000 rpm.","The aqueous phase, the upper phase, comprising the chromosomal DNA is recovered.","4) Precipitation with Sodium Acetate 1/10 volume of sodium acetate (3M, pH: 5.2), i.e.","20 μl, are added.","2.5 volumes of clean absolute ethanol, i.e.","500 μl, (bottle kept at −20° C.) are then added.","The mixture is left at −20° C. for at least 1 hour (it is possible to leave it at −20° C. overnight).","The mixture is centrifuged at 4° C. (cold room) for 30 min at 14 000 rpm.","The supernatant is removed using the vacuum pump.","400 μl of 70% ethanol are added, the mixture is centrifuged at 4° C. for 5 min at 14 000 rpm.","The supernatant is removed using the vacuum pump and the pellet is left to dry for approximately one hour on the bench.","The pellet is resuspended in 20 μl of H2O.","5) Verification of the Partial Digestions, after Precipitation with Sodium Acetate.","A 1% agarose TBE gel is prepared.","1/10 of the total volume of the precipitations, i.e.","2 μl of DNA+8 μl of H2O+2 μl of loading solution, is loaded.","In parallel, a molecular weight marker is loaded: 1 μl+9 μl of H2O+2 μl of loading solution.","6) Partial Fill-in Final volume of the reaction: approximately 50 μl 36 μl of partially digested DNA (DNA digested with 0.25, 0.125, 0.0625 or 0.03125 units of Sau3A restriction enzyme) 5 μl of synthesis buffer 10 μl of 1 mM nucleotide (A-G) mix=>final concentration: 200 μM 2 μl of Klenow (2 U/μl).","The mixture is left at ambient temperature for 30 min.","Mixture of A-G Nucleotides Mix: 2 μl of A nucleotides (100 mM) 2 μl of G nucleotides (100 mM) 16 μl of 10 mM TRIS, pH=7.5.The nucleotides are thus diluted to 1/10, giving a concentration of 10 mM.","A second dilution to 1/10 in H2O will be carried out, a concentration of 1 mM is therefore obtained.","7) Chloroform Extraction Chloroform extraction makes it possible to stop the enzyme digestion and to remove any traces of protein.","After digestion, 53 μl of chromosomal DNA are recovered.","47 μl of 10 mM TE are added.","100 μl of chloroform are added.","The mixture is vortexed and centrifuged for 1 min at 1 000 rpm.","The aqueous phase, the upper phase, comprising the chromosomal DNA is recovered.","8) Precipitation with Sodium Acetate 1/10 volume of sodium acetate (3M, pH: 5.2), i.e.","10 μl, are added.","2.5 volumes of clean absolute ethanol, i.e.","250 μl, (bottle kept at −20° C.) are then added.","The mixture is left at −20° C. for at least 1 hour (it is possible to leave it at −20° C. overnight).","The mixture is centrifuged at 4° C. (cold room) for 30 min at 14 000 rpm.","The supernatant is removed using the vacuum pump.","400 μl of 70% ethanol are added, the mixture is centrifuged at 4° C. for 5 min at 14 000 rpm.","The supernatant is removed using the vacuum pump and the pellet is left to dry for approximately one hour on the bench.","The pellet is resuspended in 20 μl of H2O.","9) Purification of the Chromosomal DNA: Preparative Gel Prepare a 1% agarose TAE gel, large wells will be provided for the samples.","Take the entire DNA, i.e.","20 μl, add 4 μl of loading solution.","In parallel, load the molecular weight marker in the following proportions: 5 μl H2O 5 μl of the 1 Kb DNA marker 2 μl of the loading solution.","Allow to migrate at 70 volts.","Cut the gel on the marker side using a scalpel and locate, by taking a photo and using a ruler, the region of interest.","Since the DNA fragments are long, SPIN X columns from COSTAR will be used to purify the DNA, this will avoid cleaving the chromosomal DNA.","10) Purification: SPIN X Column After having recovered the various pieces of agarose, in SPIN X columns, add 200 μl of 10 mM TE and then grind using a spatula.","This may be kept at 4° C. Then, centrifuge for 20 min at 5 000 rpm.","The agarose will be retained on the filters, whereas the chromosomal DNA will be found at the bottom of the tube.","11) Chloroform Extraction Chloroform extraction makes it possible to remove any traces of protein.","800 μl of chromosomal DNA are recovered.","800 μl of chloroform are added.","The mixture is vortexed and centrifuged for 1 min at 1 000 rpm.","The aqueous phase, the upper phase, comprising the chromosomal DNA is recovered.","12) Precipitation with Sodium Acetate 1/10 volume of sodium acetate (3M, ph: 5.2), i.e.","80 μl, are added.","800 μl of isopropanol are then added.","The mixture is left at −20° C. for at least 1 hour (it is possible to leave it at −20° C. overnight).","The mixture is centrifuged at 4° C. (cold room) for 30 min at 14 000 rpm.","The supernatant is removed using the vacuum pump.","400 μl of 70% ethanol are added and the mixture is centrifuged at 4° C. for 5 min at 14 000 rpm.","The supernatant is removed using the vacuum pump, and the pellet is allowed to dry for approximately half an hour on the bench and 2 min under vacuum.","The pellet is resuspended in 20 μl of TE (0.1×, i.e.","1 mM).","The suspension is incubated for 10 min at 65° C. The suspension is incubated for approximately 4 hours at 4° C., so that the DNA is well resuspended.","Verification is carried out on a 1% agarose minigel with a molecular weight marker placed in parallel.","C) Ligation of the chromosomal DNA of Photorhabdus luminescens Strain TT01 into the Vector pSYX34 Take 6 μl of chromosomal DNA.","Add 2 μl of pSYX34.Add 2 μl of ligation buffer (LB).","Add 2 μl of ligase.","Then add 8 μl of H2O (total volume: 20 μl) In parallel, prepare a ligation control by replacing the chromosomal DNA with 0.1× TE (1 mM).","Vortex, leave overnight at 16° C. Transformation of Ultracompetent XL10-Gold Kanr Cells (Stratagene) The genomic DNA library obtained in the preceding step was integrated into ultracompetent XL10 Gold Kanr cells (Stratagene) according to the conditions recommended by the manufacturer.","Analysis of the inserts of 24 clones by digesting with the SalI restriction enzyme and by sequencing of the ends was carried out and gave satisfactory results.","III) Establishing a Genomic DNA Library in the Bacterial Vector pBeloBAC11 (California Institute of Technology) Construction of the BAC Library Partial digestion of the DNA in pieces of agarose: the pieces of agarose are washed three times for 15 minutes at ambient temperature in a solution of TE (1×) with moderate agitation; the pieces of agarose are equilibrated twice in 300 μl of a Hind III digestion buffer (1×) (Boehringer or Biolabs) for 30 minutes at ambient temperature; the digestion buffer is removed and an ice-cold Hind III digestion buffer (1 ml per piece of agarose) containing 20 U of Hind III (Boehringer or Biolabs) is added; incubation is carried out for two hours in ice; the pieces of agarose are transferred to a water bath at 37° C. and incubation is carried out for 10 minutes to 30 minutes (depending on the DNA contained in the pieces of agarose); and the digestion is stopped by adding 100 μl of a 250 mM EDTA solution (pH 8).","Size Selection: Separation of the Partially Digested DNA by PFGE Using the CHEF DRIII Apparatus (Bio-Rad): gel of 1% LMP agarose in a tris-acetate-EDTA buffer (1×) at 13° C., reverse the current every 3 to 15 seconds at 6 V/cm for 16 hours, load at least two pieces of agarose and the marker, stain the marker and one lane or a part of the lane to verify the partial digestion, excise the bands (unstained part) of various sizes (i.e.","from 50 to 100 kb and from 150 to 250 kb), the agarose bands can be stored at 4° C. in a TE solution.","Preparation of the Vector: isolation of pBeloBACII by the cesium chloride method; digestion with Hind III; dephosphorylation with CIP (optionally with HK phosphatase).","Ligation and Transformation: the agarose banks containing the DNA are melted at 60° C. for 10 minutes; the molten solution of agarose/DNA is equilibrated for 15 minutes at 45° C.; gelase (Epicentre Technologies) is added in a proportion of 1 U per 100 μl of gel band (do not add digestion buffer, which causes certain problems at the time of ligation); the digestion is carried out for 1 hour at 45° C.; the ligation is carried out in 50 μl of a solution containing pBeloBACII (2 μl), T4 ligase (1 μl at 1:10), a 10× T4 buffer (5 μl ), and the DNA/agarose solution (42 μl), incubation is for 20 hours at 16° C.; the ligation medium is heated for 15 minutes at 65° C.; the ligation medium is dialyzed against a Tris-EDTA buffer, using Millipore membranes of pore size VS 0.025 mM; 1 or 2 μl of the ligation solution are introduced by electroporation into E. coli DH10B cells (Gibco BRL) using electroporation cuvettes with a width of 2 mm, with the following settings: 2.5 kV, 25 μF and 200Ω; after electroporation, the cells are resuspended in 600 μl of SOC or NYZ medium, and then incubation is carried out for 45 minutes at 37° C. with agitation; 10 and 100 μl of each cell suspension are plated out in an LB agar medium containing chloramphenicol (12.5 μg/ml), X-Gal (5-bromo-4-chloro-3-indolyl-α-D-glucuronic acid; 50 μg/ml) and IPTG (isopropyl-α-D-thiogalactopyranoside; 25 μg/ml); and the dishes thus obtained are incubated overnight.","Extraction of the Plasmid DNA and Sequencing of the Inserts The plasmid DNA was extracted by the alkaline lysis technique in 24-well plates.","The inserts of the clones were sequenced using the PE Big Dye kit according to the conditions recommended by the manufacturer, using the oligonucleotides specific for each vector.","The reactions are then introduced into the thermocycler in order to undergo 35 cycles composed of the following three steps: denaturation for 10 seconds at 96° C., hybridization for 10 seconds at 50° C., elongation for 4 minutes at 56° C. A precipitation with 76% ethanol is then carried out for 20 minutes at ambient temperature.","The plates are then centrifuged for 35 minutes at 2 200 g, then drained on absorbent paper, and then centrifuged turned upside down on absorbent paper for 1 minute at 500 g in order to leave no trace of ethanol.","The DNA pellets are then: either taken up in a solution of formamide-EDTA-dextran blue, and then denatured for 2 minutes at 96° C. The plates are then immediately placed in ice, an aliquot then being loaded onto an acrylamide gel of a PE-377 automatic sequencer, or taken up in a 0.3 mM EDTA solution, an aliquot then being automatically loaded into a PE-3700 automatic sequencer.","Assembly of the Sequences Obtained The genomic DNA sequences were assembled in a contig using the PHRED-PHRAP software and visualized using the CONSED software.","Analysis and Annotation The contigs were first of all analyzed and annotated automatically using the GMPTB software.","Example 2 The Genes of Interest The various characteristics associated with the way of life of Photorhabdus luminescens and reported in the literature (Forst et al., 1997; Hu et al., 1998, etc.)","led to the presence of genes involved in the biosynthesis of antibiotics and of toxins being sought in the genome of this bacterium.","The Operons for Biosynthesis of Antibiotics The search for peptide synthetases was undertaken in silico.","To do this, we initially used the various motifs conventionally described in the literature (Stachelhaus & Marahiel, 1995, FEMS Microbiology Letters 125:3-14) as a probe in order to locate, by sequence homology, in the Photorhabdus luminescens genome, genes which may be involved in the biosynthesis of antibiotics.","Adenylation Modules 1 - LKAGGAYYVPID 2 - YSGTTGxPKGV 3 - GELCIGGXGxARGYL 4 - YxTGD 5 - VKIRGxRIELGEIE Thioester Formation Module 6 - DNFYxLGGHSL N-Methylation Module VLE/DxGxGxG Peptide Elongation Module His-HHILxDGW Peptide Racemization Module (Optional) His-HHILxDGW A - AYxTExNDILLTAxG B - EGHGRExIIE C - RTVGWFTSMYPxxLD D - FNYLGQFD Among these various modules, some enabled us to identify, by homology search using the BLASTP—basic local alignment search tool program—(Altschul S F et al., 1990, J. Mol.","Biol., 215:403-10), genes encoding proteins containing some of these motifs.","The genes thus identified, probably encoding proteins involved in the biosynthesis of antibiotics, were cataloged and appear in particular in table I.","Emphasis was placed particularly on the genes homologous to the syr E gene of Pseudomonas syringae or to the nosD gene.","REFERENCES Eric Guenzi, Giuliano Galli, Ingeborg Grgurina, Dennis C. Gross, and Guido Grandi, 1998.Characterization of the Syringomycin Synthetase Gene Cluster.","A link between prokaryotic and eukaryotic peptide synthetases.","J. Biol.","Chem., 273:32857-32863.Bondi M, Messi P, Sabia C, Baccarani Contri M, Manicardi G., 1999.Antimicrobial properties and morphological characteristics of two Photorhabdus luminescens strains.","New Microbiol., 22:117-27.The Operons for Biosynthesis of Toxins As for the genes for biosynthesis of antibiotics, we sought, by sequence homology, in the Photorhabdus luminescens genome, genes potentially encoding toxic proteins.","We identified many genes encoding proteins having these criteria.","Entomotoxins (Tc loci).","Homologs of the Tox A gene of Clostridium difficile.","Hemolysins.","Genes potentially encoding proteins homologous to the RTX toxins of Vibrio cholerae.","Genes potentially encoding proteins homologous to the tphA and icmF genes of Legionella pneumophila (Purcell M, Shuman H A, 1998.The Legionella pneumophila icmGCDJBF genes are required for killing of human macrophages.","Infect Immun., 66:2245-55).","See annotation of these genes in table I below.","REFERENCES Ffrench-Constant R, Bowen D., 1999.Photorhabdus toxins: novel biological insecticides.","Curr Opin Microbiol., 2:284-8.Review.","Blackburn M, Golubeva E, Bowen D, Ffrench-Constant R H, 1998.A novel insecticidal toxin from Photorhabdus luminescens, toxins complex a (Tca), and its histopathological effects on the midgut of manduca sexta.","Appl Environ Microbiol., 64:3036-41.Bowen D J, Ensign J C, 1998.Purification and characterization of a high-molecular-weight insecticidal protein complex produced by the entomopathogenic bacterium Photorhabdus luminescens .","Appl Environ Microbiol., 64:3029-35.Bowen D, Rocheleau T A, Blackburn M, Andreev 0, Golubeva E, Bhartia R, Ffrench-Constant R H, 1998.Insecticidal toxins from the bacterium Photorhabdus luminescens.","Science., 280:2129-32.Guo L, Fatig R O 3rd, Orr G L, Schafer B W, Strickland J A, Sukhapinda K, Woodsworth A T, Petell J K, 1999.Photorhabdus luminescens W-14 insecticidal activity consists of at least two similar but distinct proteins.","Purification and characterization of toxin A and toxin B. J. Biol Chem., 274:9836-42.GENERAL REFERENCES 1.Altschul, S. F., T. L. Madden, A.","A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman.","1997.Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.","[Review] [90 refs].","Nucleic Acids Research.","25:3389-402.2.Birnboim, H. C. 1983.A rapid alkaline extraction method for the isolation of plasmid DNA.","Methods Enzymol.","100:243-255.3.Braun, L. F. Nato, B. Payrastre, J. C. Mazie, and P. Cossart.","1999.The 213-amino-acid leucine-rich repeat region of the Photorhabdus luminescens InIB protein is sufficient for entry into mammalian cells, stimulation of P1 3-kinase and membrane ruffling.","Mol.","Microbiol.","34:10-23.4.Buchrieser, C., C. Rusniok, L. Frangeul, E. Couvé, A. Billault, F. Kunst, E. Carniel, and P. Glaser.","1999.The 102 kb locus of Yersinia pestis: sequence analysis and comparison of selected regions among different Yersinia pestis and Yersinia pseudotuberculosis strains.","Infect.","Immun.","67:4851-4861.5.Ewing, B., and P. Green.","1998.Base-calling of automated sequencer traces using phred.","II.","Error probabilities.","Genome Res.","8:186-194.6.Fitch, W. S. 1970.Distinguishing homologous from analogous proteins.","Syst.","Zool.","19:99-113.7.Frangeul, L., K. E. Nelson, C. Buchrieser, A. Danchin, P. Glaser, and K. F. 1999.Cloning and assembly strategies in microbial genome projects.","Microbiology.","145:2625-2634.8.Gordon, D., C. Abajian, and P. Green.","1998.Consed: a graphical tool for sequence finishing.","Genome Res.","8:195-202.9.Jacquet, C., J. Bille, and J. Rocourt.","1992.Typing of Photorhabdus luminescens by restriction polymorphism of the ribonucleic acid gene region.","Zentralbl Bakteriol.","276:356-365.10.Li, P., K. C. Kupfer, C. J. Davies, D. Burbee, G. A. Evans, and H. R. Garner.","1997.PRIMO: A primer design program that applies base quality statistics for automated large-scale DNA sequencing.","Genomics.","40:476-485.11.Lukashin, A. V., and M. Borodovsky.","1998.GeneMark.hmm: new solutions for gene finding.","Nucleic Acids Res.","15:1107-1115.Scientific Description of the Photorhabdus luminescens Strain TT01 BAC Library Deposited with the CNCM [French National Collection of Cultures and Microorganisms] on May 12, 2000, Under the Number I-2478 Collection of Escherichia coli DH 10B™ clones (Calvin et al., J. Bacteriol.","170, 2796, 1988) containing genomic DNA fragments from the Photorhabdus luminescens strain TT01 bacterium, cloned into the vector pBelo BACII (Kim et al., Genomics, 34, 213, 1996) at the Hind III site.","The average insert size is 60 kb.","Databanks Local revisions of main public banks were used.","The protein bank used consists of the nonredundant fusion of these banks, such as the Genpept bank (automatic translation of GenBank and NCBI).","Use was made of the BLAST software package (public domain, Altschul et al., 1990) for searching for homologies between a sequence and protein or nucleic acid databanks.","The significance thresholds used depend on the length and on the complexity of the region tested and also on the size of the reference bank.","They were adjusted and adapted to each analysis.","The results of the search for homologies between a sequence according to the invention and protein or nucleic acid databanks are given and summarized in table I and table II below.","Table I List of putative functions (column “homology with nonredundant protein database-description”, and column “annotations” for the sequences selected as being involved in an activity or having an activity of the toxin or antibiotic type) of the proteins of sequences SEQ ID No.","42 to SEQ ID No.","3855 encoded by their respective nucleotide sequence of the Photorhabdus luminescens strain TT01 genome (genome represented by the sequences of the 41 contigs (SEQ ID No.","1 to SEQ ID No.","41)).","The nucleic acid sequences of the proteins of sequence SEQ ID No.","42 to SEQ ID No.","3855 can be easily identified by their start position (“start” column) and end position (“end” column) on each one of the contig sequences (“contig” column).","Legend of Table I: All of the putative functions associated with the proteins of sequence SEQ ID No.","42 to SEQ ID No.","3855 were obtained through a homology search using in particular BLASTP software (Altschul et al., 1990).","The significant identities, or the presence of various motifs (cf.","examples) representative of these functions, were in particular taken into account.","The description of the most probable function(s) is given in the “homology with” and “Annotation” column.","Table II List of putative functions (column “function obtained by comparison on databank”) of the proteins encoded by the sequences SEQ ID No.","5835 to SEQ ID No.","10784 of the Photorhabdus luminescens strain TT01 genome (genome here represented by the sequences of the 9 contigs (SEQ ID No.","5826 to SEQ ID No.","5834)).","The nucleic acid sequences of sequence sequences SEQ ID No.","5835 to SEQ ID No.","10784 can be easily identified by their start position (“CONTIG”, “from” column) and end position (“CONTIG” “to” column) on each one of the contig sequences.","Legend of Table II: All of the putative functions associated with the proteins encoded by the sequences SEQ ID No.","5835 to SEQ ID No.","10784 were obtained through a homology search using in particular BLASTP software (Altschul et al., 1990).","The significant identities, or the presence of various motifs (cf.","examples) representative of these functions, were in particular taken into account.","The description of the most probable function(s) is given in the “homology with” and “Annotation” column.","In the final column, “HOMOLOGOUS TO SEQUENCE SEQ ID”, the term “#N/A” means that no homology for the sequence concerned had been identified with one of the sequences SEQ ID Nos.","42 to 3855.When a homologous sequence was found among the sequences SEQ ID Nos.","42 to 3855 for the sequence concerned, this is mentioned via its SEQ ID number in this column.","LENGTHY TABLE REFERENCED HERE US20070020625A1-20070125-T00001 Please refer to the end of the specification for access instructions.","LENGTHY TABLE REFERENCED HERE US20070020625A1-20070125-T00002 Please refer to the end of the specification for access instructions.","LENGTHY TABLE The patent application contains a lengthy table section.","A copy of the table is available in electronic form from the USPTO web site () An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3)."]],"string":"[\n [\n \"Sequence of the photorhabdus luminescens strain tt01 genome and uses\",\n \"The present invention relates to the genomic sequence and to nucleotide sequences encoding polypeptides of Photorhabdus luminescens.\",\n \"The present invention further relates to polypeptides involved in operons involved in the biosynthesis of antibiotics or toxins, as well as polypeptides with activity of the antibiotic or toxin.\",\n \"Uses of the aforementioned polypeptides in pesticides, bactericides, or fungicides is provides.\",\n \"In addition, the present invention provides vectors, cells, or animals containing the sequences of the present invention.\"\n ],\n [\n \"1-56.\",\n \"(canceled) 57.An isolated nucleotide sequence derived from the Photorhabdus luminescens genome, comprising a sequence selected from the group consisting of SEQ ID No.\",\n \"1 to SEQ ID No.\",\n \"41 and SEQ ID No.\",\n \"5826 to SEQ ID No.\",\n \"5834.58.An isolated nucleotide sequence derived from the Photorhabdus luminescens genome, selected from the group consisting of: a) a nucleotide sequence comprising at least 75% identity with a sequence chosen selected from the group consisting of SEQ ID No.\",\n \"1 to SEQ ID No.\",\n \"41 and SEQ ID No.\",\n \"5826 to SEQ ID No.\",\n \"5834; b) a nucleotide sequence comprising a fragment of a sequence selected from the group consisting of SEQ ID No.\",\n \"1 to SEQ ID No.\",\n \"41 and SEQ ID No.\",\n \"5826 to SEQ ID No.\",\n \"5834; c) a nucleotide sequence complementary to a nucleotide sequence as defined in a) or b); d) a nucleotide sequence of the RNA corresponding to one of the sequences as defined in a), b) or c); and e) a nucleotide sequence as defined in a), b), c) or d), which has been modified.\",\n \"59.The nucleotide sequence as claimed in claim 58, wherein the nucleotide sequence is a fragment of a nucleotide sequence selected from the group consisting of SEQ ID No.\",\n \"1 to SEQ ID No.\",\n \"41 and SEQ ID No.\",\n \"5826 to SEQ ID No.\",\n \"5834, and wherein said fragment encodes a polypeptide selected from the group consisting of SEQ ID No.\",\n \"42 to SEQ ID No.\",\n \"3855 or a polypeptide encoded by a sequence selected from the group consisting of SEQ ID No.\",\n \"5835 to SEQ ID No.\",\n \"10784.60.The nucleotide sequence as claimed in claim 59, wherein: a) said nucleotide sequence encodes a polypeptide selected from the group consisting of SEQ ID No.\",\n \"61, SEQ ID No.\",\n \"62, SEQ ID No.\",\n \"67, SEQ ID No.\",\n \"171, SEQ ID No.\",\n \"221, SEQ ID No.\",\n \"268, SEQ ID No.\",\n \"288, SEQ ID No.\",\n \"380, SEQ ID No.\",\n \"426, SEQ ID No.\",\n \"438, SEQ ID No.\",\n \"448, SEQ ID No.\",\n \"453, SEQ ID No.\",\n \"455, SEQ ID No.\",\n \"456, SEQ ID No.\",\n \"458, SEQ ID No.\",\n \"501, SEQ ID No.\",\n \"516, SEQ ID No.\",\n \"530, SEQ ID No.\",\n \"542, SEQ ID No.\",\n \"551, SEQ ID No.\",\n \"720, SEQ ID No.\",\n \"761, SEQ ID No.\",\n \"762, SEQ ID No.\",\n \"814, SEQ ID No.\",\n \"859, SEQ ID No.\",\n \"860, SEQ ID No.\",\n \"861, SEQ ID No.\",\n \"862, SEQ ID No.\",\n \"869, SEQ ID No.\",\n \"1079, SEQ ID No.\",\n \"1168, SEQ ID No.\",\n \"1174, SEQ ID No.\",\n \"1176, SEQ ID No.\",\n \"1413, SEQ ID No.\",\n \"1414, SEQ ID No.\",\n \"1415, SEQ ID No.\",\n \"1416, SEQ ID No.\",\n \"1417, SEQ ID No.\",\n \"1457, SEQ ID No.\",\n \"1651, SEQ ID No.\",\n \"1856, SEQ ID No.\",\n \"1869, SEQ ID No.\",\n \"2021, SEQ ID No.\",\n \"2080, SEQ ID No.\",\n \"2152, SEQ ID No.\",\n \"2162, SEQ ID No.\",\n \"2173, SEQ ID No.\",\n \"2251, SEQ ID No.\",\n \"2295, SEQ ID No.\",\n \"2306, SEQ ID No.\",\n \"2317, SEQ ID No.\",\n \"2328, SEQ ID No.\",\n \"2340, SEQ ID No.\",\n \"2342, SEQ ID No.\",\n \"2351, SEQ ID No.\",\n \"2500, SEQ ID No.\",\n \"3228, SEQ ID No.\",\n \"3230, SEQ ID No.\",\n \"3311, SEQ ID No.\",\n \"3317, SEQ ID No.\",\n \"3318, SEQ ID No.\",\n \"3319, SEQ ID No.\",\n \"3320, SEQ ID No.\",\n \"3322, SEQ ID No.\",\n \"3323, SEQ ID No.\",\n \"3326, SEQ ID No.\",\n \"3327, SEQ ID No.\",\n \"3328, SEQ ID No.\",\n \"3375, SEQ ID No.\",\n \"3376, SEQ ID No.\",\n \"3377, SEQ ID No.\",\n \"3378, SEQ ID No.\",\n \"3422, SEQ ID No.\",\n \"3489, SEQ ID No.\",\n \"3503, SEQ ID No.\",\n \"3609, SEQ ID No.\",\n \"3623, SEQ ID No.\",\n \"3624, SEQ ID No.\",\n \"3772, SEQ ID No.\",\n \"3783, SEQ ID No.\",\n \"3788 and SEQ ID No.\",\n \"3794; or b) said nucleotide sequence is selected from the group consisting of SEQ ID No.\",\n \"5835 to SEQ ID No.\",\n \"10784.61.A nucleotide sequence, comprising a nucleotide sequence selected from the group consisting of: a) a nucleotide sequence as claimed in claim 59; b) a nucleotide sequence comprising at least 75% identity with a nucleotide sequence as claimed in claim 59; c) a complementary or RNA nucleotide sequence corresponding to a sequence as defined in a) or b); d) a nucleotide sequence of a representative fragment of a sequence as defined in a) or c); and e) a sequence as defined in a) or c), which has been modified.\",\n \"62.A polypeptide encoded by a nucleotide sequence as claimed in claim 58.63.A polypeptide as claimed in claim 62, wherein: a) said polypeptide is selected from the group consisting of SEQ ID No.\",\n \"61, SEQ ID No.\",\n \"62, SEQ ID No.\",\n \"67, SEQ ID No.\",\n \"171, SEQ ID No.\",\n \"221, SEQ ID No.\",\n \"268, SEQ ID No.\",\n \"288, SEQ ID No.\",\n \"380, SEQ ID No.\",\n \"426, SEQ ID No.\",\n \"438, SEQ ID No.\",\n \"448, SEQ ID No.\",\n \"453, SEQ ID No.\",\n \"455, SEQ ID No.\",\n \"456, SEQ ID No.\",\n \"458, SEQ ID No.\",\n \"501, SEQ ID No.\",\n \"516, SEQ ID No.\",\n \"530, SEQ ID No.\",\n \"542, SEQ ID No.\",\n \"551, SEQ ID No.\",\n \"720, SEQ ID No.\",\n \"761, SEQ ID No.\",\n \"762, SEQ ID No.\",\n \"814, SEQ ID No.\",\n \"859, SEQ ID No.\",\n \"860, SEQ ID No.\",\n \"861, SEQ ID No.\",\n \"862, SEQ ID No.\",\n \"869, SEQ ID No.\",\n \"1079, SEQ ID No.\",\n \"1168, SEQ ID No.\",\n \"1174, SEQ ID No.\",\n \"1176, SEQ ID No.\",\n \"1413, SEQ ID No.\",\n \"1414, SEQ ID No.\",\n \"1415, SEQ ID No.\",\n \"1416, SEQ ID No.\",\n \"1417, SEQ ID No.\",\n \"1457, SEQ ID No.\",\n \"1651, SEQ ID No.\",\n \"1856, SEQ ID No.\",\n \"1869, SEQ ID No.\",\n \"2021, SEQ ID No.\",\n \"2080, SEQ ID No.\",\n \"2152, SEQ ID No.\",\n \"2162, SEQ ID No.\",\n \"2173, SEQ ID No.\",\n \"2251, SEQ ID No.\",\n \"2295, SEQ ID No.\",\n \"2306, SEQ ID No.\",\n \"2317, SEQ ID No.\",\n \"2328, SEQ ID No.\",\n \"2340, SEQ ID No.\",\n \"2342, SEQ ID No.\",\n \"2351, SEQ ID No.\",\n \"2500, SEQ ID No.\",\n \"3228, SEQ ID No.\",\n \"3230, SEQ ID No.\",\n \"3311, SEQ ID No.\",\n \"3317, SEQ ID No.\",\n \"3318, SEQ ID No.\",\n \"3319, SEQ ID No.\",\n \"3320, SEQ ID No.\",\n \"3322, SEQ ID No.\",\n \"3323, SEQ ID No.\",\n \"3326, SEQ ID No.\",\n \"3327, SEQ ID No.\",\n \"3328, SEQ ID No.\",\n \"3375, SEQ ID No.\",\n \"3376, SEQ ID No.\",\n \"3377, SEQ ID No.\",\n \"3378, SEQ ID No.\",\n \"3422, SEQ ID No.\",\n \"3489, SEQ ID No.\",\n \"3503, SEQ ID No.\",\n \"3609, SEQ ID No.\",\n \"3623, SEQ ID No.\",\n \"3624, SEQ ID No.\",\n \"3772, SEQ ID No.\",\n \"3783, SEQ ID No.\",\n \"3788 and SEQ ID No.\",\n \"3794; or b) said polypeptide is encoded by a nucleotide sequence selected from the group consisting of SEQ ID No.\",\n \"5835 to SEQ ID No.\",\n \"10784.64.A polypeptide, comprising a polypeptide selected from the group consisting of: a) a polypeptide as claimed in claim 62; b) a polypeptide exhibiting at least 80% identity with a polypeptide of sequence selected from the group consisting of SEQ ID No.\",\n \"42 to SEQ ID No.\",\n \"3855 or with a polypeptide encoded by a sequence selected from the group consisting of SEQ ID No.\",\n \"5835 to SEQ ID No.\",\n \"10784; c) a fragment of at least 5 amino acids of a polypeptide of sequence selected from the group consisting of SEQ ID No.\",\n \"42 to SEQ ID No.\",\n \"3855 or with a polypeptide encoded by a sequence selected from the group consisting of SEQ ID No.\",\n \"5835 to SEQ ID No.\",\n \"10784; d) a biologically active fragment of a polypeptide of sequence selected from the group consisting of SEQ ID No.\",\n \"42 to SEQ ID No.\",\n \"3855 or with a polypeptide encoded by a sequence selected from the group consisting of SEQ ID No.\",\n \"5835 to SEQ ID No.\",\n \"10784; and e) a polypeptide of sequence selected from the group consisting of SEQ ID No.\",\n \"42 to SEQ ID No.\",\n \"3855 or with a polypeptide encoded by a sequence selected from the group consisting of SEQ ID No.\",\n \"5835 to SEQ ID No.\",\n \"10784, or as defined in c) or d), which has been modified.\",\n \"65.A nucleotide sequence encoding a polypeptide as claimed in claim 62.66.The nucleotide sequence as claimed in claim 58, wherein said nucleotide sequence encodes a polypeptide of P. luminescens having toxin activity or antibiotic activity, or is involved in the synthesis of toxins or antibiotics.\",\n \"67.The polypeptide as claimed in claim 62, wherein said polypeptide is a polypeptide of P. luminescens having toxin activity or antibiotic activity, or is involved in the synthesis of toxins or antibiotics, or a fragment thereof.\",\n \"68.A recording medium, comprising one or more nucleotide sequences as claimed in claim 57.69.The recording medium as claimed in claim 68, wherein said recording medium is selected from the group consisting of a CD-ROM, a computer disk and a computer server.\",\n \"70.A method of identifying primers or probes for determining genes in strains related to P. luminescens comprising inspecting the sequences recorded on a recording medium as claimed in claim 68 and identifying a primer pair or probe corresponding to a desired nucleotide or to a nucleotide sequence encoding a polypeptide with a desired function.\",\n \"71.A method of studying the genetic polymorphism of strains related to P. luminescens comprising obtaining a nucleotide sequence from a strain related to P. luminescens sequencing the nucleotide sequence comparing the nucleotide sequence to the nucleotide sequences recorded on a recording medium as claimed in claim 68.72.A method for the automatic annotation of genes originating from a genome other than P. luminescens comprising obtaining genomic DNA from a strain other than P. luminescens sequencing the genomic DNA comparing the nucleotide sequence of the genomic DNA to the nucleotide sequences recorded on a recording medium as claimed in claim 68 to assign putative function associated therewith.\",\n \"73.A recording medium, comprising one or more polypeptides sequences as claimed in claim 62.74.The recording medium as claimed in claim 73, wherein said recording medium is selected from the group consisting of a CD-ROM, a computer disk and a computer server.\",\n \"75.A method of identifying primers or probes for determining genes in strains related to P. luminescens comprising inspecting the sequences recorded on a recording medium as claimed in claim 73 and identifying a primer pair or probe corresponding to a desired nucleotide or to a nucleotide sequence encoding a polypeptide with a desired function.\",\n \"76.A method of studying the genetic polymorphism of strains related to P. luminescens comprising obtaining a nucleotide sequence from a strain related to P. luminescens sequencing the nucleotide sequence comparing the nucleotide sequence to the nucleotide sequences encoding the polypeptide sequences recorded on a recording medium as claimed in claim 73.77.A method for the automatic annotation of genes originating from a genome other than P. luminescens comprising obtaining genomic DNA from a strain other than P. luminescens sequencing the genomic DNA comparing the nucleotide sequence of the genomic DNA to the nucleotide sequences encoding the polypeptide sequences recorded on a recording medium as claimed in claim 73 to assign putative function associated therewith 78.A primer or a probe, comprising one or more sequences as claimed in claim 57.79.The nucleotide sequence as claimed in claim 78, wherein said primer or probe is labeled with a radioactive compound or with a nonradioactive compound.\",\n \"80.The nucleotide sequence as claimed in claim 78, wherein said primer or probe is immobilized on a support by a covalent or noncovalent interaction.\",\n \"81.The nucleotide sequence as claimed in claim 80, wherein said support is a high density filter or a DNA chip.\",\n \"82.A DNA chip or a filter, comprising one or more nucleotide sequences as claimed in claim 78.83.The DNA chip or the filter as claimed in claim 82, further comprising one or more nucleotide sequences from a cell from a source selected from the group consisting of a plant, an animal and a microorganism other than P. luminescens, wherein said nucleotide sequences are immobilized on the support of said chip.\",\n \"84.The DNA chip or the filter as claimed in claim 83, wherein said cell is a cell or microorganism sensitive to a toxin or an antibiotic produced by P. luminescens, a bacterium of the genus Photorhabdus, and a variant of P. luminescens.\",\n \"85.A kit for detecting and/or quantifying the expression of at least one gene of P. luminescens, comprising a DNA chip or a filter as claimed in claim 82.86.A cloning and/or expression vector, comprising a nucleotide sequence as claimed in claim 57.87.The cloning and/or expression vector as claimed in claim 86, comprising a nucleotide sequence selected from the group consisting of SEQ ID No.\",\n \"3856 to SEQ ID No.\",\n \"5825 and SEQ ID No.\",\n \"5835 to SEQ ID No.\",\n \"10784, or fragments thereof.\",\n \"88.A host cell, transformed with a vector as claimed in claim 86.89.A plant or an animal, except a human, comprising a transformed cell as claimed in claim 88.90.A method for preparing a polypeptide, comprising culturing a cell transformed with a vector as claimed in claim 88 under conditions which allow the expression of said polypeptide, and recovering the resultant recombinant polypeptide.\",\n \"91.A recombinant polypeptide obtained by the method as claimed in claim 90.92.A method for preparing a polypeptide as claimed in claim 62, comprising chemical synthesizing said polypeptide and isolating said polypeptide.\",\n \"93.An antibody selected from the group consisting of a monoclonal antibody, a polyclonal antibody, and a chimeric antibody, or a fragment thereof, wherein said antibody specifically recognizes a polypeptide as claimed in claim 62.94.The antibody as claimed in claim 93, wherein said antibody is a labeled antibody.\",\n \"95.A method for detecting and/or identifying bacteria belonging to the species P. luminescens, in a biological sample, comprising a) contacting said biological sample with an antibody as claimed in claim 93; b) detecting the formation of an antigen-antibody complex.\",\n \"96.A method for detecting the expression of a gene of P. luminescens, comprising contacting a strain of P. luminescens with an antibody as claimed in claim 93, and detecting the formation of an antigen-antibody complex.\",\n \"97.A kit for performing the method as claimed in claim 95, comprising the following elements: a) said antibody; and at least one of either b) reagents for constituting the medium suitable for an immunoreaction; or c) reagents for detecting the antigen-antibody complexes resulting from the immunoreaction.\",\n \"98.A kit for performing the method as claimed in claim 96, comprising the following elements: a) said antibody; b) reagents for constituting the medium suitable for an immunoreaction; and c) reagents for detecting the antigen-antibody complexes resulting from the immunoreaction.\",\n \"99.The polypeptide as claimed in claim 62, wherein said polypeptide is immobilized on a support 100.The polypeptide as claimed in claim 99, wherein said support a protein chip.\",\n \"101.The polypeptide as claimed in claim 91, wherein said polypeptide is immobilized on a support 102.The polypeptide as claimed in claim 101, wherein said support a protein chip.\",\n \"103.The antibody as claimed in claim 93, wherein said polypeptide is immobilized on a support 104.The antibody as claimed in claim 103, wherein said support a protein chip.\",\n \"105.A protein chip comprising one or more polypeptide as claimed in claim 62 immobilized on the support of said chip.\",\n \"106.The protein chip as claimed in claim 105, further comprising one or more polypeptides from a cell from a source selected from the group consisting of a plant, an animal and a microorganism other than P. luminescens, wherein said polypeptides are immobilized on the support of said chip.\",\n \"107.The protein chip as claimed in claim 106, wherein said cell is a cell or microorganism sensitive to a toxin or an antibiotic produced by P. luminescens, a bacterium of the genus Photorhabdus, and a variant of P. luminescens.\",\n \"108.A kit for detecting and/or quantifying the expression of at least one gene of P. luminescens, comprising a protein chip as claimed in claim 105.109.A protein chip comprising one or more polypeptide as claimed in claim 91 immobilized on the support of said chip.\",\n \"110.The protein chip as claimed in claim 109, further comprising one or more polypeptides from a cell from a source selected from the group consisting of a plant, an animal and a microorganism other than P. luminescens, wherein said polypeptides are immobilized on the support of said chip.\",\n \"111.The protein chip as claimed in claim 110, wherein said cell is a cell or microorganism sensitive to a toxin or an antibiotic produced by P. luminescens, a bacterium of the genus Photorhabdus, and a variant of P. luminescens.\",\n \"112.A kit for detecting and/or quantifying the expression of at least one gene of P. luminescens, comprising a protein chip as claimed in claim 109.113.A protein chip comprising one or more antibodies as claimed in claim 93 immobilized on the support of said chip.\",\n \"114.The protein chip as claimed in claim 113, further comprising one or more polypeptides from a cell from a source selected from the group consisting of a plant, an animal and a microorganism other than P. luminescens, wherein said polypeptides are immobilized on the support of said chip.\",\n \"115.The protein chip as claimed in claim 114, wherein said cell is a cell or microorganism sensitive to a toxin or an antibiotic produced by P. luminescens, a bacterium of the genus Photorhabdus, and a variant of P. luminescens.\",\n \"116.A kit for detecting and/or quantifying the expression of at least one gene of P. luminescens, comprising a protein chip as claimed in claim 113.117.A method for detecting and/or identifying bacteria belonging to the species P. luminescens, in a biological sample, comprising: a) isolating the DNA, or cDNA from the RNA, from the biological sample; b) specifically amplifying the DNA of bacteria belonging to the species P. luminescens using at least one primer as claimed in claim 78; c) identifying the amplification products.\",\n \"118.A kit or set for detecting and/or identifying bacteria belonging to the species P. luminescens, comprising: a) a nucleotide probe and/or primer as claimed in claim 78; and at least one of b) reagents required for carrying out a hybridization reaction; or c) reagents for a DNA amplification reaction.\",\n \"119.A composition comprising one or more nucleotide sequences as claimed in claim 57.120.A pharmaceutical composition comprising the composition as claimed in claim 119 and a pharmaceutically acceptable vehicle.\",\n \"121.A biopesticidal composition comprising the composition as claimed in claim 119.122.A composition comprising one or more polypeptides as claimed in claim 62.123.A pharmaceutical composition comprising the composition as claimed in claim 122 and a pharmaceutically acceptable vehicle.\",\n \"124.A biopesticidal composition comprising the composition as claimed in claim 122.125.A composition comprising a vector as claimed in claim 86.126.A pharmaceutical composition comprising the composition as claimed in claim 125 and a pharmaceutically acceptable vehicle.\",\n \"127.A biopesticidal composition comprising the composition as claimed in claim 125.128.A composition comprising one or more antibodies as claimed in claim 93.129.A pharmaceutical composition comprising the composition as claimed in claim 128 and a pharmaceutically acceptable vehicle.\",\n \"130.A biopesticidal composition comprising the composition as claimed in claim 128.131.A method of preparing a toxin or an antibiotic comprising culturing a cell as claimed in claim 88, and expressing a polypeptide involved in production of a toxin or an antibiotic.\",\n \"132.A genomic library of a bacterium of the genus Photorhabdus.\",\n \"133.The genomic DNA library of a bacterium of the genus Photorhabdus as claimed in claim 132, wherein said DNA library is cloned into a plasmid.\",\n \"134.The genomic DNA library as claimed in claim 132, wherein said bacterium is P. luminescens or P. luminescens strain TT01.135.The genomic DNA library as claimed in claim 132, wherein said genomic DNA library is the genomic DNA library deposited with the CNCM on May 12, 2000, under accession No.\",\n \"I-2478.136.A method for identifying at least one nucleotide sequence of P. luminescens not present in the genome of another species of bacterium or for identifying at least one nucleotide sequence of a genome of a bacterium of a species other than P. luminescens and not present in the P. luminescens genome, comprising: a) aligning the genomic sequences of the other bacterial species with the nucleotide sequences of P. luminescens as claimed in claim 57; and b) analyzing the data obtained by said aligning to identify and isolate said sequences only present in one or the other genome.\",\n \"137.A method for identifying at least one nucleotide sequence of P. luminescens not present in the genome of another species of bacterium or for identifying at least one nucleotide sequence of a genome of a bacterium of a species other than P. luminescens and not present in the P. luminescens genome, comprising: a) aligning the genomic sequences of the other bacterial species with the genomic DNA library as claimed in claim 134; and b) analyzing the data obtained by said aligning to identify and isolate said sequences only present in one or the other genome.\"\n ],\n [\n \"The invention relates to the genomic sequence and to nucleotide sequences encoding polypeptides of Photorhabdus luminescens, such as polypeptides involved in operons for biosynthesis of antibiotics, or of toxins, or polypeptides with activity of the toxin or antibiotic type which can be used as a pesticide, bactericide or fungicide, and also to vectors which include said sequences, and to cells or animals transformed with these vectors.\",\n \"Photorhabdus luminescens is an entomopathogenic, commensal intestinal bacterium of a nematode and insect parasite.\",\n \"This bacterium is both a model for studying host-parasite interactions and a bacterium which has many industrial applications because of its ability to synthesize numerous toxins (insecticides, bactericides and fungicides) and to secrete numerous enzymes.\",\n \"In order to obtain an overall understanding of the genetic determinants involved in these processes, sequencing of the Photorhabdus luminescens genome was carried out.\",\n \"The choice of the TT01 strain of Photorhabdus luminescens, subspecies laumondii, the sequencing of whose genome was carried out in the present invention, is very important since this strain has several advantages: its genome is stable; it can be cultured on a Petri dish; it is central in the phylogenetic tree, and therefore representative of the species; and its associated nematode is known and cultured (Heterorhabditis bacteriophora HP88, Trinidad).\",\n \"The present invention thus relates to the nucleotide and polypeptide sequences of Photorhabdus luminescens strain TT01.Thus, an object of the present invention is to disclose the sequence of the genome of Photorhabdus luminescens strain TT01, contained in the genomic library prepared from the genome of this strain and deposited with the CNCM [French National Collection of Cultures and Micro-organisms] on May 12, 2000, under the number I-2478, and of all the genes and noncoding regulatory sequences contained in said genome.\",\n \"Photorhabdus luminescens strain TT01 is also identified in the present application by Photorhabdus luminescens , in an interchangeable manner.\",\n \"The invention also relates to novel tools for typing Photorhabdus strains.\",\n \"These tools might be of the DNA “chip” type or of another type.\",\n \"The novel characteristics of these typing tools will be as follows: rapidity and simplicity of use; high capacity for discriminating between strains; and possibility of providing information on the genomic content of the strain analyzed.\",\n \"The present invention therefore relates to an isolated nucleotide sequence derived from the Photorhabdus luminescens genome, characterized in that it comprises a sequence chosen from the sequences SEQ ID No.\",\n \"1 to SEQ ID No.\",\n \"41 and the sequences SEQ ID No.\",\n \"5826 to SEQ ID No.\",\n \"5834.The sequences SEQ ID No.\",\n \"1 to SEQ ID No.\",\n \"41 represent the sequences of 41 contigs which altogether cover the genomic sequence of Photorhabdus luminescens TT01.It has been possible to reassemble these sequences SEQ ID No.\",\n \"1 to SEQ ID No.\",\n \"41 and to smooth them back out into 9 new contigs which altogether also cover the genomic sequence of Photorhabdus luminescens TT01.The sequences SEQ ID No.\",\n \"5826 to SEQ ID No.\",\n \"5834 represent the sequences of these 9 contigs.\",\n \"The nucleotide sequences SEQ ID No.\",\n \"1 to SEQ ID No.\",\n \"41 and SEQ ID No.\",\n \"5826 to SEQ ID No.\",\n \"5834 were obtained by sequencing the Photorhabdus luminescens TT01 genome using the “shotgun” technique (cf.\",\n \"examples).\",\n \"Despite the great precision of these sequences SEQ ID No.\",\n \"1 to SEQ ID No.\",\n \"41 or SEQ ID No.\",\n \"5826 to SEQ ID No.\",\n \"5834, it is possible that these sequences do not give a 100% perfect representation, after assembly, of the nucleotide sequence of the Photorhabdus luminescens TT01 genome, and that some rare sequencing errors or indeterminations remain in these sequences.\",\n \"In the present invention, the presence of an indetermination of an amino acid is denoted by “Xaa” and that of a nucleotide is denoted by “N” or “n” in the sequence listing hereinafter.\",\n \"These few rare errors or indeterminations may be easily demonstrated and corrected by those skilled in the art using the whole chromosome and/or its representative fragments according to the invention, and standard methods of amplification, cloning and sequencing, it being possible for the sequences obtained to be easily compared, in particular by means of computer software, and using computer-readable media for recording the sequences according to the invention, as described, for example, below.\",\n \"After correction of these possible rare errors or indeterminations, the corrected nucleotide sequence obtained would still comprise at least 97%, preferably at least 98%, 98.5%, 99% or 99.9%, identity with the genomic sequence obtained after assembly of these nucleotide sequences SEQ ID No.\",\n \"1 to SEQ ID No.\",\n \"41 or SEQ ID No.\",\n \"5826 to SEQ ID No.\",\n \"5834.The present invention also relates to an isolated nucleotide sequence derived from the Photorhabdus luminescens genome, characterized in that it is chosen from: a) a nucleotide sequence comprising at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with a sequence chosen from the sequences SEQ ID No.\",\n \"1 to SEQ ID No.\",\n \"41 or SEQ ID No.\",\n \"5826 to SEQ ID No.\",\n \"5834; b) a nucleotide sequence comprising a representative fragment of a sequence chosen from the sequences SEQ ID No.\",\n \"1 to SEQ ID No.\",\n \"41 or SEQ ID No.\",\n \"5826 to SEQ ID No.\",\n \"5834; c) a nucleotide sequence complementary to a nucleotide sequence as defined in a) or b); d) a nucleotide sequence of the RNA corresponding to one of the sequences as defined in a), b) or c); e) a nucleotide sequence as defined in a), b), c) or d), which has been modified; f) a nucleotide sequence which hybridizes, under high stringency conditions, with a sequence chosen from SEQ ID No.\",\n \"1 to SEQ ID No.\",\n \"41 or SEQ ID No.\",\n \"5826 to SEQ ID No.\",\n \"5834, and which comprises at least 20 nucleotides, preferably at least 25, 30, 50, 75, 100, 150, 200, 250, 500, 750, 1 000, 1 500, 2 000 or 2 500 nucleotides.\",\n \"More particularly, a subject of the present invention is also a nucleotide sequence included in one of the sequences SEQ ID No.\",\n \"1 to SEQ ID No.\",\n \"41 or one of the sequences SEQ ID No.\",\n \"5826 to SEQ ID No.\",\n \"5834, and in that it encodes a polypeptide chosen from the polypeptides of sequence SEQ ID No.\",\n \"42 to SEQ ID No.\",\n \"3855 or from the polypeptides encoded by a sequence SEQ ID No.\",\n \"5835 to SEQ ID No.\",\n \"10784.Preferably, the polypeptides encoded by one of the sequences SEQ ID No.\",\n \"5835 to SEQ ID No.\",\n \"10784 are the polypeptides for which the sequence of at least 5 amino acids is obtained by taking as reading frame the first nucleotide of the sequences SEQ ID No.\",\n \"5835 to SEQ ID No.\",\n \"10784.Very preferably, a subject of the invention is also a nucleotide sequence, characterized in that it encodes a polypeptide whose function annotated in table I hereinafter, final column, or in table II hereinafter, penultimate column, corresponds to an activity of the toxin and/or antibiotic type, or to an operon involved in the synthesis of a toxin and/or of an antibiotic, which polypeptide is preferably chosen from: a) the polypeptides of sequences SEQ ID No.\",\n \"61, SEQ ID No.\",\n \"62, SEQ ID No.\",\n \"67, SEQ ID No.\",\n \"171, SEQ ID No.\",\n \"221, SEQ ID No.\",\n \"268, SEQ ID No.\",\n \"288, SEQ ID No.\",\n \"380, SEQ ID No.\",\n \"426, SEQ ID No.\",\n \"438, SEQ ID No.\",\n \"448, SEQ ID No.\",\n \"453, SEQ ID No.\",\n \"455, SEQ ID No.\",\n \"456, SEQ ID No.\",\n \"458, SEQ ID No.\",\n \"501, SEQ ID No.\",\n \"516, SEQ ID No.\",\n \"530, SEQ ID No.\",\n \"542, SEQ ID No.\",\n \"551, SEQ ID No.\",\n \"720, SEQ ID No.\",\n \"761, SEQ ID No.\",\n \"762, SEQ ID No.\",\n \"814, SEQ ID No.\",\n \"859, SEQ ID No.\",\n \"860, SEQ ID No.\",\n \"861, SEQ ID No.\",\n \"862, SEQ ID No.\",\n \"869, SEQ ID No.\",\n \"1079, SEQ ID No.\",\n \"1168, SEQ ID No.\",\n \"1174, SEQ ID No.\",\n \"1176, SEQ ID No.\",\n \"1413, SEQ ID No.\",\n \"1414, SEQ ID No.\",\n \"1415, SEQ ID No.\",\n \"1416, SEQ ID No.\",\n \"1417, SEQ ID No.\",\n \"1457, SEQ ID No.\",\n \"1651, SEQ ID No.\",\n \"1856, SEQ ID No.\",\n \"1869, SEQ ID No.\",\n \"2021, SEQ ID No.\",\n \"2080, SEQ ID No.\",\n \"2152, SEQ ID No.\",\n \"2162, SEQ ID No.\",\n \"2173, SEQ ID No.\",\n \"2251, SEQ ID No.\",\n \"2295, SEQ ID No.\",\n \"2306, SEQ ID No.\",\n \"2317, SEQ ID No.\",\n \"2328, SEQ ID No.\",\n \"2340, SEQ ID No.\",\n \"2342, SEQ ID No.\",\n \"2351, SEQ ID No.\",\n \"2500, SEQ ID No.\",\n \"3228, SEQ ID No.\",\n \"3230, SEQ ID No.\",\n \"3311, SEQ ID No.\",\n \"3317, SEQ ID No.\",\n \"3318, SEQ ID No.\",\n \"3319, SEQ ID No.\",\n \"3320, SEQ ID No.\",\n \"3322, SEQ ID No.\",\n \"3323, SEQ ID No.\",\n \"3326, SEQ ID No.\",\n \"3327, SEQ ID No.\",\n \"3328, SEQ ID No.\",\n \"3375, SEQ ID No.\",\n \"3376, SEQ ID No.\",\n \"3377, SEQ ID No.\",\n \"3378, SEQ ID No.\",\n \"3422, SEQ ID No.\",\n \"3489, SEQ ID No.\",\n \"3503, SEQ ID No.\",\n \"3609, SEQ ID No.\",\n \"3623, SEQ ID No.\",\n \"3624, SEQ ID No.\",\n \"3772, SEQ ID No.\",\n \"3783, SEQ ID No.\",\n \"3788 and SEQ ID No.\",\n \"3794; or b) the polypeptides encoded by the sequences SEQ ID No.\",\n \"5835 to SEQ ID No.\",\n \"10784 homologous to the sequences as defined in a) above, as indicated in the final column of table II.\",\n \"These 82 polypeptides of sequence as defined in paragraph a) above, whose function is associated with an activity of the toxin or antibiotic type, or their homologous polypeptide of table II as defined in paragraph b) above, could be identified, for example, by the presence of a consensus motif associated with these functions or by the presence of sequences juxtaposing them on the genome and involved in this type of activity.\",\n \"More generally, the present invention also relates to the nucleotide sequences derived from SEQ ID No.\",\n \"1 to SEQ ID No.\",\n \"41 or SEQ ID No.\",\n \"5826 to SEQ ID No.\",\n \"5834, and encoding a polypeptide of P. luminescens, such that they can be isolated from SEQ ID No.\",\n \"1 to SEQ ID No.\",\n \"41 or SEQ ID No.\",\n \"5826 to SEQ ID No.\",\n \"5834.In addition, the nucleotide sequences characterized in that they comprise a nucleotide sequence chosen from: a) a nucleotide sequence encoding a polypeptide chosen from the sequences SEQ ID No.\",\n \"42 to SEQ ID No.\",\n \"3855 or from the polypeptides encoded by the sequences SEQ ID No.\",\n \"5835 to SEQ ID No.\",\n \"10784, preferably from the 82 polypeptide sequences above selected for their function associated with an activity of the toxin or antibiotic type, or their homologous peptide as defined in table II, in the final column; b) a nucleotide sequence comprising at least 75% identity with a nucleotide sequence as defined in a), preferably at least 80%, 85%, 90%, 95%, 98% or 99% identity; c) a complementary or RNA nucleotide sequence corresponding to a sequence as defined in a) or b); d) a nucleotide sequence of a representative fragment of a sequence as defined in a) or c); and e) a sequence as defined in a) or c), which has been modified are also subjects of the invention.\",\n \"The terms “nucleic acid”, “nucleic acid sequence”, “polynucleotide”, “oligonucleotide”, “polynucleotide sequence” and “nucleotide sequence”, terms which will be used indifferently in the present description, are intended to denote a precise chain of nucleotides, which may or may not be modified, making it possible to define a fragment or a region of a nucleic acid, which may or may not comprise unnatural nucleotides, and which may correspond equally to a double-stranded DNA, a single-stranded DNA and products of transcription of said DNAs.\",\n \"Thus, the nucleic acid sequences according to the invention also encompass PNAs (Peptide Nucleic Acids).\",\n \"It should be understood that the present invention does not concern the nucleotide sequences in their natural chromosomal environment, i.e.\",\n \"in their natural state.\",\n \"They are sequences which have been isolated and/or purified, i.e.\",\n \"they have been taken directly or indirectly, for example by copying, their environment having been at least partially modified.\",\n \"The nucleic acids obtained by chemical synthesis are thus also intended to be denoted.\",\n \"For the purpose of the present invention, the term “percentage identity” between two nucleic acid or amino acid sequences is intended to denote a percentage of nucleotides or of amino acid residues which are identical between the two sequences to be compared, obtained after best alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly and over their entire length.\",\n \"The term “best alignment” or “optimal alignment” is intended to denote the alignment for which the percentage identity determined as below is the highest.\",\n \"Sequence comparisons between two nucleic acid or amino acid sequences are conventionally carried out by comparing these sequences after having optimally aligned them, said comparison being carried out by segment or by “window of comparison” so as to identify and compare the local regions of sequence similarity.\",\n \"The optimal alignment of the sequences for the comparison can be carried out, besides manually, by means of the local homology algorithm of Smith and Waterman (1981, Ad.\",\n \"App.\",\n \"Math., 2:482), by means of the local homology algorithm of Neddleman and Wunsch (1970, J. Mol.\",\n \"Biol., 48:443), by means of the similarity search method of Pearson and Lipman (1988, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA, 85:2444), by means of computer programs using these algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.).\",\n \"In order to obtain the optimal alignment, the BLAST program is preferably used, with the BLOSUM 62 matrix.\",\n \"The PAM or PAM250 matrices can also be used.\",\n \"The percentage identity between two nucleic acid or amino acid sequences is determined by comparing these two sequences when optimally aligned, the nucleic acid or amino acid sequence to be compared possibly comprising additions or deletions with respect to the reference sequence, for optimal alignment between these two sequences.\",\n \"The percentage identity is calculated by determining the number of identical positions for which the nucleotide or amino acid residue is identical in the two sequences, dividing this number of identical positions by the total number of positions compared, and multiplying the result obtained by 100 so as to obtain the percentage identity between these two sequences.\",\n \"The expression “nucleic acid sequences exhibiting a percentage identity of at least 75%, preferably 80%, 85%, 90%, 95%, 98% or 99%, after optimal alignment, with a reference sequence” is intended to denote the nucleic acid sequences exhibiting, compared to the reference nucleic acid sequence, certain modifications such as in particular a deletion, a truncation, an extension, a chimeric fusion and/or a substitution, in particular of the point type, and the nucleic acid sequence of which exhibits at least 75%, preferably 80%, 85%, 90%, 95%, 98% or 99%, identity, after optimal alignment, with the reference nucleic acid sequence.\",\n \"They are preferably sequences for which the complementary sequences are capable of hybridizing specifically with the reference sequences.\",\n \"Preferably, the specific or high stringency hybridization conditions will be such that they provide at least 75%, preferably 80%, 85%, 90%, 95%, 98% or 99%, identity, after optimal alignment, between one of the two sequences and the sequence complementary thereto.\",\n \"A hybridization under high stringency conditions means that the conditions of temperature and of ionic strength are chosen such that they make it possible to maintain the hybridization between two complementary DNA fragments.\",\n \"By way of illustration, high stringency conditions for the hybridization step for the purposes of defining the polynucleotide fragments described above are advantageously as follows.\",\n \"The DNA-DNA or DNA-RNA hybridization is carried out in two steps: (1) prehybridization at 42° C. for 3 hours in phosphate buffer (20 mM, pH 7.5) containing 5×SSC (1×SSC corresponds to a solution of 0.15M NaCl+0.015M sodium citrate), 50% of formamide, 7% of sodium dodecyl sulfate (SDS), 10× Denhardt's, 5% of dextran sulfate and 1% of salmon sperm DNA; (2) hybridization per se for 20 hours at a temperature which depends on the length of the probe (i.e.\",\n \": 42° C. for a probe>100 nucleotides in length) followed by 2 washes of 20 minutes at 20° C. in 2×SSC+2% SDS and 1 wash of 20 minutes at 20° C. in 0.1×SSC+0.1% SDS.\",\n \"The final wash is carried out in 0.1×SSC+0.1% SDS for 30 minutes at 60° C. for a probe>100 nucleotides in length.\",\n \"The high stringency hybridization conditions described above for a polynucleotide of defined length can be adjusted by those skilled in the art for longer or shorter oligonucleotides, according to the teaching of Sambrook et al.\",\n \"(1989, Molecular cloning: a laboratory manual.\",\n \"2nd Ed.\",\n \"Cold Spring Harbor).\",\n \"In addition, the expression “representative fragment of sequences according to the invention” is intended to denote any nucleotide fragment having at least 15 consecutive nucleotides, preferably at least 20, 25, 30, 50, 75, 100, 150, 200, 300 and 450 consecutive nucleotides, of the sequence from which it is derived.\",\n \"The term “representative fragment” is intended to mean in particular a nucleic acid sequence encoding a biologically active fragment of a polypeptide, as defined later.\",\n \"The term “representative fragment” is also intended to mean the intergenic sequences, and in particular the nucleotide sequences carrying the regulatory signals (promoters, terminators, or even enhancers, etc).\",\n \"Among said representative fragments, preference is given to those having nucleotide sequences corresponding to open reading frames, referred to as ORF sequences, included in general between an initiation codon and a stop codon, or between two stop codons, and encoding polypeptides, preferably with at least 100 amino acids, such as, for example, without being limited thereto, the ORF sequences which will subsequently be described.\",\n \"The numbering of the ORF nucleotide sequences which will subsequently be used in the present description corresponds to the numbering of the amino acid sequences of the proteins encoded by said ORFs for the sequences SEQ ID No.\",\n \"42 to SEQ ID No.\",\n \"3855.The numbering of the ORF nucleotide sequences SEQ ID No.\",\n \"5835 to SEQ ID No.\",\n \"10784 will subsequently be used in the present description for the numbering of the amino acid sequences of the proteins encoded by said ORFs SEQ ID No.\",\n \"5835 to SEQ ID No.\",\n \"10784.The representative fragments according to the invention can be obtained, for example, by specific amplification such as PCR or after digestion, with suitable restriction enzymes, of nucleotide sequences according to the invention, this method being described in particular in the work by Sambrook et al.\",\n \"Said representative fragments can also be obtained by chemical synthesis when they are not too long, according to methods well known to those skilled in the art.\",\n \"The sequences containing sequences of the invention, or representative fragments, are also intended to include the sequences which are naturally framed by sequences which exhibit at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with the sequences according to the invention.\",\n \"The term “modified nucleotide sequence” is intended to mean any nucleotide sequence obtained by mutagenesis according to techniques well known to those skilled in the art, and comprising modifications compared to the normal sequences, preferably at most 10% of modified nucleotides compared to these normal sequences, for example mutations in the regulatory and/or promoter sequences for expression of the polypeptide, in particular leading to a modification of the level of expression or of the activity of said polypeptide.\",\n \"The term “modified nucleotide sequence” is also intended to mean any nucleotide sequence encoding a modified polypeptide as defined below.\",\n \"The representative fragments according to the invention can also be probes or primers, which can be used in methods for detecting, identifying, assaying or amplifying nucleic acid sequences.\",\n \"For the purpose of the invention, a probe or primer is defined as being a single-stranded nucleic acid fragment or a denatured double-stranded fragment comprising, for example, from 12 bases to a few kb, in particular from 15 to a few hundred bases, preferably from 15 to 50 or 100 bases, and having a specificity of hybridization under given conditions so as to form a hybridization complex with a target nucleic acid.\",\n \"The probes and primers according to the invention can be labeled directly or indirectly with a radioactive or nonradioactive compound using methods well known to those skilled in the art, in order to obtain a detectable and/or quantifiable signal (patent FR 78 10975 and bDNA of Chiron EP 225 807 and EP 510 085).\",\n \"The unlabeled sequences of polynucleotides according to the invention can be used directly as a probe or primer.\",\n \"The sequences are generally labeled to obtain sequences which can be used for many applications.\",\n \"The labeling of the primers or of the probes according to the invention is carried out with radioactive elements or with nonradioactive molecules.\",\n \"Among the radioactive isotopes used, mention may be made of 32P, 33P 35S, 3H or 125I.\",\n \"The nonradioactive entities are selected from ligands such as biotin, avidin, streptavidin or digoxigenin, haptens, dyes, and luminescent agents such as radioluminescent, chemiluminescent, bioluminescent, fluorescent or phosphorescent agents.\",\n \"The polynucleotides according to the invention can thus be used as a primer and/or a probe in methods using in particular the PCR (polymerase chain reaction) technique (Rolfs et al., 1991, Berlin: Springer-Verlag).\",\n \"This technique requires choosing pairs of oligonucleotide primers framing the fragment which must be amplified.\",\n \"Reference may, for example, be made to the technique described in U.S. Pat.\",\n \"No.\",\n \"4,683,202.The amplified fragments can be identified, for example after agarose or polyacrylamide gel electrophoresis, or after a chromatography technique such as gel filtration or ion exchange chromatography, and then sequenced.\",\n \"The specificity of the amplification can be controlled using the nucleotide sequences of polynucleotides of the invention as a matrix, plasmids containing these sequences or else the derived amplification products.\",\n \"The amplified nucleotide fragments can be used as reagents in hybridization reactions in order to demonstrate the presence, in a biological sample, of a target nucleic acid of sequence complementary to that of said amplified nucleotide fragments.\",\n \"The invention is also directed toward the nucleic acids which can be obtained by amplification using primers according to the invention.\",\n \"Other techniques for amplifying the target nucleic acid can advantageously be employed as an alternative to PCR (PCR-like), using a pair of primers of nucleotide sequences according to the invention.\",\n \"The term “PCR-like” is intended to denote all the methods using direct or indirect reproductions of nucleic acid sequences, or else in which the labeling systems have been amplified; these techniques are, of course, known.\",\n \"In general, they involve amplification of the DNA with a polymerase; when the sample of origin is an RNA, a reverse transcription should be carried out beforehand.\",\n \"A very large number of methods currently exist for this amplification, such as, for example, the SDA (strand displacement amplification) technique (Walker et al., 1992, Nucleic Acids Res., 20:1691), the TAS (transcription-based amplification system) technique described by Kwoh et al.\",\n \"(1989, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA, 86, 1173), the 3SR (self-sustained sequence replication) technique described by Guatelli et al.\",\n \"(1990, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA, 87: 1874), the NASBA (nucleic acid sequence based amplification) technique described by Kievitis et al.\",\n \"(1991, J. Virol.\",\n \"Methods, 35, 273), the TMA (transcription mediated amplification) technique, the LCR (ligase chain reaction) technique described by Landegren et al.\",\n \"(1988, Science, 241, 1077), the RCR (repair chain reaction) technique described by Segev (1992, Kessler C. Springer Verlag, Berlin, N.Y., 197-205), the CPR (cycling probe reaction) technique described by Duck et al.\",\n \"(1990, Biotechniques, 9, 142) and the Q-beta-replicase amplification technique described by Miele et al.\",\n \"(1983, J. Mol.\",\n \"Biol., 171, 281).\",\n \"Some of these techniques have since been improved.\",\n \"When the target polynucleotide to be detected is an mRNA, an enzyme of the reverse transcriptase type is advantageously used, prior to carrying out an amplification reaction using the primers according to the invention, or to carrying out a method of detection using the probes of the invention, in order to obtain a cDNA from the mRNA contained in the biological sample.\",\n \"The cDNA obtained will then serve as a target for the primers or the probes used in the method of amplification or of detection according to the invention.\",\n \"The probe hybridization technique can be carried out in various ways (Matthews et al., 1988, Anal.\",\n \"Biochem., 169, 1-25).\",\n \"The most general method consists in immobilizing the nucleic acid, extracted from the cells of various tissues or from cells in culture, on a support (such as nitrocellulose, nylon or polystyrene) and in incubating, under well-defined conditions, the immobilized target nucleic acid with the probe.\",\n \"After hybridization, the excess probe is removed and the hybrid molecules formed are detected by the appropriate method (measurement of the radioactivity, of the fluorescence or of the enzyme activity associated with the probe).\",\n \"According to another embodiment of the nucleic acid probes according to the invention, the latter can be used as capture probes.\",\n \"In this case, a probe, termed “capture probe”, is immobilized on a support and is used to capture, by specific hybridization, the target nucleic acid obtained from the biological sample to be tested, and the target nucleic acid is then detected using a second probe, termed “detection probe”, labeled with a readily detectable element.\",\n \"Among the advantageous nucleic acid fragments, mention should thus in particular be made of antisense oligonucleotides, i.e.\",\n \"oligonucleotides whose structure provides, by hybridization with the target sequence, inhibition of the expression of the corresponding product.\",\n \"Mention should also be made of sense oligonucleotides which, by interaction with proteins involved in regulating the expression of the corresponding product, will induce either inhibition or activation of this expression.\",\n \"Preferably, the probes or primers according to the invention are immobilized on a support in a covalent or noncovalent manner.\",\n \"In particular, the support can be a DNA chip or a high or medium density filter, also a subject of the present invention (patents WO 97/29212, WO 98/27317, WO 97/10365 and WO 92/10588).\",\n \"The term “DNA chip” or “high density filter” is intended to denote a support on which are attached DNA sequences, it being possible to pinpoint each one of them by its geographical location.\",\n \"These chips or filters differ mainly by their size, the material of the support and, optionally, the number of DNA sequences which are attached thereto.\",\n \"The probes or primers according to the first invention can be attached to solid supports, in particular the DNA chips, by various methods of production.\",\n \"In particular, in situ synthesis can be carried out by photochemical addressing or by inkjet.\",\n \"Other techniques consist in carrying out an ex situ synthesis and attaching the probes to the support of the DNA chip by mechanical or electronic addressing or by inkjet.\",\n \"These various methods are well known to those skilled in the art.\",\n \"A nucleotide sequence (probe or primer) according to the invention therefore makes it possible to detect and/or amplify specific nucleic acid sequences.\",\n \"In particular, the detection of these said sequences is facilitated when the probe is attached to a DNA chip or to a high density filter.\",\n \"The use of DNA chips or of high density filters in fact makes it possible to determine the gene expression in an organism having a genomic sequence close to P. luminescens, and to type the strain in question.\",\n \"The genomic sequence of P. luminescens, supplemented by the identification of the genes of these organisms, as presented in the present invention, serves as a basis for constructing these DNA chips or filters.\",\n \"The preparation of these filters or chips consists in synthesizing oligonucleotides, corresponding to the 5′ and 3′ ends of the genes or to more internal fragments, in order to amplify fragments of an appropriate length, for example of between approximately 300 and 800 bases.\",\n \"These oligonucleotides are chosen using the genomic sequence and its annotations disclosed in the present invention.\",\n \"The temperature for pairing these oligonucleotides at the corresponding places on the DNA should be approximately the same for each oligonucleotide.\",\n \"This makes it possible to prepare DNA fragments corresponding to each gene using appropriate PCR conditions in a highly automated environment.\",\n \"The amplified fragments are then immobilized on filters or supports made of glass, silicon or synthetic polymers and these media are used for the hybridization.\",\n \"The availability of such filters and/or chips and of the corresponding annotated genomic sequence makes it possible to study the expression of large sets, or even all, of the genes of Photorhabdus luminescens , in particular of P. luminescens TT01, by preparing the complementary DNAs and hybridizing them to the DNA or to the oligonucleotides immobilized on the filters or the chips.\",\n \"Similarly, the filters and/or the chips make it possible to study the strain or species variability by preparing the DNA of these organisms and hybridizing them to the DNA or to the oligonucleotides immobilized on the filters or the chips.\",\n \"The differences between the genomic sequences of the various strains or species can greatly affect the intensity of the hybridization and, consequently, disturb the interpretation of the results.\",\n \"It may therefore be necessary to have the precise sequence of the genes of the strain intended to be studied.\",\n \"The method for detecting the genes described later in detail, involving determining the sequence of random fragments of a genome and organizing them according to the sequence of the genome of P. luminescens, in particular of P. luminescens TT01, disclosed in the present invention, may be very useful.\",\n \"The nucleotide sequences according to the invention can be used in DNA chips to carry out mutation analysis.\",\n \"This analysis is based on constituting chips capable of analyzing each base of a nucleotide sequence according to the invention.\",\n \"For this purpose, use may in particular be made of the techniques of microsequencing on a DNA chip.\",\n \"The mutations are detected by extension of immobilized primers which hybridize to the matrix of the analyzed sequences, just at a position adjacent to that of the mutated nucleotide sought.\",\n \"A single-stranded, RNA or DNA matrix of the sequences to be analyzed will advantageously be prepared according to conventional methods, using products amplified according to PCR-type techniques.\",\n \"The single-stranded DNA or RNA matrices thus obtained are then deposited onto the DNA chip, under conditions which allow their specific hybridization to the immobilized primers.\",\n \"A thermostable polymerase, for example Tth or Taq DNA polymerase, specifically extends the 3′ end of the immobilized primer with a labeled nucleotide analog complementary to the nucleotide at the position of the variable site; for example, thermocycling is carried out in the presence of fluorescent dideoxyribonucleotides.\",\n \"The experimental conditions will be adjusted in particular to the chips used, to the immobilized primers, to the polymerases used and to the labeling system chosen.\",\n \"One advantage of microsequencing, compared to techniques based on probe hybridization, is that it makes it possible to identify all the variable nucleotides with optimal discrimination under homogeneous reaction conditions; when used on DNA chips, it allows optimal resolution and specificity for the routine and industrial detection of mutations in a multiplex.\",\n \"A DNA chip or a filter can be an extremely advantageous tool for determining, detecting and/or identifying a microorganism.\",\n \"Thus, the DNA chips according to the invention which also contain at least one nucleotide sequence of a microorganism other than Photorhabdus luminescens, immobilized on the support of said chip, are also preferred.\",\n \"The microorganism chosen is preferably done so from the bacteria of the genus Photorhabdus (hereinafter referred to as P. luminescens-related bacteria), or the variants of Photorhabdus luminescens TT01.A DNA chip or a filter according to the invention is a very useful element of certain kits or sets for detecting and/or identifying microorganisms, in particular bacteria belonging to the species Photorhabdus luminescens , which are also the subject of the invention.\",\n \"Moreover, the DNA chips or the filters according to the invention, containing probes or primers specific for Photorhabdus luminescens , are very advantageous elements of kits or sets for detecting and/or quantifying the expression of Photorhabdus luminescens genes.\",\n \"Specifically, the control of gene expression is a critical point for optimizing the growth and yield of a strain, either by allowing the expression of one or more new genes, or by modifying the expression of genes already present in the cell.\",\n \"The present invention provides all of the sequences naturally active in P. luminescens which allow gene expression.\",\n \"It thus makes it possible to determine all the sequences expressed in P. luminescens.\",\n \"It also provides a tool for pinpointing the genes whose expression follows a given pattern.\",\n \"To do this, the DNA of all or some of the genes of P. luminescens can be amplified using primers according to the invention, and then attached to a support such as, for example, glass or nylon or a DNA chip, in order to construct a tool for following the expression profile of these genes.\",\n \"This tool, consisting of this support containing the coding sequences, serves as a matrix of hybridization to a mixture of labeled molecules reflecting the messenger RNAs expressed in the cell (in particular the labeled probes according to the invention).\",\n \"By repeating this experiment at various moments and combining all of these data using suitable processing, the expression profiles of all these genes are then obtained.\",\n \"The knowledge of the sequences which follow a given regulatory scheme can also be exploited to search for, in a directed manner, for example by homology, other sequences following, overall, but in a slightly different way, the same regulatory scheme.\",\n \"In addition, it is possible to isolate each control sequence present upstream of the segments acting as probes and to follow the activity thereof using a suitable means such as a reporter gene (luciferase, β-galactosidase, GFP).\",\n \"These isolated sequences can then be modified and assembled by metabolic engineering with sequences of interest with a view to the optimal expression thereof.\",\n \"The invention also relates to the polypeptides encoded by a nucleotide sequence according to the invention, preferably by a representative fragment of the preceding sequences, corresponding to an ORF sequence.\",\n \"In particular, the polypeptides of Photorhabdus luminescens TT01 of SEQ ID No.\",\n \"42 to SEQ ID No.\",\n \"3855 or encoded by SEQ ID No.\",\n \"5835 to SEQ ID No.\",\n \"10784 are a subject of the invention.\",\n \"The invention also comprises the polypeptides characterized in that they comprise a polypeptide chosen from: a) a polypeptide of sequence SEQ ID No.\",\n \"42 to SEQ ID No.\",\n \"3855 or encoded by a sequence SEQ ID No.\",\n \"5835 to SEQ ID No.\",\n \"10784; b) a polypeptide exhibiting at least 80%, preferably 85%, 90%, 95% and 98%, identity with a polypeptide according to the invention; c) a fragment of at least 5 amino acids of a polypeptide as defined in a); d) a biologically active fragment of a polypeptide as defined in a); and e) a polypeptide as defined in a), b), c) or d), which has been modified.\",\n \"The nucleotide sequences encoding the polypeptides described above are also a subject of the invention.\",\n \"In the present description, the terms “polypeptides”, “polypeptide sequences”, “peptides” and “proteins” are interchangeable.\",\n \"The term “polypeptide” comprises any amino acid sequence making it possible to generate an antibody response.\",\n \"It should be understood that the invention does not concern the polypeptides in natural form, i.e.\",\n \"they are not taken in their natural environment.\",\n \"On the other hand, it concerns those which it has been possible to isolate or obtain by purification from natural sources, or else those obtained by genetic recombination or by chemical synthesis, and they can then comprise unnatural amino acids as will be described below.\",\n \"The expression “polypeptide exhibiting a certain percentage identity with another”, for which the expression “homologous polypeptide” will also be used, is intended to denote the polypeptides exhibiting, compared to the natural polypeptides, certain modifications, in particular a deletion, addition or substitution of at least one amino acid, a truncation, an extension, a chimeric solution and/or a mutation, or the polypeptides exhibiting post-translational modifications.\",\n \"Among the homologous polypeptides, preference is given to those in which the amino acid sequence exhibits at least 80%, preferably 85%, 90%, 95%, 98% or 99%, identity, after optimal alignment, with the amino acid sequences of the polypeptides according to the invention.\",\n \"In the case of a substitution, one or more consecutive or nonconsecutive amino acid(s) may be replaced with “equivalent” amino acids.\",\n \"The expression “equivalent amino acids” is here aimed at denoting any amino acid capable of being substituted for one of the amino acids of the basic structure without, however, essentially modifying the biological activities of the corresponding peptides as they will be defined subsequently.\",\n \"These equivalent amino acids can be determined either based on their structural homology with the amino acids for which they substitute, or based on results of comparative biological activity assays between the various polypeptides liable to be produced.\",\n \"By way of example, mention is made of the substitution possibilities which can be made without this resulting in a profound modification of the biological activity of the corresponding modified polypeptide.\",\n \"It is thus possible to replace leucine with valine or isoleucine, aspartic acid with glutamic acid, glutamine with asparagine, arginine with lysine, etc, it naturally being possible to envision the reverse substitutions under the same conditions.\",\n \"The homologous polypeptides also correspond to the polypeptides encoded by the nucleotide sequences which exhibit a certain percentage identity with the nucleotide sequences of the invention or which are identical, as previously defined, and thus comprise, in the present definition, mutated polypeptides or polypeptides corresponding to inter- or intraspecies variations which can exist in Photorhabdus, and which correspond in particular to truncations, substitutions, deletions and/or additions of at least one amino acid residue.\",\n \"It is understood that the percentage identity between two polypeptides is calculated in the same way as between two nucleic acid sequences.\",\n \"Thus, the percentage identity between two polypeptides is calculated, after optimal alignment of these two sequences, on a window of maximum homology.\",\n \"To define said window of maximum homology, the same algorithms as for the nucleic acid sequences can be used.\",\n \"The expression “biologically active fragment of a polypeptide according to the invention” is intended to denote in particular a polypeptide fragment, as defined below, exhibiting at least one of the biological characteristics of the polypeptides according to the invention, in particular in that it is capable of exerting, in general, even a partial activity, such as, for example: an enzymatic (metabolic) activity or an activity which may be involved in the biosynthesis or the biodegradation of organic or inorganic compounds, or preferably a toxic or antibiotic activity, in particular for insects or microorganisms (bacteria or fungi), or else an activity involved in the biosynthesis of these toxins or antibiotics; such proteins with enzymatic activity may in particular be used in methods for screening and/or selecting compounds capable of modifying this activity, in particular of inhibiting it; a structural activity (cell envelope, chaperone molecule, ribosome).\",\n \"Proteins corresponding especially to extramembrane proteins may in particular be used as an immunogen for producing mono- or polyclonal antibodies directed specifically against these extramembrane proteins; a transport activity (energy transport, ion transport); or an activity in protein secretion; an activity in the process of replication, amplification, preparation, transcription, translation or maturation, in particular of DNA, of RNA or of proteins.\",\n \"The expression “polypeptide fragment according to the invention” is intended to denote a polypeptide comprising at least 5 amino acids, preferably 10, 15, 25, 50, 100 and 150 amino acids.\",\n \"The polypeptide fragments can correspond to isolated or purified fragments naturally present in the strains of Photorhabdus, or to fragments which can be obtained by cleavage of said polypeptide with a proteolytic enzyme, such as trypsin or chymotrypsin or collagenase, or with a chemical reagent (cyanogen bromide, CNBr) or by placing said polypeptide in a very acidic environment (for example at pH=2.5).\",\n \"Polypeptide fragments can also be prepared by chemical synthesis, from hosts transformed with an expression vector according to the invention which contain a nucleic acid which allows expression of said fragment and is placed under the control of the appropriate regulatory and/or expression elements.\",\n \"The term “modified polypeptide” of a polypeptide according to the invention is intended to denote a polypeptide obtained by genetic recombination or by chemical synthesis as described later, which exhibits at least one modification compared to the normal sequence, and preferably at most 10% of modified amino acids compared to the normal sequence.\",\n \"These modifications may in particular be made on amino acids necessary for the specificity or the effectiveness of the activity, or responsible for the structural conformation, for the charge or for the hydrophobicity of the polypeptide according to the invention.\",\n \"It is thus possible to create polypeptides with equivalent, increased or decreased activity, or with equivalent, stricter or broader specificity.\",\n \"Among the modified polypeptides, mention should be made of the polypeptides in which up to five amino acids can be modified, truncated at the N- or C-terminal end, or else deleted, or added.\",\n \"As is indicated, the aim of the modifications to a polypeptide is in particular: to allow its use in methods of biosynthesis or of biodegradation of organic or inorganic compounds, or in the biosynthesis of toxins or of antibiotics, to allow its use in methods of replication, of amplification, of repair and of regulation of transcription, translation or maturation in particular of DNA, RNA or proteins; to allow its enhanced secretion, to modify its solubility, or the effectiveness or specificity of its activity, or else to facilitate its purification.\",\n \"Chemical synthesis also has the advantage of being able to use unnatural amino acids or nonpeptide bonds.\",\n \"Thus, it may be advantageous to use unnatural amino acids, for example in the D form, or amino acid analogs, in particular sulfur-containing forms.\",\n \"The invention also relates to the nucleic acid or peptide sequences according to the present invention with the exception of the nucleic acid or peptide sequences described in documents WO 99/54472, WO 99/42589, WO 99/03328, WO 98/08932 and EP 0 823 215.The present invention provides the nucleotide sequence of the Photorhabdus luminescens TT01 genome in the form of 41 contigs or in the form of 9 contigs, and also some polypeptide sequences.\",\n \"The nucleic acid or peptide sequences below, characterized by their function, can also be identified by their nucleotide and amino acid sequence with reference to table I. Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01 with activity of the toxin and/or antibiotic type, or involved in the synthesis of these toxins and/or antibiotics.\",\n \"Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in amino acid biosynthesis.\",\n \"Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the biosynthesis of cofactors, prosthetic groups and transporters.\",\n \"Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a cell envelope polypeptide or a polypeptide present at the surface of Photorhabdus luminescens TT01, or one of its fragments.\",\n \"Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the cellular machinery.\",\n \"Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in central intermediate metabolism.\",\n \"Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in energy metabolism.\",\n \"Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in fatty acid and phospholipid metabolism.\",\n \"Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the metabolism of nucleotides, purines, pyrimidines or nucleosides.\",\n \"Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in regulatory functions.\",\n \"Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the replication process.\",\n \"Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the transcription process.\",\n \"Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the translation process.\",\n \"Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the process of protein transport and binding.\",\n \"Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in adaptation to atypical conditions.\",\n \"Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, [lacuna] in sensitivity to medicinal products and analogs.\",\n \"Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in functions relating to transposons.\",\n \"Preferably, the invention relates to a nucleotide sequence according to the invention, characterized in that it encodes a polypeptide specific for Photorhabdus luminescens TT01, or one of its fragments.\",\n \"In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, [lacuna] activity of the toxin and/or antibiotic type, or involved in the synthesis of these toxins and/or antibiotics.\",\n \"In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in amino acid biosynthesis.\",\n \"In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the biosynthesis of cofactors, prosthetic groups and transporters.\",\n \"In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a cell envelope polypeptide or a surface polypeptide of Photorhabdus luminescens TT01, or one of its fragments.\",\n \"In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the cellular machinery.\",\n \"In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in central intermediate metabolism.\",\n \"In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in energy metabolism.\",\n \"In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in fatty acid and phospholipid metabolism.\",\n \"In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the metabolism of nucleotides, purines, pyrimidines or nucleosides.\",\n \"In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in regulatory functions.\",\n \"In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the replication process.\",\n \"In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the transcription process.\",\n \"In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the translation process.\",\n \"In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in the process of protein transport and binding.\",\n \"In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in adaptation to atypical conditions.\",\n \"In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, [lacuna] in sensitivity to medicinal products and analogs.\",\n \"In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TT01, or one of its fragments, involved in functions relating to transposons.\",\n \"In another aspect, a subject of the invention is preferably a polypeptide according to the invention, characterized in that it is a polypeptide specific for Photorhabdus luminescens TT01, or one of its fragments.\",\n \"A subject of the invention is also the operons involved in the synthesis of antibiotics and/or of toxins.\",\n \"Table I provides the list of some polypeptides according to the invention, and also their location in the contigs represented by SEQ ID No.\",\n \"1 to SEQ ID No.\",\n \"41, and the similarities observed after comparison in the databases.\",\n \"Table II provides the list of some polypeptides according to the invention, and also their location in the contigs represented by SEQ ID No.\",\n \"5826 to SEQ ID No.\",\n \"5834, and the similarities observed after comparison in the databases.\",\n \"In table II, contigs 1 to 9 are identified by the sequences SEQ ID No.\",\n \"5826 to SEQ ID No.\",\n \"5834.Entirely preferably, a subject of the invention is also the polypeptides whose functions annotated in table I, final column, or whose functions annotated in table II, penultimate column, correspond to activities of the toxin and/or antibiotic type, or to polypeptides involved in the synthesis of these toxins and/or antibiotics, which polypeptides are preferably chosen from: a) the polypeptides of sequence SEQ ID No.\",\n \"61, SEQ ID No.\",\n \"62, SEQ ID No.\",\n \"67, SEQ ID No.\",\n \"171, SEQ ID No.\",\n \"221, SEQ ID No.\",\n \"268, SEQ ID No.\",\n \"288, SEQ ID No.\",\n \"380, SEQ ID No.\",\n \"426, SEQ ID No.\",\n \"438, SEQ ID No.\",\n \"448, SEQ ID No.\",\n \"453, SEQ ID No.\",\n \"455, SEQ ID No.\",\n \"456, SEQ ID No.\",\n \"458, SEQ ID No.\",\n \"501, SEQ ID No.\",\n \"516, SEQ ID No.\",\n \"530, SEQ ID No.\",\n \"542, SEQ ID No.\",\n \"551, SEQ ID No.\",\n \"720, SEQ ID No.\",\n \"761, SEQ ID No.\",\n \"762, SEQ ID No.\",\n \"814, SEQ ID No.\",\n \"859, SEQ ID No.\",\n \"860, SEQ ID No.\",\n \"861, SEQ ID No.\",\n \"862, SEQ ID No.\",\n \"869, SEQ ID No.\",\n \"1079, SEQ ID No.\",\n \"1168, SEQ ID No.\",\n \"1174, SEQ ID No.\",\n \"1176, SEQ ID No.\",\n \"1413, SEQ ID No.\",\n \"1414, SEQ ID No.\",\n \"1415, SEQ ID No.\",\n \"1416, SEQ ID No.\",\n \"1417, SEQ ID No.\",\n \"1457, SEQ ID No.\",\n \"1651, SEQ ID No.\",\n \"1856, SEQ ID No.\",\n \"1869, SEQ ID No.\",\n \"2021, SEQ ID No.\",\n \"2080, SEQ ID No.\",\n \"2152, SEQ ID No.\",\n \"2162, SEQ ID No.\",\n \"2173, SEQ ID No.\",\n \"2251, SEQ ID No.\",\n \"2295, SEQ ID No.\",\n \"2306, SEQ ID No.\",\n \"2317, SEQ ID No.\",\n \"2328, SEQ ID No.\",\n \"2340, SEQ ID No.\",\n \"2342, SEQ ID No.\",\n \"2351, SEQ ID No.\",\n \"2500, SEQ ID No.\",\n \"3228, SEQ ID No.\",\n \"3230, SEQ ID No.\",\n \"3311, SEQ ID No.\",\n \"3317, SEQ ID No.\",\n \"3318, SEQ ID No.\",\n \"3319, SEQ ID No.\",\n \"3320, SEQ ID No.\",\n \"3322, SEQ ID No.\",\n \"3323, SEQ ID No.\",\n \"3326, SEQ ID No.\",\n \"3327, SEQ ID No.\",\n \"3328, SEQ ID No.\",\n \"3375, SEQ ID No.\",\n \"3376, SEQ ID No.\",\n \"3377, SEQ ID No.\",\n \"3378, SEQ ID No.\",\n \"3422, SEQ ID No.\",\n \"3489, SEQ ID No.\",\n \"3503, SEQ ID No.\",\n \"3609, SEQ ID No.\",\n \"3623, SEQ ID No.\",\n \"3624, SEQ ID No.\",\n \"3772, SEQ ID No.\",\n \"3783, SEQ ID No.\",\n \"3788 and SEQ ID No.\",\n \"3794; or b) the polypeptides encoded by the sequences SEQ ID No.\",\n \"5835 to SEQ ID No.\",\n \"10784, homologous to the sequences as defined in a), as indicated in the final column of table II.\",\n \"The subject of the present invention is also the nucleotide and/or polypeptide sequences according to the invention, characterized in that said sequences are recorded on a recording medium, the form and nature of which facilitate the reading, analysis and/or exploitation of said sequence(s).\",\n \"These media may also contain other information extracted from the present invention, in particular the similarities with already known sequences, and/or information concerning the nucleotide and/or polypeptide sequences of a cell of a plant, of an animal or of a microorganism other than P. luminescens, in particular a cell or microorganism sensitive to a toxin or an antibiotic produced by P. luminescens, a bacterium of the genus Photorhabdus, or a variant of P. luminescens, in order to facilitate the comparative analysis and the exploitation of the results obtained.\",\n \"Among these said recording media, preference is given in particular to computer-readable media, such as magnetic, optical, electrical or hybrid media, in particular computer disks, CD-ROMs and computer servers.\",\n \"Such recording media are also a subject of the invention.\",\n \"The recording media according to the invention, with the information provided, are very useful for choosing nucleotide primers or probes for determining genes in Photorhabdus luminescens TT01 or strains related to this organism.\",\n \"Similarly, the use of these media for studying the genetic polymorphism of strains related to Photorhabdus luminescens TT01, in particular by determining the regions of colinearity, is very useful insofar as these media provide not only the nucleotide sequence of the Photorhabdus luminescens TT01 genome, but also the genomic organization in said sequence.\",\n \"Thus, the uses of recording media according to the invention are also subjects of the invention.\",\n \"The analysis of homology between various sequences is in fact advantageously performed using sequence comparison programs, such as the Blast program, or the programs of the GCG package, described above.\",\n \"The invention is also directed toward the cloning and/or expression vectors which contain a nucleotide sequence according to the invention.\",\n \"The vectors according to the invention preferably comprise elements which allow the expression and/or the secretion of the nucleotide sequences in a given host cell.\",\n \"The vector should then comprise a promoter, translation initiation and termination signals, and also regions suitable for regulating transcription.\",\n \"It must be possible for it to be maintained stably in the host cell and it may optionally contain particular signals which specify secretion of the translated protein.\",\n \"These various elements are chosen and optimized by those skilled in the art as a function of the cellular host used.\",\n \"To this effect, the nucleotide sequences according to the invention can be inserted into vectors which replicate autonomously in the host chosen, or may be vectors which integrate in the host chosen.\",\n \"Such vectors are prepared by methods commonly used by those skilled in the art, and the resulting clones can be introduced into a suitable host by standard methods, such as lipofection, electroporation, heat shock or chemical methods.\",\n \"The vectors according to the invention are, for example, vectors of plasmid or viral origin.\",\n \"They are useful for transforming host cells in order to clone or express the nucleotide sequences according to the invention.\",\n \"Among these vectors, preference is also given to the cloning and/or expression vectors according to the invention, characterized in that they contain a nucleotide sequence chosen from the sequences SEQ ID No.\",\n \"3856 to SEQ ID No.\",\n \"5825, and SEQ ID No.\",\n \"5835 to SEQ ID No.\",\n \"10784, or their fragment derived from the P. luminescens genome, in particular the sequences encoding the polypeptides with toxin or antibiotic activity or involved in these activities, in particular those mentioned above whose functions annotated in table I below correspond to these activities.\",\n \"The invention also comprises the host cells transformed with a vector according to the invention.\",\n \"The cellular host may be chosen from prokaryotic or eukaryotic systems, for example bacterial cells, but also yeast cells or animal cells, in particular mammalian cells.\",\n \"Insect cells or plant cells may also be used.\",\n \"The preferred host cells according to the invention are in particular prokaryotic cells, preferably bacteria belonging to the genus Photorhabdus or to the species Photorhabdus luminescens , more particularly Photorhabdus luminescens TT01.The invention also relates to the plants and animals, except humans, which comprise a transformed cell according to the invention.\",\n \"The transformed cells according to the invention can be used in methods for preparing recombinant polypeptides according to the invention.\",\n \"The methods for preparing a polypeptide according to the invention in recombinant form, characterized in that they use a vector and/or a cell transformed with a vector according to the invention, are themselves included in the present invention.\",\n \"Preferably, a cell transformed with a vector according to the invention is cultured under conditions which allow the expression of said polypeptide, and said recombinant polypeptide is recovered.\",\n \"As has been mentioned, the cellular host can be chosen from prokaryotic or eukaryotic systems.\",\n \"In particular, it is possible to identify nucleotide sequences according to the invention which facilitate secretion in such a prokaryotic or eukaryotic system.\",\n \"A vector according to the invention carrying such a sequence can therefore be advantageously used for producing recombinant proteins intended to be secreted.\",\n \"As a result, the purification of these recombinant proteins of interest will be facilitated by the fact that they are present in the cell culture supernatant rather than inside the host cells.\",\n \"The polypeptides according to the invention may also be prepared by chemical synthesis.\",\n \"Such a method of preparation is also a subject of the invention.\",\n \"Those skilled in the art are aware of the methods of chemical synthesis, for example the techniques using solid phases (see in particular Steward et al., 1984, Solid phase peptides synthesis, Pierce Chem.\",\n \"Company, Rockford, 111, 2nd ed., (1984)) or techniques using partial solid phases, by fragment condensation or by conventional synthesis in solution.\",\n \"The polypeptides obtained by chemical synthesis, and possibly comprising corresponding unnatural amino acids, are also included in the invention.\",\n \"The hybrid polypeptides according to the invention are very useful for obtaining monoclonal or polyclonal antibodies capable of specifically recognizing the polypeptides according to the invention.\",\n \"These specific polyclonal or monoclonal antibodies can be obtained by the standard methods well known to those skilled in the art, after immunizing a mammal using these polypeptides (or their corresponding nucleic acid) or, for example, according to the conventional method of hybridoma culture described by Köhler and Milstein (1975, Nature, 256, 495) for the monoclonal antibodies.\",\n \"Such monoclonal or polyclonal antibodies, their fragments, or the chimeric antibodies, which recognize the polypeptides according to the invention, are also subjects of the invention.\",\n \"The antibodies according to the invention are, for example, chimeric antibodies, humanized antibodies, or Fab or F(ab′)2 fragments.\",\n \"They can also be in the form of immunoconjugates or of antibodies which are labeled in order to obtain a detectable and/or quantifiable signal.\",\n \"Thus, the antibodies according to the invention can be used in a method for detecting and/or identifying bacteria belonging to the genus Photorhabdus and/or to the species Photorhabdus luminescens , in a biological sample, characterized in that it comprises the following steps: a) bringing the biological sample into contact with an antibody according to the invention; b) demonstrating the antigen-antibody complex possibly formed.\",\n \"The antibodies according to the invention can also be used in order to detect expression of a gene of Photorhabdus luminescens TT01.Specifically, the presence of the expression product of a gene recognized by an antibody specific for said expression product can be detected by the presence of an antigen-antibody complex formed after the Photorhabdus luminescens strain TT01 has been brought into contact with an antibody according to the invention.\",\n \"The bacterial strain used may have been “prepared”, i.e.\",\n \"centrifuged, lyzed and/or placed in an appropriate reagent for constituting the medium suitable for the immunoreaction.\",\n \"In particular, preference is given to a method for detecting expression in the gene, corresponding to Western blotting, which may be performed after polyacrylamide gel electrophoresis of a lysate of the bacterial strain, in the presence or absence of reducing conditions (SDS-PAGE).\",\n \"After migration and separation of the proteins on the polyacrylamide gel, said proteins are transferred onto a suitable membrane (for example made of nylon) and the presence of the protein or of the polypeptide of interest is detected by bringing said membrane into contact with an antibody according to the invention.\",\n \"Thus, the present invention also comprises the kits or sets for carrying out a method as described (for detecting the expression of a gene of Photorhabdus luminescens TT01, or for detecting and/or identifying bacteria belonging to the species Photorhabdus luminescens), comprising the following elements: a) a polyclonal or monoclonal antibody according to the invention; b) optionally, the reagents for constituting the medium suitable for the immunoreaction; c) optionally, the reagents for demonstrating the antigen-antibody complexes produced by the immunoreaction.\",\n \"The polypeptides and the antibodies according to the invention can advantageously be immobilized on a support, in particular a protein chip.\",\n \"Such a protein chip is the subject of the invention and may also contain at least one polypeptide of a microorganism other than Photorhabdus luminescens , or an antibody directed against a compound of a microorganism other than Photorhabdus luminescens .\",\n \"The protein chips or high density filters containing proteins according to the invention can be constructed in the same way as the DNA chips according to the invention.\",\n \"In practice, it is possible to carry out the synthesis of the polypeptides directly attached to the protein chip, or to carry out an ex situ synthesis followed by a step of attaching the synthesized polypeptide to said chip.\",\n \"The latter method is preferable when the intention is to attach proteins of considerable size to the support, these proteins being advantageously prepared by genetic engineering.\",\n \"However, if the intention is to attach only peptides to the support of said chip, it may be more advantageous to synthesize said peptides directly in situ.\",\n \"The protein chips according to the invention can advantageously be used in kits or sets for detecting and/or identifying bacteria related to the species Photorhabdus luminescens , or to a microorganism, or more generally in kits or sets for detecting and/or identifying microorganisms.\",\n \"When the polypeptides according to the invention are attached to DNA chips, the presence of antibodies in the samples tested is sought, the attachment of an antibody according to the invention to the support of said protein chip allowing identification of the protein for which said antibody is specific.\",\n \"Preferably, an antibody according to the invention is attached to the support of the protein chip, and the presence of the corresponding antigen, specific for Photorhabdus luminescens , is detected.\",\n \"A protein chip described above can be used to detect gene products, in order to establish an expression profile for said genes, in addition to a DNA chip according to the invention.\",\n \"The protein chips according to the invention are also extremely useful for proteomic experiments which study interactions between the various proteins of a cell of a plant, of an animal, such as an insect, or of a microorganism other than P. luminescens.\",\n \"Thus, the invention also comprises a protein chip according to the invention, characterized in that it also contains at least one polypeptide of a cell of a plant, of an animal or of a microorganism other than P. luminescens, immobilized on the support of said chip, preferably said cell or other microorganism is chosen from a cell or microorganism sensitive to a toxin or an antibiotic produced by P. luminescens.\",\n \"In a simplified manner, representative peptides of the various proteins of an organism are attached to a support.\",\n \"Said support is then brought into contact with labeled proteins and, after an optional rinsing step, interactions between said labeled proteins and the peptides attached to the protein chip are detected.\",\n \"Thus, the protein chips comprising a polypeptide sequence according to the invention or an antibody according to the invention are a subject of the invention, as are the kits or sets containing them.\",\n \"The present invention also covers a method for detecting and/or identifying bacteria belonging to the species Photorhabdus luminescens , in a biological sample, which uses a nucleotide sequence according to the invention.\",\n \"It should be understood that, in the present invention, the term “biological sample” concerns samples taken from a living organism (in particular blood, tissues, organs or others taken from a mammal) or a sample containing biological material, i.e.\",\n \"DNA or RNA.\",\n \"Such a biological sample also comprises food compositions containing bacteria (for example cheeses, dairy products), but also food compositions containing yeasts (beers, breads) or others.\",\n \"The term “biological sample” also concerns bacteria isolated from these samples or food compositions.\",\n \"The method of detection and/or identification using the nucleotide sequences according to the invention may be diverse in nature.\",\n \"A method comprising the following steps is preferred: a) optionally isolating the DNA from the biological sample to be analyzed, or obtaining a cDNA from the RNA of the biological sample; b) specifically amplifying the DNA of bacteria belonging to the species Photorhabdus luminescens using at least one primer according to the invention; c) demonstrating the amplification products.\",\n \"This method is based on the specific amplification of the DNA, in particular via a chain amplification reaction.\",\n \"A method comprising the following steps is also preferred: a) bringing a nucleotide probe according to the invention into contact with a biological sample, the nucleic acid contained in the biological sample having, where appropriate, previously been made accessible to hybridization, under conditions which allow hybridization of the probe to the nucleic acid of a bacterium belonging to the species Photorhabdus luminescens ; b) demonstrating the hybrid possibly formed between the nucleotide probe and the DNA of the biological sample.\",\n \"Such a method should not be limited to detecting the presence of the DNA contained in the biological sample to be tested, it can also be used to detect the RNA contained in said sample.\",\n \"This method encompasses in particular Southern and Northern blotting.\",\n \"Another preferred method according to the invention comprises the following steps: a) bringing a nucleotide probe immobilized on a support according to the invention into contact with a biological sample, the nucleic acid of the sample having, where appropriate, previously been made accessible to hybridization, under conditions which allow hybridization of the probe to the nucleic acid of a bacterium belonging to the species Photorhabdus luminescens ; b) bringing the hybrid formed between the nucleotide probe immobilized on a support and the nucleic acid contained in the biological sample, where appropriate after removing the DNA of the biological sample which has not hybridized with the probe, into contact with a labeled nucleotide probe according to the invention; c) demonstrating the new hybrid formed in step b).\",\n \"This method is advantageously used with a DNA chip according to the invention, the nucleic acid being sought hybridizing with a probe present at the surface of said chip, and being detected using a labeled probe.\",\n \"This method is advantageously carried out by combining a prior step of amplifying the DNA or the complementary DNA optionally obtained by reverse transcription, using primers according to the invention.\",\n \"Thus, the present invention also encompasses the kits or sets for detecting and/or identifying bacteria belonging to the species Photorhabdus luminescens , characterized in that it comprises the following elements: a) a nucleotide probe according to the invention; b) optionally, the reagents required for carrying out a hybridization reaction; c) optionally, at least one primer according to the invention and also the reagents required for a DNA amplification reaction.\",\n \"Similarly, the present invention also encompasses the kits or sets for detecting and/or identifying bacteria belonging to the species Photorhabdus luminescens TT01, characterized in that it comprises the following elements: a) a nucleotide probe, termed capture probe, according to the invention; b) an oligonucleotide probe, termed detection probe, according to the invention; c) optionally, at least one primer according to the invention and also the reagents required for a DNA amplification reaction.\",\n \"Finally, the kits or sets for detecting and/or identifying bacteria belonging to the species Photorhabdus luminescens , characterized in that they comprise the following elements: a) at least one primer or one probe according to the invention; b) optionally, the reagents required to carry out a DNA amplification reaction; c) optionally, a component for verifying the sequence of the amplified fragment, more particularly an oligonucleotide probe according to the invention, are also subjects of the present invention.\",\n \"Preferably, said primers and/or probes and/or polypeptides and/or antibodies according to the present invention, used in the methods and/or kits or sets according to the present invention, are chosen from the primers and/or probes and/or polypeptides and/or antibodies specific for the species Photorhabdus luminescens.\",\n \"Preferably, these elements are chosen from the nucleotide sequences encoding a secreted protein, from the secreted polypeptides, or from the antibodies directed against secreted polypeptides of Photorhabdus luminescens.\",\n \"A subject of the present invention is also the strains of Photorhabdus luminescens TT01 containing one or more mutation(s) in a nucleotide sequence according to the invention, in particular an ORF sequence, or regulatory elements thereof (in particular promoters).\",\n \"According to the invention, preference is given to the strains of Photorhabdus luminescens TT01 exhibiting one or more mutation(s) in the nucleotide sequences encoding polypeptides preferably with activity of the toxin or antibiotic type, or involved in their biosynthesis, or else, in another aspect, involved in the cellular machinery, in particular secretion, central intermediate metabolism, energy metabolism, and processes of amino acid synthesis, of transcription and of translation, and of polypeptide synthesis.\",\n \"Said mutations may lead to inactivation of the gene or, in particular, when they are located in the regulatory elements of said gene, to overexpression of this gene.\",\n \"According to the present invention, the strains of Photorhabdus luminescens TT01 exhibiting one or more mutation(s) may be used to validate the function of a wild-type gene of Photorhabdus luminescens .\",\n \"The invention also relates to the use of a nucleotide sequence according to the invention, of a polypeptide according to the invention, of an antibody according to the invention, and/or of a cell according to the invention, for selecting an organic or inorganic compound capable of modulating, regulating, inducing or inhibiting gene expression in a plant or animal cell or in a microorganism other than P. luminescens, the resistance or sensitivity of which to at least one toxin or antibiotic produced by P. luminescens it is, for example, desired to modify.\",\n \"The invention also comprises a method for selecting compounds capable of binding to a polypeptide or one of its fragments according to the invention, capable of binding to a nucleotide sequence according to the invention, or capable of recognizing an antibody according to the invention, and/or capable of modulating, regulating, inducing or inhibiting gene expression, and/or of modifying growth or cell replication of eukaryotic or prokaryotic cells, or capable of inducing, inhibiting or increasing, in an animal or plant organism, resistance or sensitivity to at least one toxin or antibiotic produced by P. luminescens, said method comprising the following steps: a) bringing said compound into contact with said polypeptide or said nucleotide sequence and/or with a transformed cell according to the invention; b) determining the ability of said compound to bind to said polypeptide or said nucleotide sequence, or to modulate, regulate, induce or inhibit gene expression, or to modulate growth or cell replication, or to induce, inhibit or increase, in an animal or plant organism, resistance or sensitivity to at least one toxin or antibiotic produced by P. luminescens.\",\n \"The transformed cells according to the invention may advantageously be used as a model and may be used in methods for studying, identifying and/or selecting compounds liable to be responsible for resistance or sensitivity to at least one toxin or antibiotic produced by P. luminescens.\",\n \"The compounds which may be selected can be organic compounds such as polypeptides or carbohydrates or any other organic or inorganic compounds which are already known, or new organic compounds developed using molecular modeling techniques and obtained by chemical or biochemical synthesis, these techniques being known to those skilled in the art.\",\n \"The invention relates to the compounds which can be selected using a method of selection according to the invention.\",\n \"The invention also relates to a composition, in particular pesticidal or pharmaceutical composition, comprising a compound chosen from the following compounds: a) a nucleotide sequence according to the invention; b) a polypeptide according to the invention; c) a vector according to the invention; d) an antibody according to the invention; and e) a compound which can be selected using a method of selection according to the invention, optionally in combination with a pharmaceutically acceptable vehicle.\",\n \"The invention also relates to a pharmaceutical composition according to the invention, for preventing or treating an infection with a microorganism, such as a bacterium or a fungus, sensitive to at least one toxin or antibiotic produced by P. luminescens.\",\n \"The invention also relates to a pesticidal composition, in particular against insects, bacteria and/or fungi, according to the invention, for preventing or treating plants infested with animals, such as insects, or with a microorganism, such as a bacterium or a fungus, sensitive to at least one toxin or antibiotic produced by P. luminescens.\",\n \"The invention also comprises the use of a transformed cell according to the invention, for preparing a toxin or an antibiotic produced by P. luminescens.\",\n \"The expression “pharmaceutically acceptable vehicle” is intended to denote a compound or a combination of compounds making up a pharmaceutical composition causing no side effects and which makes it possible, for example, to facilitate administration of the active compound, to increase its lifetime and/or its effectiveness in the organism, to increase its solubility in solution or else to improve its conservation.\",\n \"These pharmaceutically acceptable vehicles are well known and will be adjusted by those skilled in the art as a function of the nature and of the method of administration of the active compound chosen.\",\n \"Preferably, these pharmaceutical compounds will be administered systemically, in particular intravenously, intramuscularly, intradermally or subcutaneously, or orally.\",\n \"Their methods of administration, dosages and pharmaceutical forms which are optimal can be determined according to the criteria generally taken into account in establishing a treatment suitable for a patient, such as, for example, the age or the body weight of the patient, the seriousness of his or her general condition, the tolerance to the treatment and the side effects observed.\",\n \"Finally, the invention comprises the use of a composition according to the invention, for preparing a medicinal product intended for the prevention or treatment of an infection with a microorganism, such as a bacterium or a fungus, sensitive to at least one toxin or antibiotic produced by P. luminescens.\",\n \"Moreover, a subject of the present invention is also a genomic DNA library of a bacterium of the genus Photorhabdus, preferably Photorhabdus luminescens , preferably the strain TT01.The genomic DNA libraries described in the present invention, in particular the BAC library deposited with the CNCM [French National Collection of Cultures and Microorganisms] on May 12, 2000, under No.\",\n \"I-2478 and which covers the Photorhabdus luminescens TT01 genome.\",\n \"The invention also relates to a method for identifying at least one nucleotide sequence of P. luminescens not present in the genome of another species of bacterium, in particular a pathogenic bacterium, and/or for identifying at least one nucleotide sequence of a genome of a bacterium, in particular a pathogenic bacterium, of a species other than P. luminescens and not present in the P. luminescens genome, characterized in that it comprises the following steps: a) the nucleotide sequences of P. luminescens according to the invention, or contained in a genomic library according to the invention, are aligned with the genomic sequence of the other bacterial species, or one of its fragments; and b) the data obtained with this alignment are processed in order to isolate and identify said sequence(s) only present in one or the other genome.\",\n \"Such a method, also called “subtractive genomic method”, can be used here to identify a sequence responsible for the pathogenicity of a bacterium, such as a gram-negative bacterium, for which the P. luminescens bacterium, which is not pathogenic, can serve as a model for comparison.\",\n \"The present invention thus relates to the methods for isolating a polynucleotide of interest present in a strain of Photorhabdus and absent from another strain or species, which use at least one DNA library based, for example, on a plasmid pcDNA2.1 containing the Photorhabdus genome.\",\n \"The method according to the invention for isolating a polynucleotide of interest can comprise the following steps: a) isolating at least one polynucleotide contained in a clone of the library of DNA of Photorhabdus origin; b) isolating: at least one genomic polynucleotide or cDNA of a bacterium, said bacterium belonging to a strain or species different from the Photorhabdus strain used to construct the DNA library of step a) or, alternatively, at least one polynucleotide contained in a clone of a DNA library prepared from the genome of a bacterium belonging to a strain or species different from the Photorhabdus strain used to construct the DNA library of step a), and hybridizing the polynucleotide of step a) to the polynucleotide of step b); c) selecting the polynucleotides of step a) which have not formed a hybridization complex with the polynucleotides of step b); d) characterizing the selected polynucleotide.\",\n \"The polynucleotide of step a) can be prepared by digesting at least one recombinant clone with a suitable restriction enzyme and, optionally, amplifying the polynucleotide insert which results therefrom.\",\n \"Thus, the method of the invention allows those skilled in the art to carry out comparative genomic studies between a bacterium of the genus Photorhabdus and between, for example, a pathogenic strain or species.\",\n \"In particular, it is possible to study and determine the regions of polymorphism between said strains.\",\n \"The present invention also relates to the use of the nucleic acid sequences or of the polypeptides according to the invention: for preparing biopesticides, in particular entomotoxins, antibiotics, antifungal agents or cytotoxins, for secreting proteins, as virulence factors, for control via quorum-sensing, for identifying targets for human diseases for which Photorhabdus luminescens is a model (in particular the plague or whooping cough), and for identifying targets against pathogenic Gram-negative bacteria using the subtractive genomic method (such as, for example, by comparison with E. coli or other pathogenic gram-negative bacteria).\",\n \"The present invention also relates to the use of the polypeptides according to the present invention, for screening compounds capable of modulating the activity of these polypeptides, in particular the polypeptides with enzymatic activity.\",\n \"Other characteristics and advantages of the invention appear in the following examples: EXAMPLES Example 1 Materials and Methods The strategy for sequencing the genome of Photorhabdus luminescens strain TT01 is based on random (shotgun) sequencing.\",\n \"The first step of this study consisted in cloning the genomic DNA of the Photorhabdus luminescens bacterium into various vectors (plasmids and BACs).\",\n \"Photorhabdus luminescens genomic DNA libraries used.\",\n \"Three genomic DNA libraries were prepared: I)—A genomic DNA library in a high copy number bacterial vector (pcDNA2.1, Invitrogen).\",\n \"Average insert size 1.5 kb.\",\n \"II)—A genomic DNA library in a low copy number bacterial vector (pSYX34).\",\n \"Average insert size 10 kb.\",\n \"III)—A genomic DNA library in a BAC vector (pBeloBAC11, California Institute of Technology).\",\n \"Average insert size 50 to 100 kb.\",\n \"Two batches of DNA of the TT01 strain of the Photorhabdus luminescens bacterium were extracted on Oct. 30, 1998 and Apr.\",\n \"14, 1999.I) Establishing a Genomic DNA Library in the Bacterial Vector pcDNA2.1 (Invitrogen) A) Preparation of the Vector: pcDNA2.1 We prepared the plasmid pcDNA2.1 by carrying out two midipreps (QIAGEN KIT) in parallel according to the conditions recommended by the manufacturer.\",\n \"The vector pcDNA2.1 was digested with the BstX1 restriction enzyme.\",\n \"B) Preparation of the Chromosomal DNA of Photorhabdus luminescens Strain TT01 and Ligation into the Vector pcDNA2.1 1) Dissolving of the DNA A dry pellet of genomic DNA of the strain TT01, prepared on Oct. 30, 1998, was taken up in 200 μl of 10:1 TE, and then dissolved for 30 minutes at 65° C. Its concentration was estimated at 0.15 μg/μl.\",\n \"2) Nebulization of the DNA 50 μl of genomic DNA strain TT01 in an amount of H2O sufficient for 2 ml were nebulized for 45 sec at a pressure of 1 bar of nitrogen, and centrifuged for 2 min at 600 rpm in order to recover the entire volume.\",\n \"3) Precipitation of the DNA This DNA was then precipitated with sodium acetate (2 ml of DNA+0.2 ml of 3M Na acetate, pH 5.2,+5 ml of absolute ethanol; 2 h at −20° C.), centrifuged for 30 min at 14 000 rpm and at 4° C., and then redissolved in 100 μl of water.\",\n \"4) Analysis of the DNA 4 μl were loaded onto a 1% agarose TBE gel.\",\n \"DNA fragments of the expected size of 500 bp to 3 kb were visualized.\",\n \"5) Repair of the DNA In order to blunt end the DNA fragments, repair was carried out using T4 DNA polymerase.\",\n \"The following are mixed in 2 separate tubes: 48 μl of DNA from step 3), 100 μl of H2O, 20 μl of 5× ligation buffer, 2 μl of dNTP mix (10 mM), 5 μl of T4 DNA polymerase (Boehringer).\",\n \"Incubation is carried out for 25 min at ambient temperature and the reaction is then stopped by heating for 15 min at 75° C. 6) Precipitation of the DNA This DNA was then precipitated overnight at −20° C. with sodium acetate (1/10 volume of Na acetate and 2.5 volume of absolute ethanol) and centrifuged, and the DNA pellet obtained was then air-dried and redissolved in 30 μl of water.\",\n \"7) Ligation of the DNA This DNA was ligated overnight at 16° C. with the Bstx A+Bstx B linkers (Invitrogen) in the presence of 2 μl of ligase.\",\n \"8) Preparation of the Inserts After migration of the DNA ligated to the linkers in a 1% agarose TAE gel, at 70 volts, the region of interest (between 1 and 3 Kb) was cut into four fragments.\",\n \"9) Purification of the Inserts The agarose fragments containing the regions of interest were purified using Geneclean (BIO101) according to the conditions recommended by the manufacturer.\",\n \"An aliquot was loaded onto an agarose minigel in order to validate the quality of the purification.\",\n \"10) Ligation of the Inserts into the Vector pcDNA2.1 (Invitrogen) 4 μl of DNA (insert after the Geneclean purification step, step 9), 2 μl of plasmid pcDNA2.1 (after the BstX1 digestion step and then Geneclean purification), 2 μl of ligation buffer (10×), add 2 μl of ligase, 10 μl H2O (total volume: 20 μl).\",\n \"The mixture is incubated overnight at 16° C. 11) Transformation of Ultracompetent XL2 Blue Cells (Stratagene) The genomic DNA library obtained in step 10 was integrated into ultracompetent XL2 Blue cells (Stratagene) according to the conditions recommended by the manufacturer.\",\n \"Analysis of the inserts of 24 clones by digestion with the PvuII restriction enzyme and by sequencing of the ends was carried out and gave satisfactory results.\",\n \"II) Establishing a Genomic DNA Library in the Bacterial Vector pSYX34 The PARTIAL FILL-IN technique, developed in the laboratory, allowed us to construct a library of genomic DNA of the TT01 strain of the bacterium Photorhabdus luminescens , cloned into the plasmid pSYX34 (average insert size 10 Kb).\",\n \"Note: in this case, digestion of the vector pSYX34 with the Sal I restriction enzyme frees the following ends: 5′TCGAC-G-5′ The PARTIAL FILL-IN was carried out in the presence of dCTP and dTTP deoxynucleotides.\",\n \"5′TCGAC-CTG-5′ A) Preparation of the Vector: pSYX34 1) Production of the Vector Plasmid pSYX34 was prepared by carrying out two midipreps, QIAGEN KIT, in parallel according to the conditions recommended by the manufacturer.\",\n \"2) Digestion of the Plasmid pSYX34 with the Sal I Restriction Enzyme The final volume will be 100 μl: 20 μl pSYX34, 10 μl of buffer H, 66 μl of H2O, 4 μl Sal I.\",\n \"The mixture is incubated for 2 h at 37° C. An aliquot and also a molecular weight marker were loaded onto an agarose minigel in order to validate the quality of the purification.\",\n \"3) Chloroform Extraction Chloroform extraction makes it possible to stop the enzyme digestion and to remove any traces of protein.\",\n \"After digestion, 95 μl are recovered, 105 μl of 10 mM TE are added, 200 μl of chloroform are added.\",\n \"The mixture is vortexed and centrifuged for 1 min at 1 000 rpm.\",\n \"The aqueous phase (upper phase) is recovered.\",\n \"4) Precipitation with Sodium Acetate 1/10 volume of sodium acetate (3M, pH: 5.2), i.e.\",\n \"20 μl, are added, followed by 2.5 volumes of clean absolute ethanol, i.e.\",\n \"500 μl (bottle kept at −20° C.).\",\n \"The mixture is left at −20° C. for at least 1 hour (it is possible to leave it at −20° C. overnight).\",\n \"The mixture is centrifuged at 4° C. (cold room) for 30 min at 14 000 rpm.\",\n \"The supernatant is removed using the vacuum pump.\",\n \"400 μl of 70% ethanol are added and the mixture is centrifuged at 4° C. for 5 min at 14 000 rpm.\",\n \"The supernatant is removed using the vacuum pump and the pellet is left to dry for approximately one hour on the bench.\",\n \"The pellet is resuspended in 20 μl of 10 mM TE (1/10).\",\n \"5) Partial Fill-in Final volume of the reaction: 50 μl; 20 μl of pSYX34 digested with Sal I 5 μl of synthesis buffer, 2.5 μl of 1 mM nucleotide (C-T) mix, 20.5 μl of water, 2 μl of Klenow (2 U/μl).\",\n \"The mixture is left for 30 min at ambient temperature.\",\n \"Verification is carried out on a 1% agarose minigel, followed by freezing at −20° C. Mixture of C-T Nucleotides: Mix: 2 μl of T nucleotides (100 mM), 2 μl of C nucleotides (100 mM), 16 μl of 10 mM TRIS, pH=7.5.The nucleotides are thus diluted to 1/10, for a concentration of 10 mM.\",\n \"A second dilution to 1/10 in 10 mM TRIS buffer will be carried out so as to have a concentration of 1 mM.\",\n \"In addition, during the reaction, the nucleotides are diluted to 1/20 (2.5 μl in 50 μl), thus obtaining a final concentration of nucleotides of 50 μM.\",\n \"6) Purification of the Vector pSYX34: Preparative Gel Prepare a 1% agarose TAE gel, large wells will be provided for the samples.\",\n \"Take 30 μl of pGB2 after the partial fill-in (20 μl will therefore remain in the freezer), add 6 μl of loading solution.\",\n \"In parallel, load the molecular weight marker in the following proportions: 5 μl H2O, 5 μl of the 1 Kb DNA marker, 2 μl of the loading solution.\",\n \"Allow to migrate at 70 volts.\",\n \"Cut the gel on the marker side using a scalpel and locate, by taking a photo and using a ruler, the region of interest; in our case, the band corresponding to 4 Kb will be taken.\",\n \"Each sample will be cut in half.\",\n \"The weight of each sample will indicate its approximate volume.\",\n \"There will therefore be four samples to purify by the Geneclean II technique.\",\n \"7) Purification of the Vector pSYX34 Take up in 3 volumes of sodium iodide solution (1 ml for a sample of 0.2 g), place at 49° C., shaking every 2 minutes, until the agarose has completely dissolved.\",\n \"Add 8 μl of microbeads: GLASSMILK R, leave at ambient temperature for 5-10 min, the DNA attaches to the microbeads.\",\n \"Centrifuge for 2 min at 14 000 rpm, remove the supernatant by suction.\",\n \"Wash 3 times with the New Wash solution (600 μl/eppendorf, vortex, centrifuge for a few seconds at 10 000 rpm); this solution was prepared and is kept at −20° C. Resuspend the pellet in 10 μl of H2O, vortex, leave for 5 min at 49° C. This allows the DNA to detach from the microbeads.\",\n \"Centrifuge for 2 min at 14 000 rpm.\",\n \"Recover the supernatants in new eppendorfs (No.\",\n \"1 and No.\",\n \"2, etc) Add 10 μl of H2O again to the pellets, so as to be sure to recover everything, vortex, leave for 2 min at 49° C. Centrifuge for 2 min at 14 000 rpm.\",\n \"Recover the supernatants in the preceding eppendorfs (No.\",\n \"1 and No.\",\n \"2, etc).\",\n \"There are approximately 20 μl of supernatant, centrifuge for 2 min at 14 000 rpm.\",\n \"Transfer the supernatants into new eppendorfs, so as to be sure that there are no longer any microbeads.\",\n \"Load 1 μl of each preparation (+5 μl of H2O+2 μL of loading solution) onto a 1% agarose minigel, allow to migrate at 80 volts for 2 hours.\",\n \"The most concentrated samples will be kept, they will then be frozen at −20° C. B) Preparation of the Chromosomal DNA of Photorhabdus luminescens Strain TT01 1) Dissolving of the DNA A dry pellet of genomic DNA of the strain TT01 was taken up in 200 μl of 10:1 TE and then dissolved for 30 min at 65° C. Its concentration was estimated at 0.15 μg/μl.\",\n \"2) Partial Digestion of the Genomic DNA with the Sau3A Restriction Enzyme 15 μl of DNA were digested with 2, 1, 0.5, 0.25, 0.125, 0.0625 or 0.03125 units of Sau3A restriction enzyme for 1 h at 37° C. 3) Chloroform Extraction Chloroform extraction makes it possible to stop the enzyme digestion and to remove any traces of protein.\",\n \"After digestion, 100 μl of chromosomal DNA are recovered.\",\n \"100 μl of 10 mM TE are added, 200 μl of chloroform are added.\",\n \"The mixture is vortexed and centrifuged for 1 min at 1 000 rpm.\",\n \"The aqueous phase, the upper phase, comprising the chromosomal DNA is recovered.\",\n \"4) Precipitation with Sodium Acetate 1/10 volume of sodium acetate (3M, pH: 5.2), i.e.\",\n \"20 μl, are added.\",\n \"2.5 volumes of clean absolute ethanol, i.e.\",\n \"500 μl, (bottle kept at −20° C.) are then added.\",\n \"The mixture is left at −20° C. for at least 1 hour (it is possible to leave it at −20° C. overnight).\",\n \"The mixture is centrifuged at 4° C. (cold room) for 30 min at 14 000 rpm.\",\n \"The supernatant is removed using the vacuum pump.\",\n \"400 μl of 70% ethanol are added, the mixture is centrifuged at 4° C. for 5 min at 14 000 rpm.\",\n \"The supernatant is removed using the vacuum pump and the pellet is left to dry for approximately one hour on the bench.\",\n \"The pellet is resuspended in 20 μl of H2O.\",\n \"5) Verification of the Partial Digestions, after Precipitation with Sodium Acetate.\",\n \"A 1% agarose TBE gel is prepared.\",\n \"1/10 of the total volume of the precipitations, i.e.\",\n \"2 μl of DNA+8 μl of H2O+2 μl of loading solution, is loaded.\",\n \"In parallel, a molecular weight marker is loaded: 1 μl+9 μl of H2O+2 μl of loading solution.\",\n \"6) Partial Fill-in Final volume of the reaction: approximately 50 μl 36 μl of partially digested DNA (DNA digested with 0.25, 0.125, 0.0625 or 0.03125 units of Sau3A restriction enzyme) 5 μl of synthesis buffer 10 μl of 1 mM nucleotide (A-G) mix=>final concentration: 200 μM 2 μl of Klenow (2 U/μl).\",\n \"The mixture is left at ambient temperature for 30 min.\",\n \"Mixture of A-G Nucleotides Mix: 2 μl of A nucleotides (100 mM) 2 μl of G nucleotides (100 mM) 16 μl of 10 mM TRIS, pH=7.5.The nucleotides are thus diluted to 1/10, giving a concentration of 10 mM.\",\n \"A second dilution to 1/10 in H2O will be carried out, a concentration of 1 mM is therefore obtained.\",\n \"7) Chloroform Extraction Chloroform extraction makes it possible to stop the enzyme digestion and to remove any traces of protein.\",\n \"After digestion, 53 μl of chromosomal DNA are recovered.\",\n \"47 μl of 10 mM TE are added.\",\n \"100 μl of chloroform are added.\",\n \"The mixture is vortexed and centrifuged for 1 min at 1 000 rpm.\",\n \"The aqueous phase, the upper phase, comprising the chromosomal DNA is recovered.\",\n \"8) Precipitation with Sodium Acetate 1/10 volume of sodium acetate (3M, pH: 5.2), i.e.\",\n \"10 μl, are added.\",\n \"2.5 volumes of clean absolute ethanol, i.e.\",\n \"250 μl, (bottle kept at −20° C.) are then added.\",\n \"The mixture is left at −20° C. for at least 1 hour (it is possible to leave it at −20° C. overnight).\",\n \"The mixture is centrifuged at 4° C. (cold room) for 30 min at 14 000 rpm.\",\n \"The supernatant is removed using the vacuum pump.\",\n \"400 μl of 70% ethanol are added, the mixture is centrifuged at 4° C. for 5 min at 14 000 rpm.\",\n \"The supernatant is removed using the vacuum pump and the pellet is left to dry for approximately one hour on the bench.\",\n \"The pellet is resuspended in 20 μl of H2O.\",\n \"9) Purification of the Chromosomal DNA: Preparative Gel Prepare a 1% agarose TAE gel, large wells will be provided for the samples.\",\n \"Take the entire DNA, i.e.\",\n \"20 μl, add 4 μl of loading solution.\",\n \"In parallel, load the molecular weight marker in the following proportions: 5 μl H2O 5 μl of the 1 Kb DNA marker 2 μl of the loading solution.\",\n \"Allow to migrate at 70 volts.\",\n \"Cut the gel on the marker side using a scalpel and locate, by taking a photo and using a ruler, the region of interest.\",\n \"Since the DNA fragments are long, SPIN X columns from COSTAR will be used to purify the DNA, this will avoid cleaving the chromosomal DNA.\",\n \"10) Purification: SPIN X Column After having recovered the various pieces of agarose, in SPIN X columns, add 200 μl of 10 mM TE and then grind using a spatula.\",\n \"This may be kept at 4° C. Then, centrifuge for 20 min at 5 000 rpm.\",\n \"The agarose will be retained on the filters, whereas the chromosomal DNA will be found at the bottom of the tube.\",\n \"11) Chloroform Extraction Chloroform extraction makes it possible to remove any traces of protein.\",\n \"800 μl of chromosomal DNA are recovered.\",\n \"800 μl of chloroform are added.\",\n \"The mixture is vortexed and centrifuged for 1 min at 1 000 rpm.\",\n \"The aqueous phase, the upper phase, comprising the chromosomal DNA is recovered.\",\n \"12) Precipitation with Sodium Acetate 1/10 volume of sodium acetate (3M, ph: 5.2), i.e.\",\n \"80 μl, are added.\",\n \"800 μl of isopropanol are then added.\",\n \"The mixture is left at −20° C. for at least 1 hour (it is possible to leave it at −20° C. overnight).\",\n \"The mixture is centrifuged at 4° C. (cold room) for 30 min at 14 000 rpm.\",\n \"The supernatant is removed using the vacuum pump.\",\n \"400 μl of 70% ethanol are added and the mixture is centrifuged at 4° C. for 5 min at 14 000 rpm.\",\n \"The supernatant is removed using the vacuum pump, and the pellet is allowed to dry for approximately half an hour on the bench and 2 min under vacuum.\",\n \"The pellet is resuspended in 20 μl of TE (0.1×, i.e.\",\n \"1 mM).\",\n \"The suspension is incubated for 10 min at 65° C. The suspension is incubated for approximately 4 hours at 4° C., so that the DNA is well resuspended.\",\n \"Verification is carried out on a 1% agarose minigel with a molecular weight marker placed in parallel.\",\n \"C) Ligation of the chromosomal DNA of Photorhabdus luminescens Strain TT01 into the Vector pSYX34 Take 6 μl of chromosomal DNA.\",\n \"Add 2 μl of pSYX34.Add 2 μl of ligation buffer (LB).\",\n \"Add 2 μl of ligase.\",\n \"Then add 8 μl of H2O (total volume: 20 μl) In parallel, prepare a ligation control by replacing the chromosomal DNA with 0.1× TE (1 mM).\",\n \"Vortex, leave overnight at 16° C. Transformation of Ultracompetent XL10-Gold Kanr Cells (Stratagene) The genomic DNA library obtained in the preceding step was integrated into ultracompetent XL10 Gold Kanr cells (Stratagene) according to the conditions recommended by the manufacturer.\",\n \"Analysis of the inserts of 24 clones by digesting with the SalI restriction enzyme and by sequencing of the ends was carried out and gave satisfactory results.\",\n \"III) Establishing a Genomic DNA Library in the Bacterial Vector pBeloBAC11 (California Institute of Technology) Construction of the BAC Library Partial digestion of the DNA in pieces of agarose: the pieces of agarose are washed three times for 15 minutes at ambient temperature in a solution of TE (1×) with moderate agitation; the pieces of agarose are equilibrated twice in 300 μl of a Hind III digestion buffer (1×) (Boehringer or Biolabs) for 30 minutes at ambient temperature; the digestion buffer is removed and an ice-cold Hind III digestion buffer (1 ml per piece of agarose) containing 20 U of Hind III (Boehringer or Biolabs) is added; incubation is carried out for two hours in ice; the pieces of agarose are transferred to a water bath at 37° C. and incubation is carried out for 10 minutes to 30 minutes (depending on the DNA contained in the pieces of agarose); and the digestion is stopped by adding 100 μl of a 250 mM EDTA solution (pH 8).\",\n \"Size Selection: Separation of the Partially Digested DNA by PFGE Using the CHEF DRIII Apparatus (Bio-Rad): gel of 1% LMP agarose in a tris-acetate-EDTA buffer (1×) at 13° C., reverse the current every 3 to 15 seconds at 6 V/cm for 16 hours, load at least two pieces of agarose and the marker, stain the marker and one lane or a part of the lane to verify the partial digestion, excise the bands (unstained part) of various sizes (i.e.\",\n \"from 50 to 100 kb and from 150 to 250 kb), the agarose bands can be stored at 4° C. in a TE solution.\",\n \"Preparation of the Vector: isolation of pBeloBACII by the cesium chloride method; digestion with Hind III; dephosphorylation with CIP (optionally with HK phosphatase).\",\n \"Ligation and Transformation: the agarose banks containing the DNA are melted at 60° C. for 10 minutes; the molten solution of agarose/DNA is equilibrated for 15 minutes at 45° C.; gelase (Epicentre Technologies) is added in a proportion of 1 U per 100 μl of gel band (do not add digestion buffer, which causes certain problems at the time of ligation); the digestion is carried out for 1 hour at 45° C.; the ligation is carried out in 50 μl of a solution containing pBeloBACII (2 μl), T4 ligase (1 μl at 1:10), a 10× T4 buffer (5 μl ), and the DNA/agarose solution (42 μl), incubation is for 20 hours at 16° C.; the ligation medium is heated for 15 minutes at 65° C.; the ligation medium is dialyzed against a Tris-EDTA buffer, using Millipore membranes of pore size VS 0.025 mM; 1 or 2 μl of the ligation solution are introduced by electroporation into E. coli DH10B cells (Gibco BRL) using electroporation cuvettes with a width of 2 mm, with the following settings: 2.5 kV, 25 μF and 200Ω; after electroporation, the cells are resuspended in 600 μl of SOC or NYZ medium, and then incubation is carried out for 45 minutes at 37° C. with agitation; 10 and 100 μl of each cell suspension are plated out in an LB agar medium containing chloramphenicol (12.5 μg/ml), X-Gal (5-bromo-4-chloro-3-indolyl-α-D-glucuronic acid; 50 μg/ml) and IPTG (isopropyl-α-D-thiogalactopyranoside; 25 μg/ml); and the dishes thus obtained are incubated overnight.\",\n \"Extraction of the Plasmid DNA and Sequencing of the Inserts The plasmid DNA was extracted by the alkaline lysis technique in 24-well plates.\",\n \"The inserts of the clones were sequenced using the PE Big Dye kit according to the conditions recommended by the manufacturer, using the oligonucleotides specific for each vector.\",\n \"The reactions are then introduced into the thermocycler in order to undergo 35 cycles composed of the following three steps: denaturation for 10 seconds at 96° C., hybridization for 10 seconds at 50° C., elongation for 4 minutes at 56° C. A precipitation with 76% ethanol is then carried out for 20 minutes at ambient temperature.\",\n \"The plates are then centrifuged for 35 minutes at 2 200 g, then drained on absorbent paper, and then centrifuged turned upside down on absorbent paper for 1 minute at 500 g in order to leave no trace of ethanol.\",\n \"The DNA pellets are then: either taken up in a solution of formamide-EDTA-dextran blue, and then denatured for 2 minutes at 96° C. The plates are then immediately placed in ice, an aliquot then being loaded onto an acrylamide gel of a PE-377 automatic sequencer, or taken up in a 0.3 mM EDTA solution, an aliquot then being automatically loaded into a PE-3700 automatic sequencer.\",\n \"Assembly of the Sequences Obtained The genomic DNA sequences were assembled in a contig using the PHRED-PHRAP software and visualized using the CONSED software.\",\n \"Analysis and Annotation The contigs were first of all analyzed and annotated automatically using the GMPTB software.\",\n \"Example 2 The Genes of Interest The various characteristics associated with the way of life of Photorhabdus luminescens and reported in the literature (Forst et al., 1997; Hu et al., 1998, etc.)\",\n \"led to the presence of genes involved in the biosynthesis of antibiotics and of toxins being sought in the genome of this bacterium.\",\n \"The Operons for Biosynthesis of Antibiotics The search for peptide synthetases was undertaken in silico.\",\n \"To do this, we initially used the various motifs conventionally described in the literature (Stachelhaus & Marahiel, 1995, FEMS Microbiology Letters 125:3-14) as a probe in order to locate, by sequence homology, in the Photorhabdus luminescens genome, genes which may be involved in the biosynthesis of antibiotics.\",\n \"Adenylation Modules 1 - LKAGGAYYVPID 2 - YSGTTGxPKGV 3 - GELCIGGXGxARGYL 4 - YxTGD 5 - VKIRGxRIELGEIE Thioester Formation Module 6 - DNFYxLGGHSL N-Methylation Module VLE/DxGxGxG Peptide Elongation Module His-HHILxDGW Peptide Racemization Module (Optional) His-HHILxDGW A - AYxTExNDILLTAxG B - EGHGRExIIE C - RTVGWFTSMYPxxLD D - FNYLGQFD Among these various modules, some enabled us to identify, by homology search using the BLASTP—basic local alignment search tool program—(Altschul S F et al., 1990, J. Mol.\",\n \"Biol., 215:403-10), genes encoding proteins containing some of these motifs.\",\n \"The genes thus identified, probably encoding proteins involved in the biosynthesis of antibiotics, were cataloged and appear in particular in table I.\",\n \"Emphasis was placed particularly on the genes homologous to the syr E gene of Pseudomonas syringae or to the nosD gene.\",\n \"REFERENCES Eric Guenzi, Giuliano Galli, Ingeborg Grgurina, Dennis C. Gross, and Guido Grandi, 1998.Characterization of the Syringomycin Synthetase Gene Cluster.\",\n \"A link between prokaryotic and eukaryotic peptide synthetases.\",\n \"J. Biol.\",\n \"Chem., 273:32857-32863.Bondi M, Messi P, Sabia C, Baccarani Contri M, Manicardi G., 1999.Antimicrobial properties and morphological characteristics of two Photorhabdus luminescens strains.\",\n \"New Microbiol., 22:117-27.The Operons for Biosynthesis of Toxins As for the genes for biosynthesis of antibiotics, we sought, by sequence homology, in the Photorhabdus luminescens genome, genes potentially encoding toxic proteins.\",\n \"We identified many genes encoding proteins having these criteria.\",\n \"Entomotoxins (Tc loci).\",\n \"Homologs of the Tox A gene of Clostridium difficile.\",\n \"Hemolysins.\",\n \"Genes potentially encoding proteins homologous to the RTX toxins of Vibrio cholerae.\",\n \"Genes potentially encoding proteins homologous to the tphA and icmF genes of Legionella pneumophila (Purcell M, Shuman H A, 1998.The Legionella pneumophila icmGCDJBF genes are required for killing of human macrophages.\",\n \"Infect Immun., 66:2245-55).\",\n \"See annotation of these genes in table I below.\",\n \"REFERENCES Ffrench-Constant R, Bowen D., 1999.Photorhabdus toxins: novel biological insecticides.\",\n \"Curr Opin Microbiol., 2:284-8.Review.\",\n \"Blackburn M, Golubeva E, Bowen D, Ffrench-Constant R H, 1998.A novel insecticidal toxin from Photorhabdus luminescens, toxins complex a (Tca), and its histopathological effects on the midgut of manduca sexta.\",\n \"Appl Environ Microbiol., 64:3036-41.Bowen D J, Ensign J C, 1998.Purification and characterization of a high-molecular-weight insecticidal protein complex produced by the entomopathogenic bacterium Photorhabdus luminescens .\",\n \"Appl Environ Microbiol., 64:3029-35.Bowen D, Rocheleau T A, Blackburn M, Andreev 0, Golubeva E, Bhartia R, Ffrench-Constant R H, 1998.Insecticidal toxins from the bacterium Photorhabdus luminescens.\",\n \"Science., 280:2129-32.Guo L, Fatig R O 3rd, Orr G L, Schafer B W, Strickland J A, Sukhapinda K, Woodsworth A T, Petell J K, 1999.Photorhabdus luminescens W-14 insecticidal activity consists of at least two similar but distinct proteins.\",\n \"Purification and characterization of toxin A and toxin B. J. Biol Chem., 274:9836-42.GENERAL REFERENCES 1.Altschul, S. F., T. L. Madden, A.\",\n \"A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman.\",\n \"1997.Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.\",\n \"[Review] [90 refs].\",\n \"Nucleic Acids Research.\",\n \"25:3389-402.2.Birnboim, H. C. 1983.A rapid alkaline extraction method for the isolation of plasmid DNA.\",\n \"Methods Enzymol.\",\n \"100:243-255.3.Braun, L. F. Nato, B. Payrastre, J. C. Mazie, and P. Cossart.\",\n \"1999.The 213-amino-acid leucine-rich repeat region of the Photorhabdus luminescens InIB protein is sufficient for entry into mammalian cells, stimulation of P1 3-kinase and membrane ruffling.\",\n \"Mol.\",\n \"Microbiol.\",\n \"34:10-23.4.Buchrieser, C., C. Rusniok, L. Frangeul, E. Couvé, A. Billault, F. Kunst, E. Carniel, and P. Glaser.\",\n \"1999.The 102 kb locus of Yersinia pestis: sequence analysis and comparison of selected regions among different Yersinia pestis and Yersinia pseudotuberculosis strains.\",\n \"Infect.\",\n \"Immun.\",\n \"67:4851-4861.5.Ewing, B., and P. Green.\",\n \"1998.Base-calling of automated sequencer traces using phred.\",\n \"II.\",\n \"Error probabilities.\",\n \"Genome Res.\",\n \"8:186-194.6.Fitch, W. S. 1970.Distinguishing homologous from analogous proteins.\",\n \"Syst.\",\n \"Zool.\",\n \"19:99-113.7.Frangeul, L., K. E. Nelson, C. Buchrieser, A. Danchin, P. Glaser, and K. F. 1999.Cloning and assembly strategies in microbial genome projects.\",\n \"Microbiology.\",\n \"145:2625-2634.8.Gordon, D., C. Abajian, and P. Green.\",\n \"1998.Consed: a graphical tool for sequence finishing.\",\n \"Genome Res.\",\n \"8:195-202.9.Jacquet, C., J. Bille, and J. Rocourt.\",\n \"1992.Typing of Photorhabdus luminescens by restriction polymorphism of the ribonucleic acid gene region.\",\n \"Zentralbl Bakteriol.\",\n \"276:356-365.10.Li, P., K. C. Kupfer, C. J. Davies, D. Burbee, G. A. Evans, and H. R. Garner.\",\n \"1997.PRIMO: A primer design program that applies base quality statistics for automated large-scale DNA sequencing.\",\n \"Genomics.\",\n \"40:476-485.11.Lukashin, A. V., and M. Borodovsky.\",\n \"1998.GeneMark.hmm: new solutions for gene finding.\",\n \"Nucleic Acids Res.\",\n \"15:1107-1115.Scientific Description of the Photorhabdus luminescens Strain TT01 BAC Library Deposited with the CNCM [French National Collection of Cultures and Microorganisms] on May 12, 2000, Under the Number I-2478 Collection of Escherichia coli DH 10B™ clones (Calvin et al., J. Bacteriol.\",\n \"170, 2796, 1988) containing genomic DNA fragments from the Photorhabdus luminescens strain TT01 bacterium, cloned into the vector pBelo BACII (Kim et al., Genomics, 34, 213, 1996) at the Hind III site.\",\n \"The average insert size is 60 kb.\",\n \"Databanks Local revisions of main public banks were used.\",\n \"The protein bank used consists of the nonredundant fusion of these banks, such as the Genpept bank (automatic translation of GenBank and NCBI).\",\n \"Use was made of the BLAST software package (public domain, Altschul et al., 1990) for searching for homologies between a sequence and protein or nucleic acid databanks.\",\n \"The significance thresholds used depend on the length and on the complexity of the region tested and also on the size of the reference bank.\",\n \"They were adjusted and adapted to each analysis.\",\n \"The results of the search for homologies between a sequence according to the invention and protein or nucleic acid databanks are given and summarized in table I and table II below.\",\n \"Table I List of putative functions (column “homology with nonredundant protein database-description”, and column “annotations” for the sequences selected as being involved in an activity or having an activity of the toxin or antibiotic type) of the proteins of sequences SEQ ID No.\",\n \"42 to SEQ ID No.\",\n \"3855 encoded by their respective nucleotide sequence of the Photorhabdus luminescens strain TT01 genome (genome represented by the sequences of the 41 contigs (SEQ ID No.\",\n \"1 to SEQ ID No.\",\n \"41)).\",\n \"The nucleic acid sequences of the proteins of sequence SEQ ID No.\",\n \"42 to SEQ ID No.\",\n \"3855 can be easily identified by their start position (“start” column) and end position (“end” column) on each one of the contig sequences (“contig” column).\",\n \"Legend of Table I: All of the putative functions associated with the proteins of sequence SEQ ID No.\",\n \"42 to SEQ ID No.\",\n \"3855 were obtained through a homology search using in particular BLASTP software (Altschul et al., 1990).\",\n \"The significant identities, or the presence of various motifs (cf.\",\n \"examples) representative of these functions, were in particular taken into account.\",\n \"The description of the most probable function(s) is given in the “homology with” and “Annotation” column.\",\n \"Table II List of putative functions (column “function obtained by comparison on databank”) of the proteins encoded by the sequences SEQ ID No.\",\n \"5835 to SEQ ID No.\",\n \"10784 of the Photorhabdus luminescens strain TT01 genome (genome here represented by the sequences of the 9 contigs (SEQ ID No.\",\n \"5826 to SEQ ID No.\",\n \"5834)).\",\n \"The nucleic acid sequences of sequence sequences SEQ ID No.\",\n \"5835 to SEQ ID No.\",\n \"10784 can be easily identified by their start position (“CONTIG”, “from” column) and end position (“CONTIG” “to” column) on each one of the contig sequences.\",\n \"Legend of Table II: All of the putative functions associated with the proteins encoded by the sequences SEQ ID No.\",\n \"5835 to SEQ ID No.\",\n \"10784 were obtained through a homology search using in particular BLASTP software (Altschul et al., 1990).\",\n \"The significant identities, or the presence of various motifs (cf.\",\n \"examples) representative of these functions, were in particular taken into account.\",\n \"The description of the most probable function(s) is given in the “homology with” and “Annotation” column.\",\n \"In the final column, “HOMOLOGOUS TO SEQUENCE SEQ ID”, the term “#N/A” means that no homology for the sequence concerned had been identified with one of the sequences SEQ ID Nos.\",\n \"42 to 3855.When a homologous sequence was found among the sequences SEQ ID Nos.\",\n \"42 to 3855 for the sequence concerned, this is mentioned via its SEQ ID number in this column.\",\n \"LENGTHY TABLE REFERENCED HERE US20070020625A1-20070125-T00001 Please refer to the end of the specification for access instructions.\",\n \"LENGTHY TABLE REFERENCED HERE US20070020625A1-20070125-T00002 Please refer to the end of the specification for access instructions.\",\n \"LENGTHY TABLE The patent application contains a lengthy table section.\",\n \"A copy of the table is available in electronic form from the USPTO web site () An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10467478"}}},{"rowIdx":367,"cells":{"text":{"kind":"list like","value":[["Benzo'fisoindole derivatives with affinity to the ep4 receptor","The present invention relates to a compound of formula (I): corresponding pharmaceutical compositions, preparation processes, and/or methods of using the aforementioned compounds and/or compositions."],["1.A compound of formula (I): wherein: R1 and R3 are the same or different and represent ═O, hydrogen, C1-6alkyl, C1-6-dialkyl, ═CHC1-C5alkyl, ═S, or a 5- or 6-membered aryl; R4 to R9 are the same or different and represent hydrogen, C1-6alkoxy, OCF3, OCH2CF3, O-cyclopropyl, OCH2-cyclopropyl, C1-C6alkyl, S-alkyl, NR210 where R10 is hydrogen or C1-6alkyl, halogen, NO2, OH, CH2OC1-C6alkyl, CH2OH, or CF3; Q1 is hydrogen, C1-6alkyl, C1-6dialkyl, C1-6alkoxy, NHAc, NR210 where R10 is hydrogen or C1-6alkyl, difluoro, fluoro, ═O, or OH; Q2, Q3, Q4 and Q5 are the same or different and represent hydrogen, C1-6alkoxy, OCF3, OCH2CF3, O-cyclopropyl, OCH2-cyclopropyl, C1-C6alkyl, S-alkyl, NR210 where R10 is hydrogen or C1-6alkyl, halogen, NO2, OH, CH2OC1-C6alkyl, CH2OH, or a 5- or 6-membered aryl; provided that the compound of formula (I) is not: [4-(1-oxo-1,3-dihydro-2H-benzo [f]isoindol-2-yl)phenyl]-2-propionic acid, and sodium salt and [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; and pharmaceutically acceptable derivatives thereof.","2.A compound according to claim 1 wherein R1 is ═O.","3.A compound according to claim 1 wherein R3 is methyl, dimethyl, 2-furanyl, ═O or hydrogen.","4.A compound according to claim 1 wherein R4 and R9 are each hydrogen, methoxy, ethoxy, i-propoxy, n-propoxy, n-butyloxy or n-hexyloxy.","5.A compound according to claim 1 wherein R6 and R7 are each hydrogen, methyl, methoxy, chlorine, bromine, iodine, NO2, or CF3.6.A compound according to claim 1 wherein Q1 is hydrogen, methyl, ethyl, NHAc, NH2 or methoxy.","7.A compound according to claim 1 wherein Q2, Q3, Q4 and Q5 are each hydrogen, methyl, methoxy, chlorine, bromine, iodine, 2-thiophenyl, 3-thiophenyl, 2-furanyl, 3-furanyl, or phenyl.","8.A compound of formula (I) wherein said compound is selected from the group consisting of: [4-(4,9-dimethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-di-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dipropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4,9-dibutoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dihexyloxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4-ethoxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid and [4-(9-ethoxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-isopropoxy-4-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid and [4-(4-isopropoxy-9-propoxy-1-oxo-1,3-dihydro-2H-benzo [f]isoindol-2-yl)phenyl]acetic acid; [4-(4-hexyloxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid and [4-(9-hexyloxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4,9-di-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4,9-dipropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4,9-dibutoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4,9-dihexyloxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4-ethoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(9-isopropoxy-4-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; and [4-(4-butoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid.","9.A process for preparing a compound of formula (I) and pharmaceutically acceptable derivatives thereof according to claim 1 which comprises: (A) where R1 and R3 are both ═O and R4 and R9 are the same or different and represent C1-6alkoxy, by reacting a compound of formula (II): with a 4-aminophenylacetic acid of formula (III): in glacial acetic acid at elevated temperature; (B) where one of R1 and R3 is ═O and the other is hydrogen and R4 and R9 are the same or different and represent C1-6alkoxy, by reducing a compound of formula (IV): with a suitable reducing agent, followed by separation of isomers and deprotection; (C) where one of R1 and R3 is ═O and the other is hydrogen and R4 and R9 are the same or different and represent C1-6alkoxy, by reacting a compound of formula (V): with a 4-aminophenylacetic acid of formula (III) as defined above in presence of triethylamine and dimethylformamide, followed by deprotection; (D) where one of R1 and R3 is ═O and the other is hydrogen and one of R4 and R9 is C1-6alkoxy, by reacting a compound of formula (VI): where RD is in the −4 or -9 position and is C1-6 alkoxy, with a 4-aminophenylacetate of formula (VII): in presence of sodium triacetoxyborohydride in a suitable solvent, followed by deprotection; (E) where R1 and R3 are both hydrogen and R4 and R9 are the same or different and represent C1-6 alkoxy, by reacting a compound of formula (VIII): with a 4-aminophenylacetate of formula (VII) as described above in a suitable solvent, at elevated temperature followed by deprotection; (F) where R3 is ═CHC1-5alkyl, by reacting a compound of formula (IV) as defined above with a Grignard reagent C1-C6alkyl-MgBr under conventional conditions, followed by separation of isomers and deprotection; (G) where R3 is C1-6dialkyl, by reacting a compound of formula (IX): with a 4-aminophenylacetate of formula (VII) as defined above in presence of aluminium trichloride, followed by deprotection; or (H) converting compounds of formula (I), prepared according to processes (A) to (G), into other compounds of formula (I) using conventional procedures; and optionally converting compounds of formula (I) prepared by any one of processes (A) to (H) into pharmaceutically acceptable derivatives thereof.","10.A compound of formula (I) or a pharmaceutically acceptable derivative thereof as defined in claim 1 for use in human or veterinary medicine.","11.","(Cancelled) 12.A method of treating a human or animal subject suffering from a condition which is mediated by the action of PGE2 at EP4 receptors which comprises administering to said subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable derivative thereof as defined in claim 1.13.A pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable derivative thereof, as defined in claim 1 in admixture with one or more physiologically acceptable carriers or excipients."],["This invention relates to naphthalene derivatives, to processes for their preparation, to pharmaceutical compositions containing them and to their use in medicine.","The EP4 receptor is a 7-transmembrane receptor and its natural ligand is the prostaglandin PGE2.PGE2 also has affinity for the other EP receptors (types EP1, EP2 and EP3).","The EP4 receptor is associated with smooth muscle relaxation, inflammation, lymphocyte differentiation, bone metabolism processes, allergic activities, promotion of sleep, renal regulation and gastric or enteric mucus secretion.","We have now found a novel group of compounds which bind with high affinity to the EP4 receptor.","Compounds exhibiting EP4 binding activity have been described in, for example, WO00/18744, WO00/03980, WO00/15608, WO0016760, WO00/21532, WO98/55468, EP0855389 and EP0985663.GB2330307 describes the use of EP4 antagonists in the treatment of conditions with accelerated bone resorption.","Derivatives of indoprofen, such as [4-(1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]-2-propionic acid, sodium salt shown below, have been described by Rufer et. al.","In Eur.","J. Med.","Chem.—Chimica Therapeutica, 1978, 13, no 2, pg 193-198.Accordingly, the present invention provides a compound of formula (I) wherein R1 and R3 are the same or different and represent ═O, hydrogen, C1-6alkyl, C1-6dialkyl, ═CHC1-C5alkyl, ═S, or a 5- or 6-membered aryl; R4 to R9 are the same or different and represent hydrogen, C1-6alkoxy, OCF3, OCH2CF3, O-cyclopropyl, OCH2-cyclopropyl, C1-C6alkyl, S-alkyl, NR210 where R10 is hydrogen or C1-6alkyl, halogen, NO2, OH, CH2OC1-C6alkyl, CH2OH, or CF3; Q1 is hydrogen, C1-6alkyl, C1-6dialkyl, C1-6alkoxy, NHAc, NR210 where R10 is hydrogen or C1-6alkyl, difluoro, fluoro, ═O, or OH; Q2, Q3, Q4 and Q5 are the same or different and represent hydrogen, C1-6alkoxy, OCF3, OCH2CF3, O-cyclopropyl, OCH2-cyclopropyl, C1-C6alkyl, S-alkyl, NR210 where R10 is hydrogen or C1-6alkyl, halogen, NO2, OH, CH2OC1-C6alkyl, CH2OH, or a 5- or 6-membered aryl; with the proviso that the compounds [4-(1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]-2-propionic acid, sodium salt and [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid are excluded; and pharmaceutically acceptable derivatives thereof.","By pharmaceutically acceptable derivative is meant any pharmaceutically acceptable salt, solvate or ester, or salt or solvate of such ester of the compounds of formula (I), or any other compound which upon administration to the recipient is capable of providing (directly or indirectly) a compound of formula (I) or an active metabolite or residue thereof.","It will be appreciated by those skilled in the art that the compounds of formula (I) may be modified to provide pharmaceutically acceptable derivatives thereof at any of the functional groups in the compounds, and that the compounds of formula (I) may be derivatised at more than one position.","It will be appreciated that, for pharmaceutical use, the salts referred to above will be the pharmaceutically acceptable salts, but other salts may find use, for example in the preparation of compounds of formula (I) and the pharmaceutically acceptable salts thereof.","Pharmaceutically acceptable salts include those described by Berge, Bighley and Monkhouse, J. Pharm.","Sci., 1977, 66, 1-19.Suitable pharmaceutically acceptable salts of the compounds of formula (I) include acid addition salts formed with inorganic or organic acids, preferably inorganic acids, e.g.","hydrochlorides, hydrobromides and sulphates.","Further representative examples of pharmaceutically acceptable salts include those formed from acetic, maleic, fumaric, benzoic, ascorbic, pamoic, succinic, bismethylenesalicylic, methanesulfonic, ethanedisulfonic, propionic, tartaric, salicylic, citric, gluconic, aspartic, stearic, palmitic, itaconic, glycolic, p-aminobenzoic, glutamic, benzenesulfonic, cyclohexylsulfamic, phosphoric and nitric acids.","Further representative examples of pharmaceutically acceptable salts include alkali metal salts, formed from the addition of alkali metal bases, such as alkali metal hydroxides e.g.","sodium salts.","It will be appreciated that the compound of formula (I) may be produced in vivo by metabolism of a suitable prodrug.","Such prodrugs may be for example physiologically acceptable metabolically labile esters of compounds of the general formula (I).","These may be formed by esterification of the carboxylic acid group in the parent compound of general formula (I) with, where appropriate, prior protection of any other reactive groups present in the molecule followed by deprotection if required.","Examples of such metabolically labile esters include C1-4alkyl esters e.g.","methyl ethyl or t-butyl esters esters, C3-6 alkenyl esters e.g.","allyl substituted or unsubstituted aminoalkyl esters (e.g.","aminoethyl, 2-(N,N-diethylamino) ethyl, or 2-(4-morpholino)ethyl esters or acyloxyalkyl esters such as, acyloxymethyl or 1-acyloxyethyl e.g.","pivaloyloxymethyl, 1-pivaloyloxyethyl, acetoxymethyl, 1-acetoxyethyl,1-(1-methoxy-1-methyl)ethylcarbonyloxyethyl, 1-benzoyloxyethyl, isopropoxycarbonyloxymethyl, 1-isopropoxycarbonyloxyethyl, cyclohexylcarbonyloxymethyl, 1-cyclohexylcarbonyloxyethyl ester, cyclohexyloxycarbonyloxymethyl, 1-cyclohexyloxycarbonyloxyethyl, 1-(4-tetrahydropyranyloxy)carbonyloxyethyl or 1-(4-tetrahydropyranyl)carbonyloxyethyl.","As used herein, the term halogen atom includes fluorine, and more especially chlorine, bromine or iodine.","The term C1-6alkyl, C1-6alkoxy or ═CHC1-5alkyl as used herein includes straight chain and branched chain alkyl or alkoxy groups containing 1 to 6 carbon atoms, and in particular includes methyl, ethyl, n-propyl and i-propyl or methoxy, ethoxy, i-propoxy, n-propoxy, n-butyloxy or n-hexyloxy.","The term 5-membered aryl as used herein means an aryl selected from the following: The term 6-membered aryl as used herein means an aryl selected from: A preferred subgroup of compounds of formula (I) include the compounds wherein R1 is ═O or hydrogen; R3 is ═O, hydrogen, C1-6alkyl, C1-6dialkyl, or a 5- or 6-membered aryl; R4 and R9 are the same or different and represent hydrogen or C1-6alkoxy; R5 and R8 are hydrogen, or CF3; R6 and R7 are the same or different and represent hydrogen, C1-6alkyl, C1-6alkoxy, halogen, NO2, or CF3; Q1 is hydrogen, C1-6alkyl, C1-6alkoxy, NHAc, or NR210 where R10 is hydrogen or C1-6alkyl; Q2, Q3, Q4 and Q5 are the same or different and represent hydrogen, C1-6alkyl, C1-6alkoxy, halogen, or a 5- or 6-membered aryl; with the proviso that the compounds [4-(1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]-2-propionic acid, sodium salt and [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid are excluded; and pharmaceutically acceptable derivatives thereof.","R1 is preferably ═O.","R3 is aptly selected from methyl, dimethyl, or 2-furanyl.","R3 is preferably ═O or hydrogen, and is more preferably hydrogen.","R4 and R9 are each suitably hydrogen, methoxy, ethoxy, i-propoxy, n-propoxy, n-butyloxy or n-hexyloxy.","Suitably at least one of R4 and R9 represents C1-6alkoxy when Q1 is methyl.","Preferably R4 and R9 are both C1-6alkoxy, eg ethoxy.","R6 and R7 are suitably hydrogen, methyl, methoxy, chlorine, bromine, iodine, NO2, or CF3.Preferably R6 and R7 are both hydrogen.","Q1 is suitably hydrogen, methyl, ethyl, NHAc, NH2 or methoxy.","Q1 is preferably hydrogen.","Q2, Q3, Q4 and Q5 are each suitably hydrogen, methyl, methoxy, chlorine, bromine, iodine, 2-thiophenyl, 3-thiophenyl, 2-furanyl, 3-furanyl, or phenyl.","Preferably Q2, Q3, Q4 and Q5 are all hydrogen.","A further preferred group of compounds of formula (I) are those wherein R1 is ═O; R3 is methyl, dimethyl, 2-furanyl, preferably ═O or hydrogen, and is more preferably hydrogen; R4 and R9 are each hydrogen, methoxy, ethoxy, i-propoxy, n-propoxy, n-butyloxy or n-hexyloxy, preferably R4 and R9 are both C1-6alkoxy, eg ethoxy; R5 and R8 are hydrogen; R6 and R7 are hydrogen, methyl, methoxy, chlorine, bromine, iodine, NO2, or CF3, preferably R6 and R7 are both hydrogen; Q1 is suitably hydrogen, methyl, ethyl, NHAc, NH2 or methoxy, preferably hydrogen; Q2, Q3, Q4 and Q5 are each suitably hydrogen, methyl, methoxy, chlorine, bromine, iodine, 2-thiophenyl, 3-thiophenyl, 2-furanyl, 3-furanyl, or phenyl, preferably Q2, Q3, Q4 and Q5 are all hydrogen; with the proviso that the compounds [4-(1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]-2-propionic acid, sodium salt and [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid are excluded; and pharmaceutically acceptable derivatives thereof.","It is to be understood that the present invention encompasses all isomers of the compounds of formula (I) and their pharmaceutically acceptable derivatives, including all geometric, tautomeric and optical forms, and mixtures thereof (e.g.","racemic mixtures).","Preferred compounds of the present invention include: [4-(4-methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dimethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-methoxy-9-ethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-methoxy-9-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-methoxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-ethoxy-9-methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-ethoxy-9-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-ethoxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-ethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-propoxy-9-methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-propoxy-9-ethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dipropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-isopropoxy-4-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-isopropoxy-9-methoxy-1 oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-isopropoxy-9-ethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-isopropoxy-9-propoxy-1 oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-di-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-ethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-methoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dimethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-methoxy-9-ethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-methoxy-9-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4-methoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4-ethoxy-9-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4-ethoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-ethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dipropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-isopropoxy-4-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-di-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]-2-propionic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]-2-butyric acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]-2-(N-acetylamino)acetic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]-2-aminoacetic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]-2-methoxyacetic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-3-methylphenyl]acetic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-3-methoxyphenyl]acetic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-3,5-dimethylphenyl]acetic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-3-iodophenyl]acetic acid; [3-chloro-4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-phenyl]acetic acid; [3-bromo-4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-phenyl]acetic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-2-methylphenyl]acetic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-2-methoxyphenyl]acetic acid; [2-chloro-4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-phenyl]acetic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-2-iodophenyl]acetic acid; [2-bromo-4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-phenyl]acetic acid; [4-(6,7-dichloro-4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-6-methyl-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-7-methyl-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(7-bromo-4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-7-iodo-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(6-bromo-4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-6-iodo-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-3,3-dimethyl-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-3-methyl-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-6-nitro-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-7-nitro-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-6-methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-7-methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(6-chloro-4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(7-chloro-4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dibutoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dihexyloxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-hexyloxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-hexyloxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dibutoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dihexyloxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-butoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-ethoxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; and pharmaceutically acceptable derivatives thereof.","Particularly preferred compounds according to the invention are: [4-(4,9-dimethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-di-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dipropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dibutoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dihexyloxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-ethoxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-ethoxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-isopropoxy-4-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-isopropoxy-9-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-hexyloxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-hexyloxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4-methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-di-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dipropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dibutoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dihexyloxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-ethoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-isopropoxy-4-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-butoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; and pharmaceutically acceptable derivatives thereof.","It is to be understood that the present invention covers all combinations of particular and preferred groups as described herein above.","Since the compounds of the present invention, in particular compounds of formula (I), are intended for use in pharmaceutical compositions, it will be understood that they are each provided in substantially pure form, for example at least 50% pure, more suitably at least 75% pure and preferably at least 95% pure (% are on a wt/wt basis).","Impure preparations of the compounds of formula (I) may be used for preparing the more pure forms used in the pharmaceutical compositions.","Although the purity of intermediate compounds of the present invention is less critical, it will be readily understood that the substantially pure form is preferred as for the compounds of formula (I).","Preferably, whenever possible, the compounds of the present invention are obtained in crystalline form.","When some of the compounds of this invention are allowed to crystallise or are recrystallised from organic solvents, solvent of crystallisation may be present in the crystalline product.","This invention includes within its scope such solvates.","Similarly, some of the compounds of this invention may be crystallised or recrystallised from solvents containing water.","In such cases water of hydration may be formed.","This invention includes within its scope stoichiometric hydrates as well as compounds containing variable amounts of water that may be produced by processes such as lyophilisation.","In addition, different crystallisation conditions may lead to the formation of different polymorphic forms of crystalline products.","This invention includes within its scope all polymorphic forms of the compounds of formula (I).","The compounds of the invention bind to the EP4 receptor and are therefore useful in treating EP4 receptor mediated diseases.","In view of their ability to bind to the EP4 receptor, the compounds of the invention are useful in the treatment of the disorders that follow.","Thus, the compounds of formula (I) are useful as analgesics.","For example they are useful in the treatment of chronic articular pain (e.g.","rheumatoid arthritis, osteoarthritis, rheumatoid spondylitis, gouty arthritis and juvenile arthritis) including the property of disease modification and joint structure preservation; musculoskeletal pain; lower back and neck pain; sprains and strains; neuropathic pain; sympathetically maintained pain; myositis; pain associated with cancer and fibromyalgia; pain associated with migraine; pain associated with influenza or other viral infections, such as the common cold; rheumatic fever; pain associated with functional bowel disorders such as non-ulcer dyspepsia, non-cardiac chest pain and irritable bowel syndrome; pain associated with myocardial ischemia; post operative pain; headache; toothache; and dysmenorrhea.","The compounds of the invention are particularly useful in the treatment of neuropathic pain.","Neuropathic pain syndromes can develop following neuronal injury and the resulting pain may persist for months or years, even after the original injury has healed.","Neuronal injury may occur in the peripheral nerves, dorsal roots, spinal cord or certain regions in the brain.","Neuropathic pain syndromes are traditionally classified according to the disease or event that precipitated them.","Neuropathic pain syndromes include: diabetic neuropathy; sciatica; non-specific lower back pain; multiple sclerosis pain; fibromyalgia; HIV-related neuropathy; post-herpetic neuralgia; trigeminal neuralgia; and pain resulting from physical trauma, amputation, cancer, toxins or chronic inflammatory conditions.","These conditions are difficult to treat and although several drugs are known to have limited efficacy, complete pain control is rarely achieved.","The symptoms of neuropathic pain are incredibly heterogeneous and are often described as spontaneous shooting and lancinating pain, or ongoing, burning pain.","In addition, there is pain associated with normally non-painful sensations such as “pins and needles” (paraesthesias and dysesthesias), increased sensitivity to touch (hyperesthesia), painful sensation following innocuous stimulation (dynamic, static or thermal allodynia), increased sensitivity to noxious stimuli (thermal, cold, mechanical hyperalgesia), continuing pain sensation after removal of the stimulation (hyperpathia) or an absence of or deficit in selective sensory pathways (hypoalgesia).","The compounds of formula (I) are also useful in the treatment of inflammation, for example in the treatment of skin conditions (e.g.","sunburn, burns, eczema, dermatitis, psoriasis); ophthalmic diseases such as glaucoma, retinitis, retinopathies, uveitis and of acute injury to the eye tissue (e.g.","conjunctivitis); lung disorders (e.g.","asthma, bronchitis, emphysema, allergic rhinitis, respiratory distress syndrome pigeon fanciers disease, farmer's lung, COPD); gastrointestinal tract disorders (e.g.","aphthous ulcer, Crohn's disease, atopic gastritis, gastritis varialoforme, ulcerative colitis, coeliac disease, regional ileitis, irritable bowel syndrome, inflammatory bowel disease, gastrointestinal reflux disease); organ transplantation; other conditions with an inflammatory component such as vascular disease, migraine, periarteritis nodosa, thyroiditis, aplastic anaemia, Hodgkin's disease, scierodoma, myaesthenia gravis, multiple sclerosis, sorcoidosis, nephrotic syndrome, Bechet's syndrome, polymyositis, gingivitis, myocardial ischemia, pyrexia, systemic lupus erythematosus, polymyositis, tendinitis, bursitis, and Sjogren's syndrome.","The compounds of formula (I) are also useful in the treatment of immunological diseases such as autoimmune diseases, immunological deficiency diseases or organ transplantation.","The compounds of formula (I) are also effective in increasing the latency of HIV infection.","The compounds of formula (I) are also useful in the treatment of diseases of abnormal platelet function (e.g.","occlusive vascular diseases).","The compounds of formula (I) are also useful for the preparation of a drug with diuretic action.","The compounds of formula (I) are also useful in the treatment of impotence or erectile dysfunction.","The compounds of formula (I) are also useful in the treatment of bone disease characterised by abnormal bone metabolism or resorption such as osteoporosis (especially postmenopausal osteoporosis), hyper-calcemia, hyperparathyroidism, Paget's bone diseases, osteolysis, hypercalcemia of malignancy with or without bone metastases, rheumatoid arthritis, periodontitis, osteoarthritis, ostealgia, osteopenia, cancer cacchexia, calculosis, lithiasis (especially urolithiasis), solid carcinoma, gout and ankylosing spondylitis, tendinitis and bursitis.","In a further aspect compounds of formula (I) may be useful in inhibiting bone resorption and/or promoting bone generation.","The compounds of formula (I) are also useful for attenuating the hemodynamic side effects of NSAIDs and COX-2 inhibitors.","The compounds of formula (I) are also useful in the treatment of cardiovascular diseases such as hypertension or myocardiac ischemia; functional or organic venous insufficiency; varicose therapy; haemorrhoids; and shock states associated with a marked drop in arterial pressure (e.g.","septic shock).","The compounds of formula (I) are also useful in the treatment of neurodegenerative diseases and neurodegeneration such as dementia, particularly degenerative dementia (including senile dementia, Alzheimer's disease, Pick's disease, Huntingdon's chorea, Parkinson's disease and Creutzfeldt-Jakob disease, ALS, motor neuron disease); vascular dementia (including multi-infarct dementia); as well as dementia associated with intracranial space occupying lesions; trauma; infections and related conditions (including HIV infection); metabolism; toxins; anoxia and vitamin deficiency; and mild cognitive impairment associated with ageing, particularly Age Associated Memory Impairment.","The compounds of formula (I) are also useful in the treatment of neuroprotection and in the treatment of neurodegeneration following stroke, cardiac arrest, pulmonary bypass, traumatic brain injury, spinal cord injury or the like.","The compounds of formula (I) are also useful in the treatment of tinnitus.","The compounds of formula (I) are also useful in preventing or reducing dependence on, or preventing or reducing tolerance or reverse tolerance to, a dependence—inducing agent.","Examples of dependence inducing agents include opioids (e.g.","morphine), CNS depressants (e.g.","ethanol), psychostimulants (e.g.","cocaine) and nicotine.","The compounds of formula (I) are also useful in the treatment of complications of Type 1 diabetes (e.g.","diabetic microangiopathy, diabetic retinopathy, diabetic nephropathy, macular degeneration, glaucoma), nephrotic syndrome, aplastic anaemia, uveitis, Kawasaki disease and sarcoidosis.","The compounds of formula (I) are also useful in the treatment of kidney dysfunction (nephritis, particularly mesangial proliferative glomerulonephritis, nephritic syndrome), liver dysfunction (hepatitis, cirrhosis), gastrointestinal dysfunction (diarrhoea) and colon cancer.","It is to be understood that reference to treatment includes both treatment of established symptoms and prophylactic treatment, unless explicitly stated otherwise.","According to a further aspect of the invention, we provide a compound of formula (I) or a pharmaceutically acceptable derivative thereof for use in human or veterinary medicine.","According to another aspect of the invention, we provide a compound of formula (I) or a pharmaceutically acceptable derivative thereof for use in the treatment of a condition which is mediated by the action of PGE2 at EP4 receptors.","According to a further aspect of the invention, we provide a method of treating a human or animal subject suffering from a condition which is mediated by the action of PGE2 at EP4 receptors which comprises administering to said subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable derivative thereof.","According to a further aspect of the invention we provide a method of treating a human or animal subject suffering from a pain, inflammatory, immunological, bone, neurodegenerative or renal disorder, which method comprises administering to said subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable derivative thereof.","According to another aspect of the invention, we provide the use of a compound of formula (I) or a pharmaceutically acceptable derivative thereof for the manufacture of a therapeutic agent for the treatment of a condition which is mediated by the action of PGE2 at EP4 receptors.","According to another aspect of the invention we provide the use of a compound of formula (I) or a pharmaceutically acceptable derivative thereof for the manufacture of a therapeutic agent for the treatment or prevention of a condition such as a pain, inflammatory, immunological, bone, neurodegenerative or renal disorder.","The compounds of formula (I) and their pharmaceutically acceptable derivatives are conveniently administered in the form of pharmaceutical compositions.","Such compositions may conveniently be presented for use in conventional manner in admixture with one or more physiologically acceptable carriers or excipients.","Thus, in another aspect of the invention, we provide a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable derivative thereof adapted for use in human or veterinary medicine.","While it is possible for the compounds of formula (I) or a pharmaceutically acceptable derivative thereof to be administered as the raw chemical, it is preferable to present it as a pharmaceutical formulation.","The formulations of the present invention comprise the compounds of formula (I) or a pharmaceutically acceptable derivative thereof together with one or more acceptable carriers or diluents thereof and optionally other therapeutic ingredients.","The carrier(s) must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.","The formulations include those suitable for oral, parenteral (including subcutaneous e.g.","by injection or by depot tablet, intradermal, intrathecal, intramuscular e.g.","by depot and intravenous), rectal and topical (including dermal, buccal and sublingual) administration although the most suitable route may depend upon for example the condition and disorder of the recipient.","The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.","All methods include the step of bringing into association the compound of formula (I) or a pharmaceutically acceptable acid addition salt thereof (“active ingredient”) with the carrier which constitutes one or more accessory ingredients.","In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.","Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets (e.g.","chewable tablets in particular for paediatric administration) each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.","The active ingredient may also be presented as a bolus, electuary or paste.","A tablet may be made by compression or moulding, optionally with one or more accessory ingredients.","Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active or dispersing agent.","Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.","The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein.","Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.","The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of a sterile liquid carrier, for example, water-for-injection, immediately prior to use.","Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.","Formulations for rectal administration may be presented as a suppository with the usual carriers such as cocoa butter, hard fat or polyethylene glycol.","Formulations for topical administration in the mouth, for example buccally or sublingually, include lozenges comprising the active ingredient in a flavoured basis such as sucrose and acacia or tragacanth, and pastilles comprising the active ingredient in a basis such as gelatin and glycerin or sucrose and acacia.","The compounds of the invention may also be formulated as depot preparations.","Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.","Thus, for example, the compounds of the invention may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.","In addition to the ingredients particularly mentioned above, the formulations may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.","The EP4 receptor compounds for use in the instant invention may be used in combination with other therapeutic agents, for example COX-2 inhibitors, such as celecoxib, rofecoxib, valdecoxib or parecoxib; 5-lipoxygenase inhibitors; NSAID's, such as diclofenac, indomethacin, nabumetone or ibuprofen; leukotriene receptor antagonists; DMARD's such as methotrexate; adenosine A1 receptor agonists; sodium channel blockers, such as lamotrigine; NMDA receptor modulators, such as glycine receptor antagonists; gabapentin and related compounds; tricyclic antidepressants such as amitriptyline; neurone stabilising antiepileptic drugs; mono-aminergic uptake inhibitors such as venlafaxine; opioid analgesics; local anaesthetics; 5HT1 agonists, such as triptans, for example sumatriptan, naratriptan, zolmitriptan, eletriptan, frovatriptan, almotriptan or rizatriptan; EP1 receptor ligands; EP2 receptor ligands; EP3 receptor ligands; EP1 antagonists; EP2 antagonists and EP3 antagonists.","When the compounds are used in combination with other therapeutic agents, the compounds may be administered either sequentially or simultaneously by any convenient route.","The invention thus provides, in a further aspect, a combination comprising a compound of formula (I) or a pharmaceutically acceptable derivative thereof together with a further therapeutic agent or agents.","The combinations referred to above may conveniently be presented for use in the form of a pharmaceutical formulation and thus pharmaceutical formulations comprising a combination as defined above together with a pharmaceutically acceptable carrier or excipient comprise a further aspect of the invention.","The individual components of such combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations.","When a compound of formula (I) or a pharmaceutically acceptable derivative thereof is used in combination with a second therapeutic agent active against the same disease, the dose of each compound may differ from that when the compound is used alone.","Appropriate doses will be readily appreciated by those skilled in the art.","A proposed daily dosage of compounds of formula (I) or their pharmaceutically acceptable salts for the treatment of man is from 0.01 to 10 mg/kg body weight per day and more particularly 0.1 to 3 mg/kg body weight per day, calculated as the free base, which may be administered as a single or divided dose, for example one to four times per day The dose range for adult human beings is generally from 8 to 1000 mg/day, such as from 20 to 800 mg/day, preferably 35 to 200 mg/day, calculated as the free base.","The precise amount of the compounds of formula (I) administered to a host, particularly a human patient, will be the responsibility of the attendant physician.","However, the dose employed will depend on a number of factors including the age and sex of the patient, the precise condition being treated and its severity, and the route of administration.","The present invention provides a process for preparing compounds of formula (I) and pharmaceutically acceptable derivatives thereof.","Compounds of formula (I) and pharmaceutically acceptable derivatives thereof may be prepared by any method known in the art for the preparation of compounds of analogous structure.","Suitable methods for the preparation of compounds of formula (I) and pharmaceutically acceptable derivatives thereof are described below and form a further aspect of the invention.","In the formulae that follow, R1 to R10 and Q1-Q5 are as defined in formula (I) above unless otherwise stated.","According to a first process (A), compounds of formula (I), where R1 and R3 are both ═O and R4 and R9 are the same or different and represent C1-6alkoxy, may be prepared by reacting a compound of formula (II) with a 4-aminophenylacetic acid of formula (III) in glacial acetic acid at elevated temperature.","According to another process (B) compounds of formula (I), where one of R1 and R3 is ═O and the other is hydrogen and R4 and R9 are the same or different and represent C1-6alkoxy, may be prepared by reducing a compound of formula (IV) with a suitable reducing agent, followed by separation of isomers and deprotection (eg.","with aqueous base at elevated temperature).","Suitable reducing agents include zinc in acetic acid at elevated temperature and sodium borohydride in methanol followed by trifluoroacetic acid (TFA) and triethylsilane.","According to another process (C) compounds of formula (I), where one of R1 and R3 is ═O and the other is hydrogen and R4 and R9 are the same or different and represent C1-6alkoxy, may be prepared by reacting a compound of formula (V) with a 4-aminophenylacetic acid of formula (III) as defined above in the presence of triethylamine and dimethylformamide, followed by deprotection (eg.","using acetic acid at elevated temperature).","According to a further process (D) compounds of formula (I), where one of R1 and R3 is ═O and the other is hydrogen and one of R4 and R9 is C1-6alkoxy, may be prepared by reacting a compound of formula (VI) where RD is in the −4 or −9 position and is C1-6 alkoxy, with a 4-aminophenylacetate of formula (VII) in the presence of sodium triacetoxyborohydride in a suitable solvent, such as dichloromethane, followed by deprotection (eg.","with aqueous base at elevated temperature).","According to a further process (E) compounds of formula (I), where R1 and R3 are both hydrogen and R4 and R9 are the same or different and represent C1-6 alkoxy, may be prepared by reacting a compound of formula (VIII) with a 4-aminophenylacetate of formula (VII) as described above in a suitable solvent, such as dimethylformamide, at elevated temperature followed by deprotection (eg.","using aqueous base such as lithium hydroxide in aqueous tetrahydrofuran).","According to a further process (F) compounds of formula (I), where R3 is ═CHC1-5alkyl, may be prepared by reacting a compound of formula (IV) as defined above with a Grignard reagent C1-C6alkyl-MgBr under conventional conditions, followed by separation of isomers and deprotection (eg.","with aqueous base at elevated temperature).","According to a further process (G) compounds of formula (I), where R3 is C1-6dialkyl, may be prepared by reacting a compound of formula (IX) with a 4-aminophenylacetate of formula (VII) as defined above in the presence of aluminium trichloride, followed by deprotection (eg.","with aqueous base at elevated temperature).","According to a further process (H) compounds of formula (I) prepared according to processes (A) to (G) may be converted into other compounds of formula (I) using conventional procedures.","For example, compounds of formula (I) where R3 is C1-6alkyl, may be prepared by reducing a compound of formula (I) where R3 is ═CHC1-5alkyl, protected at the carboxyl group, with a suitable reducing agent, such as hydrogen in the presence of a palladium on carbon catalyst, followed by deprotection (eg.","with aqueous base at elevated temperature).","Also, compounds of formula (I) where R3 is ═O may be converted into compounds of formula (I) where R3 is ═S by conventional methods, for example using Lawesson's reagent.","Compounds of formulae (II) to (IX) may be prepared by any method known in the art for the preparation of compounds of analogous structure.","Compounds of formula (II) may, for example be prepared according to Scheme 1 that follows.","Compounds of formula (IV) may be prepared by reacting compounds of formula (II) with a 4-aminophenylacetate of formula (VII) as defined above in the presence of acetic acid.","Compounds of formula M may, for example be prepared according to Scheme 2 that follows.","Compounds of formula (VI) may, for example, be prepared according to Scheme 3 that follows.","Compounds of formula (VIII) may be prepared, for example, by reacting a compound of formula (X) with phosphorous tribromide in an ether solvent, for example a mixture of diethyl ether and tetrahydrofuran.","Compounds of formula (IX) may be prepared by reacting a compound of formula (XI) with a Grignard reagent R3-MgBr under conventional conditions.","Compounds of formulae (III) and (VII) may be prepared according to methods known in the art for the preparation of analogous compounds, for example, compounds where Q1 is methyl may be prepared by the method of Takahashi,l et al.","Heterocycles (1996), 43(II), 2343-2346; compounds where Q1 is ethyl may be prepared by the method of Kirschenheuter, Gary P et al.","in EP465802; compounds where Q1 is NHAc may be prepared by the method described in US3479339; compounds where Q1 is NH2 may be prepared by the method of Herbert, Richard B et al.","J. Chem.","Soc., Perkin Trans.1 (1992), (1), 109-13; compounds where Q1 is MeO may be prepared from the corresponding nitro compounds prepared by the method of Tomioka, Hideo et al.","J.","Am.","Chem.","Soc.","(1990), 112(21), 7692-702; compounds where Q2 is Me or Q3 is Cl may be prepared by the method described in U.S. Pat.","No.","3,860,639 (Schultz, Everett M.); compounds where Q2 is MeO may be prepared by the method of Nannini, G et al, Arzneim.-Forsch.","(1973), 23(8), 1090-100 by hydrolysis of the ethyl ester; compounds where Q2 is Cl may be prepared by the method of Atkinson, Joseph G et al.","Tet Left.","(1979), (31), 2857-60 by reduction of the corresponding nitro compound; compounds where Q2 is I may be prepared by the method of Sindelar, Karel et al.","Collect.","Czech.","Chem.","Commun.","(1978), 43(2), 471-97 by reduction of the corresponding nitro compound; compounds where Q2 is Br may be prepared by the method of Sindelar, Karel et al.","Collect.","Czech.","Chem.","Commun.","(1978), 43(2), 471-97; compounds where Q3 is Me may be prepared by the method of Borck, Joachim et al.","in ZA 6804711; compounds where Q3 is MeO may be prepared by the method of Gallacher, Gerard et al.","Biorg.","Amines (1995), 11(1), 49-62 by reduction of the corresponding nitro compound; compounds where Q3 is I may be prepared by the method of Boehm, Marcus F et al.","J. Chem.","Soc., Chem.","Commun.","(1991), (1), 52-3; compounds where Q3 is Br may be prepared by the method of Figala, Georg et al.","in DE 2746067; compounds where Q3 and Q4 are Me may be prepared by the method of Yost, Yul et al.","Org.","Prep.","Proced.","Int.","(1985), 17(4-5), 23949 from the corresponding benzoic acid using the Arndt-Eistert reaction.","Certain intermediates described above are novel compounds, and it is to be understood that all novel intermediates herein form further aspects of the present invention.","Compounds of formula (II), (V), (VI), (VIII) and (IX) are key intermediates and represent a particular aspect of the invention.","Certain intermediates, such as compounds of formula (IV), may be prodrugs of compounds of formula (I).","Conveniently, compounds of the invention are isolated following work-up in the form of the free base.","Pharmaceutically acceptable acid addition salts of the compounds of the invention may be prepared using conventional means.","Solvates (e.g.","hydrates) of a compound of the invention may be formed during the work-up procedure of one of the aforementioned process steps.","The following Examples which should not be construed as constituting a limitation thereto are provided to illustrate the invention.","1H NMR spectra were obtained at 400 MHz on a Bruker DPX400 spectrophotometer.","J values are given in Hz.","Mass spectra were obtained on a Micromass series II MS (electrospray positive or negative).","Where HPLC retention times are given as a characterisation of intermediates or Examples this refers to an HP 1050 or a HP1100 running a 5.5 minute Gradient: Eluents: A —0.1% VN Formic Acid+10 mmol Ammonium Acetate B —95% MeCN+0.05% VN Formic Acid Flow rate: 3 ml/min Column: 3.3 cm×4.6 mm internal diameter, 3 μm ABZ+PLUS Injection Volume: 5 μl Temperature: Room temperature.","Gradient: Time % A % B 0.00 100 0.00 0.70 100 0.00 4.40 0.00 100 5.30 0.00 100 5.50 100 0.00 Intermediate 1 Ethyl 1,4-dihydroxy-2,3-naphthalenedicarboxylate Sodium (60 g, 2.6 mol) was dissolved in ethanol (1.2L) and the mixture was cooled to 40° C. Diethylphthalate (960 ml, 4.83 mol) was added and the mixture heated under nitrogen until the temperature reached 115° C. Diethyl succinate (211.3 g, 1.21 mol) was added dropwise over 45 min.","The reaction was heated at 115° C. for a further 45 min, cooled to room temperature and poured onto water (1.2L).","Ethyl acetate (1L) was added and stirred, the layers were separated and the organics were extracted with sodium hydroxide solution (2N, 1L).","The combined aqueous was acidified to pH 3 and the mixture extracted with ethyl acetate (2×1L).","The combined organics were washed with a saturated solution of sodium hydrogen carbonate (2×1.5L), then brine, dried (MgSO4), filtered and the solvent evaporated under vacuum.","The residue was purified using a 2.5 kg Biotage column eluting with 5% ethyl acetate/hexane to give ethyl 1,4-dihydroxy-2,3-naphthalenedicarboxylate as a white solid, (60 g, 16%).","δH CDCl3 10.44,(2H, s), 8.34,(2H, m), 7.68,(2H, m), 4.37,(4H, q), 1.37,(6H, t).","Intermediate 2 Ethyl 1,4-diethoxy-2,3-naphthalenedicarboxylate Ethyl 1,4-dihydroxy-2,3-naphthalenedicarboxylate (30 g, 98.6 mmol) and potassium carbonate (150 g, 1.09 mmol) were stirred in acetone (600 ml) under nitrogen.","Iodoethane (150 g, 0.96 mol) was added and the mixture was stirred at reflux overnight.","The reaction was cooled, diluted with ethyl acetate and filtered.","The filtrate was evaporated to leave a brown oil, which was dissolved in toluene and washed with potassium hydroxide solution (5%, 150 ml) and brine.","Drying over magnesium sulphate and evaporation of the solvent gave a yellow solid.","Purification using an 800 g Biotage column gave ethyl 1,4-diethoxy-2,3-naphthalenedicarboxylate as a white solid (32 g, 90%).","δH CDCl3 8.16,(2H, m), 7.60,(2H, m), 4.40,(4H, q), 4.18,(4H, q), 1.50,(6H, t), 1.40,(6H, t).","Intermediate 3 1,4-Diethoxy-2,3-naphthalenedicarboxylic Acid Ethyl 1,4-diethoxy-2,3-naphthalenedicarboxylate (32 g, 89 mmol) was added to a solution of sodium hydroxide (20 g) in ethanol (200 ml) and water (40 ml) and stirred for 1.5h at 60° C. The reaction was cooled and the thick white suspension was filtered.","The solid was dissolved in a mixture of ethyl acetate (200 ml) and water (800 ml).","The layers were separated and the aqueous was acidified with hydrochloric acid (2M, 120 ml).","The aqueous was extracted with ethyl acetate (2×) and the combined organics were dried (MgSO4).","Evaporation of the solvent under vacuum gave 1,4-diethoxy-2,3-naphthalenedicarboxylic acid as a white solid (25 g, 92%).","δH [2H6]—DMSO 13.26,(2H, s), 8.15,(2H, m), 7.72,(2H, m), 4.13,(4H, q), 1.42,(6H, t).","Intermediate 4 1,4-Diethoxy-2,3-naphthalenedicarboxylic Anhydride 1,4-Diethoxy-2,3-naphthalenedicarboxylic acid (25 g, 82 mmol) was added to a solution of thionyl chloride (23.3 g) in chloroform (150 ml) and stirred at reflux for 1 h. The resulting solution was cooled and evaporated to dryness.","Further chloroform was added and evaporation repeated to give 1,4-diethoxy-2,3-naphthalenedicarboxylic anhydride as a yellow solid (23.3 g, 99%).","δH [2H6]—DMSO 8.42,(2H, m), 7.93,(2H, m), 4.53,(4H, q), 1.46,(6H, t).","Intermediate 5 Ethyl[4-(4,9-diethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[I]isoindol-2-yl)phenyl]acetate 1,4-Diethoxy-2,3-naphthalenedicarboxylic anhydride (23.3 g, 81.5 mmol) and ethyl (4-aminophenyl)acetate (14.8 g, 82 mmol) were refluxed under nitrogen in acetic acid (160 ml) overnight.","The mixture was cooled to room temperature and poured into water (1L).","The white solid was filtered, washed with water and dissolved in dichloromethane (800 ml).","The solution was washed with water, brine and dried (MgSO4) and the solvent evaporated under vacuum to give ethyl [4-(4,9-diethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate as an off-white solid, 33 g, 96%.","δH [2H6]—DMSO 8.40,(2H, m), 7.87,(2H, m), 7.42,(4H, s), 4.47,(4H, q), 4.12,(2H, q), 3.76,(2H, s), 1.45,(6H, t), 1.21,(3H, t).","Intermediate 6 3-Hydroxy 4-methoxynaphtho[2,3-c]furan-[(3H)-one N,N,N′-Trimethylethylene diamine (0.82 ml, 6.3 mmol) was dissolved in tetrahydrofuran (16 ml) and cooled to −20° C. n-Butyl lithium (1.6M in hexanes, 3.9 ml, 6.24 mmol) was added and the reaction stirred at −20° C. for 15 min.","1-Methoxy-2-naphthaldehyde (0.969, 5.9 mmol) was added followed by n-butyl lithium (1.6M in hexanes, 11.25 ml, 18 mmol) and the reaction stirred at 25° C. for 3h.","Solid carbon dioxide was added and the reaction left until the excess carbon dioxide had sublimed.","Stirring continued for 15 min before addition of hydrochloric acid (2N, 50 ml).","The mixture was extracted with dichloromethane (50 ml, ×3).","The combined extracts were dried (MgSO4) and the solvent evaporated under vacuum.","The oily residue was preabsorbed onto silica and purified by SPE (silica, 10 g) eluting with an ethyl acetate/cyclohexane gradient to give 3-hydroxy-4-methoxynaphtho[2,3-c]furan-1 (3M-one (50 mg, 3.7%).","δH [2H6]—DMSO 8.28,(2H, m), 8.18,(2H, m), 7.70,(2H, m), 7.16,(1H, s), 4.27,(3H, s).","Intermediate 7 Ethyl 1,4-dimethoxy-2-methylnaphthalene-3-carboxylate n-Butyl lithium (1.6M in hexanes, 4.1 ml, 6.56 mmol) was added dropwise to 2-bromo-1,4-dimethoxy-3-methylnaphthalene (1.537 g, 5.47 mmol) in tetrahydrofuran (30 ml) at −50° C. The reaction was stirred for 30 min at −50° C. before dropwise addition of ethyl chloroformate (1 ml, 10.46 mmol).","The reaction was allowed to warm to 0° C. over 18h quenched by addition of hydrochloric acid (2N) and extracted with ethyl acetate.","The extract was dried (Na2SO4) and the solvent evaporated under vacuum.","The residue was purified by SPE (silica, 10 g) eluting with 10% ethyl acetate in cyclohexane to give ethyl 1,4-dimethoxy-2-methylnaphthalene-3-carboxylate (1.339, 89%).","δH CDCl3 8.08,(2H, d), 7.53,(2H, m), 4.48,(2H, q), 3.99,(3H, s), 3.88,(3H, s), 2.38,(3H, s), 1.44,(3H, t).","Intermediate 8 2,3-Bis(bromomethyl)-1,4-diethoxy-naphthalene [1,4-Diethoxy-3-(hydroxymethyl)-2-naphthyl]methanol (92 mg, 0.33 mmol) was dissolved in a mixture of diethyl ether (1.5 ml) and tetrahydrofuran (2 ml) under nitrogen at 0° C. and phosphorus tribromide (0.035 ml, 0.35 mmol) was added dropwise.","The reaction was allowed to warm to ambient temperature and stirred for 4 h before quenching with ice.","The reaction mixture was extracted with ethyl acetate (3×5 ml) and the combined organic extracts dried over magnesium sulphate.","The solvent was removed in vacuo, and the residue purified by SPE cartridge (silica), gradient elution cyclohexane to 75% cyclohexane/dichloromethane, to give 2,3-bis(bromomethyl)-1,4-diethoxy-naphthalene as a white solid (92 mg, 70%).","δH CDCl3 8.07 (2H, dd, J=6.5, 3.2), 7.54 (2H, dd, J=6.5, 3.2), 5.02 (4H, s), 4.21 (4H, q, J=7.0), 1.60 (6H, t, J=7.0); LC retention time 4.16 min.","EXAMPLE 1 (Process C) [4-(4,9-Dimethoxy-1 oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid To a solution of ethyl 1,4-dimethoxy-2-methylnaphthalene-3-carboxylate (73 mg, 0.266 mmol) in carbon tetrachloride (10 ml) was added N-bromosuccinimide (47 mg, 0.266 mmol) and dibenzylperoxide (5 mg).","The mixture was heated at reflux under nitrogen for 45 min, illuminating with a 200W lamp.","The reaction was cooled to room temperature, 4-aminophenyl acetic acid (40 mg, 0.266 mmol), triethylamine (74 μl, 0.53 mmol) and DMF (5 ml) added and the reaction stirred for 48h.","Acetic acid (glacial, 1 ml) was added to the reaction and the mixture refluxed for 3h under nitrogen.","Sodium metabisulphite solution (1 ml) was added and the reaction evaporated to dryness under vacuum.","The product was partially purified by SPE (NH2, 10 g) eluting with methanol then 5% acetic acid in methanol.","The fractions containing product were passed through a silica plug washing with 5% acetic acid in methanol.","After evaporation of the solvents under vacuum, the residue was columned on a silica gel flash column eluting with a gradient (1:1 ethyl acetate/cyclohexane to 5% methanol, 1% acetic acid in 1:1 ethyl acetate/cyclohexane) to give [4-(4,9-dimethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid (15 mg, 15%).","δH CDCl3 8.40,(1H, d), 8.17,(1H, d), 7.90.","(2H, d), 7.65,(1H, t), 7.59,(1H, t), 7.38,(2H, d), 5.02,(2H, s), 4.27,(3H, s), 4.08,(3H, s), 3.69,(2H, s).","MS 378, [MH+].","LC retention time 3.28 min.","EXAMPLE 2 Step 1 (Process E) Ethyl[4-(4,9-diethoxy-1,3-dihydro-2H-benzo[t]isoindol-2-yl)phenyl]acetate A solution of 2,3-bis(bromomethyl)-1,4-diethoxy-naphthalene (41 mg, 0.1 mmol) and 4-aminophenylacetic acid ethyl ester (20 mg, 0.11 mmol) in N,N-dimethylformamide (1 ml) was heated at 60° C. under nitrogen for 14 h. The solvent was removed in vacuo and the residue taken up in ethyl acetate (10 ml) and washed with 8% aqueous sodium bicarbonate solution (5 ml).","The organic layer was dried over magnesium sulphate and the solvent removed in vacuo.","Purification by flash column chromatography on silica gel eluting with 90% cyclohexane/ethyl acetate gave the product as a white solid (15 mg, 36%).","δH CDCl3 8.12 (2H, dd, J=6.5, 3.2), 7.50 (2H, dd, J=6.5, 3.2), 7.23 (2H, d, J=8.5), 6.73 (2H, d, J=8.5), 4.80 (4H, s), 4.17 (6H, m), 3.55 (2H, s), 1.56 (6H, m), 1.24 (3H, m); LC retention time 4.25 min, ms 420, [MH+].","EXAMPLE 2 Step 2 [4-(4,9-diethoxy-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid A solution of ethyl[4-(4,9-diethoxy-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate (10 mg, 0.024 mmol) in tetrahydrofuran (4 ml) and a solution of lithium hydroxide (6 mg, 0.24 mmol) in water (1 ml) were stirred vigorously for 14h.","The reaction mixture was diluted with water (10 ml) and extracted with ethyl acetate (2×5 ml) and the organic extracts discarded.","The aqueous phase was acidified with 2N hydrochloric acid to pH=5 and extracted with ethyl acetate (3×5 ml).","The combined organic extracts were dried over magnesium sulphate and the solvent removed in vacuo to give the product as a red solid (4 mg, 43%).","δH CDCl3 8.13 (2H, dd, J=6.5, 3.2), 7.49, (2H, dd, J=6.5, 3.2), 7.24 (2H, d, 8.3), 6.75 (2H, d, J=8.3), 4.81 (4H, s), 4.19 (4H, q, J=7.0), 3.69 (2H, s), 1.53 (6H, t, J=7.0); LC retention time 3.92 min, ms 392, [MH+].","General Methodology Method A A mixture of ethyl 1,4-dihydroxy-2,3-naphthalenedicarboxylate (1.0 g, 3.29 mmol), potassium carbonate (5 g, 36.2 mmol) and the alkyl halide (3.25 mmol) in acetone (20 ml) was heated at reflux under nitrogen for 8h.","The cooled reaction was filtered and the residue washed with acetone and ethyl acetate.","The combined filtrate and washings were evaporated to dryness under vacuum.","The residue was partitioned between hydrochloric acid (2N) and dichloromethane.","The dichloromethane extract was evaporated to dryness under vacuum.","The product was isolated by chromatography on a silica gel flash column eluting with an ethyl acetate/cyclohexane gradient (0-10% ethyl acetate).","Method B A mixture of monoalkylated material (170 mg, 0.49 mmol), potassium carbonate (850 mg, 6.15 mmol) and the alkyl halide (1.0 mmol) in acetone (5 ml) was heated at reflux under nitrogen for 4h.","The cooled reaction was filtered and the residue washed with acetone and ethyl acetate.","The combined filtrate and washings were evaporated to dryness under vacuum and the residue purified by SPE (silica) eluting with an ethyl acetate/cyclohexane gradient.","Method C A mixture of ethyl 1,4-dihydroxy-2,3-naphthalenedicarboxylate (2 g, 6.57 mmol), potassium carbonate (10 g, 72.4 mmol) and the alkyl halide (64.1 mmol) in acetone (40 ml) was heated at reflux under nitrogen for 4h.","The cooled reaction was filtered and the residue washed with acetone and ethyl acetate.","The combined filtrate and washings were evaporated to dryness under vacuum and the residue purified by SPE (silica) eluting with an ethyl acetate/cyclohexane gradient.","Method D A mixture of the diester (13.87 mmol) and sodium hydroxide solution (2N, 25 ml) in ethanol (25 ml) was heated at reflux for 4h.","The resulting solution was acidified to pH1 with hydrochloric acid (2N) and extracted with ethyl acetate (×2).","The extracts were dried (MgSO4) an d the solvent evaporated under vacuum.","Method E (Process A) A mixture of diacid (0.57 mmol) and 4-aminophenylacetic acid (1.32 mmol) in glacial acetic acid (5 ml) was heated under reflux for 24h.","The cooled reaction was diluted with water, the precipitate formed filtered off and washed with water.","Method F A mixture of diacid (0.56 mmol) and ethyl 4-aminophenylacetate (1.11 mmol) in glacial acetic acid (5 ml) was heated at reflux for 24h.","The cooled reaction was diluted with water, the precipitate formed filtered off and washed with water.","Method G (Process B—Step 1) The phthalimide ester (0.32 mmol) was dissolved in glacial acetic acid (4 ml) and zinc powder (100 mesh, 300 mg, 4.59 mmol) added.","The reaction was heated at reflux under nitrogen for 72h.","The hot reaction was filtered and the residue washed with hot acetic acid.","The combined filtrate and washings were evaporated to dryness under vacuum.","Methylamine (33% in ethanol, 2 ml) was added to the resulting solid and the suspension was stirred at room temperature for 20 min.","The methylamine solution was evaporated under vacuum and the residue purified by SPE (silica) eluting with an ethyl acetate/cyclohexane gradient to give a mixture of isomeric y-lactams.","Method H (Processes B and D—Step 2) The γ-lactams (0.06 mmol) were mixed with potassium carbonate (1.09 mmol) in ethanol (2 ml) and water (1 ml) and heated at reflux for 4h.","The cooled solution was acidified to pH1 with hydrochloric acid (2M), the precipitate filtered off and washed with water.","Dried at 40° C. under vacuum.","The following compounds were prepared using the above general methodologies: Intermediate 9 Ethyl [4-(4,9-di-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(4,9-di-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared in 49% yield from 1,4-di-isopropoxynaphthalene-2,3-dicarboxylic acid using method F. δH [2H6]—DMSO 8.41,(2H, m), 7.83,(2H, m), 7.42,(4H, s), 5.02,(2H, m), 4.12,(2H, q), 3.76,(2H, s), 1.36,(12H, d), 1.22,(3H, t).","Intermediate 10 Ethyl [4-(4,9-dipropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(4,9-dipropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared in 68% yield from 1,4-dipropoxynaphthalene-2.3-dicarboxylic acid using method F. δH [2H6]—DMSO 8.35,(2H, m), 7.82,(2H, m), 7.37,(4H, s), 4.34,(4H, t), 4.07,(2H, q), 3.71,(2H, s), 1.83,(4H, m), 1.17,(3H, t), 1.01,(6H, t).","Intermediate 11 Ethyl [4-(4,9-dibutoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(4,9-dibutoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared in 39% yield from 1,4-dibutoxynaphthalene-2,3-dicarboxylic acid using method F. δH [H6]—DMSO 8.39,(2H, m), 7.87,(2H, m), 7.42,(4H, s), 4.43,(4H, t), 4.12,(2H, q), 3.76,(2H, s), 1.85,(4H, m), 1.53,(4H, m), 1.22,(3H, t), 0.97,(6H, t).","Intermediate 12 Ethyl [444,9-dihexyloxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(4,9-dihexyloxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared from 1,4-dihexyloxynaphthalene-2,3-dicarboxylic acid using method F. δH [2H6]—DMSO 8.38,(2H, m), 7.86,(2H, m), 7.42,(4H, s), 4.41,(4H, t), 4.12,(2H, q), 3.76,(2H, s), 1.85,(4H, m), 1.49,(4H, m), 1.32,(8H, m), 1.21,(3H, t), 0.87,(6H, t).","Intermediate 13 Ethyl [4-(4-ethoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(4-ethoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared from 1-ethoxy-4-isopropoxynaphthalene-2,3-dicarboxylic acid using method F. δH [2H6]—DMSO 8.36,(2H, m), 7.79,(2H, m), 7.37,(4H, s), 4.98,(1H, m), 4.42,(2H, q), 4.07,(2H, q), 3.71,(2H, s), 1.40,(3H, t), 1.31,(6H, d), 1.17,(3H, t).","Intermediate 14 Ethyl [4-(9-isopropoxy-4-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(9-isopropoxy-4-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared from 4-isopropoxy-1-propoxynaphthalene-2,3-dicarboxylic acid using method F. δH [2H6]—DMSO 8.35,(2H, m), 7.80,(2H, m), 7.37,(4H, s), 4.97,(1H, m), 4.34,(2H, t), 4.07,(2H, q), 3.71,(2H, s), 1.83,(2H, m), 1.31,(6H, d), 1.71,(3H, t), 1.01,(3H, t).","Intermediate 15 Ethyl [4-(4-hexyloxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(4-hexyloxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared from 1-hexyloxy-4-isopropoxynaphthalene-2,3-dicarboxylic acid using method F. δH [2H6]—DMSO 8.37,(1H, m), 8.32,(1H, m), 7.80,(2H, m), 7.37,(4H, s), 4.97,(1H, m), 4.37,(2H, t), 4.07,(2H, q), 3.71,(2H, s), 1.81,(2H, m), 1.45,(2H, m), 1.35-1.26,(10H, m), 1.17,(3H, t), 0.82,(3H, t).","Intermediate 16 1,4-Di-isopropoxynaphthalene-2,3-dicarboxylic Acid 1,4-Di-isopropoxynaphthalene-2,3-dicarboxylic acid was prepared from ethyl 1,4-d]-propoxynaphthalene-2,3-dicarboxylate using method D. δH [2H6]—DMSO 8.16,(2H, m), 7.67,(2H, m), 4.43,(2H, m), 1.26,(12H, d).","Intermediate 17 1,4-Dipropoxynaphthalene-2,3-dicarboxylic Acid 1,4-Dipropoxynaphthalene-2,3-dicarboxylic acid was prepared from ethyl 1,4-dipropoxynaphthalene-2,3-dicarboxylate using method D. δH [2H6]—DMSO 8.14,(2H, m), 7.71,(2H, m), 4.01,(4H, t), 1.83,(4H, m), 1.05,(6H, t).","Intermediate 18 1,4-Dibutoxynaphthalene-2,3-dicarboxylic Acid 1,4-Dibutoxynaphthalene-2,3-dicarboxylic acid was prepared from ethyl 1,4-dibutoxynaphthalene-2,3-dicarboxylate using method D. δH [2H6]—DMSO 8.12,(2H, m), 7.12,(2H, m), 4.05,(4H, t), 1.80,(4H, m), 1.52,(4H, m), 0.97,(6H, t).","Intermediate 19 1,4-Dihexyloxynaphthalene-2,3-dicarboxylic Acid 1,4-Dihexyloxynaphthalene-2,3-dicarboxylic acid was prepared from ethyl 1,4-dihexyloxynaphthalene-2,3-dicarboxylate using method D. δH [2H6]—DMSO 8.39,(2H, m), 7.92,(2H, m), 4.46,(4H, t), 1.85,(4H, m), 1.49,(4H, m), 1.32,(8H, m), 0.88,(6H, t).","Intermediate 20 1-Ethoxy-4-isopropoxynaphthalene-2,3-dicarboxylic Acid 1-Ethoxy-4-isopropoxynaphthalene-2,3-dicarboxylic acid was prepared from ethyl 1-ethoxy-4-isopropoxynaphthalene-2,3-dicarboxylate using method D. δH [2H6]—DMSO 8.18,(1H, m), 8.12,(1H, m), 7.69,(2H, m), 4.40,(1H, m), 4.11,(2H, q), 1.41,(3H, t), 1.27,(6H, d).","Intermediate 21 4-Isopropoxy-1-propoxynaphthalene-2,3-dicarboxylic Acid 4-Isopropoxy-1-propoxynaphthalene-2,3-dicarboxylic acid was prepared from ethyl 4-isopropoxy-1-propoxynaphthalene-2,3-dicarboxylate using method D. δH [2H6]—DMSO 8.18,(1H, m), 8.11,(1H, m), 7.70,(2H, m), 4.41,(1H, m), 4.02,(2H, t), 1.83,(2H, m), 1.27,(6H, d), 1.06,(3H, t).","Intermediate 22 1-Butoxy-4-isopropoxynaphthalene-2,3-dicarboxylic Acid 1-Butoxy-4-isopropoxynaphthalene-2,3-dicarboxylic acid was prepared from ethyl 1-butoxy-4-isopropoxynaphthalene-2,3-dicarboxylate using method D. δH [2H6]—DMSO 8.18,(1H, m), 8.09,(1H, m), 7.69,(2H, m), 4.40,(1H, m), 4.05,(2H, t), 180,(2H, m), 1.52,(2H, m), 1.26,(6H, d), 0, 97,(3H, t).","Intermediate 23 1-Hexyloxy-4-isopropoxynaphthalene-2,3-dicarboxylic Acid 1-Hexyloxy-4-isopropoxynaphthalene-2,3-dicarboxylic acid was prepared from ethyl 1-hexyloxy-4-isopropoxynaphthalene-2,3-dicarboxylate using method D. δH [2H6]—DMSO 8.18,(1H, m), 8.09,(1H, m), 7.70,(2H, m), 4.40,(1H, m), 4.04,(2H, t), 1.81,(2H, m), 1.49,(2H, m), 1.34,(4H, m), 1.27,(6H, d), 0.90,(3H, t).","Intermediate 24 Ethyl 1,4-di-isopropoxynaphthalene-2,3-dicarboxylate Ethyl 1,4-di-isopropoxynaphthalene-2,3-dicarboxylate was prepared from ethyl 1,4-dihydroxynaphthalene-2,3-dicarboxylate and 2-iodopropane using method C. δH [2H6]—DMSO 8.18,(2H, m), 7.73,(2H, m), 4.37,(2H, m), 4.28,(4H, q), 1.30,(6H, t), 1.25,(12H, d).","Intermediate 25 Ethyl 1,4-dipropoxynaphthalene-2,3-dicarboxylate Ethyl 1,4-dipropoxynaphthalene-2,3-dicarboxylate was prepared from ethyl 1,4-dihydroxynaphthalene-2,3-dicarboxylate and 1-iodopropane using method C. δH [2H6]—DMSO 8.16,(2H, m), 7.77,(2H, m), 4.29,(4H, q), 4.00,(4H, t), 1.83,(4H, m), 1.30,(4H, t), 1.04,(4H, t).","Intermediate 26 Ethyl 1,4-dibutoxynaphthalene-2,3-dicarboxylate Ethyl 1,4-dibutoxynaphthalene-2,3-dicarboxylate was prepared from ethyl 1,4-dihydroxynaphthalene-2,3-dicarboxylate and 1-iodobutane using method C. δH [2H6]—DMSO 8.15,(2H, m), 7.76,(2H, m), 4.29,(4H, q), 4.03,(4H, t), 1.79,(4H, m), 1.50,(4H, m), 1.30,(6H, t), 0.96,(6H, t).","Intermediate 27 Ethyl 1,4-dihexyloxynaphthalene-2,3-dicarboxylate Ethyl 1,4-dihexyloxynaphthalene-2,3-dicarboxylate was prepared from ethyl 1,4-dihydroxynaphthalene-2,3-dicarboxylate and 2-iodohexane using method C. δH [2H6]—DMSO 8.14,(2H, m), 7.76,(2H, m), 4.29,(4H, q), 4.03,(4H, t), 1.81,(4H, m), 1.47,(4H, m), 1.38-1.21,(14H, m), 0.88,(6H, t).","Intermediate 28 Ethyl 1-ethoxy-4-isopropoxynaphthalene-2,3-dicarboxylate Ethyl 1-ethoxy-4-isopropoxynaphthalene-2,3-dicarboxylate was prepared in 91% yield from ethyl 1-hydroxy-4-isopropoxynaphthalene-2,3-dicarboxylate and iodoethane using method B. δH CDCl3 8.22,(1H, m), 8.13,(1H, m), 7.59,(2H, m), 4.40,(5H, m), 4.18,(2H, q), 1.49,(3H, t), 1.43-1.38,(6H, m), 1.34,(6H, d).","Intermediate 29 Ethyl 4-isopropoxy-1-propoxynaphthalene-2,3-dicarboxylate Ethyl 4-isopropoxy-1-propoxynaphthalene-2,3-dicarboxylate was prepared in 91% yield from ethyl 1-hydroxy-4-isopropoxynaphthalene-2,3-dicarboxylate and 1-iodopropane using method B. δH CDCl3 8.22,(1H, m), 8.14,(1H, m), 7.60,(2H, m), 4.40,(5H, m), 4.07,(2H, t), 1.92,(2H, m), 1.40,(6H, m), 1.34,(6H, d), 1.10,(3H, t).","Intermediate 30 Ethyl 1-butoxy-4-isopropoxynaphthalene-2,3-dicarboxylate Ethyl 1-butoxy-4-isopropoxynaphthalene-2,3-dicarboxylate was prepared from ethyl 1-hydroxy-4-isopropoxynaphthalene-2,3-dicarboxylate and 1-iodobutane using method B. δH [2H6]—DMSO 8.20,(1H, m), 8.12,(1H, m), 7.75,(2H, m), 4.38-4.25,(5H, m), 4.04,(2H, t), 1.80,(2H, m), 1.50,(2H, m), 1.30,(6H, t), 1.25,(6H, d), 0.96,(3H, t).","Intermediate 31 Ethyl 1-hexyloxy-4-isopropoxynaphthalene-2,3-dicarboxylate Ethyl 1-hexyloxy-4-isopropoxynaphthalene-2,3-dicarboxylate was prepared in 80 yield from ethyl 1-hydroxy-4-isopropoxynaphthalene-2,3-dicarboxylate and 1-iodohexane using method B. δH CDCl3 8.21,(1H, m), 8.13,(1H, m), 7.59,(2H, m), 4.40,(5H, m), 4.10,(2H, t), 1.90,(2H, m), 1.53,(2H, m), 1.45-1.31,(16H, m), 0.92,(3H, t).","Intermediate 32 Ethyl 1-ethoxy-4-hydroxynaphthalene-2,3-dicarboxylate Ethyl 1-ethoxy-4-hydroxynaphthalene-2,3-dicarboxylate was prepared from ethyl 1,4-dihydroxynaphthalene-2,3-dicarboxylate and iodoethane using method A. δH [2H6]—DMSO 8.36,(1H, d), 8.06,(1H, d), 7.83,(1H, m), 7.72,(1H, m), 4.35,(4H, m), 4.03,(2H, q), 1.39,(3H, t), 1.32,(6H, m).","Intermediate 33 Ethyl 1-hydroxy-4-isopropoxynaphthalene-2,3-dicarboxylate Ethyl 1-hydroxy-4-isopropoxynaphthalene-2,3-dicarboxylate was prepared in 36% yield from ethyl 1,4-dihydroxynaphthalene-2,3-dicarboxylate and 2-iodopropane using method A. δH CDCl3 12.3,(1H, s), 8.45,(1H, d), 8.10,(1H, d), 7.65,(1H, m), 7.55,(1H, m), 4.42,(1H, m), 1.41,(6H, t), 1.32,(6H, d).","EXAMPLE 4 Step 1 (Process B) Ethyl [4-(4,9-di-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(4,9-di-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared in 16% yield from ethyl [4-(4,9-di-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method G. δH [2H6]—DMSO 8.34,(1H, d), 8.16,(1H, d), 7.93,(2H, d), 7.68,(1H, t), 7.60,(1H, t), 7.34,(2H, d), 5.10,(2H, s), 5.01,(1H, m), 4.68,(1H, m), 4.10,(2H, q), 3.69,(2H, s), 1.40,(3H, d), 1.35,(3H, d), 1.19,(3H, t).","EXAMPLE 4 Step 2 [4-(4,9-Di-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4,9-Di-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared in 81% yield from ethyl [4-(4,9-di-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method H. δH [2H6]—DMSO 8.34,(1H, d), 8.17,(1H, d), 7.91,(2H, d), 7.69,(1H, m), 7.61,(1H, m), 7.34,(2H, d), 5.10,(2H, s), 5.02,(1H, m), 4.68,(1H, m), 3.59,(2H, s), 1.38,(6H, d), 1.34,(6H, d).","MS 434, [MH+].","LC retention time 3.91 min.","EXAMPLE 5 Step 1 (Process B) Ethyl [4-(4,9-dipropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(4,9-dipropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared in 13% yield from ethyl [4-(4,9-dipropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method G. δH [2H6]—DMSO 8.32,(1H, d), 8.19,(1H, d), 7.93,(2H, d), 7.72,(1H, t), 7.65,(1H, t), 7.36,(2H, d), 5.19,(2H, s), 4.30,(2H, t), 4.22,(2H, t), 4.10,(2H, q), 3.69,(2H, s), 1.87,(4H, m), 1.20,(3H, t), 1.13-1.05,(6H, m).","EXAMPLE 5 Step 2 [4-(4,9-Dipropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4,9-Dipropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared in 89% yield from ethyl [4-(4,9-dipropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method H. δH [2H6]—DMSO 8.32,(1H, d), 8.19,(1H, d), 7.91,(2H, d), 7.71,(1H, t), 7.65,(1H, t), 7.65,(2H, d), 5.19,(2H, s), 4.30,(2H, t), 4.22,(2H, t), 3.59,(2H, s), 1.89,(4H, m), 1.09,(6H, m).","MS 434, [MH+].","LC retention time 3.97 min.","EXAMPLE 6 Step 1 (Process B) Ethyl [4-(4,9-dibutoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(4,9-dibutoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared in 26% yield from ethyl [4-(4,9-dibutoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method G. δH [2H6]—DMSO 8.22,(1H, d), 8.09,(1H, d), 7.84,(2H, d), 7.63,(1H, t), 7.57,(1H, t), 7.27,(2H, d), 5.10,(2H, s), 4.26,(2H, t), 4.17,(2H, t), 4.02,(2H, q), 3.60,(2H, s), 1.77,(4H, quintet), 1.49,(4H, m), 1.12,(3H, t), 0.91,(6H, q).","EXAMPLE 6 Step 2 [4-(4,9-Dibutoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4,9-Dibutoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared in 98% yield from ethyl [4-(4,9-dibutoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method H. δH [2H6]—DMSO 8.30,(1H, d), 8.18,(1H, d), 7.91,(2H, d), 7.71,(1H, t), 7.65,(1H, t), 7.35,(2H, d), 5.19,(2H, s), 4.34,(2H, t), 4.26,(2H, t), 3.59,(2H, s), 1.85,(4H, m), 1.57,(4H, m), 0.99,(6H, m).","MS 462, [MH+].","LC retention time 4.25 min.","EXAMPLE 7 Step 1 (Process B) Ethyl [44,9-dihexyloxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(4,9-dihexyloxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared in 28% yield from ethyl [4-(4,9-dihexyloxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method G. δH [2H6]—DMSO 8.19,(1H, d), 8.07,(1H, d), 7.82,(2H, d), 7.60,(1H, t), 7.54,(1H, t), 7.25,(2H, d), 5.08,(2H, s), 4.23,(2H, t), 4.15,(2H, t), 3.99,(2H, q), 3.58,(2H, s), 1.75,(4H, quintet), 1.50-1.36,(4H, m), 1.29-1.21,(8H, m), 1.09,(3H, t), 0.79,(6H, q).","EXAMPLE 7 Step 2 [4-(4,9-Dihexyloxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4,9-Dihexyloxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared in 97% yield from ethyl [4-(4,9-dihexyloxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method H. δH [2H6]—DMSO 8.30,(1H, d), 8.17,(1H, d), 7.91,(2H, d), 7.71,(1H, t), 7.64,(1H, t), 7.34,(2H, d), 5.18,(2H, s), 4.33,(2H, t), 4.25,(2H, t), 3.59,(2H, s), 1.86,(4H, m), 1.54,(4H, m), 1.35,(8H, m), 0.90,(6H, m).","MS 518, [MH+].","LC retention time 4.76 min.","EXAMPLE 8 Step 1 (Process B) Ethyl [4-(4-ethoxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate and ethyl [4-(9-thoxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate A mixture of ethyl [4-(4-ethoxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate and ethyl [4-(9-ethoxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate (60:40) was prepared in 20% yield from ethyl [4-(4-ethoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method G. δH [2H6]—DMSO 8.25,(1H, m), 8.09,(1H, m), 7.85,(2H, d), 7.64-7.58,(2H, m), 7.26,(2H, d), 5.09,(1.2H, s), 5.02,(0.8H, s), 4.90,(0.6H, m), 4.60,(0.4H, m), 4.32,(0.8H, q), 4.22,(1.2H, q), 4.01,(2H, q), 3.60,(2H, s), 1.38,(3H, m), 1.29,(2.4H, d), 1.24,(3.6H, d), 1.11,(3H, t).","EXAMPLE 8 Step 2 [4-(4-Ethoxy-9-isopropoxy-1 oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid and [4-(9-ethoxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid A mixture of [4-(4-ethoxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid and [4-(9-ethoxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid (60:40) was prepared in 98% yield from ethyl [4-(4-ethoxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate and ethyl [4-(9-ethoxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate (60:40) using method H. δH [2H6]—DMSO 8.34,(1H, m), 8.18,(1H, m), 7.92,(2H, d), 7.70,(1H, t), 7.68,(1H, t), 7.34,(2H, d), 5.18,(1.2H, s), 5.11,(0.8H, s), 5.01,(0.6H, m), 4.69,(0.4H, m), 4.40,(0.8H, q), 4.31,(1.2H, q), 3.59,(2H, s), 1.46,(3H, m), 1.37,(2.4H, d), 1.33,(3.6H, d).","MS 420, [MH+].","LC retention time 3.73 min.","EXAMPLE 9 Step 1 (Process B) Ethyl [4-(9-isopropoxy-4-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate and ethyl [4-(4-isopropoxy-9-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate A mixture of ethyl [4-(9-isopropoxy-4-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate and ethyl [4-(4-isopropoxy-9-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate (60:40) was prepared in 21% yield from ethyl [4-(9-isopropoxy-4-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method G. δH [2H6]—DMSO 8.25,(1H, m), 8.10,(1H, m), 7.85,(2H, m), 7.62,(1H, m), 7.53,(1H, m), 7.27,(2H, d), 5.10,(1.2H, s), 5.02,(0.8H, s), 4.91,(0.6H, m), 4.58,(0.4H, m), 4.23,(0.8H, t), 4.13,(1.2H, t), 4.01,(2H, q), 3.60,(2H, s), 1.80,(2H, m), 1.29,(2.4H, d), 1.24,(3.6H, d), 1.11,(3H, t), 1.01,(3H, m).","EXAMPLE 9 Step 2 [4-(9-isopropoxy-4-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid and [4-(4-isopropoxy-9-propoxy-1 oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid A mixture of [4-(9-isopropoxy-4-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid and [4-(4-isopropoxy-9-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid (60:40) was prepared in quantitative yield from ethyl [4-(9-isopropoxy-4-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate and ethyl [4-(4-isopropoxy-9-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate (60:40) using method H. δH [2H6]—DMSO 8.34,(1H, m), 8.18,(1H, m), 7.92,(2H, m), 7.70.","(1H, t), 7.63,(1H, m), 7.34,(2H, d), 5.18,(1.2H, s), 5.11,(0.8H, s), 5.01,(0.6H, m), 4.68,(0.4H, m), 4.32,(0.8H, q), 4.22,(1.2H, q), 3.59,(2H, s), 1.89,(2H, m), 1.37,(2.4H, d), 1.33,(3.6H, d), 1.10,(3H, m).","MS 434, [MH+].","LC retention time 3.89 min.","EXAMPLE 10 Step 1 (Process B) Ethyl [4-(4-hexyloxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate and ethyl [4-(9-hexyloxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate A mixture of ethyl [4-(4-hexyloxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate and ethyl [4-(9-hexyloxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate (60:40) was prepared in 16% yield from ethyl [4-(4-hexyloxy-9-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method G. δH [2H6]—DMSO 8.33,(1H, m), 8.17,(1H, m), 7.94,(2H, m), 7.70,(1H, m), 7.64,(1H, m), 7.35,(2H, d), 5.18,(1.2H, s), 5.11,(0.8H, s), 5.00,(0.6H, m), 4.69,(0.4H, m), 4.35,(0.8H, t), 4.25,(1.2H, t), 4.10,(2H, q), 3.69,(2H, s), 1.87,(2H, m), 1.60-1.45,(2H, m), 1.37,(2.4H, d), 1.33,(3.6H, d), 1.20,(3H, t), 0.91,(3H, m).","EXAMPLE 10 Step 2 [4-(4-Hexyloxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid and [4-(9-hexyloxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid A mixture of [4-(4-hexyloxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid and [4-(9-hexyloxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid (60:40) was prepared in 78% yield from ethyl [4-(4-hexyloxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate and ethyl [4-(9-hexyloxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate (60:40) using method H. δH [2H6]—DMSO 8.35,(0.6H, d), 8.30,(0.4H, d), 8.17,(2H, m), 7.91,(2H, m), 7.70,(1H, t), 7.63,(1H, m), 7.34,(2H, d), 5.18,(1.2H, s), 5.10,(0.8H, s), 5.00,(0.6H, m), 4.68,(0.4H, m), 4.35,(0.8H, q), 4.25,(1.2H, q), 3.59,(2H, s), 1.87,(2H, m), 1.54,(2H, m), 1.35,(10H, m), 0.91,(3H, m).","MS 476, [MH+].","LC retention time 4.32 min.","EXAMPLE 11 Step 1 (Process D) Ethyl [44-methoxy-1 oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate 3-Hydroxy-4-methoxynaphtho[2,3-c]furan-1 (3H-one (50 mg, 0.22 mmol) and ethyl 4-aminophenylacetate (47 mg, 0.26 mmol) in dichloromethane (5 ml) were stirred at room temperature under nitrogen for 1 hour.","Sodium triacetoxyborohydride (138 mg, 0.66 mmol) was added and stirring continued for 24h.","The reaction was adjusted to pH14 with sodium hydroxide solution (2N) and extracted with dichloromethane (3×5 ml).","The combined extracts were evaporated under vacuum and the residue triturated with cyclohexane/ether to give ethyl [4-(4-methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate as a white solid (45 mg, 54%).","δH [2H6]—DMSO 8.24,(1H, d), 8.16,(1H, d), 8.13,(1H, s), 7.99,(2H, d), 7.64,(2H, m), 7.37,(2H, d), 5.44,(2H, s), 4.26,(3H, s), 4.10,(2H, q), 3.69,(2H, s), 1.20,(3H, t).","EXAMPLE 11 Step 2 [4-(4-Methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4-Methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared in quantitative yield from ethyl [4-(4-methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method H. δH [2H6]—DMSO 8.19,(1H, d), 8.09,(2H, m), 7.92,(2H, d), 7.59,(2H, m), 7.31,(2H, d), 5.39,(2H, s), 4.21,(3H, s), 3.56,(2H, s).","MS 348, [MH+].","LC retention time 3.50 min.","EXAMPLE 12 [44,9-Diethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4,9-Diethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared from 1,4-diethoxynaphthalene-2,3-dicarboxylic acid using method E. δH [2H6]—DMSO 8.35,(2H, m), 7.81,(2H, m), 7.36,(4H, s), 4.43,(4H, q), 3.62,(2H, s), 1.40,(6H, t).","MS 420, [MH+].","LC retention time 3.58 min.","EXAMPLE 13 [4-(4,9-Di-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4,9-Di-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared from 1,4-di-isopropoxynaphthalene-2,3-dicarboxylic acid using method E. δH [2H6]—DMSO 8.41,(2H, m), 7.83,(2H, m), 7.41,(4H, s), 5.02,(2H, m), 3.66,(2H, s), 1.36,(12H, d).","MS 448, [MH+].","LC retention time 3.74 min.","EXAMPLE 14 [4-(4,9-Dipropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4,9-Dipropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared from 1,4-dipropoxynaphthalene-2,3-dicarboxylic acid using method E. δH [2H6]—DMSO 8.39,(2H, m), 7.87,(2H, m), 7.41,(4H, s), 4.39,(4H, t), 3.67,(2H, s), 1.87,(4H, m), 1.05,(6H, t).).","MS 448, [MH+].","LC retention time 3.89 min.","EXAMPLE 15 [4-(4,9-Dibutoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4,9-Dibutoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared from 1,4-dibutoxynaphthalene-2,3-dicarboxylic acid using method E. δH [2H6]—DMSO 8.39,(2H, m), 7.88,(2H, m), 7.41,(4H, s), 4.43,(4H, t), 3.67,(2H, s), 1.84,(4H, m), 1.52,(4H, m), 0.96,(6H, t).","MS 476, [MH+].","LC retention time 4.15 min.","EXAMPLE 16 [4-(4,9-Dihexyloxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4,9-Dihexyloxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared from 1,4-dihexyloxynaphthalene-2,3-dicarboxylic acid using method E. δH [2H6]—DMSO 8.38,(2H, m), 7.86,(2H, m), 7.41,(4H, s), 4.42,(4H, t), 3.67,(2H, s), 1.85,(4H, m), 1.49,(4H, m), 1.32,(8H, m), 0.87,(6H, t).).","MS 532, [MH+].","LC retention time 4.62 min.","EXAMPLE 17 [4-(4-Ethoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4-Ethoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared from 1-ethoxy-4-isopropoxynaphthalene-2,3-dicarboxylic acid using method E. δH [2H6]—DMSO 8.41,(2H, m), 7.84,(2H, m), 7.41,(4H, s), 5.03,(1H, m), 4.48,(2H, q), 3.67,(2H, s), 1.45,(3H, t), 1.36,(6H, d).","MS 434, [MH+].","LC retention time 3.66 min.","EXAMPLE 18 [4-(9-isopropoxy-4-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(9-isopropoxy-4-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared from 4-isopropoxy-1-propoxynaphthalene-2,3-dicarboxylic acid using method E. δH [2H6]—DMSO 8.41,(2H, m), 7.84,(2H, m), 7.41,(4H, s), 5.02,(1H, m), 4.39,(2H, t), 3.66,(2H, s), 1.88,(2H, m), 1.36,(6H, d), 1.05,(3H, t).","MS 448, [MH+].","LC retention time 3.81 min.","EXAMPLE 19 [4-(4-Butoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4-Butoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared from 1-butoxy-4-isopropoxynaphthalene-2,3-dicarboxylic acid using method E. δH [2H6]—DMSO 8.42,(1H, m), 8.37,(1H, m), 7.85,(2H, m), 7.41,(4H, s), 5.02,(1H, m), 4.43,(2H, t), 3.66,(2H, s), 1.85,(2H, m), 1.53,(2H, m), 1.36,(6H, d), 0.97,(3H, t).","MS 461, [MH+].","LC retention time 3.94 min.","Biological Data The ability of the compounds of the invention to bind to EP4 receptors has been demonstrated in the Human EP4 Scintillation Proximity Assay.","Quantification of radioligand binding by scintillation proximity assay (SPA) is a long-established principle.","Briefly, the affinity of novel compounds for a receptor is assessed by the specific competition between known quantities of radiolabelled ligand and novel compound for that receptor.","Increasing concentrations of novel compound reduce the amount of radiolabel that binds to the receptor.","This gives rise to a diminishing scintillation signal from SPA beads coated with membranes that bear the receptor.","The signal may be detected with a suitable scintillation counter and the data generated may be analysed with suitable curve-fitting software.","The human EP4 SPA assay (hereafter referred to as ‘the assay’) utilises membranes prepared from Chinese Hamster Ovary (CHO cells) infected with Semliki Forest Virus (SFV).","Genetically engineered SFV-1 viral particles containing the genetic sequence of the human EP4 receptor were used to infect CHO cells resulting in expression of the receptor protein in cellular membranes.","Cells washed free of media are homogenised in a pH-buffered medium containing peptidase inhibitors.","A suitable buffer is of the following composition: 50 mM HEPES, 1 mM EDTA, 25 μg/ml bacitracin, 100 μM leupeptin, 1 mM PMSF, 2 μM Pepstatin A, pH adjusted to 7.4 with KOH.","Following removal of cell debris by a low-speed centrifugation, a pellet of membranes is prepared by a high-speed (48000 g) centrifugation of the resulting supernatant.","Membrane suspensions such as that described may be stored at −80° C. until used.","For assay, membranes expressing human EP4 receptors are diluted in a pH-buffered medium and mixed with SPA beads coated with a suitable substance to facilitate the adhesion of membranes to the beads.","The concentrations of membrane protein and SPA beads chosen should result in SPA binding signal of at least 300 corrected counts per minute (CCPM) when tritiated radioligand at a concentration close to its Kd (affinity value) is combined with the mixture.","Non-specific binding (nsb) may be determined by competition between the radiolabelled ligand and a saturating concentration of unlabelled ligand.","In order to quantify the affinity of novel EP4 receptor ligands, compounds are diluted in a stepwise manner across the wells of a 96-well plate.","Radioligand, novel compound, and unlabelled ligand are then added to a 96-well plate suitable for the measurement of SPA binding signals prior to the addition of bead/membrane mixture to initiate the binding reaction.","Equilibrium may be achieved by incubation at room temperature for 120 minutes prior to scintillation counting.","The data so generated may be analysed by means of a computerised curve-fitting routine in order to quantify the concentration of compound that displaces 50% of the specific radioligand binding (IC50).","The affinity (pKi) of the novel compound may be calculated from the IC50 by application of the Cheng-Prusoff correction.","Suitable reagents and protocols are: reaction buffer containing 50 mM HEPES, 10 mM MgCl2, pH adjusted to 7.4 with KOH; SPA beads coated with wheatgerm agglutinin; 1.25 nM [3H]-prostaglandin E2 as radioligand; 10 μM prostaglandin E2 as unlabelled ligand; a three-fold dilution series of novel compound starting at 10 μM and ending at 0.3 nM is adequate.","The following examples have a pKi of 6.0 or greater at EP4 receptors as determined using the above-mentioned procedure: 1, 4, 5, 8, 9, 12, 13, 14, 17."]],"string":"[\n [\n \"Benzo'fisoindole derivatives with affinity to the ep4 receptor\",\n \"The present invention relates to a compound of formula (I): corresponding pharmaceutical compositions, preparation processes, and/or methods of using the aforementioned compounds and/or compositions.\"\n ],\n [\n \"1.A compound of formula (I): wherein: R1 and R3 are the same or different and represent ═O, hydrogen, C1-6alkyl, C1-6-dialkyl, ═CHC1-C5alkyl, ═S, or a 5- or 6-membered aryl; R4 to R9 are the same or different and represent hydrogen, C1-6alkoxy, OCF3, OCH2CF3, O-cyclopropyl, OCH2-cyclopropyl, C1-C6alkyl, S-alkyl, NR210 where R10 is hydrogen or C1-6alkyl, halogen, NO2, OH, CH2OC1-C6alkyl, CH2OH, or CF3; Q1 is hydrogen, C1-6alkyl, C1-6dialkyl, C1-6alkoxy, NHAc, NR210 where R10 is hydrogen or C1-6alkyl, difluoro, fluoro, ═O, or OH; Q2, Q3, Q4 and Q5 are the same or different and represent hydrogen, C1-6alkoxy, OCF3, OCH2CF3, O-cyclopropyl, OCH2-cyclopropyl, C1-C6alkyl, S-alkyl, NR210 where R10 is hydrogen or C1-6alkyl, halogen, NO2, OH, CH2OC1-C6alkyl, CH2OH, or a 5- or 6-membered aryl; provided that the compound of formula (I) is not: [4-(1-oxo-1,3-dihydro-2H-benzo [f]isoindol-2-yl)phenyl]-2-propionic acid, and sodium salt and [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; and pharmaceutically acceptable derivatives thereof.\",\n \"2.A compound according to claim 1 wherein R1 is ═O.\",\n \"3.A compound according to claim 1 wherein R3 is methyl, dimethyl, 2-furanyl, ═O or hydrogen.\",\n \"4.A compound according to claim 1 wherein R4 and R9 are each hydrogen, methoxy, ethoxy, i-propoxy, n-propoxy, n-butyloxy or n-hexyloxy.\",\n \"5.A compound according to claim 1 wherein R6 and R7 are each hydrogen, methyl, methoxy, chlorine, bromine, iodine, NO2, or CF3.6.A compound according to claim 1 wherein Q1 is hydrogen, methyl, ethyl, NHAc, NH2 or methoxy.\",\n \"7.A compound according to claim 1 wherein Q2, Q3, Q4 and Q5 are each hydrogen, methyl, methoxy, chlorine, bromine, iodine, 2-thiophenyl, 3-thiophenyl, 2-furanyl, 3-furanyl, or phenyl.\",\n \"8.A compound of formula (I) wherein said compound is selected from the group consisting of: [4-(4,9-dimethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-di-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dipropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4,9-dibutoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dihexyloxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4-ethoxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid and [4-(9-ethoxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-isopropoxy-4-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid and [4-(4-isopropoxy-9-propoxy-1-oxo-1,3-dihydro-2H-benzo [f]isoindol-2-yl)phenyl]acetic acid; [4-(4-hexyloxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid and [4-(9-hexyloxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4,9-di-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4,9-dipropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4,9-dibutoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4,9-dihexyloxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4-ethoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(9-isopropoxy-4-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; and [4-(4-butoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid.\",\n \"9.A process for preparing a compound of formula (I) and pharmaceutically acceptable derivatives thereof according to claim 1 which comprises: (A) where R1 and R3 are both ═O and R4 and R9 are the same or different and represent C1-6alkoxy, by reacting a compound of formula (II): with a 4-aminophenylacetic acid of formula (III): in glacial acetic acid at elevated temperature; (B) where one of R1 and R3 is ═O and the other is hydrogen and R4 and R9 are the same or different and represent C1-6alkoxy, by reducing a compound of formula (IV): with a suitable reducing agent, followed by separation of isomers and deprotection; (C) where one of R1 and R3 is ═O and the other is hydrogen and R4 and R9 are the same or different and represent C1-6alkoxy, by reacting a compound of formula (V): with a 4-aminophenylacetic acid of formula (III) as defined above in presence of triethylamine and dimethylformamide, followed by deprotection; (D) where one of R1 and R3 is ═O and the other is hydrogen and one of R4 and R9 is C1-6alkoxy, by reacting a compound of formula (VI): where RD is in the −4 or -9 position and is C1-6 alkoxy, with a 4-aminophenylacetate of formula (VII): in presence of sodium triacetoxyborohydride in a suitable solvent, followed by deprotection; (E) where R1 and R3 are both hydrogen and R4 and R9 are the same or different and represent C1-6 alkoxy, by reacting a compound of formula (VIII): with a 4-aminophenylacetate of formula (VII) as described above in a suitable solvent, at elevated temperature followed by deprotection; (F) where R3 is ═CHC1-5alkyl, by reacting a compound of formula (IV) as defined above with a Grignard reagent C1-C6alkyl-MgBr under conventional conditions, followed by separation of isomers and deprotection; (G) where R3 is C1-6dialkyl, by reacting a compound of formula (IX): with a 4-aminophenylacetate of formula (VII) as defined above in presence of aluminium trichloride, followed by deprotection; or (H) converting compounds of formula (I), prepared according to processes (A) to (G), into other compounds of formula (I) using conventional procedures; and optionally converting compounds of formula (I) prepared by any one of processes (A) to (H) into pharmaceutically acceptable derivatives thereof.\",\n \"10.A compound of formula (I) or a pharmaceutically acceptable derivative thereof as defined in claim 1 for use in human or veterinary medicine.\",\n \"11.\",\n \"(Cancelled) 12.A method of treating a human or animal subject suffering from a condition which is mediated by the action of PGE2 at EP4 receptors which comprises administering to said subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable derivative thereof as defined in claim 1.13.A pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable derivative thereof, as defined in claim 1 in admixture with one or more physiologically acceptable carriers or excipients.\"\n ],\n [\n \"This invention relates to naphthalene derivatives, to processes for their preparation, to pharmaceutical compositions containing them and to their use in medicine.\",\n \"The EP4 receptor is a 7-transmembrane receptor and its natural ligand is the prostaglandin PGE2.PGE2 also has affinity for the other EP receptors (types EP1, EP2 and EP3).\",\n \"The EP4 receptor is associated with smooth muscle relaxation, inflammation, lymphocyte differentiation, bone metabolism processes, allergic activities, promotion of sleep, renal regulation and gastric or enteric mucus secretion.\",\n \"We have now found a novel group of compounds which bind with high affinity to the EP4 receptor.\",\n \"Compounds exhibiting EP4 binding activity have been described in, for example, WO00/18744, WO00/03980, WO00/15608, WO0016760, WO00/21532, WO98/55468, EP0855389 and EP0985663.GB2330307 describes the use of EP4 antagonists in the treatment of conditions with accelerated bone resorption.\",\n \"Derivatives of indoprofen, such as [4-(1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]-2-propionic acid, sodium salt shown below, have been described by Rufer et. al.\",\n \"In Eur.\",\n \"J. Med.\",\n \"Chem.—Chimica Therapeutica, 1978, 13, no 2, pg 193-198.Accordingly, the present invention provides a compound of formula (I) wherein R1 and R3 are the same or different and represent ═O, hydrogen, C1-6alkyl, C1-6dialkyl, ═CHC1-C5alkyl, ═S, or a 5- or 6-membered aryl; R4 to R9 are the same or different and represent hydrogen, C1-6alkoxy, OCF3, OCH2CF3, O-cyclopropyl, OCH2-cyclopropyl, C1-C6alkyl, S-alkyl, NR210 where R10 is hydrogen or C1-6alkyl, halogen, NO2, OH, CH2OC1-C6alkyl, CH2OH, or CF3; Q1 is hydrogen, C1-6alkyl, C1-6dialkyl, C1-6alkoxy, NHAc, NR210 where R10 is hydrogen or C1-6alkyl, difluoro, fluoro, ═O, or OH; Q2, Q3, Q4 and Q5 are the same or different and represent hydrogen, C1-6alkoxy, OCF3, OCH2CF3, O-cyclopropyl, OCH2-cyclopropyl, C1-C6alkyl, S-alkyl, NR210 where R10 is hydrogen or C1-6alkyl, halogen, NO2, OH, CH2OC1-C6alkyl, CH2OH, or a 5- or 6-membered aryl; with the proviso that the compounds [4-(1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]-2-propionic acid, sodium salt and [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid are excluded; and pharmaceutically acceptable derivatives thereof.\",\n \"By pharmaceutically acceptable derivative is meant any pharmaceutically acceptable salt, solvate or ester, or salt or solvate of such ester of the compounds of formula (I), or any other compound which upon administration to the recipient is capable of providing (directly or indirectly) a compound of formula (I) or an active metabolite or residue thereof.\",\n \"It will be appreciated by those skilled in the art that the compounds of formula (I) may be modified to provide pharmaceutically acceptable derivatives thereof at any of the functional groups in the compounds, and that the compounds of formula (I) may be derivatised at more than one position.\",\n \"It will be appreciated that, for pharmaceutical use, the salts referred to above will be the pharmaceutically acceptable salts, but other salts may find use, for example in the preparation of compounds of formula (I) and the pharmaceutically acceptable salts thereof.\",\n \"Pharmaceutically acceptable salts include those described by Berge, Bighley and Monkhouse, J. Pharm.\",\n \"Sci., 1977, 66, 1-19.Suitable pharmaceutically acceptable salts of the compounds of formula (I) include acid addition salts formed with inorganic or organic acids, preferably inorganic acids, e.g.\",\n \"hydrochlorides, hydrobromides and sulphates.\",\n \"Further representative examples of pharmaceutically acceptable salts include those formed from acetic, maleic, fumaric, benzoic, ascorbic, pamoic, succinic, bismethylenesalicylic, methanesulfonic, ethanedisulfonic, propionic, tartaric, salicylic, citric, gluconic, aspartic, stearic, palmitic, itaconic, glycolic, p-aminobenzoic, glutamic, benzenesulfonic, cyclohexylsulfamic, phosphoric and nitric acids.\",\n \"Further representative examples of pharmaceutically acceptable salts include alkali metal salts, formed from the addition of alkali metal bases, such as alkali metal hydroxides e.g.\",\n \"sodium salts.\",\n \"It will be appreciated that the compound of formula (I) may be produced in vivo by metabolism of a suitable prodrug.\",\n \"Such prodrugs may be for example physiologically acceptable metabolically labile esters of compounds of the general formula (I).\",\n \"These may be formed by esterification of the carboxylic acid group in the parent compound of general formula (I) with, where appropriate, prior protection of any other reactive groups present in the molecule followed by deprotection if required.\",\n \"Examples of such metabolically labile esters include C1-4alkyl esters e.g.\",\n \"methyl ethyl or t-butyl esters esters, C3-6 alkenyl esters e.g.\",\n \"allyl substituted or unsubstituted aminoalkyl esters (e.g.\",\n \"aminoethyl, 2-(N,N-diethylamino) ethyl, or 2-(4-morpholino)ethyl esters or acyloxyalkyl esters such as, acyloxymethyl or 1-acyloxyethyl e.g.\",\n \"pivaloyloxymethyl, 1-pivaloyloxyethyl, acetoxymethyl, 1-acetoxyethyl,1-(1-methoxy-1-methyl)ethylcarbonyloxyethyl, 1-benzoyloxyethyl, isopropoxycarbonyloxymethyl, 1-isopropoxycarbonyloxyethyl, cyclohexylcarbonyloxymethyl, 1-cyclohexylcarbonyloxyethyl ester, cyclohexyloxycarbonyloxymethyl, 1-cyclohexyloxycarbonyloxyethyl, 1-(4-tetrahydropyranyloxy)carbonyloxyethyl or 1-(4-tetrahydropyranyl)carbonyloxyethyl.\",\n \"As used herein, the term halogen atom includes fluorine, and more especially chlorine, bromine or iodine.\",\n \"The term C1-6alkyl, C1-6alkoxy or ═CHC1-5alkyl as used herein includes straight chain and branched chain alkyl or alkoxy groups containing 1 to 6 carbon atoms, and in particular includes methyl, ethyl, n-propyl and i-propyl or methoxy, ethoxy, i-propoxy, n-propoxy, n-butyloxy or n-hexyloxy.\",\n \"The term 5-membered aryl as used herein means an aryl selected from the following: The term 6-membered aryl as used herein means an aryl selected from: A preferred subgroup of compounds of formula (I) include the compounds wherein R1 is ═O or hydrogen; R3 is ═O, hydrogen, C1-6alkyl, C1-6dialkyl, or a 5- or 6-membered aryl; R4 and R9 are the same or different and represent hydrogen or C1-6alkoxy; R5 and R8 are hydrogen, or CF3; R6 and R7 are the same or different and represent hydrogen, C1-6alkyl, C1-6alkoxy, halogen, NO2, or CF3; Q1 is hydrogen, C1-6alkyl, C1-6alkoxy, NHAc, or NR210 where R10 is hydrogen or C1-6alkyl; Q2, Q3, Q4 and Q5 are the same or different and represent hydrogen, C1-6alkyl, C1-6alkoxy, halogen, or a 5- or 6-membered aryl; with the proviso that the compounds [4-(1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]-2-propionic acid, sodium salt and [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid are excluded; and pharmaceutically acceptable derivatives thereof.\",\n \"R1 is preferably ═O.\",\n \"R3 is aptly selected from methyl, dimethyl, or 2-furanyl.\",\n \"R3 is preferably ═O or hydrogen, and is more preferably hydrogen.\",\n \"R4 and R9 are each suitably hydrogen, methoxy, ethoxy, i-propoxy, n-propoxy, n-butyloxy or n-hexyloxy.\",\n \"Suitably at least one of R4 and R9 represents C1-6alkoxy when Q1 is methyl.\",\n \"Preferably R4 and R9 are both C1-6alkoxy, eg ethoxy.\",\n \"R6 and R7 are suitably hydrogen, methyl, methoxy, chlorine, bromine, iodine, NO2, or CF3.Preferably R6 and R7 are both hydrogen.\",\n \"Q1 is suitably hydrogen, methyl, ethyl, NHAc, NH2 or methoxy.\",\n \"Q1 is preferably hydrogen.\",\n \"Q2, Q3, Q4 and Q5 are each suitably hydrogen, methyl, methoxy, chlorine, bromine, iodine, 2-thiophenyl, 3-thiophenyl, 2-furanyl, 3-furanyl, or phenyl.\",\n \"Preferably Q2, Q3, Q4 and Q5 are all hydrogen.\",\n \"A further preferred group of compounds of formula (I) are those wherein R1 is ═O; R3 is methyl, dimethyl, 2-furanyl, preferably ═O or hydrogen, and is more preferably hydrogen; R4 and R9 are each hydrogen, methoxy, ethoxy, i-propoxy, n-propoxy, n-butyloxy or n-hexyloxy, preferably R4 and R9 are both C1-6alkoxy, eg ethoxy; R5 and R8 are hydrogen; R6 and R7 are hydrogen, methyl, methoxy, chlorine, bromine, iodine, NO2, or CF3, preferably R6 and R7 are both hydrogen; Q1 is suitably hydrogen, methyl, ethyl, NHAc, NH2 or methoxy, preferably hydrogen; Q2, Q3, Q4 and Q5 are each suitably hydrogen, methyl, methoxy, chlorine, bromine, iodine, 2-thiophenyl, 3-thiophenyl, 2-furanyl, 3-furanyl, or phenyl, preferably Q2, Q3, Q4 and Q5 are all hydrogen; with the proviso that the compounds [4-(1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]-2-propionic acid, sodium salt and [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid are excluded; and pharmaceutically acceptable derivatives thereof.\",\n \"It is to be understood that the present invention encompasses all isomers of the compounds of formula (I) and their pharmaceutically acceptable derivatives, including all geometric, tautomeric and optical forms, and mixtures thereof (e.g.\",\n \"racemic mixtures).\",\n \"Preferred compounds of the present invention include: [4-(4-methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dimethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-methoxy-9-ethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-methoxy-9-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-methoxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-ethoxy-9-methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-ethoxy-9-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-ethoxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-ethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-propoxy-9-methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-propoxy-9-ethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dipropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-isopropoxy-4-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-isopropoxy-9-methoxy-1 oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-isopropoxy-9-ethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-isopropoxy-9-propoxy-1 oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-di-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-ethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-methoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dimethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-methoxy-9-ethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-methoxy-9-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4-methoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4-ethoxy-9-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4-ethoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-ethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dipropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-isopropoxy-4-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-di-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]-2-propionic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]-2-butyric acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]-2-(N-acetylamino)acetic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]-2-aminoacetic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]-2-methoxyacetic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-3-methylphenyl]acetic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-3-methoxyphenyl]acetic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-3,5-dimethylphenyl]acetic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-3-iodophenyl]acetic acid; [3-chloro-4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-phenyl]acetic acid; [3-bromo-4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-phenyl]acetic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-2-methylphenyl]acetic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-2-methoxyphenyl]acetic acid; [2-chloro-4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-phenyl]acetic acid; [4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-2-iodophenyl]acetic acid; [2-bromo-4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)-phenyl]acetic acid; [4-(6,7-dichloro-4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-6-methyl-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-7-methyl-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(7-bromo-4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-7-iodo-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(6-bromo-4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-6-iodo-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-3,3-dimethyl-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-3-methyl-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-6-nitro-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-7-nitro-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-6-methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-7-methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(6-chloro-4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(7-chloro-4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dibutoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dihexyloxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-hexyloxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-hexyloxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dibutoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dihexyloxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-butoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-ethoxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; and pharmaceutically acceptable derivatives thereof.\",\n \"Particularly preferred compounds according to the invention are: [4-(4,9-dimethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-di-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dipropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dibutoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dihexyloxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-ethoxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-ethoxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-isopropoxy-4-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-isopropoxy-9-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-hexyloxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-hexyloxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl) phenyl]acetic acid; [4-(4-methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-diethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-di-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dipropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dibutoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4,9-dihexyloxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-ethoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(9-isopropoxy-4-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; [4-(4-butoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid; and pharmaceutically acceptable derivatives thereof.\",\n \"It is to be understood that the present invention covers all combinations of particular and preferred groups as described herein above.\",\n \"Since the compounds of the present invention, in particular compounds of formula (I), are intended for use in pharmaceutical compositions, it will be understood that they are each provided in substantially pure form, for example at least 50% pure, more suitably at least 75% pure and preferably at least 95% pure (% are on a wt/wt basis).\",\n \"Impure preparations of the compounds of formula (I) may be used for preparing the more pure forms used in the pharmaceutical compositions.\",\n \"Although the purity of intermediate compounds of the present invention is less critical, it will be readily understood that the substantially pure form is preferred as for the compounds of formula (I).\",\n \"Preferably, whenever possible, the compounds of the present invention are obtained in crystalline form.\",\n \"When some of the compounds of this invention are allowed to crystallise or are recrystallised from organic solvents, solvent of crystallisation may be present in the crystalline product.\",\n \"This invention includes within its scope such solvates.\",\n \"Similarly, some of the compounds of this invention may be crystallised or recrystallised from solvents containing water.\",\n \"In such cases water of hydration may be formed.\",\n \"This invention includes within its scope stoichiometric hydrates as well as compounds containing variable amounts of water that may be produced by processes such as lyophilisation.\",\n \"In addition, different crystallisation conditions may lead to the formation of different polymorphic forms of crystalline products.\",\n \"This invention includes within its scope all polymorphic forms of the compounds of formula (I).\",\n \"The compounds of the invention bind to the EP4 receptor and are therefore useful in treating EP4 receptor mediated diseases.\",\n \"In view of their ability to bind to the EP4 receptor, the compounds of the invention are useful in the treatment of the disorders that follow.\",\n \"Thus, the compounds of formula (I) are useful as analgesics.\",\n \"For example they are useful in the treatment of chronic articular pain (e.g.\",\n \"rheumatoid arthritis, osteoarthritis, rheumatoid spondylitis, gouty arthritis and juvenile arthritis) including the property of disease modification and joint structure preservation; musculoskeletal pain; lower back and neck pain; sprains and strains; neuropathic pain; sympathetically maintained pain; myositis; pain associated with cancer and fibromyalgia; pain associated with migraine; pain associated with influenza or other viral infections, such as the common cold; rheumatic fever; pain associated with functional bowel disorders such as non-ulcer dyspepsia, non-cardiac chest pain and irritable bowel syndrome; pain associated with myocardial ischemia; post operative pain; headache; toothache; and dysmenorrhea.\",\n \"The compounds of the invention are particularly useful in the treatment of neuropathic pain.\",\n \"Neuropathic pain syndromes can develop following neuronal injury and the resulting pain may persist for months or years, even after the original injury has healed.\",\n \"Neuronal injury may occur in the peripheral nerves, dorsal roots, spinal cord or certain regions in the brain.\",\n \"Neuropathic pain syndromes are traditionally classified according to the disease or event that precipitated them.\",\n \"Neuropathic pain syndromes include: diabetic neuropathy; sciatica; non-specific lower back pain; multiple sclerosis pain; fibromyalgia; HIV-related neuropathy; post-herpetic neuralgia; trigeminal neuralgia; and pain resulting from physical trauma, amputation, cancer, toxins or chronic inflammatory conditions.\",\n \"These conditions are difficult to treat and although several drugs are known to have limited efficacy, complete pain control is rarely achieved.\",\n \"The symptoms of neuropathic pain are incredibly heterogeneous and are often described as spontaneous shooting and lancinating pain, or ongoing, burning pain.\",\n \"In addition, there is pain associated with normally non-painful sensations such as “pins and needles” (paraesthesias and dysesthesias), increased sensitivity to touch (hyperesthesia), painful sensation following innocuous stimulation (dynamic, static or thermal allodynia), increased sensitivity to noxious stimuli (thermal, cold, mechanical hyperalgesia), continuing pain sensation after removal of the stimulation (hyperpathia) or an absence of or deficit in selective sensory pathways (hypoalgesia).\",\n \"The compounds of formula (I) are also useful in the treatment of inflammation, for example in the treatment of skin conditions (e.g.\",\n \"sunburn, burns, eczema, dermatitis, psoriasis); ophthalmic diseases such as glaucoma, retinitis, retinopathies, uveitis and of acute injury to the eye tissue (e.g.\",\n \"conjunctivitis); lung disorders (e.g.\",\n \"asthma, bronchitis, emphysema, allergic rhinitis, respiratory distress syndrome pigeon fanciers disease, farmer's lung, COPD); gastrointestinal tract disorders (e.g.\",\n \"aphthous ulcer, Crohn's disease, atopic gastritis, gastritis varialoforme, ulcerative colitis, coeliac disease, regional ileitis, irritable bowel syndrome, inflammatory bowel disease, gastrointestinal reflux disease); organ transplantation; other conditions with an inflammatory component such as vascular disease, migraine, periarteritis nodosa, thyroiditis, aplastic anaemia, Hodgkin's disease, scierodoma, myaesthenia gravis, multiple sclerosis, sorcoidosis, nephrotic syndrome, Bechet's syndrome, polymyositis, gingivitis, myocardial ischemia, pyrexia, systemic lupus erythematosus, polymyositis, tendinitis, bursitis, and Sjogren's syndrome.\",\n \"The compounds of formula (I) are also useful in the treatment of immunological diseases such as autoimmune diseases, immunological deficiency diseases or organ transplantation.\",\n \"The compounds of formula (I) are also effective in increasing the latency of HIV infection.\",\n \"The compounds of formula (I) are also useful in the treatment of diseases of abnormal platelet function (e.g.\",\n \"occlusive vascular diseases).\",\n \"The compounds of formula (I) are also useful for the preparation of a drug with diuretic action.\",\n \"The compounds of formula (I) are also useful in the treatment of impotence or erectile dysfunction.\",\n \"The compounds of formula (I) are also useful in the treatment of bone disease characterised by abnormal bone metabolism or resorption such as osteoporosis (especially postmenopausal osteoporosis), hyper-calcemia, hyperparathyroidism, Paget's bone diseases, osteolysis, hypercalcemia of malignancy with or without bone metastases, rheumatoid arthritis, periodontitis, osteoarthritis, ostealgia, osteopenia, cancer cacchexia, calculosis, lithiasis (especially urolithiasis), solid carcinoma, gout and ankylosing spondylitis, tendinitis and bursitis.\",\n \"In a further aspect compounds of formula (I) may be useful in inhibiting bone resorption and/or promoting bone generation.\",\n \"The compounds of formula (I) are also useful for attenuating the hemodynamic side effects of NSAIDs and COX-2 inhibitors.\",\n \"The compounds of formula (I) are also useful in the treatment of cardiovascular diseases such as hypertension or myocardiac ischemia; functional or organic venous insufficiency; varicose therapy; haemorrhoids; and shock states associated with a marked drop in arterial pressure (e.g.\",\n \"septic shock).\",\n \"The compounds of formula (I) are also useful in the treatment of neurodegenerative diseases and neurodegeneration such as dementia, particularly degenerative dementia (including senile dementia, Alzheimer's disease, Pick's disease, Huntingdon's chorea, Parkinson's disease and Creutzfeldt-Jakob disease, ALS, motor neuron disease); vascular dementia (including multi-infarct dementia); as well as dementia associated with intracranial space occupying lesions; trauma; infections and related conditions (including HIV infection); metabolism; toxins; anoxia and vitamin deficiency; and mild cognitive impairment associated with ageing, particularly Age Associated Memory Impairment.\",\n \"The compounds of formula (I) are also useful in the treatment of neuroprotection and in the treatment of neurodegeneration following stroke, cardiac arrest, pulmonary bypass, traumatic brain injury, spinal cord injury or the like.\",\n \"The compounds of formula (I) are also useful in the treatment of tinnitus.\",\n \"The compounds of formula (I) are also useful in preventing or reducing dependence on, or preventing or reducing tolerance or reverse tolerance to, a dependence—inducing agent.\",\n \"Examples of dependence inducing agents include opioids (e.g.\",\n \"morphine), CNS depressants (e.g.\",\n \"ethanol), psychostimulants (e.g.\",\n \"cocaine) and nicotine.\",\n \"The compounds of formula (I) are also useful in the treatment of complications of Type 1 diabetes (e.g.\",\n \"diabetic microangiopathy, diabetic retinopathy, diabetic nephropathy, macular degeneration, glaucoma), nephrotic syndrome, aplastic anaemia, uveitis, Kawasaki disease and sarcoidosis.\",\n \"The compounds of formula (I) are also useful in the treatment of kidney dysfunction (nephritis, particularly mesangial proliferative glomerulonephritis, nephritic syndrome), liver dysfunction (hepatitis, cirrhosis), gastrointestinal dysfunction (diarrhoea) and colon cancer.\",\n \"It is to be understood that reference to treatment includes both treatment of established symptoms and prophylactic treatment, unless explicitly stated otherwise.\",\n \"According to a further aspect of the invention, we provide a compound of formula (I) or a pharmaceutically acceptable derivative thereof for use in human or veterinary medicine.\",\n \"According to another aspect of the invention, we provide a compound of formula (I) or a pharmaceutically acceptable derivative thereof for use in the treatment of a condition which is mediated by the action of PGE2 at EP4 receptors.\",\n \"According to a further aspect of the invention, we provide a method of treating a human or animal subject suffering from a condition which is mediated by the action of PGE2 at EP4 receptors which comprises administering to said subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable derivative thereof.\",\n \"According to a further aspect of the invention we provide a method of treating a human or animal subject suffering from a pain, inflammatory, immunological, bone, neurodegenerative or renal disorder, which method comprises administering to said subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable derivative thereof.\",\n \"According to another aspect of the invention, we provide the use of a compound of formula (I) or a pharmaceutically acceptable derivative thereof for the manufacture of a therapeutic agent for the treatment of a condition which is mediated by the action of PGE2 at EP4 receptors.\",\n \"According to another aspect of the invention we provide the use of a compound of formula (I) or a pharmaceutically acceptable derivative thereof for the manufacture of a therapeutic agent for the treatment or prevention of a condition such as a pain, inflammatory, immunological, bone, neurodegenerative or renal disorder.\",\n \"The compounds of formula (I) and their pharmaceutically acceptable derivatives are conveniently administered in the form of pharmaceutical compositions.\",\n \"Such compositions may conveniently be presented for use in conventional manner in admixture with one or more physiologically acceptable carriers or excipients.\",\n \"Thus, in another aspect of the invention, we provide a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable derivative thereof adapted for use in human or veterinary medicine.\",\n \"While it is possible for the compounds of formula (I) or a pharmaceutically acceptable derivative thereof to be administered as the raw chemical, it is preferable to present it as a pharmaceutical formulation.\",\n \"The formulations of the present invention comprise the compounds of formula (I) or a pharmaceutically acceptable derivative thereof together with one or more acceptable carriers or diluents thereof and optionally other therapeutic ingredients.\",\n \"The carrier(s) must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.\",\n \"The formulations include those suitable for oral, parenteral (including subcutaneous e.g.\",\n \"by injection or by depot tablet, intradermal, intrathecal, intramuscular e.g.\",\n \"by depot and intravenous), rectal and topical (including dermal, buccal and sublingual) administration although the most suitable route may depend upon for example the condition and disorder of the recipient.\",\n \"The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.\",\n \"All methods include the step of bringing into association the compound of formula (I) or a pharmaceutically acceptable acid addition salt thereof (“active ingredient”) with the carrier which constitutes one or more accessory ingredients.\",\n \"In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.\",\n \"Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets (e.g.\",\n \"chewable tablets in particular for paediatric administration) each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.\",\n \"The active ingredient may also be presented as a bolus, electuary or paste.\",\n \"A tablet may be made by compression or moulding, optionally with one or more accessory ingredients.\",\n \"Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active or dispersing agent.\",\n \"Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.\",\n \"The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein.\",\n \"Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.\",\n \"The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of a sterile liquid carrier, for example, water-for-injection, immediately prior to use.\",\n \"Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.\",\n \"Formulations for rectal administration may be presented as a suppository with the usual carriers such as cocoa butter, hard fat or polyethylene glycol.\",\n \"Formulations for topical administration in the mouth, for example buccally or sublingually, include lozenges comprising the active ingredient in a flavoured basis such as sucrose and acacia or tragacanth, and pastilles comprising the active ingredient in a basis such as gelatin and glycerin or sucrose and acacia.\",\n \"The compounds of the invention may also be formulated as depot preparations.\",\n \"Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.\",\n \"Thus, for example, the compounds of the invention may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.\",\n \"In addition to the ingredients particularly mentioned above, the formulations may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.\",\n \"The EP4 receptor compounds for use in the instant invention may be used in combination with other therapeutic agents, for example COX-2 inhibitors, such as celecoxib, rofecoxib, valdecoxib or parecoxib; 5-lipoxygenase inhibitors; NSAID's, such as diclofenac, indomethacin, nabumetone or ibuprofen; leukotriene receptor antagonists; DMARD's such as methotrexate; adenosine A1 receptor agonists; sodium channel blockers, such as lamotrigine; NMDA receptor modulators, such as glycine receptor antagonists; gabapentin and related compounds; tricyclic antidepressants such as amitriptyline; neurone stabilising antiepileptic drugs; mono-aminergic uptake inhibitors such as venlafaxine; opioid analgesics; local anaesthetics; 5HT1 agonists, such as triptans, for example sumatriptan, naratriptan, zolmitriptan, eletriptan, frovatriptan, almotriptan or rizatriptan; EP1 receptor ligands; EP2 receptor ligands; EP3 receptor ligands; EP1 antagonists; EP2 antagonists and EP3 antagonists.\",\n \"When the compounds are used in combination with other therapeutic agents, the compounds may be administered either sequentially or simultaneously by any convenient route.\",\n \"The invention thus provides, in a further aspect, a combination comprising a compound of formula (I) or a pharmaceutically acceptable derivative thereof together with a further therapeutic agent or agents.\",\n \"The combinations referred to above may conveniently be presented for use in the form of a pharmaceutical formulation and thus pharmaceutical formulations comprising a combination as defined above together with a pharmaceutically acceptable carrier or excipient comprise a further aspect of the invention.\",\n \"The individual components of such combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations.\",\n \"When a compound of formula (I) or a pharmaceutically acceptable derivative thereof is used in combination with a second therapeutic agent active against the same disease, the dose of each compound may differ from that when the compound is used alone.\",\n \"Appropriate doses will be readily appreciated by those skilled in the art.\",\n \"A proposed daily dosage of compounds of formula (I) or their pharmaceutically acceptable salts for the treatment of man is from 0.01 to 10 mg/kg body weight per day and more particularly 0.1 to 3 mg/kg body weight per day, calculated as the free base, which may be administered as a single or divided dose, for example one to four times per day The dose range for adult human beings is generally from 8 to 1000 mg/day, such as from 20 to 800 mg/day, preferably 35 to 200 mg/day, calculated as the free base.\",\n \"The precise amount of the compounds of formula (I) administered to a host, particularly a human patient, will be the responsibility of the attendant physician.\",\n \"However, the dose employed will depend on a number of factors including the age and sex of the patient, the precise condition being treated and its severity, and the route of administration.\",\n \"The present invention provides a process for preparing compounds of formula (I) and pharmaceutically acceptable derivatives thereof.\",\n \"Compounds of formula (I) and pharmaceutically acceptable derivatives thereof may be prepared by any method known in the art for the preparation of compounds of analogous structure.\",\n \"Suitable methods for the preparation of compounds of formula (I) and pharmaceutically acceptable derivatives thereof are described below and form a further aspect of the invention.\",\n \"In the formulae that follow, R1 to R10 and Q1-Q5 are as defined in formula (I) above unless otherwise stated.\",\n \"According to a first process (A), compounds of formula (I), where R1 and R3 are both ═O and R4 and R9 are the same or different and represent C1-6alkoxy, may be prepared by reacting a compound of formula (II) with a 4-aminophenylacetic acid of formula (III) in glacial acetic acid at elevated temperature.\",\n \"According to another process (B) compounds of formula (I), where one of R1 and R3 is ═O and the other is hydrogen and R4 and R9 are the same or different and represent C1-6alkoxy, may be prepared by reducing a compound of formula (IV) with a suitable reducing agent, followed by separation of isomers and deprotection (eg.\",\n \"with aqueous base at elevated temperature).\",\n \"Suitable reducing agents include zinc in acetic acid at elevated temperature and sodium borohydride in methanol followed by trifluoroacetic acid (TFA) and triethylsilane.\",\n \"According to another process (C) compounds of formula (I), where one of R1 and R3 is ═O and the other is hydrogen and R4 and R9 are the same or different and represent C1-6alkoxy, may be prepared by reacting a compound of formula (V) with a 4-aminophenylacetic acid of formula (III) as defined above in the presence of triethylamine and dimethylformamide, followed by deprotection (eg.\",\n \"using acetic acid at elevated temperature).\",\n \"According to a further process (D) compounds of formula (I), where one of R1 and R3 is ═O and the other is hydrogen and one of R4 and R9 is C1-6alkoxy, may be prepared by reacting a compound of formula (VI) where RD is in the −4 or −9 position and is C1-6 alkoxy, with a 4-aminophenylacetate of formula (VII) in the presence of sodium triacetoxyborohydride in a suitable solvent, such as dichloromethane, followed by deprotection (eg.\",\n \"with aqueous base at elevated temperature).\",\n \"According to a further process (E) compounds of formula (I), where R1 and R3 are both hydrogen and R4 and R9 are the same or different and represent C1-6 alkoxy, may be prepared by reacting a compound of formula (VIII) with a 4-aminophenylacetate of formula (VII) as described above in a suitable solvent, such as dimethylformamide, at elevated temperature followed by deprotection (eg.\",\n \"using aqueous base such as lithium hydroxide in aqueous tetrahydrofuran).\",\n \"According to a further process (F) compounds of formula (I), where R3 is ═CHC1-5alkyl, may be prepared by reacting a compound of formula (IV) as defined above with a Grignard reagent C1-C6alkyl-MgBr under conventional conditions, followed by separation of isomers and deprotection (eg.\",\n \"with aqueous base at elevated temperature).\",\n \"According to a further process (G) compounds of formula (I), where R3 is C1-6dialkyl, may be prepared by reacting a compound of formula (IX) with a 4-aminophenylacetate of formula (VII) as defined above in the presence of aluminium trichloride, followed by deprotection (eg.\",\n \"with aqueous base at elevated temperature).\",\n \"According to a further process (H) compounds of formula (I) prepared according to processes (A) to (G) may be converted into other compounds of formula (I) using conventional procedures.\",\n \"For example, compounds of formula (I) where R3 is C1-6alkyl, may be prepared by reducing a compound of formula (I) where R3 is ═CHC1-5alkyl, protected at the carboxyl group, with a suitable reducing agent, such as hydrogen in the presence of a palladium on carbon catalyst, followed by deprotection (eg.\",\n \"with aqueous base at elevated temperature).\",\n \"Also, compounds of formula (I) where R3 is ═O may be converted into compounds of formula (I) where R3 is ═S by conventional methods, for example using Lawesson's reagent.\",\n \"Compounds of formulae (II) to (IX) may be prepared by any method known in the art for the preparation of compounds of analogous structure.\",\n \"Compounds of formula (II) may, for example be prepared according to Scheme 1 that follows.\",\n \"Compounds of formula (IV) may be prepared by reacting compounds of formula (II) with a 4-aminophenylacetate of formula (VII) as defined above in the presence of acetic acid.\",\n \"Compounds of formula M may, for example be prepared according to Scheme 2 that follows.\",\n \"Compounds of formula (VI) may, for example, be prepared according to Scheme 3 that follows.\",\n \"Compounds of formula (VIII) may be prepared, for example, by reacting a compound of formula (X) with phosphorous tribromide in an ether solvent, for example a mixture of diethyl ether and tetrahydrofuran.\",\n \"Compounds of formula (IX) may be prepared by reacting a compound of formula (XI) with a Grignard reagent R3-MgBr under conventional conditions.\",\n \"Compounds of formulae (III) and (VII) may be prepared according to methods known in the art for the preparation of analogous compounds, for example, compounds where Q1 is methyl may be prepared by the method of Takahashi,l et al.\",\n \"Heterocycles (1996), 43(II), 2343-2346; compounds where Q1 is ethyl may be prepared by the method of Kirschenheuter, Gary P et al.\",\n \"in EP465802; compounds where Q1 is NHAc may be prepared by the method described in US3479339; compounds where Q1 is NH2 may be prepared by the method of Herbert, Richard B et al.\",\n \"J. Chem.\",\n \"Soc., Perkin Trans.1 (1992), (1), 109-13; compounds where Q1 is MeO may be prepared from the corresponding nitro compounds prepared by the method of Tomioka, Hideo et al.\",\n \"J.\",\n \"Am.\",\n \"Chem.\",\n \"Soc.\",\n \"(1990), 112(21), 7692-702; compounds where Q2 is Me or Q3 is Cl may be prepared by the method described in U.S. Pat.\",\n \"No.\",\n \"3,860,639 (Schultz, Everett M.); compounds where Q2 is MeO may be prepared by the method of Nannini, G et al, Arzneim.-Forsch.\",\n \"(1973), 23(8), 1090-100 by hydrolysis of the ethyl ester; compounds where Q2 is Cl may be prepared by the method of Atkinson, Joseph G et al.\",\n \"Tet Left.\",\n \"(1979), (31), 2857-60 by reduction of the corresponding nitro compound; compounds where Q2 is I may be prepared by the method of Sindelar, Karel et al.\",\n \"Collect.\",\n \"Czech.\",\n \"Chem.\",\n \"Commun.\",\n \"(1978), 43(2), 471-97 by reduction of the corresponding nitro compound; compounds where Q2 is Br may be prepared by the method of Sindelar, Karel et al.\",\n \"Collect.\",\n \"Czech.\",\n \"Chem.\",\n \"Commun.\",\n \"(1978), 43(2), 471-97; compounds where Q3 is Me may be prepared by the method of Borck, Joachim et al.\",\n \"in ZA 6804711; compounds where Q3 is MeO may be prepared by the method of Gallacher, Gerard et al.\",\n \"Biorg.\",\n \"Amines (1995), 11(1), 49-62 by reduction of the corresponding nitro compound; compounds where Q3 is I may be prepared by the method of Boehm, Marcus F et al.\",\n \"J. Chem.\",\n \"Soc., Chem.\",\n \"Commun.\",\n \"(1991), (1), 52-3; compounds where Q3 is Br may be prepared by the method of Figala, Georg et al.\",\n \"in DE 2746067; compounds where Q3 and Q4 are Me may be prepared by the method of Yost, Yul et al.\",\n \"Org.\",\n \"Prep.\",\n \"Proced.\",\n \"Int.\",\n \"(1985), 17(4-5), 23949 from the corresponding benzoic acid using the Arndt-Eistert reaction.\",\n \"Certain intermediates described above are novel compounds, and it is to be understood that all novel intermediates herein form further aspects of the present invention.\",\n \"Compounds of formula (II), (V), (VI), (VIII) and (IX) are key intermediates and represent a particular aspect of the invention.\",\n \"Certain intermediates, such as compounds of formula (IV), may be prodrugs of compounds of formula (I).\",\n \"Conveniently, compounds of the invention are isolated following work-up in the form of the free base.\",\n \"Pharmaceutically acceptable acid addition salts of the compounds of the invention may be prepared using conventional means.\",\n \"Solvates (e.g.\",\n \"hydrates) of a compound of the invention may be formed during the work-up procedure of one of the aforementioned process steps.\",\n \"The following Examples which should not be construed as constituting a limitation thereto are provided to illustrate the invention.\",\n \"1H NMR spectra were obtained at 400 MHz on a Bruker DPX400 spectrophotometer.\",\n \"J values are given in Hz.\",\n \"Mass spectra were obtained on a Micromass series II MS (electrospray positive or negative).\",\n \"Where HPLC retention times are given as a characterisation of intermediates or Examples this refers to an HP 1050 or a HP1100 running a 5.5 minute Gradient: Eluents: A —0.1% VN Formic Acid+10 mmol Ammonium Acetate B —95% MeCN+0.05% VN Formic Acid Flow rate: 3 ml/min Column: 3.3 cm×4.6 mm internal diameter, 3 μm ABZ+PLUS Injection Volume: 5 μl Temperature: Room temperature.\",\n \"Gradient: Time % A % B 0.00 100 0.00 0.70 100 0.00 4.40 0.00 100 5.30 0.00 100 5.50 100 0.00 Intermediate 1 Ethyl 1,4-dihydroxy-2,3-naphthalenedicarboxylate Sodium (60 g, 2.6 mol) was dissolved in ethanol (1.2L) and the mixture was cooled to 40° C. Diethylphthalate (960 ml, 4.83 mol) was added and the mixture heated under nitrogen until the temperature reached 115° C. Diethyl succinate (211.3 g, 1.21 mol) was added dropwise over 45 min.\",\n \"The reaction was heated at 115° C. for a further 45 min, cooled to room temperature and poured onto water (1.2L).\",\n \"Ethyl acetate (1L) was added and stirred, the layers were separated and the organics were extracted with sodium hydroxide solution (2N, 1L).\",\n \"The combined aqueous was acidified to pH 3 and the mixture extracted with ethyl acetate (2×1L).\",\n \"The combined organics were washed with a saturated solution of sodium hydrogen carbonate (2×1.5L), then brine, dried (MgSO4), filtered and the solvent evaporated under vacuum.\",\n \"The residue was purified using a 2.5 kg Biotage column eluting with 5% ethyl acetate/hexane to give ethyl 1,4-dihydroxy-2,3-naphthalenedicarboxylate as a white solid, (60 g, 16%).\",\n \"δH CDCl3 10.44,(2H, s), 8.34,(2H, m), 7.68,(2H, m), 4.37,(4H, q), 1.37,(6H, t).\",\n \"Intermediate 2 Ethyl 1,4-diethoxy-2,3-naphthalenedicarboxylate Ethyl 1,4-dihydroxy-2,3-naphthalenedicarboxylate (30 g, 98.6 mmol) and potassium carbonate (150 g, 1.09 mmol) were stirred in acetone (600 ml) under nitrogen.\",\n \"Iodoethane (150 g, 0.96 mol) was added and the mixture was stirred at reflux overnight.\",\n \"The reaction was cooled, diluted with ethyl acetate and filtered.\",\n \"The filtrate was evaporated to leave a brown oil, which was dissolved in toluene and washed with potassium hydroxide solution (5%, 150 ml) and brine.\",\n \"Drying over magnesium sulphate and evaporation of the solvent gave a yellow solid.\",\n \"Purification using an 800 g Biotage column gave ethyl 1,4-diethoxy-2,3-naphthalenedicarboxylate as a white solid (32 g, 90%).\",\n \"δH CDCl3 8.16,(2H, m), 7.60,(2H, m), 4.40,(4H, q), 4.18,(4H, q), 1.50,(6H, t), 1.40,(6H, t).\",\n \"Intermediate 3 1,4-Diethoxy-2,3-naphthalenedicarboxylic Acid Ethyl 1,4-diethoxy-2,3-naphthalenedicarboxylate (32 g, 89 mmol) was added to a solution of sodium hydroxide (20 g) in ethanol (200 ml) and water (40 ml) and stirred for 1.5h at 60° C. The reaction was cooled and the thick white suspension was filtered.\",\n \"The solid was dissolved in a mixture of ethyl acetate (200 ml) and water (800 ml).\",\n \"The layers were separated and the aqueous was acidified with hydrochloric acid (2M, 120 ml).\",\n \"The aqueous was extracted with ethyl acetate (2×) and the combined organics were dried (MgSO4).\",\n \"Evaporation of the solvent under vacuum gave 1,4-diethoxy-2,3-naphthalenedicarboxylic acid as a white solid (25 g, 92%).\",\n \"δH [2H6]—DMSO 13.26,(2H, s), 8.15,(2H, m), 7.72,(2H, m), 4.13,(4H, q), 1.42,(6H, t).\",\n \"Intermediate 4 1,4-Diethoxy-2,3-naphthalenedicarboxylic Anhydride 1,4-Diethoxy-2,3-naphthalenedicarboxylic acid (25 g, 82 mmol) was added to a solution of thionyl chloride (23.3 g) in chloroform (150 ml) and stirred at reflux for 1 h. The resulting solution was cooled and evaporated to dryness.\",\n \"Further chloroform was added and evaporation repeated to give 1,4-diethoxy-2,3-naphthalenedicarboxylic anhydride as a yellow solid (23.3 g, 99%).\",\n \"δH [2H6]—DMSO 8.42,(2H, m), 7.93,(2H, m), 4.53,(4H, q), 1.46,(6H, t).\",\n \"Intermediate 5 Ethyl[4-(4,9-diethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[I]isoindol-2-yl)phenyl]acetate 1,4-Diethoxy-2,3-naphthalenedicarboxylic anhydride (23.3 g, 81.5 mmol) and ethyl (4-aminophenyl)acetate (14.8 g, 82 mmol) were refluxed under nitrogen in acetic acid (160 ml) overnight.\",\n \"The mixture was cooled to room temperature and poured into water (1L).\",\n \"The white solid was filtered, washed with water and dissolved in dichloromethane (800 ml).\",\n \"The solution was washed with water, brine and dried (MgSO4) and the solvent evaporated under vacuum to give ethyl [4-(4,9-diethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate as an off-white solid, 33 g, 96%.\",\n \"δH [2H6]—DMSO 8.40,(2H, m), 7.87,(2H, m), 7.42,(4H, s), 4.47,(4H, q), 4.12,(2H, q), 3.76,(2H, s), 1.45,(6H, t), 1.21,(3H, t).\",\n \"Intermediate 6 3-Hydroxy 4-methoxynaphtho[2,3-c]furan-[(3H)-one N,N,N′-Trimethylethylene diamine (0.82 ml, 6.3 mmol) was dissolved in tetrahydrofuran (16 ml) and cooled to −20° C. n-Butyl lithium (1.6M in hexanes, 3.9 ml, 6.24 mmol) was added and the reaction stirred at −20° C. for 15 min.\",\n \"1-Methoxy-2-naphthaldehyde (0.969, 5.9 mmol) was added followed by n-butyl lithium (1.6M in hexanes, 11.25 ml, 18 mmol) and the reaction stirred at 25° C. for 3h.\",\n \"Solid carbon dioxide was added and the reaction left until the excess carbon dioxide had sublimed.\",\n \"Stirring continued for 15 min before addition of hydrochloric acid (2N, 50 ml).\",\n \"The mixture was extracted with dichloromethane (50 ml, ×3).\",\n \"The combined extracts were dried (MgSO4) and the solvent evaporated under vacuum.\",\n \"The oily residue was preabsorbed onto silica and purified by SPE (silica, 10 g) eluting with an ethyl acetate/cyclohexane gradient to give 3-hydroxy-4-methoxynaphtho[2,3-c]furan-1 (3M-one (50 mg, 3.7%).\",\n \"δH [2H6]—DMSO 8.28,(2H, m), 8.18,(2H, m), 7.70,(2H, m), 7.16,(1H, s), 4.27,(3H, s).\",\n \"Intermediate 7 Ethyl 1,4-dimethoxy-2-methylnaphthalene-3-carboxylate n-Butyl lithium (1.6M in hexanes, 4.1 ml, 6.56 mmol) was added dropwise to 2-bromo-1,4-dimethoxy-3-methylnaphthalene (1.537 g, 5.47 mmol) in tetrahydrofuran (30 ml) at −50° C. The reaction was stirred for 30 min at −50° C. before dropwise addition of ethyl chloroformate (1 ml, 10.46 mmol).\",\n \"The reaction was allowed to warm to 0° C. over 18h quenched by addition of hydrochloric acid (2N) and extracted with ethyl acetate.\",\n \"The extract was dried (Na2SO4) and the solvent evaporated under vacuum.\",\n \"The residue was purified by SPE (silica, 10 g) eluting with 10% ethyl acetate in cyclohexane to give ethyl 1,4-dimethoxy-2-methylnaphthalene-3-carboxylate (1.339, 89%).\",\n \"δH CDCl3 8.08,(2H, d), 7.53,(2H, m), 4.48,(2H, q), 3.99,(3H, s), 3.88,(3H, s), 2.38,(3H, s), 1.44,(3H, t).\",\n \"Intermediate 8 2,3-Bis(bromomethyl)-1,4-diethoxy-naphthalene [1,4-Diethoxy-3-(hydroxymethyl)-2-naphthyl]methanol (92 mg, 0.33 mmol) was dissolved in a mixture of diethyl ether (1.5 ml) and tetrahydrofuran (2 ml) under nitrogen at 0° C. and phosphorus tribromide (0.035 ml, 0.35 mmol) was added dropwise.\",\n \"The reaction was allowed to warm to ambient temperature and stirred for 4 h before quenching with ice.\",\n \"The reaction mixture was extracted with ethyl acetate (3×5 ml) and the combined organic extracts dried over magnesium sulphate.\",\n \"The solvent was removed in vacuo, and the residue purified by SPE cartridge (silica), gradient elution cyclohexane to 75% cyclohexane/dichloromethane, to give 2,3-bis(bromomethyl)-1,4-diethoxy-naphthalene as a white solid (92 mg, 70%).\",\n \"δH CDCl3 8.07 (2H, dd, J=6.5, 3.2), 7.54 (2H, dd, J=6.5, 3.2), 5.02 (4H, s), 4.21 (4H, q, J=7.0), 1.60 (6H, t, J=7.0); LC retention time 4.16 min.\",\n \"EXAMPLE 1 (Process C) [4-(4,9-Dimethoxy-1 oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid To a solution of ethyl 1,4-dimethoxy-2-methylnaphthalene-3-carboxylate (73 mg, 0.266 mmol) in carbon tetrachloride (10 ml) was added N-bromosuccinimide (47 mg, 0.266 mmol) and dibenzylperoxide (5 mg).\",\n \"The mixture was heated at reflux under nitrogen for 45 min, illuminating with a 200W lamp.\",\n \"The reaction was cooled to room temperature, 4-aminophenyl acetic acid (40 mg, 0.266 mmol), triethylamine (74 μl, 0.53 mmol) and DMF (5 ml) added and the reaction stirred for 48h.\",\n \"Acetic acid (glacial, 1 ml) was added to the reaction and the mixture refluxed for 3h under nitrogen.\",\n \"Sodium metabisulphite solution (1 ml) was added and the reaction evaporated to dryness under vacuum.\",\n \"The product was partially purified by SPE (NH2, 10 g) eluting with methanol then 5% acetic acid in methanol.\",\n \"The fractions containing product were passed through a silica plug washing with 5% acetic acid in methanol.\",\n \"After evaporation of the solvents under vacuum, the residue was columned on a silica gel flash column eluting with a gradient (1:1 ethyl acetate/cyclohexane to 5% methanol, 1% acetic acid in 1:1 ethyl acetate/cyclohexane) to give [4-(4,9-dimethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid (15 mg, 15%).\",\n \"δH CDCl3 8.40,(1H, d), 8.17,(1H, d), 7.90.\",\n \"(2H, d), 7.65,(1H, t), 7.59,(1H, t), 7.38,(2H, d), 5.02,(2H, s), 4.27,(3H, s), 4.08,(3H, s), 3.69,(2H, s).\",\n \"MS 378, [MH+].\",\n \"LC retention time 3.28 min.\",\n \"EXAMPLE 2 Step 1 (Process E) Ethyl[4-(4,9-diethoxy-1,3-dihydro-2H-benzo[t]isoindol-2-yl)phenyl]acetate A solution of 2,3-bis(bromomethyl)-1,4-diethoxy-naphthalene (41 mg, 0.1 mmol) and 4-aminophenylacetic acid ethyl ester (20 mg, 0.11 mmol) in N,N-dimethylformamide (1 ml) was heated at 60° C. under nitrogen for 14 h. The solvent was removed in vacuo and the residue taken up in ethyl acetate (10 ml) and washed with 8% aqueous sodium bicarbonate solution (5 ml).\",\n \"The organic layer was dried over magnesium sulphate and the solvent removed in vacuo.\",\n \"Purification by flash column chromatography on silica gel eluting with 90% cyclohexane/ethyl acetate gave the product as a white solid (15 mg, 36%).\",\n \"δH CDCl3 8.12 (2H, dd, J=6.5, 3.2), 7.50 (2H, dd, J=6.5, 3.2), 7.23 (2H, d, J=8.5), 6.73 (2H, d, J=8.5), 4.80 (4H, s), 4.17 (6H, m), 3.55 (2H, s), 1.56 (6H, m), 1.24 (3H, m); LC retention time 4.25 min, ms 420, [MH+].\",\n \"EXAMPLE 2 Step 2 [4-(4,9-diethoxy-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid A solution of ethyl[4-(4,9-diethoxy-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate (10 mg, 0.024 mmol) in tetrahydrofuran (4 ml) and a solution of lithium hydroxide (6 mg, 0.24 mmol) in water (1 ml) were stirred vigorously for 14h.\",\n \"The reaction mixture was diluted with water (10 ml) and extracted with ethyl acetate (2×5 ml) and the organic extracts discarded.\",\n \"The aqueous phase was acidified with 2N hydrochloric acid to pH=5 and extracted with ethyl acetate (3×5 ml).\",\n \"The combined organic extracts were dried over magnesium sulphate and the solvent removed in vacuo to give the product as a red solid (4 mg, 43%).\",\n \"δH CDCl3 8.13 (2H, dd, J=6.5, 3.2), 7.49, (2H, dd, J=6.5, 3.2), 7.24 (2H, d, 8.3), 6.75 (2H, d, J=8.3), 4.81 (4H, s), 4.19 (4H, q, J=7.0), 3.69 (2H, s), 1.53 (6H, t, J=7.0); LC retention time 3.92 min, ms 392, [MH+].\",\n \"General Methodology Method A A mixture of ethyl 1,4-dihydroxy-2,3-naphthalenedicarboxylate (1.0 g, 3.29 mmol), potassium carbonate (5 g, 36.2 mmol) and the alkyl halide (3.25 mmol) in acetone (20 ml) was heated at reflux under nitrogen for 8h.\",\n \"The cooled reaction was filtered and the residue washed with acetone and ethyl acetate.\",\n \"The combined filtrate and washings were evaporated to dryness under vacuum.\",\n \"The residue was partitioned between hydrochloric acid (2N) and dichloromethane.\",\n \"The dichloromethane extract was evaporated to dryness under vacuum.\",\n \"The product was isolated by chromatography on a silica gel flash column eluting with an ethyl acetate/cyclohexane gradient (0-10% ethyl acetate).\",\n \"Method B A mixture of monoalkylated material (170 mg, 0.49 mmol), potassium carbonate (850 mg, 6.15 mmol) and the alkyl halide (1.0 mmol) in acetone (5 ml) was heated at reflux under nitrogen for 4h.\",\n \"The cooled reaction was filtered and the residue washed with acetone and ethyl acetate.\",\n \"The combined filtrate and washings were evaporated to dryness under vacuum and the residue purified by SPE (silica) eluting with an ethyl acetate/cyclohexane gradient.\",\n \"Method C A mixture of ethyl 1,4-dihydroxy-2,3-naphthalenedicarboxylate (2 g, 6.57 mmol), potassium carbonate (10 g, 72.4 mmol) and the alkyl halide (64.1 mmol) in acetone (40 ml) was heated at reflux under nitrogen for 4h.\",\n \"The cooled reaction was filtered and the residue washed with acetone and ethyl acetate.\",\n \"The combined filtrate and washings were evaporated to dryness under vacuum and the residue purified by SPE (silica) eluting with an ethyl acetate/cyclohexane gradient.\",\n \"Method D A mixture of the diester (13.87 mmol) and sodium hydroxide solution (2N, 25 ml) in ethanol (25 ml) was heated at reflux for 4h.\",\n \"The resulting solution was acidified to pH1 with hydrochloric acid (2N) and extracted with ethyl acetate (×2).\",\n \"The extracts were dried (MgSO4) an d the solvent evaporated under vacuum.\",\n \"Method E (Process A) A mixture of diacid (0.57 mmol) and 4-aminophenylacetic acid (1.32 mmol) in glacial acetic acid (5 ml) was heated under reflux for 24h.\",\n \"The cooled reaction was diluted with water, the precipitate formed filtered off and washed with water.\",\n \"Method F A mixture of diacid (0.56 mmol) and ethyl 4-aminophenylacetate (1.11 mmol) in glacial acetic acid (5 ml) was heated at reflux for 24h.\",\n \"The cooled reaction was diluted with water, the precipitate formed filtered off and washed with water.\",\n \"Method G (Process B—Step 1) The phthalimide ester (0.32 mmol) was dissolved in glacial acetic acid (4 ml) and zinc powder (100 mesh, 300 mg, 4.59 mmol) added.\",\n \"The reaction was heated at reflux under nitrogen for 72h.\",\n \"The hot reaction was filtered and the residue washed with hot acetic acid.\",\n \"The combined filtrate and washings were evaporated to dryness under vacuum.\",\n \"Methylamine (33% in ethanol, 2 ml) was added to the resulting solid and the suspension was stirred at room temperature for 20 min.\",\n \"The methylamine solution was evaporated under vacuum and the residue purified by SPE (silica) eluting with an ethyl acetate/cyclohexane gradient to give a mixture of isomeric y-lactams.\",\n \"Method H (Processes B and D—Step 2) The γ-lactams (0.06 mmol) were mixed with potassium carbonate (1.09 mmol) in ethanol (2 ml) and water (1 ml) and heated at reflux for 4h.\",\n \"The cooled solution was acidified to pH1 with hydrochloric acid (2M), the precipitate filtered off and washed with water.\",\n \"Dried at 40° C. under vacuum.\",\n \"The following compounds were prepared using the above general methodologies: Intermediate 9 Ethyl [4-(4,9-di-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(4,9-di-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared in 49% yield from 1,4-di-isopropoxynaphthalene-2,3-dicarboxylic acid using method F. δH [2H6]—DMSO 8.41,(2H, m), 7.83,(2H, m), 7.42,(4H, s), 5.02,(2H, m), 4.12,(2H, q), 3.76,(2H, s), 1.36,(12H, d), 1.22,(3H, t).\",\n \"Intermediate 10 Ethyl [4-(4,9-dipropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(4,9-dipropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared in 68% yield from 1,4-dipropoxynaphthalene-2.3-dicarboxylic acid using method F. δH [2H6]—DMSO 8.35,(2H, m), 7.82,(2H, m), 7.37,(4H, s), 4.34,(4H, t), 4.07,(2H, q), 3.71,(2H, s), 1.83,(4H, m), 1.17,(3H, t), 1.01,(6H, t).\",\n \"Intermediate 11 Ethyl [4-(4,9-dibutoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(4,9-dibutoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared in 39% yield from 1,4-dibutoxynaphthalene-2,3-dicarboxylic acid using method F. δH [H6]—DMSO 8.39,(2H, m), 7.87,(2H, m), 7.42,(4H, s), 4.43,(4H, t), 4.12,(2H, q), 3.76,(2H, s), 1.85,(4H, m), 1.53,(4H, m), 1.22,(3H, t), 0.97,(6H, t).\",\n \"Intermediate 12 Ethyl [444,9-dihexyloxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(4,9-dihexyloxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared from 1,4-dihexyloxynaphthalene-2,3-dicarboxylic acid using method F. δH [2H6]—DMSO 8.38,(2H, m), 7.86,(2H, m), 7.42,(4H, s), 4.41,(4H, t), 4.12,(2H, q), 3.76,(2H, s), 1.85,(4H, m), 1.49,(4H, m), 1.32,(8H, m), 1.21,(3H, t), 0.87,(6H, t).\",\n \"Intermediate 13 Ethyl [4-(4-ethoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(4-ethoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared from 1-ethoxy-4-isopropoxynaphthalene-2,3-dicarboxylic acid using method F. δH [2H6]—DMSO 8.36,(2H, m), 7.79,(2H, m), 7.37,(4H, s), 4.98,(1H, m), 4.42,(2H, q), 4.07,(2H, q), 3.71,(2H, s), 1.40,(3H, t), 1.31,(6H, d), 1.17,(3H, t).\",\n \"Intermediate 14 Ethyl [4-(9-isopropoxy-4-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(9-isopropoxy-4-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared from 4-isopropoxy-1-propoxynaphthalene-2,3-dicarboxylic acid using method F. δH [2H6]—DMSO 8.35,(2H, m), 7.80,(2H, m), 7.37,(4H, s), 4.97,(1H, m), 4.34,(2H, t), 4.07,(2H, q), 3.71,(2H, s), 1.83,(2H, m), 1.31,(6H, d), 1.71,(3H, t), 1.01,(3H, t).\",\n \"Intermediate 15 Ethyl [4-(4-hexyloxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(4-hexyloxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared from 1-hexyloxy-4-isopropoxynaphthalene-2,3-dicarboxylic acid using method F. δH [2H6]—DMSO 8.37,(1H, m), 8.32,(1H, m), 7.80,(2H, m), 7.37,(4H, s), 4.97,(1H, m), 4.37,(2H, t), 4.07,(2H, q), 3.71,(2H, s), 1.81,(2H, m), 1.45,(2H, m), 1.35-1.26,(10H, m), 1.17,(3H, t), 0.82,(3H, t).\",\n \"Intermediate 16 1,4-Di-isopropoxynaphthalene-2,3-dicarboxylic Acid 1,4-Di-isopropoxynaphthalene-2,3-dicarboxylic acid was prepared from ethyl 1,4-d]-propoxynaphthalene-2,3-dicarboxylate using method D. δH [2H6]—DMSO 8.16,(2H, m), 7.67,(2H, m), 4.43,(2H, m), 1.26,(12H, d).\",\n \"Intermediate 17 1,4-Dipropoxynaphthalene-2,3-dicarboxylic Acid 1,4-Dipropoxynaphthalene-2,3-dicarboxylic acid was prepared from ethyl 1,4-dipropoxynaphthalene-2,3-dicarboxylate using method D. δH [2H6]—DMSO 8.14,(2H, m), 7.71,(2H, m), 4.01,(4H, t), 1.83,(4H, m), 1.05,(6H, t).\",\n \"Intermediate 18 1,4-Dibutoxynaphthalene-2,3-dicarboxylic Acid 1,4-Dibutoxynaphthalene-2,3-dicarboxylic acid was prepared from ethyl 1,4-dibutoxynaphthalene-2,3-dicarboxylate using method D. δH [2H6]—DMSO 8.12,(2H, m), 7.12,(2H, m), 4.05,(4H, t), 1.80,(4H, m), 1.52,(4H, m), 0.97,(6H, t).\",\n \"Intermediate 19 1,4-Dihexyloxynaphthalene-2,3-dicarboxylic Acid 1,4-Dihexyloxynaphthalene-2,3-dicarboxylic acid was prepared from ethyl 1,4-dihexyloxynaphthalene-2,3-dicarboxylate using method D. δH [2H6]—DMSO 8.39,(2H, m), 7.92,(2H, m), 4.46,(4H, t), 1.85,(4H, m), 1.49,(4H, m), 1.32,(8H, m), 0.88,(6H, t).\",\n \"Intermediate 20 1-Ethoxy-4-isopropoxynaphthalene-2,3-dicarboxylic Acid 1-Ethoxy-4-isopropoxynaphthalene-2,3-dicarboxylic acid was prepared from ethyl 1-ethoxy-4-isopropoxynaphthalene-2,3-dicarboxylate using method D. δH [2H6]—DMSO 8.18,(1H, m), 8.12,(1H, m), 7.69,(2H, m), 4.40,(1H, m), 4.11,(2H, q), 1.41,(3H, t), 1.27,(6H, d).\",\n \"Intermediate 21 4-Isopropoxy-1-propoxynaphthalene-2,3-dicarboxylic Acid 4-Isopropoxy-1-propoxynaphthalene-2,3-dicarboxylic acid was prepared from ethyl 4-isopropoxy-1-propoxynaphthalene-2,3-dicarboxylate using method D. δH [2H6]—DMSO 8.18,(1H, m), 8.11,(1H, m), 7.70,(2H, m), 4.41,(1H, m), 4.02,(2H, t), 1.83,(2H, m), 1.27,(6H, d), 1.06,(3H, t).\",\n \"Intermediate 22 1-Butoxy-4-isopropoxynaphthalene-2,3-dicarboxylic Acid 1-Butoxy-4-isopropoxynaphthalene-2,3-dicarboxylic acid was prepared from ethyl 1-butoxy-4-isopropoxynaphthalene-2,3-dicarboxylate using method D. δH [2H6]—DMSO 8.18,(1H, m), 8.09,(1H, m), 7.69,(2H, m), 4.40,(1H, m), 4.05,(2H, t), 180,(2H, m), 1.52,(2H, m), 1.26,(6H, d), 0, 97,(3H, t).\",\n \"Intermediate 23 1-Hexyloxy-4-isopropoxynaphthalene-2,3-dicarboxylic Acid 1-Hexyloxy-4-isopropoxynaphthalene-2,3-dicarboxylic acid was prepared from ethyl 1-hexyloxy-4-isopropoxynaphthalene-2,3-dicarboxylate using method D. δH [2H6]—DMSO 8.18,(1H, m), 8.09,(1H, m), 7.70,(2H, m), 4.40,(1H, m), 4.04,(2H, t), 1.81,(2H, m), 1.49,(2H, m), 1.34,(4H, m), 1.27,(6H, d), 0.90,(3H, t).\",\n \"Intermediate 24 Ethyl 1,4-di-isopropoxynaphthalene-2,3-dicarboxylate Ethyl 1,4-di-isopropoxynaphthalene-2,3-dicarboxylate was prepared from ethyl 1,4-dihydroxynaphthalene-2,3-dicarboxylate and 2-iodopropane using method C. δH [2H6]—DMSO 8.18,(2H, m), 7.73,(2H, m), 4.37,(2H, m), 4.28,(4H, q), 1.30,(6H, t), 1.25,(12H, d).\",\n \"Intermediate 25 Ethyl 1,4-dipropoxynaphthalene-2,3-dicarboxylate Ethyl 1,4-dipropoxynaphthalene-2,3-dicarboxylate was prepared from ethyl 1,4-dihydroxynaphthalene-2,3-dicarboxylate and 1-iodopropane using method C. δH [2H6]—DMSO 8.16,(2H, m), 7.77,(2H, m), 4.29,(4H, q), 4.00,(4H, t), 1.83,(4H, m), 1.30,(4H, t), 1.04,(4H, t).\",\n \"Intermediate 26 Ethyl 1,4-dibutoxynaphthalene-2,3-dicarboxylate Ethyl 1,4-dibutoxynaphthalene-2,3-dicarboxylate was prepared from ethyl 1,4-dihydroxynaphthalene-2,3-dicarboxylate and 1-iodobutane using method C. δH [2H6]—DMSO 8.15,(2H, m), 7.76,(2H, m), 4.29,(4H, q), 4.03,(4H, t), 1.79,(4H, m), 1.50,(4H, m), 1.30,(6H, t), 0.96,(6H, t).\",\n \"Intermediate 27 Ethyl 1,4-dihexyloxynaphthalene-2,3-dicarboxylate Ethyl 1,4-dihexyloxynaphthalene-2,3-dicarboxylate was prepared from ethyl 1,4-dihydroxynaphthalene-2,3-dicarboxylate and 2-iodohexane using method C. δH [2H6]—DMSO 8.14,(2H, m), 7.76,(2H, m), 4.29,(4H, q), 4.03,(4H, t), 1.81,(4H, m), 1.47,(4H, m), 1.38-1.21,(14H, m), 0.88,(6H, t).\",\n \"Intermediate 28 Ethyl 1-ethoxy-4-isopropoxynaphthalene-2,3-dicarboxylate Ethyl 1-ethoxy-4-isopropoxynaphthalene-2,3-dicarboxylate was prepared in 91% yield from ethyl 1-hydroxy-4-isopropoxynaphthalene-2,3-dicarboxylate and iodoethane using method B. δH CDCl3 8.22,(1H, m), 8.13,(1H, m), 7.59,(2H, m), 4.40,(5H, m), 4.18,(2H, q), 1.49,(3H, t), 1.43-1.38,(6H, m), 1.34,(6H, d).\",\n \"Intermediate 29 Ethyl 4-isopropoxy-1-propoxynaphthalene-2,3-dicarboxylate Ethyl 4-isopropoxy-1-propoxynaphthalene-2,3-dicarboxylate was prepared in 91% yield from ethyl 1-hydroxy-4-isopropoxynaphthalene-2,3-dicarboxylate and 1-iodopropane using method B. δH CDCl3 8.22,(1H, m), 8.14,(1H, m), 7.60,(2H, m), 4.40,(5H, m), 4.07,(2H, t), 1.92,(2H, m), 1.40,(6H, m), 1.34,(6H, d), 1.10,(3H, t).\",\n \"Intermediate 30 Ethyl 1-butoxy-4-isopropoxynaphthalene-2,3-dicarboxylate Ethyl 1-butoxy-4-isopropoxynaphthalene-2,3-dicarboxylate was prepared from ethyl 1-hydroxy-4-isopropoxynaphthalene-2,3-dicarboxylate and 1-iodobutane using method B. δH [2H6]—DMSO 8.20,(1H, m), 8.12,(1H, m), 7.75,(2H, m), 4.38-4.25,(5H, m), 4.04,(2H, t), 1.80,(2H, m), 1.50,(2H, m), 1.30,(6H, t), 1.25,(6H, d), 0.96,(3H, t).\",\n \"Intermediate 31 Ethyl 1-hexyloxy-4-isopropoxynaphthalene-2,3-dicarboxylate Ethyl 1-hexyloxy-4-isopropoxynaphthalene-2,3-dicarboxylate was prepared in 80 yield from ethyl 1-hydroxy-4-isopropoxynaphthalene-2,3-dicarboxylate and 1-iodohexane using method B. δH CDCl3 8.21,(1H, m), 8.13,(1H, m), 7.59,(2H, m), 4.40,(5H, m), 4.10,(2H, t), 1.90,(2H, m), 1.53,(2H, m), 1.45-1.31,(16H, m), 0.92,(3H, t).\",\n \"Intermediate 32 Ethyl 1-ethoxy-4-hydroxynaphthalene-2,3-dicarboxylate Ethyl 1-ethoxy-4-hydroxynaphthalene-2,3-dicarboxylate was prepared from ethyl 1,4-dihydroxynaphthalene-2,3-dicarboxylate and iodoethane using method A. δH [2H6]—DMSO 8.36,(1H, d), 8.06,(1H, d), 7.83,(1H, m), 7.72,(1H, m), 4.35,(4H, m), 4.03,(2H, q), 1.39,(3H, t), 1.32,(6H, m).\",\n \"Intermediate 33 Ethyl 1-hydroxy-4-isopropoxynaphthalene-2,3-dicarboxylate Ethyl 1-hydroxy-4-isopropoxynaphthalene-2,3-dicarboxylate was prepared in 36% yield from ethyl 1,4-dihydroxynaphthalene-2,3-dicarboxylate and 2-iodopropane using method A. δH CDCl3 12.3,(1H, s), 8.45,(1H, d), 8.10,(1H, d), 7.65,(1H, m), 7.55,(1H, m), 4.42,(1H, m), 1.41,(6H, t), 1.32,(6H, d).\",\n \"EXAMPLE 4 Step 1 (Process B) Ethyl [4-(4,9-di-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(4,9-di-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared in 16% yield from ethyl [4-(4,9-di-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method G. δH [2H6]—DMSO 8.34,(1H, d), 8.16,(1H, d), 7.93,(2H, d), 7.68,(1H, t), 7.60,(1H, t), 7.34,(2H, d), 5.10,(2H, s), 5.01,(1H, m), 4.68,(1H, m), 4.10,(2H, q), 3.69,(2H, s), 1.40,(3H, d), 1.35,(3H, d), 1.19,(3H, t).\",\n \"EXAMPLE 4 Step 2 [4-(4,9-Di-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4,9-Di-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared in 81% yield from ethyl [4-(4,9-di-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method H. δH [2H6]—DMSO 8.34,(1H, d), 8.17,(1H, d), 7.91,(2H, d), 7.69,(1H, m), 7.61,(1H, m), 7.34,(2H, d), 5.10,(2H, s), 5.02,(1H, m), 4.68,(1H, m), 3.59,(2H, s), 1.38,(6H, d), 1.34,(6H, d).\",\n \"MS 434, [MH+].\",\n \"LC retention time 3.91 min.\",\n \"EXAMPLE 5 Step 1 (Process B) Ethyl [4-(4,9-dipropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(4,9-dipropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared in 13% yield from ethyl [4-(4,9-dipropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method G. δH [2H6]—DMSO 8.32,(1H, d), 8.19,(1H, d), 7.93,(2H, d), 7.72,(1H, t), 7.65,(1H, t), 7.36,(2H, d), 5.19,(2H, s), 4.30,(2H, t), 4.22,(2H, t), 4.10,(2H, q), 3.69,(2H, s), 1.87,(4H, m), 1.20,(3H, t), 1.13-1.05,(6H, m).\",\n \"EXAMPLE 5 Step 2 [4-(4,9-Dipropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4,9-Dipropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared in 89% yield from ethyl [4-(4,9-dipropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method H. δH [2H6]—DMSO 8.32,(1H, d), 8.19,(1H, d), 7.91,(2H, d), 7.71,(1H, t), 7.65,(1H, t), 7.65,(2H, d), 5.19,(2H, s), 4.30,(2H, t), 4.22,(2H, t), 3.59,(2H, s), 1.89,(4H, m), 1.09,(6H, m).\",\n \"MS 434, [MH+].\",\n \"LC retention time 3.97 min.\",\n \"EXAMPLE 6 Step 1 (Process B) Ethyl [4-(4,9-dibutoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(4,9-dibutoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared in 26% yield from ethyl [4-(4,9-dibutoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method G. δH [2H6]—DMSO 8.22,(1H, d), 8.09,(1H, d), 7.84,(2H, d), 7.63,(1H, t), 7.57,(1H, t), 7.27,(2H, d), 5.10,(2H, s), 4.26,(2H, t), 4.17,(2H, t), 4.02,(2H, q), 3.60,(2H, s), 1.77,(4H, quintet), 1.49,(4H, m), 1.12,(3H, t), 0.91,(6H, q).\",\n \"EXAMPLE 6 Step 2 [4-(4,9-Dibutoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4,9-Dibutoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared in 98% yield from ethyl [4-(4,9-dibutoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method H. δH [2H6]—DMSO 8.30,(1H, d), 8.18,(1H, d), 7.91,(2H, d), 7.71,(1H, t), 7.65,(1H, t), 7.35,(2H, d), 5.19,(2H, s), 4.34,(2H, t), 4.26,(2H, t), 3.59,(2H, s), 1.85,(4H, m), 1.57,(4H, m), 0.99,(6H, m).\",\n \"MS 462, [MH+].\",\n \"LC retention time 4.25 min.\",\n \"EXAMPLE 7 Step 1 (Process B) Ethyl [44,9-dihexyloxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate Ethyl [4-(4,9-dihexyloxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate was prepared in 28% yield from ethyl [4-(4,9-dihexyloxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method G. δH [2H6]—DMSO 8.19,(1H, d), 8.07,(1H, d), 7.82,(2H, d), 7.60,(1H, t), 7.54,(1H, t), 7.25,(2H, d), 5.08,(2H, s), 4.23,(2H, t), 4.15,(2H, t), 3.99,(2H, q), 3.58,(2H, s), 1.75,(4H, quintet), 1.50-1.36,(4H, m), 1.29-1.21,(8H, m), 1.09,(3H, t), 0.79,(6H, q).\",\n \"EXAMPLE 7 Step 2 [4-(4,9-Dihexyloxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4,9-Dihexyloxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared in 97% yield from ethyl [4-(4,9-dihexyloxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method H. δH [2H6]—DMSO 8.30,(1H, d), 8.17,(1H, d), 7.91,(2H, d), 7.71,(1H, t), 7.64,(1H, t), 7.34,(2H, d), 5.18,(2H, s), 4.33,(2H, t), 4.25,(2H, t), 3.59,(2H, s), 1.86,(4H, m), 1.54,(4H, m), 1.35,(8H, m), 0.90,(6H, m).\",\n \"MS 518, [MH+].\",\n \"LC retention time 4.76 min.\",\n \"EXAMPLE 8 Step 1 (Process B) Ethyl [4-(4-ethoxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate and ethyl [4-(9-thoxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate A mixture of ethyl [4-(4-ethoxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate and ethyl [4-(9-ethoxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate (60:40) was prepared in 20% yield from ethyl [4-(4-ethoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method G. δH [2H6]—DMSO 8.25,(1H, m), 8.09,(1H, m), 7.85,(2H, d), 7.64-7.58,(2H, m), 7.26,(2H, d), 5.09,(1.2H, s), 5.02,(0.8H, s), 4.90,(0.6H, m), 4.60,(0.4H, m), 4.32,(0.8H, q), 4.22,(1.2H, q), 4.01,(2H, q), 3.60,(2H, s), 1.38,(3H, m), 1.29,(2.4H, d), 1.24,(3.6H, d), 1.11,(3H, t).\",\n \"EXAMPLE 8 Step 2 [4-(4-Ethoxy-9-isopropoxy-1 oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid and [4-(9-ethoxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid A mixture of [4-(4-ethoxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid and [4-(9-ethoxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid (60:40) was prepared in 98% yield from ethyl [4-(4-ethoxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate and ethyl [4-(9-ethoxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate (60:40) using method H. δH [2H6]—DMSO 8.34,(1H, m), 8.18,(1H, m), 7.92,(2H, d), 7.70,(1H, t), 7.68,(1H, t), 7.34,(2H, d), 5.18,(1.2H, s), 5.11,(0.8H, s), 5.01,(0.6H, m), 4.69,(0.4H, m), 4.40,(0.8H, q), 4.31,(1.2H, q), 3.59,(2H, s), 1.46,(3H, m), 1.37,(2.4H, d), 1.33,(3.6H, d).\",\n \"MS 420, [MH+].\",\n \"LC retention time 3.73 min.\",\n \"EXAMPLE 9 Step 1 (Process B) Ethyl [4-(9-isopropoxy-4-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate and ethyl [4-(4-isopropoxy-9-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate A mixture of ethyl [4-(9-isopropoxy-4-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate and ethyl [4-(4-isopropoxy-9-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate (60:40) was prepared in 21% yield from ethyl [4-(9-isopropoxy-4-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method G. δH [2H6]—DMSO 8.25,(1H, m), 8.10,(1H, m), 7.85,(2H, m), 7.62,(1H, m), 7.53,(1H, m), 7.27,(2H, d), 5.10,(1.2H, s), 5.02,(0.8H, s), 4.91,(0.6H, m), 4.58,(0.4H, m), 4.23,(0.8H, t), 4.13,(1.2H, t), 4.01,(2H, q), 3.60,(2H, s), 1.80,(2H, m), 1.29,(2.4H, d), 1.24,(3.6H, d), 1.11,(3H, t), 1.01,(3H, m).\",\n \"EXAMPLE 9 Step 2 [4-(9-isopropoxy-4-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid and [4-(4-isopropoxy-9-propoxy-1 oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid A mixture of [4-(9-isopropoxy-4-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid and [4-(4-isopropoxy-9-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid (60:40) was prepared in quantitative yield from ethyl [4-(9-isopropoxy-4-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate and ethyl [4-(4-isopropoxy-9-propoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate (60:40) using method H. δH [2H6]—DMSO 8.34,(1H, m), 8.18,(1H, m), 7.92,(2H, m), 7.70.\",\n \"(1H, t), 7.63,(1H, m), 7.34,(2H, d), 5.18,(1.2H, s), 5.11,(0.8H, s), 5.01,(0.6H, m), 4.68,(0.4H, m), 4.32,(0.8H, q), 4.22,(1.2H, q), 3.59,(2H, s), 1.89,(2H, m), 1.37,(2.4H, d), 1.33,(3.6H, d), 1.10,(3H, m).\",\n \"MS 434, [MH+].\",\n \"LC retention time 3.89 min.\",\n \"EXAMPLE 10 Step 1 (Process B) Ethyl [4-(4-hexyloxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate and ethyl [4-(9-hexyloxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate A mixture of ethyl [4-(4-hexyloxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate and ethyl [4-(9-hexyloxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate (60:40) was prepared in 16% yield from ethyl [4-(4-hexyloxy-9-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method G. δH [2H6]—DMSO 8.33,(1H, m), 8.17,(1H, m), 7.94,(2H, m), 7.70,(1H, m), 7.64,(1H, m), 7.35,(2H, d), 5.18,(1.2H, s), 5.11,(0.8H, s), 5.00,(0.6H, m), 4.69,(0.4H, m), 4.35,(0.8H, t), 4.25,(1.2H, t), 4.10,(2H, q), 3.69,(2H, s), 1.87,(2H, m), 1.60-1.45,(2H, m), 1.37,(2.4H, d), 1.33,(3.6H, d), 1.20,(3H, t), 0.91,(3H, m).\",\n \"EXAMPLE 10 Step 2 [4-(4-Hexyloxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid and [4-(9-hexyloxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid A mixture of [4-(4-hexyloxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid and [4-(9-hexyloxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid (60:40) was prepared in 78% yield from ethyl [4-(4-hexyloxy-9-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate and ethyl [4-(9-hexyloxy-4-isopropoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate (60:40) using method H. δH [2H6]—DMSO 8.35,(0.6H, d), 8.30,(0.4H, d), 8.17,(2H, m), 7.91,(2H, m), 7.70,(1H, t), 7.63,(1H, m), 7.34,(2H, d), 5.18,(1.2H, s), 5.10,(0.8H, s), 5.00,(0.6H, m), 4.68,(0.4H, m), 4.35,(0.8H, q), 4.25,(1.2H, q), 3.59,(2H, s), 1.87,(2H, m), 1.54,(2H, m), 1.35,(10H, m), 0.91,(3H, m).\",\n \"MS 476, [MH+].\",\n \"LC retention time 4.32 min.\",\n \"EXAMPLE 11 Step 1 (Process D) Ethyl [44-methoxy-1 oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate 3-Hydroxy-4-methoxynaphtho[2,3-c]furan-1 (3H-one (50 mg, 0.22 mmol) and ethyl 4-aminophenylacetate (47 mg, 0.26 mmol) in dichloromethane (5 ml) were stirred at room temperature under nitrogen for 1 hour.\",\n \"Sodium triacetoxyborohydride (138 mg, 0.66 mmol) was added and stirring continued for 24h.\",\n \"The reaction was adjusted to pH14 with sodium hydroxide solution (2N) and extracted with dichloromethane (3×5 ml).\",\n \"The combined extracts were evaporated under vacuum and the residue triturated with cyclohexane/ether to give ethyl [4-(4-methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate as a white solid (45 mg, 54%).\",\n \"δH [2H6]—DMSO 8.24,(1H, d), 8.16,(1H, d), 8.13,(1H, s), 7.99,(2H, d), 7.64,(2H, m), 7.37,(2H, d), 5.44,(2H, s), 4.26,(3H, s), 4.10,(2H, q), 3.69,(2H, s), 1.20,(3H, t).\",\n \"EXAMPLE 11 Step 2 [4-(4-Methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4-Methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared in quantitative yield from ethyl [4-(4-methoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetate using method H. δH [2H6]—DMSO 8.19,(1H, d), 8.09,(2H, m), 7.92,(2H, d), 7.59,(2H, m), 7.31,(2H, d), 5.39,(2H, s), 4.21,(3H, s), 3.56,(2H, s).\",\n \"MS 348, [MH+].\",\n \"LC retention time 3.50 min.\",\n \"EXAMPLE 12 [44,9-Diethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4,9-Diethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared from 1,4-diethoxynaphthalene-2,3-dicarboxylic acid using method E. δH [2H6]—DMSO 8.35,(2H, m), 7.81,(2H, m), 7.36,(4H, s), 4.43,(4H, q), 3.62,(2H, s), 1.40,(6H, t).\",\n \"MS 420, [MH+].\",\n \"LC retention time 3.58 min.\",\n \"EXAMPLE 13 [4-(4,9-Di-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4,9-Di-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared from 1,4-di-isopropoxynaphthalene-2,3-dicarboxylic acid using method E. δH [2H6]—DMSO 8.41,(2H, m), 7.83,(2H, m), 7.41,(4H, s), 5.02,(2H, m), 3.66,(2H, s), 1.36,(12H, d).\",\n \"MS 448, [MH+].\",\n \"LC retention time 3.74 min.\",\n \"EXAMPLE 14 [4-(4,9-Dipropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4,9-Dipropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared from 1,4-dipropoxynaphthalene-2,3-dicarboxylic acid using method E. δH [2H6]—DMSO 8.39,(2H, m), 7.87,(2H, m), 7.41,(4H, s), 4.39,(4H, t), 3.67,(2H, s), 1.87,(4H, m), 1.05,(6H, t).).\",\n \"MS 448, [MH+].\",\n \"LC retention time 3.89 min.\",\n \"EXAMPLE 15 [4-(4,9-Dibutoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4,9-Dibutoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared from 1,4-dibutoxynaphthalene-2,3-dicarboxylic acid using method E. δH [2H6]—DMSO 8.39,(2H, m), 7.88,(2H, m), 7.41,(4H, s), 4.43,(4H, t), 3.67,(2H, s), 1.84,(4H, m), 1.52,(4H, m), 0.96,(6H, t).\",\n \"MS 476, [MH+].\",\n \"LC retention time 4.15 min.\",\n \"EXAMPLE 16 [4-(4,9-Dihexyloxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4,9-Dihexyloxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared from 1,4-dihexyloxynaphthalene-2,3-dicarboxylic acid using method E. δH [2H6]—DMSO 8.38,(2H, m), 7.86,(2H, m), 7.41,(4H, s), 4.42,(4H, t), 3.67,(2H, s), 1.85,(4H, m), 1.49,(4H, m), 1.32,(8H, m), 0.87,(6H, t).).\",\n \"MS 532, [MH+].\",\n \"LC retention time 4.62 min.\",\n \"EXAMPLE 17 [4-(4-Ethoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4-Ethoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared from 1-ethoxy-4-isopropoxynaphthalene-2,3-dicarboxylic acid using method E. δH [2H6]—DMSO 8.41,(2H, m), 7.84,(2H, m), 7.41,(4H, s), 5.03,(1H, m), 4.48,(2H, q), 3.67,(2H, s), 1.45,(3H, t), 1.36,(6H, d).\",\n \"MS 434, [MH+].\",\n \"LC retention time 3.66 min.\",\n \"EXAMPLE 18 [4-(9-isopropoxy-4-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(9-isopropoxy-4-propoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared from 4-isopropoxy-1-propoxynaphthalene-2,3-dicarboxylic acid using method E. δH [2H6]—DMSO 8.41,(2H, m), 7.84,(2H, m), 7.41,(4H, s), 5.02,(1H, m), 4.39,(2H, t), 3.66,(2H, s), 1.88,(2H, m), 1.36,(6H, d), 1.05,(3H, t).\",\n \"MS 448, [MH+].\",\n \"LC retention time 3.81 min.\",\n \"EXAMPLE 19 [4-(4-Butoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic Acid [4-(4-Butoxy-9-isopropoxy-1,3-dioxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetic acid was prepared from 1-butoxy-4-isopropoxynaphthalene-2,3-dicarboxylic acid using method E. δH [2H6]—DMSO 8.42,(1H, m), 8.37,(1H, m), 7.85,(2H, m), 7.41,(4H, s), 5.02,(1H, m), 4.43,(2H, t), 3.66,(2H, s), 1.85,(2H, m), 1.53,(2H, m), 1.36,(6H, d), 0.97,(3H, t).\",\n \"MS 461, [MH+].\",\n \"LC retention time 3.94 min.\",\n \"Biological Data The ability of the compounds of the invention to bind to EP4 receptors has been demonstrated in the Human EP4 Scintillation Proximity Assay.\",\n \"Quantification of radioligand binding by scintillation proximity assay (SPA) is a long-established principle.\",\n \"Briefly, the affinity of novel compounds for a receptor is assessed by the specific competition between known quantities of radiolabelled ligand and novel compound for that receptor.\",\n \"Increasing concentrations of novel compound reduce the amount of radiolabel that binds to the receptor.\",\n \"This gives rise to a diminishing scintillation signal from SPA beads coated with membranes that bear the receptor.\",\n \"The signal may be detected with a suitable scintillation counter and the data generated may be analysed with suitable curve-fitting software.\",\n \"The human EP4 SPA assay (hereafter referred to as ‘the assay’) utilises membranes prepared from Chinese Hamster Ovary (CHO cells) infected with Semliki Forest Virus (SFV).\",\n \"Genetically engineered SFV-1 viral particles containing the genetic sequence of the human EP4 receptor were used to infect CHO cells resulting in expression of the receptor protein in cellular membranes.\",\n \"Cells washed free of media are homogenised in a pH-buffered medium containing peptidase inhibitors.\",\n \"A suitable buffer is of the following composition: 50 mM HEPES, 1 mM EDTA, 25 μg/ml bacitracin, 100 μM leupeptin, 1 mM PMSF, 2 μM Pepstatin A, pH adjusted to 7.4 with KOH.\",\n \"Following removal of cell debris by a low-speed centrifugation, a pellet of membranes is prepared by a high-speed (48000 g) centrifugation of the resulting supernatant.\",\n \"Membrane suspensions such as that described may be stored at −80° C. until used.\",\n \"For assay, membranes expressing human EP4 receptors are diluted in a pH-buffered medium and mixed with SPA beads coated with a suitable substance to facilitate the adhesion of membranes to the beads.\",\n \"The concentrations of membrane protein and SPA beads chosen should result in SPA binding signal of at least 300 corrected counts per minute (CCPM) when tritiated radioligand at a concentration close to its Kd (affinity value) is combined with the mixture.\",\n \"Non-specific binding (nsb) may be determined by competition between the radiolabelled ligand and a saturating concentration of unlabelled ligand.\",\n \"In order to quantify the affinity of novel EP4 receptor ligands, compounds are diluted in a stepwise manner across the wells of a 96-well plate.\",\n \"Radioligand, novel compound, and unlabelled ligand are then added to a 96-well plate suitable for the measurement of SPA binding signals prior to the addition of bead/membrane mixture to initiate the binding reaction.\",\n \"Equilibrium may be achieved by incubation at room temperature for 120 minutes prior to scintillation counting.\",\n \"The data so generated may be analysed by means of a computerised curve-fitting routine in order to quantify the concentration of compound that displaces 50% of the specific radioligand binding (IC50).\",\n \"The affinity (pKi) of the novel compound may be calculated from the IC50 by application of the Cheng-Prusoff correction.\",\n \"Suitable reagents and protocols are: reaction buffer containing 50 mM HEPES, 10 mM MgCl2, pH adjusted to 7.4 with KOH; SPA beads coated with wheatgerm agglutinin; 1.25 nM [3H]-prostaglandin E2 as radioligand; 10 μM prostaglandin E2 as unlabelled ligand; a three-fold dilution series of novel compound starting at 10 μM and ending at 0.3 nM is adequate.\",\n \"The following examples have a pKi of 6.0 or greater at EP4 receptors as determined using the above-mentioned procedure: 1, 4, 5, 8, 9, 12, 13, 14, 17.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10467487"}}},{"rowIdx":368,"cells":{"text":{"kind":"list like","value":[["Protein-coupled receptor","The present application provides a gene encoding a G protein-coupled receptor termed nGPCR-14; constructs and recombinant host cells incorporating the genes; the nGPCR-14 polypeptides encoded by the gene; antibodies to the nGPCR-x polypeptides; and methods of making and using all of the foregoing."],["1.An isolated nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence homologous to a sequence of SEQ ID NO: 192, and fragments thereof; said nucleic acid molecule encoding at least a portion of nGPCR-14, wherein the isolated nucleic acid comprises at least one or more nucleotides from one or more of the following regions of SEQ ID NO: 191: nucleotides 1 to 193 of SEQ ID NO: 191; nucleotides 612 to 644 of SEQ ID NO:191; nucleotides 697 to 706 of SEQ ID NO:191; nucleotides 1011 to 1049 of SEQ ID NO: 191; nucleotides 1051 to 1057 of SEQ ID NO:191; nucleotides 1090 to 1096 of SEQ ID NO:191; or nucleotides 1141 to 1642 of SEQ ID NO:191.2.The isolated nucleic acid molecule of claim 1 comprising a sequence that encodes a polypeptide comprising a sequences of SEQ ID NO: 192, and fragments thereof.","3.The isolated nucleic acid molecule of claim 1 comprising a sequence homologous to a sequence of SEQ ID NO: 191, and fragments thereof.","4.The isolated nucleic acid molecule of claim 1 comprising a sequence of SEQ ID NO:191, and fragments thereof.","5.The isolated nucleic acid molecule of claim 1 wherein said nucleic acid molecule is DNA.","6.The isolated nucleic acid molecule of claim 1 wherein said nucleic acid molecule is RNA.","7.An expression vector comprising a nucleic acid molecule of any one of claims 1 to 5.8.The expression vector of claim 7 wherein said nucleic acid molecule comprises a sequence of SEQ ID NO:191.9.The expression vector of claim 7 wherein said vector is a plasmid.","10.The expression vector of claim 7 wherein said vector is a viral particle.","11.The expression vector of claim 10 wherein said vector is selected from the group consisting of adenoviruses, baculoviruses, parvoviruses, herpesviruses, poxviruses, adeno-associated viruses, Semliki Forest viruses, vaccinia viruses, and retroviruses.","12.The expression vector of claim 7 wherein said nucleic acid molecule is operably connected to a promoter selected from the group consisting of simian virus 40, mouse mammary tumor virus, long terminal repeat of human immunodeficiency virus, maloney virus, cytomegalovirus immediate early promoter, Epstein Barr virus, rous sarcoma virus, human actin, human myosin, human hemoglobin, human muscle creatine, and human metalothionein.","13.A host cell transformed with an expression vector of claim 7.14.The transformed host cell of claim 13 wherein said cell is a bacterial cell.","15.The transformed host cell of claim 14 wherein said bacterial cell is E. coli.","16.The transformed host cell of claim 13 wherein said cell is yeast.","17.The transformed host cell of claim 16 wherein said yeast is S. cerevisiae.","18.The transformed host cell of claim 13 wherein said cell is an insect cell.","19.The transformed host cell of claim 18 wherein said insect cell is S. frugiperda.","20.The transformed host cell of claim 13 wherein said cell is a mammalian cell.","21.The transformed host cell of claim 20 wherein mammalian cell is selected from the group consisting of chinese hamster ovary cells, HeLa cells, African green monkey kidney cells, human 293 cells, and murine 3T3 fibroblasts.","22.An isolated nucleic acid molecule comprising a nucleotide sequence complementary to at least a portion of a sequence of SEQ ID NO:191, said portion comprising at least 10 nucleotides, wherein said isolated nucleic acid comprises at least one or more nucleotides from one or more of the following regions of SEQ ID NO: 191: nucleotides 1 to 193 of SEQ ID NO: 191; nucleotides 612 to 644 of SEQ ID NO:191; nucleotides 697 to 706 of SEQ ID NO: 191; nucleotides 1011 to 1049 of SEQ ID NO: 191; nucleotides 1051 to 1057 of SEQ ID NO:191; nucleotides 1090 to 1096 of SEQ ID NO:191; or nucleotides 1141 to 1642 of SEQ ID NO:191.23.The nucleic acid molecule of claim 22 wherein said molecule is an antisense oligonucleotide directed to a region of a sequence of SEQ ID NO: 191.24.The nucleic acid molecule of claim 23 wherein said oligonucleotide is directed to a regulatory region of a sequence of SEQ ID NO: 191.25.A composition comprising a nucleic acid molecule of any one of claims 1 to 5 or 22 and an acceptable carrier or diluent.","26.A composition comprising a recombinant expression vector of claim 7 and an acceptable carrier or diluent.","27.A method of producing a polypeptide that comprises a sequence of SEQ ID NO: 192, and homologs and fragments thereof, said method comprising the steps of: a) introducing a recombinant expression vector of claim 7 into a compatible host cell; b) growing said host cell under conditions for expression of said polypeptide; and c) recovering said polypeptide, wherein the polypeptide comprising a sequence of SEQ ID NO:192 comprises at least one or more amino acid residues from one or more of the following regions of SEQ ID NO: 192: amino acid residues 1 to 42 of SEQ ID NO:192; amino acid residues 68 to 77 of SEQ ID NO:192; amino acid residues 185 to 197 of SEQ ID NO: 192; or amino acid residues 293 to 513 of SEQ ID NO: 192.28.The method of claim 27 wherein said host cell is lysed and said polypeptide is recovered from the lysate of said host cell.","29.The method of claim 27 wherein said polypeptide is recovered by purifying the culture medium without lysing said host cell.","30.An isolated polypeptide encoded by a nucleic acid molecule of claim 1, wherein the polynucleotide comprises at least one or more amino acid residues from one or more of the following regions of SEQ ID NO: 192: amino acid residues 1 to 42 of SEQ ID NO:192; amino acid residues 68 to 77 of SEQ ID NO:192; amino acid residues 185 to 197 of SEQ ID NO: 192; or amino acid residues 293 to 513 of SEQ ID NO: 192.31.The polypeptide of claim 30 wherein said polypeptide comprises a fragment of SEQ ID NO:192.32.The polypeptide of claim 30 wherein said polypeptide comprises an amino acid sequence homologous to a sequence of SEQ ID NO: 192.33.The polypeptide of claim 30 wherein said sequence homologous to a sequence of SEQ ID NO:192 comprises at least one conservative amino acid substitution compared to the sequence of SEQ ID NO: 192.34.The polypeptide of claim 30 wherein said polypeptide comprises a fragment of a polypeptide with a sequence of SEQ ID NO:192.35.A composition comprising a polypeptide of claim 30 and an acceptable carrier or diluent.","36.An isolated antibody which binds to an epitope on a polypeptide of claim 30.37.The antibody of claim 36 wherein said antibody is a monoclonal antibody.","38.A composition comprising an antibody of claim 36 and an acceptable carrier or diluent.","39.A method of inducing an immune response in a mammal against a polypeptide of claim 30 comprising administering to said mammal an amount of said polypeptide sufficient to induce said immune response.","40.A method for identifying a compound which binds nGPCR-14 comprising the steps of: a) contacting nGPCR-14 with a compound; and b) determining whether said compound binds nGPCR-14.41.The method of claim 40 wherein the nGPCR-14 comprises an amino acid sequence of SEQ ID NO: 192.42.The method of claim 40 wherein binding of said compound to nGPCR-14 is determined by a protein binding assay.","43.The method of claim 40 wherein said protein binding assay is selected from the group consisting of a gel-shift assay, Western blot, radiolabeled competition assay, phage-based expression cloning, co-fractionation by chromatography, co-precipitation, cross linking, interaction trap/two-hybrid analysis, southwestern analysis, and ELISA.","44.A compound identified by the method of claim 40.45.A method for identifying a compound which binds a nucleic acid molecule encoding nGPCR-14 comprising the steps of: a) contacting said nucleic acid molecule encoding nGPCR-14 with a compound; and b) determining whether said compound binds said nucleic acid molecule.","46.The method of claim 45 wherein binding is determined by a gel-shift assay.","47.A compound identified by the method of claim 45.48.A method for identifying a compound which modulates the activity of nGPCR-14 comprising the steps of: a) contacting nGPCR-14 with a compound; and b) determining whether nGPCR-14 activity has been modulated.","49.The method of claim 48 wherein the nGPCR-14 comprises an amino acid sequence of SEQ ID NO: 192.50.The method of claim 48 wherein said activity is neuropeptide binding.","51.The method of claim 48 wherein said activity is neuropeptide signaling.","52.A compound identified by the method of claim 48.53.A method of identifying an animal homolog of nGPCR-14 comprising the steps: a) comparing the nucleic acid sequences of the animal with a sequence of SEQ ID NO: 191, and portions thereof, said portions being at least 10 nucleotides; and b) identifying nucleic acid sequences of the animal that are homologous to said sequence of SEQ ID NO: 191, and portions thereof.","54.The method of claim 53 wherein comparing the nucleic acid sequences of the animal with a sequence selected of SEQ ID NO: 191, and portions thereof, said portions being at least 10 nucleotides, is performed by DNA hybridization.","55.The method of claim 53 wherein comparing the nucleic acid sequences of the animal with a sequence selected of SEQ ID NO: 191, and portions thereof, said portions being at least 10 nucleotides, is performed by computer homology search.","56.A method of screening a human subject to diagnose a disorder affecting the brain or genetic predisposition therefor, comprising the steps of: (a) assaying nucleic acid of a human subject to determine a presence or an absence of a mutation altering an amino acid sequence, expression, or biological activity of at least one nGPCR that is expressed in the brain, wherein the nGPCR comprises an amino acid sequence of SEQ ID NO:192, and allelic variants thereof, and wherein the nucleic acid corresponds to a gene encoding the nGPCR; and (b) diagnosing the disorder or predisposition from the presence or absence of said mutation, wherein the presence of a mutation altering the amino acid sequence, expression, or biological activity of the nGPCR in the nucleic acid correlates with an increased risk of developing the disorder.","57.A method according to claim 56, wherein the nGPCR is nGPCR-14 comprising an amino acid sequence set forth in SEQ ID NO:192 or an allelic variant thereof.","58.A method according to claim 56, wherein the assaying step comprises at least one procedure selected from the group consisting of: a) comparing nucleotide sequences from the human subject and reference sequences and determining a difference of at least a nucleotide of at least one codon between is the nucleotide sequences from the human subject that encodes an nGPCR-14 allele and an nGPCR-14 reference sequence; (b) performing a hybridization assay to determine whether nucleic acid from the human subject has a nucleotide sequence identical to or different from one or more reference sequences; (c) performing a polynucleotide migration assay to determine whether nucleic acid from the human subject has a nucleotide sequence identical to or different from one or more reference sequences; and (d) performing a restriction endonuclease digestion to determine whether nucleic acid from the human subject has a nucleotide sequence identical to or different from one or more reference sequences.","59.A method according to claim 58 wherein the assaying step comprises: performing a polymerase chain reaction assay to amplify nucleic acid comprising nGPCR-14 coding sequence, and determining nucleotide sequence of the amplified nucleic acid.","60.A method of screening for an nGPCR-14 mental disorder genotype in a human patient, comprising the steps of: (a) providing a biological sample comprising nucleic acid from said patient, said nucleic acid including sequences corresponding to allelles of nGPCR-14; and (b) detecting the presence of one or more mutations in the nGPCR-14 allelle; wherein the presence of a mutation in an nGPCR-40 allelle or nGPCR-54 allele is indicative of a mental disorder genotype.","61.The method according to claim 59 wherein said biological sample is a cell sample.","62.The method according to claim 59 wherein said detecting the presence of a mutation comprises sequencing at least a portion of said nucleic acid, said portion comprising at least one codon of said nGPCR-14 alleles.","63.The method according to claim 59 wherein said nucleic acid is DNA.","64.The method according to claim 59 wherein said nucleic acid is RNA.","65.A kit for screening a human subject to diagnose a mental disorder or a genetic predisposition therefor, comprising, in association: (a) an oligonucleotide useful as a probe for identifying polymorphisms in a human nGPCR-14 gene, the oligonucleotide comprising 6-50 nucleotides in a sequence that is identical or complementary to a sequence of a wild type human nGPCR-14 gene sequence or nGPCR-14 coding sequence, except for one sequence difference selected from the group consisting of a nucleotide addition, a nucleotide deletion, or nucleotide substitution; and (b) a media packaged with the oligonucleotide, said media containing information for identifying polymorphisms that correlate with schizophrenia or a genetic predisposition therefor, the polymorphisms being identifiable using the oligonucleotide as a probe.","66.A method of identifying a nGPCR allelic variant that correlates with a mental disorder, comprising steps of: (a) providing a biological sample comprising nucleic acid from a human patient diagnosed with a mental disorder, or from the patient's genetic progenitors or progeny; (b) detecting in the nucleic acid the presence of one or more mutations in an nGPCR that is expressed in the brain, wherein the nGPCR comprises an amino acid sequence of SEQ ID NO: 192, and allelic variants thereof, and wherein the nucleic acid includes sequence corresponding to the gene or genes encoding nGPCR; wherein the one or more mutations detected indicates an allelic variant that correlates with a mental disorder.","67.A method according to claim 66 wherein the at least one nGPCR is nGPCR-14, or an allelic variant thereof.","68.A purified and isolated polynucleotide comprising a nucleotide sequence encoding a nGPCR-14 allelic variant identified according to claim 67.69.A host cell transformed or transfected with a polynucleotide according to claim 68 or with a vector comprising the polynucleotide.","70.A purified polynucleotide comprising a nucleotide sequence encoding nGPCR-14 of a human with a mental disorder; wherein said polynucleotide hybridizes to the complement of SEQ ID NO:191 under the following hybridization conditions: (a) hybridization for 16 hours at 42° C. in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% dextran sulfate and (b) washing 2 times for 30 minutes at 60° C. in a wash solution comprising 0.1×SSC and 1% SDS; and wherein the polynucleotide that encodes the nGPCR-14 amino acid sequence of the human differs from SEQ ID NO:192 by at least one residue and comprises at least one or more amino acid residues from one or more of the following regions of SEQ ID NO: 192: amino acid residues 1 to 42 of SEQ ID NO: 192; amino acid residues 68 to 77 of SEQ ID NO: 192; amino acid residues 185 to 197 of SEQ ID NO: 192; or amino acid residues 293 to 513 of SEQ ID NO: 192.71.A vector comprising a polynucleotide according to claim 70.72.A host cell that has been transformed or transected with a polynucleotide according to claim 70 and that expresses the nGPCR-14 protein encoded by the polynucleotide.","73.A host cell according to claim 72 that has been co-transfected with a polynucleotide encoding the nGPCR-14 amino acid sequence set forth in SEQ ID NO:192 and that expresses the nGPCR-14 protein having the amino acid sequence set forth in SEQ ID NO:192.74.A method for identifying a modulator of biological activity of nGPCR-14 comprising the steps of: a) contacting a cell according to claim 72 in the presence and in the absence of a putative modulator compound; b) measuring nGPCR-14 biological activity in the cell; wherein decreased or increased nGPCR-14 biological activity in the presence versus absence of the putative modulator is indicative of a modulator of biological activity.","75.A method to identify compounds useful for the treatment of a mental disorder, said method comprising steps of: (a) contacting a composition comprising nGPCR-14 with a compound suspected of binding nGPCR-14; (b) detecting binding between nGPCR-14 and the compound suspected of binding nGPCR-14; wherein compounds identified as binding nGPCR-14 are candidate compounds useful for the treatment of a mental disorder.","76.A method for identifying a compound useful as a modulator of binding between nGPCR-14 and a binding partner of nGPCR-14 comprising the steps of: (a) contacting the binding partner and a composition comprising nGPCR-14 in the presence and in the absence of a putative modulator compound; (b) detecting binding between the binding partner and nGPCR-14; wherein decreased or increased binding between the binding partner and nGPCR-14 in the presence of the putative modulator, as compared to binding in the absence of the putative modulator is indicative a modulator compound useful for the treatment of schizophrenia.","77.A method according to claim 75 or 76 wherein the composition comprises a cell expressing nGPCR-14 on its surface.","78.A method according to claim 77 wherein the composition comprises a cell transformed or transfected with a polynucleotide that encodes nGPCR-14.79.A method of purifying a G protein from a sample containing said G protein comprising the steps of: a) contacting said sample with a polypeptide of claim 1 for a time sufficient to allow said G protein to form a complex with said polypeptide; b) isolating said complex from remaining components of said sample; c) maintaining said complex under conditions which result in dissociation of said G protein from said polypeptide; and d) isolating said G protein from said polypeptide.","80.The method of claim 79 wherein said sample comprises an amino acid sequence of SEQ ID NO: 192.81.The method of claim 79 wherein said polypeptide comprises an amino acid sequence homologous to a sequence of SEQ ID NO:192."],["<SOH> BACKGROUND OF THE INVENTION <EOH>The G protein-coupled receptors (GPCRs) form a vast superfamily of cell surface receptors which are characterized by an amino-terminal extracellular domain, a carboxyl terminal intracellular domain, and a serpentine structure that passes through the cell membrane seven times.","Hence, such receptors are sometimes also referred to as seven transmembrane (7TM) receptors.","These seven transmembrane domains define three extracellular loops and three intracellular loops, in addition to the amino- and carboxy-terminal domains.","The extracellular portions of the receptor have a role in recognizing and binding one or more extracellular binding partners (e.g., ligands), whereas the intracellular portions have a role in recognizing and communicating with downstream molecules in the signal transduction cascade.","The G protein-coupled receptors bind a variety of ligands including calcium ions, hormones, chemokines, neuropeptides, neurotransmitters, nucleotides, lipids, odorants, and even photons, and are important in the normal (and sometimes the aberrant) function of many cell types.","[See generally Strosberg, Eur.","J. Biochem.","196:1-10 (1991) and Bohm et al., Biochem J.","322:1-18 (1997).]","When a specific ligand binds to its corresponding receptor, the ligand typically stimulates the receptor to activate a specific heterotrimeric guanine-nucleotide-binding regulatory protein (G-protein) that is coupled to the intracellular portion of the receptor.","The G protein in turn transmits a signal to an effector molecule within the cell, by either stimulating or inhibiting the activity of that effector molecule.","These effector molecules include adenylate cyclase, phospholipases and ion channels.","Adenylate cyclase and phospholipases are enzymes that are involved in the production of the second messenger molecules cAMP, inositol triphosphate and diacyglycerol.","It is through this sequence of events that an extracellular ligand stimuli exerts intracellular changes through a G protein-coupled receptor.","Each such receptor has its own characteristic primary structure, expression pattern, ligand-binding profile, and intracellular effector system.","Because of the vital role of G protein-coupled receptors in the communication between cells and their environment, such receptors are attractive targets for therapeutic intervention, for example by activating or antagonizing such receptors.","For receptors having a known ligand, the identification of agonists or antagonists may be sought specifically to enhance or inhibit the action of the ligand.","Some G protein-coupled receptors have roles in disease pathogenesis (e.g., certain chemokine receptors that act as HIV co-receptors may have a role in AIDS pathogenesis), and are attractive targets for therapeutic intervention even in the absence of knowledge of the natural ligand of the receptor.","Other receptors are attractive targets for therapeutic intervention by virtue of their expression pattern in tissues or cell types that are themselves attractive targets for therapeutic intervention.","Examples of this latter category of receptors include receptors expressed in immune cells, which can be targeted to either inhibit autoimmune responses or to enhance immune responses to fight pathogens or cancer; and receptors expressed in the brain or other neural organs and tissues, which are likely targets in the treatment of schizophrenia, depression, bipolar disease, or other neurological disorders.","This latter category of receptor is also useful as a marker for identifying and/or purifying (e.g., via fluorescence-activated cell sorting) cellular subtypes that express the receptor.","Unfortunately, only a limited number of G protein receptors from the central nervous system (CNS) are known.","Thus, a need exists for G protein-coupled receptors that have been identified and show promise as targets for therapeutic intervention in a variety of animals, including humans."],["<SOH> SUMMARY OF THE INVENTION <EOH>The present invention relates to an isolated nucleic acid molecule that comprises a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence homologous to even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a fragment thereof.","The nucleic acid molecule encodes at least a portion of nGPCR-x.","In some embodiments, the nucleic acid molecule comprises a sequence that encodes a polypeptide comprising even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a fragment thereof.","In some embodiments, the nucleic acid molecule comprises a sequence homologous to odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or a fragment thereof.","In some embodiments, the nucleic acid molecule comprises a sequence selected from the group consisting of odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, and fragments thereof.","According to some embodiments, the present invention provides vectors which comprise the nucleic acid molecule of the invention.","In some embodiments, the vector is an expression vector.","According to some embodiments, the present invention provides host cells which comprise the vectors of the invention.","In some embodiments, the host cells comprise expression vectors.","The present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence complementary to at least a portion of a sequence from an odd numbered sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, said portion comprising at least 10 nucleotides.","The present invention provides a method of producing a polypeptide comprising a sequence from an even numbered sequence ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a homolog or fragment thereof.","The method comprising the steps of introducing a recombinant expression vector that includes a nucleotide sequence that encodes the polypeptide into a compatible host cell, growing the host cell under conditions for expression of the polypeptide and recovering the polypeptide.","The present invention provides an isolated antibody which binds to an epitope on a polypeptide comprising a sequence from an even numbered sequence ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a homolog or fragment thereof.","The present invention provides an method of inducing an immune response in a mammal against a polypeptide comprising a sequence from an even numbered sequence ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a homolog or fragment thereof.","The method comprises administering to a mammal an amount of the polypeptide sufficient to induce said immune response.","The present invention provides a method for identifying a compound which binds nGPCR-x.","The method comprises the steps of: contacting nGPCR-x with a compound and determining whether the compound binds nGPCR-x.","The present invention provides a method for identifying a compound which binds a nucleic acid molecule encoding nGPCR-x.","The method comprises the steps of contacting said nucleic acid molecule encoding nGPCR-x with a compound and determining whether said compound binds said nucleic acid molecule.","The present invention provides a method for identifying a compound which modulates the activity of nGPCR-x.","The method comprises the steps of contacting nGPCR-x with a compound and determining whether nGPCR-x activity has been modulated.","The present invention provides a method of identifying an animal homolog of nGPCR-x.","The method comprises the steps screening a nucleic acid database of the animal with an odd numbered sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or a portion thereof and determining whether a portion of said library or database is homologous to said odd numbered sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or portion thereof.","The present invention provides a method of identifying an animal homolog of nGPCR-x.","The methods comprises the steps screening a nucleic acid library of the animal with a nucleic acid molecule having an odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or a portion thereof; and determining whether a portion of said library or database is homologous to said odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or a portion thereof.","Another aspect of the present invention relates to methods of screening a human subject to diagnose a disorder affecting the brain or genetic predisposition therefor.","The methods comprise the steps of assaying nucleic acid of a human subject to determine a presence or an absence of a mutation altering an amino acid sequence, expression, or biological activity of at least one nGPCR that is expressed in the brain.","The nGPCR comprise an amino acid sequence selected from the group consisting of: SEQ ID NO:74, SEQ ID NO: 186, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:90, and SEQ ID NO:94, and allelic variants thereof.","A diagnosis of the disorder or predisposition is made from the presence or absence of the mutation.","The presence of a mutation altering the amino acid sequence, expression, or biological activity of the nGPCR in the nucleic acid correlates with an increased risk of developing the disorder.","The present invention further relates to methods of screening for a nGPCR-40 or nGPCR-54 hereditary schizophrenia genotype in a human patient.","The methods comprise the steps of providing a biological sample comprising nucleic acid from the patient, in which the nucleic acid includes sequences corresponding to alleles of nGPCR-40 or nGPCR-54.The presence of one or more mutations in the nGPCR-40 allele or the nGPCR-54 allele is detected indicative of a hereditary schizophrenia genotype.","The present invention provides kits for screening a human subject to diagnose schizophrenia or a genetic predisposition therefor.","The kits include an oligonucleotide useful as a probe for identifying polymorphisms in a human nGPCR-40 gene or a human nGPCR-54 gene.","The oligonucleotide comprises 6-50 nucleotides in a sequence that is identical or complementary to a sequence of a wild type human nGPCR-40 or nGPCR-54 gene sequence or nGPCR-40 or nGPCR-54 coding sequence, except for one sequence difference selected from the group consisting of a nucleotide addition, a nucleotide deletion, or nucleotide substitution.","The kit also includes a media packaged with the oligonucleotide.","The media contains information for identifying polymorphisms that correlate with schizophrenia or a genetic predisposition therefor, the polymorphisms being identifiable using the oligonucleotide as a probe.","The present invention further relates to methods of identifying nGPCR allelic variants that correlates with mental disorders.","The methods comprise the steps of providing biological samples that comprise nucleic acid from a human patient diagnosed with a mental disorder, or from the patient's genetic progenitors or progeny, and detecting in the nucleic acid the presence of one or more mutations in an nGPCR that is expressed in the brain.","The nGPCR comprises an amino acid sequence selected from the group consisting of SEQ ID NO:74, SEQ ID NO: 186, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:90, and SEQ ID NO:94, and allelic variants thereof.","The nucleic acid includes sequences corresponding to the gene or genes encoding nGPCR.","The one or more mutations detected indicate an allelic variant that correlates with a mental disorder.","The present invention further relates to purified polynucleotides comprising nucleotide sequences encoding alleles of nGPCR-40 or nGPCR-54 from a human with schizophrenia.","The polynucleotide hybridizes to the complement of SEQ ID NO:83 or of SEQ ID NO:85 under the following hybridization conditions: (a) hybridization for 16 hours at 42° C. in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% dextran sulfate and (b) washing 2 times for 30 minutes at 60° C. in a wash solution comprising 0.1×SSC and 1% SDS.","The polynucleotide that encodes nGPCR-40 or nGPCR-54 amino acid sequence of the human differs from SEQ ID NO:84 or SEQ ID NO:86 by at least one residue.","The present invention also provides methods for identifying a modulator of biological activity of nGPCR-40 or nGPCR-54 comprising the steps of contacting a cell that expresses nGPCR-40 or nGPCR-54 in the presence and in the absence of a putative modulator compound and measuring nGPCR-40 or nGPCR-54 biological activity in the cell.","The decreased or increased nGPCR-40 or nGPCR-54 biological activity in the presence versus absence of the putative modulator is indicative of a modulator of biological activity.","The present invention further provides methods to identify compounds useful for the treatment of schizophrenia.","The methods comprise the steps of contacting a composition comprising nGPCR-40 with a compound suspected of binding nGPCR-40 or contacting a composition comprising nGPCR-54 with a compound suspected of binding nGPCR-54.The binding between nGPCR-40 and the compound suspected of binding nGPCR-40 or between nGPCR-54 and the compound suspected of binding nGPCR-54 is detected.","Compounds identified as binding nGPCR-40 or nGPCR-54 are candidate compounds useful for the treatment of schizophrenia.","The present invention further provides methods for identifying a compound useful as a modulator of binding between nGPCR-40 and a binding partner of nGPCR-40 or between nGPCR-54 and a binding partner of nGPCR-54.The methods comprise the steps of contacting the binding partner and a composition comprising nGPCR-40 or nGPCR-54 in the presence and in the absence of a putative modulator compound and detecting binding between the binding partner and nGPCR-40 or nGPCR-54.Decreased or increased binding between the binding partner and nGPCR-40 or nGPCR-54 in the presence of the putative modulator, as compared to binding in the absence of the putative modulator is indicative a modulator compound useful for the treatment of schizophrenia.","Another aspect of the present invention relates to methods of purifying a G protein from a sample containing a G protein.","The methods comprise the steps of contacting the sample with an nGPCR for a time sufficient to allow the G protein to form a complex with the nGPCR; isolating the complex from remaining components of the sample; maintaining the complex under conditions which result in dissociation of the G protein from the nGPCR; and isolating said G protein from the nGPCR.","detailed-description description=\"Detailed Description\" end=\"lead\"?"],["FIELD OF THE INVENTION The present invention relates generally to the fields of genetics and cellular and molecular biology.","More particularly, the invention relates to novel G protein coupled receptors, to polynucleotides that encode such novel receptors, to reagents such as antibodies, probes, primers and kits comprising such antibodies, probes, primers related to the same, and to methods which use the novel G protein coupled receptors, polynucleotides or reagents.","BACKGROUND OF THE INVENTION The G protein-coupled receptors (GPCRs) form a vast superfamily of cell surface receptors which are characterized by an amino-terminal extracellular domain, a carboxyl terminal intracellular domain, and a serpentine structure that passes through the cell membrane seven times.","Hence, such receptors are sometimes also referred to as seven transmembrane (7TM) receptors.","These seven transmembrane domains define three extracellular loops and three intracellular loops, in addition to the amino- and carboxy-terminal domains.","The extracellular portions of the receptor have a role in recognizing and binding one or more extracellular binding partners (e.g., ligands), whereas the intracellular portions have a role in recognizing and communicating with downstream molecules in the signal transduction cascade.","The G protein-coupled receptors bind a variety of ligands including calcium ions, hormones, chemokines, neuropeptides, neurotransmitters, nucleotides, lipids, odorants, and even photons, and are important in the normal (and sometimes the aberrant) function of many cell types.","[See generally Strosberg, Eur.","J. Biochem.","196:1-10 (1991) and Bohm et al., Biochem J.","322:1-18 (1997).]","When a specific ligand binds to its corresponding receptor, the ligand typically stimulates the receptor to activate a specific heterotrimeric guanine-nucleotide-binding regulatory protein (G-protein) that is coupled to the intracellular portion of the receptor.","The G protein in turn transmits a signal to an effector molecule within the cell, by either stimulating or inhibiting the activity of that effector molecule.","These effector molecules include adenylate cyclase, phospholipases and ion channels.","Adenylate cyclase and phospholipases are enzymes that are involved in the production of the second messenger molecules cAMP, inositol triphosphate and diacyglycerol.","It is through this sequence of events that an extracellular ligand stimuli exerts intracellular changes through a G protein-coupled receptor.","Each such receptor has its own characteristic primary structure, expression pattern, ligand-binding profile, and intracellular effector system.","Because of the vital role of G protein-coupled receptors in the communication between cells and their environment, such receptors are attractive targets for therapeutic intervention, for example by activating or antagonizing such receptors.","For receptors having a known ligand, the identification of agonists or antagonists may be sought specifically to enhance or inhibit the action of the ligand.","Some G protein-coupled receptors have roles in disease pathogenesis (e.g., certain chemokine receptors that act as HIV co-receptors may have a role in AIDS pathogenesis), and are attractive targets for therapeutic intervention even in the absence of knowledge of the natural ligand of the receptor.","Other receptors are attractive targets for therapeutic intervention by virtue of their expression pattern in tissues or cell types that are themselves attractive targets for therapeutic intervention.","Examples of this latter category of receptors include receptors expressed in immune cells, which can be targeted to either inhibit autoimmune responses or to enhance immune responses to fight pathogens or cancer; and receptors expressed in the brain or other neural organs and tissues, which are likely targets in the treatment of schizophrenia, depression, bipolar disease, or other neurological disorders.","This latter category of receptor is also useful as a marker for identifying and/or purifying (e.g., via fluorescence-activated cell sorting) cellular subtypes that express the receptor.","Unfortunately, only a limited number of G protein receptors from the central nervous system (CNS) are known.","Thus, a need exists for G protein-coupled receptors that have been identified and show promise as targets for therapeutic intervention in a variety of animals, including humans.","SUMMARY OF THE INVENTION The present invention relates to an isolated nucleic acid molecule that comprises a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence homologous to even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a fragment thereof.","The nucleic acid molecule encodes at least a portion of nGPCR-x.","In some embodiments, the nucleic acid molecule comprises a sequence that encodes a polypeptide comprising even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a fragment thereof.","In some embodiments, the nucleic acid molecule comprises a sequence homologous to odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or a fragment thereof.","In some embodiments, the nucleic acid molecule comprises a sequence selected from the group consisting of odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, and fragments thereof.","According to some embodiments, the present invention provides vectors which comprise the nucleic acid molecule of the invention.","In some embodiments, the vector is an expression vector.","According to some embodiments, the present invention provides host cells which comprise the vectors of the invention.","In some embodiments, the host cells comprise expression vectors.","The present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence complementary to at least a portion of a sequence from an odd numbered sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, said portion comprising at least 10 nucleotides.","The present invention provides a method of producing a polypeptide comprising a sequence from an even numbered sequence ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a homolog or fragment thereof.","The method comprising the steps of introducing a recombinant expression vector that includes a nucleotide sequence that encodes the polypeptide into a compatible host cell, growing the host cell under conditions for expression of the polypeptide and recovering the polypeptide.","The present invention provides an isolated antibody which binds to an epitope on a polypeptide comprising a sequence from an even numbered sequence ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a homolog or fragment thereof.","The present invention provides an method of inducing an immune response in a mammal against a polypeptide comprising a sequence from an even numbered sequence ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a homolog or fragment thereof.","The method comprises administering to a mammal an amount of the polypeptide sufficient to induce said immune response.","The present invention provides a method for identifying a compound which binds nGPCR-x.","The method comprises the steps of: contacting nGPCR-x with a compound and determining whether the compound binds nGPCR-x.","The present invention provides a method for identifying a compound which binds a nucleic acid molecule encoding nGPCR-x.","The method comprises the steps of contacting said nucleic acid molecule encoding nGPCR-x with a compound and determining whether said compound binds said nucleic acid molecule.","The present invention provides a method for identifying a compound which modulates the activity of nGPCR-x.","The method comprises the steps of contacting nGPCR-x with a compound and determining whether nGPCR-x activity has been modulated.","The present invention provides a method of identifying an animal homolog of nGPCR-x.","The method comprises the steps screening a nucleic acid database of the animal with an odd numbered sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or a portion thereof and determining whether a portion of said library or database is homologous to said odd numbered sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or portion thereof.","The present invention provides a method of identifying an animal homolog of nGPCR-x.","The methods comprises the steps screening a nucleic acid library of the animal with a nucleic acid molecule having an odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or a portion thereof; and determining whether a portion of said library or database is homologous to said odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or a portion thereof.","Another aspect of the present invention relates to methods of screening a human subject to diagnose a disorder affecting the brain or genetic predisposition therefor.","The methods comprise the steps of assaying nucleic acid of a human subject to determine a presence or an absence of a mutation altering an amino acid sequence, expression, or biological activity of at least one nGPCR that is expressed in the brain.","The nGPCR comprise an amino acid sequence selected from the group consisting of: SEQ ID NO:74, SEQ ID NO: 186, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:90, and SEQ ID NO:94, and allelic variants thereof.","A diagnosis of the disorder or predisposition is made from the presence or absence of the mutation.","The presence of a mutation altering the amino acid sequence, expression, or biological activity of the nGPCR in the nucleic acid correlates with an increased risk of developing the disorder.","The present invention further relates to methods of screening for a nGPCR-40 or nGPCR-54 hereditary schizophrenia genotype in a human patient.","The methods comprise the steps of providing a biological sample comprising nucleic acid from the patient, in which the nucleic acid includes sequences corresponding to alleles of nGPCR-40 or nGPCR-54.The presence of one or more mutations in the nGPCR-40 allele or the nGPCR-54 allele is detected indicative of a hereditary schizophrenia genotype.","The present invention provides kits for screening a human subject to diagnose schizophrenia or a genetic predisposition therefor.","The kits include an oligonucleotide useful as a probe for identifying polymorphisms in a human nGPCR-40 gene or a human nGPCR-54 gene.","The oligonucleotide comprises 6-50 nucleotides in a sequence that is identical or complementary to a sequence of a wild type human nGPCR-40 or nGPCR-54 gene sequence or nGPCR-40 or nGPCR-54 coding sequence, except for one sequence difference selected from the group consisting of a nucleotide addition, a nucleotide deletion, or nucleotide substitution.","The kit also includes a media packaged with the oligonucleotide.","The media contains information for identifying polymorphisms that correlate with schizophrenia or a genetic predisposition therefor, the polymorphisms being identifiable using the oligonucleotide as a probe.","The present invention further relates to methods of identifying nGPCR allelic variants that correlates with mental disorders.","The methods comprise the steps of providing biological samples that comprise nucleic acid from a human patient diagnosed with a mental disorder, or from the patient's genetic progenitors or progeny, and detecting in the nucleic acid the presence of one or more mutations in an nGPCR that is expressed in the brain.","The nGPCR comprises an amino acid sequence selected from the group consisting of SEQ ID NO:74, SEQ ID NO: 186, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:90, and SEQ ID NO:94, and allelic variants thereof.","The nucleic acid includes sequences corresponding to the gene or genes encoding nGPCR.","The one or more mutations detected indicate an allelic variant that correlates with a mental disorder.","The present invention further relates to purified polynucleotides comprising nucleotide sequences encoding alleles of nGPCR-40 or nGPCR-54 from a human with schizophrenia.","The polynucleotide hybridizes to the complement of SEQ ID NO:83 or of SEQ ID NO:85 under the following hybridization conditions: (a) hybridization for 16 hours at 42° C. in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% dextran sulfate and (b) washing 2 times for 30 minutes at 60° C. in a wash solution comprising 0.1×SSC and 1% SDS.","The polynucleotide that encodes nGPCR-40 or nGPCR-54 amino acid sequence of the human differs from SEQ ID NO:84 or SEQ ID NO:86 by at least one residue.","The present invention also provides methods for identifying a modulator of biological activity of nGPCR-40 or nGPCR-54 comprising the steps of contacting a cell that expresses nGPCR-40 or nGPCR-54 in the presence and in the absence of a putative modulator compound and measuring nGPCR-40 or nGPCR-54 biological activity in the cell.","The decreased or increased nGPCR-40 or nGPCR-54 biological activity in the presence versus absence of the putative modulator is indicative of a modulator of biological activity.","The present invention further provides methods to identify compounds useful for the treatment of schizophrenia.","The methods comprise the steps of contacting a composition comprising nGPCR-40 with a compound suspected of binding nGPCR-40 or contacting a composition comprising nGPCR-54 with a compound suspected of binding nGPCR-54.The binding between nGPCR-40 and the compound suspected of binding nGPCR-40 or between nGPCR-54 and the compound suspected of binding nGPCR-54 is detected.","Compounds identified as binding nGPCR-40 or nGPCR-54 are candidate compounds useful for the treatment of schizophrenia.","The present invention further provides methods for identifying a compound useful as a modulator of binding between nGPCR-40 and a binding partner of nGPCR-40 or between nGPCR-54 and a binding partner of nGPCR-54.The methods comprise the steps of contacting the binding partner and a composition comprising nGPCR-40 or nGPCR-54 in the presence and in the absence of a putative modulator compound and detecting binding between the binding partner and nGPCR-40 or nGPCR-54.Decreased or increased binding between the binding partner and nGPCR-40 or nGPCR-54 in the presence of the putative modulator, as compared to binding in the absence of the putative modulator is indicative a modulator compound useful for the treatment of schizophrenia.","Another aspect of the present invention relates to methods of purifying a G protein from a sample containing a G protein.","The methods comprise the steps of contacting the sample with an nGPCR for a time sufficient to allow the G protein to form a complex with the nGPCR; isolating the complex from remaining components of the sample; maintaining the complex under conditions which result in dissociation of the G protein from the nGPCR; and isolating said G protein from the nGPCR.","DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Definitions Various definitions are made throughout this document.","Most words have the meaning that would be attributed to those words by one skilled in the art.","Words specifically defined either below or elsewhere in this document have the meaning provided in the context of the present invention as a whole and as are typically understood by those skilled in the art.","“Synthesized” as used herein and understood in the art, refers to polynucleotides produced by purely chemical, as opposed to enzymatic, methods.","“Wholly” synthesized DNA sequences are therefore produced entirely by chemical means, and “partially” synthesized DNAs embrace those wherein only portions of the resulting DNA were produced by chemical means.","By the term “region” is meant a physically contiguous portion of the primary structure of a biomolecule.","In the case of proteins, a region is defined by a contiguous portion of the amino acid sequence of that protein.","The term “domain” is herein defined as referring to a structural part of a biomolecule that contributes to a known or suspected function of the biomolecule.","Domains may be co-extensive with regions or portions thereof; domains may also incorporate a portion of a biomolecule that is distinct from a particular region, in addition to all or part of that region.","Examples of GPCR protein domains include, but are not limited to, the extracellular (i.e., N-terminal), transmembrane and cytoplasmic (i.e., C-terminal) domains, which are co-extensive with like-named regions of GPCRS; each of the seven transmembrane segments of a GPCR; and each of the loop segments (both extracellular and intracellular loops) connecting adjacent transmembrane segments.","As used herein, the term “activity” refers to a variety of measurable indicia suggesting or revealing binding, either direct or indirect; affecting a response, i.e.","having a measurable affect in response to some exposure or stimulus, including, for example, the affinity of a compound for directly binding a polypeptide or polynucleotide of the invention, or, for example, measurement of amounts of upstream or downstream proteins or other similar functions after some stimulus or event.","Unless indicated otherwise, as used herein, the abbreviation in lower case (gpcr) refers to a gene, cDNA, RNA or nucleic acid sequence, while the upper case version (GPCR) refers to a protein, polypeptide, peptide, oligopeptide, or amino acid sequence.","The term “nGPCR-x” refers to any of the nGPCRs taught herein, while specific reference to a nGPCR (for example nGPCR-5) refers only to that specific nGPCR.","As used herein, the term “antibody” is meant to refer to complete, intact antibodies, and Fab, Fab′, F(ab)2, and other fragments thereof.","Complete, intact antibodies include monoclonal antibodies such as murine monoclonal antibodies, chimeric antibodies and humanized antibodies.","As used herein, the term “binding” means the physical or chemical interaction between two proteins or compounds or associated proteins or compounds or combinations thereof.","Binding includes ionic, nononic, Hydrogen bonds, Van der Waals, hydrophobic interactions, etc.","The physical interaction, the binding, can be either direct or indirect, indirect being through or due to the effects of another protein or compound.","Direct binding refers to interactions that do not take place through or due to the effect of another protein or compound but instead are without other substantial chemical intermediates.","Binding may be detected in many different manners.","As a non-limiting example, the physical binding interaction between a nGPCR-x of the invention and a compound can be detected using a labeled compound.","Alternatively, functional evidence of binding can be detected using, for example, a cell transfected with and expressing a nGPCR-x of the invention.","Binding of the transfected cell to a ligand of the nGPCR that was transfected into the cell provides functional evidence of binding.","Other methods of detecting binding are well-known to those of skill in the art.","As used herein, the term “compound” means any identifiable chemical or molecule, including, but not limited to, small molecule, peptide, protein, sugar, nucleotide, or nucleic acid, and such compound can be natural or synthetic.","As used herein, the term “complementary” refers to Watson-Crick basepairing between nucleotide units of a nucleic acid molecule.","As used herein, the term “contacting” means bringing together, either directly or indirectly, a compound into physical proximity to a polypeptide or polynucleotide of the invention.","The polypeptide or polynucleotide can be in any number of buffers, salts, solutions etc.","Contacting includes, for example, placing the compound into a beaker, microtiter plate, cell culture flask, or a microarray, such as a gene chip, or the like, which contains the nucleic acid molecule, or polypeptide encoding the nGPCR or fragment thereof.","As used herein, the phrase “homologous nucleotide sequence,” or “homologous amino acid sequence,” or variations thereof, refers to sequences characterized by a homology, at the nucleotide level or amino acid level, of at least the specified percentage.","Homologous nucleotide sequences include those sequences coding for isoforms of proteins.","Such isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA.","Alternatively, isoforms can be encoded by different genes.","Homologous nucleotide sequences include nucleotide sequences encoding for a protein of a species other than humans, including, but not limited to, mammals.","Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein.","A homologous nucleotide sequence does not, however, include the nucleotide sequence encoding other known GPCRs.","Homologous amino acid sequences include those amino acid sequences which contain conservative amino acid substitutions and which polypeptides have the same binding and/or activity.","A homologous amino acid sequence does not, however, include the amino acid sequence encoding other known GPCRs.","Percent homology can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using the default settings, which uses the algorithm of Smith and Waterman (Adv.","Appl.","Math., 1981, 2, 482 489, which is incorporated herein by reference in its entirety).","As used herein, the term “isolated” nucleic acid molecule refers to a nucleic acid molecule (DNA or RNA) that has been removed from its native environment.","Examples of isolated nucleic acid molecules include, but are not limited to, recombinant DNA molecules contained in a vector, recombinant DNA molecules maintained in a heterologous host cell, partially or substantially purified nucleic acid molecules, and synthetic DNA or RNA molecules.","As used herein, the terms “modulates” or “modifies” means an increase or decrease in the amount, quality, or effect of a particular activity or protein.","As used herein, the term “oligonucleotide” refers to a series of linked nucleotide residues which has a sufficient number of bases to be used in a polymerase chain reaction (PCR).","This short sequence is based on (or designed from) a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.","Oligonucleotides comprise portions of a DNA sequence having at least about 10 nucleotides and as many as about 50 nucleotides, preferably about 15 to 30 nucleotides.","They are chemically synthesized and may be used as probes.","As used herein, the term “probe” refers to nucleic acid sequences of variable length, preferably between at least about 10 and as many as about 6,000 nucleotides, depending on use.","They are used in the detection of identical, similar, or complementary nucleic acid sequences.","Longer length probes are usually obtained from a natural or recombinant source, are highly specific and much slower to hybridize than oligomers.","They may be single- or double-stranded and carefully designed to have specificity in PCR, hybridization membrane based, or ELISA-like technologies.","The term “preventing” refers to decreasing the probability that an organism contracts or develops an abnormal condition.","The term “treating” refers to having a therapeutic effect and at least partially alleviating or abrogating an abnormal condition in the organism.","The term “therapeutic effect” refers to the inhibition or activation factors causing or contributing to the abnormal condition.","A therapeutic effect relieves to some extent one or more of the symptoms of the abnormal condition.","In reference to the treatment of abnormal conditions, a therapeutic effect can refer to one or more of the following: (a) an increase in the proliferation, growth, and/or differentiation of cells; (b) inhibition (i.e., slowing or stopping) of cell death; (c) inhibition of degeneration; (d) relieving to some extent one or more of the symptoms associated with the abnormal condition; and (e) enhancing the function of the affected population of cells.","Compounds demonstrating efficacy against abnormal conditions can be identified as described herein.","The term “abnormal condition” refers to a function in the cells or tissues of an organism that deviates from their normal functions in that organism.","An abnormal condition can relate to cell proliferation, cell differentiation, cell signaling, or cell survival.","An abnormal condition may also include obesity, diabetic complications such as retinal degeneration, and irregularities in glucose uptake and metabolism, and fatty acid uptake and metabolism.","Abnormal cell proliferative conditions include cancers such as fibrotic and mesangial disorders, abnormal angiogenesis and vasculogenesis, wound healing, psoriasis, diabetes mellitus, and inflammation.","Abnormal differentiation conditions include, but are not limited to, neurodegenerative disorders, slow wound healing rates, and slow tissue grafting healing rates.","Abnormal cell signaling conditions include, but are not limited to, psychiatric disorders involving excess neurotransmitter activity.","Abnormal cell survival conditions may also relate to conditions in which programmed cell death (apoptosis) pathways are activated or abrogated.","A number of protein kinases are associated with the apoptosis pathways.","Aberrations in the function of any one of the protein kinases could lead to cell immortality or premature cell death.","The term “administering” relates to a method of incorporating a compound into cells or tissues of an organism.","The abnormal condition can be prevented or treated when the cells or tissues of the organism exist within the organism or outside of the organism.","Cells existing outside the organism can be maintained or grown in cell culture dishes.","For cells harbored within the organism, many techniques exist in the art to administer compounds, including (but not limited to) oral, parenteral, dermal, injection, and aerosol applications.","For cells outside of the organism, multiple techniques exist in the art to administer the compounds, including (but not limited to) cell microinjection techniques, transformation techniques and carrier techniques.","The abnormal condition can also be prevented or treated by administering a compound to a group of cells having an aberration in a signal transduction pathway to an organism.","The effect of administering a compound on organism function can then be monitored.","The organism is preferably a mouse, rat, rabbit, guinea pig or goat, more preferably a monkey or ape, and most preferably a human.","By “amplification” it is meant increased numbers of DNA or RNA in a cell compared with normal cells.","“Amplification” as it refers to RNA can be the detectable presence of RNA in cells, since in some normal cells there is no basal expression of RNA.","In other normal cells, a basal level of expression exists, therefore in these cases amplification is the detection of at least 1 to 2-fold, and preferably more, compared to the basal level.","As used herein, the phrase “stringent hybridization conditions” or “stringent conditions” refers to conditions under which a probe, primer, or oligonucleotide will hybridize to its target sequence, but to no other sequences.","Stringent conditions are sequence-dependent and will be different in different circumstances.","Longer sequences hybridize specifically at higher temperatures.","Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.","The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium.","Since the target sequences are generally present in excess, at Tm, 50% of the probes are occupied at equilibrium.","Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes, primers or oligonucleotides (e.g.","10 to 50 nucleotides) and at least about 60° C. for longer probes, primers or oligonucleotides.","Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.","The amino acid sequences are presented in the amino to carboxy direction, from left to right.","The amino and carboxy groups are not presented in the sequence.","The nucleotide sequences are presented by single strand only, in the 5′ to 3′ direction, from left to right.","Nucleotides and amino acids are represented in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission or (for amino acids) by three letters code.","Polynucleotides The present invention provides purified and isolated polynucleotides (e.g., DNA sequences and RNA transcripts, both sense and complementary antisense strands, both single- and double-stranded, including splice variants thereof) that encode unknown G protein-coupled receptors heretofore termed novel GPCRs, or nGPCRs.","These genes are described herein and designated herein collectively as nGPCR-x (where x is 1, 3, 4, 5, 9, 11, 12, 14, 15, 18, 16, 17, 20, 21, 22, 24, 27, 28, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 53, 54, 55, 56, 57, 58, 59, or 60).","That is, these genes are described herein and designated herein as nGPCR-1 (also referred to as beGPCR-1), nGPCR-3 (also referred to as beGPCR-3), nGPCR-4 (also referred to as beGPCR-4), nGPCR-5 (also referred to as beGPCR-5 and TL-GPCR-5), nGPCR-9 (also referred to as beGPCR-9), nGPCR-11 (also referred to as beGPCR-11), nGPCR-12 (also referred to as beGPCR-12), nGPCR-14 (also referred to as beGPCR-14), nGPCR-15 (also referred to as beGPCR-15), nGPCR-18 (also referred to as beGPCR-18), nGPCR-16 (also referred to as beGPCR-16), nGPCR-17 (also referred to as beGPCR-17), nGPCR-20 (also referred to as beGPCR-20), nGPCR-21 (also referred to as beGPCR-21), nGPCR-22 (also referred to as beGPCR-22), nGPCR-24 (also referred to as beGPCR-24), nGPCR-27 (also referred to as beGPCR-27), nGPCR-28 (also referred to as beGPCR-28), nGPCR-31 (also referred to as beGPCR-31), nGPCR-32 (also referred to as beGPCR-32), nGPCR-33 (also referred to as beGPCR-33), nGPCR-34 (also referred to as beGPCR-34), nGPCR-35 (also referred to as beGPCR-35), nGPCR-36 (also referred to as beGPCR-36), nGPCR-37 (also referred to as beGPCR-37), nGPCR-38 (also referred to as beGPCR-38), nGPCR-40 (also referred to as beGPCR-40), nGPCR-1 (also referred to as beGPCR-41), nGPCR-53, nGPCR-54, nGPCR-55, nGPCR-56, nGPCR-57, nGPCR-58, nGPCR-59, and nGPCR-60.Table 1 below identifies the novel gene sequence nGPCR-x designation, the SEQ ID NO: of the gene sequence, the SEQ ID NO: of the polypeptide hereby, and the U.S.","Provisional Application in which the gene sequence has been TABLE 1 Nucleotide Amino acid Sequence Sequence (SEQ ID (SEQ ID Originally nGPCR NO:) NO:) filed in: 1 1 2 A 1 73 74 E 3 3 4 A 3 185 186 P 4 5 6 A 5 7 8 A 5 75 76 F 9 9 10 A 9 77 78 G 11 11 12 A 11 79 80 H 12 13 14 A 14 15 16 A 14 191 192 herein 15 17 18 A 18 19 20 A 16 21 22 B 16 81 82 I 17 23 24 B 20 25 26 B 21 27 28 B 22 29 30 B 24 31 32 B 27 33 34 B 28 35 36 B 31 37 38 B 32 39 40 B 33 41 42 C 34 43 44 C 35 45 46 C 36 47 48 C 37 49 50 C 38 51 52 C 40 53 54 C 40 83 84 J 41 55 56 C 53 57 58 D 54 59 60 D 54 85 86 K 55 61 62 D 56 63 64 D 56 87 88 L 56 89 90 M 57 65 66 D 58 67 68 D 58 91 92 N 58 93 94 O 59 69 70 D 60 71 72 D Legend A = Ser.","No.","60/165,838 B = Ser.","No.","60/166,701 C = Ser.","No.","60/166,678 D = Ser.","No.","60/173,396 E = Ser.","No.","60/184,129 F = Ser.","No.","60/188,114 G = Ser.","No.","60/185,421 H = Ser.","No.","60/186,811 I = Ser.","No.","60/186,530 J = Ser.","No.","60/207,094 K = Ser.","No.","60/203,111 L = Ser.","No.","60/190,310 M = Ser.","No.","60/201,190 N = Ser.","No.","60/185554 O = Ser.","No.","60/190,800 P = Ser.","No.","60/198,568 When a specific nGPCR is identified (for example nGPCR-5), it is understood that only that specific nGPCR is being referred to.","As described in Example 4 below, the genes encoding nGPCR-1 (nucleic acid sequence SEQ ID NO: 1, SEQ ID NO: 73, amino acid sequence SEQ ID NO: 2, SEQ ID NO:74), nGPCR-9 (nucleic acid sequence SEQ ID NO:9, SEQ ID NO:77, amino acid sequence SEQ ID NO:10, SEQ ID NO:78), nGPCR-11 (nucleic acid sequence SEQ ID NO:11, SEQ ID NO:79, amino acid sequence SEQ ID NO:12, SEQ ID NO:80), nGPCR-16 (nucleic acid sequence SEQ ID NO: 21, SEQ ID NO:81, amino acid sequence SEQ ID NO: 22, SEQ ID NO:82), nGPCR-40 (nucleic acids sequence SEQ ID NO:53, SEQ ID NO:83, amino acid sequence SEQ ID NO:54, SEQ ID NO:84), nGPCR-54 (nucleic acid sequence SEQ ID NO:59, SEQ ID NO:85, amino acid sequence SEQ ID NO:60, SEQ ID NO: 86), nGPCR-56 (nucleic acid sequence SEQ ID NO:63, SEQ ID NO:87, SEQ ID NO:89, amino acid sequence SEQ ID NO:64, SEQ ID NO: 88, SEQ ID NO:90), nGPCR-58 (nucleic acid sequence SEQ ID NO:67, SEQ ID NO:91, SEQ ID NO:93, amino acid sequence SEQ ID NO:68, SEQ ID NO: 92, SEQ ID NO:94) and nGPCR-3 (nucleic acid sequence SEQ ID NO:3, SEQ ID NO:185, amino acid sequence SEQ ID NO:4, SEQ ID NO: 186) have been detected in brain tissue indicating that these n-GPCR-x proteins are neuroreceptors.","The invention provides purified and isolated polynucleotides (e.g., cDNA, genomic DNA, synthetic DNA, RNA, or combinations thereof, whether single or double-stranded) that comprise a nucleotide sequence encoding the amino acid sequence of the polypeptides of the invention.","Such polynucleotides are useful for recombinantly expressing the receptor and also for detecting expression of the receptor in cells (e.g., using Northern hybridization and in situ hybridization assays).","Such polynucleotides also are useful in the design of antisense and other molecules for the suppression of the expression of nGPCR-x in a cultured cell, a tissue, or an animal; for therapeutic purposes; or to provide a model for diseases or conditions characterized by aberrant nGPCR-x expression.","Specifically excluded from the definition of polynucleotides of the invention are entire isolated, non-recombinant native chromosomes of host cells.","A preferred polynucleotide has the sequence of the sequence set forth in odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191, which correspond to naturally occurring nGPCR-x sequences.","It will be appreciated that numerous other polynucleotide sequences exist that also encode nGPCR-x having the sequence set forth in even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO:192, due to the well-known degeneracy of the universal genetic code.","The invention also provides a purified and isolated polynucleotide comprising a nucleotide sequence that encodes a mammalian polypeptide, wherein the polynucleotide hybridizes to a polynucleotide having the sequence set forth in odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185, and SEQ ID NO:191, or the non-coding strand complementary thereto, under the following hybridization conditions: (a) hybridization for 16 hours at 42° C. in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% dextran sulfate; and (b) washing 2 times for 30 minutes each at 60° C. in a wash solution comprising 0.1% SSC, 1% SDS.","Polynucleotides that encode a human allelic variant are highly preferred.","The present invention relates to molecules which comprise the gene sequences that encode the nGPCRs; constructs and recombinant host cells incorporating the gene sequences; the novel GPCR polypeptides encoded by the gene sequences; antibodies to the polypeptides and homologs; kits employing the polynucleotides and polypeptides, and methods of making and using all of the foregoing.","In addition, the present invention relates to homologs of the gene sequences and of the polypeptides and methods of making and using the same.","Genomic DNA of the invention comprises the protein-coding region for a polypeptide of the invention and is also intended to include allelic variants thereof.","It is widely understood that, for many genes, genomic DNA is transcribed into RNA transcripts that undergo one or more splicing events wherein intron (i.e., non-coding regions) of the transcripts are removed, or “spliced out.” RNA transcripts that can be spliced by alternative mechanisms, and therefore be subject to removal of different RNA sequences but still encode a nGPCR-x polypeptide, are referred to in the art as splice variants which are embraced by the invention.","Splice variants comprehended by the invention therefore are encoded by the same original genomic DNA sequences but arise from distinct mRNA transcripts Allelic variants are modified forms of a wild-type gene sequence, the modification resulting from recombination during chromosomal segregation or exposure to conditions which give rise to genetic mutation.","Allelic variants, like wild type genes, are naturally occurring sequences (as opposed to non-naturally occurring variants that arise from in vitro manipulation).","The invention also comprehends cDNA that is obtained through reverse transcription of an RNA polynucleotide encoding nGPCR-x (conventionally followed by second strand synthesis of a complementary strand to provide a double-stranded DNA).","Preferred DNA sequences encoding human nGPCR-x polypeptides are set out in odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191.A preferred DNA of the invention comprises a double stranded molecule along with the complementary molecule (the “non-coding strand” or “complement”) having a sequence unambiguously deducible from the coding strand according to Watson-Crick base-pairing rules for DNA.","Also preferred are other polynucleotides encoding the nGPCR-x polypeptide of even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO:192 which differ in sequence from the polynucleotides of odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 192, by virtue of the well-known degeneracy of the universal nuclear genetic code.","In a preferred embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence which encodes a fragment of polypeptide comprising a sequence of SEQ ID NO: 192.The fragment of the polypeptide comprising a sequence of SEQ ID NO: 192 comprises at least one or more amino acid residues from one or more of the following regions of SEQ ID NO: 192: amino acid residues 1 to 42 of SEQ ID NO: 192; amino acid residues 68 to 77 of SEQ ID NO: 192; amino acid residues 185 to 197 of SEQ ID NO: 192; or amino acid residues 293 to 513 of SEQ ID NO: 192.In a preferred embodiment, the isolated nucleic acid comprises a nucleotide sequence of SEQ ID NO: 191, and fragments thereof, that encode a polypeptide having a sequence of SEQ ID NO: 192, or fragments thereof.","The fragment of the nucleotide sequence of SEQ ID NO: 191 comprises at least one or more nucleotides from one or more of the following regions of SEQ ID NO: 191: nucleotides 1 to 193 of SEQ ID NO: 191; nucleotides 612 to 644 of SEQ ID NO:191; nucleotides 697 to 706 of SEQ ID NO:191; nucleotides 1011 to 1049 of SEQ ID NO: 191; nucleotides 1051 to 1057 of SEQ ID NO:191; nucleotides 1090 to 1096 of SEQ ID NO:191; or nucleotides 1141 to 1642 of SEQ ID NO:191.The invention further embraces other species, preferably mammalian, homologs of the human nGPCR-x DNA.","Species homologs, sometimes referred to as “orthologs,” in general, share at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homology with human DNA of the invention.","Generally, percent sequence “homology” with respect to polynucleotides of the invention may be calculated as the percentage of nucleotide bases in the candidate sequence that are identical to nucleotides in the nGPCR-x sequence set forth in odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.","Polynucleotides of the invention permit identification and isolation of polynucleotides encoding related nGPCR-x polypeptides, such as human allelic variants and species homologs, by well-known techniques including Southern and/or Northern hybridization, and polymerase chain reaction (PCR).","Examples of related polynucleotides include human and non-human genomic sequences, including allelic variants, as well as polynucleotides encoding polypeptides homologous to nGPCR-x and structurally related polypeptides sharing one or more biological, immunological, and/or physical properties of nGPCR-x.","Non-human species genes encoding proteins homologous to nGPCR-x can also be identified by Southern and/or PCR analysis and are useful in animal models for nGPCR-x disorders.","Knowledge of the sequence of a human nGPCR-x DNA also makes possible through use of Southern hybridization or polymerase chain reaction (PCR) the identification of genomic DNA sequences encoding nGPCR-x expression control regulatory sequences such as promoters, operators, enhancers, repressors, and the like.","Polynucleotides of the invention are also useful in hybridization assays to detect the capacity of cells to express nGPCR-x.","Polynucleotides of the invention may also provide a basis for diagnostic methods useful for identifying a genetic alteration(s) in a nGPCR-x locus that underlies a disease state or states, which information is useful both for diagnosis and for selection of therapeutic strategies.","According to the present invention, the nGPCR-x nucleotide sequences disclosed herein may be used to identify homologs of the nGPCR-x, in other animals, including but not limited to humans and other mammals, and invertebrates.","Any of the nucleotide sequences disclosed herein, or any portion thereof, can be used, for example, as probes to screen databases or nucleic acid libraries, such as, for example, genomic or cDNA libraries, to identify homologs, using screening procedures well known to those skilled in the art.","Accordingly, homologs having at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably at least 100% homology with nGPCR-x sequences can be identified.","The disclosure herein of full-length polynucleotides encoding nGPCR-x polypeptides makes readily available to the worker of ordinary skill in the art every possible fragment of the full-length polynucleotide.","One preferred embodiment of the present invention provides an isolated nucleic acid molecule comprising a sequence homologous to odd numbered sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:93, SEQ ID NO: 185 and SEQ ID NO: 191, and fragments thereof.","Another preferred embodiment provides an isolated nucleic acid molecule comprising a sequence selected from the group of odd numbered sequences consisting of SEQ ID NO: 1 to SEQ ID NO:93, SEQ ID NO: 185 and SEQ ID NO: 191, and fragments thereof.","As used in the present invention, fragments of nGPCR-x-encoding polynucleotides comprise at least 10, and preferably at least 12, 14, 16, 18, 20, 25, 50, or 75 consecutive nucleotides of a polynucleotide encoding nGPCR-x.","Preferably, fragment polynucleotides of the invention comprise sequences unique to the nGPCR-x-encoding polynucleotide sequence, and therefore hybridize under highly stringent or moderately stringent conditions only (i.e., “specifically”) to polynucleotides encoding nGPCR-x (or fragments thereof).","Polynucleotide fragments of genomic sequences of the invention comprise not only sequences unique to the coding region, but also include fragments of the full-length sequence derived from introns, regulatory regions, and/or other non-translated sequences.","Sequences unique to polynucleotides of the invention are recognizable through sequence comparison to other known polynucleotides, and can be identified through use of alignment programs routinely utilized in the art, e.g., those made available in public sequence databases.","Such sequences also are recognizable from Southern hybridization analyses to determine the number of fragments of genomic DNA to which a polynucleotide will hybridize.","Polynucleotides of the invention can be labeled in a manner that permits their detection, including radioactive, fluorescent, and enzymatic labeling.","Fragment polynucleotides are particularly useful as probes for detection of full-length or fragments of nGPCR-x polynucleotides.","One or more polynucleotides can be included in kits that are used to detect the presence of a polynucleotide encoding nGPCR-x, or used to detect variations in a polynucleotide sequence encoding nGPCR-x.","The invention also embraces DNAs encoding nGPCR-x polypeptides that hybridize under moderately stringent or high stringency conditions to the non-coding strand, or complement, of the polynucleotides set forth in odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:192.Exemplary highly stringent hybridization conditions are as follows: hybridization at 42° C. in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% Dextran sulfate, and washing twice for 30 minutes at 60° C. in a wash solution comprising 0.1×SSC and 1% SDS.","It is understood in the art that conditions of equivalent stringency can be achieved through variation of temperature and buffer, or salt concentration as described Ausubel et al.","(Eds.","), Protocols in Molecular Biology, John Wiley & Sons (1994), pp.","6.0.3 to 6.4.10.Modifications in hybridization conditions can be empirically determined or precisely calculated based on the length and the percentage of guanosine/cytosine (GC) base pairing of the probe.","The hybridization conditions can be calculated as described in Sambrook, et al., (Eds.","), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y. (1989), pp.","9.47 to 9.51.With the knowledge of the nucleotide sequence information disclosed in the present invention, one skilled in the art can identify and obtain nucleotide sequences which encode nGPCR-x from different sources (i.e., different tissues or different organisms) through a variety of means well known to the skilled artisan and as disclosed by, for example, Sambrook et al., “Molecular cloning: a laboratory manual”, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (19.89), which is incorporated herein by reference in its entirety.","For example, DNA that encodes nGPCR-x may be obtained by screening of mRNA, cDNA, or genomic DNA with oligonucleotide probes generated from the nGPCR-x gene sequence information provided herein.","Probes may be labeled with a detectable group, such as a fluorescent group, a radioactive atom or a chemiluminescent group in accordance with procedures known to the skilled artisan and used in conventional hybridization assays, as described by, for example, Sambrook et al.","A nucleic acid molecule comprising any of the nGPCR-x nucleotide sequences described above can alternatively be synthesized by use of the polymerase chain reaction (PCR) procedure, with the PCR oligonucleotide primers produced from the nucleotide sequences provided herein.","See U.S. Pat.","No.","4,683,195 to Mulliset al.","and U.S. Pat.","No.","4,683,202 to Mullis.","The PCR reaction provides a method for selectively increasing the concentration of a particular nucleic acid sequence even when that sequence has not been previously purified and is present only in a single copy in a particular sample.","The method can be used to amplify either single- or double-stranded DNA.","The essence of the method involves the use of two oligonucleotide probes to serve as primers for the template dependent, polymerase mediated replication of a desired nucleic acid molecule.","A wide variety of alternative cloning and in vitro amplification methodologies are well known to those skilled in the art Examples of these techniques are found in, for example, Berger et al., Guide to Molecular Cloning Techniques, Methods in Enzymology 152, Academic Press, Inc., San Diego, Calif. (Berger), which is incorporated herein by reference in its entirety.","Automated sequencing methods can be used to obtain or verify the nucleotide sequence of nGPCR-x.","The nGPCR-x nucleotide sequences of the present invention are believed to be 100% accurate.","However, as is known in the art, nucleotide sequence obtained by automated methods may contain some errors.","Nucleotide sequences determined by automation are typically at least about 90%, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of a given nucleic acid molecule.","The actual sequence may be more precisely determined using manual sequencing methods, which are well known in the art.","An error in a sequence which results in an insertion or deletion of one or more nucleotides may result in a frame shift in translation such that the predicted amino acid sequence will differ from that which would be predicted from the actual nucleotide sequence of the nucleic acid molecule, starting at the point of the mutation.","The nucleic acid molecules of the present invention, and fragments derived therefrom, are useful for screening for restriction fragment length polymorphism (RFLP) associated with certain disorders, as well as for genetic mapping.","The polynucleotide sequence information provided by the invention makes possible large-scale expression of the encoded polypeptide by techniques well known and routinely practiced in the art.","Vectors Another aspect of the present invention is directed to vectors, or recombinant expression vectors, comprising any of the nucleic acid molecules described above.","Vectors are used herein either to amplify DNA or RNA encoding nGPCR-x and/or to express DNA which encodes nGPCR-x.","Preferred vectors include, but are not limited to, plasmids, phages, cosmids, episomes, viral particles or viruses, and integratable DNA fragments (i.e., fragments integratable into the host genome by homologous recombination).","Preferred viral particles include, but are not limited to, adenoviruses, baculoviruses, parvoviruses, herpesviruses, poxviruses, adeno-associated viruses, Semliki Forest viruses, vaccinia viruses, and retroviruses.","Preferred expression vectors include, but are not limited to, pcDNA3 (Invitrogen) and pSVL (Pharmacia Biotech).","Other expression vectors include, but are not limited to, pSPORT™ vectors, pGEM™ vectors (Promega), pPROEX vectors™ (LTI, Bethesda, Md.","), Bluescript™ vectors (Stratagene), pQE™ vectors (Qiagen), pSE420™ (Invitrogen), and pYES2™ (Invitrogen).","Expression constructs preferably comprise GPCR-x-encoding polynucleotides operatively linked to an endogenous or exogenous expression control DNA sequence and a transcription terminator.","Expression control DNA sequences include promoters, enhancers, operators, and regulatory element binding sites generally, and ore typically selected based on the expression systems in which the expression construct is to be utilized.","Preferred promoter and enhancer sequences are generally selected for the ability to increase gene expression, while operator sequences are generally selected for the ability to regulate gene expression.","Expression constructs of the invention may also include sequences encoding one or more selectable markers that permit identification of host cells bearing the construct.","Expression constructs may also include sequences that facilitate, and preferably promote, homologous recombination in a host cell.","Preferred constructs of the invention also include sequences necessary for replication in a host cell.","Expression constructs are preferably utilized for production of an encoded protein, but may also be utilized simply to amplify a nGPCR-x-encoding polynucleotide sequence.","In preferred embodiments, the vector is an expression vector wherein the polynucleotide of the invention is operatively linked to a polynucleotide comprising an expression control sequence.","Autonomously replicating recombinant expression constructs such as plasmid and viral DNA vectors incorporating polynucleotides of the invention are also provided.","Preferred expression vectors are replicable DNA constructs in which a DNA sequence encoding nGPCR-x is operably linked or connected to suitable control sequences capable of effecting the expression of the nGPCR-x in a suitable host DNA regions are operably linked or connected when they are functionally related to each other.","For example, a promoter is operably linked or connected to a coding sequence if it controls the transcription of the sequence.","Amplification vectors do not require expression control domains, but rather need only the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants.","The need for control sequences in the expression vector will vary depending upon the host selected and the transformation method chosen.","Generally, control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding and sequences which control the termination of transcription and translation.","Preferred vectors preferably contain a promoter that is recognized by the host organism.","The promoter sequences of the present invention may be prokaryotic, eukaryotic or viral.","Examples of suitable prokaryotic sequences include the PR and PL promoters of bacteriophage lambda (The bacteriophage Lambda, Hershey, A. D., Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1973), which is incorporated herein by reference in its entirety; Lambda II, Hendrix, R. W., Ed., Cold Spring Habor Press, Cold Spring Harbor, N.Y. (1980), which is incorporated herein by reference in its entirety); the trp, recA, heat shock, and lacZ promoters of E. coli and the SV40 early promoter (Benoist et al.","Nature, 1981, 290, 304-310, which is incorporated herein by reference in its entirety).","Additional promoters include, but are not limited to, mouse mammary tumor virus, long terminal repeat of human immunodeficiency virus, maloney virus, cytomegalovirus immediate early promoter, Epstein Barr virus, Rous sarcoma virus, human actin, human myosin, human hemoglobin, human muscle creatine, and human metalothionein.","Additional regulatory sequences can also be included in preferred vectors.","Preferred examples of suitable regulatory sequences are represented by the Shine-Dalgarno of the replicase gene of the phage MS-2 and of the gene cII of bacteriophage lambda.","The Shine-Dalgarno sequence may be directly followed by DNA encoding nGPCR-x and result in the expression of the mature nGPCR-x protein.","Moreover, suitable expression vectors can include an appropriate marker that allows the screening of the transformed host cells.","The transformation of the selected host is carried out using any one of the various techniques well known to the expert in the art and described in Sambrook et al., supra.","An origin of replication can also be provided either by construction of the vector to include an exogenous origin or may be provided by the host cell chromosomal replication mechanism.","If the vector is integrated into the host cell chromosome, the latter may be sufficient.","Alternatively, rather than using vectors which contain viral origins of replication, one skilled in the art can transform mammalian cells by the method of co-transformation with a selectable marker and nGPCR-x DNA.","An example of a suitable marker is dihydrofolate reductase (DHFR) or thymidine kinase (see, U.S. Pat.","No.","4,399,216).","Nucleotide sequences encoding GPCR-x may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesiderable joining, and ligation with appropriate ligases.","Techniques for such manipulation are disclosed by Sambrook et al., supra and are well known in the art Methods for construction of mammalian expression vectors are disclosed in, for example, Okayama et al, Mol.","Cell.","Biol., 1983, 3, 280, Cosman et al., Mol.","Immutol., 1986, 23, 935, Cosman et al., Nature, 1984, 312, 768, EP-A-0367566, and WO 91/18982, each of which is incorporated herein by reference in its entirety.","Host Cells According to another aspect of the invention, host cells are provided, including prokaryotic and eukaryotic cells, comprising a polynucleotide of the invention (or vector of the invention) in a manner that permits expression of the encoded nGPCR-x polypeptide.","Polynucleotides of the invention may be introduced into the host cell as part of a circular plasmid, or as linear DNA comprising an isolated protein coding region or a viral vector.","Methods for introducing DNA into the host cell that are well known and routinely practiced in the art include transformation, transfection, electroporation, nuclear injection, or fusion with carriers such as liposomes, micelles, ghost cells, and protoplasts.","Expression systems of the invention include bacterial, yeast, fungal, plant, insect, invertebrate, vertebrate, and mammalian cells systems.","The invention provides host cells that are transformed or transfected (stably or transiently) with polynucleotides of the invention or vectors of the invention.","As stated above, such host cells are useful for amplifying the polynucleotides and also for expressing the nGPCR-x polypeptide or fragment thereof encoded by the polynucleotide.","In still another related embodiment, the invention provides a method for producing a nGPCR-x polypeptide (or fragment thereof) comprising the steps of growing a host cell of the invention in a nutrient medium and isolating the polypeptide or variant thereof from the cell or the medium.","Because nGPCR-x is a seven transmembrane receptor, it will be appreciated that, for some applications, such as certain activity assays, the preferable isolation may involve isolation of cell membranes containing the polypeptide embedded therein, whereas for other applications a more complete isolation may be preferable.","According to some aspects of the present invention, transformed host cells having an expression vector comprising any of the nucleic acid molecules described above are provided.","Expression of the nucleotide sequence occurs when the expression vector is introduced into an appropriate host cell.","Suitable host cells for expression of the polypeptides of the invention include, but are not limited to, prokaryotes, yeast, and eukaryotes.","If a prokaryotic expression vector is employed, then the appropriate host cell would be any prokaryotic cell capable of expressing the cloned sequences.","Suitable prokaryotic cells include, but are not limited to, bacteria of the genera Escherichia, Bacillus, Salmonella, Pseudomonas, Streptomyces, and Staphylococcus.","If an eukaryotic expression vector is employed, then the appropriate host cell would be any eukaryotic cell capable of expressing the cloned sequence.","Preferably, eukaryotic cells are cells of higher eukaryotes.","Suitable eukaryotic cells include, but are not limited to, non-human mammalian tissue culture cells and human tissue culture cells.","Preferred host cells include, but are not limited to, insect cells, HeLa cells, Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (COS cells), human 293 cells, and murine 3T3 fibroblasts.","Propagation of such cells in cell culture has become a routine procedure (see, Tissue Culture, Academic Press, Kruse and Patterson, eds.","(1973), which is incorporated herein by reference in its entirety).","In addition, a yeast host may be employed as a host cell.","Preferred yeast cells include, but are not limited to, the genera Saccharomyces, Pichia, and Kluveromyces.","Preferred yeast hosts are S. cerevisiae and P. pastoris.","Preferred yeast vectors can contain an origin of replication sequence from a 2T yeast plasmid, an autonomously replication sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene.","Shuttle vectors for replication in both yeast and E. coli are also included herein.","Alternatively, insect cells may be used as host cells.","In a preferred embodiment, the polypeptides of the invention are expressed using a baculovirus expression system (sea Luckow et al., Bio/Technology, 1988, 6, 47, Baculovirus Expression Vectors: A Laboratory Manual, O'Rielly et al (Eds.","), W.H.","Freeman and Company, New York, 1992, and U.S. Pat.","No.","4,879,236, each of which is incorporated herein by reference in its entirety).","In addition, the MAXBAC™ complete baculovirus expression system (Invitrogen) can, for example, be used for production in insect cells.","Host cells of the invention are a valuable source of immunogen for development of antibodies specifically immunoreactive with nGPCR-x.","Host cells of the invention are also useful in methods for the large-scale production of nGPCR-x polypeptides wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated from the cells, or from the medium in which the cells are grown, by purification methods known in the art, e.g., conventional chromatographic methods including immunoaffinity chromatography, receptor affinity chromatography, hydrophobic interaction chromatography, lectin affinity chromatography, size exclusion filtration, cation or anion exchange chromatography, high pressure liquid chromatography (HPLC), reverse phase HPLC, and the like.","Still other methods of purification include those methods wherein the desired protein is expressed and purified as a fusion protein having a specific tag, label, or chelating moiety that is recognized by a specific binding partner or agent.","The purified protein can be cleaved to yield the desired protein, or can be left as an intact fusion protein.","Cleavage of the fusion component may produce a form of the desired protein having additional amino acid residues as a result of the cleavage process.","Knowledge of nGPCR-x DNA sequences allows for modification of cells to permit, or increase, expression of endogenous nGPCR-x.","Cells can be modified (e.g., by homologous recombination) to provide increased expression by replacing, in whole or in part, the naturally occurring nGPCR-x promoter with all or part of a heterologous promoter so that the cells express nGPCR-x at higher levels.","The heterologous promoter is inserted in such a manner that it is operatively linked to endogenous nGPCR-x encoding sequences.","(See, for example, PCT International Publication No.","WO 94/12650, PCT International Publication No.","WO 92/20808, and PCT International Publication No.","WO 91/09955.)","It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamoyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA.","If linked to the nGPCR-x coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the nGPCR-x coding sequences in the cells.","Knockouts The DNA sequence information provided by the present invention also makes possible the development (e.g., by homologous recombination or “knock-out” strategies; see Capecchi, Science 244:1288-1292 (1989), which is incorporated herein by reference) of animals that fail to express functional nGPCR-x or that express a variant of nGPCR-x.","Such animals (especially small laboratory animals such as rats, rabbits, and mice) are useful as models for studying the in vivo activities of nGPCR-x and modulators of nGPCR-x.","Antisense Also made available by the invention are anti-sense polynucleotides that recognize and hybridize to polynucleotides encoding nGPCR-x.","Full-length and fragment anti-sense polynucleotides are provided.","Fragment antisense molecules of the invention include (i) those that specifically recognize and hybridize to nGPCR-x RNA (as determined by sequence comparison of DNA encoding nGPCR-x to DNA encoding other known molecules).","Identification of sequences unique to nGPCR-x encoding polynucleotides can be deduced through use of any publicly available sequence database, and/or through use of commercially available sequence comparison programs.","After identification of the desired sequences, isolation through restriction digestion or amplification using any of the various polymerase chain reaction techniques well known in the art can be performed.","Antisense polynucleotides are particularly relevant to regulating expression of nGPCR-x by those cells expressing nGPCR-x mRNA.","Antisense nucleic acids (preferably 10 to 30 base-pair oligonucleotides) capable of specifically binding to nGPCR-x expression control sequences or nGPCR-x RNA are introduced into cells (e.g., by a viral vector or colloidal dispersion system such as a liposome).","The antisense nucleic acid binds to the nGPCR-x target nucleotide sequence in the cell and prevents transcription and/or translation of the target sequence.","Phosphorothioate and methylphosphonate antisense oligonucleotides are specifically contemplated for therapeutic use by the invention.","The antisense oligonucleotides may be further modified by adding poly-L-lysine, transferrin polylysine, or cholesterol moieties at their 5′ end.","Suppression of nGPCR-x expression at either the transcriptional or translational level is useful to generate cellular or animal models for diseases/conditions characterized by aberrant nGPCR-x expression.","Antisense oligonucleotides, or fragments of odd numbered nucleotide sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191, or sequences complementary or homologous thereto, derived from the nucleotide sequences of the present invention encoding nGPCR-x are useful as diagnostic tools for probing gene expression in various tissues.","For example, tissue can be probed in situ with oligonucleotide probes carrying detectable groups by conventional autoradiography techniques to investigate native expression of this enzyme or pathological conditions relating thereto.","Antisense oligonucleotides are preferably directed to regulatory regions of odd numbered nucleotide sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191, or mRNA corresponding thereto, including, but not limited to, the initiation codon, TATA box, enhancer sequences, and the like.","Transcription Factors The nGPCR-x sequences taught in the present invention facilitate the design of novel transcription factors for modulating nGPCR-x expression in native cells and animals, and cells transformed or transfected with nGPCR-x polynucleotides.","For example, the Cys2-His2 zinc finger proteins, which bind DNA via their zinc finger domains, have been shown to be amenable to structural changes that lead to the recognition of different target sequences.","These artificial zinc finger proteins recognize specific target sites with high affinity and low dissociation constants, and are able to act as gene switches to modulate gene expression.","Knowledge of the particular nGPCR-x target sequence of the present invention facilitates the engineering of zinc finger proteins specific for the target sequence using known methods such as a combination of structure-based modeling and screening of phage display libraries (Segal et al., Proc.","Natl.","Acad.","Sci.","(USA) 96:2758-2763 (1999); Liu et al, Proc.","Natl.","Acad.","Sci.","(USA) 94:5525-5530 (1997); Greisman et al., Science 275:657-661 (1997); Choo et al., J. Mol.","Biol.","273:525-532 (1997)).","Each zinc finger domain usually recognizes three or more base pairs.","Since a recognition sequence of 18 base pairs is generally sufficient in length to render it unique in any known genome, a zinc finger protein consisting of 6 tandem repeats of zinc fingers would be expected to ensure specificity for a particular sequence (Segal et al.)","The artificial zinc finger repeats, designed based on nGPCR-x sequences, are fused to activation or repression domains to promote or suppress nGPCR-x expression (Liu et al.)","Alternatively, the zinc finger domains can be fused to the TATA box-binding factor (TBP) with varying lengths of linker region between the zinc finger peptide and the TBP to create either transcriptional activators or repressors (Kim et al., Proc.","Natl.","Acad.","Sci.","(USA) 94:3616-3620 (1997).","Such proteins and polynucleotides that encode them, have utility for modulating nGPCR-x expression in vivo in both native cells, animals and humans; and/or cells transfected with nGPCR-x-encoding sequences.","The novel transcription factor can be delivered to the target cells by transfecting constructs that express the transcription factor (gene therapy), or by introducing the protein.","Engineered zinc finger proteins can also be designed to bind RNA sequences for use in therapeutics as alternatives to antisense or catalytic RNA methods (McColl et al., Proc.","Natl.","Acad.","Sci.","(USA) 96:9521-9526 (1997); Wu et al., Proc.","Natl.","Acad.","Sci.","(USA) 92:344-348 (1995)).","The present invention contemplates methods of designing such transcription factors based on the gene sequence of the invention, as well as customized zinc finger proteins, that are useful to modulate nGPCR-x expression in cells (native or transformed) whose genetic complement includes these sequences.","Polypeptides The invention also provides purified and isolated mammalian nGPCR-x polypeptides encoded by a polynucleotide of the invention.","Presently preferred is a human nGPCR-x polypeptide comprising the amino acid sequence set out in even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO:192, or fragments thereof comprising an epitope specific to the polypeptide.","By “epitope specific to” is meant a portion of the nGPCR receptor that is recognizable by an antibody that is specific for the nGPCR, as defined in detail below.","Although the sequences provided are particular human sequences, the invention is intended to include within its scope other human allelic variants; non-human mammalian forms of nGPCR-x, and other vertebrate forms of nGPCR-x.","It will be appreciated that extracellular epitopes are particularly useful for generating and screening for antibodies and other binding compounds that bind to receptors such as nGPCR-x.","Thus, in another preferred embodiment, the invention provides a purified and isolated polypeptide comprising at least one extracellular domain (e.g., the N-terminal extracellular domain or one of the three extracellular loops) of nGPCR-x.","Purified and isolated polypeptides comprising the N-terminal extracellular domain of nGPCR-x are highly preferred.","Also preferred is a purified and isolated polypeptide comprising a nGPCR-x fragment selected from the group consisting of the N-terminal extracellular domain of nGPCR-x, transmembrane domains of nGPCR-x, an extracellular loop connecting transmembrane domains of nGPCR-x, an intracellular loop connecting transmembrane domains of nGPCR-x, the C-terminal cytoplasmic region of nGPCR-x, and fusions thereof.","Such fragments may be continuous portions of the native receptor.","However, it will also be appreciated that knowledge of the nGPCR-x gene and protein sequences as provided herein permits recombining of various domains that are not contiguous in the native protein.","Using a FORTRAN computer program called “tmtrest all” [Parodi et al., Comput.","Appl.","Biosci.","5:527-535 (1994)], nGPCR-x was shown to contain transmembrane-spanning domains.","The invention also embraces polypeptides that have at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55% or at least 50% identity and/or homology to the preferred polypeptide of the invention.","Percent amino acid sequence “identity” with respect to the preferred polypeptide of the invention is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues in the nGPCR-x sequence after aligning both sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.","Percent sequence “homology” with respect to the preferred polypeptide of the invention is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues in the nGPCR-x sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and also considering any conservative substitutions as part of the sequence identity.","In one aspect, percent homology is calculated as the percentage of amino acid residues in the smaller of two sequences which align with identical amino acid residue in the sequence being compared, when four gaps in a length of 100 amino acids may be introduced to maximize alignment [Dayhoff, in Atlas of Protein Sequence and Structure, Vol.","5, p. 124, National Biochemical Research Foundation, Washington, D.C. (1972), incorporated herein by reference].","Polypeptides of the invention may be isolated from natural cell sources or may be chemically synthesized, but are preferably produced by recombinant procedures involving host cells of the invention.","Use of mammalian host cells is expected to provide for such post-translational modifications (e.g., glycosylation, truncation, lipidation, and phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the invention.","Glycosylated and non-glycosylated forms of nGPCR-x polypeptides are embraced by the invention.","The invention also embraces variant (or analog) nGPCR-x polypeptides.","In one example, insertion variants are provided wherein one or more amino acid residues supplement a nGPCR-x amino acid sequence.","Insertions may be located at either or both termini of the protein, or may be positioned within internal regions of the nGPCR-x amino acid sequence.","Insertional variants with additional residues at either or both termini can include, for example, fusion proteins and proteins including amino acid tags or labels.","Insertion variants include nGPCR-x polypeptides wherein one or more amino acid residues are added to a nGPCR-x acid sequence or to a biologically active fragment thereof.","Variant products of the invention also include mature nGPCR-x products, ie., nGPCR-x products wherein leader or signal sequences are removed, with additional amino terminal residues.","The additional amino terminal residues may be derived from another protein, or may include one or more residues that are not identifiable as being derived from specific proteins.","nGPCR-x products with an additional methionine residue at position −1 (Met−1-nGPCR-x) are contemplated, as are variants with additional methionine and lysine residues at positions −2 and −1 (Met−2-Lys−1-nGPCR-x).","Variants of nGPCR-x with additional Met, Met-Lys, Lys residues (or, one or more basic residues in general) are particularly useful for enhanced recombinant protein production in bacterial host cells.","The invention also embraces nGPCR-x variants having additional amino acid residues that result from use of specific expression systems.","For example, use of commercially available vectors that express a desired polypeptide as part of a glutathione-S-transferase (GST) fusion product provides the desired polypeptide having an additional glycine residue at position −1 after cleavage of the GST component from the desired polypeptide.","Variants that result from expression in other vector systems are also contemplated.","Insertional variants also include fusion proteins wherein the amino terminus and/or the carboxy terminus of nGPCR-x is/are fused to another polypeptide.","In another aspect, the invention provides deletion variants wherein one or more amino acid residues in a nGPCR-x polypeptide are removed.","Deletions can be effected at one or both termini of the nGPCR-x polypeptide, or with removal of one or more non-terminal amino acid residues of nGPCR-x.","Deletion variants, therefore, include all fragments of a nGPCR-x polypeptide.","The invention also embraces polypeptide fragments of the even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, wherein the fragments maintain biological (e.g., ligand binding and/or intracellular signaling) immunological properties of a nGPCR-x polypeptide.","In one preferred embodiment of the invention, an isolated nucleic acid molecule comprises a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence homologous to even numbered sequences selected from the group consisting of: SEQ ID NO:2 to SEQ ID NO:94, SEQ ID NO: 186 and SEQ ID NO:192, and fragments thereof, wherein the nucleic acid molecule encoding at least a portion of nGPCR-x.","In a more preferred embodiment, the isolated nucleic acid molecule comprises a sequence that encodes a polypeptide comprising even numbered sequences selected from the group consisting of SEQ ID NO:2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, and fragments thereof.","As used in the present invention, polypeptide fragments comprise at least 5, 10, 15, 20, 25, 30, 35, or 40 consecutive amino acids of the even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO:192.Preferred polypeptide fragments display antigenic properties unique to, or specific for, human nGPCR-x and its allelic and species homologs.","Fragments of the invention having the desired biological and immunological properties can be prepared by any of the methods well known and routinely practiced in the art.","In still another aspect, the invention provides substitution variants of nGPCR-x polypeptides.","Substitution variants include those polypeptides wherein one or more amino acid residues of a nGPCR-x polypeptide are removed and replaced with alternative residues.","In one aspect, the substitutions are conservative in nature; however, the invention embraces substitutions that are also non-conservative.","Conservative substitutions for this purpose may be defined as set out in Tables 2, 3, or 4 below.","Variant polypeptides include those wherein conservative substitutions have been introduced by modification of polynucleotides encoding polypeptides of the invention.","Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure.","A conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties.","Exemplary conservative substitutions are set out in Table 2 (from WO 97/09433, page 10, published Mar.","13, 1997 (PCT/GB96/02197, filed Sep. 6, 1996), immediately below.","TABLE 2 Conservative Substitutions I SIDE CHAIN CHARACTERISTIC AMINO ACID Aliphatic Non-polar GAP ILV Polar - uncharged CSTM NQ Polar - charged DE KR Aromatic HFWY Other NQDE Alternatively, conservative amino acids can be grouped as described in Lehninger, [Biochemistry, Second Edition; Worth Publishers, Inc. NY, N.Y. (1975), pp.71-77] as set out in Table 3, below.","Table 3 Conservative Substitutions II TABLE 3 Conservative Substitutions II SIDE CHAIN CHARACTERISTIC AMINO ACID Non-polar (hydrophobic) A. Aliphatic: ALIVP B. Aromatic: FW C. Sulfur-containing: M D. Borderline: G Uncharged-polar A. Hydroxyl: STY B. Amides: NQ C. Sulfhydryl: C D. Borderline: G Positively Charged (Basic): KRH Negatively Charged (Acidic): DE As still another alternative, exemplary conservative substitutions are set out in Table 4, below.","TABLE 4 Conservative Substitutions III Original Residue Exemplary Substitution Ala (A) Val, Leu, Ile Arg (R) Lys, Gln, Asn Asn (N) Gln, His, Lys, Arg Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp His (H) Asn, Gln, Lys, Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu (L) Ile, Val, Met, Ala, Phe Lys (K) Arg, Gln, Asn Met (M) Leu, Phe, Ile Phe (F) Leu, Val, Ile, Ala Pro (P) Gly Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp, Phe, Thr, Ser Val (V) Ile, Leu, Met, Phe, Ala It should be understood that the definition of polypeptides of the invention is intended to include polypeptides bearing modifications other than insertion, deletion, or substitution of amino acid residues.","By way of example, the modifications may be covalent in nature, and include for example, chemical bonding with polymers, lipids, other organic, and inorganic moieties.","Such derivatives may be prepared to increase circulating half-life of a polypeptide, or may be designed to improve the targeting capacity of the polypeptide for desired cells, tissues, or organs.","Similarly, the invention further embraces nGPCR-x polypeptides that have been covalently modified to include one or more water-soluble polymer attachments such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.","Variants that display ligand binding properties of native nGPCR-x and are expressed at higher levels, as well as variants that provide for constitutively active receptors, are particularly useful in assays of the invention; the variants are also useful in providing cellular, tissue and animal models of diseases/conditions characterized by aberrant nGPCR-x activity.","In a related embodiment, the present invention provides compositions comprising purified polypeptides of the invention.","Preferred compositions comprise, in addition to the polypeptide of the invention, a pharmaceutically acceptable (i.e., sterile and non-toxic) liquid, semisolid, or solid diluent that serves as a pharmaceutical vehicle, excipient, or medium.","Any diluent known in the art may be used.","Exemplary diluents include, but are not limited to, water, saline solutions, polyoxyethylene sorbitan monolaurate, magnesium stearate, methyl- and propylhydroxybenzoate, talc, alginates, starches, lactose, sucrose, dextrose, sorbitol, mannitol, glycerol, calcium phosphate, mineral oil, and cocoa butter.","Variants that display ligand binding properties of native nGPCR-x and are expressed at higher levels, as well as variants that provide for constitutively active receptors, are particularly useful in assays of the invention; the variants are also useful in assays of the invention and in providing cellular, tissue and animal models of diseases/conditions characterized by aberrant nGPCR-x activity.","The G protein-coupled receptor functions through a specific heterotrimeric guanine-nucleotide-binding regulatory protein (G-protein) coupled to the intracellular portion of the G protein-coupled receptor molecule.","Accordingly, the G protein-coupled receptor has a specific affinity to G protein.","G proteins specifically bind to guanine nucleotides.","Isolation of G proteins provides a means to isolate guanine nucleotides.","G Proteins may be isolated using commercially available anti-G protein antibodies or isolated G protein-coupled receptors.","Similarly, G proteins may be detected in a sample isolated using commercially available detectable anti-G protein antibodies or isolated G protein-coupled receptors.","According to the present invention, the isolated n-GPCR-x proteins of the present invention are useful to isolate and purify G proteins from samples such as cell lysates.","Example 15 below sets forth an example of isolation of G proteins using isolated nGPCR-x proteins.","Such methodolgy may be used in place of the use of commercially available ant-G protein antibodies which are used to isolate G proteins.","Moreover, G proteins may be detected using nGPCR-x proteins in place of commercially available detectable anti-G protein antibodies.","Since nGPCR-x proteins specifically bind to G proteins, they can be employed in any specific use where G protein specific affinity is required, such as those uses where commercially available anti-G protein antibodies are employed.","Antibodies Also comprehended by the present invention are antibodies (e.g.","monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifunctional/bispecific antibodies, humanized antibodies, human antibodies, and complementary determining region (CDR)-grafted antibodies, including compounds which include CDR sequences which specifically recognize a polypeptide of the invention) specific for nGPCR-x or fragments thereof.","Preferred antibodies of the invention are human antibodies that are produced and identified according to methods described in WO93/11236, published Jun.","20, 1993, which is incorporated herein by reference in its entirety.","Antibody fragments, including Fab, Fab′, F(ab′)2, and Fv, are also provided by the invention.","The term “specific for,” when used to describe antibodies of the invention, indicates that the variable regions of the antibodies of the invention recognize and bind nGPCR-x polypeptides exclusively (i.e., are able to distinguish nGPCR-x polypeptides from other known GPCR polypeptides by virtue of measurable differences in binding affinity, despite the possible existence of localized sequence identity, homology, or similarity between nGPCR-x and such polypeptides).","It will be understood that specific antibodies may also interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and, in particular, in the constant region of the molecule.","Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art.","For a comprehensive discussion of such assays, see Harlow et al.","(Eds.","), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988), Chapter 6.Antibodies that recognize and bind fragments of the nGPCR-x polypeptides of the invention are also contemplated, provided that the antibodies are specific for nGPCR-x polypeptides.","Antibodies of the invention can be produced using any method well known and routinely practiced in the art.","The invention provides an antibody that is specific for the nGPCR-x of the invention.","Antibody specificity is described in greater detail below.","However, it should be emphasized that antibodies that can be generated from polypeptides that have previously been described in the literature and that are capable of fortuitously cross-reacting with nGPCR-x (e.g., due to the fortuitous existence of a similar epitope in both polypeptides) are considered “cross-reactive” antibodies.","Such cross-reactive antibodies are not antibodies that are “specific” for nGPCR-x.","The determination of whether an antibody is specific for nGPCR-x or is cross-reactive with another known receptor is made using any of several assays, such as Western blotting assays, that are well known in the art.","For identifying cells that express nGPCR-x and also for modulating nGPCR-x-ligand binding activity, antibodies that specifically bind to an extracellular epitope of the nGPCR-x are preferred.","In one preferred variation, the invention provides monoclonal antibodies.","Hybridomas that produce such antibodies also are intended as aspects of the invention.","In yet another variation, the invention provides a humanized antibody.","Humanized antibodies are useful for in vivo therapeutic indications.","In another variation, the invention provides a cell-free composition comprising polyclonal antibodies, wherein at least one of the antibodies is an antibody of the invention specific for nGPCR-x.","Antisera isolated from an animal is an exemplary composition, as is a composition comprising an antibody fraction of an antisera that has been resuspended in water or in another diluent, excipient, or carrier.","In still another related embodiment, the invention provides an anti-idiotypic antibody specific for an antibody that is specific for nGPCR-x.","It is well known that antibodies contain relatively small antigen binding domains that can be isolated chemically or by recombinant techniques.","Such domains are useful nGPCR-x binding molecules themselves, and also may be reintroduced into human antibodies, or fused to toxins or other polypeptides.","Thus, in still another embodiment, the invention provides a polypeptide comprising a fragment of a nGPCR-x-specific antibody, wherein the fragment and the polypeptide bind to the nGPCR-x.","By way of non-limiting example, the invention provides polypeptides that are single chain antibodies and CDR-grafted antibodies.","Non-human antibodies may be humanized by any of the methods known in the art.","In one method, the non-human CDRs are inserted into a human antibody or consensus antibody framework sequence.","Further changes can then be introduced into the antibody framework to modulate affinity or immunogenicity.","Antibodies of the invention are useful for, e.g., therapeutic purposes (by modulating activity of nGPCR-x), diagnostic purposes to detect or quantitate nGPCR-x, and purification of nGPCR-x.","Kits comprising an antibody of the invention for any of the purposes described herein are also comprehended.","In general, a kit of the invention also includes a control antigen for which the antibody is immunospecific.","Compositions Mutations in the nGPCR-x gene that result in loss of normal function of the nGPCR-x gene product underlie nGPCR-x-related human disease states.","The invention comprehends gene therapy to restore nGPCR-x activity to treat those disease states.","Delivery of a functional nGPCR-x gene to appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), or er vivo by use of physical DNA transfer methods (erg, liposomes or chemical treatments).","See, for example, Anderson, Nature, supplement to vol.","392, no.","6679, pp.25-20 (1998).","For additional reviews of gene therapy technology see Friedmann, Science, 244: 1275-1281 (1989); Verna, Scientific American: 68-84 (1990); and Miller, Nature, 357: 455-460 (1992).","Alternatively, it is contemplated that in other human disease states, preventing the expression of, or inhibiting the activity of, nGPCR-x will be useful in treating disease states.","It is contemplated that antisense therapy or gene therapy could be applied to negatively regulate the expression of nGPCR-x.","Another aspect of the present invention is directed to compositions, including pharmaceutical compositions, comprising any of the nucleic acid molecules or recombinant expression vectors described above and an acceptable carrier or diluent.","Preferably, the carrier or diluent is pharmaceutically acceptable.","Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field, which is incorporated herein by reference in its entirety.","Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin.","Liposomes and nonaqueous vehicles such as fixed oils may also be used.","The formulations are sterilized by commonly used techniques.","Also within the scope of the invention are compositions comprising polypeptides, polynucleotides, or antibodies of the invention that have been formulated with, e.g., a pharmaceutically acceptable carrier.","The invention also provides methods of using antibodies of the invention.","For example, the invention provides a method for modulating ligand binding of a nGPCR-x comprising the step of contacting the nGPCR-x with an antibody specific for the nGPCR-x, under conditions wherein the antibody binds the receptor.","GPCRs that may be expressed in the brain, such as nGPCR-x, provide an indication that aberrant nGPCR-x signaling activity may correlate with one or more neurological or psychological disorders.","The invention also provides a method for treating a neurological or psychiatric disorder comprising the step of administering to a mammal in need of such treatment an amount of an antibody-like polypeptide of the invention that is sufficient to modulate ligand binding to a nGPCR-x in neurons of the mammal.","nGPCR-x may also be expressed in other tissues, including but not limited to, peripheral blood lymphocytes, pancreas, ovary, uterus, testis, salivary gland, thyroid gland, kidney, adrenal gland, liver, bone marrow, prostate, fetal liver, colon, muscle, and fetal brain, and may be found in many other tissues.","Within the brain, nGPCR-x mRNA transcripts may be found in many tissues, including, but not limited to, frontal lobe, hypothalamus, pons, cerebellum, caudate nucleus, and medulla.","Tissues and brain regions where specific nGPCRs of the present invention are expressed are identified in the Examples below.","Kits The present invention is also directed to kits, including pharmaceutical kits.","The kits can comprise any of the nucleic acid molecules described above, any of the polypeptides described above, or any antibody which binds to a polypeptide of the invention as described above, as well as a negative control.","The kit preferably comprises additional components, such as, for example, instructions, solid support, reagents helpful for quantification, and the like.","In another aspect, the invention features methods for detection of a polypeptide in a sample as a diagnostic tool for diseases or disorders, wherein the method comprises the steps of: (a) contacting the sample with a nucleic acid probe which hybridizes under hybridization assay conditions to a nucleic acid target region of a polypeptide having the sequence of even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, said probe comprising the nucleic acid sequence encoding the polypeptide, fragments thereof, and the complements of the sequences and fragments; and (b) detecting the presence or amount of the probe:target region hybrid as an indication of the disease.","In preferred embodiments of the invention, the disease is selected from the group consisting of thyroid disorders (e.g.","thyreotoxicosis, myxoedema); renal failure; inflammatory conditions (e.g., Crohn's disease); diseases related to cell differentiation and homeostasis; rheumatoid arthritis; autoimmune disorders; movement disorders; CNS disorders (e.g., pain including migraine; stroke; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, anxiety, generalized anxiety disorder, post-traumatic-stress disorder, depression, bipolar disorder, delirium, dementia, severe mental retardation; dyskinesias, such as Huntington's disease or Tourette's Syndrome; attention disorders including ADD and ADHD, and degenerative disorders such as Parkinson's, Alzheimer's; movement disorders, including ataxias, supranuclear palsy, etc.","); infections, such as viral infections caused by HIV-1 or HIV-2; metabolic and cardiovascular diseases and disorders (e.g., type 2 diabetes, obesity, anorexia, hypotension, hypertension, thrombosis, myocardial infarction, cardiomyopathies, atherosclerosis, etc.","); proliferative diseases and cancers (e.g., different cancers such as breast, colon, lung, etc., and hyperproliferative disorders such as psoriasis, prostate hyperplasia, etc.","); hormonal disorders (e.g., male/female hormonal replacement, polycystic ovarian syndrome, alopecia, etc); and sexual dysfunction, among others.","As described above and in Example 4 below, the genes encoding nGPCR-1 (nucleic acid sequence SEQ ID NO: 1, SEQ ID NO: 73, amino acid sequence SEQ ID NO: 2, SEQ ID NO:74), nGPCR-9 (nucleic acid sequence SEQ ID NO:9, SEQ ID NO:77, amino acid sequence SEQ ID NO:10, SEQ ID NO:78), nGPCR-11 (nucleic acid sequence SEQ ID NO:11, SEQ ID NO:79, amino acid sequence SEQ ID NO:12, SEQ ID NO:80), nGPCR-16 (nucleic acid sequence SEQ ID NO: 21, SEQ ID NO:81, amino acid sequence SEQ ID NO: 22, SEQ ID NO:82), nGPCR-40 (nucleic acid sequence SEQ ID NO:53, SEQ ID NO:83, amino acid sequence SEQ ID NO:54, SEQ ID NO:84), nGPCR-54 (nucleic acid sequence SEQ ID NO:59, SEQ ID NO:85, amino acid sequence SEQ ID NO:60, SEQ ID NO: 86), nGPCR-56 (nucleic acid sequence SEQ ID NO:63, SEQ ID NO:87, SEQ ID NO:89, amino acid sequence SEQ ID NO:64, SEQ ID NO: 88, SEQ ID NO:90), nGPCR-58 (nucleic acid sequence SEQ ID NO:67, SEQ ID NO:91, SEQ ID NO:93, amino acid sequence SEQ ID NO:68, SEQ ID NO: 92, SEQ ID NO:94) and nGPCR-3 (nucleic acid sequence SEQ ID NO:3, SEQ ID NO:185, amino acid sequence SEQ ID NO:4, SEQ ID NO: 186) have been detected in brain tissue indicating that these n-GPCR-x proteins are neuroreceptors.","Kits may be designed to detect either expression of polynucleotides encoding these proteins or the proteins themselves in order to identify tissue as being neurological.","For example, oligonucleotide hybridization kits can be provided which include a container having an oligonucleotide probe specific for the n-GPCR-x-specific DNA and optionally, containers with positive and negative controls and/or instructions.","Similarly, PCR kits can be provided which include a container having primers specific for the n-GPCR-x-specific sequences, DNA and optionally, containers with size markers, positive and negative controls and/or instructions.","Hybridization conditions should be such that hybridization occurs only with the genes in the presence of other nucleic acid molecules.","Under stringent hybridization conditions only highly complementary nucleic acid sequences hybridize.","Preferably, such conditions prevent hybridization of nucleic acids having 1 or 2 mismatches out of 20 contiguous nucleotides.","Such conditions are defined supra.","The diseases for which detection of genes in a sample could be diagnostic include diseases in which nucleic acid (DNA and/or RNA) is amplified in comparison to normal cells.","By “amplification” is meant increased numbers of DNA or RNA in a cell compared with normal cells.","The diseases that could be diagnosed by detection of nucleic acid in a sample preferably include central nervous system and metabolic diseases.","The test samples suitable for nucleic acid probing methods of the present invention include, for example, cells or nucleic acid extracts of cells, or biological fluids.","The samples used in the above-described methods will vary based on the assay format, the detection method and the nature of the tissues, cells or extracts to be assayed.","Methods for preparing nucleic acid extracts of cells are well known in the art and can be readily adapted in order to obtain a sample that is compatible with the method utilized.","Alternatively, immunoassay kits can be provided which have containers container having antibodies specific for the n-GPCR-x-protein and optionally, containers with positive and negative controls and/or instructions.","Kits may also be provided useful in the identification of GPCR binding partners such as natural ligands or modulators (agonists or antagonists).","Substances useful for treatment of disorders or diseases preferably show positive results in one or more in vitro assays for an activity corresponding to treatment of the disease or disorder in question.","Substances that modulate the activity of the polypeptides preferably include, but are not limited to, antisense oligonucleotides, agonists and antagonists, and inhibitors of protein kinases.","Methods of Inducing Immune Response Another aspect of the present invention is directed to methods of inducing an immune response in a mammal against a polypeptide of the invention by administering to the mammal an amount of the polypeptide sufficient to induce an immune response.","The amount will be dependent on the animal species, size of the animal, and the like but can be determined by those skilled in the art.","Methods of Identifying Ligands The invention also provides assays to identify compounds that bind nGPCR-x.","One such assay comprises the steps of: (a) contacting a composition comprising a nGPCR-x with a compound suspected of binding nGPCR-x; and (b) measuring binding between the compound and nGPCR-x.","In one variation, the composition comprises a cell expressing nGPCR-x on its surface.","In another variation, isolated nGPCR-x or cell membranes comprising nGPCR-x are employed.","The binding may be measured directly, e.g., by using a labeled compound, or may be measured indirectly by several techniques, including measuring intracellular signaling of nGPCR-x induced by the compound (or measuring changes in the level of nGPCR-x signaling).","Specific binding molecules, including natural ligands and synthetic compounds, can be identified or developed using isolated or recombinant nGPCR-x products, nGPCR-x variants, or preferably, cells expressing such products.","Binding partners are useful for purifying nGPCR-x products and detection or quantification of nGPCR-x products in fluid and tissue samples using known immunological procedures.","Binding molecules are also manifestly useful in modulating (i.e., blocking, inhibiting or stimulating) biological activities of nGPCR-x, especially those activities involved in signal transduction.","The DNA and amino acid sequence information provided by the present invention also makes possible identification of binding partner compounds with which a nGPCR-x polypeptide or polynucleotide will interact.","Methods to identify binding partner compounds include solution assays, in vitro assays wherein nGPCR-x polypeptides are immobilized, and cell-based assays.","Identification of binding partner compounds of nGPCR-x polypeptides provides candidates for therapeutic or prophylactic intervention in pathologies associated with nGPCR-x normal and aberrant biological activity.","The invention includes several assay systems for identifying nGPCR-x binding partners.","In solution assays, methods of the invention comprise the steps of (a) contacting a nGPCR-x polypeptide with one or more candidate binding partner compounds and (b) identifying the compounds that bind to the nGPCR-x polypeptide.","Identification of the compounds that bind the nGPCR-x polypeptide can be achieved by isolating the nGPCR-x polypeptide/binding partner complex, and separating the binding partner compound from the nGPCR-x polypeptide.","An additional step of characterizing the physical, biological, and/or biochemical properties of the binding partner compound is also comprehended in another embodiment of the invention.","In one aspect, the nGPCR-x polypeptide/binding partner complex is isolated using an antibody immunospecific for either the nGPCR-x polypeptide or the candidate binding partner compound.","In still other embodiments, either the nGPCR-x polypeptide or the candidate binding partner compound comprises a label or tag that facilitates its isolation, and methods of the invention to identify binding partner compounds include a step of isolating the nGPCR-x polypeptide/binding partner complex through interaction with the label or tag.","An exemplary tag of this type is a poly-histidine sequence, generally around six histidine residues, that permits isolation of a compound so labeled using nickel chelation.","Other labels and tags, such as the FLAG® tag (Eastman Kodak, Rochester, N.Y.), well known and routinely used in the art, are embraced by the invention.","In one variation of an in vitro assay, the invention provides a method comprising the steps of (a) contacting an immobilized nGPCR-x polypeptide with a candidate binding partner compound and (b) detecting binding of the candidate compound to the nGPCR-x polypeptide.","In an alternative embodiment, the candidate binding partner compound is immobilized and binding of nGPCR-x is detected.","Immobilization is accomplished using any of the methods well known in the art, including covalent bonding to a support, a bead, or a chromatographic resin, as well as non-covalent, high affinity interactions such as antibody binding, or use of streptavidin/biotin binding wherein the immobilized compound includes a biotin moiety.","Detection of binding can be accomplished (i) using a radioactive label on the compound that is not immobilized, (ii) using of a fluorescent label on the non-immobilized compound, (iii) using an antibody immunospecific for the non-immobilized compound, (iv) using a label on the non-immobilized compound that excites a fluorescent support to which the immobilized compound is attached, as well as other techniques well known and routinely practiced in the art.","The invention also provides cell-based assays to identify binding partner compounds of a nGPCR-x polypeptide.","In one embodiment, the invention provides a method comprising the steps of contacting a nGPCR-x polypeptide expressed on the surface of a cell with a candidate binding partner compound and detecting binding of the candidate binding partner compound to the nGPCR-x polypeptide.","In a preferred embodiment, the detection comprises detecting a calcium flux or other physiological event in the cell caused by the binding of the molecule.","Another aspect of the present invention is directed to methods of identifying compounds that bind to either nGPCR-x or nucleic acid molecules encoding nGPCR-x, comprising contacting nGPCR-x, or a nucleic acid molecule encoding the same, with a compound, and determining whether the compound binds nGPCR-x or a nucleic acid molecule encoding the same.","Binding can be determined by binding assays which are well known to the skilled artisan, including, but not limited to, gel-shift assays, Western blots, radiolabeled competition assay, phage-based expression cloning, co-fractionation by chromatography, co-precipitation, cross linking, interaction trap/two-hybrid analysis, southwestern analysis, ELISA, and the like, which are described in, for example, Current Protocols in Molecular Biology, 1999, John Wiley & Sons, NY, which is incorporated herein by reference in its entirety.","The compounds to be screened include (which may include compounds which are suspected to bind nGPCR-x, or a nucleic acid molecule encoding the same), but are not limited to, extracellular, intracellular, biologic or chemical origin.","The methods of the invention also embrace ligands, especially neuropeptides, that are attached to a label, such as a radiolabel (e.g., 125I, 35S, 32P, 33P, 3H), a fluorescence label, a chemiluminescent label, an enzymic label and an immunogenic label.","Modulators falling within the scope of the invention include, but are not limited to, non-peptide molecules such as non-peptide mimetics, non-peptide allosteric effectors, and peptides.","The nGPCR-x polypeptide or polynucleotide employed in such a test may either be free in solution, attached to a solid support, borne on a cell surface or located intracellularly or associated with a portion of a cell.","One skilled in the art can, for example, measure the formation of complexes between nGPCR-x and the compound being tested.","Alternatively, one skilled in the art can examine the diminution in complex formation between nGPCR-x and its substrate caused by the compound being tested.","In another embodiment of the invention, high throughput screening for compounds having suitable binding affinity to nGPCR-x is employed.","Briefly, large numbers of different small peptide test compounds are synthesized on a solid substrate.","The peptide test compounds are contacted with nGPCR-x and washed.","Bound nGPCR-x is then detected by methods well known in the art.","Purified polypeptides of the invention can also be coated directly onto plates for use in the aforementioned drug screening techniques.","In addition, non-neutralizing antibodies can be used to capture the protein and immobilize it on the solid support.","Generally, an expressed nGPCR-x can be used for HTS binding assays in conjunction with its defined ligand, in this case the corresponding neuropeptide that activates it.","The identified peptide is labeled with a suitable radioisotope, including, but not limited to, 125I, 3H, 35S or 32P, by methods that are well known to those skilled in the art.","Alternatively, the peptides may be labeled by well-known methods with a suitable fluorescent derivative (Baindur et al., Drug Dev.","Res., 1994, 33, 373-398; Rogers, Drug Discovery Today, 1997, 2, 156-160).","Radioactive ligand specifically bound to the receptor in membrane preparations made from the cell line expressing the recombinant protein can be detected in HTS assays in one of several standard ways, including filtration of the receptor-ligand complex to separate bound ligand from unbound ligand (Williams, Med.","Res.","Rev., 1991, 11, 147-184; Sweetnam et al., J.","Natural Products, 1993, 56, 441455).","Alternative methods include a scintillation proximity assay (SPA) or a FlashPlate format in which such separation is unnecessary (Nakayama, Cur.","Opinion Drug Disc.","Dev., 1998,1,85-91 Bosse et al., J. Biomolecular Screening, 1998, 3, 285-292.).","Binding of fluorescent ligands can be detected in various ways, including fluorescence energy transfer (FRET), direct spectrophotofluorometric analysis of bound ligand, or fluorescence polarization (Rogers, Drug Discovery Today, 1997, 2, 156-160; Hill, Cur.","Opinion Drug Disc.","Dev, 1998, 1, 92-97).","Other assays may be used to identify specific ligands of a nGPCR-x receptor, including assays that identify ligands of the target protein through measuring direct binding of test ligands to the target protein, as well as assays that identify ligands of target proteins through affinity ultrafiltration with ion spray mass spectroscopy/HPLC methods or other physical and analytical methods.","Alternatively, such binding interactions are evaluated indirectly using the yeast two-hybrid system described in Fields et al., Nature, 340:245-246 (1989), and Fields et al., Trends in Genetics, 10:286-292 (1994), both of which are incorporated herein by reference.","The two-hybrid system is a genetic assay for detecting interactions between two proteins or polypeptides.","It can be used to identify proteins that bind to a known protein of interest, or to delineate domains or residues critical for an interaction.","Variations on this methodology have been developed to clone genes that encode DNA binding proteins, to identify peptides that bind to a protein, and to screen for drugs.","The two-hybrid system exploits the ability of a pair of interacting proteins to bring a transcription activation domain into close proximity with a DNA binding domain that binds to an upstream activation sequence (UAS) of a reporter gene, and is generally performed in yeast.","The assay requires the construction of two hybrid genes encoding (1) a DNA-binding domain that is fused to a first protein and (2) an activation domain fused to a second protein.","The DNA-binding domain targets the first hybrid protein to the UAS of the reporter gene; however, because most proteins lack an activation domain, this DNA-binding hybrid protein does not activate transcription of the reporter gene.","The second hybrid protein, which contains the activation domain, cannot by itself activate expression of the reporter gene because it does not bind the UAS.","However, when both hybrid proteins are present, the noncovalent interaction of the first and second proteins tethers the activation domain to the UAS, activating transcription of the reporter gene.","For example, when the first protein is a GPCR gene product, or fragment thereof, that is known to interact with another protein or nucleic acid, this assay can be used to detect agents that interfere with the binding interaction.","Expression of the reporter gene is monitored as different test agents are added to the system.","The presence of an inhibitory agent results in lack of a reporter signal.","The function of nGPCR-x gene products is unclear and no ligands have yet been found which bind the gene product.","The yeast two-hybrid assay can also be used to identify proteins that bind to the gene product.","In an assay to identify proteins that bind to a nGPCR-x receptor, or fragment thereof, a fusion polynucleotide encoding both a nGPCR-x receptor (or fragment) and a UAS binding domain (i.e., a first protein) may be used.","In addition, a large number of hybrid genes each encoding a different second protein fused to an activation domain are produced and screened in the assay.","Typically, the second protein is encoded by one or more members of a total cDNA or genomic DNA fusion library, with each second protein-coding region being fused to the activation domain.","This system is applicable to a wide variety of proteins, and it is not even necessary to know the identity or function of the second binding protein.","The system is highly sensitive and can detect interactions not revealed by other methods; even transient interactions may trigger transcription to produce a stable mRNA that can be repeatedly translated to yield the reporter protein.","Other assays may be used to search for agents that bind to the target protein.","One such screening method to identify direct binding of test ligands to a target protein is described in U.S. Pat.","No.","5,585,277, incorporated herein by reference.","This method relies on the principle that proteins generally exist as a mixture of folded and unfolded states, and continually alternate between the two states.","When a test ligand binds to the folded form of a target protein (i.e., when the test ligand is a ligand of the target protein), the target protein molecule bound by the ligand remains in its folded state.","Thus, the folded target protein is present to a greater extent in the presence of a test ligand which binds the target protein, than in the absence of a ligand.","Binding of the ligand to the target protein can be determined by any method that distinguishes between the folded and unfolded states of the target protein.","The function of the target protein need not be known in order for this assay to be performed.","Virtually any agent can be assessed by this method as a test ligand, including, but not limited to, metals, polypeptides, proteins, lipids, polysaccharides, polynucleotides and small organic molecules.","Another method for identifying ligands of a target protein is described in Wieboldt et al., Anal.","Chem., 69:1683-1691 (1997), incorporated herein by reference.","This technique screens combinatorial libraries of 20-30 agents at a time in solution phase for binding to the target protein.","Agents that bind to the target protein are separated from other library components by simple membrane washing.","The specifically selected molecules that are retained on the filter are subsequently liberated from the target protein and analyzed by HPLC and pneumatically assisted electrospray (ion spray) ionization mass spectroscopy.","This procedure selects library components with the greatest affinity for the target protein, and is particularly useful for small molecule libraries.","Other embodiments of the invention comprise using competitive screening assays in which neutralizing antibodies capable of binding a polypeptide of the invention specifically compete with a test compound for binding to the polypeptide.","In this manner, the antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants with nGPCR-x.","Radiolabeled competitive binding studies are described in A. H. Lin et al.","Antimicrobial Agents and Chemotherapy, 1997, vol.","41, no.","10.pp.","2127-2131, the disclosure of which is incorporated herein by reference in its entirety.","As described above and in Example 4 below, the genes encoding nGPCR-1 (nucleic acid sequence SEQ ID NO: 1, SEQ ID NO: 73, amino acid sequence SEQ ID NO: 2, SEQ ID NO:74), nGPCR-9 (nucleic acid sequence SEQ ID NO:9, SEQ ID NO:77, amino acid sequence SEQ ID NO:10, SEQ ID NO:78), nGPCR-11 (nucleic acid sequence SEQ ID NO:11, SEQ ID NO:79, amino acid sequence SEQ ID NO:12, SEQ ID NO:80), nGPCR-16 (nucleic acid sequence SEQ ID NO: 21, SEQ ID NO:81, amino acid sequence SEQ ID NO: 22, SEQ ID NO:82), nGPCR-40 (nucleic acid sequence SEQ ID NO:53, SEQ ID NO:83, amino acid sequence SEQ ID NO:54, SEQ ID NO:84), nGPCR-54 (nucleic acid sequence SEQ ID NO:59, SEQ ID NO:85, amino acid sequence SEQ ID NO:60, SEQ ID NO: 86), nGPCR-56 (nucleic acid sequence SEQ ID NO:63, SEQ ID NO:87, SEQ ID NO:89, amino acid sequence SEQ ID NO:64, SEQ ID NO: 88, SEQ ID NO:90), nGPCR-58 (nucleic acid sequence SEQ ID NO:67, SEQ ID NO:91, SEQ ID NO:93, amino acid sequence SEQ ID NO:68, SEQ ID NO: 92, SEQ ID NO:94), and nGPCR-3 (nucleic acid sequence SEQ ID NO:3, SEQ ID NO:185, amino acid sequence SEQ ID NO:4, SEQ ID NO: 186) have been detected in brain tissue indicating that these nGPCR-x proteins are neuroreceptors.","Accordingly, natural binding partners of these molecules include neurotransmitters.","Identification of Modulating Agents The invention also provides methods for identifying a modulator of binding between a nGPCR-x and a nGPCR-x binding partner, comprising the steps of: (a) contacting a nGPCR-x binding partner and a composition comprising a nGPCR-x in the presence and in the absence of a putative modulator compound; (b) detecting binding between the binding partner and the nGPCR-x; and (c) identifying a putative modulator compound or a modulator compound in view of decreased or increased binding between the binding partner and the nGPCR-x in the presence of the putative modulator, as compared to binding in the absence of the putative modulator.","nGPCR-x binding partners that stimulate nGPCR-x activity are useful as agonists in disease states or conditions characterized by insufficient nGPCR-x signaling (e.g., as a result of insufficient activity of a nGPCR-x ligand).","nGPCR-x binding partners that block ligand-mediated nGPCR-x signaling are useful as nGPCR-x antagonists to treat disease states or conditions characterized by excessive nGPCR-x signaling.","In addition nGPCR-x modulators in general, as well as nGPCR-x polynucleotides and polypeptides, are useful in diagnostic assays for such diseases or conditions.","In another aspect, the invention provides methods for treating a disease or abnormal condition by administering to a patient in need of such treatment a substance that modulates the activity or expression of a polypeptide having the sequence of even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192.Agents that modulate (i.e., increase, decrease, or block) nGPCR-x activity or expression may be identified by incubating a putative modulator with a cell containing a nGPCR-x polypeptide or polynucleotide and determining the effect of the putative modulator on nGPCR-x activity or expression.","The selectivity of a compound that modulates the activity of nGPCR-x can be evaluated by comparing its effects on nGPCR-x to its effect on other GPCR compounds.","Selective modulators may include, for example, antibodies and other proteins, peptides, or organic molecules that specifically bind to a nGPCR-x polypeptide or a nGPCR-x-encoding nucleic acid.","Modulators of nGPCR-x activity will be therapeutically useful in treatment of diseases and physiological conditions in which normal or aberrant nGPCR-x activity is involved.","nGPCR-x polynucleotides, polypeptides, and modulators may be used in the treatment of such diseases and conditions as infections, such as viral infections caused by HIV-1 or HIV-2; pain; cancers; Parkinson's disease; hypotension; hypertension; and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Tourette's Syndrome, among others.","nGPCR-x polynucleotides and polypeptides, as well as nGPCR-x modulators, may also be used in diagnostic assays for such diseases or conditions.","Methods of the invention to identify modulators include variations on any of the methods described above to identify binding partner compounds, the variations including techniques wherein a binding partner compound has been identified and the binding assay is carried out in the presence and absence of a candidate modulator.","A modulator is identified in those instances where binding between the nGPCR-x polypeptide and the binding partner compound changes in the presence of the candidate modulator compared to binding in the absence of the candidate modulator compound.","A modulator that increases binding between the nGPCR-x polypeptide and the binding partner compound is described as an enhancer or activator, and a modulator that decreases binding between the nGPCR-x polypeptide and the binding partner compound is described as an inhibitor.","The invention also comprehends high-throughput screening (HTS) assays to identify compounds that interact with or inhibit biological activity (i.e., affect enzymatic activity, binding activity, etc.)","of a nGPCR-x polypeptide.","HTS assays permit screening of large numbers of compounds in an efficient manner.","Cell-based HTS systems are contemplated to investigate nGPCR-x receptor-ligand interaction.","HTS assays are designed to identify “hits” or “lead compounds” having the desired property, from which modifications can be designed to improve the desired property.","Chemical modification of the “hit” or “lead compound” is often based on an identifiable structure/activity relationship between the “hit” and the nGPCR-x polypeptide.","Another aspect of the present invention is directed to methods of identifying compounds which modulate (i.e., increase or decrease) activity of nGPCR-x comprising contacting nGPCR-x with a compound, and determining whether the compound modifies activity of nGPCR-x.","The activity in the presence of the test compared is measured to the activity in the absence of the test compound.","Where the activity of the sample containing the test compound is higher than the activity in the sample lacking the test compound, the compound will have increased activity.","Similarly, where the activity of the sample containing the test compound is lower than the activity in the sample lacking the test compound, the compound will have inhibited activity.","The present invention is particularly useful for screening compounds by using nGPCR-x in any of a variety of drug screening techniques.","The compounds to be screened include (which may include compounds which are suspected to modulate nGPCR-x activity), but are not limited to, extracellular, intracellular, biologic or chemical origin.","The nGPCR-x polypeptide employed in such a test may be in any form, preferably, free in solution, attached to a solid support, borne on a cell surface or located intracellularly.","One skilled in the art can, for example, measure the formation of complexes between nGPCR-x and the compound being tested.","Alternatively, one skilled in the art can examine the diminution in complex formation between nGPCR-x and its substrate caused by the compound being tested.","The activity of nGPCR-x polypeptides of the invention can be determined by, for example, examining the ability to bind or be activated by chemically synthesized peptide ligands.","Alternatively, the activity of nGPCR-x polypeptides can be assayed by examining their ability to bind calcium ions, hormones, chemokines, neuropeptides, neurotransmitters, nucleotides, lipids, odorants, and photons.","Alternatively, the activity of the nGPCR-x polypeptides can be determined by examining the activity of effector molecules including, but not limited to, adenylate cyclase, phospholipases and ion channels.","Thus, modulators of nGPCR-x polypeptide activity may alter a GPCR receptor function, such as a binding property of a receptor or an activity such as G protein-mediated signal transduction or membrane localization.","In various embodiments of the method, the assay may take the form of an ion flux assay, a yeast growth assay, a non-hydrolyzable GTP assay such as a [35S]-GTP S assay, a cAMP assay, an inositol triphosphate assay, a diacylglycerol assay, an Aequorin assay, a Luciferase assay, a FLIPR assay for intracellular Ca2+ concentration, a mitogenesis assay, a MAP Kinase activity assay, an arachidonic acid release assay (e.g., using [3H]-arachidonic acid), and an assay for extracellular acidification rates, as well as other binding or function-based assays of nGPCR-x activity that are generally known in the art.","In several of these embodiments, the invention comprehends the inclusion of any of the G proteins known in the art, such as G16, G15, or chimeric Gqd5, Gqs5, Gqo5, Gq25, and the like.","nGPCR-x activity can be determined by methodologies that are used to assay for FaRP activity, which is well known to those skilled in the art.","Biological activities of nGPCR-x receptors according to the invention include, but are not limited to, the binding of a natural or an unnatural ligand, as well as any one of the functional activities of GPCRs known in the art.","Non-limiting examples of GPCR activities include transmembrane signaling of various forms, which may involve G protein association and/or the exertion of an influence over G protein binding of various guanidylate nucleotides; another exemplary activity of GPCRs is the binding of accessory proteins or polypeptides that differ from known G proteins.","The modulators of the invention exhibit a variety of chemical structures, which can be generally grouped into non-peptide mimetics of natural GPCR receptor ligands, peptide and non-peptide allosteric effectors of GPCR receptors, and peptides that may function as activators or inhibitors (competitive, uncompetitive and non-competitive) (e.g., antibody products) of GPCR receptors.","The invention does not restrict the sources for suitable modulators, which may be obtained from natural sources such as plant, animal or mineral extracts, or non-natural sources such as small molecule libraries, including the products of combinatorial chemical approaches to library construction, and peptide libraries.","Examples of peptide modulators of GPCR receptors exhibit the following primary structures: GLGPRPLRFamide, GNSFLRFamide, GGPQGPLRFamide, GPSGPLRFamide, PDVDHVFLRFamide, and pyro-EDVDHVFLRFamide.","Other assays can be used to examine enzymatic activity including, but not limited to, photometric, radiometric, HPLC, electrochemical, and the like, which are described in, for example, Enzyme Assays: A Practical Approach, eds.","R. Eisenthal and M. J. Danson, 1992, Oxford University Press, which is incorporated herein by reference in its entirety.","The use of cDNAs encoding GPCRs in drug discovery programs is well-known; assays capable of testing thousands of unknown compounds per day in high-throughput screens (HTSs) are thoroughly documented.","The literature is replete with examples of the use of radiolabelled ligands in HTS binding assays for drug discovery (see Williams, Medicinal Research Reviews, 1991, 11, 147-184.; Sweetnam, et al., J.","Natural Products, 1993, 56, 441-455 for review).","Recombinant receptors are preferred for binding assay HTS because they allow for better specificity (higher relative purity), provide the ability to generate large amounts of receptor material, and can be used in a broad variety of formats (see Hodgson, Bio/Technology, 1992, 10, 973-980; each of which is incorporated herein by reference in its entirety).","A variety of heterologous systems is available for functional expression of recombinant receptors that are well known to those skilled in the art.","Such systems include bacteria (Strosberg, et al., Trends in Pharmacological Sciences, 1992, 13, 95-98), yeast (Pausch, Trends in Biotechnology, 1997, 15, 487-494), several kinds of insect cells (Vanden Broeck, Int.","Rev.","Cytology, 1996, 164, 189-268), amphibian cells (Jayawickreme et al., Current Opinion in Biotechnology, 1997, 8, 629634) and several mammalian cell lines (CHO, BEK293, COS, etc.","; see Gerhardt, et al., Eur.","J. Pharmacology, 1997, 334, 1-23).","These examples do not preclude the use of other possible cell expression systems, including cell lines obtained from nematodes (PCT application WO 98/37177).","In preferred embodiments of the invention, methods of screening for compounds that modulate nGPCR-x activity comprise contacting test compounds with nGPCR-x and assaying for the presence of a complex between the compound and nGPCR-x.","In such assays, the ligand is typically labeled.","After suitable incubation, free ligand is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of the particular compound to bind to nGPCR-x.","It is well known that activation of heterologous receptors expressed in recombinant systems results in a variety of biological responses, which are mediated by G proteins expressed in the host cells.","Occupation of a GPCR by an agonist results in exchange of bound GDP for GIT at a binding site on the Ga subunit; one can use a radioactive, non-hydrolyzable derivative of GTP, GTPγ[35S], to measure binding of an agonist to the receptor (Sim et al., Neuroreport, 1996, 7, 729-733).","One can also use this binding to measure the ability of antagonists to bind to the receptor by decreasing binding of GTPγ[35S] in the presence of a known agonist.","One could therefore construct a HTS based on GTPγ[35S] binding, though this is not the preferred method.","The G proteins required for functional expression of heterologous GPCRs can be native constituents of the host cell or can be introduced through well-known recombinant technology.","The G proteins can be intact or chimeric.","Often, a nearly universally competent G protein (e.g., Gα16) is used to couple any given receptor to a detectable response pathway.","G protein activation results in the stimulation or inhibition of other native proteins, events that can be linked to a measurable response.","Examples of such biological responses include, but are not limited to, the following: the ability to survive in the absence of a limiting nutrient in specifically engineered yeast cells (Pausch, Trends in Biotechnology, 1997, 15, 487-494); changes in intracellular Ca2+ concentration as measured by fluorescent dyes (Murphy, et al, Cur.","Opinion Drug Disc.","Dev., 1998, 1, 192-199).","Fluorescence changes can also be used to monitor ligand-induced changes in membrane potential or intracellular pH; an automated system suitable for HTS has been described for these purposes (Schroeder, et al., J. Biomolecular Screening, 1996, 1, 75-80).","Melanophores prepared from Xenopus laevis show a ligand-dependent change in pigment organization in response to heterologous GPCR activation; this response is adaptable to HTS formats (Jayawickreme et al, Cur.","Opinion Biotechnology, 1997, 8, 629634).","Assays are also available for the measurement of common second messengers, including cAMP, phosphoinositides and arachidonic acid, but these are not generally preferred for HTS.","Preferred methods of HTS employing these receptors include permanently transfected CHO cells, in which agonists and antagonists can be identified by the ability to specifically alter the binding of GTPγ[35S] in membranes prepared from these cells.","In another embodiment of the invention, permanently transfected CHO cells could be used for the preparation of membranes which contain significant amounts of the recombinant receptor proteins; these membrane preparations would then be used in receptor binding assays, employing the radiolabelled ligand specific for the particular receptor.","Alternatively, a functional assay, such as fluorescent monitoring of ligand-induced changes in internal Ca2+ concentration or membrane potential in permanently transfected CHO cells containing each of these receptors individually or in combination would be preferred for HTS.","Equally preferred would be an alternative type of mammalian cell, such as HEK293 or COS cells, in similar formats.","More preferred would be permanently transfected insect cell lines, such as Drosophila S2 cells.","Even more preferred would be recombinant yeast cells expressing the Drosophila melanogaster receptors in HTS formats well known to those skilled in the art (e.g., Pausch, Trends in Biotechnology, 1997, 15, 487-494).","The invention contemplates a multitude of assays to screen and identify inhibitors of ligand binding to nGPCR-x receptors.","In one example, the nGPCR-x receptor is immobilized and interaction with a binding partner is assessed in the presence and absence of a candidate modulator such as an inhibitor compound.","In another example, interaction between the nGPCR-x receptor and its binding partner is assessed in a solution assay, both in the presence and absence of a candidate inhibitor compound.","In either assay, an inhibitor is identified as a compound that decreases binding between the nGPCR-x receptor and its binding partner.","Another contemplated assay involves a variation of the dihybrid assay wherein an inhibitor of protein/protein interactions is identified by detection of a positive signal in a transformed or transfected host cell, as described in PCT publication number WO 95/20652, published Aug. 3, 1995.Candidate modulators contemplated by the invention include compounds selected from libraries of either potential activators or potential inhibitors.","There are a number of different libraries used for the identification of small molecule modulators, including: (1) chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules.","Chemical libraries consist of random chemical structures, some of which are analogs of known compounds or analogs of compounds that have been identified as “hits” or “leads” in other drug discovery screens, some of which are derived from natural products, and some of which arise from non-directed synthetic organic chemistry.","Natural product libraries are collections of microorganisms, animals, plants, or marine organisms which are used to create mixtures for screening by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of plants or marine organisms.","Natural product libraries include polyketides, non-ribosomal peptides, and variants (non-naturally occurring) thereof.","For a review, see Science 282:63-68 (1998).","Combinatorial libraries are composed of large numbers of peptides, oligonucleotides, or organic compounds as a mixture.","These libraries are relatively easy to prepare by traditional automated synthesis methods, PCR, cloning, or proprietary synthetic methods.","Of particular interest are non-peptide combinatorial libraries.","Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries.","For a review of combinatorial chemistry and libraries created therefrom, see Myers, Curr.","Opin.","Biotechnol.","8:701-707 (1997).","Identification of modulators through use of the various libraries described herein permits modification of the candidate “hit” (or “lead”) to optimize the capacity of the “hit” to modulate activity.","Still other candidate inhibitors contemplated by the invention can be designed and include soluble forms of binding partners, as well as such binding partners as chimeric, or fusion, proteins.","A “binding partner” as used herein broadly encompasses non-peptide modulators, as well as such peptide modulators as neuropeptides other than natural ligands, antibodies, antibody fragments, and modified compounds comprising antibody domains that are immunospecific for the expression product of the identified nGPCR-x gene.","The polypeptides of the invention are employed as a research tool for identification, characterization and purification of interacting, regulatory proteins.","Appropriate labels are incorporated into the polypeptides of the invention by various methods known in the art and the polypeptides are used to capture interacting molecules.","For example, molecules are incubated with the labeled polypeptides, washed to remove unbound polypeptides, and the polypeptide complex is quantified.","Data obtained using different concentrations of polypeptide are used to calculate values for the number, affinity, and association of polypeptide with the protein complex.","Labeled polypeptides are also useful as reagents for the purification of molecules with which the polypeptide interacts including, but not limited to, inhibitors.","In one embodiment of affinity purification, a polypeptide is covalently coupled to a chromatography column.","Cells and their membranes are extracted, and various cellular subcomponents are passed over the column.","Molecules bind to the column by virtue of their affinity to the polypeptide.","The polypeptide-complex is recovered from the column, dissociated and the recovered molecule is subjected to protein sequencing.","This amino acid sequence is then used to identify the captured molecule or to design degenerate oligonucleotides for cloning the corresponding gene from an appropriate cDNA library.","Alternatively, compounds may be identified which exhibit similar properties to the ligand for the nGPCR-x of the invention, but which are smaller and exhibit a longer half time than the endogenous ligand in a human or animal body.","When an organic compound is designed, a molecule according to the invention is used as a “lead” compound.","The design of mimetics to known pharmaceutically active compounds is a well-known approach in the development of pharmaceuticals based on such “lead” compounds.","Mimetic design, synthesis and testing are generally used to avoid randomly screening a large number of molecules for a target property.","Furthermore, structural data deriving from the analysis of the deduced amino acid sequences encoded by the DNAs of the present invention are useful to design new drugs, more specific and therefore with a higher pharmacological potency.","Comparison of the protein sequence of the present invention with the sequences present in all the available databases showed a significant homology with the transmembrane portion of G protein coupled receptors.","Accordingly, computer modeling can be used to develop a putative tertiary structure of the proteins of the invention based on the available information of the transmembrane domain of other proteins.","Thus, novel ligands based on the predicted structure of nGPCR-x can be designed.","In a particular embodiment, the novel molecules identified by the screening methods according to the invention are low molecular weight organic molecules, in which case a composition or pharmaceutical composition can be prepared thereof for oral intake, such as in tablets.","The compositions, or pharmaceutical compositions, comprising the nucleic acid molecules, vectors, polypeptides, antibodies and compounds identified by the screening methods described herein, can be prepared for any route of administration including, but not limited to, oral, intravenous, cutaneous, subcutaneous, nasal, intramuscular or intraperitoneal.","The nature of the carrier or other ingredients will depend on the specific route of administration and particular embodiment of the invention to be administered.","Examples of techniques and protocols that are useful in this context are, inter alia, found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A (ed.","), 1980, which is incorporated herein by reference in its entirety.","The dosage of these low molecular weight compounds will depend on the disease state or condition to be treated and other clinical factors such as weight and condition of the human or animal and the route of administration of the compound.","For treating human or animals, between approximately 0.5 mg/kg of body weight to 500 mg/kg of body weight of the compound can be administered.","Therapy is typically administered at lower dosages and is continued until the desired therapeutic outcome is observed.","The present compounds and methods, including nucleic acid molecules, polypeptides, antibodies, compounds identified by the screening methods described herein, have a variety of pharmaceutical applications and may be used, for example, to treat or prevent unregulated cellular growth, such as cancer cell and tumor growth.","In a particular embodiment, the present molecules are used in gene therapy.","For a review of gene therapy procedures, see e.g.","Anderson, Science, 1992, 256, 808-813, which is incorporated herein by reference in its entirety.","The present invention also encompasses a method of agonizing (stimulating) or antagonizing a nGPCR-x natural binding partner associated activity in a mammal comprising administering to said mammal an agonist or antagonist to one of the above disclosed polypeptides in an amount sufficient to effect said agonism or antagonism.","One embodiment of the present invention, then, is a method of treating diseases in a mammal with an agonist or antagonist of the protein of the present invention comprises administering the agonist or antagonist to a mammal in an amount sufficient to agonize or antagonize nGPCR-x-associated functions.","In an effort to discover novel treatments for diseases, biomedical researchers and chemists have designed, synthesized, and tested molecules that inhibit the function of protein polypeptides.","Some small organic molecules form a class of compounds that modulate the function of protein polypeptides.","Examples of molecules that have been reported to inhibit the function of protein kinases include, but are not limited to, bis monocyclic, bicyclic or heterocyclic aryl compounds (PCT WO 92/20642, published Nov. 26, 1992 by Maguire et al.","), vinylene-azaindole derivatives (PCT WO 94/14808, published Jul.","7, 1994 by Ballinari et al.","), 1-cyclopropyl-4-pyridyluinolones (U.S. Pat.","No.","5,330,992), styryl compounds (U.S. Pat.","No.","5,217,999), styryl-substituted pyridyl compounds (U.S. Pat.","No.","5,302,606), certain quinazoline derivatives (EP Application No.","0 566 266 A1), seleoindoles and selenides (PCT WO 94/03427, published Feb. 17, 1994 by Denny et al.","), tricyclic polyhydroxylic compounds (PCT WO 92/21660, published Dec. 10, 1992 by Dow), and benzylphosphonic acid compounds (PCT WO 91/15495, published Oct. 17, 1991 by Dow et al), all of which are incorporated by reference herein, including any drawings.","Exemplary diseases and conditions amenable to treatment based on the present invention include, but are not limited to, thyroid disorders (e.g.","thyreotoxicosis, myxoedema); renal failure; inflammatory conditions (e g., Chron's disease); diseases related to cell differentiation and homeostasis; rheumatoid arthritis; autoimmune disorders; movement disorders; CNS disorders (e.g., pain including migraine; stroke; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, anxiety, generalized anxiety disorder, post-traumatic-stress disorder, depression, bipolar disorder, delirium, dementia, severe mental retardation; dyskinesias, such as Huntington's disease or Tourette's Syndrome; attention disorders including ADD and ADHD, and degenerative disorders such as Parkinson's, Alzheimer's; movement disorders, including ataxias, supranuclear palsy, etc); infections, such as viral infections caused by HIV-1 or HIV-2; metabolic and cardiovascular diseases and disorders (e.g., type 2 diabetes, obesity, anorexia, hypotension, hypertension, thrombosis, myocardial infarction, cardiomyopathies, atherosclerosis, etc.","); proliferative diseases and cancers (e.g., different cancers such as breast, colon, lung, etc., and hyperproliferative disorders such as psoriasis, prostate hyperplasia, etc); hormonal disorders (e.g., male/female hormonal replacement, polycystic ovarian syndrome, alopecia, etc.","); sexual dysfunction, among others.","Compounds that can traverse cell membranes and are resistant to acid hydrolysis are potentially advantageous as therapeutics as they can become highly bioavailable after being administered orally to patients.","However, many of these protein inhibitors only weakly inhibit function.","In addition, many inhibit a variety of protein kinases and will therefore cause multiple side effects as therapeutics for diseases.","Some indolinone compounds, however, form classes of acid resistant and membrane permeable organic molecules.","WO 96/22976 (published Aug. 1, 1996 by Ballinari et at) describes hydrosoluble indolinone compounds that harbor tetralin, naphthalene, quinoline, and indole substituents fused to the oxindole ring.","These bicyclic substituents are in turn substituted with polar groups including hydroxylated alkyl, phosphate, and ether substituents.","U.S. patent application Ser.","No.","08/702,232, filed Aug. 23, 1996, entitled “Indolinone Combinatorial Libraries and Related Products and Methods for the Treatment of Disease” by Tang et al.","(Lyon & Lyon Docket No.","221/187) and 08/485,323, filed Jun.","7, 1995, entitled “Benzylidene-Z-Indoline Compounds for the Treatment of Disease” by Tang et al.","(Lyon & Lyon Docket No.","223/298) and International Patent Publication WO 96/22976, published Aug. 1, 1996 by Ballinari et al., all of which are incorporated herein by reference in their entirety, including any drawings, describe indolinone chemical libraries of indolinone compounds harboring other bicyclic moieties as well as monocyclic moieties fused to the oxindole ring.","Application Ser.","Nos.","08/02,232, filed Aug. 23, 1996, entitled “Indolinone Combinatorial Libraries and Related Products and Methods for the Treatment of Disease” by Tang et al.","(Lyon & Lyon Docket No.","221/187), 08/485,323, filed Jun.","7, 1995, entitled “Benzylidene-Z-Indoline Compounds for the Treatment of Disease” by Tang et al.","(Lyon & Lyon Docket No.","223/298), and WO 96/22976, published Aug. 1, 1996 by Ballinari et al.","teach methods of indolinone synthesis, methods of testing the biological activity of indolinone compounds in cells, and inhibition patterns of indolinone derivatives, both of which are incorporated by reference herein, including any drawings.","Other examples of substances capable of modulating kinase activity include, but are not limited to, tyrphostins, quinazolines, quinoxolines, and quinolines.","The quinazolines, tyrphostins, quinolines, and quinoxolines referred to above include well-known compounds such as those described in the literature.","For example, representative publications describing quinazolines include Barker et al., EPO Publication No.","0 520 722 A1; Jones et al., U.S. Pat.","No.","4,447,608; Kabbe et al., U.S. Pat.","No.","4,757,072; Kaul and Vougioukas, U.S. Pat.","No.","5,316,553; Kreighbaum and Corner, U.S. Pat.","No.","4,343,940; Pegg and Wardleworth, EPO Publication No.","0 562 734 A1; Barker et al., Proc.","of Am.","Assoc.","for Cancer Research 32:327 (1991); Bertino, J. R., Cancer Research 3:293-304 (1979); Bertino, J. R., Cancer Research 9(2 part 1):293-304 (1979); Curtinet al., Br.","J.","Cancer 53:361-368 (1986); Fernandes et al., Cancer Research 43:1117-1123 (1983); Ferris et al.","J. Org.","Chem.","44(2):173-178; Fry et al., Science 265:1093-1095 (1994); Jackman et al., Cancer Research 51:5579-5586 (1981); Jones et al.","J. Med.","Chem.","29(6):1114-1118; Lee and Skibo, Biochemistry 26(23):7355-7362 (1987); Lemus et al., J. Org.","Chem.","54:3511-3518 (1989); Ley and Seng, Synthesis 1975:415-522 (1975); Maxwell et al., Magnetic Resonance in Medicine 17:189-196 (1991); Mini et al., Cancer Research 45:325-330 (1985); Phillips and Castle, J. Heterocyclic Chem.","17(19):1489-1596 (1980); Reece et al., Cancer Research 47(11):2996-2999 (1977); Sculier et al., Cancer Immunol.","and Immunother.","23:A65 (1986); Sikora et al., Cancer Letters 23:289-295 (1984); and Sikora et al., Analytical Biochem.","172:344-355 (1988), all of which are incorporated herein by reference in their entirety, including any drawings.","Quinoxaline is described in Kaul and Vougioukas, U.S. Pat.","No.","5,316,553, incorporated herein by reference in its entirety, including any drawings.","Quinolines are described in Dolle et al., J. Med.","Chem.","37:2627-2629 (1994); MaGuire, J. Med.","Chem.","37:2129-2131 (1994); Burke et al., J. Med.","Chem.","36:425-432 (1993); and Burke et al.","BioOrganic Med.","Chem.","Letters 2:1771-1774 (1992), all of which are incorporated by reference in their entirety, including any drawings.","Tyrphostins are described in Allen et al., Clin.","Exp.","Immunol.","91:141-156 (1993); Anafi et al., Blood 82:12:3524-3529 (1993); Baker et al., J.","Cell Sci.","102:543-555 (1992); Bilder et al., Amer.","Physiol.","Soc.","pp.","6363-6143:C721-C730 (1991); Brunton et al., Proceedings of Amer.","Assoc.","Cancer Rsch.","33:558 (1992); Bryckaert et al., Experimental Cell Research 199:255-261 (1992); Dong et al., J. Leukocyte Biology 53:53-60 (1993); Dong et al., J. Immunol.","151(5):2717-2724 (1993); Gazit et al., J. Med.","Chem.","32:2344-2352 (1989); Gazit et al., J. Med.","Chem.","36:3556-3564 (1993); Kaur et al., Anti-Cancer Drugs 5:213-222 (1994); King et al., Biochem.","J.","275:413418 (1991); Kuo et al., Cancer Letters 74:197-202 (1993); Levitzkd, A., The FASEB J.","6:3275-3282 (1992); Lyall et al., J. Biol.","Chem.","264:14503-14509 (1989); Peterson et al., The Prostate 22:335-345 (1993); Pillemer et al., Int.","J.","Cancer 50:80-85 (1992); Posner et al., Molecular Pharmacology 45:673-683 (1993); Rendu et al., Biol.","Pharmacology 44(5):881-888 (1992); Sauro and Thomas, Life Sciences 53:371-376 (1993); Sauro and Thomas, J. Pharm.","and Experimental Therapeutics 267(3):119-1125 (1993); Wolbring et al., J. Biol.","Chem.","269(36):22470-22472 (1994); and Yoneda et al., Cancer Research 51:44304435 (1991); all of which are incorporated herein by reference in their entirety, including any drawings.","Other compounds that could be used as modulators include oxindolinones such as those described in U.S. patent application Ser.","No.","08/702,232 filed Aug. 23, 1996, incorporated herein by reference in its entirety, including any drawings.","Methods of determining the dosages of compounds to be administered to a patient and modes of administering compounds to an organism are disclosed in U.S. application Ser.","No.","08/702,282, filed Aug. 23, 1996 and International patent publication number WO 96/22976, published Aug. 1, 1996, both of which are incorporated herein by reference in their entirety, including any drawings, figures or tables.","Those skilled in the art will appreciate that such descriptions are applicable to the present invention and can be easily adapted to it.","The proper dosage depends on various factors such as the type of disease being treated, the particular composition being used and the size and physiological condition of the patient.","Therapeutically effective doses for the compounds described herein can be estimated initially from cell culture and animal models.","For example, a dose can be formulated in animal models to achieve a circulating concentration range that initially takes into account the IC50 as determined in cell culture assays.","The animal model data can be used to more accurately determine useful doses in humans.","Plasma half-life and biodistribution of the drug and metabolites in the plasma, tumors and major organs can also be determined to facilitate the selection of drugs most appropriate to inhibit a disorder.","Such measurements can be carried out.","For example, HPLC analysis can be performed on the plasma of animals treated with the drug and the location of radiolabeled compounds can be determined using detection methods such as X-ray, CAT scan and MRI.","Compounds that show potent inhibitory activity in the screening assays, but have poor pharmacokinetic characteristics, can be optimized by altering the chemical structure and retesting.","In this regard, compounds displaying good pharmacokinetic characteristics can be used as a model.","Toxicity studies can also be carried out by measuring the blood cell composition.","For example, toxicity studies can be carried out in a suitable animal model as follows: 1) the compound is administered to mice (an untreated control mouse should also be used); 2) blood samples are periodically obtained via the tail vein from one mouse in each treatment group; and 3) the samples are analyzed for red and white blood cell counts, blood cell composition and the percent of lymphocytes versus polymorphonuclear cells.","A comparison of results for each dosing regime with the controls indicates if toxicity is present.","At the termination of each toxicity study, further studies can be carried out by sacrificing the animals (preferably, in accordance with the American Veterinary Medical Association guidelines Report of the American Veterinary Medical Assoc.","Panel on Euthanasia, Journal of American Veterinary Medical Assoc., 202:229-249, 1993).","Representative animals from each treatment group can then be examined by gross necropsy for immediate evidence of metastasis, unusual illness or toxicity.","Gross abnormalities in tissue are noted and tissues are examined histologically.","Compounds causing a reduction in body weight or blood components are less preferred, as are compounds having an adverse effect on major organs.","In general, the greater the adverse effect the less preferred the compound.","For the treatment of cancers the expected daily dose of a hydrophobic pharmaceutical agent is between 1 to 500 mg/day, preferably 1 to 250 mg/day, and most preferably 1 to 50 mg/day.","Drugs can be delivered less frequently provided plasma levels of the active moiety are sufficient to maintain therapeutic effectiveness.","Plasma levels should reflect the potency of the drug.","Generally, the more potent the compound the lower the plasma levels necessary to achieve efficacy.","nGPCR-x mRNA transcripts may found in many tissues, including, but not limited to, brain, peripheral blood lymphocytes, pancreas, ovary, uterus, testis, salivary gland, kidney, adrenal gland, liver, bone marrow, prostate, fetal liver, colon, muscle, and fetal brain, and may be found in many other tissues.","Within the brain, nGPCR-x mRNA transcripts may be found in many tissues, including, but not limited to, frontal lobe, hypothalamus, pons, cerebellum, caudate nucleus, and medulla.","Tissues and brain regions where specific nGPCR mRNA transcripts are expressed are identified in the Examples, below.","Odd numbered nucleotide sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191 will, as detailed above, enable screening the endogenous neurotransmitters/hormones/ligands which activate, agonize, or antagonize nGPCR-x and for compounds with potential utility in treating disorders including, but not limited to, thyroid disorders (e.g.","thyreotoxicosis, myxoedema); renal failure; inflammatory conditions (e.g., Chron's disease); diseases related to cell differentiation and homeostasis; rheumatoid arthritis; autoimmune disorders; movement disorders; CNS disorders (e.g., pain including migraine; stroke; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, anxiety, generalized anxiety disorder, post-traumatic-stress disorder, depression, bipolar disorder, delirium, dementia, severe mental retardation; dyskinesias, such as Huntington's disease or Tourette's Syndrome; attention disorders including ADD and ADHD, and degenerative disorders such as Parkinson's, Alzheimer's; movement disorders, including ataxias, supranuclear palsy, etc.","); infections, such as viral infections caused by HIV-1 or HIV-2; metabolic and cardiovascular diseases and disorders (e.g., type 2 diabetes, obesity, anorexia, hypotension, hypertension, thrombosis, myocardial infarction, cardiomyopathies, atherosclerosis, etc.","); proliferative diseases and cancers (e.g., different cancers such as breast, colon, lung, etc, and hyperproliferative disorders such as psoriasis, prostate hyperplasia, etc.","); hormonal disorders (e.g., male/female hormonal replacement, polycystic ovarian syndrome, alopecia, etc.","); sexual dysfunction, among others.","For example, nGPCR-x may be useful in the treatment of respiratory ailments such as asthma, where T cells are implicated by the disease.","Contraction of airway smooth muscle is stimulated by thrombin.","Cicala et al (1999) Br J Pharmacol 126:478484.Additionally, in bronchiolitis obliterans, it has been noted that activation of thrombin receptors may be deleterious.","Hauck et al.","(1999) Am J Physiol 277:L22-L29.Furthermore, mast cells have also been shown to have thrombin receptors.","Cirino et al (1996) J Exp Med 183:821-827.nGPCR-x may also be useful in remodeling of airway structure s in chronic pulmonary inflammation via stimulation of fibroblast procollagen synthesis.","See, e.g., Chambers et al.","(1998) Biochem J 333:121-127; Trejo et al.","(1996) J Biol Chem 271:21536-21541.In another example, increased release of sCD40L and expression of CD40L by T cells after activation of thrombin receptors suggests that nGPCR-x may be useful in the treatment of unstable angina due to the role of T cells and inflammation.","See Aukrust et al.","(1999) Circulation 100:614-620.A further example is the treatment of inflammatory diseases, such as psoriasis, inflammatory bowel disease, multiple sclerosis, rheumatoid arthritis, and thyroiditis.","Due to the tissue expression profile of nGPCR-x, inhibition of thrombin receptors may be beneficial for these diseases.","See, e.g., Morris et al.","(1996) Ann Rheum Dis 55:841-843.In addition to T cells, NK cells and monocytes are also critical cell types which contribute to the pathogenesis of these diseases.","See, e.g., Naldini & Carney (1996) Cell Immunol 172:3542; Hoffman & Cooper (1995) Blood Cells Mol Dis 21:156-167; Colotta et al.","(1994) Am J Pathol 144:975-985.Expression of nGPCR-x in bone marrow and spleen may suggest that it may play a role in the proliferation of hematopoietic progenitor cells.","See DiCuccio et al.","(1996) Exp Hematol 24:914-918.As another example, nGPCR-x may be useful in the treatment of acute and/or traumatic brain injury.","Astrocytes have been demonstrated to express thrombin receptors.","Activation of thrombin receptors may be involved in astrogliosis following brain injury.","Therefore, inhibition of receptor activity may be beneficial for limiting neuroinflammation.","Scar formation mediated by astrocytes may also be limited by inhibiting thrombin receptors.","See, e.g., Pindon et al.","(1998) Eur J Biochem 255:766-774; Ubl & Reiser.","(1997) Glia 21:361-369; Grabham & Cunningham (1995) J Neurochem 64:583-591.nGPCR-x receptor activation may mediate neuronal and astrocyte apoptosis and prevention of neurite outgrowth.","Inhibition would be beneficial in both chronic and acute brain injury.","See, e.g., Donovan et al.","(1997) J Neurosci 17:5316-5326; Turgeon et al (1998) J Neurosci 18:6882-6891; Smith-Swintosky et al.","(1997) J Neurochem 69:1890-1896; Gill et al.","(1998) Brain Res 797:321-327; Suidan et al.","(1996) Semin Thromb Hemost 22:125-133.The attached Sequence Listing contains the sequences of the polynucleotides and polypeptides of the invention and is incorporated herein by reference in its entirety.","As described above and in Example 4 below, the genes encoding nGPCR-1 (nucleic acid sequence SEQ ID NO: 1, SEQ ID NO: 73, amino acid sequence SEQ ID NO: 2, SEQ ID NO:74), nGPCR-9 (nucleic acid sequence SEQ ID NO:9, SEQ ID NO:77, amino acid sequence SEQ ID NO: 10, SEQ ID NO:78), nGPCR-11 (nucleic acid sequence SEQ ID NO:11, SEQ ID NO:79, amino acid sequence SEQ ID NO:12, SEQ ID NO:80), nGPCR-16 (nucleic acid sequence SEQ ID NO: 21, SEQ.","ID NO:81, amino acid sequence SEQ ID NO: 22, SEQ ID NO:82), nGPCR-40 (nucleic acid sequence SEQ ID NO:53, SEQ ID NO:83, amino acid sequence SEQ ID NO: 54, SEQ ID NO:84), nGPCR-54 (nucleic acid sequence SEQ ID NO:59, SEQ ID NO:85, amino acid sequence SEQ ID NO:60, SEQ ID NO: 86), nGPCR-56 (nucleic acid sequence SEQ ID NO:63, SEQ ID NO:87, SEQ ID NO:89, amino acid sequence SEQ ID NO:64, SEQ ID NO: 88, SEQ ID NO:90), nGPCR-58 (nucleic acid sequence SEQ ID NO:3, SEQ ID NO: 185, amino acid sequence SEQ ID NO:4, SEQ ID NO: 186) have been detected in brain tissue indicating that these nGPCR-x proteins are neuroreceptors.","The identification of modulators such as agonists and antagonists is therefore useful for the identification of compounds useful to treat neurological diseases and disorders.","Such neurological diseases and disorders, including but are not limited to, schizophrenia, affective disorders, ADHD/ADD (i.e., Attention Deficit-Hyperactivity Disorder/Attention Deficit Disorder), and neural disorders such as Alzheimer's disease, Parkinson's disease, migraine, and senile dementia as well as depression, anxiety, bipolar disease, epilepsy, neuritis, neurasthenia, neuropathy, neuroses, and the like.","Methods of Screening Human Subjects Thus in yet another embodiment, the invention provides genetic screening procedures that entail analyzing a person's genome—in particular their alleles for GPCRs of the invention—to determine whether the individual possesses a genetic characteristic found in other individuals that are considered to be afflicted with, or at risk for, developing a mental disorder or disease of the brain that is suspected of having a hereditary component.","For example, in one embodiment, the invention provides a method for determining a potential for developing a disorder affecting the brain in a human subject comprising the steps of analyzing the coding sequence of one or more GPCR genes from the human subject; and determining development potential for the disorder in said human subject from the analyzing step.","More particularly, the invention provides a method of screening a human subject to diagnose a disorder affecting the brain or genetic predisposition therefor, comprising the steps of: (a) assaying nucleic acid of a human subject to determine a presence or an absence of a mutation altering the amino acid sequence, expression, or biological activity of at least one seven transmembrane receptor that is expressed in the brain, wherein the seven transmembrane receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 74, 186, 78, 80, 82, 84, 86, 90, and 94, or an allelic variant thereof, and wherein the nucleic acid corresponds to the gene encoding the seven transmembrane receptor; and (b) diagnosing the disorder or predisposition from the presence or absence of said mutation, wherein the presence of a mutation altering the amino acid sequence, expression, or biological activity of allele in the nucleic acid correlates with an increased risk of developing the disorder.","In preferred variations, the seven transmembrane receptor is nGPCR-40 or nGPCR-54 comprising amino acid sequences set forth in SEQ ID NO: 84 for nGPCR-40 and SEQ ID NO: 86 for nGPCR-54, or an allelic variant thereof, and the disease is schizophrenia.","By “human subject” is meant any human being, human embryo, or human fetus.","It will be apparent that methods of the present invention will be of particular interest to individuals that have themselves been diagnosed with a disorder affecting the brain or have relatives that have been diagnosed with a disorder affecting the brain.","By “screening for an increased risk” is meant determination of whether a genetic variation exists in the human subject that correlates with a greater likelihood of developing a disorder affecting the brain than exists for the human population as a whole, or for a relevant racial or ethnic human sub-population to which the individual belongs.","Both positive and negative determinations (i.e., determinations that a genetic predisposition marker is present or is absent) are intended to fall within the scope of screening methods of the invention.","In preferred embodiments, the presence of a mutation altering the sequence or expression of at least one nGPCR-40 or nGPCR-54 seven transmembrane receptor allele in the nucleic acid is correlated with an increased risk of developing schizophrenia, whereas the absence of such a mutation is reported as a negative determination.","The “assaying” step of the invention may involve any techniques available for analyzing nucleic acid to determine its characteristics, including but not limited to well-known techniques such as single-strand conformation polymorphism analysis (SSCP) [Orita et al., Proc Natl.","Acad.","Sci.","USA, 86: 2766-2770 (1989)]; heteroduplex analysis [White et al., Genomics, 12: 301-306 (1992)]; denaturing gradient gel electrophoresis analysis [Fischer et al., Proc.","Natl.","Acad.","Sci.","USA, 80: 1579-1583 (1983); and Riesner et al., Electrophoresis, 10: 377-389 (1989)]; DNA sequencing; RNase cleavage [Myers et al., Science, 230: 1242-1246 (1985)]; chemical cleavage of mismatch techniques [Rowley et al., Genomics, 30: 574-582 (1995); and Roberts et al., Nucl.","Acids Res., 25: 3377-3378 (1997)]; restriction fragment length polymorphism analysis; single nucleotide primer extension analysis [Shumaker et al., Hum.","Mutat., 7: 346-354 (1996); and Pastinen et al., Genome Res., 7: 606-614 (1997)]; 5′ nuclease assays [Pease et al., Proc.","Natl.","Acad.","Sci.","USA, 91:5022-5026 (1994)]; DNA Microchip analysis [Raisay, G., Nature Biotechnology, 16: 4048 (1999); and Chee et al., U.S. Pat.","No.","5,837,832]; and ligase chain reaction [Whiteley et al., U.S. Pat.","No.","5,521,065].","[See generally, Schafer and Hawkins, Nature Biotechnology, 16: 33-39 (1998).]","All of the foregoing documents are hereby incorporated by reference in their entirety.","Thus, in one preferred embodiment involving screening nGPCR-40 or nGPCR-54 sequences, for example, the assaying step comprises at least one procedure selected from the group consisting of: (a) determining a nucleotide sequence of at least one codon of at least one nGPCR-40 or nGPCR-54 allele of the human subject; (b) performing a hybridization assay to determine whether nucleic acid from the human subject has a nucleotide sequence identical to or different from one or more reference sequences; (c) performing a polynucleotide migration assay to determine whether nucleic acid from the human subject has a nucleotide sequence identical to or different from one or more reference sequences; and (d) performing a restriction endonuclease digestion to determine whether nucleic acid from the human subject has a nucleotide sequence identical to or different from one or more reference sequences.","In a highly preferred embodiment, the assaying involves sequencing of nucleic acid to determine nucleotide sequence thereof, using any available sequencing technique.","[See, e.g., Sanger et al., Proc.","Natl.","Acad.","Sci.","(USA), 74: 5463-5467 (1977) (dideoxy chain termination method); Mirzabekov, TIBTECH, 12: 27-32 (1994) (sequencing by hybridization); Drmanac et al., Nature Biotechnology, 16: 54-58 (1998); U.S. Pat.","No.","5,202,231; and Science, 260: 1649-1652 (1993) (sequencing by hybridization); Kieleczawa et al., Science, 258: 1787-1791 (1992) (sequencing by primer walking); (Douglas et al., Biotechniques, 14: 824828 (1993) (Direct sequencing of PCR products); and Akane et al., Biotechniques 16: 238-241 (1994); Maxam and Gilbert, Meth.","Enzymol., 65: 499-560 (1977) (chemical termination sequencing), all incorporated herein by reference.]","The analysis may entail sequencing of the entire nGPCR gene genomic DNA sequence, or portions thereof, or sequencing of the entire seven transmembrane receptor coding sequence or portions thereof.","In some circumstances, the analysis may involve a determination of whether an individual possesses a particular allelic variant, in which case sequencing of only a small portion of nucleic acid—enough to determine the sequence of a particular codon characterizing the allelic variant—is sufficient This approach is appropriate, for example, when assaying to determine whether one family member inherited the same allelic variant that has been previously characterized for another family member, or, more generally, whether a person's genome contains an allelic variant that has been previously characterized and correlated with a mental disorder having a heritable component.","In another highly preferred embodiment, the assaying step comprises performing a hybridization assay to determine whether nucleic acid from the human subject has a nucleotide sequence identical to or different from one or more reference sequences.","In a preferred embodiment, the hybridization involves a determination of whether nucleic acid derived from the human subject will hybridize with one or more oligonucleotides, wherein the oligonucleotides have nucleotide sequences that correspond identically to a portion of the GPCR gene sequence taught herein, such as the nGPCR-40 or nGPCR-54 coding sequence set forth in SEQ ID NOS: 83 for nGPCR-40 or 85 for nGPCR-54, or that correspond identically except for one mismatch.","The hybridization conditions are selected to differentiate between perfect sequence complementarity and imperfect matches differing by one or more bases.","Such hybridization experiments thereby can provide single nucleotide polymorphism sequence information about the nucleic acid from the human subject, by virtue of knowing the sequences of the oligonucleotides used in the experiments.","Several of the techniques outlined above involve an analysis wherein one performs a polynucleotide migration assay, e.g., on a polyacrylamide electrophoresis gel (or in a capillary electrophoresis system), under denaturing or non-denaturing conditions.","Nucleic acid derived from the human subject is subjected to gel electrophoresis, usually adjacent to (or co-loaded with) one or more reference nucleic acids, such as reference GPCR-encoding sequences having a coding sequence identical to all or a portion of SEQ ID NOS: 83 or 85 (or identical except for one known polymorphism).","The nucleic acid from the human subject and the reference sequence(s) are subjected to similar chemical or enzymatic treatments and then electrophoresed under conditions whereby the polynucleotides will show a differential migration pattern, unless they contain identical sequences.","[See generally Ausubel et al.","(eds.","), Current Protocols in Molecular Biology, New York: John Wiley & Sons, Inc. (1987-1999); and Sambrook et al., (eds.","), Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press (1989), both incorporated herein by reference in their entirety.]","In the context of assaying, the term “nucleic acid of a human subject” is intended to include nucleic acid obtained directly from the human subject (e.g., DNA or RNA obtained from a biological sample such as a blood, tissue, or other cell or fluid sample); and also nucleic acid derived from nucleic acid obtained directly from the human subject.","By way of non-limiting examples, well known procedures exist for creating cDNA that is complementary to RNA derived from a biological sample from a human subject, and for amplifying (e.g., via polymerase chain reaction (PCR)) DNA or RNA derived from a biological sample obtained from a human subject.","Any such derived polynucleotide which retains relevant nucleotide sequence information of the human subject's own DNA/RNA is intended to fall within the definition of “nucleic acid of a human subject” for the purposes of the present invention.","In the context of assaying, the term “mutation” includes addition, deletion, and/or substitution of one or more nucleotides in the GPCR gene sequence (e.g., as compared to the seven transmembrane receptor-encoding sequences set forth of SEQ ID NOS: 74, 186, 78, 80, 82, 84, 86, 90, and 94) and other polymorphisms that occur in introns (where introns exist) and that are identifiable via sequencing, restriction fragment length polymorphism, or other techniques.","The various activity examples provided herein permit determination of whether a mutation modulates activity of the relevant receptor in the presence or absence of various test substances.","In a related embodiment, the invention provides methods of screening a person's genotype with respect to GPCR's of the invention, and correlating such genotypes with diagnoses for disease or with predisposition for disease (for genetic counseling).","For example, the invention provides a method of screening for an nGPCR-40 or nGPCR-54 hereditary schizophrenia genotype in a human patient, comprising the steps of: (a) providing a biological sample comprising nucleic acid from the patient, the nucleic acid including sequences corresponding to said patients nGPCR-40 or nGPCR-54 alleles; (b) analyzing the nucleic acid for the presence of a mutation or mutations; (c) determining an nGPCR-40 or nGPCR-54 genotype from the analyzing step; and (d) correlating the presence of a mutation in an nGPCR-40 or nGPCR-54 allele with a hereditary schizophrenia genotype.","In a preferred embodiment, the biological sample is a cell sample containing human cells that contain genomic DNA of the human subject.","The analyzing can be performed analogously to the assaying described in preceding paragraphs.","For example, the analyzing comprises sequencing a portion of the nucleic acid (e.g., DNA or RNA), the portion comprising at least one codon of the nGPCR-40 or nGPCR-54 alleles.","Although more time consuming and expensive than methods involving nucleic acid analysis, the invention also may be practiced by assaying protein of a human subject to determine the presence or absence of an amino acid sequence variation in GPCR protein from the human subject.","Such protein analyses may be performed, e.g., by fragmenting GPCR protein via chemical or enzymatic methods and sequencing the resultant peptides; or by Western analyses using an antibody having specificity for a particular allelic variant of the GPCR.","The invention also provides materials that are useful for performing methods of the invention.","For example, the present invention provides oligonucleotides useful as probes in the many analyzing techniques described above.","In general, such oligonucleotide probes comprise 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides that have a sequence that is identical, or exactly complementary, to a portion of a human GPCR gene sequence taught herein (or allelic variant thereof), or that is identical or exactly complementary except for one nucleotide substitution.","In a preferred embodiment, the oligonucleotides have a sequence that corresponds in the foregoing manner to a human GPCR coding sequence taught herein, and in particular, the coding sequences set forth in SEQ ID NO: 83 and 85.In one variation, an oligonucleotide probe of the invention is purified and isolated.","In another variation, the oligonucleotide probe is labeled, e.g., with a radioisotope, chromophore, or fluorophore.","In yet another variation, the probe is covalently attached to a solid support.","[See generally Ausubel et al.","And Sambrook et al., supra.]","In a related embodiment, the invention provides kits comprising reagents that are useful for practicing methods of the invention.","For example, the invention provides a kit for screening a human subject to diagnose schizophrenia or a genetic predisposition therefor, comprising, in association: (a) an oligonucleotide useful as a probe for identifying polymorphisms in a human nGPCR-40 or nGPCR-54 seven transmembrane receptor gene, the oligonucleotide comprising 6-50 nucleotides that have a sequence that is identical or exactly complementary to a portion of a human nGPCR-40 or nGPCR-54 gene sequence or nGPCR-40 or nGPCR-54 coding sequence, except for one sequence difference selected from the group consisting of a nucleotide addition, a nucleotide deletion, or nucleotide substitution; and (b) a media packaged with the oligonucleotide containing information identifying polymorphisms identifiable with the probe that correlate with schizophrenia or a genetic predisposition therefor.","Exemplary information-containing media include printed paper package inserts or packaging labels; and magnetic and optical storage media that are readable by computers or machines used by practitioners who perform genetic screening and counseling services.","The practitioner uses the information provided in the media to correlate the results of the analysis with the oligonucleotide with a diagnosis.","In a preferred variation, the oligonucleotide is labeled.","In still another embodiment, the invention provides methods of identifying those allelic variants of GPCRs of the invention that correlate with mental disorders.","For example, the invention provides a method of identifying a seven transmembrane allelic variant that correlates with a mental disorder, comprising steps of: (a) providing a biological sample comprising nucleic acid from a human patient diagnosed with a mental disorder, or from the patient's genetic progenitors or progeny; (b) analyzing the nucleic acid for the presence of a mutation or mutations in at least one seven transmembrane receptor that is expressed in the brain, wherein the at least one seven transmembrane receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 74, 186, 78, 80, 82, 84, 86, 90, and 94 or an allelic variant thereof, and wherein the nucleic acid includes sequence corresponding to the gene or genes encoding the at least one seven transmembrane receptor, (c) determining a genotype for the patient for the at least one seven transmembrane receptor from said analyzing step; and (d) identifying an allelic variant that correlates with the mental disorder from the determining step.","To expedite this process, it may be desirable to perform linkage studies in the patients (and possibly their families) to correlate chromosomal markers with disease states.","The chromosomal localization data provided herein facilitates identifying an involved GPCR with a chromosomal marker.","The foregoing method can be performed to correlate GPCR's of the invention to a number of disorders having hereditary components that are causative or that predispose persons to the disorder.","For example, in one preferred variation, the disorder is schizophrenia, and the at least one seven transmembrane receptor comprises nGPCR-40 having an amino acid sequence set forth in SEQ ID NO: 84 or an allelic variant thereof.","Also contemplated as part of the invention are polynucleotides that comprise the allelic variant sequences identified by such methods, and polypeptides encoded by the allelic variant sequences, and oligonucleotide and oligopeptide fragments thereof that embody the mutations that have been identified.","Such materials are useful in in vitro cell-free and cell-based assays for identifying lead compounds and therapeutics for treatment of the disorders.","For example, the variants are used in activity assays, binding assays, and assays to screen for activity modulators described herein.","In one preferred embodiment, the invention provides a purified and isolated polynucleotide comprising a nucleotide sequence encoding a nGPCR-40 or nGPCR-54 receptor allelic variant identified according to the methods described above; and an oligonucleotide that comprises the sequences that differentiate the allelic variant from the nGPCR-40 or nGPCR-54 sequences set forth in SEQ ID NOS: 83 and 88.The invention also provides a vector comprising the polynucleotide (preferably an expression vector); and a host cell transformed or transfected with the polynucleotide or vector.","The invention also provides an isolated cell line that is expressing the allelic variant GPCR polypeptide; purified cell membranes from such cells; purified polypeptide; and synthetic peptides that embody the allelic variation amino acid sequence.","In one particular embodiment, the invention provides a purified polynucleotide comprising a nucleotide sequence encoding a nGPCR-40 seven transmembrane receptor protein of a human that is affected with schizophrenia; wherein said polynucleotide hybridizes to the complement of SEQ ID NO: 83 under the following hybridization conditions: (a) hybridization for 16 hours at 42° C. in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% dextran sulfate and (b) washing 2 times for 30 minutes at 60° C. in a wash solution comprising 0.1×SSC and 1% SDS; and wherein the polynucleotide encodes a nGPCR-40 amino acid sequence that differs from SEQ ID NO: 84 by at least one residue.","An exemplary assay for using the allelic variants is a method for identifying a modulator of nGPCR-x biological activity, comprising the steps of: (a) contacting a cell expressing the allelic variant in the presence and in the absence of a putative modulator compound; (b) measuring nGPCR-x biological activity in the cell; and (c) identifying a putative modulator compound in view of decreased or increased nGPCR-x biological activity in the presence versus absence of the putative modulator.","Additional features of the invention will be apparent from the following Examples.","Examples 1, 2, 4, 11, 12, and 13 are actual, while the remaining Examples are prophetic.","Additional features and variations of the invention will be apparent to those skilled in the art from the entirety of this application, including the detailed description, and all such features are intended as aspects of the invention.","Likewise, features of the invention described herein can be re-combined into additional embodiments that also are intended as aspects of the invention, irrespective of whether the combination of features is specifically mentioned above as an aspect or embodiment of the invention.","Also, only such limitations which are described herein as critical to the invention should be viewed as such; variations of the invention lacking limitations which have not been described herein as critical are intended as aspects of the invention.","EXAMPLES Example 1 Identification of nGPCR-X A.","Database Search The Celera database was searched using known GPCR receptors as query sequences to find patterns suggestive of novel G protein-coupled receptors.","Positive hits were further analyzed with the GCG program BLAST to determine which ones were the most likely candidates to encode G protein-coupled receptors, using the standard (default) alignment produced by BLAST as a guide.","Briefly, the BLAST algorithm, which stands for Basic Local Alignment Search Tool is suitable for determining sequence similarity (Altschul et al., J. Molec.","Biol., 1990, 215, 403-410, which is incorporated herein by reference in its entirety).","Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).","This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence.","T is referred to as the neighborhood word score threshold (Altschul et al., supra).","These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing them.","The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased.","Extension for the word hits in each direction are halted when: 1) the cumulative alignment score falls off by the quantity X from its maximum achieved value; 2) the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or 3) the end of either sequence is reached.","The Blast algorithm parameters W, T and X determine the sensitivity and speed of the alignment.","The Blast program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff et al., Proc.","Natl.","Acad.","Sci.","USA, 1992, 89, 10915-10919, which is incorporated herein by reference in its entirety) alignments (13) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.","The BLAST algorithm (Karlin et al., Proc.","Natl.","Acad.","Sci.","USA, 1993, 90, 5873-5787, which is incorporated herein by reference in its entirety) and Gapped BLAST perform a statistical analysis of the similarity between two sequences.","One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.","For example, a nucleic acid is considered similar to a GPCR gene or cDNA if the smallest sum probability in comparison of the test nucleic acid to a GPCR nucleic acid is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.Homology searches were performed with the program BLAST version 2.08.A collection of 340 query amino acid sequences derived from GPCR's was used to search the genomic DNA sequence using TBLASTN and alignments with an E-value lower than 0.01 were collected from each BLAST search.","The amino acid sequences have been edited to remove regions in the sequence that produce non-significant alignments with proteins that are not related to GPCR's.","Multiple query sequences may have a significant alignment to the same genomic region, although each alignment may not cover exactly the same DNA region.","A procedure is used to determine the region of maximum common overlap between the alignments from several query sequences.","This region is called the consensus DNA region.","The procedure for determining this consensus involves the automatic parsing of the BLAST output files using the program MSPcrunch to produce a tabular report.","From this tabular report the start and end of each alignment in the genomic DNA is extracted.","This information was used by a PERL script to derive the maximum common overlap.","These regions were reported in the form of a unique sequence identifier, a start and the end position in the sequence.","The sequences defined by these regions were extracted from the original genomic sequence file using the program fetchdb.","The consensus regions were assembled into a non-redundant set by using the program phrap.","After assembly with phrap a set of contigs and singletons was defined as candidate DNA regions coding for nGPCR-x.","These sequences were then submitted for further sequence analysis.","Further sequence analysis involved the removal of sequences previously isolated and removal of sequences related to olfactory GPCRs.","The transmembrane regions for the sequences that remained were determined using a FORTRAN computer program called “tmtrest.all” [Parodi et al., Comput.","Appl.","Biosci.","5:527-535(1994)].","Only sequences that contained transmembrane regions in a pattern found in GPCRs were retained.","cDNAs were sequenced directly using an ABI377 fluorescence-based sequencer (Perkin-Elmer/Applied Biosystems Division, PE/ABD, Foster City, Calif.) and the ABI PRISM™ Ready Dye-Deoxy Terminator kit with Taq FS™ polymerase.","Each ABI cycle sequencing reaction contained about 0.5 μg of plasmid DNA.","Cycle-sequencing was performed using an initial denaturation at 98° C. for 1 minute, followed by 50 cycles using the following parameters: 98° C. for 30 seconds, annealing at 50° C. for 30 seconds, and extension at 60° C. for 4 minutes.","Temperature cycles and times were controlled by a Perkin-Elmer 9600 thermocycler.","Extension products were purified using Centriflex™ gel filtration cartridges (Advanced Genetic Technologies Corp., Gaithersburg, Md.).","Each reaction product was loaded by pipette onto the column, which is then centrifuged in a swinging bucket centrifuge (Sorvall model RT6000B tabletop centrifuge) at 1500×g for 4 minutes at room temperature.","Column-purified samples were dried under vacuum for about 40 minutes and then dissolved in 5 μl of a DNA loading solution (83% deionized formamide, 8.3 mM EDTA, and 1.6 mg/ml Blue Dextran).","The samples were then heated to 90° C. for three minutes and loaded into the gel sample wells for sequence analysis using the ABI377 sequencer.","Sequence analysis was performed by importing ABI377 files into the Sequencer program (Gene Codes, Ann Arbor, Mich.).","Generally, sequence reads of 700 bp were obtained.","Potential sequencing errors were minimized by obtaining sequence information from both DNA strands and by re-sequencing difficult areas using primers annealing at different locations until all sequencing ambiguities were removed.","The following Table 5 contains the sequences of the polynucleotides and polypeptides of the invention.","Start and stop codons within the polynucleotide sequence are identified by boldface type.","The transmembrane domains within the polypeptide sequence are identified by underlining.","TABLE 5 The following DNA sequence beGPCR-seq1 <SEQ ID NO.","1> was identified in H. sapiens: GTCTGGGGGTGGGGGATGCTGGGACAGGGGTCAATTGCCTGAAGCAAGTGCTCTCATCCCCCTAGCTCCTGC TGATCTAGTTGGGGCTCCAGAGTGGGGAGGAGAAAGGCACTTTGAAACTTCTCTGCCCTTACCGTCTTAGCC ATCAAACTCTGAGCTGGAGATAGTGACGATGTGACAGGAACTTTCCCTGGGCCTCTCTGGGCCACAATTCCT GGCCGAGAGAAAGAGGAGGAATGAGGTGAGCACCTTCTTCACTCCTAGGGCCATGTGGTAGAGCTGCAGTCG CACCTCCTTCTGCCAATAGGCATAGATGAGTGGGTTGAGCAGGGAGTTGCCCACGCCGAGCAGCCACAGGTA CCGTTCCAGCACTAGGTAGAGGTGACACTCCTGGCAGGCCACCTGCACAATGCCAGTGATAAGGAAGGGGGT CCAGGATAGAGCAAAGCTCCCAATGAGAACAGACACAGTACGGAGAGCTTTGAAGTCGCTGGGAGTCCGTGG GGATCGATAACCTCCAGCCATGGCTCCTGCATGTTCCATCTTTCGAATCTGCTGGCTGTGCATGGAGGCAAT TTGAGCATGTCGCAGTAGAAGAAGACAAAGAGGAGCATGGCTGGGAAGAAGCCAACGCAGGAGAGGGTCAGC ACGAAGTGAGGGTGAAATACAGCAAAGAAGCTGCACTGCCCTTTGTAGGCAGTCTGCTGGAACATGGGGATT CCGAGTGGGAGGAAGCCAATGAGGTAAGACACTAACCACAGCCCGGCAATGCAGGCCCCGGCCACGAACCCA CTCATGATCTTCAAGTAGCGGAAGGGCTGCTTGATGGCAAGGTACCTGTCAAAGGTGATCAGCATGACCGTG AGGACAGAGGCAGCTGCGGAGGAAGTGACAAATGCCATCCGCAGGCTGCACAGGGTCTTCTGTGTGGGCCGA GAAGGGCTGGAGAGCTGGTCTGTGAGTAGGCCAGAGATGGCCACACCAATCAAGGTGTCAGCCACAGCCAGA TTCAAGGTGAAGCAGAGACTGACACCATCATTCTTGTGGATCAACAGCAGCACAGCCACAGCCACTAGTGTG TTAGTAGCAATGATGAGGGAGGCCAGGACAGCAAGGATCACTCCAAATGAGAAAGATGATTCCATGTCTCGA AGTGGCAGGACTTCACTTACCAGGGCATG The following amino acid sequence <SEQ ID NO.","2> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","1: MESSFSFGVILAVLASLIIATNTLVAVAVLLLIHKNDGVSLCFTLNLAVADTLIGVAISGLLTDQLSSPSRPT QKTLCSLRMAFVTSSAAASVLTVMLITFDRYLAIKQPFRYLKIMSGFVAGACIAGLWLVSYLIGFLPLGIPMF QQTAYKGQCSFFAVFHPHFVLTLSCVGFFPAMLLFVFFYCDMLKIASMHSQQIRKMEHAGAMAGGYRSPRTPS DFKALRTVSVLIGSFALSWTPFLITGIVQVACQECHLYLVLERYLWLLGVGNSLLNPLIYAYWQKEVRLQLYH MALGVKKVLTSFLLFLSARNCGPERPRESSCHIVTISSSEFDG The following DNA sequence beGPCR-seq3 <SEQ ID NO.","3> was identified in H. sapiens: CAGCGCGAGCGCCTTCATGGTGACGGTGTCCATGCGCTGGCAGTGTCTGCGTGCCACCCGGTGCACCTGGAG CGAGGTGAGGCAGAGCACCGCCAGCGGCAGCACGAAGCCCACGGCATGGAGCGTGGCGGTGAAGGCTGCGAA GCGCGGACGCTCAGGCTCGGGCGGCAGGCGCAGCGAACAGGACGCGAAGGCGCTGCTGTAGCCAAGCCACGA GCAGCCAAGTGCAGCGCCTGAGAAGGCCAGCGACTGTCCCCAGGCACAGCCCAGCAGCAGGCCGGCATAGCG CGGTCGCAGGCGTCCGGCGTAGCGCAGTGGGAAGCCCACTGCCAGCCACTGGTCTGCGCTCAGCGCCGCCAC GCTCAGCGCCGCGTTGGACGCCAGGAAGGTGTCCAGGAAGCCAATGACTTGGCATGCGCCGGGCGCCGACGG TGTCCGCCCGCGCATCACACCGAGCAGCGTGAAGGGCATGTCCAGCGCCGCCAGCAGCAGGTGGCCCAGAGA CAGATTCACCAGGAGGACGCCTGAGGCTCGAGTGCGGAGCTCAGCGCTGTAGGCGCAACAAAGCAGCACCAG TGCGTTGGATAGCAGCGCCACGGCCAGTACCATCACCAGGAGACCCGCCAGCAGCGCCTCGCCGGGGCCCAT GGCGCTAGC The following amino acid sequence <SEQ ID NO.","4> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","3: SAMGPGEALLAGLLVMVLAVALLSNALVLLCCAYSAELRTRASGVLLVNLSLGHLLLAALDMPFTLLGVMRGR TPSAPGACQVIGFLDTFLASNAALSVAALSADQWLAVGFPLRYAGRLRPRYAGLLLGCAWGQSLAFSGAALGC SWLGYSSAFASCSLRLPPEPERPRFAAFTATLHAVGFVLPLAVLCLTSLQVHRVARRHCQRMDTVTMKALA The following DNA sequence beGPCR-seq4 <SEQ ID NO.","5> was identified in H. sapiens: TGTGCAGGTGTGATCTCCATTCCTTTGTACATCCCTCACACGCTGTTCGATGGGATTTTGGAAAGGAAATCT GTGTATTTTGGCTCACTACTGACTATCTGTTATGTACAGCATCTGTATATAACATTGTCCTCATCAGCTATG ATCGATACCTGTCAGTCTCAAATGCTGTAAGTCGAACACATTAATTTATCCCCCTTAGAAGATTATGTAAAT GTATA The following amino acid sequence <SEQ ID NO.","6> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","5: CAGVISIPLYIPHTLFEWDFGKEICVFWLTTDYLLCTASVYNIVLISYDRYLSVSNAVSRTHFIPLR RLCKCI The following DNA sequence beGPCR-seq5 <SEQ ID NO.","7> was identified in H. sapiens: GACGTCGAAGCAGGTGATGATGCCCAGGGCGTGCACCGGGTAGGTGAGATCGGTGCGCGCCAGCGGGGACAGG GCGGTCAGGAGCAGCAGCCAGGTCCCTGCACACGCGGCCACCGCGTAACGACGGCGGCGCCAGCGCTTGGAGC TGAGCGGGTACAGGATCCCCAGGAAGCGCTCCACGCTGATACAGGTCATGGTGAGGATGCTGGAATACATGTT TGCGTAAAAGGCCACGGTCACCACGTTGCAAAGCAGCACCCCGAATACCCAGTGGTGGCGGTTGCAATGGTAG TAGATTTGGAAAGGCAACACGCTGGCCAGCATCAGGTCCGTGACGCTCAGGTTGATCATGAAGATGACCGACG GGGATCTGGGCCCCATGCGCCGGCACAGCACCCACAGAGAGAAGAGGTTGCCCGGGATGCTGACCGCCGCCAC CAGCGAGTACACCACGGGCAGGGCCACCGCGATCGCCGGGTTCCGCAGCATCTGCAGCGTCGCGTTGTC The following amino acid sequence <SEQ ID NO.","8> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","7: DNATLQMLRNPAIAVALPVVYSLVAAVSIPGNLFSLWVLCRRMGPRSPSVIFMINLSVTDLMLASVLPFQIYY HCNRHHWVFGVLCNLVVTVAFYANMYSSILTMTCISVERFLGILYPLSSKRWRRRRYAVAACAGTWLLLLTAL SPLARTDLTYPVHALGIITCFDV The following DNA sequence beGPCR-seq9 <SEQ ID NO.","9> was identified in H. sapiens: CCCATGTTCCTGCTCCTGGGCAGCCTCACGTTGTCGGATCTGCTGGCAGGCGCCGCCTACGCCGCCAACAT CCTACTGTCGGGGCCGCTCACGCTGAAACTGTCCCCCGCGCTCTGGTTCGCACGGGAGGGAGGCGTCTTCG TGGCACTCACTGCGTCCGTGCTGAGCCTCCTGGGCATCGCGCTGGAGCGCAGCCTCACCATGGCGCGCAGG GGGCCCGCGCCCGTCTCCAGTCGGGGGCGCACGCTGGCGATGGCAGCCGCGGCCTGG The following amino acid sequence <SEQ ID NO.","10> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","9: PMFLLLGSLTLSDLLAGAAYAANILLSGPLTLKLSPALWFAREGGVFVALTASVLSLLGIALERSLTMARRGP APVSSRGRTLAMAAAAW The following DNA sequence beGPCR-seq11 <SEQ ID NO.","11> was identified in H. sapiens: CTGCTCATTGTGGCCTTTGTGCTGGGCGCACTAGGCAATGGGGTCGCCCTGTGTGGTTTCTGCTTCCACAT GAAGACCTGGAAGCCCAGCACTGTTTACCTTTTCAATTTGGCCGTGGCTGATTTCCTCCTTATGATCTGCC TGCCTTTTCGGACAGACTATTACCTCAGACGTAGACACTGGGCTTTTGGGGACATTCCCTGCCGAGTGGGG CTCTTCACGTTGGCCATGAACAGGGCCGGGAGCATCGTGTTCCTTACGGTGGTGGCTGCGGACAGGTATTT CAAAGTGGTCCACCCCCACCACGCGGTGAACACTATCTCCACCCGGGTGGCGGCTGGCATCGTCTGCACCC TGTGGGCCCTGGTCATCCTGGGAACAGTGTATCTTTTGCTGGAGAACCATCTCTGCGTGCAAGAGACGGCC GTCTCCTGTGAGAGCTTCATCATGGAGTCGGCCAATGGCTGGCATGACATCATGTTCCAGCTGGAGTTCTT TATGCCCCTCGGCATCATCTTATTTTGCTCCTTCAAGATTGTTTGGAGCCTGAGGCGGAGGCAGCAGCTGG CCAGACAGGCTCGGATGAAGAAGGCGACCCGGTTCATCATGGTGGTGGCAATTGTGTTCATCACATGCTAC CTGCCCAGCGTGTCTGCTAGACTCTATTTCCTCTGGACGGTGCCCTCGAGTGCCTGCGATCCCTCTGTCCA TGGGGCCCTGCACATAACCCTCAGCTTCACCTACATGAACAGCATGCTGGATCCCCTGGTGTATTATTTTT CAAGCCCCTCCTTTCCCAAATTCTACAACAAGCTCAAAATCTGCAGTCTGAAACCCAAGCAGCCAGGACAC TCAAAAACACAAAGGCCGGAAGAGATGCCAATTTCG The following amino acid sequence <SEQ ID NO.","12> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","11: LLIVAFVLGALGNGVALCGFCFHMKTWKPSTVYLFNLAVADFLLMICLPFRTDYYLRRRHWAFGDIPCRVGLF TLAMNRAGSIVFLTVVAADRYFKVVHPHHAVNTISTRVAAGIVCTLWALVILGTVYLLLENHLCVQETAVSCE SFIMESANGWHDIMFQLEFFMPLGIILFCSFKIVWSLRRRQQLARQARMKKATRFIMVVAIVFITCYLPSVSA RLYFLWTVPSSACDPSVHGALHITLSFTYMNSMLDPLVYYFSSPSFPKFYNKLKICSLKPKQPGHSKTQRPEE MPIS The following DNA sequence beGPCR-seq12 <SEQ ID NO.","13> was identified in H. sapiens: TGGAGCTGTGCCACCACCTATCTGGTGAACCTGATGGTGGCCGACCTGCTTTATGTGCTATTGCCCTTCCT CATCATCACCTACTCACTAGATGACAGGTGGCCCTTCGGGGAGCTGCTCTGCAAGCTGGTGCACTTCCTGT TCTATATCAACCTTTACGGCAGCATCCTGCTGCTGACCTGCATCTCTGTGCACCAGTTCCTAGGTGTGTGC CACCCACTGTGTTCGCTGCCCTACCGGACCCGCAGGCATGCCTGGCTGGGCACCAGCACCACCTGGGCCCT GGTGGTCCTCCAGCTGCTGCCCACACTGGCCTTCTCCCACACGGACTACATCAATGGCCAGATGATCTGGT ATGACATGACCAGCCAAGAGAATTTTGATCGGCTTTTTGCCTACGGCATAGTTCTGACATTGTCTGGCTTT CTTTCCCTCCTTGGTCATTTTGGTGTGCTATTCACTGATGGTCAGGAGCCTGATCAAGCCAGAGGAGAACC TCATGAGGACAGG The following amino acid sequence <SEQ ID NO.","14> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","13: WSCATTYLVNLMVADLLYVLLPFLIITYSLDDRWPFGELLCKLVHFLFYINLYGSILLLTCISVHQFLGVCHP LCSLPYRTRRHAWLGTSTTWALVVLQLLPTLAFSHTDYINGQMIWYDMTSQENFDRLFAYGIVLTLSGFLSLL GHFGVLFTDGQEPDQARGEPHEDR The following DNA sequence beGPCR-seq14 <SEQ ID NO.","15> was identified in H. sapiens: CCACCACGCGCAGCACGCCGACAGGGCCTCTCCCTCCCATTCTCCCGCAGGCCCGGACGACCACGCTGCCT CCAGCCGGTCGGCAAACTAGGGCAGCTCGCAGCCCACGAACAGCAGCCCCAGCAGCTGGCTCATCTTCAGG CTCTGCACCTTGGCGCGGGGCATCGCGCTGGGCGCACGGGCTCCACCTGGGCTCGCCGACCAGGCCGCTGC ACCCGCTGGGGCCTTCAGCCGGTGCCGCCACCAGACGGAGAGTAGGTGGCCACAAGCGACACCCATGATCT TAACAGGCGCGACGAAGCCCGCGACGGCCTCATAGAACGCGTACACCTGCACGTGCCAGCGCTGCAGGAGC GCGAAGATCCAGTGGCAGCGACGCATCCCCGGCCAGGCTCGGGCGGAGAGTGGCGCGCCTGGCTGCAGAGA CGTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAGTACTAGCGCACCACAAACCCCGACCCCCGCGCCA GCAGCAGTGCCAGCAGCCAGCCCAGGGCGGCGAGGGCACGCGCGGGCAGCGGCCGGCCGTGCGGAAGACGC ACCGCGCGCCGGCGCTCGAGGGCGATGAGCACCACGAGGTGGGCCGAGGCGCCCCGCCCGGATGCCTGCAG CAGCTGCAGGAAGCGGCACGCCAGGTCCCCCGTGGCCGCGCGGGGCTCGCCCAGCAGTTCCCAGGCCAGCT GTGACAGCGCCGTGCCCCCGCACGCGTACAGGTCCGCCAGGGCCAGCTGCACCAGCAGGAAGTCCATCTTG CGACGCTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNACAGGCGGCACAGCACTGTGGTGTTGCCTGCCAC CGCCACCACCAGGATGACCCCCAGGAACACCAGGCGGACGCG The following amino acid sequence <SEQ ID NO.","16> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","15: RVRLVFLGVILVVAVAGNTTVLCRLXXXXXXXXXXKRRKMDFLLVQLALADLYACGGTALSQLAWELLGEPRA ATGDLACRFLQLLQASGRGASAHLVVLIALERRRAVRLPHGRPLPARALAALGWLLALLLARGSGFVVRYXXX XXXXXXXXTSLQPGAPLSARAWPGMRRCHWIFALLQRWHVQVYAFYEAVAGFVAPVKIMGVACGHLLSVWWRH RLKAPAGAAAWSASPGGARAPSAMPRAKVQSLKMSQLLGLLFVGCELPFADRLEAAWSSGPAGEWEGEALSAC CAWW The following DNA sequence beGPCR-seq15 <SEQ ID NO.","17> was identified in H. sapiens: TCTAAGTTTTTCTCTGAACTTTGAGCCTGTGAAAAAAGAAGGGATGCTGCCTCAGGCCACCCCAGCCTAGA TACTCACTCTGAGTGCCATGAGGTAGTAGAGGACACTGATGACAGTCATGGGGAGGAGGTAGAATAGGAAG GAGGTGACCTGGATGATGAAATTGTAGATCCACATGGGCTTGATGACCGTACAGGTGGCCGAACCTGGGAC CAGGGACCCATTGGGGAAGTAGTGGAACTTGATGCCATGGATGCTGGTGTTGGGCAGGGAGAAGAGCACGG AGAAGCCCCAGACGATGCCGAGGATCCTGAGGGCCCGGCGCCGGGTGCTCTGCAGTTTGGCGCGGAACGGG TGTAGGATGGCCACGTAGCGCTCCACGCTGACGGTGGTGATGCTGAGGATGGAGGCGAAGCACACGGTCTC AAAGAGGGCCGTCTTGAAGTAGCAGCCCACGGGCCCGAACAAGAAAGGGTAGTTGCGCCACATCTCATAGA CCTCCAGGGGCATTCCAAGGAGCAGGACCAGGAGGTCAGAGACCGCCAGGCTGAAGAGGTAGTAGTTGGTG GGCGTCTTCATAGCCTGGTGCTGCAGAATCACCAGGCACACCAGGACATTGCCAATGACCCCCACCACAAA AATTGGCACATACACCACAGACACGGGGAGGAAGAAGTGGCTGCGCCGAGGTCCGCAGAGGAAGGCCAGAT ACTCCTCGGTGCTGTTCAGGTGTTTCTGGAATGGATCTTCTAGTTTCTGCTGGTAGATCCAGGAAGCATTC TGAAGTTTTTCCATCCCTGA The following amino acid sequence <SEQ ID NO.","18> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","17: SGMEKLQNASWIYQQKLEDPFQKHLNSTEEYLAFLCGPRRSHFFLPVSVVYVPIFVVGVIGNVLVCLVILQHQ AMKTPNTYYLFSLAVSDLLVLLLGMPLEVYEMWRNYPFLFGPVGCYFKTALFETVCFASILSITTVSVERYVA ILHPFRAKLQSTRRRALRILGIVWGFSVLFSLPNTSIHGIKFHYFPNGSLVPGSATCTVIKPMWIYNFIIQVT SFLFYLLPMTVISVLYYLMALRVSIAGVAG The following DNA sequence beGPCR-seq18 <SEQ ID NO.","19> was identified in H. sapiens: ATCAAGATGATTTTTGCTATCGTGCAAATTATTGGATTTTCCAACTCCATCTGTAATCCCATTGTCTATGC ATTTATGAATGAAAACTTCAAAAAAAATGTTTTGTCTGCAGTTTGTTATTGCATAGTAAATAAAACCTTCT CTCCAGCACAAAGGCATGGAAATTCAGGAATTACAATGATGCGGAAGAAAGCAAAGTTTTCCCTCAGAGAG AATCCAGTG The following amino acid sequence <SEQ ID NO.","20> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","19: IKMIFAIVQIIGFSNSICNPIVYAFMNENFKKNVLSAVCYCIVNKTFSPAQRHGNSGITMMRKKAKFSLRENP The following DNA sequence beGPCR-seq16 <SEQ ID NO.","21> was identified in H. sapiens: GCCACAGCATGCAGTTTTCTGTAGAATTCCACTTTGTCTTTGCACTTGAAGAAGATGAGGTATCTGGTGAC CAGGATCACCACATAGAATAGGAACCGTGAGGTACATGTGGATGTGCAGCATGGCACTCACAAATTTGCAG AAGGGCAGCCCAAACATCCAAGTCTTCTTGATGAGGTAGGTCAAGCGAAATGGCACTGTCAGCAGAAAAAC GCTGTGGACCACCACCAAGTTAATGACCGCCATGGTGGTCACTGACCGGGTGTTCATTTTCACCAGGAGGA AAAGAATGGAAATGACACCCACCAGCCCGCCAATAAGCACTATGAAGTAGAGGCTGATTAAGTGGGGTGTC ACTATAGGATCGCAAGAGGAATTCCTGGAGGTATTGTGGCCAGGCATACTTGGGAAGTCACCTGGAGGAGA AAAAGCACCAGAGTAACTGAC The following amino acid sequence <SEQ ID NO.","22> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","21: VSYSGAFSPPGDFPSMPGHNTSRNSSCDPIVTPHLISLYFIVLIGGLVGVISILFLLVKMNTRSVTTMAVINL VVVHSVFLLTVPFRLTYLIKKTWMFGLPFCKFVSAMLHIHMYLTVPILCGDPGHQIPHLLQVQRQSGILQKTA CCG The following DNA sequence beGPCR-seq17 <SEQ ID NO.","23> was identified in H. sapiens: ACTGACCAAGGTCAGGGCATCGACTGAGGCTAGAAGGCCACAGGAAATGCCAGTCAAGGTGTTGGCGCCTG CAATCGCACCTACCACAAACTTGACCGGGGGCAGGGGGGCAGGCCCGCCAGCGAACACGGTCAGCAGCACC AGTCCATTGCAGAGCACGGAGAGCAACACGATGGCCCACACGGCCAGGCGGATGCCCCAGCTTTCAAAGAG GTACTCACA The following amino acid sequence <SEQ ID NO.","24> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","23: CEYLFESWGIRLAVWAIVLLSVLCNGLVLLTVFAGGPAPLPPVKFVVGAIAGANTLTGISCGLLASVDALTLV S The following DNA sequence beGPCR-seq20 <SEQ ID NO.","25> was identified in H. sapiens: AACCCCATCATCTACACGCTCACCAACCGCGACCTGCGCCACGCGCTCCTGCGCCTGGTCTGCTGCGGACG CCACTCCTGCGGCAGAGACCCGAGTGGCTCCCAGCAGTCGGCGAGCGCGGCTGAGGCTTCCGGGGGCCTGC GCCGCTGCCTGCCCCCGGGCCTTGATGGGAGCTTCAGCGGCTCGGAGCGCTCATCGCCCCAGCGCGACGGG CTGGACACCAGCGGCTCCACAGGCAGCCCCGGT The following amino acid sequence <SEQ ID NO.","26> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","25: NPIIYTLTNRDLRHALLRLVCCGRHSCGRDPSGSQQSASAAEASGGLRRCLPPGLDGSFSGSERSSPQRDGLD TSGSTGSPG The following DNA sequence beGPCR-seq21 <SEQ ID NO.","27> was identified in H. sapiens: CGTGAAGAACAGCGCCACCATGACCAGCATGTGCACCACGCGCGCTCTGCGCCGCGATGCTCGCGGGTCCG CAGCCTCCTNNNNNNNNNNNNNNNNNNNNNNNNNNTGGCAGAGCTTGCGCGCGATGCGGGCGTACATGACC ACGATGAGCGCCAGCGGCGCCAGGTAGATGTGCGAGAAGAGCACAGTGGTGTAGACCCTGCGCATGCCCTT CTCGGGCCAGGCCTCCCAGCAGGAGTAGAGAGGGTAGGAGCGGTTGCGGGCGTCCACCATGAAGTGGTGCT CCTCACGGGTGACGGTCAGCGTGACGGCCGAGGGACACATGATGAGCAGCGCCAGGGCCCAGATGACGGCG ATGGTGACGAGCGCCTTCCGCAGGGTCAGCTTCTCGCGGAAAGGGTGCACGATGCAGCGGAACCT The following amino acid sequence <SEQ ID NO.","28> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","27: FRCIVHPFREKLTLRKALVTIAVIWALALLIMCPSAVTLTVTREEHHFMVDARNRSYPLYSCWEAWPEKGM RRVYTTVLFSHIYLAPLALIVVMYARIARKLCXXXXXXXXXXEAADPRASRRRARVVHMLVMVALFFT The following DNA sequence beGPCR-seq22 <SEQ ID NO.","29> was identified in H. sapiens: GCAGGGGGCGTGAGTCCTCAGGCACTTCTTGAGGTCCTTGTTGAGCAGGAAGCAGACAATTGGGTTGACGG CAGCCTGGGCGAAGCTCATCCAAACAGCATGGCCAGGTAGCGGTGGGGCACAGCACAGGCTTTCACAAACA CTCGCCAGTAGCAGGCCACGATGTAGGGTGACCAGAGGAGCAGAAAGAGCAGTGTGATCGCGTAGAACATG CGGCCCAGCTGCTTTTCACCCTTGACCTCGTCCATGCCCAGTAGCCGCCGGCTGGCTGCATGCCCATTCTG CCGGATACCCAGCAGGGTTGGTGGCATGGGCCC The following amino acid sequence <SEQ ID NO.","30> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","29: GPMPPTLLGIRQNGHASRRLLGMDEVKGEKQLGRMFYAITLLFLLLWSPYIVACYWRVFVKACAVPHRYLAT AVWMSFAQAAVNPIVCFLLNKDLKKCLRTHAPC The following DNA sequence beGPCR-seq24 <SEQ ID NO.","31> was identified in H. sapiens: TATTCTGTAATGAAGAATGTCATTCACACTGCCATTGGCACATCCAGTGGCCTCACCTAGCATTGTGAAAG CCCTTCGGTTGGTGTATTGCCACTTCATTTTAAAAGGATGCACAAGTCCCTGGTGCCTTTCCACAGCAATG CAGGTCATAGTGAGGATTTCTGTCACAACAGCGGTAGACTGGACAAATGGCACCATCTTGCAAATGAAAGC ACCTGCAGTAAGGAAATAGGATAAATCATACATCAAAACAAAAAGAATAAAGGTTTCATCTGTGTCTTTGT AATTATCACTATCAGTCCATTCTGAGCCTCTGCCAAAAAGTTTGATAATTGTAATTACTCTGTAGACACA The following amino acid sequence <SEQ ID NO.","32> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","31: VYRVITIIKLFGRGSEWTDSDNYKDTDETFILFVLMYDLSYFLTAGAFICKMVPFVQSTAVVTEILTMTCIAV ERHQGLVHPFKMKWQYTNRRAFTMLGEATGCANGSVNDILHYRI The following DNA sequence beGPCR-seq27 <SEQ ID NO.","33> was identified in H. sapiens: GAGCAACATGATCTTTTTGAAGTACTTGACGGTGTCGTTCTTGACGGTCACGAAGCACAGAGTGTTGATCA TGCTGTTGCTCATGGCGATGCACTCGACGATGTAGAAGGCAGTGAGGTAGTGCTTCTCCTTCACAAACACG GTGGGGAAGAAGTCGCGCACGATGGTGAAGCCGTAGAAGGGCGCCCAGCATAGCACGTAGGCGGTGAGGAT GCACATGAGCACCAGGACCGTCTTCCTGCGGCAGCGCAGCCTCTTGCGGATCTGCTCTGTCTGGAATCCAG GGACCGCCTTGAACCAGAGCTCCCGGGAGATCCTGGCATAGCACAGGGTCATGGTGACCACGGGGCCCACG AATTCTATGCCAAAGATAAAGAGGAAGTAGGACTTGTAGTAGAGCTGCTGGTCCACAGGCCAGATCTGGCC GCAGAAGATCTTTTCCTGGCTCTTGACAATGACGAGGACCGTCTCGGTGGTGAAGTAGGCGGAAGGGATGG CGATCAGGATGGACACCGTCCACACCAAGGCAATCAGGCCAGTGGCTGTTTGGCACTTCATTCGTGGTCTC AGCGGATGGACAATAGCCAGATACCTAGGGCAAGAACACAAGTGGAGGCAGCC The following amino acid sequence <SEQ ID NO.","34> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","33: GCLHLCSCPRYLAIVHPLRPRMKCQTATGLIALVWTVSILIAIPSAYFTTETVLVIVKSQEKIFCGQIWPVDQ QLYYKSYFLFIFGIEFVGPVVTMTLCYARISRELWFKAVPGFQTEQIRKRLRCRRKTVLVLMCILTAYVLCWA PFYGFTIVRDFFPTVFVKEKHYLTAFYIVECIAMSNSMINTLCFVTVKNDTVKYFKKIMLL The following DNA sequence beGPCR-seq28 <SEQ ID NO.","35> was identified in H. sapiens: CAGCCACACTGCAGTGATGAAATCAAATGTCCAACACCAACCATAGTCACCATTACTAACTAAGAAGCCAC AAAACTTCCCTTCCAGGGTGTTCAGCAGCAGGGACAGGGCCCAGGGCAGGGCACACATGACAGTTGACAGG TTTCTTGGGCAGCAGCAGCAGTACCAGATAGGCCGCAGGACAGACAGGCAGCACTCAGTACTGATGGCACT CAGCATGCTCAGGCCTACAAGGTAGGCAAAGGTCATCACGCTGGTGAAGAAGCTAGGGAAATTGATGGAGA TGGAACAGAAGAAGTTACTGAGGTACACCAGGCAATTTATAATCTGGAAGCAGAGGAAGAGGAAGTCGGCC CCGGCCAGGCTGAGGACGTAGACAGAGAAGGCGTTCCTGCGCATGCGGAAGCCCAGGAGCCAGAGCACAAA CCCGTTTCCTACCAGCCCGACCAGGGCAATGAAAAGGATCAGGAAGACCGGGATCAG The following amino acid sequence <SEQ ID NO.","36> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","35: LIPVFLILFIALVGLVGNGFVLWLLGFRMRRNAFSVYVLSLAGADFLFLCFQIINCLVYLSNFFCSISINFPS FFTSVMTFAYLVGLSMLSAISTECCLSVLRPIWYCCCCPRNLSTVMCALPWALSLLLNTLEGKFCGFLVSNGD YGWCWTFDFITAVWL The following DNA sequence beGPCR-seq31 <SEQ ID NO.","37> was identified in H. sapiens: GAGAGTCTGATTCTGACTTACATCACATATGTAGGCCTGGGCATTTCTATTTGCAGCCTGATCCTTTGCTTGT CCGTTGAGGTCCTAGTCTGGAGCCAAGTGACAAAGACAGAGATCACCTATTTACGCCATGTGTGCATTGTTAA CATTGCAGCCACTTTGCTGATGGCAGATGTGTGGTTCATTGTGGCTTCCTTTCTTAGTGGCCCAATAACACAC CACAAGGGATGTGTGGCAGCCACATTTTTTGGTCATTTCTTTTACCTTTCTGTATTTTTCTGGATGCTTGCCA AGGCACTCCTTATCCTCTATGGAATCATGATTGTTTTC The following amino acid sequence <SEQ ID NO.","38> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","37: ESLILTYITYVGLGISICSLILCLSVEVLVWSQVTKTEITYLRHVCIVNIAATLLMADVWFIVASFLSGPITH HKGCVAATFFGHFFYLSVFFWMLAKALLILYGIMIVF The following DNA sequence beGPCR-seq32 <SEQ ID NO.","39> was identified in H. sapiens: TTGTGTGGCAGTAGAGAGATGTCAGGCTTCAGAGTCAACAAGAACTGGATTTCAAACTGGATTTGAGGACCCC CACCTTTGGTAAGTGACTTATTATCTGCGAGCCTCTGTTTCTCTCTTCTTTAAATGAGGACAGTAAATCCCAT ACGGCAGGGTGGTGGGGAGAATCAGAGATGATACAGCTGGTGATCACATCTGGTTTGTGTTCCCAGGGGCACC AGACTAGGGTTTCTGAGCATGGATCCAACCGTCCCAGTCTTCGGTACAAAACTGACACCAATCAACGGACGTG AGGAGACTCCTTGCTACAATCAGACCCTGAGCTTCACGGTGCTGACGTGCATCATTTCCCTTGTCGGACTGAC AGGAAACGCGGTAGTGCTCTGGCTCCTGGGCTACCGCATGCGCAGGAACGCTGTCTCCATCTACATCCTCAAC CTGGCCGCAGCAGACTTCCTCTTCCTCAGCTTCCAGATTATACGTTCGCCATTACGCCTCATCAATATCAGCC ATCTCATCCGCAAAATCCTCGTTTCTGTGATGACCTTTCCCTACTTTACAGGCCTGAGTATGCTGAGCGCCAT CAGCACCGAGCGCTGCCTGTCTGTTCTGTGGCCCATCTGGTACC The following amino acid sequence <SEQ ID NO.","40> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","39: LCGSREMSGFRVNKNWISNWIGPPPLVSDLLSASLCFSLLMRTVNPIRQGGGENQRYSWSHLVCVPRGTRLGF LSMDPTVPVFGTKLTPINGREETPCYNQTLSFTVLTCIISLVGLTGNAVVLWLLGYRMRRNAVSIYILNLAAA DFLFLSFQIIRSPLRLINISHLIRKILVSVMTFPYFTGLSMLSAISTERCLSVLWPIWY The following DNA sequence beGPCR-seq33 <SEQ ID NO.","41> was identified in H. sapiens: ACAGAAAGCAAGGCCACCAGGACCTTAGGCATAGTCATGGGAGTGTTTGTGTTGTGCTGGCTGCCCTTCTTTG TCTTGACGATCACAGATCCTTTCATTAATTTTACAACCCTTGAAGATCTGTACAATGTCTTCCTCTGGCTAGG CTATTTCAACTCTGCTTTCAATCCCATTTTATATGGCATGCTTTATCCTTGGTTTCGCAAGGCATTGAGGATG ATTGTCACAGGCATGATCTTCCACCCTGACTCTTCCACCCTAAGCCTGTTTTCTGCCCATGCTTAGGCTGTGT TCATCATTCAATAGGACTCTTTTCTGG The following amino acid sequence <SEQ ID NO.","42> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","41: TESKATRTLGIVMGVFVLCWLPFFVLTITDPFINFTTLEDLYNVFLWLGYFNSAFNPILYGMLYPWFRKALRM IVTGMIFHPDSSTLSLFSAHAAVFIIQDSF The following DNA sequence beGPCR-seq34 <SEQ ID NO.","43> was identified in H. sapiens: TAGGAATCTCAGAGAAGAAAGTAAGGAACCAGAAAACCATAAAAGAATGTAAATGGAAAAGAATCAGCAAATC TTATTCACTTATCACTAAATCTAAAATATGTCAAAATACATGAAGACAACAAATGCTTTAGAACAACTGTTGA ATGTATTGTCCTACAACTTGGCATATGATCATGCTTGCCTCTCTATGTCCAAGTGTTTATTTTTGCAGTTGAC CTTAATTTCAAGTTAGTTTTGAGGTCTCTACAGTAATGTTTTTAATCTGTCTCTACTTCTTCAGAAAATAAAT TAGTTGTTGACGAATCAGTCCTTAAGACCTTGCCGCTTACAATAAGTTTTATTGCCTTCCCAAACCATTGGTA AAAGAAAGCATAAATCAAGGGGTTCATAGCTGAATTATAATAAACACACCAAACTAAAATCTCATAAACATAA GGAGGAGTTATAAAATTCATATAAGCATCAATCACTGCATCAACGAGGTATGGTAGCCAAGAGACAAGAAATG CTGC The following amino acid sequence <SEQ ID NO.","44> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","43: LHQRGMVAKRQEMLAAFLVSWLPYLVDAVIDAYMNFITPPYVYEILVWCVYYNSAMNPLIYAFFYQWFGKAIK LIVSGKVLRTDSSTTNLFSEEVETDKHYCRDLKTNLKLRSTAKINTWTRGKHDHMPSCRTIHSTVVLKHLLSS CI The following DNA sequence beGPCR-seq35 <SEQ ID NO.","45> was identified in H. sapiens: CTGGAAAGAGGTCCTCGATCTATCCTCTACGCCGTCCTTGGTTTTGGGGCTGTGCTGGCAGCGTTTGGAAACT TACTGGTCATGATTGCTATCCTTCACTTCTAACAACTGCACACACCTACAAACTTTCTGATTGCGTCGCTGGC CTGTGCTGACTTCTTGGTGGGAGTCACTGTGATGCCCTTCAGCACAGTGAGGTCTGTGGAGAGCTGTTGGTAC TTTGGGGACAGTTACTGTAAATTCCATACATGTTTTGACACATCTTTCTGTTTTGCTTCTTTATTTCATTTAT GCTGTATCTCTGTTGATAGATACATTGCTGTTACTGATCCTCTGACCTATCCAACCAAGTTTACTGTGTCAGT TTCAGGGATATGCATTGTTCTTTCCTGGTTCTTTTCTGTCACATACAGCTTTTCGATCTTTTACACGGGAGCC AACGAAGAAGGAATTGAGGAATTAGTAGTTGCTCTAACCTGTGTAGGAGGCTGCCAGGCTCCACTGAATCAAA ACTGGGTCCTACTTTGTTTTCTTCTATTCTTTATACCCAATGTCGCCATGGTGTTTATATACAGTAAGATATT TTTGGTGGCCAAGCATCAGGCTAGGAAGATAGAAAGTACAGCCAGCCAAGCTCAGTCCTTCTCAGAGAGTTAC AAGGAAAGAGTAGCAAAAAGAGAGAGAAAGGCTGCCAAAACCTTGGGAATTGCTATGGCAGCATTTCTT The following amino acid sequence <SEQ ID NO.","46> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","45: LERGPRSILYAVLGFGAVLAAFGNLLVMIAILHFQLHTPTNFLIASLACADFLVGVTVMPFSTVRSVESCWYF GDSYCKFHTCFDTSFCFASLFHLCCISVDRYIAVTDPLTYPTKFTVSVSGICIVLSWFFSVTYSFSIFYTGAN EEGIEELVVALTCVGGCQAPLNQNWVLLCFLLFFIPNVAMVFIYSKIFLVAKHQARKIESTASQAQSFSESYK ERVAKRERKAAKTLGIAMAAFL The following DNA sequence beGPCR-seq36 <SEQ ID NO.","47> was identified in H. sapiens: AACCAGGTGGCCTTACTCCTAAGACCCCTGGCCTTGTCTATGGCCTTTATCAACAGCTGTCTCAATCCAGTTC TCTATGTCTTCATTGGGCATGACTTCTGGGAGCACTTGCTCCACTCCCTGCTAGCTGCCTTAGAACGGGCACT TAGCGAGGAGCCAGATAGTGCCTGAATCCCAGCTCCCAGGCAGATGAGTCCTTTATAACATGACCCAATTTCC TACTCCATTTTCCCACCACTCAATCCTCTTCCCAAACAGCTCTACCATAATCCAACATCCAACAGAATTTAAG AGAATAAACCACAACTTTTAAGTGAGCTCTATGTGCTAGGTCATGTTTTAGAATACAACCTTAAGTGCCTGGA AGATGGAGGCAAGAAACAAACAAGGTCTCATTCTTTAGAGGAAGACAGTTCACCAAGACTCAAACAGAAAAAA AGATAGTTATCTTGTGACAAAACAAGTCATAAAATTGGGTCAGGACCTGCAGCAATGACTTTATGCTAGAATC CAGAGCACTAGCAGGAAACTGCTTAAATTTTACTTAATCAAAGTCAAGTTTGGACATACATGTCAGGTAAAAC CTAGCAGAGATGAGCTACCTTGATTTTAAAACTTCAAGGGATAGCTCAATGTCATCAAGATCCTTTTGATGAC TTG The following amino acid sequence <SEQ ID NO.","48> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","47: NQVALLLRPLALSMAFINSCLNPVLYVFIGHDFWEHLLHSLLAALERALSEEPDSAIPAPRQMSPLHDPISYS IFPPLNPLPKQLYHNPTSNRIENKPQLLSELYVLGHVLEYNLKCLEDGGKKQTRSHSLEEDSSPRLKQKKRLS CDKTSHKIGSGPAAMTLCNPEHQETAILLNQSQVWTYMSGKTQRATLILKLQGIAQCHQDPFDDL The following DNA sequence beGPCR-seq37 <SEQ ID NO.","49> was identified in H. sapiens: GCTTGTTCACGGCCACCATCCTCAAGCTGTTGCGCACGGAGGAGGCGCACGGCCGGGAGCAGCGGAGGCGCGC GGTGGGCCTGGCCGCGGTGGTCTTGCTGGCCTTTGTCACCTGCTTCGCCCCCAACAACTTCGTGCTCCTGGCG CACATCGTGAGCCGCCTGTTCTACGGCAAGAGCTACTACCACGTGTACAAGCTCACGCTGTGTCTCAGCTGCC TCAACAACTGTCTGGACCCGTTTGTTTATTACTTTGCGTCCCGGGAATTCCAGCTGCGCCTGCGGGAATATTT GGGCTGCCGCCGGGTGCCCAGAGACACCCTGGACACGCGCCGCGAGAGCCTCTTCTCCGCCAGGACCACGTCC GTGCGCTCCGAGGCCGGTGCGCACCCTGAAGGGATGGAGGGAGCCACCAGGCCCGGCCTCCAGAGGCAGGAGA GTGTGTTCTGAGTCCCGGGGGCGCAGC The following amino acid sequence <SEQ ID NO.","50> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","49: LFTATILKLLRTEEAHGREQRRRAVGLAAVVLLAFVTCFAPNNFVLLAHIVSRLFYGKSYYHVYKLTLCLSCL NNCLDPFVYYFASREFQLRLREYLGCRRVPRDTLDTRRESLFSARTTSVRSEAGAHPEGMEGATRPGLQRQES VFVPGAQAAPPGLR The following DNA sequence beGPCR-seq38 <SEQ ID NO.","51> was identified in H. sapiens: TTACTTATTCTGCCCTTTATCCAACTTTTAATTCCCTTTGCTATTCTCCTGCCTCATTTTCTGGCCTCATTTT CCCTATTATCCTGCCTCACATTGATCAAGGGATGAGGCTGGCAGGATCCGGAACCCACAGGGCCCCGTGGGCC ATGAGAGGCTCCTGGACTTGAACCTCAGGACACTCCCACTCTGGCTGCCGGCAGGGATGGAAGCTGGATGAGC AGGCAGGAGCTGGCAGTGGGGGTGGAGAGCCATAGGCTATTGGGGTGGACAGGCTTGGGTGCCTCATGGGAGC TCCCCATGGGAGCTGTGGCCCCTTGGGGCCTCTTATTTCTCACCCCAGGCTTTCCCGGGAGAGGTTCAAGTCA GAAGATGCCCCAAAGATCCACGTGGCCCTGGGTGGCAGCCTGTTCCTCCTGAATCTGGCCTTCTTGGTCAATG TGGGGAGTGGCTCAAAGGGGTCTGATGCTGCCTGCTGGGCCCGGGGGGCTGTCTTCCACTACTTCCTGCTCTG TGCCTTCACCTGGATGGGCCTTGAAGCCTTCCACCTCTACCTGCTCGCTGTCAGGGTCTTCAACACCTACTTC GGGCACTACTTCCTGAAGC The following amino acid sequence <SEQ ID NO.","52> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","51: ETYSALYPTFNSLCYSPASFSGLIFPIILPHIDQGMRLAGSGTHRAPWAMRGSWTTSGHSHSGCRQGWKLDEQ AGAGSGGGEPAIGVDRLGCLMGAPHGSCGPLGPLISHPRLSRERFKSEDAPKIHVALGGSLFLLNLAFLVNVG SGSKGSDAACWARGAVFHYFLLCAFTWMGLEAFHLYLLAVRVFNTYFGHYFL The following DNA sequence beGPCR-seq40 <SEQ ID NO.","53> was identified in H. sapiens: AATTGGTCGGAGAGTGCAGCTGCTTGAAATGGAGGATTGAAATCATCACCAGGAGGTTTCCAAACACAGCCAG CACAGCCCCAAAGCCAAACACTATGTACAGAATCACCCGGGATCCCGGCGAGAAGGGGATTTTCACACAGGAC CCATTCACGTTCGCGTAGCACAGCTGCACAGCCACCAGCAGGGATGAATTGCTGCTCATAACGCTGGTATTTA CATATGGAGAAATTTTGTCCTTGTTGATTATCACAAAAAATACAGGATTGTTCCTGATTTTCATTGCTCCTGC GGAAAAAAACACATATTCACCAGGATGCCAGAGGAAATGATCA The following amino acid sequence <SEQ ID NO.","54> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","53: DHFLWHPGEYVFFSAGAMKIRNNPVFFVIINKDKISPYVNTSVMSSNSSLLVAVQLCYANVNGSCVKIPFSPG SRVILYIVFGFGAVLAVFGNLLVMISILHFKQLHSPTN The following DNA sequence beGPCR-seq41 <SEQ ID NO.","55> was identified in H. sapiens: CACATCTTAACAAGACTGAAAAACATTGATTTGTTTTTAATTTGAAGAGCAATTTATTTGCTATTCATTCATA GTCTTACTTGATTTTTAAAAACTCATTTCGCTTGGTAATTTTAAAGGTATCCTGAACTTCGTCTATCCAACTG CTTATATATGTTCAGAAAACAAATTCATGGTTGCTGAACTGTTCTTTAAAACCTGACCAGTTACAATAACTTT TATTGCTTTCCTAAACCATGGGTAAAATAAAGCATAAATCAAAGGATTCATGGCTGAGTTATAATAAGCACAC CAACAGCATCATAAATACAGGCAGGGGTTATAAAGCCCATAAAGGCATCAATTAATGAATCAATGCTATATGG TAACCATGAAATCATAAATGCTACCACTGTGACCCCCAGGGTTTTAGCTGCTTTTCTCTCTCTCCTGGCCACT CTGGCTTTGTAACTCTCTGAGGATGATTCTGTCTTGCTACCAGTATTTTCTATCTTTTTCGCCTGTCGTCTAG CCACAAGAAATATGTTACCATACAGAATTATCATAATAAAGGTAGGTATAAAGAAGGATAGAAAATCTGTCAA CA The following amino acid sequence <SEQ ID NO.","56> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","55: LTDFLSFFIPTFIMIILYGNIFLVARRQAKKIENTGSKTESSSESYKARVARRERKAAKTLGVTVVAFMISWL PYSIDSLIDAFMGFITPACIYEICCWCAYYNSAMNPLIYALFYPWFRKAIKVIVTGQVLKNSSATMNLFSEHI AVGTKFRIPLKLPSEMSFKSSKTMNEQINCSSNKQINVFQSCDV The following DNA sequence nGPCR-seq53 <SEQ ID NO.","57> was identified in H. sapiens: TTTGTGGCAAGGAGACCCTGATCCCGGTCTTCCTGATCCTTTTCATTGCCCTGGTCGGGCTGGTAGGAAACGG GTTTGTGCTCTGGCTCCTGGGCTTCCGCATGCGCAGGAACGCCTTCTCTGTCTACGTCCTCAGCCTGGCCGGG GCCGACTTCCTCTTCCTCTGCTTCCAGATTATAAATTGCCTGGTGTACCTCAGTAACTTCTTCTGTTCCATCT CCATCAATTTCCCTAGCTTCTTCACCACTGTGATGACCTGTGCCTACCTTGCAGGCCTGAGCATGCTGAGCAC CGTCAGCACCGAGCGCTGCCTGTCCGTCCTGTGGCCCATCTGGTATCGCTGCCGCCGCCCCAGACACCTGTCA GCGGTCGTGTGTGTCCTGCTCTGGGCCCTGTCCCTACTGCTGAGCATCTTGGAAGGGAAGTTCTGTGGCTTCT TATTTAGTGATGGTGACTCTGGTTGGTGTCAGACATTTGATTTCATCACTGCAGCGTGGCTGATTTTTTTATT CATGGTTCTCTGTGGGTCCAGTCTGGCCCTGCTGGTCAGGATCCTCTGTGGCTCCAGGGGTCTGCCACTGACC AGGCTGTACCTGACCATCCTGCTCACAGTGCTGGTGTCCCTCCTCTGCGGCCTGCCCTTTGGCATTCAGTGGT TCCTAATATTATGGATCTGGAAGGATTCTGATGTCTTATTTTGTCATATTCATCCAGTTTCAGTTGTCCTGTC ATCTCTTAACAGCAGTGCCAACCCCATCATTTACTTCTTCGTGGGCTCTTTTAGGAAGCAGTGGCGGSTGCAG CACCCGATCCTCAAGCTGGCTCTCCAGAGGGCTCTGCAGGACATTGCTGAGGTGGATCACAGTGAAGGATGCT TCCGTCAGGGCACCCGGAGATTCAAAGAAGCATTCTGGTGTAGGGATGGACCCCTCTACTTCCATCATATATA TGTGGCTTTGAGAGGCAACTTTGCCCC The following amino acid sequence <SEQ ID NO.","58> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","57: CGKETLIPVFLILFIALVGLVGNGFVLWLLGFRMRRNAFSVYVLSLAGADFLFLCFQIINCLVYLSNFFCSIS INFPSFFTTVMTCAYLAGLSMLSTVSTERCLSVLWPIWYRCRRPRHLSAVVCVLLWALSLLLSILEGKFCGFL FSDGDSGWCQTFDFITAAWLIFLFMVLCGSSLALLVRILCGSRGLPLTRLYLTILLTVLVSLLCGLPFGIQWF LILWIWKDSDVLFCHIHPVSVVLSSLNSSANPIIYFFVGSFRKQWRXQHPILKLALQRALQDIAEVDHSEGCF RQGTRRFKEAFWCRDGPLYFHHIYVALRGNFA The following DNA sequence nGPCR-seq54 <SEQ ID NO.","59> was identified in H. sapiens: CTTTGCATCTCACTGTTGAGCAGACAGCCTGCTGAAAGTTGTCGCTGACCACCACATATAGTAACAGGTTACC AAAGGTGTTCAGAGCAGCATAATGGTCTAGAAACGATGTAAGCTTCATGGATCTGATTCTCAATGGAACAACT GATTGAAAGCAGGCTGAGATTCGATCCTGAATGACCCTCAAGATATGGAAGGGTAAAAAACATACGTAAAATG CAAGGAGTAGCAGAATGGTTAGCCTTCGTGCTTTCTGCTTAAGGCAGCTGTCAGTTTGCAGTCCATGGGTCAA AGTGTGGATAATCGTGGTATAGCAAAGTGTCACTATCACCAAGGGGAGGCAGAAAGTACTTGCAGTCAAAATC AGGTTGTACCACTTAATAGTATTGAGTTCATCCGAACTGGTGAGGTCGAGACAGGCTGATCTGTTGGTCCTGT TGGTTGATGTGATCAAGAAGGTCATCGGAATGACAGCTACCAGTGAAATGATCCACACCACAGCACAGGCTAC AACTGCACATCGAGTTTTGTGAATGGAAAAGCAGCTCATTGGGTGAATGATCACACAGTAGCGGAAG The following amino acid sequence <SEQ ID NO.","60> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","59: FRYCVIIHPMSCFSIHKTRCAVVACAVVWIISLVAVIPMTFLITSTNRTNRSACLDLTSSDELNTIKWYNLIL TASTFCLPLVIVTLCYTTIIHTLTHGLQTDSCLKQKARRLTILLLLAFYVCFLPFHILRVIQDRISACFQSVV PLRIRSMKLTSFLDHYAALNTFGNLLLYVVVSDNFQQAVCSTVRCK The following DNA sequence nGPCR-seq55 <SEQ ID NO.","61> was identified in H. sapiens, where the underlined ATG identifies a probable start codon: GGGAGGGCTCGTAGACACACTAACCCTACCCTTTCTGTTTCTTCCTCATCTTTCCTTTCCATCTGTTTCTCAT GGTCTCCTGTCTGTCTCTCTCTCTCTCCCCTCTTTCTCTCTCCTCGCTCTTTCTCATCCCCTCCATTTCTGTG TCAATCTCAATCCATTTATATCGGTGGCCACTTTTCTATCTCTTTGTTCTATCTCTCTCTCTCTCTCTTTCCC ACTTTGTCTCTGCACGCCTGTTGTGTTTTTCTGCCTGTCTCTCTCTTGCCCTCATCTCTCTGTCTCTCTCTTG CCCTCATCTCTCTGTCTCTCTGTGTCTGTGTCTCCCCCGCTCATTCCCATTTGCAGGTGCAATGTAGCAGGAC AACTCATGGAGCCCCCCCGGGCCCATCGAGTACCGGACTGGCTGACCCCCTAGGGTTGGCAGTAGCCCCTGAC CCTCAGTATGGCCAACACTACCGGAGAGCCTGAGGAGGTGAGCGGCGCTCTGTCCCCACCGTCCGCATCAGCT TATGTGAAGCTGGTACTGCTGGGACTGATTATGTGCGTGAGCCTGGCGGGTAACGCCATCTTGTCCCTGCTGG TGCTCAAGGAGCGGGCCCTGCACAAGGCTCCTTACTACTTCCTGCTGGACCTGTGCCTGGCCGATGGCATACG CTCTGCCGTCTGCTTCCCCTTTGTGCTGGCTTCTGTGCGCCACGGCTCTTCATGGACCTTCAGTGCACTCAGC TGCAAGATTGTGGCCTTTATGGCCGTGCTCTTTTGCTTCCATGCGGCCTTCATGCTGTTCTGCATCAGCGTCA CCCGCTACATGGCCATCGCCCACCACCGCTTCTACGCCAAGCGCATGACACTCTGGACATGCGCGGCTG The following amino acid sequence <SEQ ID NO.","62> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","61: MANTTGEPEEVSGALSPPSASAYVKLVLLGLIMCVSLAGNAILSLLVLKERALHKAPYYFLLDLCLADGIRSA VCFPFVLASVRHGSSWTFSALSCKIVAFMAVLFCFHAAFMLFCISVTRYMAIAHHRFYAKRMTLWTCAAE The following DNA sequence nGPCR-seq56 <SEQ ID NO.","63> was identified in H. sapiens: AAAAATTGCTGTACTGAACTATTGAATGGAACTTGGAAATAAAGTCCCTTCCAAAATAACTATTCTTCAACAG AGAGTAATAGGTAAATGTTTTAGAAGTGAGAGGACTCAAATTGCCAATGATTTACTCTTTTATTTTTCCTCCT AGGTTTCTGGGATAAGTATGTGCAAATAAAAAATAAACATGAGAAGGAACTGTAACCTGATTATGGATTTGGG AAAAAGATAAATCAACACACAAAGGGAAAAGTAAACTGATTGACAGCCCTCAGGAATGATGCCCTTTTGCCAC AATATAATTAATATTTCCTGTGTGAAAAACAACTGGTCAAATGATGTCCGTGCTTCCCTGTACAGTTTAATGG TGCTCATAATTCTGACCACACTCGTTGGCAATCTGATAGTTATTGTTTCTATATCACACTTCAAACAACTTCA TACCCCAACAAATTGGCTCATTCATTCCATGGCCACTGTGGACTTTCTTCTGGGGTGTCTGGTCATGCCTTAC AGTATGGTGAGATCTGCTGAGCACTGTTGGTATTTTGGAGAAGTCTTCTGTAAAATTCACACAAGCACCGACA TTATGCTGAGCTCAGCCTCCATTTTCCATTTGTCTTTCATCTCCATTGACCGCTACTATGCTGTGTGTGATCC ACTGAGATATAAAGCCAAGATGAATATCTTGGTTATTTGTGTGATGATCTTCATTAGTTGGAGTGTCCCTGCT GTTTTTGCATTTGGAATGATCTTTCTGGAGCTAAACTTCAAAGGCGCTGAAGAGATATATTACAAACATGTTC ACTGCAGAGGAGGTTGCTCTGTCTTCTTTAGCAAAATATCTGGGGTACTGACCTTTATGACTTCTTTTTATAT ACCTGGATCTATTATGTTATGTGTCTATTACAGAATATATCTTATCGCTAAAGAACAGGCAAGATTAATTAGT GATGCCAATCAGA The following amino acid sequence <SEQ ID NO.","64> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","63: REKTDQPSGMMPFCRNIINISCVKNNWSNDVRASLYSLMVLIILTTLVGNLIVIVSISHFKQLHTPTNWLIHS MATVDFLLGCLVMPYSMVRSAEHCWYFGEVFCKIHTSTDIMLSSASIFHLSFISIDRYYAVCDPLRYKAKMNI LVICVMIFISWSVPAVFAFGMIFLELNFKGAEEIYYKHVHCRGGCSVFFSKISGVLTFMTSFYIPGSIMLCVY YRIYLIAKEQARLISDANQ The following DNA sequence nGPCR-seq57 <SEQ ID NO.","65> was identified in H. sapiens: AACAGTCCCGGGTGGAACCTGGGCATGTATATTTTGATTGTTTTATGCATACTCCTAGTGAAGAACCAATGTC TTGCTCAGATAGAAGCAAGATACTCAGACTTAGTTTCTCTGTAGCTCCTGCTTTTTATTATTCCTGGTTGGAT TGCACCACTACTCAGTTTCTATTTTATAATACTGATTATAAAACATGGGAGGGAAATAACTTTGTATTGGTTT TTATGGATAATTTATTATGTGTCCTAGACTCTGGCCTTGTCAAAAGAAGGACGTAAGAAGGCACGATGTATTA TACTTGGGAATGATAGAAGAGACTGACCTGGTATTTCCACCCGGAAGAGGGAAAGGATTTTAACTACAAATAC AGGAATCCAGCAGATGGCATCAGAGAACACTATAAAAAAGAAACGATTTGCAACAGCCACCTCTCTTCCAAAA CAATTCCTTACTTCTGTGGTCTGCAAGGCGGTTTTTTGAATGGAACAGAACATAGTAATATAGGAAAACACAA TGATGAGAAAAGCCAGCAAGTTCACACCTGTTGGGGAAAAGCACACTTTTAACATCTCAGGCGTAAAAGTCAA CAGTAAAATTACTGTGGTACAGGTTGAGTATCCCTTACCCAAAATGTTTGAAACCAGAAATGTTTTGGATTTC GGATTTCGGAATATTTACACATTCATAATGATATATCTTGGAAATGGTTCCCAAGTCTAAACACAAAATTTAT TTATGTTTCATATACACCTTATACACATAGTCTGAAAGTAATTTTGTACAATATTTTAAATAATTTTGGGCAT GAAACAAAGTTTGCATACATTGAACCATCAGACAGCAAAAGCTTCAGGTGTGGAATTTTCCACTTGTGGCATC ATGTTGATGCTCAAAAAGTTCCATATTTTAGAGCATTTCAAATTTTGGATTTTCAAATTACAAATGCTTAACC TGTACTTAGATGTTAAATACAGTGCCTCTTCCACGGGCACTTTCAGGAAGCATTCTTTTATATAAGCCC The following amino acid sequence <SEQ ID NO.","66> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","65: YIKECFLKVPVEEALYLTSKYRLSICNLKIQNLKCSKIWNFLSINMMPQVENSTPEAFAVWFNVCKLCFMPKI INIVQNYFQTMCIRCININKFCVTWEPFPRYIIMNVIFRNPKSKTFLVSNILGKGYSTCTTVILLLTFTPEML KVCFSPTGVNLLAFLIIVFSYITMFCSIQKTALQTTEVRNCFGREVAVANRFFFIVFSDAICWIPVFVVKILS LFRVEIPGQSLLSFPSIIHRAFLRPSFDKARVDTIIHKNQYKVISLPCFIISIIKKLSSGAIQPGIIKSRSYR ETKSEYLASIARHWFFTRSMHKTIKIYMPRFHPGL The following DNA sequence nGPCR-seq58 <SEQ ID NO.","67> was identified in H. sapiens: ACTACCATGGAAGCTGACCTGGGTGCCACTGGCCACAGGCCCCGCACAGAGCTTGATGATGAGGACTCCTACC CCCAAGGTGGCTGGGACACGGTCTTCCTGGTGGCCCTGCTGCTCCTTGGGCTGCCAGCCAATGGGTTGATGGC GTGGCTGGCCGGCTCCCAGGCCCGGCATGGAGCTGGCACGCGTCTGGCGCTGCTCCTGCTCAGCCTGGCCCTC TCTGACTTCTTGTTCCTGGCAGCAGCGGCCTTCCAGATCCTAGAGATCCGGCATGGGGGACACTGGCCGCTGG GGACAGCTGCCTGCCGCTTCTACTACTTCCTATGGGGCGTGTCCTACTCCTCCGGCCTCTTCCTGCTGGCCGC CCTCAGCCTCGACCGCTGCCTGCTGGCGCTGTGCCCACACTGGTACCCTGGGCACCGCCCAGTCCGCCTGCCC CTCTGGGTCTGCGCCGGTGTCTGGGTGCTGGCCACACTCTTCAGCGTGCCCTGGCTGGTCTTCCCCGAGGCTG CCGTCTGGTGGTACGACCTGGTCATCTGCCTGGACTTCTGGGACAGCGAGGAGCTGTCGCTGAGGATGCTGGA GGTCCTGGGGGGCTTCCTGCCTTTCCTCCTGCTGCTCGTCTGCCACGTGCTCACCCAGGCCACAGCCTGTCGC ACCTGCCACCGCCAACAGCAGCCCGCAGCCTGCCGGGGCTTCGCCCGTGTGGCCAGGACCATTCTGTCAGCCT ATGTGGTCCTGAGGCTGCCCTACCAGCTGGCCCAGCTGCTCTACCTGGCCTTCCTGTGGGACGTCTACTCTGG CTACCTGCTCTGGGAGGCCCTGGTCTACTCCGACTACCTGATCCTACTCAACAGCTGCCTCAGCCCCTTCCTC TGCCTCATGGCCAGTGCCGACCTCCGGACCCTGCTGCGCTCCGTGCTCTCGTCCTTCGCGGCAGCTCTCTGCG AGGAGCGGCCGGGCAGCTTCACGCCCACTGAGCCACAGACCCAGCTAGATTCTGAGGGTCCAACTCTGCCAGA GCCGATGGCAGAGGCCCAGTCACAGATGGATCCTGTGGCCCAGCCTCAGGTGAACCCCACACTCCAGCCACGA TCGGATCCCACAGCTCAGCCACAGCTGAACCCTACGGCCCAGCCACAGTCGGATCCCACAGCCCAGCCACAGC TGAACCTCATGGCCCAGCCACAGTCAGATTCTGTGGCCCAGCCACAGGCAGACACTAACGTCCAGACCCCTGC ACCTGCTGCC The following amino acid sequence <SEQ ID NO.","68> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","67: TTMEADLGATGHRPRTELDDEDSYPQGGWDTVFLVALLLLGLPANGLMAWLAGSQARHGAGTRLALLL LSLALSDFLFLAAAAFQILEIRHGGHWPLGTAACRFYYFLWGVSYSSGLFLLAALSLDRCLLALCPHW YPGHRPVRLPLWVCAGVWVLATLFSVPWLVFPEAAVWWYDLVICLDFWDSEELSLRMLEVLGGFLPFL LLLVCHVLTQATACRTCHRQQQPAACRGFARVARTILSAYVVLRLPYQLAQLLYLAFLWDVYSGYLLW EALVYSDYLILLNSCLSPFLCLMASADLRTLLRSVLSSFAAALCEERPGSFTPTEPQTQLDSEGPTLP EPMAEAQSQMDPVAQPQVNPTLQPRSDPTAQPQLNPTAQPQSDPTAQPQLNLMAQPQSDSVAQPQADT NVQTPAPAA The following DNA sequence nGPCR-seq59 <SEQ ID NO.","69> was identified in H. sapiens: TACAGGCCTGAGCATGCTGGGCTCCATCAGCACCAAGCACTGCCTGTCCATCCTGTGGCCCATCTAGTACCGC TGCCACCACCCCACACACCTGTCAGCAGTCGTGTGTCCTGCTCTGGGCCCTGTCCCTGCTGCAGAGCATCCTG GAATGGATGTTCTGTGGCTTCCTGTCTAGTGGTGCTGATTCTGTTTGGTGTGAAACATCAGATTTCATCACAG TCACATGGCTGATTTTTTTATGTGTGGTTCTCTGCGGGTCCAGCCCGGTTCTGCTGGTCAGGATCCTTTGTGG ATCCCGGAAGATGCCCTTGACCAGGCTGTACATGACCATCCTGCTCAGAGTGCTGGTCTTCCTCCTCTGTGAC CTGCCCTTTGGCATTCAGTGATTCCTATTTTTCTGGATCCACGTGGATTTGTCACGTTCGTCTAGTTTCCATT TTCCTGTCCACTCTTAACAGCAGTGCCAACCCCATTATTTACTTCTTCATGGGCTCCTTTAGGCAGCTTCAAA ACAGGAAGACTCTCTAGCTGGTTCTCCAGAGGGCTCTGCAGGACACGCCTGAGGTGGAAGAAGGCAGATGGCG GCTTTCTGAGGAAACCCTGGAGCTGTCATGAAGCAGATTGGGGCCATGAGGAAGAGCCTCTGCCCTGTCAGTC AG The following amino acid sequence <SEQ ID NO.","70> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","69: YRPEHAGLHQHQALPVHPVAHLVPLPPPHTPVSSRVSCSGPCPCCRASWNGCSVASCLVVLILFGVKHQISSQ SHGFFYVWFSAGPARFCWSGSFVDPGRCPPGCTPSCSECWSSSSVTCPLAFSDSYFSGSTWICHVRLVSIFLS TLNSSANPIIYFFMGSFRQLQNRKTLLVLQRALQDTPEVEEGRWRLSEETLELSSRLGPGRASALSV The following DNA sequence nGPCR-seq60 <SEQ ID NO.","71> was identified in H. sapiens: ATGCCGAAGGCAGGCCGCAGAAGAGAAGAGGAGGACGGTGAGGAGGATGAGCCCAGGGAAGCCCCGGGGTGGG GGCCGCTGGGGGCCTCGCTCCACCCGCAGCAGCAGCATAAGGCTGGCCCCACACATGGTGCAACACAGCAGAG CCAGCAGCACCGCTGCCACCAGCCACAGCGTCCGGCACAAGTGGCGGCTGGGCTCCCCGAAGAACTGGGTGCA GGCGCCGCTGAGCAGCAGGTGCAGCAGCAGGCAGAGGGCCCAGGTGAGGGCGCACACACAGGTGGTCAGGTGG CGTGGGCGGCGGCACGAGTACCAGGCTGGGAAGAGGGCGGCCAGGCACTGCTCCACGCTGACGGCCGCCAGGA GACTCAGGCCCACGATGTAGCAGAAGAAGCGCAGCGTTGCCAGGCTGGTCTGCACGAAGCCCGGGAAGTCCAG CCGGCCTTGCAGCAAGTCGGGGACGATGGCCACCATGTGGCAGCCAAGGAAGATGAGATCCGCGCAGGCCACG TCCAGGAGGTAGATGGCGAAAGGGTTTCTGTAGACATTGGAGCTGAGC The following amino acid sequence <SEQ ID NO.","72> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","71: LSSNVYRNPFAIYLLDVACADLIFLGCHMVAIVPDLLQGRLDFPGFVQTSLATLRFFCYIVGLSLLA AVSVEQCLAALFPAWYSCRRPRHLTTCVCALTWALCLLLHLTTCVCALTWALCLLLHLLLSGACTLL LSGACTQFFGEPSRHLCRTLWLVAAVLLALLCCTMCGASLMLLLRVERGPQRPPPRGFPGLILLTVL LFSSAACLRH The following DNA sequence nGPCR-1 <SEQ ID NO.","73> was identified in H. sapiens: ATGGAATCATCTTTCTCATTTGGAGTGATCCTTGCTGTCCTGGCCTCCCTCATCATTGCTACTAACACACTAG TGGCTGTGGCTGTGCTGCTGTTGATCCACAAGAATGATGGTGTCAGTCTCTGCTTCACCTTGAATCTGGCTGT GGCTGACACCTTGATTGGTGTGGCCATCTCTGGCCTACTCACAGACCAGCTCTCCAGCCCTTCTCGGCCCACA CAGAAGACCCTGTGCAGCCTGCGGATGGCATTTGTCACTTCCTCCGCAGCTGCCTCTGTCCTCACGGTCATGC TGATCACCTTTGACAGGTACCTTGCCATCAAGCAGCCCTTCCGCTACTTGAAGATCATGAGTGGGTTCGTGGC CGGGGCCTGCATTGCCGGGCTGTGGTTAGTGTCTTACCTCATTGGCTTCCTCCCACTCGGAATCCCCATGTTC CAGCAGACTGCCTACAAAGGGCAGTGCAGCTTCTTTGCTGTATTTCACCCTCACTTCGTGCTGACCCTCTCCT GCGTTGGCTTCTTCCCAGCCATGCTCCTCTTTGTCTTCTTCTACTGCGACATGCTCAAGATTGCCTCCATGCA CAGCCAGCAGATTCGAAAGATGGAACATGCAGGAGCCATGGCTGGAGGTTATCGATCCCCACGGACTCCCAGC GACTTCAAAGCTCTCCGTACTGTGTCTGTTCTCATTGGGAGCTTTGCTCTATCCTGGACCCCCTTCCTTATCA CTGGCATTGTGCAGGTGGCCTGCCAGGAGTGTCACCTCTACCTAGTGCTGGAACGGTACCTGTGGCTGCTCGG CGTGGGCAACTCCCTGCTCAACCCACTCATCTATGCCTATTGGCAGAAGGAGGTGCGACTGCAGCTCTACCAC ATGGCCCTAGGAGTGAAGAAGGTGCTCACCTCATTCCTCCTCTTTCTCTCGGCCAGGAATTGTGGCCCAGAGA GGCCCAGGGAAAGTTCCTGTCACATCGTCACTATCTCCAGCTCAGAGTTTGATGGCTAA The following amino acid sequence <SEQ ID NO.","74> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","73: MESSFSFGVILAVLASLIIATNTLVAVAVLLLIHKNDGVSLCFTLNLAVADTLIGVAISGLLTDQLSSPSRPT QKTLCSLRMAFVTSSAAASVLTVMLITFDRYLAIKQPFRYLKIMSGFVAGACIAGLWLVSYLIGFLPLGIPMF QQTAYKGQCSFFAVFHPHFVLTLSCVGFFPAMLLFVFFYCDMLKIASMHSQQIRKMEHAGAMAGGYRSPRTPS DFKALRTVSVLIGSFALSWTPFLITGIVQVACQECHLYLVLERYLWLLGVGNSLLNPLIYAYWQKEVRLQLY HMALGVKKVLTSFLLFLSARNCGPERPRESSCHIVTISSSEFDG The following DNA sequence TL-GPCR-seq5 <SEQ ID NO.","75> was identified in H. sapiens.","AACTGGAAGGGCAGCCGTCTGCCGCCCACGAACACCTTCTCAAGCACTTTGAGTGACCACGGCTTGCAAGCTG GTGGCTGGCCCCCCGAGTCCCGGGCTCTGAGGCACGGCCGTCGACTTAAGCGTTGCATCCTGTTACCTGGAGA CCCTCTGAGCTCTCACCTGCTACTTCTGCCGCTGCTTCTGCACAGAGCCCGGGCGAGGACCCCTCCAGGATGC AGGTCCCGAACAGCACCGGCCCGGACAACGCGACGCTGCAGATGCTGCGGAACCCGGCGATCGCGGTGGCCCT GCCCGTGGTGTACTCGCTGGTGGCGGCGGTCAGCATCCCGGGCAACCTCTTCTCTCTGTGGGTGCTGTGCCGG CGCATGGGGCCCAGATCCCCGTCGGTCATCTTCATGATCAACCTGAGCGTCACGGACCTGATGCTGGCCAGCG TGTTGCCTTTCCAAATCTACTACCATTGCAACCGCCACCACTGGGTATTCGGGGTGCTGCTTTGCAACGTGGT GACCGTGGCCTTTTACGCAAACATGTATTCCAGCATCCTCACCATGACCTGTATCAGCGTGGAGCGCTTCCTG GGGGTCCTGTACCCGCTCAGCTCCAAGCGCTGGCGCCGCCGTCGTTACGCGGTGGCCGCGTGTGCAGGGACCT GGCTGCTGCTCCTGACCGCCCTGTCCCCGCTGGCGCGCACCGATCTCACCTACCCGGTGCACGCCCTGGGCAT CATCACCTGCTTCGACGTCCTCAAGTGGACGATGCTCCCCAGCGTGGCCATGTGGGCCGTGTTCCTCTTCACC ATCTTCATCCTGCTGTTCCTCATCCCGTTCGTGATCACCGTGGCTTGTTACACGGCCACCATCCTCAAGCTGT TGCGCACGGAGGAGGCGCACGGCCGGGAGCAGCGGAGGCGCGCGGTGGGCCTGGCCGCGGTGGTCTTGCTGGC CTTTGTCACCTGCTTCGCCCCCAACAACTTCGTGCTCCTGGCGCACATCGTGAGCCGCCTGTTCTACGGCAAG AGCTACTACCACGTGTACAAGCTCACGCTGTGTCTCAGCTGCCTCAACAACTGTCTGGACCCGTTTGTTTATT ACTTTGCGTCCCGGGAATTCCAGCTGCGCCTGCGGGAATATTTGGGCTGCCGCCGGGTGCCCAGAGACACCCT GGACACGCGCCGCGAGAGCCTCTTCTCCGCCAGGACCACGTCCGTGCGCTCCGAGGCCGGTGCGCACCCTGAA GGGATGGAGGGAGCCACCAGGCCCGGCCTCCAGAGGCAGGAGAGTGTGTTCTGAGTCCCGGGGGCGCAGCTTG GAGAGCCGGGGGCGCAGCTTGGAGGATCCAGGGGCGCATGGAGAGGCCACGGTGCCAGAGGTTCAGGGAGAAC AGCTGCGTTGCTCCCAGGCACTGCAGAGGCCCGGTGGGGAAGGGTCTCCAGGCTTTATTCCTCCCAGGCACTG CAGAGGCACCGGTGAGGAAGGGTCTCCAGGCTTCACTCAGGGTAGAGAAACAAGCAAAGCCCAGCAGCGCACA GGGTGCTTGTTATCCTGCAGAGGGTGCCTCTGCCTCTCTGTGTCAGGGGACAGCTTGTGTCACCACGCCCGGC TAATTTTTGTATTTTTTTTAGTAGAGCTGGGCTGTCACCCCCGAGCTCCTTAGACACTCCTCACACCTGTCCA TACCCGAGGATGGATATTCAACCAGCCCCACCGCCTACCCGACTCGGTTTCTGGATATCCTCTGTGGGCGAAC TGCGAGCCCCATTCCCAGCTCTTCTCCCTGCTGACATCGTCCCTTAGCACACCTGTCCATACCCGAGGATGGA TATTCAACCAGCCCCACCGCCTACCCGACTCGGTTTCTGGATATCCTCTGTGGGCGAACTGCGAGCCCCATTC CCAGCTCTTCTCCCTGCTGACATCGTCCCTTAGTTGTGGTTCTGGCCTTCTCCATTCTCCTCCAGGGGTTCTG GTCTCCGTAGCCCGGTGCACGCCGAAATTTCTGTTTATTTCACTCAGGGGCACTGTGGTTGCTGTGGTTGGAA TTCTTCTTTCAGAGGAGCGCCTGGGGCTCCTGCAAGTCAGCTACTCTCCGTGCCCACTTCCCCTCACACACAC ACCCCCCTCGTGCCGAATTC The following amino acid sequence <SEQ ID NO.","76> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","75.MQVPNSTGPDNATLQMLRNPAIAVALPVVYSLVAAVSIPGNLFSLWVLCRRMGPRSPSVIFMINLSVTDLMLA SVLPFQIYYHCNRHHWVFGVLLCNVVTVAFYANMYSSILTMTCISVERFLGVLYPLSSKRWRRRRYAVAACAG TWLLLLTALSPLARTDLTYPVHALGIITCFDVLKWTMLPSVAMWAVFLFTIFILLFLIPFVITVACYTATILK LLRTEEAHGREQRRRAVGLAAVVLLAFVTCFAPNNFVLLAHIVSRLFYGKSYYHVYKLTLCLSCLNNCLDPFV YYFASREFQLRLREYLGCRRVPRDTLDTRRESLFSARTTSVRSEAGAHPEGMEGATRPGLQRQESVF The following DNA sequence nGPCR-9 <SEQ ID NO.","77> was identified in H. sapiens: ATGGAGTCGGGGCTGCTGCGGCCGGCGCCGGTGAGCGAGGTCATCGTCCTGCATTACAACTACACCGGCAAGC TCCGCGGTGCGCGCTACCAGCCGGGTGCCGGCCTGCGCGCCGACGCCGTGGTGTGCCTGGCGGTGTGCGCCTT CATCGTGCTAGAGAATCTAGCCGTGTTGTTGGTGCTCGGACGCCACCCGCGCTTCCACGCTCCCATGTTCCTG CTCCTGGGCAGCCTCACGTTGTCGGATCTGCTGGCAGGCGCCGCCTACGCCGCCAACATCCTACTGTCGGGGC CGCTCACGCTGAAACTGTCCCCCGCGCTCTGGTTCGCACGGGAGGGAGGCGTCTTCGTGGCACTCACTGCGTC CGTGCTGAGCCTCCTGGCCATCGCGCTGGAGCGCAGCCTCACCATGGCGCGCAGGGGGCCCGCGCCCGTCTCC AGTCGGGGGCGCACGCTGGCGATGGCAGCCGCGGCCTGGGGCGTGTCGCTGCTCCTCGGGCTCCTGCCAGCGC TGGGCTGGAATTGCCTGGGTCGCCTGGACGCTTGCTCCACTGTCTTGCCGCTCTACGCCAAGGCCTACGTGCT CTTCTGCGTGCTCGCCTTCGTGGGCATCCTGGCCGCTATCTGTGCACTCTACGCGCGCATCTACTGCCAGGTA CGCGCCAACGCGCGGCGCCTGCCGGCACGGCCCGGGACTGCGGGGACCACCTCGACCCGGGCGCGTCGCAAGC CGCGCTCGCTGGCCTTGCTGCGCACGCTCAGCGTGGTGCTCCTGGCCTTTGTGGCATGTTGGGGCCCCCTCTT CCTGCTGCTGTTGCTCGACGTGGCGTGCCCGGCGCGCACCTGTCCTGTACTCCTGCAGGCCGATCCCTTCCTG GGACTGGCCATGGCCAACTCACTTCTGAACCCCATCATCTACACGCTCACCAACCGCGACCTGCGCCACGCGC TCCTGCGCCTGGTCTGCTGCGGACGCCACTCCTGCGGCAGAGACCCGAGTGGCTCCCAGCAGTCGGCGAGCGC GGCTGAGGCTTCCGGGGGCCTGCGCCGCTGCCTGCCCCCGGGCCTTGATGGGAGCTTCAGCGGCTCGGAGCGC TCATCGCCCCAGCGCGACGGGCTGGACACCAGCGGCTCCACAGGCAGCCCCGGTGCACCCACAGCCGCCCGGA CTCTGGTATCAGAACCGGCTGCAGACTGA The following amino acid sequence <SEQ ID NO.","78> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","77: MESGLLRPAPVSEVIVLHYNYTGKLRGARYQPGAGLRADAVVCLAVCAFIVLENLAVLLVLGRHPRFHAPMFL LLGSLTLSDLLAGAAYAANILLSGPLTLKLSPALWFAREGGVFVALTASVLSLLAIALERSLTMARRGPAPVS SRGRTLAMAAAAWGVSLLLGLLPALGWNCLGRLDACSTVLPLYAKAYVLFCVLAFVGILAAICALYARIYCQV RANARRLPARPGTAGTTSTRARRKPRSLALLRTLSVVLLAFVACWGPLFLLLLLDVACPARTCPVLLQADPFL GLAMANSLLNPIIYTLTNRDLRHALLRLVCCGRHSCGRDPSGSQQSASAAEASGGLRRCLPPGLDGSFSGSER SSPQRDGLDTSGSTGSPGAPTAARTLVSEPAAD The following DNA sequence nGPcR-11 <SEQ ID NO.","79> was identified in H. sapiens: ATGTACAACGGGTCGTGCTGCCGCATCGAGGGGGACACCATCTCCCAGGTGATGCCGCCGCTGCTCATTGTGG CCTTTGTGCTGGGCGCACTAGGCAATGGGGTCGCCCTGTGTGGTTTCTGCTTCCACATGAAGACCTGGAAGCC CAGCACTGTTTACCTTTTCAATTTGGCCGTGGCTGATTTCCTCCTTATGATCTGCCTGCCTTTTCGGACAGAC TATTACCTCAGACGTAGACACTGGGCTTTTGGGGACATTCCCTGCCGAGTGGGGCTCTTCACGTTGGCCATGA ACAGGGCCGGGAGCATCGTGTTCCTTACGGTGGTGGCTGCGGACAGGTATTTCAAAGTGGTCCACCCCCACCA CGCGGTGAACACTATCTCCACCCGGGTGGCGGCTGGCATCGTCTGCACCCTGTGGGCCCTGGTCATCCTGGGA ACAGTGTATCTTTTGCTGGAGAACCATCTCTGCGTGCAAGAGACGGCCGTCTCCTGTGAGAGCTTCATCATGG AGTCGGCCAATGGCTGGCATGACATCATGTTCCAGCTGGAGTTCTTTATGCCCCTCGGCATCATCTTATTTTG CTCCTTCAAGATTGTTTGGAGCCTGAGGCGGAGGCAGCAGCTGGCCAGACAGGCTCGGATGAAGAAGGCGACC CGGTTCATCATGGTGGTGGCAATTGTGTTCATCACATGCTACCTGCCCAGCGTGTCTGCTAGACTCTATTTCC TCTGGACGGTGCCCTCGAGTGCCTGCGATCCCTCTGTCCATGGGGCCCTGCACATAACCCTCAGCTTCACCTA CATGAACAGCATGCTGGATCCCCTGGTGTATTATTTTTCAAGCCCCTCCTTTCCCAAATTCTACAACAAGCTC AAAATCTGCAGTCTGAAACCCAAGCAGCCAGGACACTCAAAAACACAAAGGCCGGAAGAGATGCCAATTTCGA ACCTCGGTCGCAGGAGTTGCATCAGTGTGGCAAATAGTTTCCAAAGCCAGTCTGATGGGCAATGGGATCCCCA CATTGTTGAGTGGCACTGA The following amino acid sequence <SEQ ID NO.","80> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","79: MYNGSCCRIEGDTISQVMPPLLIVAFVLGALGNGVALCGFCFHMKTWKPSTVYLFNLAVADFLLMICLPFRTD YYLRRRHWAFGDIPCRVGLFTLAMNRAGSIVFLTVVAADRYFKVVHPHHAVNTISTRVAAGIVCTLWALVILG TVYLLLENHLCVQETAVSCESFIMESANGWHDIMFQLEFFMPLGIILFCSFKIVWSLRRRQQLARQARMKKAT RFIMVVAIVFITCYLPSVSARLYFLWTVPSSACDPSVHGALHITLSFTYMNSMLDPLVYYFSSPSFPKFYNKL KICSLKPKQPGHSKTQRPEEMPISNLGRRSCISVANSFQSQSDGQWDPHIVEWH The following DNA sequence nGPCR-16 <SEQ ID NO.","81> was identified in H. sapiens: ATGACAGGTGACTTCCCAAGTATGCCTGGCCACAATACCTCCAGGAATTCCTCTTGCGATCCTATAGACACCC CACTTAATCAGCCTCTACTTCATAGTGCTTATTGGCGGGCTGGTGGGTGTCATTTCCATTCTTTTCCTCCTGG TGAAAATGAACACCCGGTCAGTGACCACCATGGCGGTCATTAACTTGGTGGTGGTCCACAGCGTTTTTCTGCT GACAGTGCCATTTCGCTTGACCTACCTCATCAAGAAGACTTGGATGTTTGGGCTGCCCTTCTGCAAATTTGTG AGTGCCATGCTGCACATCCACATGTACCTCACGTTCCTATTCTATGTGGTGATCCTGGTCACCAGATACCTCA TCTTCTTCAAGTGCAAAGACAAAGTGGAATTCTACAGAAAACTGCATGCTGTGGCTGCCAGTGCTGGCATGTG GACGCTGGTGATTGTCATTGTGGTACCCCTGGTTGTCTCCCGGTATGGAATCCATGAGGAATACAATGAGGAG CACTGTTTTAAATTTCACAAAGAGCTTGCTTACACATATGTGAAAATCATCAACTATATGATAGTCATTTTTG TCATAGCCGTTGCTGTGATTCTGTTGGTCTTCCAGGTCTTCATCATTATGTTGATGGTGCAGAAGCTACGCCA CTCTTTACTATCCCACCAGGAGTTCTGGGCTCAGCTGAAAAACCTATTTTTTATAGGGGTCATCCTTGTTTGT TTCCTTCCCTACCAGTTCTTTAGGATCTATTACTTGAATGTTGTGACGCATTCCAATGCCTGTAACAGCAAGG TTGCATTTTATAACGAAATCTTCTTGAGTGTAACAGCAATTAGCTGCTATGATTTGCTTCTCTTTGTCTTTGG GGGAAGCCATTGGTTTAAGCAAAAGATAATTGGCTTATGGAATTGTGTTTTGTGCCGTTAGCCACAAACTACA GTATTCATATTTGCTTCCTTTATATTGGGAATAAAAATGGGTATAGGGGAGGTAAGAATGGTATTTCATTACT TGATCAAAACCATGCCTTGATGTACCCAAAACAAAAGGACTATAAAATGCAAGAGCCCTCATTGTAGTCCTTA TGGGATCCCTCCCATCTCTGAGTGATGGCCGTACAAAGACCAGTGTTGTTGAATCCACCTGGAGTTGCAATAT TACATTATTTTCCAGTACAGAATGTCTGTGTGGCCCATGAAAGCAACATAGGTTTTAAGAGTTTTAGAGTTTC ATTAGCTCATTCTAAGTTCCTCTGTTTGAAGCATGGTCTCTTAGGTTTTGGACTGAACTCAGACCTTTAGTTC TTTTCATCCCACTTCACCTTAGGTAAGTAAATTCTGGCCACCACCCAGCTCCAAAGACACAAACTCTCCTTCG CTAACCAGGTTAGATGTCCCATTCATCTCATGCCCTGATAAAAACTGATAAGGGGAGAGAATAGTTAAAAATT TTTCTAGGGTATCATAACTCTGGTAGGAAGTCATCTGTCTAGAAATCAAGAGAAAAAGAACGTGTGGCCTCCT GTTATAACAAGGGTTTCTAGATTTGTCCTGTGAAAGGTCGTTTAAGGACTTGGGGATCAACTTCCTCAATTAT CACCAATTGCACTGTTGCTCCAAAAATCATTTAAAAGCTTACTGGACATATCTACATAATGGTGAAACTGTAA TTTAGAGACTATCCCTGACTAATGTGCTGGTAGGCATTAAAATGAGTTCCCAAGGGAAGTGATTAAAATTTTT TTCTCTTCTGTTTTTTGAGAGAATTTCTAGATGTCCTGGGCCACAGTTAATTAAGATTTTTAGGGGGGACAGA AAGTTATACTGAAATCTTTAGAGCTCCCTTCCGCCGTTAAAATTATATATATATATATTTAAATTATACCTTA AGTTCTGGGGTACATGTGCAGAATGTGCAGGTTTGTTACATAGGTATACACGTGCCATGGTGGTTTGCGGCAC CTGTCAACCCATCTACATTAGGTATTTCTCCTAATGCTCTCCCTCCCCTAGCCCCCCACCCCTGGACAGGCCC CATTGTGTGATGTTCCCCTCCCTGTGTCCATGTGTTTTCATTGTTCAACTCCCACTTCTAAGTGAGAACATGC GGTGTTTGGTTTTCTGTTCCTGTGTTAGTTTGCTGAGAATGATGGTTTCCAGGTTAAAATTATATATTTTTAA ATAAATGAAAACTGTGTTTTTAAAAGAGGACTTTTGAGAAGTATATAGAAAAACCATTAATTTAGACTCTGTG AGATTAGGTTGCATGAAGAAGGTTTTCTGAATATTTGAAGAGTGGATAAATAAATGTCCCCCAAAGCAATAAA ATCATAATCCTTTAAAATATAGGAAAAATAACTAATGGGAACTAGGCTTAATACTCGGGATGAAATAATCTGT ACAACAAACTCCCATGACACATGTTTACCTATGTAACAAACCTGCACATGTACCCCTGAACTTAAAATAAAAT TTAAAGTATAATAATAAAATAATATGGATTTTCTTT The following amino acid sequence <SEQ ID NO.","82> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","81: MTGDFPSMPGHNTSRNSSCDPIVTPHLISLYFIVLIGGLVGVISILFLLVKMNTRSVTTMAVINLVVVHSVFL LTVPFRLTYLIKKTWMFGLPFCKFVSAMLHIHMYLTFLFYVVILVTRYLIFFKCKDKVEFYRKLHAVAASAGM WTLVIVIVVPLVVSRYGIHEEYNEEHCFKFHKELAYTYVKIINYMIVIFVIAVAVILLVFQVFIIMLMVQKLR HSLLSHQEFWAQLKNLFFIGVILVCFLPYQFFRIYYLNVVTHSNACNSKVAFYNEIFLSVTAISCYDLLLFVF GGSHWFKQKIIGLWNCVLCR The following DNA sequence nGPcR-40 <SEQ ID NO.","83> was identified in H. sapiens: GCAGGAGCACTGAAAATCAGGAACAATCCTGTATTTTTTGTGATAATCAACAAGGACAAAACTTCTCCATATG TAAATAACAGCGTTATGAGCAGCAATTCATCCCTGCTGGTGGCTGTGCAGCTGTGCTACGCGAACGTGAATGG GTCCTGTGTGAAAATCCCCTTCTCGCCGGGATCCCGGGTGATTCTGTACATAGTGTTTGGCTTTGGGGCTGTG CTGGCTGTGTTTGGAAACCTCCTGGTGATGATTTCAATCCTCCATTTCAAGCAGCTGCACTCTCCGACCAATT TTCTCGTTGCCTCTCTGGCCTGCGCTGATTTCTTGGTGGGTGTGACTGTGATGCCCTTCAGCATGGTCAGGAC GGTGGAGAGCTGCTGGTATTTTGGGAGGAGTTTTTGTACTTTCCACACCTGCTGTGATGTGGCATTTTGTTAC TCTTCTCTCTTTCACTTGTGCTTCATCTCCATCGACAGGTACATTGCGGTTACTGACCCCCTGGTCTATCCTA CCAAGTTCACCGTATCTGTGTCAGGAATTTGCATCAGCGTGTCCTGGATCCTGCCCCTCATGTACAGCGGTGC TGTGTTCTACACAGGTGTCTATGACGATGGGCTGGAGGAATTATCTGATGCCCTAAACTGTATAGGAGGTTGT CAGACCGTTGTAAATCAAAACTGGGTGTTGACAGATTTTCTATCCTTCTTTATACCTACCTTTATTATGATAA TTCTGTATGGTAACATATTTCTTGTGGCTAGACGACAGGCGAAAAAGATAGAAAATACTGGTAGCAAGACAGA ATCATCCTCAGAGAGTTACAAAGCCAGAGTGGCCAGGAGAGAGAGAAAAGCAGCTAAAACCCTGGGGGTCACA GTGGTAGCATTTATGATTTCATGGTTACCATATAGCATTGATTCATTAATTGATGCCTTTATGGGCTTTATAA CCCCTGCCTGTATTTATGAGATTTGCTGTTGGTGTGCTTATTATAACTCAGCCATGAATCCTTTGATTTATGC TTTATTTTACCCATGGTTTAGGAAAGCAATAAAAGTTATTGTAACTGGTCAGGTTTTAAAGAACAGTTCAGCA ACCATGAATTTGTTTTCTGAACATATATAA The following amino acid sequence <SEQ ID NO.","84> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","83: MSSNSSLLVAVQLCYANVNGSCVKIPFSPGSRVILYIVFGFGAVLAVFGNLLVMISILHFKQLHSPTNFLVAS LACADFLVGVTVMPFSMVRTVESCWYFGRSFCTFHTCCDVAFCYSSLFHLCFISIDRYIAVTDPLVYPTKFTV SVSGICISVSWILPLMYSGAVFYTGVYDDGLEELSDALNCIGGCQTVVNQNWVLTDFLSFFIPTFIMIILYGN IFLVARRQAKKIENTGSKTESSSESYKARVARRERKAAKTLGVTVVAFMISWLPYSIDSLIDAFMGFITPACI YEICCWCAYYNSAMNPLIYALFYPWFRKAIKVIVTGQVLKNSSATMNLFSEHI The following DNA sequence nGPCR-54 <SEQ ID NO.","85> was identified in H. sapiens: ACCATGAATGAGCCACTAGACTATTTAGCAAATGCTTCTGATTTCCCCGATTATGCAGCTGCTTTTGGAAATT GCACTGATGAAAACATCCCACTCAAGATGCACTACCTCCCTGTTATTTATGGCATTATCTTCCTCGTGGGATT TCCAGGCAATGCAGTAGTGATATCCACTTACATTTTCAAAATGAGACCTTGGAAGAGCAGCACCATCATTATG CTGAACCTGGCCTGCACAGATCTGCTGTATCTGACCAGCCTCCCCTTCCTGATTCACTACTATGCCAGTGGCG AAAACTGGATCTTTGGAGATTTCATGTGTAAGTTTATCCGCTTCAGCTTCCATTTCAACCTGTATAGCAGCAT CCTCTTCCTCACCTGTTTCAGCATCTTCCGCTACTGTGTGATCATTCACCCAATGAGCTGCTTTTCCATTCAC AAAACTCGATGTGCAGTTGTAGCCTGTGCTGTGGTGTGGATCATTTCACTGGTAGCTGTCATTCCGATGACCT TCTTGATCACATCAACCAACAGGACCAACAGATCAGCCTGTCTCGACCTCACCAGTTCGGATGAACTCAATAC TATTAAGTGGTACAACCTGATTTTGACTGCAAGTACTTTCTGCCTCCCCTTGGTGATAGTGACACTTTGCTAT ACCACGATTATCCACACTTTGACCCATGGACTGCAAACTGACAGCTGCCTTAAGCAGAAAGCACGAAGGCTAA CCATTCTGCTACTCCTTGCATTTTACGTATGTTTTTTACCCTTCCATATCTTGAGGGTCATTCAGGATCGAAT CTCAGCCTGCTTTCAATCAGTTGTTCCATTGAGAATCAGATCCATGAAGCTTACATCGTTTCTAGACCATTAT GCTGCTCTGAACACCTTTGGTAACCTGTTACTATATGTGGTGGTCAGCGACAACTTTCAGCAGGCTGTCTGCT CAACAGTGAGATGCAAAGTAAGCGGGAACCTTGAGCAAGCAAAGAAAATTAGTTACTCAAACAACCCTTGA The following amino acid sequence <SEQ ID NO.","86> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","85: MNEPLDYLANASDFPDYAAAFGNCTDENIPLKMHYLPVIYGIIFLVGFPGNAVVISTYIFKMRPWKSSTIIML NLACTDLLYLTSLPFLIHYYASGENWIFGDFMCKFIRFSFHFNLYSSILFLTCFSIFRYCVIIHPMSCFSIHK TRCAVVACAVVWIISLVAVIPMTFLITSTNRTNRSACLDLTSSDELNTIKWYNLILTASTFCLPLVIVTLCYT TIIHTLTHGLQTDSCLKQKARRLTILLLLAFYVCFLPFHILRVIQDRISACFQSVVPLRIRSMKLTSFLDHYA ALNTFGNLLLYVVVSDNFQQAVCSTVRCKVSGNLEQAKKISYSN The following DNA sequence nGPCR-56 <SEQ ID NO.","87> was identified in H. sapiens: AAAAATTGCTGTACTGAACTATTGAATGGAACTTGGAAATAAAGTCCCTTCCAAAATAACTATTCTTCAACAG AGAGTAATAGGTAAATGTTTTAGAAGTGAGAGGACTCAAATTGCCAATGATTTACTCTTTTATTTTTCCTCCT AGGTTTCTGGGATAAGTATGTGCAAATAAAAAATAAACATGAGAAGGAACTGTAACCTGATTATGGATTTGGG AAAAAGATAAATCAACACACAAAGGGAAAAGTAAACTGATTGACAGCCCTCAGGAATGATGCCCTTTTGCCAC AATATAATTAATATTTCCTGTGTGAAAAACAACTGGTCAAATGATGTCCGTGCTTCCCTGTACAGTTTAATGG TGCTCATAATTCTGACCACACTCGTTGGCAATCTGATAGTTATTGTTTCTATATCACACTTCAAACAACTTCA TACCCCAACAAATTGGCTCATTCATTCCATGGCCACTGTGGACTTTCTTCTGGGGTGTCTGGTCATGCCTTAC AGTATGGTGAGATCTGCTGAGCACTGTTGGTATTTTGGAGAAGTCTTCTGTAAAATTCACACAAGCACCGACA TTATGCTGAGCTCAGCCTCCATTTTCCATTTGTCTTTCATCTCCATTGACCGCTACTATGCTGTGTGTGATCC ACTGAGATATAAAGCCAAGATGAATATCTTGGTTATTTGTGTGATGATCTTCATTAGTTGGAGTGTCCCTGCT GTTTTTGCATTTGGAATGATCTTTCTGGAGCTAAACTTCAAAGGCGCTGAAGAGATATATTACAAACATGTTC ACTGCAGAGGAGGTTGCTCTGTCTTCTTTAGCAAAATATCTGGGGTACTGACCTTTATGACTTCTTTTTATAT ACCTGGATCTATTATGTTATGTGTCTATTACAGAATATATCTTATCGCTAAAGAACAGGCAAGATTAATTAGT GATGCCAATCAGAAGCTCCAAATTGGATTGGAAATGAAAAATGGAATTTCACAAAGCAAAGAAAGGAAAGCTG TGAAGACATTGGGGATTGTGATGGGAGTTTTCCTAATATGCTGGTGCCCTTTCTTTATCTGTACAGTCATGGA CCCTTTTCTTCACTACATTATTCCACCTACTTTGAATGATGTA The following amino acid sequence <SEQ ID NO.","88> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","87: MMPFCHNIINISCVKNNWSNDVRASLYSLMVLIILTTLVGNLIVIVSISHFKQLHTPTNWLIHSMATVDFLLG CLVMPYSMVRSAEHCWYFGEVFCKIHTSTDIMLSSASIFHLSFISIDRYYAVCDPLRYKAKMNILVICVMIFI SWSVPAVFAFGMIFLELNFKGAEEIYYKHVHCRGGCSVFFSKISGVLTFMTSFYIPGSIMLCVYYRIYLIAKE QARLISDANQKLQIGLEMKNGISQSKERKAVKTLGIVMGVFLICWCPFFICTVMDPFLHYIIPPTLNDARGSR ANSA The following DNA sequence nGPCR-56 <SEQ ID NO.","89> was identified in H. sapiens: GGAATGATGCCCTTTTGCCACAATATAATTAATATTTCCTGTGTGAAAAACAACTGGTCAAATGATGTCCGTG CTTCCCTGTACAGTTTAATGGTGCTCATAATTCTGACCACACTCGTTGGCAATCTGATAGTTATTGTTTCTAT ATCACACTTCAAACAACTTCATACCCCAACAAATTGGCTCATTCATTCCATGGCCACTGTGGACTTTCTTCTG GGGTGTCTGGTCATGCCTTACAGTATGGTGAGATCTGCTGAGCACTGTTGGTATTTTGGAGAAGTCTTCTGTA AAATTCACACAAGCACCGACATTATGCTGAGCTCAGCCTCCATTTTCCATTTGTCTTTCATCTCCATTGACCG CTACTATGCTGTGTGTGATCCACTGAGATATAAAGCCAAGATGAATATCTTGGTTATTTGTGTGATGATCTTC ATTAGTTGGAGTGTCCCTGCTGTTTTTGCATTTGGAATGATCTTTCTGGAGCTAAACTTCAAAGGCGCTGAAG AGATATATTACAAACATGTTCACTGCAGAGGAGGTTGCTCTGTCTTCTTTAGCAAAATATCTGGGGTACTGAC CTTTATGACTTCTTTTTATATACCTGGATCTATTATGTTATGTGTCTATTACAGAATATATCTTATCGCTAAA GAACAGGCAAGATTAATTAGTGATGCCAATCAGAAGCTCCAAATTGGATTGGAAATGAAAAATGGAATTTCAC AAAGCAAAGAAAGGAAAGCTGTGAAGACATTGGGGATTGTGATGGGAGTTTTCCTAATATGCTGGTGCCCTTT CTTTATCTGTACAGTCATGGACCCTTTTCTTCACTACATTATTCCACCTACTTTGAATGATGTATTGATTTGG TTTGGCTACTTGAACTCTACATTTAATCCAATGGTTTATGCATTTTTCTATCCTTGGTTTAGAAAAGCACTGA AGATGATGCTGTTTGGTAAAATTTTCCAAAAAGATTCATCCAGGTGTAAATTATTTTTGGAATTGAGTTCATA G The following amino acid sequence <SEQ ID NO.","90> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","89: MMPFCHNIINISCVKNNWSNDVRASLYSLMVLIILTTLVGNLIVIVSISHFKQLHTPTNWLIHSMATVDFLLG CLVMPYSMVRSAEHCWYFGEVFCKIHTSTDIMLSSASIFHLSFISIDRYYAVCDPLRYKAKMNILVICVMIFI SWSVPAVFAFGMIFLELNFKGAEEIYYKHVHCRGGCSVFFSKISGVLTFMTSFYIPGSIMLCVYYRIYLIAKE QARLISDANQKLQIGLEMKNGISQSKERKAVKTLGIVMGVFLICWCPFFICTVMDPFLHYIIPPTLNDVLIWF GYLNSTFNPMVYAFFYPWFRKALKMMLFGKIFQKDSSRCKLFLELSS The following DNA sequence nGPCR-58 <SEQ ID NO.","91> was identified in H. sapiens: CTGTAAAGTAGATTGTATGAGGACTCCATGAGGTCATCCACTTCAAGTCCTTGGCATAGGATAATTACTCAAA AGGTGATGACAATGGCGCAGGGAGGGATGGTGACTTGCCTGGAGATGCACAGCACCGTCTCTCCCATACTCGG TCATTCACACCATCATTGATTCACCAGGCACCACTCCGTGTCCAGCAGGACTCTGGGGACCCCAAATGGACAC TACCATGGAAGCTGACCTGGGTGCCACTGGCCACAGGCCCCGCACAGAGCTTGATGATGAGGACTCCTACCCC CAAGGTGGCTGGGACACGGTCTTCCTGGTGGCCCTGCTGCTCCTTGGGCTGCCAGCCAATGGGTTGATGGCGT GGCTGGCCGGCTCCCAGGCCCGGCATGGAGCTGGCACGCGTCTGGCGCTGCTCCTGCTCAGCCTGGCCCTCTC TGACTTCTTGTTCCTGGCAGCAGCGGCCTTCCAGATCCTAGAGATCCGGCATGGGGGACACTGGCCGCTGGGG ACAGCTGCCTGCCGCTTCTACTACTTCCTATGGGGCGTGTCCTACTCCTCCGGCCTCTTCCTGCTGGCCGCCC TCAGCCTCGACCGCTGCCTGCTGGCGCTGTGCCCACACTGGTACCCTGGGCACCGCCCAGTCCGCCTGCCCCT CTGGGTCTGCGCCGGTGTCTGGGTGCTGGCCACACTCTTCAGCGTGCCCTGGCTGGTCTTCCCCGAGGCTGCC GTCTGGTGGTACGACCTGGTCATCTGCCTGGACTTCTGGGACAGCGAGGAGCTGTCGCTGAGGATGCTGGAGG TCCTGGGGGGCTTCCTGCCTTTCCTCCTGCTGCTCGTCTGCCACGTGCTCACCCAGGCCACAGCCTGTCGCAC CTGCCACCGCCAACAGCAGCCCGCAGCCTGCCGGGGCTTCGCCCGTGTGGCCAGGACCATTCTGTCAGCCTAT GTGGTCCTGAGGCTGCCCTACCAGCTGGCCCAGCTGCTCTACCTGGCCTTCCTGTGGGACGTCTACTCTGGCT ACCTGCTCTGGGAGGCCCTGGTCTACTCCGACTACCTGATCCTACTCAACAGCTGCCTCAGCCCCTTCCTCTG CCTCATGGCCAGTGCCGACCTCCGGACCCTGCTGCGCTCCGTGCTCTCGTCCTTCGCGGCAGCTCTCTGCGAG GAGCGGCCGGGCAGCTTCACGCCCACTGAGCCACAGACCCAGCTAGATTCTGAGGGTCCAACTCTGCCAGAGC CGATGGCAGAGGCCCAGTCACAGATGGATCCTGTGGCCCAGCCTCAGGTGAACCCCACACTCCAGCCACGATC GGATCCCACAGCTCAGCCACAGCTGAACCCTACGGCCCAGCCACAGTCGGATCCCACAGCCCAGCCACAGCTG AACCTCATGGCCCAGCCACAGTCAGATTCTGTGGCCCAGCCACAGGCAGACACTAACGTCCAGACCCCTGCAC CTGCTGCCAGTTCTGTGCCCAGTCCCTGTGATGAAGCTTCCCCAACCCCATCCTCGCATCCTACCCCAGGGGC CCTTGAGGACCCAGCCACACCTCCTGCCTCTGAAGGAGAAAGCCCCAGCAGCACCCCGCCAGAGGCGGCCCCG GGCGCAGGCCCCACGTGAGGGTCCAGGAACACGCAGGCCCACCAGAGCAGTGAAAGAGCCCAGGGCAGACAGA GGAACCAGCCAGTCAGA The following amino acid seqence <SEQ ID NO.","92> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","91: LAWRCTAPSLPYSVIHTIIDSPGTTPCPAGLWGPQMDTTMEADLGATGHRPRTELDDEDSYPQGGWDTVFLVA LLLLGLPANGLMAWLAGSQARHGAGTRLALLLLSLALSDFLFLAAAAFQILEIRHGGHWPLGTAACRFYYFLW GVSYSSGLFLLAALSLDRCLLALCPHWYPGHRPVRLPLWVCAGVWVLATLFSVPWLVFPEAAVWWYDLVICLD FWDSEELSLRMLEVLGGFLPFLLLLVCHVLTQATACRTCHRQQQPAACRGFARVARTILSAYVVLRLPYQLAQ LLYLAFIMDVYSGYLLWEALVYSDYLILLNSCLSPFLCLMASADLRTLLRSVLSSFAAALCEERPGSFTPTEP QTQLDSEGPTLPEPMAEAQSQMDPVAQPQVNPTLQPRSDPTAQPQLNPTAQPQSDPTAQPQLNLMAQPQSDSV AQPQADTNVQTPAPAASSVPSPCDEASPTPSSHPTPGALEDPATPPASEGESPSSTPPEAAPGAGPT The following DNA sequence nGPCR-58 <SEQ ID NO.","93> was identified in H. sapiens: ATGGACACTACCATGGAAGCTGACCTGGGTGCCACTGGCCACAGGCCCCGCACAGAGCTTGATGATGAGGACT CCTACCCCCAAGGTGGCTGGGACACGGTCTTCCTGGTGGCCCTGCTGCTCCTTGGGCTGCCAGCCA ATGGGTTGATGGCGTGGCTGGCCGGCTCCCAGGCCCGGCATGGAGCTGGCACGCGTCTGGCGCTGCTCCTGCT CAGCCTGGCCCTCTCTGACTTCTTGTTCCTGGCAGCAGCGGCCTTCCAGATCCTAGAGATCCGGCATGGGGGA CACTGGCCGCTGGGGACAGCTGCCTGCCGCTTCTACTACTTCCTATGGGGCGTGTCCTACTCCTCCGGCCTCT TCCTGCTGGCCGCCCTCAGCCTCGACCGCTGCCTGCTGGCGCTGTGCCCACACTGGTACCCTGGGCACCGCCC AGTCCGCCTGCCCCTCTGGGTCTGCGCCGGTGTCTGGGTGCTGGCCACACTCTTCAGCGTGCCCTGGCTGGTC TTCCCCGAGGCTGCCGTCTGGTGGTACGACCTGGTCATCTGCCTGGACTTCTGGGACAGCGAGGAGCTGTCGC TGAGGATGCTGGAGGTCCTGGGGGGCTTCCTGCCTTTCCTCCTGCTGCTCGTCTGCCACGTGCTCACCCAGGC CACAGCCTGTCGCACCTGCCACCGCCAACAGCAGCCCGCAGCCTGCCGGGGCTTCGCCCGTGTGGCCAGGACC ATTCTGTCAGCCTATGTGGTCCTGAGGCTGCCCTACCAGCTGGCCCAGCTGCTCTACCTGGCCTTCCTGTGGG ACGTCTACTCTGGCTACCTGCTCTGGGAGGCCCTGGTCTACTCCGACTACCTGATCCTACTCAACAGCTGCCT CAGCCCCTTCCTCTGCCTCATGGCCAGTGCCGACCTCCGGACCCTGCTGCGCTCCGTGCTCTCGTCCTTCGCG GCAGCTCTCTGCGAGGAGCGGCCGGGCAGCTTCACGCCCACTGAGCCACAGACCCAGCTAGATTCTGAGGGTC CAACTCTGCCAGAGCCGATGGCAGAGGCCCAGTCACAGATGGATCCTGTGGCCCAGCCTCAGGTGAACCCCAC ACTCCAGCCACGATCGGATCCCACAGCTCAGCCACAGCTGAACCCTACGGCCCAGCCACAGTCGGATCCCACA GCCCAGCCACAGCTGAACCTCATGGCCCAGCCACAGTCAGACTCTGTGGCCCAGCCACAGGCAGACACTAACG TCCAGACCCCTGCACCTGCTGCCAGTTCTGTGCCCAGTCCCTGTGATGAAGCTTCCCCAACCCCATCCTCGCA TCCTACCCCAGGGGCCCTTGAGGACCCAGCCACACCTCCTGCCTCTGAAGGAGAAAGCCCCAGCAGCACCCCG CCAGAGGCGGCCCCGGGCGCAGGCCCCACGTGA The following amino acid sequence <SEQ ID NO.","94> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","93: MDTTMEADLGATGHRPRTELDDEDSYPQGGWDTVFLVALLLLGLPANGLMAWLAGSQARHGAGTRLALLLLSL ALSDFLFLAAAAFQILEIRHGGHWPLGTAACRFYYFLWGVSYSSGLFLLAALSLDRCLLALCPHWYPGHRPVR LPLWVCAGVWVLATLFSVPWLVFPEAAVWWYDLVICLDFWDSEELSLRMLEVLGGFLPFLLLLVCHVLTQATA CRTCHRQQQPAACRGFARVARTILSAYVVLRLPYQLAQLLYLAFLWDVYSGYLLWEALVYSDYLILLNSCLSP FLCLMASADLRTLLRSVLSSFAAALCEERPGSFTPTEPQTQLDSEGPTLPEPMAEAQSQMDPVAQPQVNPTLQ PRSDPTAQPQLNPTAQPQSDPTAQPQLNLMAQPQSDSVAQPQADTNVQTPAPAA The following DNA sequence nGPCR-3 <SEQ ID NO.","185> was identified in H. sapiens: AGGCTCGCGCCCGAAGCAGAGCCATGAGAACCCCAGGGTGCCTGGCGAGCCGCTAGCGCCATGGGCCCCGGCG AGGCGCTGCTGGCGGGTCTCCTGGTGATGGTACTGGCCGTGGCGCTGCTATCCAACGCACTGGTGCTGCTTTG TTGCGCCTACAGCGCTGAGCTCCGCACTCGAGCCTCAGGCGTCCTCCTGGTGAATCTGTCTCTGGGCCACCTG CTGCTGGCGGCGCTGGACATGCCCTTCACGCTGCTCGGTGTGATGCGCGGGCGGACACCGTCGGCGCCCGGCG CATGCCAAGTCATTGGCTTCCTGGACACCTTCCTGGCGTCCAACGCGGCGCTGAGCGTGGCGGCGCTGAGCGC AGACCAGTGGCTGGCAGTGGGCTTCCCACTGCGCTACGCCGGACGCCTGCGACCGCGCTATGCCGGCCTGCTG CTGGGCTGTGCCTGGGGACAGTCGCTGGCCTTCTCAGGCGCTGCACTTGGCTGCTCGTGGCTTGGCTACAGCA GCGCCTTCGCGTCCTGTTCGCTGCGCCTGCCGCCCGAGCCTGAGCGTCCGCGCTTCGCAGCCTTCACCGCCAC GCTCCATGCCGTGGGCTTCGTGCTGCCGCTGGCGGTGCTCTGCCTCACCTCGCTCCAGGTGCACCGGGTGGCA CGCAGACACTGCCAGCGCATGGACACCGTCACCATGAAGGCGCTCGCGCTGCTCGCCGACCTGCACCCCAGTG TGCGGCAGCGCTGCCTCATCCAGCAGAAGCGGCGCCGCCACCGCGCCACCAGGAAGATTGGCATTGCTATTGC GACCTTCCTCATCTGCTTTGCCCCGTATGTCATGACCAGGCTGGCGGAGCTCGTGCCCTTCGTCACCGTGAAC GCCCAGTGGGGCATCCTCAGCAAGTGCCTGACCTACAGCAAGGCGGTGGCCGACCCGTTCACGTACTCTCTGC TCCGCCGGCCGTTCCGCCAAGTCCTGGCCGGCATGGTGCACCGGCTGCTGAAGAGAACCCCGCGCCCAGCATC CACCCATGACAGCTCTCTGGATGTGGCCGGCATGGTGCACCAGCTGCTGAAGAGAACCCCGCGCCCAGCGTCC ACCCACAACGGCTCTGTGGACACAGAGAATGATTCCTGCCTGCAGCAGACACACTGAGGGCCTGGCAGGGCTC ATCGCCCCCACCTTCTAAGA The following amino acid sequence <SEQ ID NO.","186> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","185: MGPGEALLAGLLVMVLAVALLSNALVLLCCAYSAELRTRASGVLLVNLSLGHLLLAALDMPFTLLGVMRGRTP SAPGACQVIGFLDTFLASNAALSVAALSADQWLAVGFPLRYAGRLRPRYAGLLLGCAWGQSLAFSGAALGCSW LGYSSAFASCSLRLPPEPERPRFAAFTATLHAVGFVLPLAVLCLTSLQVHRVARRHCQRMDTVTMKALALLAD LHPSVRQRCLIQQKRRRHRATRKIGIAIATFLICFAPYVMTRLAELVPFVTVNAQWGILSKCLTYSKAVADPF TYSLLRRPFRQVLAGMVHRLLKRTPRPASTHDSSLDVAGMVHQLLKRTPRPASTHNGSVDTENDSCLQQTH The following DNA sequence nGPCR-58 <SEQ ID NO.","93> was identified in H. sapiens: ATGGACACTACCATGGAAGCTGACCTGGGTGCCACTGGCCACAGGCCCCGCACAGAGCTTGATGATGAGGACT CCTACCCCCAAGGTGGCTGGGACACGGTCTTCCTGGTGGCCCTGCTGCTCCTTGGGCTGCCAGCCA ATGGGTTGATGGCGTGGCTGGCCGGCTCCCAGGCCCGGCATGGAGCTGGCACGCGTCTGGCGCTGCTCCTGCT CAGCCTGGCCCTCTCTGACTTCTTGTTCCTGGCAGCAGCGGCCTTCCAGATCCTAGAGATCCGGCATGGGGGA CACTGGCCGCTGGGGACAGCTGCCTGCCGCTTCTACTACTTCCTATGGGGCGTGTCCTACTCCTCCGGCCTCT TCCTGCTGGCCGCCCTCAGCCTCGACCGCTGCCTGCTGGCGCTGTGCCCACACTGGTACCCTGGGCACCGCCC AGTCCGCCTGCCCCTCTGGGTCTGCGCCGGTGTCTGGGTGCTGGCCACACTCTTCAGCGTGCCCTGGCTGGTC TTCCCCGAGGCTGCCGTCTGGTGGTACGACCTGGTCATCTGCCTGGACTTCTGGGACAGCGAGGAGCTGTCGC TGAGGATGCTGGAGGTCCTGGGGGGCTTCCTGCCTTTCCTCCTGCTGCTCGTCTGCCACGTGCTCACCCAGGC CACAGCCTGTCGCACCTGCCACCGCCAACAGCAGCCCGCAGCCTGCCGGGGCTTCGCCCGTGTGGCCAGGACC ATTCTGTCAGCCTATGTGGTCCTGAGGCTGCCCTACCAGCTGGCCCAGCTGCTCTACCTGGCCTTCCTGTGGG ACGTCTACTCTGGCTACCTGCTCTGGGAGGCCCTGGTCTACTCCGACTACCTGATCCTACTCAACAGCTGCCT CAGCCCCTTCCTCTGCCTCATGGCCAGTGCCGACCTCCGGACCCTGCTGCGCTCCGTGCTCTCGTCCTTCGCG GCAGCTCTCTGCGAGGAGCGGCCGGGCAGCTTCACGCCCACTGAGCCACAGACCCAGCTAGATTCTGAGGGTC CAACTCTGCCAGAGCCGATGGCAGAGGCCCAGTCACAGATGGATCCTGTGGCCCAGCCTCAGGTGAACCCCAC ACTCCAGCCACGATCGGATCCCACAGCTCAGCCACAGCTGAACCCTACGGCCCAGCCACAGTCGGATCCCACA GCCCAGCCACAGCTGAACCTCATGGCCCAGCCACAGTCAGACTCTGTGGCCCAGCCACAGGCAGACACTAACG TCCAGACCCCTGCACCTGCTGCCAGTTCTGTGCCCAGTCCCTGTGATGAAGCTTCCCCAACCCCATCCTCGCA TCCTACCCCAGGGGCCCTTGAGGACCCAGCCACACCTCCTGCCTCTGAAGGAGAAAGCCCCAGCAGCACCCCG CCAGAGGCGGCCCCGGGCGCAGGCCCCACGTGA The following amino acid sequence <SEQ ID NO.","94> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","93: MDTTMEADLGATGHRPRTELDDEDSYPQGGWDTVFLVALLLLGLPANGLMAWLAGSQARHGAGTRLALLLLSL ALSDFLFLAAAAFQILEIRHGGHWPLGTAACRFYYFLWGVSYSSGLFLLAALSLDRCLLALCPHWYPGHRPVR LPLWVCAGVWVLATLFSVPWLVFPEAAVWWYDLVICLDFWDSEELSLRMLEVLGGFLPFLLLLVCHVLTQATA CRTCHRQQQPAACRGFARVARTILSAYVVLRLPYQLAQLLYLAFLWDVYSGYLLWEALVYSDYLILLNSCLSP FLCLMASADLRTLLRSVLSSFAAALCEERPGSFTPTEPQTQLDSEGPTLPEPMAEAQSQMDPVAQPQVNPTLQ PRSDPTAQPQLNPTAQPQSDPTAQPQLNLMAQPQSDSVAQPQADTNVQTPAPAA The following DNA sequence nGPCR-14 <SEQ ID NO.","191> was identified in H. sapiens: ACTAACTTTGGGAACTCGTATAGACCCAGCGTCGCTCCCCGCGCCGCCTCGCCTCCACTTTGGTTTCCCGCGT CCTGCCCGCCCTCTTCGGTGCCTCCTCTTCCTCCGGGACAAGGATGGAGGATCTCTTTAGCCCCTCAATTCTG CCGCCGGCGCCCAACATTTCCGTGCCCATCTTGCTGGGCTGGGGTCTCAACCTGACCTTGGGGCAAGGACCCC CTGCCTCTGGGCCGCCCAGCCCGCGTGCGGGGGCACGGCGCTGTCACAGCTGGCCTGGGAACTGCTGGGCGAG CCCCGCGCGGCCACGGGGGACCTGGCGTGCCGCTTCCTGCAGCTGCTGCAGGCATCCGGGCGGGGCGCCTCGG CCCACCTAGTGGTGCTCATCGCCCTCGAGCGCCGGCGCGCGGTGCGTCTTCCGCACGGCCGGCCGCTGCCCGC GCGTGCCCTCGCCGCCCTGGGCTGGCTGCTGGCACTGCTGCTGGCGCTGCCCCCGGCCTTCGTGGTGCGCGGG GACTCCCCCTCGCCGCTGCCGCCGCCGCCGCCGCCAACGTCCCTGCAGCCAGGCGCGCCCCCGGCCGCCCGCG CCTGGCCGGGGGAGCGTCGCTGCCACGGGATCTTCGCGCCCCTGCCGCGCTGGCACCTGCAGGTCTACGCGTT CTACGAGGCCGTCGCGGGCTTCGTCGCGCCTGTTACGGTCCTGGGCGTCGCTTGCGGCCACCTACTCTCCGTC TGGTGGCGGCACCGGCCGCAGGCCCCCGCGGCTGCAGCGCCCTGGTCGGCGAGCCCAGGTCGAGCCCCTGCGC CCAGCGCGCTGCCCCGCGCCAAGGTGCAGAGCCTGAAGATGAGCCTGCTGCTGGCGCTGCTGTTCGTGGGCTG CGAGCTGCCCTACTTTGCCGCCCGGCTGGCGGCCGCGTGGTCGTCCGGGCCCGCGGGAGACTGGGAGGGAGAG GGCCTGTCGGCGGCGCTGCGCGTGgTGGCGATGGCCAACAGCGCTCTCAATCCCTTCGTCTACCTCTTCTTCC AGGCGGGCGACTGCTGGCTCCGGCGACAGCTGCGGAAGCGGCTGGGCTCTCTGTGCTGCGCGCCGCAGGGAGG CGCGGAGGACGAGGAGGGGCCCCGGGGCCACCAGGCGCTCTACCGCCAACGCTGGCCCCACCCTCATTATCAC CATGCTCGGCGGGAACCCGCTGGACGAGGGCGGCTTGCGCCCACCCCCTCCGCGCCCCAGACCCCTGCCTTGC TCCTGCGAAAGTGCCTTCTAGGTGCTTGGTGGTCAGAGACGGGTCATCTGTCGCTAAGGCGCAACCTCCAGGG AACTCGAGGCCTGCCAGGGTCTGTCCAGATCACAAGGGGCAGGAGAGTCTGTGAGAGAGTGACACTGAAGTTG TCCCCTTCCTCCACTCTCCTATTCCCTTCTCATGTTTACATTTCCCTATGCTCTTCCAGTTTCTCTTCTTCCC TACAGTTCCTCTCATATCTCCCCATTTGGAGACAGTGAGCCACTGGAAAGTTGTAAAAACAAAAACAGTTATT TTTGCAGTTTTCTTTCACGCATTTATAGTGCTCTGGATAATGCCATTTATTTTTGCTGATTACCCAACTTTCA GTATTTGCTGTGTTATCATCTGTATTTACTTATTTTGA The following amino acid sequence <SEQ ID NO.","192> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.","191: MEDLFSPSILPPAPNISVPILLGWGLNLTLGQGAPASGPPSRRVRLVFLGVILVVAVAGNTTVLCRLCGGGG PWAGPKRRKMDFLLVQLALADLYACGGTALSQLAWELLGEPRAATGDLACRFLQLLQASGRGASAHLVVLIA LERRRAVRLPHGRPLPARALAALGWLLALLLALPPAFVVRGDSPSPLPPPPPPTSLQPGAPPAARAWPGERR CHGIFAPLPRWHLQVYAFYEAVAGFVAPVTVLGVACGHLLSVWWRHRPQAPAAAAPWSASPGRAPAPSALPR AKVQSLKMSLLLALLFVGCELPYFAARLAAAWSSGPAGDWEGEGLSAALRVVAMANSALNPFVYLFFQAGDC WLRRQLRKRLGSLCCAPQGGAEDEEGPRGHQALYRQRWPHPHYHHARREPAGRGRLAPTPSAPQTPALLLRK CLLGAWWSETGHLSLRRNLQGTRGLPGSVQITRGRRVCERVTLKLSPSSTLLFPSHVYISLCSSSFSSSLQF LSYLPIWRQ Example 2 Cloning of nGPCR-x To isolate a cDNA clone encoding full length nGPCR-x, a DNA fragment corresponding to a nucleotide sequence set forth in odd numbered nucleotide sequences ranging from SEQ ID NO: 1-93, or a portion thereof, can be used as a probe for hybridization screening of a phage cDNA library.","The DNA fragment is amplified by the polymerase chain reaction (PCR) method.","The PCR reaction mixture of 50 μl contains polymerase mixture (0.2 mM dNTPs, 1×PCR Buffer and 0.75 μl Expand High Fidelity Polymerase (Roche Biochemicals)), 1 μg of plasmid, and 50 pmoles of forward primer and 50 pmoles of reverse primer.","The primers are preferably 10 to 25 nucleotides in length and are determined by procedures well known to those skilled in the art.","Amplification is performed in an Applied Biosystems PE2400 thermocycler, using the following program: 95° C. for 15 seconds, 52° C. for 30 seconds and 72° C. for 90 seconds; repeated for 25 cycles.","The amplified product is separated from the plasmid by agarose gel electrophoresis, and purified by Qiaquick™ gel extraction kit (Qiagen).","A lambda phage library containing cDNAs cloned into lambda ZAPII phagovector is plated with E. coli XL-1 blue host, on 15 cm LB-agar plates at a density of 50,000 pfu per plate, and grown overnight at 37° C.; (plated as described by Sambrook et al., supra).","Phage plaques are transferred to nylon membranes (Amersham Hybond NJ), denatured for 2 minutes in denaturation solution (0.5 M NaOH, 1.5 M NaCl), renatured for 5 minutes in renaturation solution (1 M Tris pH 7.5, 1.5 M NaCl), and washed briefly in 2×SSC (20×SSC: 3 M NaCl, 0.3 M Na-citrate).","Filter membranes are dried and incubated at 80° C. for 120 minutes to cross-link the phage DNA to the membranes.","The membranes are hybridized with a DNA probe prepared as described above.","A DNA fragment (25 ng) is labeled with α-32P-dCTP (NEN) using Rediprime™ random priming (Amersham Pharmacia Biotech), according to manufacturers instructions.","Labeled DNA is separated from unincorporated nucleotides by S200 spin columns (Amersham Pharmacia Biotech), denatured at 95° C. for 5 minutes and kept on ice.","The DNA-containing membranes (above) are pre-hybridized in 50 ml ExpressHyb™ (Clontech) solution at 68° C. for 90 minutes.","Subsequently, the labeled DNA probe is added to the hybridization solution, and the probe is left to hybridize to the membranes at 68° C. for 70 minutes.","The membranes are washed five times in 2×SSC, 0.1% SDS at 42° C. for 5 minutes each, and finally washed 30 minutes in 0.1×SSC, 0.2% SDS.","Filters are exposed to Kodak XAR™ film (Eastman Kodak Company, Rochester, N.Y., USA) with an intensifying screen at −80° C. for 16 hours.","One positive colony is isolated from the plates, and replated with about 1000 pfu on a 15 cm LB plate.","Plating, plaque lift to filters and hybridization are performed as described above.","About four positive phage plaques are isolated form this secondary screening.","cDNA containing plasmids (pBluescript SK−) are rescued from the isolated phages by in vivo excision by culturing XL-1 blue cells co-infected with the isolated phages and with the Excision helper phage, as described by manufacturer (Stratagene).","XL-blue cells containing the plasmids are plated on LB plates and grown at 37° C. for 16 hours.","Colonies (18) from each plate are replated on LB plates and grown.","One colony from each plate is stricken onto a nylon filter in an ordered array, and the filter is placed on a LB plate to raise the colonies.","The filter is then hybridized with a labeled probe as described above.","About three positive colonies are selected and grown up in LB medium.","Plasmid DNA is isolated from the three clones by Qiagen Midi Kit™ (Qiagen) according to the manufacturer's instructions.","The size of the insert is determined by digesting the plasmid with the restriction enzymes NotI and SalI, which establishes an insert size.","The sequence of the entire insert is determined by automated sequencing on both strands of the plasmids.","nGPCR-1: PCR and Subcloning cDNAs were sequenced directly using an AB1377 fluorescence-based sequencer (Perkin Elmer/Applied Biosystems Division, PE/ABD, Foster City, Calif.) and the ABI PRISM Ready Dye-Deoxy Terminator kit with Taq FS polymerase.","Each ABI cycle sequencing reaction contained about 0.5 μg of plasmid DNA.","Cycle-sequencing was performed using an initial denaturation at 98° C. for 1 min, followed by 50 cycles: 99° C. for 30 sec, annealing at 50° C. for 30 sec, and extension at 60° C. for 4 min.","Temperature cycles and times were controlled by a Perkin-Elmer 9600 thermocycler.","Extension products were purified using AGTC® gel filtration block (Edge BiosSystems, Gaithersburg, Md.).","Each reaction product was loaded by pipette onto the column, which was then centrifuged in a swinging bucket centrifuge (Sorvall model RT6000B table top centrifuge) at 1500×g for 4 min at room temperature.","Column-purified samples were dried under vacuum for about 40 min and then dissolved in 5 μl of a DNA loading solution (83% deionized formamide, 8.3 mM EDTA, and 1.6 mg/ml Blue Dextran).","The samples were then heated to 90° C. for three min and loaded into the gel sample wells for sequence analysis by the ABI377 sequencer.","Sequence analysis was performed by importing ABI373A files into the Sequencher program (Gene Codes, Ann Arbor, Mich.).","The PCR reaction was performed in 50 μL samples containing 41.9 μL H2O, 5 μL 10× Buffer containing 15 mM MgCl2 (Boehringer Mannheim Expand High Fidelity PCR System), 0.5 μL 10 mM dNTP mix, 1.5 μL human genomic DNA (Clontech #6550-1, 0.1 μg/μL), 0.3 μL primer VR1A (1 μg/μL), 0.3 μL primer VR1B (1 μg/μL), and 0.5 μL High Fidelity Taq polymerase (Boehringer Mannheim, 3.5U/μl).","The primer sequences for and, respectively were: 5′TCAAAGCTTATGGAATCATCTTTCTCATTTGGAGTGATCCTTGCTGTC, (VR1A) (SEQ ID NO: 95) corresponding to the 5′ end of the coding region and containing a HindIII restriction site, and: 5′ TTCACTCGAGTTAGCCATCAAACTCTGAGCTGGAGATAGTGACGATGTG (VR1B) (SEQ ID NO: 96) corresponding to the 3′ end of the coding region and containing an XhoI restriction site (Genosys).","The PCR reaction was carried out using a GeneAmp PCR9700 thermocycler (Perkin Elmer Applied Biosystems) and started with 1 cycle of 94° C. for 2 min followed by 5 cycles at 94° C. for 30 sec, 60° C. for 2 min, 72° C. for 1.5 min, followed by 20 cycles at 94° C. for 30 sec, 60° C. for 30 sec, 72° C. for 1.5 min.","The PCR reaction was loaded onto a 0.75% agarose gel.","The DNA band was excised from the gel and the DNA eluted from the agarose using a QIAquick gel extraction kit (Qiagen).","The eluted DNA was ethanol-precipitated and resuspended in 4 μL H2O for ligation.","The ligation reaction consisted of 4 μL of fresh ethanol-precipitated PCR product and 1 μL of pCRII-TOPO vector (Invitrogen).","The reaction was gently mixed and allowed to incubate for 5 min.","at room temperature followed by the addition of 1 μL of 6×TOPO cloning stop solution and mixing for 10 sec.","at room temperature.","The sample was then placed on ice and 2 μL was transformed in 50 μL of One Shot cells (Invitrogen) and plated onto ampicillin plates.","Four white colonies were chosen and the presence of an insert was verified by PCR in the following manner.","Each colony was resuspended in 2 ml LB broth for 2 hrs.","A 500 μL aliquot was spun down in the microfuge, the supernatant discarded, and the pellet resuspended in 25 μL of H2O.","A 16 μL aliquot was removed and boiled for 5 min and the sample was placed on ice.","The sample was microfuged briefly to pellet any bacterial debris and PCR was carried out with 15 μL sample using primers VR1A and VR1B, described above.","Colonies from positive clones identified by PCR were used to inoculate a 4 ml culture of LB medium containing 100 μg/ml ampicillin.","Plasmid DNA was purified using the Wizard Plus Minipreps DNA purification system (Promega).","Since the primers used to amplify the fragment of nGPCR-1 from genomic DNA were engineered to have HindIII and XhoI sites, the cDNA obtained from the minipreps was digested with these restriction enzymes.","One clone was verified by gel electrophoresis to give a DNA band of the correct size.","cDNA from this clone was then sequenced, yielding the sequence of SEQ ID NO: 73.nGPCR-3: PCR and Subcloning First-strand cDNA synthesis was performed following the directions for 3′-RACE ready cDNA from the SMART™ RACE cDNA Amplification Kit (Clontech).","First 3 μl of H2O, 1 μl human whole brain poly A+ RNA (1 μg/μl) (Clontech, 6516-1) and 1 μl 3′-CDS primer were mixed together, incubated at 70° C. for 2 minutes, then placed on ice for 2 minutes.","Added to the tube was 2 μl 5× First-Strand buffer, 1 μl 20 mM DTT, 1 μl dNTP mix (10 mM) and 1 μl Superscript II RT (200 units/μl) (GIBCO/BRL).","The tube was incubated at 42° C. for 1.5 hours then the reaction was diluted with 250 μl of Tricine-EDTA buffer.","PCR was performed in a 50 μl reaction using components that come with the Advantage®-GC cDNA PCR Kit.","The PCR reaction contained 22.4 μl H2O, 10 μl 5×GC cDNA PCR Reaction buffer, 10 μl 5M GC Melt, 1 μl 50×dNTP mix (10 mM each), 5 μl human brain cDNA, 0.3 μl of LW1649 (SEQ ID NO: 187) (1 μg/μl), 0.3 μl of LW1650 (SEQ ID NO: 188) (1 μg/μl), 1 μl 50× Advantage-GC cDNA polymerase mix.","The PCR reaction was performed in a Perkin-Elmer 9600 GeneAmp PCR System starting with 1 cycle of 94° C. for 2 min then 8 cycles at 94° C. for 15 sec, 72° C. for 2 min (decreasing 1° C. with each cycle), 72° C. for 3 min, followed by 30 cycles of 94° C. for 15 sec, 68° C. for 3 min.","The PCR reaction was loaded onto a 1.2% agarose gel.","The DNA band was excised from the gel, placed in GenElute Agarose spin column (Supelco) and spun for 10 min at maximum speed in a microcentrifuge.","The eluted DNA was EtOH precipitated and resuspended in 4H2O for ligation.","The PCR primer sequence for LW1649 was: GCATAAGCTTGCCATGGGCCCCGGCGAGG (SEQ ID NO: 187) and for LW1650 was: GCATTCTAGACCTCAGTGTGTCTGCTGC (SEQ ID NO: 188).","The underlined portion of the primers matches the 5′ and 3′ areas, respectively, of the coding region.","The ligation reaction used solutions from the TOPO TA Cloning Kit (Invitrogen) which consisted of 4 μl PCR product DNA, 1 μl Salt Solution and 1 μl pCRII-TOPO vector that was incubated for 5 minutes at room temperature and then placed on ice.","Two microliters of the ligation reaction was transformed in One-Shot TOP10 cells (Invitrogen), and placed on ice for 30 minutes.","The cells were heat-shocked for 30 seconds at 42° C., placed on ice for two minutes, 250 μl of SOC was added, then incubated at 37° C. with shaking for one hour and then plated onto ampicillin plates.","A single colony containing an insert was used to inoculate a 5 ml culture of LB medium.","Plasmid DNA was purified using a Concert Rapid Plasmid Miniprep System (GibcoBRL) and then sequenced.","The DNA subcloned into pCRII-TOPO was sequenced using the ABI PRISM™ 310 Genetic Analyzer (PE Applied Biosystems) which uses advanced capillary electrophoresis technology and the ABI PRISM™ BigDye™ Terminator Cycle Sequencing Ready Reaction Kit.","Each cycle-sequencing reaction contained 6 μl of H2O, 8 μl of BigDye Terminator mix, 5 μl mini-prep DNA (0.1 μg/μl), and 1 μl primer (25 ng/μl) and was performed in a Perkin-Elmer 9600 thermocycler with 25 cycles of 96° C. for 10 sec, 50° C. for 10 sec, and 60° C. for 4 min.","The product was purified using a Centriflex™ gel filtration cartridge, dried under vacuum, then dissolved in 16 μl of Template Suppression Reagent (PE Applied Biosystems).","The samples were heated at 95° C. for 5 min then placed in the 310 Genetic Analyzer, yielding the sequence of SEQ ID NO: 95.nGPCR-9: PCR and Subcloning The PCR reaction was performed in 50 μl containing 34.5 μl H2O, 5 μl Buffer II (PE Applied Biosystems AmpliTaq Gold system), 6 μl 25 mM MgCl2, 2 μl 10 mM dNTP mix, 1.5 μl human genomic DNA (Clontech #6550-1, 0.1 μg/μl), 0.3 μl primer VR9A (1 μg/μl), 0.3 μl primer VR9B (1 μg/μl), and 0.4 μl AmpliTaq Gold™ DNA Polymerase.","The primer sequences for VR9A and VR9B were as follows: VR9A 5′TTCAAAGCTTATGGAGTCGGGGCTGCTG 3′ (SEQ ID NO: 101), corresponding to the 5′ end of the coding region and containing a HindIII restriction site, and the reverse primer was: VR9B 5′ TTCACTCGAGTCAGTCTGCAGCCGGTTCTG 3′, (SEQ ID NO: 102), corresponding to the 3′ end of the coding region and containing an XhoI restriction site (Genosys).","The PCR reaction was carried out using a GeneAmp PCR 9700 thermocycler (Perkin Elmer Applied Biosystems) and started with 1 cycle of 95° C. for 10 min, then 10 cycles at 95° C. for 30 sec, 72° C. for 2 min decreasing 1° C. each cycle, 72° C. for 1 min.","followed by 30 cycles at 95° C. for 30 sec, 60° C. for 30 sec, 72° C. for 1 min.","The PCR reaction was loaded on a 0.75% gel.","The DNA band was excised from the gel and the DNA was eluted from the agarose using a QIAquick gel extraction kit (Qiagen).","The eluted DNA was ethanol-precipitated and resuspended in 4 μl H2O for ligation.","The ligation reaction consisted of 4 μl of fresh ethanol-precipitated PCR product and 1 μl of pCRII-TOPO vector (Invitrogen).","The reaction was gently mixed and allowed to incubate for 5 min at room temperature followed by the addition of 1 μl of 6×TOPO cloning stop solution and mixing for 10 sec at room temperature.","The sample was then placed on ice and 2 μl was transformed in 50 μl of One Shot cells (Invitrogen) and plated onto ampicillin plates.","Five white colonies were chosen and were used to inoculate a 4 ml culture of LB medium containing 100 μg/ml ampicillin.","Plasmid DNA was purified using the Wizard Plus Minipreps DNA purification system (Promega).","Since the primers used to PCR SEQ-9 from genomic DNA were engineered to have HindIII and XhoI sites, the cDNA obtained from the minipreps was digested with these restriction enzymes.","One clone was verified by gel electrophoresis to give a DNA band of the correct size.","cDNA from this clone was then submitted for sequencing.","One mutation was found (bp 621 T→G) and repaired as described as below.","The mutation in the identified clone was repaired using the QuikChange Site-Directed Mutagenesis Kit (Stratagene).","The PCR reaction contained 39.3 μl H2O, 5 μl 10× reaction buffer, 50 ng mini-prep cDNA, 1.25 μl primer VR9E (100 ng/μl), 1.25 μl primer VR9F (100 ng/μl), 1 μl 20 mM dNTP mix, 1 μl Pfu DNA polymerase.","The cycle conditions were 95° C. for 30 sec, then 12 cycles at 95° C. for 30 sec, 55° C. for 1 min, 68° C. for 10 min.","One μl of DpnI was added and the tube incubated at 37° C. for 1 hr.","One μl of the DpnI-treated DNA was transformed into 50 μl Epicurian coli XL1-Blue supercompetent cells and the entire insert was re-sequenced.","The primer sequences used were: VR9E: 5′ GCATCCTGGCCGCTATCTGTGCACTCTACG 3′ (SEQ ID NO: 103) and VR9F: 5′ CGTAGAGTGCACAGATAGCGGCCAGGATGC 3′ (SEQ ID NO: 104) where the base underlined was the base being corrected.","The clone described above was sequenced directly using an ABI377 fluorescence-based sequencer (Perkin Elmer/Applied Biosystems Division, PE/ABD, Foster City, Calif.) and the ABI BigDye™ Terminator Cycle Sequencing Ready Reaction kit with Taq FS™ polymerase.","Each ABI cycle sequencing reaction contained 0.5 μg of plasmid DNA.","Cycle-sequencing was performed using an initial denaturation at 98° C. for 1 min, followed by 50 cycles: 96° C. for 30 sec, annealing at 50° C. for 30 sec, and extension at 60° C. for 4 min.","Temperature cycles and times were controlled by a Perkin-Elmer 9600 thermocycler.","Extension products were purified using AGTC (R) gel filtration block (Edge BiosSystems, Gaithersburg, Md.).","Each reaction product was loaded by pipette onto the column, which was then centrifuged in a swinging bucket centrifuge (Sorvall model RT6000B tabletop centrifuge) at 1500×g for 4 min at room temperature.","Column-purified samples were dried under vacuum for about 40 min and then dissolved in 3 μl of a DNA loading solution (83% deionized formamide, 8.3 mM EDTA, and 1.6 mg/ml Blue Dextran).","The samples were then heated to 90° C. for 3.5 min and loaded into the gel sample wells for sequence analysis by the ABI377 sequencer.","Sequence analysis was performed by importing ABI377 files into the 310 Genetic Analyzer, yielding the sequence of SEQ ID NO: 77.nGPCR-11: PCR and Subcloning PCR was performed in a 50 g reaction containing 32 μl H2O, 5 A 10×TT buffer (140 mM Ammonium Sulfate, 0.1% gelatin, 0.6 M Tris-tricine pH 8.4), 5 μl 15 mM MgSO4, 2 μl 10 mM dNTP, 5 μl human genomic DNA (0.3 μg/μL) (Clontech), 0.3 μl of LW1564 (1 μg/μl), 0.3 μl of LW1565 (1 μg/μl), 0.4 μl High Fidelity Taq polymerase (Boehringer Mannheim).","The PCR reaction was performed in a GeneAmp 9600 PCR thermocycler (PE Applied Biosystems) starting with 1 cycle of 94° C. for 2 min followed by 17 cycles at 94° C. for 30 sec, 72° C. for 2 min decreasing 1° C. each cycle, 68° C. for 2 min, then 25 cycles of 94° C. for 30 sec, 55° C. for 30 sec, 68° C. for 2 min.","The PCR reaction was loaded onto a 1.2% agarose gel.","The DNA band was excised from the gel, placed in GenElute Agarose spin column (Supelco) and spun for 10 min at maximum speed in a microcentrifuge.","The eluted DNA was EtOH precipitated and resuspended in 4 μl H2O for ligation.","The forward PCR primer sequence was: LW1564: GCATAAGCTTCCATGTACAACGGGTCGTGCTGC (SEQ ID NO: 107), and the reverse PCR primer was: LW1565: GCATTCTAGATCAGTGCCACTCAACAATGTGGG (SEQ ID NO: 108).","The ligation reaction used solutions from the TOPO TA Cloning Kit (vitrogen) which consisted of 4 μl PCR product DNA and 1 μl pCkII-TOPO vector that was incubated for 5 minutes at room temperature.","To the ligation reaction one microliter of 6×TOPO Cloning Stop Solution was added then the reaction was placed on ice.","Two microliters of the ligation reaction was transformed in One Shot TOP10 cells (Invitrogen), and placed on ice for 30 minutes.","The cells were heat-shocked for 30 seconds at 42° C., placed on ice for two minutes, 250 Tl of SOC was added, then incubated at 37° C. with shaking for one hour and then plated onto ampicillin plates.","A single colony containing an insert was used to inoculate a 5 ml culture of LB medium.","Plasmid DNA was purified using a Concert Rapid Plasmid Miniprep System (GibcoBRL) and then sequenced.","The DNA subcloned into pCRII was sequenced using the ABI PRISM™ 310 Genetic Analyzer (PE Applied Biosystems) which uses advanced capillary electrophoresis technology and the ABI PRISM™ BigDye™ Terminator Cycle Sequencing Ready Reaction Kit.","Each cycle-sequencing reaction contained 6 μl of H2O, 8 μl of BigDye Terminator mix, 5 μl mini-prep DNA (0.1 μg/μl), and 1 μl primer (25 ng/11) and was performed in a Perkin-Elmer 9600 thermocycler with 25 cycles of 96° C. for 10 sec, 50° C. for 10 sec, and 60° C. for 4 min.","The product was purified using a Centriflex™ gel filtration cartridge, dried under vacuum, then dissolved in 16 μl of Template Suppression Reagent (PE Applied Biosystems).","The samples were heated at 95° C. for 5 min then placed in the 310 Genetic Analyzer, yielding the sequence of SEQ ID NO: 79.nGPCR-16: PCR and Subcloning PCR was performed in a 50 μl reaction containing 32 μl H2O, 5 μl 10×TT buffer (140 mM Ammonium Sulfate, 0.1% gelatin, 0.6 M Tris-tricine pH 8.4), 5 μl 15 mM MgSO4, 2 μl 10 mM dNTP, 5 μl 2445704H1 DNA (0.17 Tg/Tl), 0.3 μl of LW1587 (1 μg/μl), 0.3 μl of LW1588 (1 μg/μl), 0.4 μl High Fidelity Taq polymerase (Boehringer Mannheim).","The PCR reaction was performed on a Robocycler thermocycler (Stratagene) starting with 1 cycle of 94° C. for 2 min followed by 15 cycles of 94° C. for 30 sec, 55° C. for 1.3 min, 68° C. for 2 min.","The PCR reaction was loaded onto a 1.2% agarose gel.","The DNA band was excised from the gel, placed in GenElute Agarose spin column (Supelco) and spun for 10 min at maximum speed in a microcentrifuge.","The eluted DNA was EtOH precipitated and resuspended in 12 μl H2O for ligation.","The PCR primer sequence for the forward primer was: LW1587: GATCAAGCTTATGACAGGTGACTTCCCAAGTATGC (SEQ ID NO: 111), and the sequence for the reverse primer was: LW1588: GATCCTCGAGGCTAACGGCACAAAACACAATTCC (SEQ ID NO: 112).","The ligation reaction used solutions from the TOPO TA Cloning Kit (Invitrogen) which consisted of 4 μl PCR product DNA and 1 μl pCRII-TOPO vector that was incubated for 5 minutes at room temperature.","To the ligation reaction one microliter of 6×TOPO Cloning Stop Solution was added then the reaction was placed on ice.","Two microliters of the ligation reaction was transformed in One-Shot TOP10 cells (Invitrogen), and placed on ice for 30 minutes.","The cells were heat-shocked for 30 seconds at 42° C., placed on ice for two minutes, 250 μl of SOC was added, then incubated at 37° C. with shaking for one hour and then plated onto ampicillin plates.","A single colony containing an insert was used to inoculate a 5 ml culture of LB medium.","Plasmid DNA was purified using a Concert Rapid Plasmid Miniprep System (GibcoBRL) and then sequenced.","The DNA subcloned into pCRII was sequenced using the ABI PRISM™ 310 Genetic Analyzer (PE Applied Biosystems) which uses advanced capillary electrophoresis technology and the ABI PRISM™ BigDye™ Terminator Cycle Sequencing Ready Reaction Kit.","Each cycle-sequencing reaction contained 6 μl of H2O, 8 μl of BigDye Terminator mix, 5 μl mini-prep DNA (0.1 μg/μl), and 1 μl primer (25 ng/μl) and was performed in a Perkin-Elmer 9600 thermocycler with 25 cycles of 96° C. for 10 sec, 50° C. for 10 sec, and 60° C. for 4 min.","The product was purified using a Centriflex™ gel filtration cartridge, dried under vacuum, then dissolved in 16 μl of Template Suppression Reagent (PE Applied Biosystems).","The samples were heated at 95° C. for 5 min then placed in the 310 Genetic Analyzer, yielding the sequence of SEQ ID NO: 81.nGPCR-40: PCR and Subcloning PCR was performed in a 50 μl reaction containing utilizing Herculase DNA Polymerase blend (Stratagene), using the buffer recommendations provided by the manufacturer, 200 ng each of primers PSK 18 and 19 (SEQ ID NOS: 115 and 116), 150 ng of human genomic DNA (Clontech), and 2% DMSO.","The PCR reaction was performed on a Robocycler thermocycler (Stratagene) starting with 1 cycle of 94° C. for 2 min followed by 35 cycles of 94° C. for 30 sec, 65° C. for.","30 sec, 72° C. for 2 min.","The PCR reaction was purified using the QiaQuick PCR Purification Kit (Qiagen), and then eluted in TE.","The PCR primer sequences were: PSK 18 GATC GAATTCGCAGGAGCAATG AAAATCAGGAAC (SEQ ID NO: 115), and: PSK19: GATCGAATTCTTATATATGTTCAGAAAACAAATTCATGG (SEQ ID NO: 116)).","The underlined portion of the primer matches the 5′ and 3′ areas, respectively, of a portion of the 5′ untranslated region and coding region.","Initiation and termination codons are shown above in bold.","The PCR product was ligated into the pCR-BluntII-TOPO vector (Invitrogen) using the Zero Blunt Topo PCR TA cloning kit as follow: 3 μl PCR product DNA, 1 μl pCRII-TOPO vector, and 1 μl TOPOII salt solution (1.2M NaCl, 0.06M MgCl2).","The mixture was incubated for 5 minutes at room temperature.","To the ligation reaction one microliter of 6×TOPO Cloning Stop Solution was added, and then the reaction was placed on ice.","Two microliters of the ligation reaction was transformed in One-Shot TOP10 cells (Invitrogen), and placed on ice for 30 minutes.","The cells were heat-shocked for 30 seconds at 42° C., placed on ice for two minutes, 250 μl of SOC was added, then incubated at 37° C. with shaking for one hour and then plated onto ampicillin plates supplemented with Xgal and IPTG.","Single colonies were screened by PCR for the presence of the insert, and a plasmid DNA from colony 58 was purified using a Qiagen Endo-Free plasmid purification kit.","nGPCR-40 was sequenced directly using an ABI377 fluorescence-based sequencer (Perkin Elmer/Applied Biosystems Division, PE/ABD, Foster City, Calif.) and the ABI BigDye™ Terminator Cycle Sequencing Ready Reaction kit with Taq FS™ polymerase.","Each ABI cycle sequencing reaction contained about 0.5 μg of plasmid DNA.","Cycle-sequencing was performed using an initial denaturation at 98° C. for 1 min, followed by 50 cycles: 96° C. for 30 sec, annealing at 50° C. for 30 sec, and extension at 60° C. for 4 min.","Temperature cycles and times were controlled by a Perkin-Elmer 9600 thermocycler.","Extension products were purified using AGTC® gel filtration block (Edge BiosSystems, Gaithersburg, Md.).","Each reaction product was loaded by pipette onto the column, which was then centrifuged in a swinging bucket centrifuge (Sorvall model RT6000B tabletop centrifuge) at 1500×g for 4 min at room temperature.","Column-purified samples were dried under vacuum for about 40 min and then dissolved in 3 μl of DNA loading solution (83% deionized formamide, 8.3 mM EDTA, and 1.6 mg/ml Blue Dextran).","The samples were then heated to 90° C. for 3.5 min and loaded into the gel sample wells for sequence analysis by the ABI377 sequencer.","Sequence analysis was performed by importing ABI377 files into the Sequencher program (Gene Codes, Aim Arbor, M), which yielded a sequence identical to SEQ ID NO:83 with the exception that the nucleotide at position 10 was identified as an “A” which incorrectly indicated the presence of an initiation codon at that position.","Subsequent analysis of genomic DNA samples indicated that this position was incorrectly assigned and that the correct nucleotide at that position was a “C”.","The sequence reported at SEQ ID NO.","83 correctly identifies the nucleotide at position 10 and indicates that the first initiation codon occurs at position 88-90.nGPCR-54: PCR and Subcloning Two microliters of a human genomic library (˜108 PFU/ml) (Clontech) was added to 6 ml of an overnight culture of K802 cells (Clontech), then distributed as 250 μl aliquots into each of 24 tubes.","The tubes were incubated at 37° C. for 15 min.","Seven milliliters of 0.8% agarose was added to each tube, mixed, then poured onto LB agar+10 mM MgSO4 plates and incubated overnight at 37° C. To each plate 5 ml of SM (0.1M NaCl, 8.1 mM MgSO4-7H2O, 50 mM Tris-Cl (pH 7.5), 0.0001% gelatin) phage buffer was added and the top agarose was removed with a microscope slide and placed in a 50 ml centrifuge tube.","A drop of chloroform was added and the tube was place in a 37° C. shaker for 15 min, then centrifuged for 20 min at 4000 RPM (Sorvall RT6000 table top centrifuge) and the supernatant stored at 4° C. as a stock solution.","Two μl of phage from each tube was heated to 99° C. for 4 min then cooled to 10° C. Added to the phage was a PCR mix containing 8.8 μl H2O, 4 μl 5× Rapid-load Buffer (Origene), 2 μl 10×PCR buffer II (Perkin-Elmer), 2 μl 25 mM MgCl2, 0.8 μl 10 mM dNTP, 0.12 μl LW1634 (1 μg/μl) (SEQ ID NO: 119), 0.12 μl LW1635 (1 μg/μl) (SEQ ID NO: 120), 0.2 μl AmpliTaq Gold polymerase (Perlin Elmer).","The PCR reaction involved 1 cycle at 95° C. for 10 min followed by 35 cycles at 95° C. for 45 sec, 53.5° C. for 2 min, 72° C. for 45 sec.","The reaction was loaded onto a 2% agarose gel.","From the tube that gave a PCR product of the correct size, 10 μl was used to set up five 1:10 dilutions that were plated onto LB agar+10 mM MgSO4 plates and incubated overnight.","A BA85 nitrocellulose filter (Schleicher & Schuell) was placed on top of each plate for 1 hour.","The filter was removed, placed phage side up in a petri dish, and covered with 4 ml of SM for 15 min to elute the phage.","One milliliter of SM was removed from each plate and used to set up a PCR reaction as above.","The plate of the lowest dilution to give a PCR product was subdivided, filter-lifted and the PCR reaction was repeated.","The series of dilutions and subdividing of the plate was continued until a single plaque was isolated that gave a positive PCR band.","Once a single plaque was isolated, 10 μl phage supernatant was added to 100 μl SM and 200 μl of K802 cells per plate with a total of 8 plates set up.","The plates were incubated overnight at 37° C. The top agarose was removed by adding 8 ml of SM then scrapping off the agarose with a microscope slide and collected in a centrifuge tube.","To the tube, 3 drops of chloroform was added, vortexed, incubated at 37° C. for 15 min then centrifuged for 20 min at 4000 RPM (Sorvall RT6000 table top centrifuge) to recover the phage, which was used to isolate genomic phage DNA using the Qiagen Lambda Midi Kit.","The sequence for primer LW1634 was: CTGAAAGTTGTCGCTGACC (SEQ ID NO: 119), and for primer LW1635 was: CGATTATCCACACTTTGACCC (SEQ ID NO: 120).","The PCR reaction for the coding region was performed in a 50 μl reaction containing 33 μl H2O, 5 μl 10×TT buffer (140 mM Ammonium Sulfate, 0.1% gelatin, 0.6 M Tris tricine pH 8.4), 5 μl 15 mM MgSO4, 2 μl 10 mM DNTP, 4 μl genomic phage DNA (0.25 μg/μl), 0.3 μl LW1698 (1 μg/μl) (SEQ ID NO: 121), 0.3 μl LW1699 (1 μg/μl) (SEQ ID NO: 122), 0.4 μl High Fidelity Taq polymerase (Boehringer Mannheim).","The PCR reaction was started with 1 cycle of 94° C. for 2 min followed by 30 cycles at 94° C. for 30 sec, 55° C. for 30 sec., 68° C. for 2 min.","The PCR reaction was loaded onto a 2% agarose gel.","The DNA band was excised from the gel, placed in GenElute Agarose spin column (Supelco) and spun for 10 min at maximum speed.","The eluted DNA was EtOH precipitated and resuspended in 8 μl H2O.","The PCR primer sequence for primer LW1698 was: GCATACCATGAATGAGCCACTAGAC (SEQ ID NO: 121), and for primer LW1699 was: GCATCTCGAGTCAAGGGTTGTTTGAGTAAC (SEQ ID NO: 122).","The underlined portion of the primer matches the 5′ and 3′ areas, respectively, of the coding region of nGPCR-54.The ligation reaction used solutions from the TOPO TA Cloning Kit (Invitrogen) which consisted of 4 μl PCR product DNA, 1 μl of salt solution and 1 μl pCRII-TOPO vector that was incubated for 5 minutes at room temperature then the reaction was placed on ice.","Two microliters of the ligation reaction was transformed in One-Shot TOP10 cells (Invitrogen), and placed on ice for 30 minutes.","The cells were heat-shocked for 30 seconds at 42° C., placed on ice for two minutes, 250 μl of SOC was added, then incubated at 37° C. with shaking for one hour and then plated onto ampicillin plates.","A single colony containing an insert was used to inoculate a 5 ml culture of LB medium.","Plasmid DNA was purified using a Concert Rapid Plasmid Miniprep System (GibcoBRL) and then sequenced.","nGPCR-54 genomic phage DNA was sequenced using the ABI PRISM™ 310 Genetic Analyzer (PE Applied Biosystems) which uses advanced capillary electrophoresis technology and the ABI PRISM™ BigDye™ Terminator Cycle Sequencing Ready Reaction Kit.","The cycle-sequencing reaction contained 14 μl of H2O, 16 μl of BigDye Terminator mix, 7 μl genomic phage DNA (0.1 μg/μl), and 3 μl primer (25 ng/μl).","The reaction was performed in a Perkin-Elmer 9600 thermocycler at 95° C. for 5 min, followed by 99 cycles of 95° C. for 30 sec, 55° C. for 20 sec, and 60° C. for 4 min.","The product was purified using a Centriflex™ gel filtration cartridges, dried under vacuum, then dissolved in 16 μl of Template Suppression Reagent.","The samples were heated at 95° C. for 5 min then placed in the 310 Genetic Analyzer.","The DNA subcloned into pCRII was sequenced using the ABI PRISM™ 310 Genetic Analyzer (PE Applied Biosystems) which uses advanced capillary electrophoresis technology and the ABI PRISM™ BigDye™ Terminator Cycle Sequencing Ready Reaction Kit.","Each cycle-sequencing reaction contained 6 μl of H2O, 8 μl of BigDye Terminator mix, 5 μl mini-prep DNA (0.1 μg/μl), and 1 μl primer (25 ng/μl) and was performed in a Perkin-Elmer 9600 thermocycler with 25 cycles of 96° C. for 10 sec, 50° C. for 10 sec, and 60° C. for 4 min.","The product was purified using a Centriflex™ gel filtration cartridge, dried under vacuum, then dissolved in 16 μl of Template Suppression Reagent (PE Applied Biosystems).","The samples were heated at 95° C. for 5 min then placed in the 310 Genetic Analyzer, yielding the sequence of SEQ ID NO: 85.nGPCR-56: PCR and Subcloning The PCR reaction for the coding region of nGPCR-56 used components that come with PLATINUM® Pfx DNA Polymerase (GibcoBRL) containing 35.5 μl H2O, 5 μl 10×Pfx Amplification buffer, 1.5 μl 50 mM MgSO4, 2 μl 10 mM &M, 5 μl human genomic DNA (0.3 μg/μl) (Clontech), 0.3 μl of LW1603 (1 μg/μl) (SEQ ID NO: 152), 0.3 μl of LW1604 (1 μg/μl) (SEQ ID NO: 153), 0.4 μl PLATINUM® Pfa DNA Polymerase (2.5 U/Tl).","The PCR reaction was performed in a Robocycler Gradient 96 (Stratagene) starting with 1 cycle of 94° C. for 5 min followed by 30 cycles at 94° C. for 40 sec, 55° C. for 2 min, 68° C. for 3 min.","Following the final cycle, 0.5 μl of AmpliTaq DNA Polymerase (5 U/μl) was added and the tube was incubated at 72° C. for 5 min.","The sequence of LW1603 is: GATCAAGCTTGGAATGATGCCCTITTGCCAC (SEQ ID NO: 152), and for LW1604 is: GATCCTCGAGCATCATTCAAAGTAGGTGG.","(SEQ ID NO: 153).","The underlined portion of the primer matches the 5′ and 3′ areas, respectively, of a portion of the coding region of nGPCR-56.The PCR reaction for the coding region was performed in a 50 μl reaction containing 32 μl H2O, 5 μl 10×TT buffer (140 mM Ammonium Sulfate, 0.1% gelatin, 0.6 M Tris tricine pH 8.4), 5 μl 15 mM MgSO4, 2 μl 10 mM DNTP, 5 μl human genomic DNA (0.3 μg/μl) (Clontech), 0.3 μl LW1603 (1 μg/μl) (SEQ ID NO: 152), 0.3 μl LW1696 (1 μg/μl) (SEQ ID NO: 154), 0.4 μl High Fidelity Taq polymerase (Boehringer Mannheim).","The PCR reaction was started with 1 cycle of 94° C. for 2 min followed by 25 cycles at 94° C. for 40 sec, 55° C. for 60 sec., 68° C. for 2 min.","The PCR reaction was loaded onto a 2% agarose gel.","The DNA band was excised from the gel, placed in GenElute Agarose spin column (Supelco) and spun for 10 min at maximum speed.","The eluted DNA was EtOH precipitated and resuspended in 12 μl H2O for ligation.","The PCR primer sequence for LW1603 is: GATCAAGCTTGGAATGATGCCCTTTTGCCAC (SEQ ID NO: 152), and LW1696: GATCCTCGAGCTATGAACTCAATTCCAAAAATAATTTACACC (SEQ ID NO: 154).","The underlined portion of the primer matches the 5′ and 3′ areas, respectively, of a portion of the coding region.","The ligation reaction used solutions from the TOPO TA Cloning Kit (Invitrogen) which consisted of 4 μl PCR product DNA, 1 μl of salt solution and 1 μl pCRH-TOPO vector that was incubated for 5 minutes at room temperature then the reaction was placed on ice.","Two microliters of the ligation reaction was transformed in One-Shot TOP10 cells (Invitrogen), and placed on ice for 30 minutes.","The cells were heat-shocked for 30 seconds at 42° C., placed on ice for two minutes, 250 μl of SOC was added, then incubated at 37° C. with shaking for one hour and then plated onto ampicillin plates.","A single colony containing an insert was used to inoculate a 5 ml culture of LB medium.","Plasmid DNA was purified using a Concert Rapid Plasmid Miniprep System (GibcoBRL) and then sequenced.","The mutation in nGPCR-56 was repaired using the QuikChange Site-Directed Mutagenesis Kit (Stratagene).","The PCR reaction contained 40 μl H2O, 5 μl 10× Reaction buffer, 1 μl mini-prep DNA, 1 μl LW1700 (125 ng/μl) (SEQ ID NO: 155), 1 μl LW1701 (125 ng/μl) (SEQ ID NO: 156), 1 μl 10 mM dNTP, 1 μl Pfu DNA polymerase.","The cycle conditions were 95° C. for 30 sec then 14 cycles at 95° C. for 30 sec, 55° C. for 1 min, 68° C. for 12 min.","The tube was placed on ice for 2 min, then 1 μl of DpnI was added and the tube incubated at 37° C. for one hour.","One microliter of the DpnI-treated DNA was transformed into Epicurian coli XL1-Blue supercompetent cells and the entire insert was re sequenced.","The primer sequences are: GCTACTTGAACTCTACATTTAATCCAATGGTTTATGCATITTTCTATCC (LW1700) (SEQ ID NO: 155), and: GGATAGAAAAATGCATAAACCATTGGATTAAATGTAGAGTTCAAGTAGC (LW1701) (SEQ ID NO: 156).","The DNA subcloned into pCRII was sequenced using the ABI PRISM™ 310 Genetic Analyzer (PE Applied Biosystems) which uses advanced capillary electrophoresis technology and the ABI PRISM™ BigDye™ Terminator Cycle Sequencing Ready Reaction Kit.","Each cycle-sequencing reaction contained 6 μl of H2O, 8 μl of BigDye Terminator mix, 5 μl mini-prep DNA (0.1 μg/μl), and 1 μl primer (25 ng/μl) and was performed in a Perkin-Elmer 9600 thermocycler with 25 cycles of 96° C. for 10 sec, 50° C. for 10 sec, and 60° C. for 4 min.","The product was purified using a Centriflex™ gel filtration cartridge, dried under vacuum, then dissolved in 16 μl of Template Suppression Reagent (PE Applied Biosystems).","The samples were heated at 95° C. for 5 min then placed in the 310 Genetic Analyzer, yielding the sequence of SEQ ID NO: 89.nGPCR-58: PCR and Subcloning Isolation of a clone for nGPCR-58 from genomic DNA was performed by PCR in a 50 μl reaction containing Herculase DNA Polymerse blend (Stratagene), with buffer recommendations as supplied by the manufacturer, 200 ng each primers PSK14 (SEQ ID NO: 157) and PSK15 (SEQ ID NO: 158), 150 ng of human genomic DNA (Clontech) and 6% DMSO.","The PCR reaction was performed on a Robocycler thermocycler (Stratagene) starting with 1 cycle of 94° C. for 2 min followed by 35 cycles of 94° C. for 30 sec, 65° C. for 30 sec, 72° C. for 2 min.","The PCR reaction was purified by the QiaQuick PCR Purification Kit (Qiagen) and eluted in Th.","The PCR primer sequences were: PSK14: 5′GATCGAATTCATGGACACTACCATGGAAGCTGACC (SEQ ID NO: 157), and: PSK15: 5′GATCCTCGAGTCACGTGGGGCCTGCGCCCGG (SEQ ID NO: 158).","The underlined portion of the primers match the 5′ and 3′ areas, respectively, of a portion of the 5′ untranslated region and coding region.","Translation initiation and termination codons are shown above in bold.","The blunt ended PCR product was prepared for cloning by the addition of a single base “A” residue by AmpliTaq Gold (Perkin Elmer) in a reaction with 1×PCR Buffer II, 1 mM MgCl2, 200 uM each dATP, dGTP, dCTP, and dTTP.","The reaction was incubated at 94° C. for 10 minutes followed by 72° C. for 10 minutes.","The products were cloned into the pCRII-TOPO vector (Invitrogen) using the TOPO TA cloning kit as follows: 3 μl PCR product DNA, 1 μl pCRII-TOPO vector, and 1 μl TOPOII salt solution (1.2M NaCl, 0.06M MgCl2) was incubated for 5 minutes at room temperature.","To the ligation reaction one microliter of 6×TOPO Cloning Stop Solution was added then the reaction was placed on ice.","Two microliters of the ligation reaction was transformed in One-Shot TOP10 cells (Invitrogen), and placed on ice for 30 minutes.","The cells were heat-shocked for 30 seconds at 42° C., placed on ice for two minutes, 250 μl of SOC was added, then incubated at 37° C. with shaking for one hour and then plated onto ampicillin plates supplemented with X-gal and IPTG.","Single colonies were screened by PCR for the presence of the insert, and a plasmid DNA from colony 58-6 was purified using a Qiagen Endo-Free plasmid purification kit and deposited as nGPCR-58.nGPCR-58 was sequenced directly using an ABI377 fluorescence-based sequencer (Perkin Elmer/Applied Biosystems Division, PE/ABD, Foster City, Calif.) and the ABI BigDye™ Terminator Cycle Sequencing Ready Reaction kit with Taq FS™ polymerase.","Each ABI cycle sequencing reaction contained about 0.5 μg of plasmid DNA.","Cycle-sequencing was performed using an initial denaturation at 98° C. for 1 min, followed by 50 cycles: 96° C. for 30 sec, annealing at 50° C. for 30 sec, and extension at 60° C. for 4 min.","Temperature cycles and times were controlled by a Perkin-Elmer 9600 thermocycler.","Extension products were purified using AGTC (R) gel filtration block (Edge RiosSystems, Gaithersburg, Md.).","Each reaction product was loaded by pipette onto the column, which was then centrifuged in a swinging bucket centrifuge (Sorvall model RT6000B tabletop centrifuge) at 1500×g for 4 min at room temperature.","Column-purified samples were dried under vacuum for about 40 min and then dissolved in 3 μl of a DNA loading solution (83% deionized formamide, 8.3 mM EDTA, and 1.6 mg/ml Blue Dextran).","The samples were then heated to 90° C. for 3.5 min and loaded into the gel sample wells for sequence analysis by the ABI377 sequencer.","Sequence analysis was performed by importing ABI377 files into the Sequencer program (Gene Codes, Ann Arbor, Mich.), yielding the sequence of SEQ ID NO: 93.Example 3 Hybridization Analysis to Demonstrate nGPCR-X Expression in Brain The expression of nGPCR-x in mammals, such as the rat, may be investigated by in situ hybridization histochemistry.","To investigate expression in the brain, for example, coronal and sagittal rat brain cryosections (20 pun thick) are prepared using a Reichert-Jung cryostat.","Individual sections are thaw-mounted onto silanized, nuclease-free slides (CEL Associates, Inc., Houston, Tex.","), and stored at −80° C. Sections are processed starting with post-fixation in cold 4% paraformaldehyde, rinsed in cold phosphate-buffered saline (PBS), acetylated using acetic anhydride in triethanolamine buffer, and dehydrated through a series of alcohol washes in 70%, 95%, and 100% alcohol at room temperature.","Subsequently, sections are delipidated in chloroform, followed by rehydration through successive exposure to 100% and 95% alcohol at room temperature.","Microscope slides containing processed cryosections are allowed to air dry prior to hybridization.","Other tissues may be assayed in a similar fashion.","A nGPCR-x-specific probe is generated using PCR.","Following PCR amplification, the fragment is digested with restriction enzymes and cloned into pBluescript II cleaved with the same enzymes.","For production of a probe specific for the sense strand of nGPCR-x, the nGPCR-x clone in pBluescript II is linearized with a suitable restriction enzyme, which provides a substrate for labeled run-off transcripts (i.e., cRNA riboprobes) using the vector-borne T7 promoter and commercially available T7 RNA polymerase.","A probe specific for the antisense strand of nGPCR-x is also readily prepared using the nGPCR-x clone in pBluescript II by cleaving the recombinant plasmid with a suitable restriction enzyme to generate a linearized substrate for the production of labeled run-off cRNA transcripts using the T3 promoter and cognate polymerase.","The riboprobes are labeled with [35S]-UTP to yield a specific activity of about 0.40×106 cpm/pmol for antisense riboprobes and about 0.65×106 cpm/pmol for sense-strand riboprobes.","Each riboprobe is subsequently denatured and added (2 pmol/ml) to hybridization buffer which contained 50% formamide, 10% dextran, 0.3 M NaCl, 10 mM Tris (pH 8.0), 1 mM EDTA, 1× Denhardt's Solution, and 10 mM dithiothreitol.","Microscope slides containing sequential brain cryosections are independently exposed to 45 μl of hybridization solution per slide and silanized cover slips are placed over the sections being exposed to hybridization solution.","Sections are incubated overnight (15-18 hours) at 52° C. to allow hybridization to occur.","Equivalent series of cryosections are exposed to sense or antisense nGPCR-40-specific cRNA riboprobes.","Following the hybridization period, coverslips are washed off the slides in 1×SSC, followed by RNase A treatment involving the exposure of slides to 20 μg/ml RNase A in a buffer containing 10 mM Tris-HCl (pH 7.4), 0.5 M EDTA, and 0.5 M NaCl for 45 minutes at 37° C. The cryosections are then subjected to three high-stringency washes in 0.1×SSC at 52° C. for 20 minutes each.","Following the series of washes, cryosections are dehydrated by consecutive exposure to 70%, 95%, and 100% ammonium acetate in alcohol, followed by air drying and exposure to Kodak BioMax™ MR-1 film.","After 13 days of exposure, the film is developed.","Based on these results, slides containing tissue that hybridized, as shown by film autoradiograms, are coated with Kodak NTB-2 nuclear track emulsion and the slides are stored in the dark for 32 days.","The slides are then developed and counterstained with hematoxylin.","Emulsion-coated sections are analyzed microscopically to determine the specificity of labeling.","The signal is determined to be specific if autoradiographic grains (generated by antisense probe hybridization) are clearly associated with cresyl violate-stained cell bodies.","Autoradiographic grains found between cell bodies indicates non-specific binding of the probe.","Expression of nGPCR-x in the brain provides an indication that modulators of nGPCR-x activity have utility for treating neurological disorders, including but not limited to, schizophrenia, affective disorders, ADHD/ADD (i.e., Attention Deficit-Hyperactivity Disorder/Attention Deficit Disorder), and neural disorders such as Alzheimer's disease, Parkinson's disease, migraine, and senile dementia.","Some other diseases for which modulators of nGPCR-x may have utility include depression, anxiety, bipolar disease, epilepsy, neuritis, neurasthenia, neuropathy, neuroses, and the like.","Use of nGPCR-x modulators, including nGPCR-x ligands and anti-nGPCR-x antibodies, to treat individuals having such disease states is intended as an aspect of the invention.","Example 4 Tissue Expression Profiling Tissue specific expression of the cDNAs encoding nGPCR-1, nGPCR-3, nGPCR-9, nGPCR-11, nGPCR-16, nGPCR-40, nGPCR-54, nGPCR-56, and nGPCR-58 was detected using a PCR-based system.","Tissue specific expression of cDNAs encoding nGPCR-x may be accomplished using similar methods.","Primers were synthesized by Genosys Corp., The Woodlands, Tex.","PCR reactions were assembled using the components of the Expand Hi-Fi PCR System™ (Roche Molecular Biochemicals, Indianapolis, Ind.).","nGPCR-1 The RapidScan™ Gene Expression Panel was used to generate a comprehensive expression profile of the putative GPCR in human tissues.","Human tissues in the array may include: brain, heart, kidney, spleen, liver, colon, lung, small intestine, muscle, stomach, testis, placenta, salivary gland, thyroid, adrenal gland, pancreas, ovary, uterus, prostate, skin, PBL, bone marrow, fetal brain, fetal liver.","Human brain regions in the array may include: frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.","Expression of the nGPCR-1 in the various tissues was detected by using PCR primers designed based on the available sequence of the receptor that will prime the synthesis of a 212 bp fragment in the presence of the appropriate cDNA.","The forward primer was: GCTCAACCCACTCATCTATGCC (SEQ ID NO: 97), and the reverse primer was: AAACTTCTCTGCCCTTACCGTC (SEQ ID NO: 98) The PCR reaction mixture was added to each well of the PCR plate.","The plate was placed in a GeneAmp PCR9700 PCR thermocycler (Perkin Elmer Applied Biosystems).","The plate was then exposed to the following cycling parameters: Pre-soak 94° C. for 3 min; denaturation at 94° C. for 30 seconds; annealing at primer Tm for 45 seconds; extension 72° C. for 2 minutes; for 35 cycles.","PCR products were then separated and analyzed by electrophoresis on a 1.5-% agarose gel.","The 4-log dilution range of cDNA deposited on the plate ensured that the amplification reaction is within the linear range and, hence, facilitated the semi-quantitative determination of relative mRNA accumulation in the various tissues or brain regions examined.","Expression of nGPCR-1 was found to be highest in the testis, adrenal gland and heart.","Significant levels of expression were also found in the brain, kidney, spleen ovary, prostate, muscle, PBL, stomach and bone marrow.","Within the brain, expression levels were highest in the cerebellum, amygdala, thalamus and spinal cord, with significant levels of expression in the frontal lobe, hippocampus, substantia nigra, hypothalamus and pons.","Expression of nGPCR-1 in the brain provided an indication that modulators of nGPCR-1 activity have utility for treating neurological disorders, including but not limited to, schizophrenia, affective disorders, ADHD/ADD (i.e., Attention Deficit-Hyperactivity Disorder/Attention Deficit Disorder), and neural disorders such as Alzheimer's disease, Parkinson's disease, migraine, and senile dementia.","Some other diseases for which modulators of nGPCR-1 may have utility include depression, anxiety, bipolar disease, epilepsy, neuritis, neurasthenia, neuropathy, neuroses, and the like.","Use of nGPCR-1 modulators, including nGPCR-1 ligands and ant-nGPCR-1 antibodies, to treat individuals having such disease states is intended as an aspect of the invention.","nGPCR-3 Tissue specific expression of the cDNA encoding nGPCR-3 was detected using a PCR-based method.","Multiple Choice™ first strand cDNAs (OriGene Technologies, Rockville, Md.)","from 6 human tissues were serially diluted over a 3-log range and arrayed into a multi-well PCR plate.","This array was used to generate a comprehensive expression profile of the putative GPCR in human tissues.","Human tissues arrayed included: brain, heart, kidney, peripheral blood leukocytes, lung and testis.","PCR primers were designed based on the available sequence of the putative GPCR.","The sequence of the forward primer used was: 5′TGCTGCTTTGTTGCGCCTAC3′ (SEQ ID NO: 189), corresponding to base pairs 77 through 96 of the predicted coding sequence of nGPCR-3.The sequence of the reverse primer used was: 5′TTGGACGCCAGGAAGGTG3′ (SEQ ID NO: 190), corresponding to base pairs 258 through 285 of the predicted coding sequence of nGPCR-3.This primer set primes the synthesis of a 298 base pair fragment in the presence of the appropriate cDNA.","For detection of expression within brain regions, the same primer set was used with the Human Brain Rapid Scan™ Panel (OriGene Technologies, Rockville, Md.).","This panel represents serial dilutions over a 3 log range of first strand cDNA from the following brain regions arrayed in a 96 well format: frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.","Primers were synthesized by Genosys Corp., The Woodlands, Tex.","PCR reactions were assembled using the components of the Expand Hi-Fi PCR System (Roche Molecular Biochemicals, Indianapolis, Ind.).","Twenty-five microliters of the PCR reaction mixture was added to each well of the RapidScan PCR plate.","The plate was placed in a GeneAmp 9700 PCR thermocycler (Perkin Elmer Applied Biosystems).","The following cycling program was executed: Pre-soak at (94° C. for 3 min.)","followed by 35 cycles of [(94° C. for 45 sec.","), (53° C. for 2 min.","), and (72° C. for 45 sec.)].","PCR reaction products were then separated and analyzed by electrophoresis on a 2.0% agarose gel stained with ethidium bromide.","The results indicated that nGPCR-3 was expressed in the brain, heart, kidney, peripheral blood lymphocytes, lung, and testis.","In the brain, nGPCR-3 was expressed in frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla, as well as in the spinal cord.","nGPCR-9 The RapidScan™ Gene Expression Panel was used to generate a comprehensive expression profile of the putative GPCR in human tissues.","Human tissues arrayed include: brain, heart, kidney, spleen, liver, colon, lung, small intestine, muscle, stomach, testis, placenta, salivary gland, thyroid, adrenal gland, pancreas, ovary, uterus, prostate, skin, PBL, bone marrow, fetal brain, fetal liver.","The forward primer used was to detect expression of nGPCR-9 was: 5′ AACCCCATCATCTACACGC 3′(SEQ ID NO: 105), and, the reverse primer was: 5′ TGCCTGTGGAGCCGCTGG 3′(SEQ ID NO: 106).","This primer set will prime the synthesis of a 238 base pair fragment in the presence of the appropriate cDNA.","For detection of expression within brain regions, the same primer set was used with the Human Brain Rapid Scan™ Panel (OriGene Technologies, Rockville, Md.).","This panel represents serial dilutions over a 2-log range of first strand cDNA from the following brain regions arrayed in a 96 well format: frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.","Twenty-five microliters of the PCR reaction mixture was added to each well of the PCR plate.","The plate was placed in a GeneAmp 9700 PCR thermocycler (Perkin Elmer Applied Biosystems).","The following cycling program was executed: Pre-soak at (94° C. for 3 min.)","followed by 35 cycles of [(94° C. for 45 sec.)","(52° C. for 2 min.)","(72° C. for 45 sec.)].","PCR reaction products were then separated and analyzed by electrophoresis on a 2.0% agarose gel and stained with ethidium bromide.","nGPCR-9 was expressed in the brain, peripheral blood leukocytes, heart, kidney, adrenal gland, spleen, pancreas, liver, lung, skin, bone marrow, testis, placenta, salivary gland, uterus, small intestine, muscle, stomach, and fetal liver.","Within the brain, nGPCR-9 was expressed in all areas examined including the frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.","Expression of nGPCR-9 in the brain provided an indication that modulators of nGPCR-9 activity have utility for treating disorders, including but not limited to, schizophrenia, affective disorders, movement disorders, metabolic disorders, inflammatory disorders, cancers, ADHD/ADD (i.e., Attention Deficit-Hyperactivity Disorder/Attention Deficit Disorder), and neural disorders such as Alzheimer's disease, Parkinson's disease, migraine, and senile dementia.","Use of nGPCR-9 modulators, including nGPCR-9 ligands and anti-nGPCR-9 antibodies, to treat individuals having such disease states is intended as an aspect of the invention.","nGPCR-41 The RapidScan™ Gene Expression Panel was used to generate a comprehensive expression profile of the putative GPCR in human tissues.","Human tissues in the array included, inter alia: brain, heart, kidney, spleen, liver, colon, lung, small intestine, muscle, stomach, testis, placenta, salivary gland, thyroid, adrenal gland, pancreas, ovary, uterus, prostate, skin, PBL, bone marrow, fetal brain, fetal liver.","Human brain regions in the array included, inter alia: frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.","Expression of nGPCR-11 in the various tissues was detected by using PCR primers designed based on the available sequence of the receptor that will prime the synthesis of a 206 bp fragment in the presence of the appropriate cDNA.","The forward primer used to detect expression of nGPCR-11 was: 5′-GAAGCCCAGCACTGTTTACC-3′ (SEQ ID NO: 109), and the reverse primer was: 5′-TGAAATACCTGTCCGCAGCC-3 (SEQ ID NO: 10).","Twenty-five microliters of the PCR reaction mixture was added to each well of the RapidScan PCR plate.","The plate was placed in a GeneAmp 9700 PCR thermocycler (PE Applied Biosystems).","The following cycling program was executed: Pre-soak 94° C. for 3 min; denaturation at 94° C. for 30 seconds; annealing at primer Tm for 45 seconds; extension at 72° C. for 2 minutes; for 35 cycles.","PCR reaction products were then separated and analyzed by electrophoresis on a 2.0% agarose gel stained with ethidium bromide.","The 4-log dilution range of cDNA deposited on the plate ensured that the amplification reaction was within the linear range and, facilitated semi-quantitative determination of relative mRNA accumulation in the various tissues or brain regions examined.","nGPCR-11 was expressed in the thyroid gland, brain, heart, kidney, adrenal gland, spleen, liver, ovary, muscle, testis, salivary gland, colon, prostate, small intestine, skin stomach, bone marrow, fetal brain and placenta.","Within the brain, nGPCR-11 was expressed in the temporal lobe, amygdala, substantia nigra, pons, spinal cord, frontal lobe, and cerebellum.","Expression of the nGPCR-11 in the brain provided an indication that modulators of nGPCR-11 activity have utility for treating disorders, including but not limited to, schizophrenia, affective disorders, metabolic disorders, inflammatory disorders, cancers, ADHD/ADD (i.e., Attention Deficit-Hyperactivity Disorder/Attention Deficit Disorder), and neural disorders such as Alzheimer's disease, Parkinson's disease, migraine, and senile dementia.","Some other diseases for which modulators of nGPCR-11 may have utility include depression, anxiety, bipolar disease, epilepsy, neuritis, neurasthenia, neuropathy, neuroses, and the lice.","Use of nGPCR-11 modulators, including nGPCR1-11 ligands and anti-nGPCR-11 antibodies, to treat individuals having such disease states is intended as an aspect of the invention.","Expression of nGPCR-11 in the thyroid gland, indicates that agonists or antagonists could be of use in the treatment of thyroid dysfunction such as thyreotoxicosis and myxoedema.","They could also be of use in the stimulation of thyroid hormone release leading to overall increase in metabolic rate and weight reduction.","The expression of nGPCR-11 in liver and muscle indicate a use for agonists or antagonists in regulation of glucose metabolism applicable in diabetes type II.","nGCPR-16 The RapidScan™ Gene Expression Panel was used to generate a comprehensive expression profile of the putative GPCR in human tissues.","Human tissues in the array included, inter alia: brain, heart, kidney, spleen, liver, colon, lung, small intestine, muscle, stomach, testis, placenta, salivary gland, thyroid, adrenal gland, pancreas, ovary, uterus, prostate, skin, PBL, bone marrow, fetal brain, fetal liver.","Human brain regions in the array included, inter alia: frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.","Expression of nGPCR-16 in the various tissues was detected by using PCR primers designed based on the available sequence of the receptor that will prime the synthesis of a 205 bp fragment in the presence of the appropriate cDNA.","The forward primer used to detect expression of nGPCR-16 was: 5′ CAGCCCAAACATCCAAGTC 3′.","(SEQ ID NO: 113).","The reverse primer used to detect expression of nGPCR-16 was: 5′ ACCCCACTTAATCAGCCTC 3′ (SEQ ID NO: 114).","For detection of expression within brain regions, the same primer set was used with the Human Brain Rapid Scan™ Panel (OriGene Technologies, Rockville, Md.).","This panel represents serial dilutions over a 2 log range of first strand cDNA from the following brain regions arrayed in a 96 well format: frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.","Twenty-five microliters of the PCR reaction mixture was added to each well of the RapidScan PCR plate.","The plate was placed in a GeneAmp 9700 PCR thermocycler (Perkin Elmer Applied Biosystems).","The following cycling program was executed: Pre-soak at (94° for 3 min.)","followed by 35 cycles of [(94° C. for 45 sec.)","(53° C. for 2 min.)","(72° C. for 45 sec.)].","PCR reaction products were then separated and analyzed by electrophoresis on a 2.0% agarose gel, and stained with ethidium bromide.","The 4-log dilution range of cDNA deposited on the plate ensured that the amplification reaction was within the linear range and, facilitated semi-quantitative determination of relative mRNA accumulation in the various tissues or brain regions examined.","nGPCR-16 was expressed in the ovary, lung, prostate, bone marrow, salivary gland, heart, adrenal gland, spleen, liver, small intestine, skin, muscle, peripheral blood leukocytes, testis, placenta, fetal liver, brain, thyroid gland, kidney, pancreas, colon, uterus, and stomach.","Within the brain, nGPCR-16 was expressed in all areas examined including the frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.","Expression of nGPCR-16 in the brain provides an indication that modulators of nGPCR-16 activity have utility for treating neurological disorders, including but not limited to, schizophrenia, affective disorders, ADHD/ADD (i.e., Attention Deficit-Hyperactivity Disorder/Attention Deficit Disorder), and neural disorders such as Alzheimer's disease, Parkinson's disease, migraine, and senile dementia.","Some other diseases for which modulators of nGPCR-16 may have utility include depression, anxiety, bipolar disease, epilepsy, neuritis, neurasthenia, neuropathy, neuroses, and the like.","Use of nGPCR-16 modulators, including nGPCR-16 ligands and anti-nGPCR-16 antibodies, to treat individuals having such disease states is intended as an aspect of the invention.","nGPCR-40 The RapidScan™ Gene Expression Panel (OriGene Technologies, Rockville, Md.)","was used to generate a comprehensive expression profile of the putative GPCR in human tissues.","Human tissues arrayed include: brain, heart, kidney, spleen, liver, colon, lung, small intestine, muscle, stomach, testis, placenta, salivary gland, thyroid, adrenal gland, pancreas, ovary, uterus, prostate, skin, PBL, bone marrow, fetal brain, fetal liver.","The forward primer used was: 5′ACAGCCCCAAAGCCAAACAC3′, (SEQ ID NO: 117), and the reverse primer was: 5′CCGCAGGAGCAATGAAAATCAG3′, (SEQ ID NO: 118).","This primer set primed the synthesis of a 220 base pair fragment in the presence of the appropriate cDNA.","For detection of expression within brain regions, the same primer set was used with the Human Brain RapidScan™ Panel (OriGene Technologies, Rockville, Md.).","This panel represents serial dilutions over a 2 log range of first strand cDNA from the following brain regions arrayed in a 96 well format: frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.","Twenty-five microliters of the PCR reaction mixture was added to each well of the RapidScan PCR plate.","The plate was placed in a GeneAmp 9700 PCR thermocycler (Perkin Elmer Applied Biosystems).","The following cycling program was executed: Pre-soak at (94° C. for 3 min.)","followed by 35 cycles of [(94° for 45 sec.)","(54° C. for 2 min.)","(72° for 45 sec.)].","PCR reaction products were then separated and analyzed by electrophoresis on a 2.0% agarose gel stained with ethidium bromide.","The dilution range of cDNA deposited on the plates ensured that the amplification reaction was within the linear range and, hence, facilitated semi-quantitative determination of relative mRNA accumulation in the various tissues or brain regions examined.","nGPCR-40 was expressed in the brain, peripheral blood lymphocytes, pancreas, ovary, uterus, testis, salivary gland, kidney, adrenal gland, liver, bone marrow, prostate, fetal liver, colon, muscle, and fetal brain, may be found in many other tissues, including, but not limited to, lung, small intestine, fetal brain cord, and bone.","Within the brain, nGPCR-40 was expressed in the frontal lobe, hypothalamus, pons, cerebellum, caudate nucleus, and medulla.","Expression of nGPCR-40 in the brain provides an indication that modulators of nGPCR-40 activity have utility for treating neurological disorders, including but not limited to, movement disorders, affective disorders, metabolic disorders, inflammatory disorders and cancers.","Use of nGPCR-40 modulators, including nGPCR-40 ligands and anti-nGPCR-40 antibodies, to treat individuals having such disease states is intended as an aspect of the invention.","nGPCR-54 Multiple Choice™ first strand cDNAs (OriGene Technologies, Rockville, Md.)","from 12 human tissues were serially diluted over a 3-log range and arrayed into a multi-well PCR plate.","Human tissues arrayed include: brain, heart, kidney, peripheral blood leukocytes, liver, lung, muscle, ovary, prostate, small intestine, spleen and testis.","PCR primers were designed based on the sequence of nGPCR-54 provided herein.","The forward primer used was: 5′CTGTCTCTCTGTCCTCTTCC3′, (SEQ ID NO: 123).","The reverse primer used was: 5′GCACCGATCTTCATTGAATITC3′, (SEQ ID NO: 124).","This primer set primes the synthesis of a 145 base pair fragment in the presence of the appropriate cDNA.","For detection of expression within brain regions, the same primer set was used with the Human Brain Rapid Scan™ Panel (OriGene Technologies, Rockville, Md.).","This panel represents serial dilutions over a 3 log range of first strand cDNA from the following brain regions arrayed in a 96 well format: frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.","Twenty-five microliters of the PCR reaction mixture was added to each well of the RapidScan PCR plate.","The plate was placed in a GeneAmp 9700 PCR thermocycler (Perkin Elmer Applied Biosystems).","The following cycling program was executed: Pre-soak at (94° C. for 3 min.)","followed by 35 cycles of [(94° C. for 45 sec.)","(52.5° C. for 2 min.)","(72° C. for 45 sec.)].","PCR reaction products were then separated and analyzed by electrophoresis on a 2.0% agarose gel stained with ethidium bromide.","nGPCR-54 was expressed in the brain, kidney, lung, muscle, testis, heart, liver, ovary, prostate, small intestine, spleen, and peripheral blood leukocytes.","Within the brain, nGPCR-54 was expressed in the cerebellum, hippocampus, substantia nigra, thalamus, hypothalamus, pons, frontal lobe, temporal lobe, caudate nucleus, medulla, spinal cord, and amygdala.","Expression of the nGPCR-54 in the brain provides an indication that modulators of nGPCR-54 activity have utility for treating neurological disorders, including but not limited to, movement disorders, affective disorders, metabolic disorders, inflammatory disorders and cancers.","Use of nGPCR-54 modulators, including nGPCR-54 ligands and anti-nGPCR-54 antibodies, to treat individuals having such disease states is intended as an aspect of the invention.","nGPCR-56 The RapidScan™ Gene Expression Panel was used to generate a comprehensive expression profile of the putative GPCR in human tissues.","Human tissues arrayed include: brain, heart, kidney, spleen, liver, colon, lung, small intestine, muscle, stomach, testis, placenta, salivary gland, thyroid, adrenal gland, pancreas, ovary, uterus, prostate, skin, PBL, bone marrow, fetal brain, fetal liver.","The forward primer used was: 5′ ACTRCAAACAACTTCATACCCC 3′ (SEQ ID NO: 125), and the reverse primer used was: 5′ACACACAGCATAGTAGCG 3′ (SEQ ID NO: 126).","This primer set will prime the synthesis of a 231 base pair fragment in the presence of the appropriate cDNA.","For detection of expression within brain regions, the same primer set was used with the Human Brain Rapid Scan™ Panel (OriGene Technologies, Rockville, Md.).","This panel represents serial dilutions over a 2 log range of first strand cDNA from the following brain regions arrayed in a 96 well format: frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.","Twenty-five microliters of the PCR reaction mixture was added to each well of the RapidScan PCR plate.","The plate was placed in a GeneAmp 9700 PCR thermocycler (Perkin Elmer Applied Biosystems).","The following cycling program was executed: Pre-soak at (94° C. for 3 min.)","followed by 35 cycles of [(94° C. for 45 sec.)","(53° C. for 2 min.)","(72° C. for 45 sec.)].","PCR reaction products were then separated and analyzed by electrophoresis on a 2.0% agarose gel stained with ethidium bromide.","nGPCR-56 was expressed in peripheral blood lymphocytes, testis, salivary gland, kidney, spleen, skin, stomach, placenta, ovary, bone marrow, fetal liver, small intestine, and fetal brain.","Expression of nGPCR-56 in the brain provides an indication that modulators of nGPCR-56 activity have utility for treating neurological disorders, including but not limited to, movement disorders, affective disorders, metabolic disorders, inflammatory disorders and cancers.","Use of nGPCR-56 modulators, including nGPCR-56 ligands and anti-nGPCR-56 antibodies, to treat individuals having such disease states is intended as an aspect of the invention.","nGPCR-58 The RapidScan™ Gene Expression Panel was used to generate a comprehensive expression profile of the putative GPCR in human tissues.","Human tissues in the array included: brain, heart, kidney, spleen, liver, lung, small intestine, muscle, testis, ovary, prostate, and PBL.","Human brain regions in the array included: frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.","Expression of the nGPCR-58 in the various tissues was detected by using PCR primers designed based on the available sequence of the receptor that will prime the synthesis of a 282 bp fragment in the presence of the appropriate cDNA.","The forward primer was: CAGAGCTTGATGATGAGGAC (SEQ ID NO: 127), and the reverse primer was: CCCATAGGAAGTAGTAGAAG (SEQ ID NO: 128).","The PCR reaction mixture was added to each well of the PCR plate.","The plate was placed in a GeneAmp PCR9700 PCR thermocycler (Perkin Elmer Applied Biosystems).","The plate was then exposed to the following cycling parameters: Pre-soak 94° for 3 min; denaturation at 94° for 30 seconds; annealing at primer Tm for 45 seconds; extension at 72° for 2 minutes; for 35 cycles.","PCR productions were then separated and analyzed by electrophoresis on a 1.5-% agarose gel.","The 4-log dilution range of cDNA deposited on the plate ensured that the amplification reaction was within the linear range and, hence, facilitated semi-quantitative determination of relative mRNA accumulation in the various tissues or brain regions examined.","nGPCR-58 was expressed in all tissues included on the array, including brain, muscle, prostate, kidney, peripheral blood lymphocytes, liver, lung, small intestine, spleen, testis, heart, and ovary.","Within the brain, nGPCR-58 was expressed in many regions including, but not limited to cerebellum, substantia nigra, thalamus, pons, spinal cord, frontal lobe, temporal lobe, hippocampus, caudate nucleus, amygdala, hypothalamus, and medulla.","Expression of the nGPCR-58 in the brain provided an indication that modulators of nGPCR-58 activity have utility for treating disorders, including but not limited to, schizophrenia, affective disorders, ADHD/ADD (i.e., Attention Deficit-Hyperactivity Disorder/Attention Deficit Disorder), neural disorders such as Alzheimer's disease, Parkinson's disease, migraine, senile dementia, depression, anxiety, bipolar disease, epilepsy, neuritis, neurasthenia, neuropathy, neuroses, metabolic disorders, inflammatory disorders, cancers and the like.","Use of nGPCR-58 modulators, including nGPCR-58 ligands and anti-nGPCR-58 antibodies, to treat individuals having such disease states is intended as an aspect of the invention.","Example 5 Northern Blot Analysis Northern blots are performed to examine the expression of nGPCR-x mRNA.","The sense orientation oligonucleotide and the antisense-orientation oligonucleotide, described above, are used as primers to amplify a portion of the GPCR-x cDNA sequence of an odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191.Multiple human tissue northern blots from Clontech (Human II # 7767-1) are hybridized with the probe.","Pre-hybridization is carried out at 42 C for 4 hours in 5×SSC, 1× Denhardt's reagent, 0.1% SDS, 50% formamide, 250 mg/ml salmon sperm DNA.","Hybridization is performed overnight at 42° C. in the same mixture with the addition of about 1.5×106 cpm/ml of labeled probe.","The probe is labeled with α-32P-DCTP by Rediprime™ DNA labeling system (Amersham Pharmacia), purified on Nick Column (Amersham Pharmacia) and added to the hybridization solution.","The filters are washed several times at 42 C in 0.2×SSC, 0.1% SDS.","Filters are exposed to Kodak XAR film (Eastman Kodak Company, Rochester, N.Y., USA) with intensifying screen at −80° C. Example 6 Recombinant Expression of nGPCR-x in Eukaryotic Host Cells A.","Expression of nGPCR-x in Mammalian Cells To produce nGPCR-x protein, a nGPCR-x-encoding polynucleotide is expressed in a suitable host cell using a suitable expression vector and standard genetic engineering techniques.","For example, the nGPCR-x-encoding sequence described in Example 1 is subcloned into the commercial expression vector pzeoSV2 (Invitrogen, San Diego, Calif.) and transfected into Chinese Hamster Ovary (CHO) cells using the transfection reagent FuGENE6™ (Boehringer-Mannheim) and the transfection protocol provided in the product insert.","Other eukaryotic cell lines, including human embryonic kidney (HEK 293) and COS cells, are suitable as well.","Cells stably expressing nGPCR-x are selected by growth in the presence of 100 μg/ml zeocin (Stratagene, LaJolla, Calif.).","Optionally, nGPCR-x may be purified from the cells using standard chromatographic techniques.","To facilitate purification, antisera is raised against one or more synthetic peptide sequences that correspond to portions of the nGPCR-x amino acid sequence, and the antisera is used to affinity purify nGPCR-x.","The nGPCR-x also may be expressed in-frame with a tag sequence (e.g.","polyhistidine, hemagluttinin, FLAG) to facilitate purification.","Moreover, it will be appreciated that many of the uses for nGPCR-x polypeptides, such as assays described below, do not require purification of nGPCR-x from the host cell.","B.","Expression of nGPCR-x in 293 Cells For expression of nGPCR-x in mammalian cells 293 (transformed human, primary embryonic kidney cells), a plasmid bearing the relevant nGPCR-x coding sequence is prepared, using vector pSecTag2A (Invitrogen).","Vector pSecTag2A contains the murine IgK chain leader sequence for secretion, the c-myc epitope for detection of the recombinant protein with the anti-myc antibody, a C-terminal polyhistidine for purification with nickel chelate chromatography, and a Zeocin resistant gene for selection of stable transfectants.","The forward primer for amplification of this GPCR cDNA is determined by routine procedures and preferably contains a 5′ extension of nucleotides to introduce the HindIII cloning site and nucleotides matching the GPCR sequence.","The reverse primer is also determined by routine procedures and preferably contains a 5′ extension of nucleotides to introduce an XhoI restriction site for cloning and nucleotides corresponding to the reverse complement of the nGPCR-x sequence.","The PCR conditions are 55° C. as the annealing temperature.","The PCR product is gel purified and cloned into the HindIII-XhoI sites of the vector.","The DNA is purified using Qiagen chromatography columns and transfected into 293 cells using DOTAP™ transfection media (Boehringer Mannheim, Indianapolis, Ind.).","Transiently transfected cells are tested for expression after 24 hours of transfection, using western blots probed with anti-His and anti-nGPCR-x peptide antibodies.","Permanently transfected cells are selected with Zeocin and propagated.","Production of the recombinant protein is detected from both cells and media by western blots probed with anti-His, anti-Myc or anti-GPCR peptide antibodies.","C. Expression of nGPCR-x in COS Cells For expression of the nGPCR-x in COS7 cells, a polynucleotide molecule having an odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191 can be cloned into vector p3-CI.","This vector is a pUC18-derived plasmid that contains the HCMV (human cytomegalovirus) promoter-intron located upstream from the bGH (bovine growth hormone) polyadenylation sequence and a multiple cloning site.","In addition, the plasmid contains the dhrf (dihydrofolate reductase) gene which provides selection in the presence of the drug methotrexane (WIX) for selection of stable transformants.","The forward primer is determined by routine procedures and preferably contains a 5′ extension which introduces an XbaI restriction site for cloning, followed by nucleotides which correspond to an odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191.The reverse primer is also determined by routine procedures and preferably contains 5′-extension of nucleotides which introduces a SalI cloning site followed by nucleotides which correspond to the reverse complement of an odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191.The PCR consists of an initial denaturation step of 5 min at 95° C. 30 cycles of 30 sec denaturation at 95° C., 30 sec annealing at 58° C. and 30 sec extension at 72° C., followed by 5 min extension at 72° C. The PCR product is gel purified and ligated into the XbaI and SalI sites of vector p3-CI.","This construct is transformed into E. coli cells for amplification and DNA purification.","The DNA is purified with Qiagen chromatography columns and transfected into COS 7 cells using Lipofectamine™ reagent from BRL, following the manufacturer's protocols.","Forty-eight and 72 hours after transfection, the media and the cells are tested for recombinant protein expression.","nGPCR-x expressed from a COS cell culture can be purified by concentrating the cell-growth media to about 10 mg of protein/ml, and purifying the protein by, for example, chromatography.","Purified nGPCR-x is concentrated to 0.5 mg/ml in an Amicon concentrator fitted with a YM-10 membrane and stored at −80° C. D. Expression of nGPCR-x in Insect Cells For expression of nGPCR-x in a baculovirus system, a polynucleotide molecule having an odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191 can be amplified by PCR.","The forward primer is determined by routine procedures and preferably contains a 5′ extension which adds the NdeI cloning site, followed by nucleotides which correspond to an odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191.The reverse primer is also determined by routine procedures and preferably contains a 5′ extension which introduces the KpnI cloning site, followed by nucleotides which correspond to the reverse complement of an odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191.The PCR product is gel purified, digested with NdeI and KpnI, and cloned into the corresponding sites of vector pACHTL-A (Pharmingen, San Diego, Calif.).","The pAcHTL expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV), and a 6×His tag upstream from the multiple cloning site.","A protein kinase site for phosphorylation and a thrombin site for excision of the recombinant protein precede the multiple cloning site is also present.","Of course, many other baculovirus vectors could be used in place of pAcHTL-A, such as pAc373, pVL941 and pAcIM1.Other suitable vectors for the expression of GPCR polypeptides can be used, provided that the vector construct includes appropriately located signals for transcription, translation, and trafficking, such as an in-frame AUG and a signal peptide, as required.","Such vectors are described in Luckow et al., Virology 170:31-39, among others.","The virus is grown and isolated using standard baculovirus expression methods, such as those described in Summers et al.","(A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experimental Station Bulletin No.","1555 (1987)).","In a preferred embodiment, pAcHLT-A containing nGPCR-x gene is introduced into baculovirus using the “BaculoGold™” transfection kit (Pharmingen, San Diego, Calif.) using methods established by the manufacturer.","Individual virus isolates are analyzed for protein production by radiolabeling infected cells with 35S-methionine at 24 hours post infection.","Infected cells are harvested at 48 hours post infection, and the labeled proteins are visualized by SDS-PAGE.","Viruses exhibiting high expression levels can be isolated and used for scaled up expression.","For expression of a nGPCR-x polypeptide in a Sf9 cells, a polynucleotide molecule having the sequence of an odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191 can be amplified by PCR using the primers and methods described above for baculovirus expression.","The nGPCR-x cDNA is cloned into vector pAcHLT-A (Pharmingen) for expression in Sf9 insect.","The insert is cloned into the NdeI and KpnI sites, after elimination of an internal NdeI site (using the same primers described above for expression in baculovirus).","DNA is purified with Qiagen chromatography columns and expressed in Sf9 cells.","Preliminary Western blot experiments from non-purified plaques are tested for the presence of the recombinant protein of the expected size which reacted with the GPCR-specific antibody.","These results are confirmed after further purification and expression optimization in HiG5 cells.","Example 7 Interaction Trap/Two-Hybrid System In order to assay for nGPCR-x-interacting proteins, the interaction trap/two-hybrid library screening method can be used.","This assay was first described in Fields et al., Nature, 1989, 340, 245, which is incorporated herein by reference in its entirety.","A protocol is published in Current Protocols in Molecular Biology 1999, John Wiley & Sons, NY, and Ausubel, F. M et al.","1992, Short protocols in molecular biology, Fourth edition, Greene and Wiley-interscience, NY, each of which is incorporated herein by reference in its entirety.","Kits are available from Clontech, Palo Alto, Calif. (Matchmaker Two-Hybrid System 3).","A fusion of the nucleotide sequences encoding all or partial nGPCR-x and the yeast transcription factor GAL4 DNA-binding domain (DNA-BD) is constructed in an appropriate plasmid (ie., pGBKT7) using standard subcloning techniques.","Similarly, a GALA active domain (AD) fusion library is constructed in a second plasmid (i.e., pGADT7) from cDNA of potential GPCR-binding proteins (for protocols on forming cDNA libraries, see Sambrook et al.","1989, Molecular cloning: a laboratory manual, second edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.), which is incorporated herein by reference in its entirety.","The DNA-BD/nGPCR-x fusion construct is verified by sequencing, and tested for autonomous reporter gene activation and cell toxicity, both of which would prevent a successful two-hybrid analysis.","Similar controls are performed with the AD/library fusion construct to ensure expression in host cells and lack of transcriptional activity.","Yeast cells are transformed (ca.","105 transformants/mg DNA) with both the nGPCR-x and library fusion plasmids according to standard procedures (Ausubel et al., 1992, Short protocols in molecular biology, fourth edition, Greene and Wiley-interscience, NY, which is incorporated herein by reference in its entirety).","In vivo binding of DNA-BD/nGPCR-x with AD/library proteins results in transcription of specific yeast plasmid reporter genes (i.e., lacZ, HIS3, ADE2, LEU2).","Yeast cells are plated on nutrient-deficient media to screen for expression of reporter genes.","Colonies are dually assayed for β-galactosidase activity upon growth in Xgal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) supplemented media (filter assay for β-galactosidase activity is described in Breeden et al., Cold Spring Harb.","Symp.","Quant.","Biol., 1985, 50, 643, which is incorporated herein by reference in its entirety).","Positive AD-library plasmids are rescued from transformants and reintroduced into the original yeast strain as well as other strains containing unrelated DNA-BD fusion proteins to confirm specific nGPCR-x/library protein interactions.","Insert DNA is sequenced to verify the presence of an open reading frame fused to GAL4 AD and to determine the identity of the nGPCR-x-binding protein.","Example 8 Mobility Shift DNA-Binding Assay Using Gel Electrophoresis A gel electrophoresis mobility shift assay can rapidly detect specific protein-DNA interactions.","Protocols are widely available in such manuals as Sambrook et al.","1989, Molecular cloning: a laboratory manual, second edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. and Ausubel, F. M. et al., 1992, Short Protocols in Molecular Biology, fourth edition, Greene and Wiley-interscience, NY, each of which is incorporated herein by reference in its entirety.","Probe DNA (<300 bp) is obtained from synthetic oligonucleotides, restriction endonuclease fragments, or PCR fragments and end-labeled with 32P.","An aliquot of purified nGPCR-x (ca.","15 μg) or crude nGPCR-x extract (ca.","15 ng) is incubated at constant temperature (in the range 22-37 C) for at least 30 minutes in 10-15 μl of buffer (i.e.","TAE or TBE, pH 8.0-8.5) containing radiolabeled probe DNA, nonspecific carrier DNA (ca 1 μg), BSA (300 μg/ml), and 10% (v/v) glycerol.","The reaction mixture is then loaded onto a polyacrylamide gel and run at 30-35 mA until good separation of free probe DNA from protein-DNA complexes occurs.","The gel is then dried and bands corresponding to free DNA and protein-DNA complexes are detected by autoradiography.","Example 9 Antibodies to nGPCR-X Standard techniques are employed to generate polyclonal or monoclonal antibodies to the nGPCR-x receptor, and to generate useful antigen-binding fragments thereof or variants thereof, including “humanized” variants.","Such protocols can be found, for example, in Sambrook et al.","(1989) and Harlow et al.","(Eds.","), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988).","In one embodiment, recombinant nGPCR-x polypeptides (or cells or cell membranes containing such polypeptides) are used as antigen to generate the antibodies.","In another embodiment, one or more peptides having amino acid sequences corresponding to an immunogenic portion of nGPCR-x (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids) are used as antigen.","Peptides corresponding to extracellular portions of nGPCR-x, especially hydrophilic extracellular portions, are preferred.","The antigen may be mixed with an adjuvant or linked to a hapten to increase antibody production.","A. Polyclonal or Monoclonal Antibodies As one exemplary protocol, recombinant nGPCR-x or a synthetic fragment thereof is used to immunize a mouse for generation of monoclonal antibodies (or larger mammal, such as a rabbit, for polyclonal antibodies).","To increase antigenicity, peptides are conjugated to Keyhole Lympet Hemocyanin (Pierce), according to the manufacturer's recommendations.","For an initial injection, the antigen is emulsified with Freund's Complete Adjuvant and injected subcutaneously.","At intervals of two to three weeks, additional aliquots of nGPCR-x antigen are emulsified with Freund's Incomplete Adjuvant and injected subcutaneously.","Prior to the final booster injection, a serum sample is taken from the immunized mice and assayed by western blot to confirm the presence of antibodies that immunoreact with nGPCR-x.","Serum from the immunized animals may be used as polyclonal antisera or used to isolate polyclonal antibodies that recognize nGPCR-x.","Alternatively, the mice are sacrificed and their spleen removed for generation of monoclonal antibodies.","To generate monoclonal antibodies, the spleens are placed in 10 ml serum-free RPMI 1640, and single cell suspensions are formed by grinding the spleens in serum-free RPMI 1640, supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, and 100 μg/ml streptomycin (RPMI) (Gibco, Canada).","The cell suspensions are filtered and washed by centrifugation and resuspended in serum-free RPMI.","Thymocytes taken from three naive Balb/c mice are prepared in a similar manner and used as a Feeder Layer.","NS-1 myeloma cells, kept in log phase in RPMI with 10% fetal bovine serum (FBS) (Hyclone Laboratories, Inc., Logan, Utah) for three days prior to fusion, are centrifuged and washed as well.","To produce hybridoma fusions, spleen cells from the immunized mice are combined with NS-1 cells and centrifuged, and the supernatant is aspirated.","The cell pellet is dislodged by tapping the tube, and 2 ml of 37° C. PEG 1500 (50% in 75 mM HEPES, pH 8.0) (Boehringer-Mannheim) is stirred into the pellet, followed by the addition of serum-free RPMI.","Thereafter, the cells are centrifuged, resuspended in RPMI containing 15% FBS, 100 μM sodium hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine (HAT) (Gibco), 25 units/ml IL-6 (Boehringer-Mannheim) and 1.5×106 thymocytes/ml, and plated into 10 Corning flat-bottom 96-well tissue culture plates (Corning, Corning N.Y.).","On days 2, 4, and 6 after the fusion, 100 μl of medium is removed from the wells of the fusion plates and replaced with fresh medium.","On day 8, the fusions are screened by ELISA, testing for the presence of mouse IgG that binds to nGPCR-x.","Selected fusion wells are further cloned by dilution until monoclonal cultures producing anti-nGPCR-x antibodies are obtained.","B. Humanization of Anti-nGPCR-x Monoclonal Antibodies The expression pattern of nGPCR-x as reported herein and the proven track record of GPCRs as targets for therapeutic intervention suggest therapeutic indications for nGPCR-x inhibitors (antagonists).","nGPCR-x-neutralizing antibodies comprise one class of therapeutics useful as nGPCR-x antagonists.","Following are protocols to improve the utility of anti-nGPCR-x monoclonal antibodies as therapeutics in humans by “humanizing” the monoclonal antibodies to improve their serum half-life and render them less immunogenic in human hosts (i.e., to prevent human antibody response to non-human anti-nGPCR-x antibodies).","The principles of humanization have been described in the literature and are facilitated by the modular arrangement of antibody proteins.","To minimize the possibility of binding complement, a humanized antibody of the IgG4 isotype is preferred.","For example, a level of humanization is achieved by generating chimeric antibodies comprising the variable domains of non-human antibody proteins of interest with the constant domains of human antibody molecules.","(See, e.g., Morrison et al., Adv.","Immunol., 44:65-92 (1989)).","The variable domains of nGPCR-x-neutralizing anti-nGPCR-x antibodies are cloned from the genomic DNA of a B-cell hybridoma or from cDNA generated from mRNA isolated from the hybridoma of interest.","The V region gene fragments are linked to exons encoding human antibody constant domains, and the resultant construct is expressed in suitable mammalian host cells (e.g., myeloma or CHO cells).","To achieve an even greater level of humanization, only those portions of the variable region gene fragments that encode antigen-binding complementarity determining regions (“CDR”) of the non-human monoclonal antibody genes are cloned into human antibody sequences.","(See, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-36 (1988); and Tempest et al., Bio/Technology 9: 266-71 (1991)).","If necessary, the P-sheet framework of the human antibody surrounding the CDR3 regions also is modified to more closely mirror the three dimensional structure of the antigen-binding domain of the original monoclonal antibody.","(See Kettleborough et al., Protein Engin., 4:773-783 (1991); and Foote et al., J. Mol.","Biol., 224:487-499 (1992)).","In an alternative approach, the surface of a non-human monoclonal antibody of interest is humanized by altering selected surface residues of the non-human antibody, e.g., by site-directed mutagenesis, while retaining all of the interior and contacting residues of the non-human antibody.","See Padlan, Molecular Immunol., 28(4/5):489-98 (1991).","The foregoing approaches are employed using nGPCR-x-neutralizing anti-nGPCR-x monoclonal antibodies and the hybridomas that produce them to generate humanized nGPCR-x-neutralizing antibodies useful as therapeutics to treat or palliate conditions wherein nGPCR-x expression or ligand-mediated nGPCR-x signaling is detrimental.","C. Human nGPCR-x-Neutralizing Antibodies from Phage Display Human nGPCR-x-neutralizing antibodies are generated by phage display techniques such as those described in Aujame et al., Human Antibodies 8(4):155-168 (1997); Hoogenboom, TIBTECH 15:62-70 (1997); and Rader et al., Curr.","Opin.","Biotechnol.","8:503-508 (1997), all of which are incorporated by reference.","For example, antibody variable regions in the form of Fab fragments or linked single chain Fv fragments are fused to the amino terminus of filamentous phage minor coat protein pIII.","Expression of the fusion protein and incorporation thereof into the mature phage coat results in phage particles that present an antibody on their surface and contain the genetic material encoding the antibody.","A phage library comprising such constructs is expressed in bacteria, and the library is screened for nGPCR-x-specific phage-antibodies using labeled or immobilized nGPCR-x as antigen-probe.","D. Human nGPCR-x-Neutralizing Antibodies from Transgenic Mice Human nGPCR-x-neutralizing antibodies are generated in transgenic mice essentially as described in Bruggemann et al., Immunol.","Today 17(8):391-97 (1996) and Bruggemann et al., Curr.","Opin.","Biotechnol.","8:455-58 (1997).","Transgenic mice carrying human V-gene segments in germline configuration and that express these transgenes in their lymphoid tissue are immunized with a nGPCR-x composition using conventional immunization protocols.","Hybridomas are generated using B cells from the immunized mice using conventional protocols and screened to identify hybridomas secreting anti-nGPCR-x human antibodies (e.g., as described above).","Example 10 Assays to Identify Modulators of nGPCR-x Activity Set forth below are several nonlimiting assays for identifying modulators (agonists and antagonists) of nGPCR-x activity.","Among the modulators that can be identified by these assays are natural ligand compounds of the receptor, synthetic analogs and derivatives of natural ligands; antibodies, antibody fragments, and/or antibody-like compounds derived from natural antibodies or from antibody-like combinatorial libraries; and/or synthetic compounds identified by high-throughput screening of libraries; and the like.","All modulators that bind nGPCR-x are useful for identifying nGPCR-x in tissue samples (e.g., for diagnostic purposes, pathological purposes, and the like).","Agonist and antagonist modulators are useful for up-regulating and down-regulating nGPCR-x activity, respectively, to treat disease states characterized by abnormal levels of nGPCR-x activity.","The assays may be performed using single putative modulators, and/or may be performed using a known agonist in combination with candidate antagonists (or visa versa).","A. cAMP Assays In one type of assay, levels of cyclic adenosine monophosphate (cAMP) are measured in nGPCR-x-transfected cells that have been exposed to candidate modulator compounds.","Protocols for cAMP assays have been described in the literature.","(See, e.g., Sutherland et al., Circulation 37: 279 (1968); Frandsen et al., Life Sciences 18: 529-541 (1976); Dooley et al., Journal of Pharmacology and Experimental Therapeutics 283 (2): 735-41 (1997); and George et al., Journal of Biomolecular Screening 2 (4): 23540 (1997)).","An exemplary protocol for such an assay, using an Adenylyl Cyclase Activation FlashPlate® Assay from NEN™ Life Science Products, is set forth below.","Briefly, the nGPCR-x coding sequence (e.g., a cDNA or intronless genomic DNA) is subcloned into a commercial expression vector, such as pzeoSV2 (Invitrogen), and transiently transfected into Chinese Hamster Ovary (CHO) cells using known methods, such as the transfection protocol provided by Boehringer-Mannheim when supplying the FuGENE 6 transfection reagent.","Transfected CHO cells are seeded into 96-well microplates from the FlashPlate® assay kit, which are coated with solid scintillant to which antisera to cAMP has been bound.","For a control, some wells are seeded with wild type (untransfected) CHO cells.","Other wells in the plate receive various amounts of acAMP standard solution for use in creating a standard curve.","One or more test compounds (i.e., candidate modulators) are added to the cells in each well, with water and/or compound-free medium/diluent serving as a control or controls.","After treatment, cAMP is allowed to accumulate in the cells for exactly 15 minutes at room temperature.","The assay is terminated by the addition of lysis buffer containing [125I]-labeled cAMP, and the plate is counted using a Packard Topcount™ 96-well microplate scintillation counter.","Unlabeled cAMP from the lysed cells (or from standards) and fixed amounts of [125I]-cAMP compete for antibody bound to the plate.","A standard curve is constructed, and cAMP values for the unknowns are obtained by interpolation.","Changes in intracellular cAMP levels of cells in response to exposure to a test compound are indicative of nGPCR-x modulating activity.","Modulators that act as agonists of receptors which couple to the Gs subtype of G proteins will stimulate production of cAMP, leading to a measurable 3-10 fold increase in cAMP levels.","Agonists of receptors which couple to the Gi/o subtype of G proteins will inhibit forskolin-stimulated cAMP production, leading to a measurable decrease in cAMP levels of 50-100%.","Modulators that act as inverse agonists will reverse these effects at receptors that are either constitutively active or activated by known agonists.","B. Aequorin Assays In another assay, cells (e.g., CHO cells) are transiently co-transfected with both a nGPCR-x expression construct and a construct that encodes the photoprotein apoaquorin.","In the presence of the cofactor coelenterazine, apoaquorin will emit a measurable luminescence that is proportional to the amount of intracellular (cytoplasmic) free calcium.","(See generally, Cobbold, et al.","“Aequorin measurements of cytoplasmic free calcium,” In: McCormack J. G. and Cobbold P. H., eds., Cellular Calcium: A Practical Approach.","Oxford:IRL Press (1991); Stables et al., Analytical Biochemistry 252: 115-26 (1997); and Haugland, Handbook of Fluorescent Probes and Research Chemicals.","Sixth edition.","Eugene Oreg.",": Molecular Probes (1996).)","In one exemplary assay, nGPCR-x is subcloned into the commercial expression vector pzeoSV2 (Invitrogen) and transiently co-transfected along with a construct that encodes the photoprotein apoaquorin (Molecular Probes, Eugene, Oreg.)","into CHO cells using the transfection reagent FuGENE 6 (Boehringer-Mannheim) and the transfection protocol provided in the product insert.","The cells are cultured for 24 hours at 37° C. in MEM (Gibco/BRL, Gaithersburg, Md.)","supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin and 10 μg/ml streptomycin, at which time the medium is changed to serum-free MEM containing 5 μM coelenterazine (Molecular Probes, Eugene, Oreg.).","Culturing is then continued for two additional hours at 37° C. Subsequently, cells are detached from the plate using VERSEN (Gibco/BRL), washed, and resuspended at 200,000 cells/ml in serum-free MEM.","Dilutions of candidate nGPCR-x modulator compounds are prepared in serum-free MEM and dispensed into wells of an opaque 96-well assay plate at 50 μl/well.","Plates are then loaded onto an MLX microtiter plate luminometer (Dynex Technologies, Inc., Chantilly, Va.).","The instrument is programmed to dispense 50 μl cell suspensions into each well, one well at a time, and immediately read luminescence for 15 seconds.","Dos response curves for the candidate modulators are constructed using the area under the curve for each light signal peak.","Data are analyzed with SlideWrite, using the equation for a one-site ligand, and EC50 values are obtained.","Changes in luminescence caused by the compounds are considered indicative of modulatory activity.","Modulators that act as agonists at receptors which couple to the Gq subtype of G proteins give an increase in luminescence of up to 100 fold.","Modulators that act as inverse agonists will reverse this effect at receptors that are either constitutively active or activated by known agonists.","C. Luciferase Reporter Gene Assay The photoprotein luciferase provides another useful tool for assaying for modulators of nGPCR-x activity.","Cells (e.g., CHO cells or COS 7 cells) are transiently co-transfected with both a nGPCR-x expression construct (e.g., nGPCR-x in pzeoSV2) and a reporter construct which includes a gene for the luciferase protein downstream from a transcription factor binding site, such as the cAMP-response element (CRE), AP-1, or NF-kappa B. Agonist binding to receptors coupled to the G. subtype of G proteins leads to increases in cAMP, thereby activating the CRE transcription factor and resulting in expression of the luciferase gene.","Agonist binding to receptors coupled to the Gq subtype of G protein leads to production of diacylglycerol that activates protein kinase C, which activates the AP-1 or NF-kappa B transcription factors, in turn resulting in expression of the luciferase gene.","Expression levels of luciferase reflect the activation status of the signaling events.","(See generally, George et al, Journal of Biomolecular Screening 2(4): 235-240 (1997); and Stratowa et al, Current Opinion in Biotechnology 6: 574-581 (1995)).","Luciferase activity may be quantitatively measured using, e.g., luciferase assay reagents that are commercially available from Promega (Madison, Wis.).","In one exemplary assay, CHO cells are plated in 24-well culture dishes at a density of 100,000 cells/well one day prior to transfection and cultured at 37° C. in MEM (Gibco/BRL) supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin and 10 μg/ml streptomycin.","Cells are transiently co-transfected with both a nGPCR-x expression construct and a reporter construct containing the luciferase gene.","The reporter plasmids CRE-luciferase, AP-1-luciferase and NF-kappaB-luciferase may be purchased from Stratagene (LaJolla, Calif.).","Transfections are performed using the FuGENE 6 transfection reagent (Boehringer-Mannheim) according to the supplier's instructions.","Cells transfected with the reporter construct alone are used as a control.","Twenty-four hours after transfection, cells are washed once with PBS pre-warmed to 37° C. Serum-free MEM is then added to the cells either alone (control) or with one or more candidate modulators and the cells are incubated at 37° C. for five hours.","Thereafter, cells are washed once with ice-cold PBS and lysed by the addition of 100 μl of lysis buffer per well from the luciferase assay kit supplied by Promega.","After incubation for 15 minutes at room temperature, 15 μl of the lysate is mixed with 50 μl of substrate solution (Promega) in an opaque-white, 96-well plate, and the luminescence is read immediately on a Wallace model 1450 MicroBeta scintillation and luminescence counter (Wallace Instruments, Gaithersburg, Md.).","Differences in luminescence in the presence versus the absence of a candidate modulator compound are indicative of modulatory activity.","Receptors that are either constitutively active or activated by agonists typically give a 3 to 20-fold stimulation of luminescence compared to cells transfected with the reporter gene alone.","Modulators that act as inverse agonists will reverse this effect.","D. Intracellular Calcium Measurement Using FLIPR Changes in intracellular calcium levels are another recognized indicator of G protein-coupled receptor activity, and such assays can be employed to screen for modulators of nGPCR-x activity.","For example, CHO cells stably transfected with a nGPCR-x expression vector are plated at a density of 4×104 cells/well in Packard black-walled, 96-well plates specially designed to discriminate fluorescence signals emanating from the various wells on the plate.","The cells are incubated for 60 minutes at 37° C. in modified Dulbecco's PBS (D-PBS) containing 36 mg/L pyruvate and 1 g/L glucose with the addition of 1% fetal bovine serum and one of four calcium indicator dyes (Fluo-3™ AM, Fluo-4™ AM, Calcium Green™-1 AM, or Oregon Green™ 488 BAPTA-1 AM), each at a concentration of 4 μM.","Plates are washed once with modified D-PBS without 1% fetal bovine serum and incubated for 10 minutes at 37° C. to remove residual dye from the cellular membrane.","In addition, a series of washes with modified D-PBS without 1% fetal bovine serum is performed immediately prior to activation of the calcium response.","A calcium response is initiated by the addition of one or more candidate receptor agonist compounds, calcium ionophore A23187 (10 μM; positive control), or ATP (4 μM; positive control).","Fluorescence is measured by Molecular Device's FLIPR with an argon laser (excitation at 488 nm).","(See, e.g., Kuntzweiler et al., Drug Development Research, 44(1): 14-20 (1998)).","The F-stop for the detector camera was set at 2.5 and the length of exposure was 0.4 milliseconds.","Basal fluorescence of cells was measured for 20 seconds prior to addition of candidate agonist, ATP, or A23187, and the basal fluorescence level was subtracted from the response signal.","The calcium signal is measured for approximately 200 seconds, taking readings every two seconds.","Calcium ionophore A23187 and ATP increase the calcium signal 200% above baseline levels.","In general, activated GPCRs increase the calcium signal approximately 10-15% above baseline signal.","E. Mitogenesis Assay In a mitogenesis assay, the ability of candidate modulators to induce or inhibit nGPCR-x-mediated cell division is determined.","(See, e.g., Lajiness et al., Journal of Pharmacology and Experimental Therapeutics 267(3): 1573-1581 (1993)).","For example, CHO cells stably expressing nGPCR-x are seeded into 96-well plates at a density of 5000 cells/well and grown at 37° C. in MEM with 10% fetal calf serum for 48 hours, at which time the cells are rinsed twice with serum-free MEM.","After rinsing, 80 μl of fresh MEM, or MEM containing a known mitogen, is added along with 20 μl MEM containing varying concentrations of one or more candidate modulators or test compounds diluted in serum-free medium.","As controls, some wells on each plate receive serum-free medium alone, and some receive medium containing 10% fetal bovine serum.","Untransfected cells or cells transfected with vector alone also may serve as controls.","After culture for 16-18 hours, 1 μCi of [3H]-thymidine (2 Ci/mmol) is added to the wells and cells are incubated for an additional 2 hours at 37° C. The cells are trypsinized and collected on filter mats with a cell harvester (Tomtec); the filters are then counted in a Betaplate counter.","The incorporation of [3H]-thymidine in serum-free test wells is compared to the results achieved in cells stimulated with serum (positive control).","Use of multiple concentrations of test compounds permits creation and analysis of dose-response curves using the non-linear, least squares fit equation: A=B×[C/(D+C)]+G where A is the percent of serum stimulation; B is the maximal effect minus baseline; C is the EC50; D is the concentration of the compound; and G is the maximal effect.","Parameters B, C and G are determined by Simplex optimization.","Agonists that bind to the receptor are expected to increase [3H]-thymidine incorporation into cells, showing up to 80% of the response to serum.","Antagonists that bind to the receptor will inhibit the stimulation seen with a known agonist by up to 100%.","F. [35S]GTPγS Binding Assay Because G protein-coupled receptors signal through intracellular G proteins whose activity involves GTP binding and hydrolysis to yield bound GDP, measurement of binding of the non-hydrolyzable GTP analog [35S]GTPγS in the presence and absence of candidate modulators provides another assay for modulator activity.","(See, e.g., Kowal et al., Neuropharmacology 37:179-187 (1998).)","In one exemplary assay, cells stably transfected with a nGPCR-x expression vector are grown in 10 cm tissue culture dishes to subconfluence, rinsed once with 5 ml of ice-cold Ca2+/Mg2+-free phosphate-buffered saline, and scraped into 5 ml of the same buffer.","Cells are pelleted by centrifugation (500×g, 5 minutes), resuspended in TEE buffer (25 mM Tris, pH 7.5, 5 in M EDTA, 5 mM EGTA), and frozen in liquid nitrogen.","After thawing, the cells are homogenized using a Dounce homogenizer (one ml TEE per plate of cells), and centrifuged at 1,000×g for 5 minutes to remove nuclei and unbroken cells.","The homogenate supernatant is centrifuged at 20,000×g for 20 minutes to isolate the membrane fraction, and the membrane pellet is washed once with TEE and resuspended in binding buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 1 mM EDTA).","The resuspended membranes can be frozen in liquid nitrogen and stored at −70° C. until use.","Aliquots of cell membranes prepared as described above and stored at −70° C. are thawed, homogenized, and diluted into buffer containing 20 mM HEPES, 10 mM MgCl, 1 mM EDTA, 120 mM NaCl, 10 μM GDP, and 0.2 mM ascorbate, at a concentration of 10-50 μg/ml.","In a final volume of 90 μl, homogenates are incubated with varying concentrations of candidate modulator compounds or 100 μM GTP for 30 minutes at 30° C. and then placed on ice.","To each sample, 10 μl guanosine 5′-O-(3[35S]thio) triphosphate (NEN, 1200 Ci/mmol; [35S]-GTPγS), was added to a final concentration of 100-200 pM.","Samples are incubated at 30° C. for an additional 30 minutes, 1 ml of 10 mM HEPES, pH 7.4, 10 mM MgCl2, at 4° C. is added and the reaction is stopped by filtration.","Samples are filtered over Whatman GF/B filters and the filters are washed with 20 ml ice-cold 10 mM HEPES, pH 7.4, 10 mM MgCl2.Filters are counted by liquid scintillation spectroscopy.","Nonspecific binding of [35S]-GTPγS is measured in the presence of 100 μM GTP and subtracted from the total.","Compounds are selected that modulate the amount of [35]-GTPγS binding in the cells, compared to untransfected control cells.","Activation of receptors by agonists gives up to a five-fold increase in [35S]GTPγS binding.","This response is blocked by antagonists.","G. MAP Kinase Activity Assay Evaluation of MAP kinase activity in cells expressing a GPCR provides another assay to identify modulators of GPCR activity.","(See, e.g.","Lajiness et al., Journal of Pharmacology and Experimental Therapeutics 267(3):1573-1581 (1993) and Boulton et al., Cell 65:663-675 (1991).)","In one embodiment, CHO cells stably transfected with nGPCR-x are seeded into 6-well plates at a density of 70,000 cells/well 48 hours prior to the assay.","During this 48-hour period, the cells are cultured at 37° C. in MEM medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin and 10 μg/ml streptomycin.","The cells are serum-starved for 1-2 hours prior to the addition of stimulants.","For the assay, the cells are treated with medium alone or medium containing either a candidate agonist or 200 nM Phorbol ester-myristoyl acetate (i.e., PMA, a positive control), and the cells are incubated at 37° C. for varying times.","To stop the reaction, the plates are placed on ice, the medium is aspirated, and the cells are rinsed with 1 ml of ice-cold PBS containing 1 mM EDTA.","Thereafter, 200 μl of cell lysis buffer (12.5 mM MOPS, pH 7.3, 12.5 mM glycerophosphate, 7.5 mM MgCl2, 0.5 MM EGTA, 0.5 mM sodium vanadate, 1 mM benzamidine, 1 mM dithiothreitol, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 2 μg/ml pepstatin A, and 1 μM okadaic acid) is added to the cells.","The cells are scraped from the plates and homogenized by 10 passages through a 23 3/4 G needle, and the cytosol fraction is prepared by centrifugation at 20,000×g for 15 minutes.","Aliquots (5-10 μl containing 1-5 μg protein) of cytosol are mixed with 1 mM MAPK Substrate Peptide (APRTPGGRR (SEQ ID NO: 129), Upstate Biotechnology, Inc., N.Y.) and 50 μM [γ-32P]ATP (NEN, 3000 Ci/mmol), diluted to a final specific activity of ˜2000 cpm/pmol, in a total volume of 25 μl.","The samples are incubated for 5 minutes at 30° C., and reactions are stopped by spotting 20 μl on 2 cm2 squares of Whatman P81 phosphocellulose paper.","The filter squares are washed in 4 changes of 1% H3PO4, and the squares are subjected to liquid scintillation spectroscopy to quantitate bound label.","Equivalent cytosolic extracts are incubated without MAPK substrate peptide, and the bound label from these samples are subtracted from the matched samples with the substrate peptide.","The cytosolic extract from each well is used as a separate point.","Protein concentrations are determined by a dye binding protein assay (Bio-Rad Laboratories).","Agonist activation of the receptor is expected to result in up to a five-fold increase in MAPK enzyme activity.","This increase is blocked by antagonists.","H. [3H]Anachidonic Acid Release The activation of GPCRs also has been observed to potentiate arachidonic acid release in cells, providing yet another useful assay for modulators of GPCR activity.","(See, e.g., Kanterman et al., Molecular Pharmacology 39:364-369 (1991).)","For example, CHO cells that are stably transfected with a nGPCR-x expression vector are plated in 24-well plates at a density of 15,000 cells/well and grown in MEM medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin and 10 μg/ml streptomycin for 48 hours at 37° C. before use.","Cells of each well are labeled by incubation with [3H]-arachidonic acid (Amersham Corp., 210 Ci/mmol) at 0.5 μCi/ml in 1 ml MEM supplemented with 10 mM HEPES, pH 7.5, and 0.5% fatty-acid-free bovine serum albumin for 2 hours at 37° C. The cells are then washed twice with 1 ml of the same buffer.","Candidate modulator compounds are added in 1 ml of the same buffer, either alone or with 10 μM ATP and the cells are incubated at 37° C. for 30 minutes.","Buffer alone and mock-transfected cells are used as controls.","Samples (0.5 ml) from each well are counted by liquid scintillation spectroscopy.","Agonists which activate the receptor will lead to potentiation of the ATP-stimulated release of [3H]-arachidonic acid.","This potentiation is blocked by antagonists.","I. Extracellular Acidification Rate In yet another assay, the effects of candidate modulators of nGFCR-x activity are assayed by monitoring extracellular changes in pH induced by the test compounds.","(See, e.g., Dunlop et al, Journal of Pharmacological and Toxicological Methods 40(1):47-55 (1998).)","In one embodiment, CHO cells transfected with a nGPCR-x expression vector are seeded into 12 mm capsule cups (Molecular Devices Corp.) at 4×105 cells/cup in MEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 10 U/ml penicillin, and 10 μg/ml streptomycin.","The cells are incubated in this medium at 37° C. in 5% CO2 for 24 hours.","Extracellular acidification rates are measured using a Cytosensor microphysiometer (Molecular Devices Corp.).","The capsule cups are loaded into the sensor chambers of the microphysiometer and the chambers are perfused with running buffer (bicarbonate-free MEM supplemented with 4 mM L-glutamine, 10 units/ml penicillin, 10 μg/ml streptomycin, 26 mM NaCl) at a flow rate of 100 μl/minute.","Candidate agonists or other agents are diluted into the running buffer and perfused through a second fluid path.","During each 60-second pump cycle, the pump is run for 38 seconds and is off for the remaining 22 seconds.","The pH of the running buffer in the sensor chamber is recorded during the cycle from 43-58 seconds, and the pump is re-started at 60 seconds to start the next cycle.","The rate of acidification of the running buffer during the recording time is calculated by the Cytosoft program.","Changes in the rate of acidification are calculated by subtracting the baseline value (the average of 4 rate measurements immediately before addition of a modulator candidate) from the highest rate measurement obtained after addition of a modulator candidate.","The selected instrument detects 61 mV/pH unit.","Modulators that act as agonists of the receptor result in an increase in the rate of extracellular acidification compared to the rate in the absence of agonist.","This response is blocked by modulators which act as antagonists of the receptor.","Example 11 In Situ Hybridization DNA Probe Preparation for nGPCR-11, -16, 40, -54, and -56 DNA probes for in situ hybridization were prepared as follows.","Two sets of primer pairs were prepared.","The first set has the sequence for T7 polymerase promoter on the 5′ primer to make the sense RNA, and the second set has the T7 polymerase promoter sequence on the 3′ primer to make the antisense RNA.","PCR was performed in a 50 μl reaction containing 36.5 μl H2O, 5 μl 10×TT buffer (140 mM Ammonium Sulfate, 0.1% gelatine, 0.6 M Tris-tricine pH 8.4), 5 μl 25 mM MgCl2, 2 μl 10 mM dNTP, 0.4 μl Incyte clone 1722192 DNA, 0.5 μl AmpliTaq (PE Applied Biosystems), and 0.3 μl oligo1 (1 mg/ml) and 0.3 μl oligo2 (1 mg/ml) [to make the sense RNA], or 0.3 μl oligo3 (1 mg/ml) and 0.3 μl oligo4 (1 mg/ml) [to make the antisense RNA].","The PCR reaction involved one cycle at 94° C. for 2 min followed by 35 cycles at 94° C. for 30 sec, 60° C. for 30 sec, 72° C. for 30 sec.","The two PCR reactions were loaded onto a 1.2% agarose gel.","The DNA band was excised from the gel, placed in a GenElute Agarose spin column (Supelco) and spun for 10 min at maximum speed.","The eluted DNA was EtOH precipitated and resuspended in transcription buffer.","The primer sequences for each nGPCR tested are listed below.","For nGPCR-11, the sense primers were: GCGTAATACGACTCACTATAGGGAGACCGCGTGTCTGCTAGACTCTATTTCC 3′(LW1658) (SEQ ID NO: 159), and: 5′ TGCCACACTGATGCAACTCC 3′ (LW661) (SEQ ID NO: 160).","The antisense primers were: GCGTAATACGACTCACTATAGGGAGACCTGCCACACTGATGCAACTCC (LW1659) SEQ ID NO: 161) and: 5′GCGTGTCTGCTAGACTCTATITCC 3′ (LW1660) (SEQ ID NO: 162).","The primer pairs yielded a product of 275 bp.","For nGPCR-16, the sense primers were: 5′GCGTAATACGACTCACTATAGGGAGACCGCACGCCACTCTTTACTATCCC (LW1645) (SEQ ID NO: 163), and: 5′ GCACAAAACACAATFCCATAAGCC 3′ (LW1648) (SEQ ID NO: 164).","The antisense primers were: 5′GCGTAATACGACTCACTATAGGGAGACCGCACAAAACACAATTCCATAAGCC 3′ (LW1646) (SEQ ID NO: 165), and: 5′ GCTACGCCACTCTTrACTATCCC 3′ (LW1647) (SEQ ID NO: 166).","The primer pairs yielded a product of 283 bp.","For nGPCR-40, the sense primers were: 5′GCGTAATACGACTCACTATAGGGAGACCTTATGAGCAGCAATTCATCCC 3′(LW1704) (SEQ ID NO: 167), and: 5′CACACCCACCAAGAAATCAG 3′(LW1707) (SEQ ID NO: 168).","The antisense primers were: 5′GCGTAATACGACTCACTATAGGGAGACCCACACCCACCAAGAAATCAG 3′(LW1705) (SEQ ID NO: 169), and: 5′ TTATGAGCAGCAATTCATCCC 3′ (LW1706) (SEQ ID NO: 170).","The primer pairs yielded a product of 25 lbp.","For nGPCR-54, the sense primers were: 5′GCGTAATACGACTCACTATAGGGAGACCCGATTATCCACACTTTGACCC 3′ (LW1803) (SEQ ID NO: 171), and: 5′CTGAAAGTrGTCGCTGACC 3′ (LW1634) (SEQ ID NO: 172).","The anti-sense primers were: GCGTAATACGACTCACTATAGGGAGACCCTGCTGAAAGTTGTCGCTGACC 3′ (LW1804) (SEQ ID NO: 173), and: 5′CGATTATCCACACTTTGACGC 3′ (LW1635) (SEQ ID NO: 174).","The primer pairs yielded a product of 286 bp.","For nGPCR-56, the sense primers were: GCGTAATACGACTCACTATAGGGAGACCCTGTAAAATTCACACAAGCACC 3′ (LW1763) (SEQ ID NO: 175), and: 5′AGAAGACAGAGCAACCTCC 3′ (L[W1766) (SEQ ID NO: 176).","The anti-sense primers were: GCGTAATACGACTCACTATAGGGAGACCAGAAGACAGAGCAACCTCC (LW1764) (SEQ ID NO: 177) and: CTGTAAAATTCACACAAGCACC (LW1765) (SEQ ID NO: 178).","The primer pairs yielded a product of 272 bp.","DNA Probe Preparation For nGPCR-1 Probes for nGPCR-1 were prepared as above with the following modifications.","Using a sense primer: GCATGGATCCTCTTTTGCTGTATTTCACCCTC) (LW1595) (SEQ ID NO: 179) and an antisense primer: 5′GCATGAATTCACAATGCCAGTGATAAGGAAG 3′ (LW1596) (SEQ ID NO: 180), a 271 bp fragment was generated by PCR.","The fragment was digested with BamHI and EcoRI and ligated into a BluescriptII vector that had been cut with BamHI and EcoRI.","The orientation of the insert was such that T7 polymerase generates the anti-sense strand and T3 polymerase generates the sense strand.","Histochemistry Coronal and sagittal oriented rat brain sections were cryosectioned (20 μm thick) using a Reichert-Jung cryostat.","The individual sections were thaw-mounted onto silanated, nuclease-free slides (CEL Associates, Inc., Houston, Tex.","), and stored at −80° C. The sections were processed starting with post-fixation in cold 4% paraformaldehyde, rinsed in cold PBS, acetylated using acetic anhydride in triethanolamine buffer and dehydrated through 70%, 95%, and 100% alcohols at room temperature (RT).","This was followed with delipidation in chloroform then rehydration in 100% and 95% alcohol at room temperature.","Sections were air-dried prior to hybridization.","Two PCR fragments (˜250 bp) were generated, one that contained T7 polymerase on the 5′ end (sense) and the other with T7 polymerase on the 3′ end (antisense).","The PCR fragments were labeled with 35S-UTP to yield a specific activity of 0.655×106 cpm/pmol for antisense and 0.675×106 cpm/pmol for sense probe.","Both riboprobes were denatured and added to hybridization buffer containing 50% formamide, 10% dextran, 0.3M NaCl, 10 mM Tris, 1 mM EDTA, 1× Denhardts, and 10 mM DTT.","Sequential brain cryosections were hybridized with 45 μl/slide of the sense and antisense riboprobe hybridization mixture, then covered with silanized glass coverslips.","The sections were hybridized overnight (15-18 hrs) at 42° C. in an incubator.","Coverslips were washed off the slides in 1×SSC, followed by RNase A treatment, and high temperature stringency washes (3×, 20 mins at 41° C.) in 0.1×SSC.","Slides were dehydrated with 70%, 95% NH4OAc, and 100% NH4OAc alcohols, air-dried and exposed to Kodak BioMax MR-1 film.","After 9 days of exposure, the film was developed.","This was followed with coating selected tissue slides with Kodak NTB-2 nuclear track emulsion and storing the slides in the dark for 23 days.","The slides were then developed and counterstained with hematoxylin.","Emulsion-coated sections were analyzed microscopically to determine the specificity of labeling.","Presence of autoradiographic grains (generated by antisense probe hybridization) over cell bodies (versus between cell bodies) was used as an index of specific hybridization.","Results In Situ Hybridization Results Indicated Localization in the Following Brain Areas: nGPCR-1 was localized to the dentate gyrus of hippocampus, piriform cortex, and red nucleus.","nGPCR-11 was localized to the piriform cortex, hippocampus, red nucleus, subthalamic nuclei, dorsal raphe, interpeduncular nucleus, and habenula.","nGPCR-16 was localized to the cortex, piriform cortex, hippocampus, thalamus, subthalamic nuclei, hypothalamus, bed nucleus stria terminals and posterior striatum.","nGPCR-40 was localized to the cortex, piriform cortex, hippocampus, substantia nigra compacta, hypothalamus, laterial septus, bed nucleus stria terminalis, thalamus, ventral tegmental area, interpeduncular nucleus, dorsal raphe, medical geniculate, islands of Calleja, subthamalmic nuclei, choroid plexus.","nGPCR-54 was localized to the piriform cortex and hippocampus, including the dentate gyms, CA1 and CA3.nGPCR-56 was localized to the piriform cortex, cortex, interpeduncular nuceus, red nucleus, hippocampus, habenula, substantia nigra pars compacta, mamillary body stria terminalis, hypothalamus, subthamalmic nuclei, corsal raphe, and ventral tegmental area.","Example 12 Chromosomal Localization Methods Chromosomal location of the genes encoding nGPCRs was determined using the Stanford G3 Radiation Hybrid Panel (Research Genetics, Inc., Huntsville, Ala.).","This panel contains 83 radiation hybrid clones of the entire human genome created by the Stanford Human Genome Center.","PCR reactions were assembled containing 25 ng of DNA from each clone and the components of the Expand Hi-Fi PCR System™ (Roche Molecular Biochemicals, Indianapolis, Ind.)","in a final reaction volume of 15 μl.","PCR primers were synthesized by Genosys Corp., The Woodlands, Tex.","PCR reactions were incubated in a GeneAmp 9700 PCR thermocycler (Perkin Elmer Applied Biosystems).","The following cycling program was executed: Pre-soak at (940 for 3 min.)","(94° for 30 sec.)","(52° C. for 60 sec.)","(72′ for 2 min.)]","for 35 cycles.","PCR reaction products were then separated and analyzed by electrophoresis on a 2.0% agarose gel, and stained with ethidium bromide.","Lanes were scored for the presence or absence of the expected PCR product and the results submitted to the Stanford Human Genome Center via e-mail for analysis (http://wwwshgc.stanford.edu./RH/rhserverformnew.html).","nGPCR-40 PCR primers were designed based on the available sequence of the Celera sequence HUM_IDS|Contig|11000258115466.The forward primer used was: 5′ACAGCCCCAAAGCCAAACAC3′ (SEQ ID NO: 181).","The reverse primer was: 5′CCGCAGGAGCAATG-AAAATCAG3′ (SEQ ID NO: 182).","This prier set will prime the synthesis of a 220 base pair fragment in the presence of the appropriate genomic DNA.","G3 Radiation Hybrid Panel Analysis places nGPCR-40 on chromosome 6, most nearly linked to Stanford marker SHGC-1836 with a LOD score of 11.84.This marker lies at position 6q21.In a genome scanning data set, Cao et al.","(Genomics 1997 Jul.","1: 43(1): 1-8) found excess allele sharing for markers on 6q13-q26.Greatest allele sharing was at interval 6q21-q22.3 with a maximum multipoint MLS value of 3.06 close to marker D6S278.Replication data from a second data set found maximum multipoint MLS at the interval D6S424-D6S275.These results provide suggestive evidence for a susceptibility locus for schizophrenia in chromosome 6q from two independent data sets.","nGPCR-54 PCR primers were designed based on the available sequence of the Celera sequence GA—11824020.The forward primer used was: 5′CTGTCTCTCTGTCCTCTTCC3′, (SEQ ID NO: 183).","The reverse primer used was: 5′GCACCGATCTTCATTGAATTTC3′, (SEQ ID NO: 184).","This primer set will prime the synthesis of a 145 base pair fragment in the presence of the appropriate genomic DNA.","G3 Radiation Hybrid Panel Analysis places nGPCR-54 on chromosome 13, most nearly linked to Stanford marker SHGC-68276 with a LOD score of 6.31.This marker lies at position 13q32.Numerous investigations have found significant suggestion of linkage of schizophrenia to this region of chromosome 13q32.See, for example, Brzustowicz et al., Am J Hum Genet 1999 October; 65(4): 1096-1103; Blouinet al., Nat Genet 1998 September; 20(1): 70-3; Shaw et al., Am J Med Genet.","1998 Sep. 7; 81(5): 364-76; Lin et al., Hum Genet 1997 March, 99(3): 417-20; Pulver et al., Cold Spring Harb Symp Quant Biol 1996; 61:797-814.Genes localized to chromosomal regions in linkage with schizophrenia are candidate genes for disease susceptibility.","Genes in these regions with the potential to play a biochemical/functional role in the disease process (like G protein coupled receptors) have a high probability of being a disease-modifying locus.","nGPCR-40 and -54, because of their chromosomal location, are attractive targets therefore for screening ligands useful in modulating cellular processes involved in schizophrenia.","Example 13 Clone Deposit Information In accordance with the Budapest Treaty, clones of the present invention have been deposited at the Agricultural Research Culture Collection (NRRL) International Depository Authority, 1815 N. University Street, Peoria, Ill. 61604, U.S.A. Accession numbers and deposit dates are provided below in Table 6.TABLE 6 DEPOSIT INFORMATION Accession Number Nudapest Treaty Clone NRRL Deposit Date nGPCR-1 (SEQ ID NO:73) B-30243 Jan. 18, 2000 nGPCR-5 (SEQ ID NO: 75) B-30244 Jan. 18, 2000 nGPCR-16 (SEQ ID NO: 81) B-30245 Jan. 18, 2000 nGPCR-11 (SEQ ID NO: 79) B-30258 Feb. 2, 2000 nGPCR-17 (SEQ ID NO: 23) B-30259 Feb. 3, 2000 nGPCR-9 (SEQ ID NO: 77) B-30262 Feb. 22, 2000 nGPCR-58 (SEQ ID NO: 91) B-30274 Mar.","23, 2000 nGPCR-56 (SEQ ID NO: 89) B-30288 May 5, 2000 nGPCR-3 (SEQ ID NO: 185) B-30290 May 5, 2000 nGPCR-54 (SEQ ID NO: 85) B-30291 May 5, 2000 nGPCR-40 (SEQ ID NO: 83*) B-30299N Jun.","2, 2000 *The clone deposited with NRLL Accession Number N30299N comprises a sequence identical to SEQ ID NO: 83 but with the substitution of an “A” at neucleotide position 10.Example 14 Using nGPCR-x Proteins to Isolate Neurotransmitters The isolated nGPCR-x proteins can be used to isolate novel or known neurotransmitters (Saito et al., Nature 400: 265-269, 1999).","The cDNAs that encode the isolated nGPCR-x can be cloned into mammalian expression vectors and used to stably or transiently transfect mammalian cells including CHO, Cos or HEK293 cells.","Receptor expression can be determined by Northern blot analysis of transfected cells and identification of an appropriately sized mRNA band (predicted size from the cDNA).","Brain regions shown by mRNA analysis to express each of the nGPCR-x proteins could be processed for peptide extraction using any of several protocols ((Reinsheidk R. K. et al., Science 270: 243-247, 1996; Sakurai, T., et al., Cell 92; 573-585, 1998; Hinuma, S., et al., Nature 398: 272-276, 1998).","Chromotographic fractions of brain extracts could be tested for ability to activate nGPCR-x proteins by measuring second messenger production such as changes in cAMP production in the presence or absence of forskolin, changes in inositol 3-phosphate levels, changes in intracellular calcium levels or by indirect measures of receptor activation including receptor stimulated mitogenesis, receptor mediated changes in extracellular acidification or receptor mediated changes in reporter gene activation in response to cAMP or calcium (these methods should all be referenced in other sections of the patent).","Receptor activation could also be monitored by co-transfecting cells with a chimeric GIq/i3 to force receptor coupling to a calcium stimulating pathway (Conklin et al., Nature 363; 274-276, 1993).","Neurotransmitter mediated activation of receptors could also be monitored by measuring changes in [35S]-GTPKS binding in membrane fractions prepared from transfected mammalian cells.","This assay could also be performed using baculoviruses containing nGPCR-x proteins infected into SF9 insect cells.","The neurotransmitter which activates nGPCR-x proteins can be purified to homogeneity through successive rounds of purification using nGPCR-x proteins activation as a measurement of neurotransmitter activity.","The composition of the neurotransmitter can be determined by mass spectrometry and Edman degradation if peptidergic.","Neurotramitters isolated in this manner will be bioactive materials which will alter neurotransmission in the central nervous system and will produce behavioral and biochemical changes.","Example 15 Using nGPCR-x Proteins to Isolate and Purify G Proteins cDNAs encoding nGPCR-x proteins are epitope-tagged at the amino terminus end of the cDNA with the cleavable influenza-hemagglutinin signal sequence followed by the FLAG epitope (IBI, New Haven, Conn.).","Additionally, these sequences are tagged at the carboxyl terminus with DNA encoding six histidine residues.","(Amino and Carboxyl Terminal Modifications to Facilitate the Production and Purification of a G Protein-Coupled Receptor, B. K. Kobilka, Analytical Biochemistry, Vol.","231, No.","1, October 1995, pp.","269-271).","The resulting sequences are cloned into a baculovirus expression vector such as pVL1392 (Invitrogen).","The baculovirus expression vectors are used to infect SF-9 insect cells as described (Guan, X. M., Kobilka, T. S., and Kobilka, B. K. (1992) J. Biol.","Chem.","267, 21995-21998).","Infected SF-9 cells could be grown in 1000-ml cultures in SF900 II medium (Life Technologies, Inc.) containing 5% fetal calf serum (Gemini, Calabasas, Calif.) and 0.1 mg/ml gentamicin (Life Technologies, Inc.) for 48 hours at which time the cells could be harvested.","Cell membrane preparations could be separated from soluble proteins following cell lysis.","nGPCR-x protein purification is carried out as described for purification of the θ2 receptor (Kobilka, Anal.","Biochem., 231 (1): 269-271, 1995) including solubilization of the membranes in 0.8-1.0% n-dodecyl-D-maltoside (DM) (CalBiochem, La Jolla, Calif.) in buffer containing protease inhibitors followed by Ni-column chromatography using chelating Sepharose™ (Pharmacia, Uppsala, Sweden).","The eluate from the Ni-column is further purified on an M1 anti-FLAG antibody column (IBI).","Receptor containing fractions are monitored by using receptor specific antibodies following western blot analysis or by SDS-PAGE analysis to look for an appropriate sized protein band (appropriate size would be the predicted molecular weight of the protein).","This method of purifying G protein is particularly useful to isolate G proteins that bind to the nGPCR-x proteins in the absence of an activating ligand.","Some of the preferred embodiments of the invention described above are outlined below and include, but are not limited to, the following embodiments.","As those skilled in the art will appreciate, numerous changes and modifications may be made to the preferred embodiments of the invention without departing from the spirit of the invention.","It is intended that all such variations fall within the scope of the invention.","The entire disclosure of each publication cited herein is hereby incorporated by reference."]],"string":"[\n [\n \"Protein-coupled receptor\",\n \"The present application provides a gene encoding a G protein-coupled receptor termed nGPCR-14; constructs and recombinant host cells incorporating the genes; the nGPCR-14 polypeptides encoded by the gene; antibodies to the nGPCR-x polypeptides; and methods of making and using all of the foregoing.\"\n ],\n [\n \"1.An isolated nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence homologous to a sequence of SEQ ID NO: 192, and fragments thereof; said nucleic acid molecule encoding at least a portion of nGPCR-14, wherein the isolated nucleic acid comprises at least one or more nucleotides from one or more of the following regions of SEQ ID NO: 191: nucleotides 1 to 193 of SEQ ID NO: 191; nucleotides 612 to 644 of SEQ ID NO:191; nucleotides 697 to 706 of SEQ ID NO:191; nucleotides 1011 to 1049 of SEQ ID NO: 191; nucleotides 1051 to 1057 of SEQ ID NO:191; nucleotides 1090 to 1096 of SEQ ID NO:191; or nucleotides 1141 to 1642 of SEQ ID NO:191.2.The isolated nucleic acid molecule of claim 1 comprising a sequence that encodes a polypeptide comprising a sequences of SEQ ID NO: 192, and fragments thereof.\",\n \"3.The isolated nucleic acid molecule of claim 1 comprising a sequence homologous to a sequence of SEQ ID NO: 191, and fragments thereof.\",\n \"4.The isolated nucleic acid molecule of claim 1 comprising a sequence of SEQ ID NO:191, and fragments thereof.\",\n \"5.The isolated nucleic acid molecule of claim 1 wherein said nucleic acid molecule is DNA.\",\n \"6.The isolated nucleic acid molecule of claim 1 wherein said nucleic acid molecule is RNA.\",\n \"7.An expression vector comprising a nucleic acid molecule of any one of claims 1 to 5.8.The expression vector of claim 7 wherein said nucleic acid molecule comprises a sequence of SEQ ID NO:191.9.The expression vector of claim 7 wherein said vector is a plasmid.\",\n \"10.The expression vector of claim 7 wherein said vector is a viral particle.\",\n \"11.The expression vector of claim 10 wherein said vector is selected from the group consisting of adenoviruses, baculoviruses, parvoviruses, herpesviruses, poxviruses, adeno-associated viruses, Semliki Forest viruses, vaccinia viruses, and retroviruses.\",\n \"12.The expression vector of claim 7 wherein said nucleic acid molecule is operably connected to a promoter selected from the group consisting of simian virus 40, mouse mammary tumor virus, long terminal repeat of human immunodeficiency virus, maloney virus, cytomegalovirus immediate early promoter, Epstein Barr virus, rous sarcoma virus, human actin, human myosin, human hemoglobin, human muscle creatine, and human metalothionein.\",\n \"13.A host cell transformed with an expression vector of claim 7.14.The transformed host cell of claim 13 wherein said cell is a bacterial cell.\",\n \"15.The transformed host cell of claim 14 wherein said bacterial cell is E. coli.\",\n \"16.The transformed host cell of claim 13 wherein said cell is yeast.\",\n \"17.The transformed host cell of claim 16 wherein said yeast is S. cerevisiae.\",\n \"18.The transformed host cell of claim 13 wherein said cell is an insect cell.\",\n \"19.The transformed host cell of claim 18 wherein said insect cell is S. frugiperda.\",\n \"20.The transformed host cell of claim 13 wherein said cell is a mammalian cell.\",\n \"21.The transformed host cell of claim 20 wherein mammalian cell is selected from the group consisting of chinese hamster ovary cells, HeLa cells, African green monkey kidney cells, human 293 cells, and murine 3T3 fibroblasts.\",\n \"22.An isolated nucleic acid molecule comprising a nucleotide sequence complementary to at least a portion of a sequence of SEQ ID NO:191, said portion comprising at least 10 nucleotides, wherein said isolated nucleic acid comprises at least one or more nucleotides from one or more of the following regions of SEQ ID NO: 191: nucleotides 1 to 193 of SEQ ID NO: 191; nucleotides 612 to 644 of SEQ ID NO:191; nucleotides 697 to 706 of SEQ ID NO: 191; nucleotides 1011 to 1049 of SEQ ID NO: 191; nucleotides 1051 to 1057 of SEQ ID NO:191; nucleotides 1090 to 1096 of SEQ ID NO:191; or nucleotides 1141 to 1642 of SEQ ID NO:191.23.The nucleic acid molecule of claim 22 wherein said molecule is an antisense oligonucleotide directed to a region of a sequence of SEQ ID NO: 191.24.The nucleic acid molecule of claim 23 wherein said oligonucleotide is directed to a regulatory region of a sequence of SEQ ID NO: 191.25.A composition comprising a nucleic acid molecule of any one of claims 1 to 5 or 22 and an acceptable carrier or diluent.\",\n \"26.A composition comprising a recombinant expression vector of claim 7 and an acceptable carrier or diluent.\",\n \"27.A method of producing a polypeptide that comprises a sequence of SEQ ID NO: 192, and homologs and fragments thereof, said method comprising the steps of: a) introducing a recombinant expression vector of claim 7 into a compatible host cell; b) growing said host cell under conditions for expression of said polypeptide; and c) recovering said polypeptide, wherein the polypeptide comprising a sequence of SEQ ID NO:192 comprises at least one or more amino acid residues from one or more of the following regions of SEQ ID NO: 192: amino acid residues 1 to 42 of SEQ ID NO:192; amino acid residues 68 to 77 of SEQ ID NO:192; amino acid residues 185 to 197 of SEQ ID NO: 192; or amino acid residues 293 to 513 of SEQ ID NO: 192.28.The method of claim 27 wherein said host cell is lysed and said polypeptide is recovered from the lysate of said host cell.\",\n \"29.The method of claim 27 wherein said polypeptide is recovered by purifying the culture medium without lysing said host cell.\",\n \"30.An isolated polypeptide encoded by a nucleic acid molecule of claim 1, wherein the polynucleotide comprises at least one or more amino acid residues from one or more of the following regions of SEQ ID NO: 192: amino acid residues 1 to 42 of SEQ ID NO:192; amino acid residues 68 to 77 of SEQ ID NO:192; amino acid residues 185 to 197 of SEQ ID NO: 192; or amino acid residues 293 to 513 of SEQ ID NO: 192.31.The polypeptide of claim 30 wherein said polypeptide comprises a fragment of SEQ ID NO:192.32.The polypeptide of claim 30 wherein said polypeptide comprises an amino acid sequence homologous to a sequence of SEQ ID NO: 192.33.The polypeptide of claim 30 wherein said sequence homologous to a sequence of SEQ ID NO:192 comprises at least one conservative amino acid substitution compared to the sequence of SEQ ID NO: 192.34.The polypeptide of claim 30 wherein said polypeptide comprises a fragment of a polypeptide with a sequence of SEQ ID NO:192.35.A composition comprising a polypeptide of claim 30 and an acceptable carrier or diluent.\",\n \"36.An isolated antibody which binds to an epitope on a polypeptide of claim 30.37.The antibody of claim 36 wherein said antibody is a monoclonal antibody.\",\n \"38.A composition comprising an antibody of claim 36 and an acceptable carrier or diluent.\",\n \"39.A method of inducing an immune response in a mammal against a polypeptide of claim 30 comprising administering to said mammal an amount of said polypeptide sufficient to induce said immune response.\",\n \"40.A method for identifying a compound which binds nGPCR-14 comprising the steps of: a) contacting nGPCR-14 with a compound; and b) determining whether said compound binds nGPCR-14.41.The method of claim 40 wherein the nGPCR-14 comprises an amino acid sequence of SEQ ID NO: 192.42.The method of claim 40 wherein binding of said compound to nGPCR-14 is determined by a protein binding assay.\",\n \"43.The method of claim 40 wherein said protein binding assay is selected from the group consisting of a gel-shift assay, Western blot, radiolabeled competition assay, phage-based expression cloning, co-fractionation by chromatography, co-precipitation, cross linking, interaction trap/two-hybrid analysis, southwestern analysis, and ELISA.\",\n \"44.A compound identified by the method of claim 40.45.A method for identifying a compound which binds a nucleic acid molecule encoding nGPCR-14 comprising the steps of: a) contacting said nucleic acid molecule encoding nGPCR-14 with a compound; and b) determining whether said compound binds said nucleic acid molecule.\",\n \"46.The method of claim 45 wherein binding is determined by a gel-shift assay.\",\n \"47.A compound identified by the method of claim 45.48.A method for identifying a compound which modulates the activity of nGPCR-14 comprising the steps of: a) contacting nGPCR-14 with a compound; and b) determining whether nGPCR-14 activity has been modulated.\",\n \"49.The method of claim 48 wherein the nGPCR-14 comprises an amino acid sequence of SEQ ID NO: 192.50.The method of claim 48 wherein said activity is neuropeptide binding.\",\n \"51.The method of claim 48 wherein said activity is neuropeptide signaling.\",\n \"52.A compound identified by the method of claim 48.53.A method of identifying an animal homolog of nGPCR-14 comprising the steps: a) comparing the nucleic acid sequences of the animal with a sequence of SEQ ID NO: 191, and portions thereof, said portions being at least 10 nucleotides; and b) identifying nucleic acid sequences of the animal that are homologous to said sequence of SEQ ID NO: 191, and portions thereof.\",\n \"54.The method of claim 53 wherein comparing the nucleic acid sequences of the animal with a sequence selected of SEQ ID NO: 191, and portions thereof, said portions being at least 10 nucleotides, is performed by DNA hybridization.\",\n \"55.The method of claim 53 wherein comparing the nucleic acid sequences of the animal with a sequence selected of SEQ ID NO: 191, and portions thereof, said portions being at least 10 nucleotides, is performed by computer homology search.\",\n \"56.A method of screening a human subject to diagnose a disorder affecting the brain or genetic predisposition therefor, comprising the steps of: (a) assaying nucleic acid of a human subject to determine a presence or an absence of a mutation altering an amino acid sequence, expression, or biological activity of at least one nGPCR that is expressed in the brain, wherein the nGPCR comprises an amino acid sequence of SEQ ID NO:192, and allelic variants thereof, and wherein the nucleic acid corresponds to a gene encoding the nGPCR; and (b) diagnosing the disorder or predisposition from the presence or absence of said mutation, wherein the presence of a mutation altering the amino acid sequence, expression, or biological activity of the nGPCR in the nucleic acid correlates with an increased risk of developing the disorder.\",\n \"57.A method according to claim 56, wherein the nGPCR is nGPCR-14 comprising an amino acid sequence set forth in SEQ ID NO:192 or an allelic variant thereof.\",\n \"58.A method according to claim 56, wherein the assaying step comprises at least one procedure selected from the group consisting of: a) comparing nucleotide sequences from the human subject and reference sequences and determining a difference of at least a nucleotide of at least one codon between is the nucleotide sequences from the human subject that encodes an nGPCR-14 allele and an nGPCR-14 reference sequence; (b) performing a hybridization assay to determine whether nucleic acid from the human subject has a nucleotide sequence identical to or different from one or more reference sequences; (c) performing a polynucleotide migration assay to determine whether nucleic acid from the human subject has a nucleotide sequence identical to or different from one or more reference sequences; and (d) performing a restriction endonuclease digestion to determine whether nucleic acid from the human subject has a nucleotide sequence identical to or different from one or more reference sequences.\",\n \"59.A method according to claim 58 wherein the assaying step comprises: performing a polymerase chain reaction assay to amplify nucleic acid comprising nGPCR-14 coding sequence, and determining nucleotide sequence of the amplified nucleic acid.\",\n \"60.A method of screening for an nGPCR-14 mental disorder genotype in a human patient, comprising the steps of: (a) providing a biological sample comprising nucleic acid from said patient, said nucleic acid including sequences corresponding to allelles of nGPCR-14; and (b) detecting the presence of one or more mutations in the nGPCR-14 allelle; wherein the presence of a mutation in an nGPCR-40 allelle or nGPCR-54 allele is indicative of a mental disorder genotype.\",\n \"61.The method according to claim 59 wherein said biological sample is a cell sample.\",\n \"62.The method according to claim 59 wherein said detecting the presence of a mutation comprises sequencing at least a portion of said nucleic acid, said portion comprising at least one codon of said nGPCR-14 alleles.\",\n \"63.The method according to claim 59 wherein said nucleic acid is DNA.\",\n \"64.The method according to claim 59 wherein said nucleic acid is RNA.\",\n \"65.A kit for screening a human subject to diagnose a mental disorder or a genetic predisposition therefor, comprising, in association: (a) an oligonucleotide useful as a probe for identifying polymorphisms in a human nGPCR-14 gene, the oligonucleotide comprising 6-50 nucleotides in a sequence that is identical or complementary to a sequence of a wild type human nGPCR-14 gene sequence or nGPCR-14 coding sequence, except for one sequence difference selected from the group consisting of a nucleotide addition, a nucleotide deletion, or nucleotide substitution; and (b) a media packaged with the oligonucleotide, said media containing information for identifying polymorphisms that correlate with schizophrenia or a genetic predisposition therefor, the polymorphisms being identifiable using the oligonucleotide as a probe.\",\n \"66.A method of identifying a nGPCR allelic variant that correlates with a mental disorder, comprising steps of: (a) providing a biological sample comprising nucleic acid from a human patient diagnosed with a mental disorder, or from the patient's genetic progenitors or progeny; (b) detecting in the nucleic acid the presence of one or more mutations in an nGPCR that is expressed in the brain, wherein the nGPCR comprises an amino acid sequence of SEQ ID NO: 192, and allelic variants thereof, and wherein the nucleic acid includes sequence corresponding to the gene or genes encoding nGPCR; wherein the one or more mutations detected indicates an allelic variant that correlates with a mental disorder.\",\n \"67.A method according to claim 66 wherein the at least one nGPCR is nGPCR-14, or an allelic variant thereof.\",\n \"68.A purified and isolated polynucleotide comprising a nucleotide sequence encoding a nGPCR-14 allelic variant identified according to claim 67.69.A host cell transformed or transfected with a polynucleotide according to claim 68 or with a vector comprising the polynucleotide.\",\n \"70.A purified polynucleotide comprising a nucleotide sequence encoding nGPCR-14 of a human with a mental disorder; wherein said polynucleotide hybridizes to the complement of SEQ ID NO:191 under the following hybridization conditions: (a) hybridization for 16 hours at 42° C. in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% dextran sulfate and (b) washing 2 times for 30 minutes at 60° C. in a wash solution comprising 0.1×SSC and 1% SDS; and wherein the polynucleotide that encodes the nGPCR-14 amino acid sequence of the human differs from SEQ ID NO:192 by at least one residue and comprises at least one or more amino acid residues from one or more of the following regions of SEQ ID NO: 192: amino acid residues 1 to 42 of SEQ ID NO: 192; amino acid residues 68 to 77 of SEQ ID NO: 192; amino acid residues 185 to 197 of SEQ ID NO: 192; or amino acid residues 293 to 513 of SEQ ID NO: 192.71.A vector comprising a polynucleotide according to claim 70.72.A host cell that has been transformed or transected with a polynucleotide according to claim 70 and that expresses the nGPCR-14 protein encoded by the polynucleotide.\",\n \"73.A host cell according to claim 72 that has been co-transfected with a polynucleotide encoding the nGPCR-14 amino acid sequence set forth in SEQ ID NO:192 and that expresses the nGPCR-14 protein having the amino acid sequence set forth in SEQ ID NO:192.74.A method for identifying a modulator of biological activity of nGPCR-14 comprising the steps of: a) contacting a cell according to claim 72 in the presence and in the absence of a putative modulator compound; b) measuring nGPCR-14 biological activity in the cell; wherein decreased or increased nGPCR-14 biological activity in the presence versus absence of the putative modulator is indicative of a modulator of biological activity.\",\n \"75.A method to identify compounds useful for the treatment of a mental disorder, said method comprising steps of: (a) contacting a composition comprising nGPCR-14 with a compound suspected of binding nGPCR-14; (b) detecting binding between nGPCR-14 and the compound suspected of binding nGPCR-14; wherein compounds identified as binding nGPCR-14 are candidate compounds useful for the treatment of a mental disorder.\",\n \"76.A method for identifying a compound useful as a modulator of binding between nGPCR-14 and a binding partner of nGPCR-14 comprising the steps of: (a) contacting the binding partner and a composition comprising nGPCR-14 in the presence and in the absence of a putative modulator compound; (b) detecting binding between the binding partner and nGPCR-14; wherein decreased or increased binding between the binding partner and nGPCR-14 in the presence of the putative modulator, as compared to binding in the absence of the putative modulator is indicative a modulator compound useful for the treatment of schizophrenia.\",\n \"77.A method according to claim 75 or 76 wherein the composition comprises a cell expressing nGPCR-14 on its surface.\",\n \"78.A method according to claim 77 wherein the composition comprises a cell transformed or transfected with a polynucleotide that encodes nGPCR-14.79.A method of purifying a G protein from a sample containing said G protein comprising the steps of: a) contacting said sample with a polypeptide of claim 1 for a time sufficient to allow said G protein to form a complex with said polypeptide; b) isolating said complex from remaining components of said sample; c) maintaining said complex under conditions which result in dissociation of said G protein from said polypeptide; and d) isolating said G protein from said polypeptide.\",\n \"80.The method of claim 79 wherein said sample comprises an amino acid sequence of SEQ ID NO: 192.81.The method of claim 79 wherein said polypeptide comprises an amino acid sequence homologous to a sequence of SEQ ID NO:192.\"\n ],\n [\n \"<SOH> BACKGROUND OF THE INVENTION <EOH>The G protein-coupled receptors (GPCRs) form a vast superfamily of cell surface receptors which are characterized by an amino-terminal extracellular domain, a carboxyl terminal intracellular domain, and a serpentine structure that passes through the cell membrane seven times.\",\n \"Hence, such receptors are sometimes also referred to as seven transmembrane (7TM) receptors.\",\n \"These seven transmembrane domains define three extracellular loops and three intracellular loops, in addition to the amino- and carboxy-terminal domains.\",\n \"The extracellular portions of the receptor have a role in recognizing and binding one or more extracellular binding partners (e.g., ligands), whereas the intracellular portions have a role in recognizing and communicating with downstream molecules in the signal transduction cascade.\",\n \"The G protein-coupled receptors bind a variety of ligands including calcium ions, hormones, chemokines, neuropeptides, neurotransmitters, nucleotides, lipids, odorants, and even photons, and are important in the normal (and sometimes the aberrant) function of many cell types.\",\n \"[See generally Strosberg, Eur.\",\n \"J. Biochem.\",\n \"196:1-10 (1991) and Bohm et al., Biochem J.\",\n \"322:1-18 (1997).]\",\n \"When a specific ligand binds to its corresponding receptor, the ligand typically stimulates the receptor to activate a specific heterotrimeric guanine-nucleotide-binding regulatory protein (G-protein) that is coupled to the intracellular portion of the receptor.\",\n \"The G protein in turn transmits a signal to an effector molecule within the cell, by either stimulating or inhibiting the activity of that effector molecule.\",\n \"These effector molecules include adenylate cyclase, phospholipases and ion channels.\",\n \"Adenylate cyclase and phospholipases are enzymes that are involved in the production of the second messenger molecules cAMP, inositol triphosphate and diacyglycerol.\",\n \"It is through this sequence of events that an extracellular ligand stimuli exerts intracellular changes through a G protein-coupled receptor.\",\n \"Each such receptor has its own characteristic primary structure, expression pattern, ligand-binding profile, and intracellular effector system.\",\n \"Because of the vital role of G protein-coupled receptors in the communication between cells and their environment, such receptors are attractive targets for therapeutic intervention, for example by activating or antagonizing such receptors.\",\n \"For receptors having a known ligand, the identification of agonists or antagonists may be sought specifically to enhance or inhibit the action of the ligand.\",\n \"Some G protein-coupled receptors have roles in disease pathogenesis (e.g., certain chemokine receptors that act as HIV co-receptors may have a role in AIDS pathogenesis), and are attractive targets for therapeutic intervention even in the absence of knowledge of the natural ligand of the receptor.\",\n \"Other receptors are attractive targets for therapeutic intervention by virtue of their expression pattern in tissues or cell types that are themselves attractive targets for therapeutic intervention.\",\n \"Examples of this latter category of receptors include receptors expressed in immune cells, which can be targeted to either inhibit autoimmune responses or to enhance immune responses to fight pathogens or cancer; and receptors expressed in the brain or other neural organs and tissues, which are likely targets in the treatment of schizophrenia, depression, bipolar disease, or other neurological disorders.\",\n \"This latter category of receptor is also useful as a marker for identifying and/or purifying (e.g., via fluorescence-activated cell sorting) cellular subtypes that express the receptor.\",\n \"Unfortunately, only a limited number of G protein receptors from the central nervous system (CNS) are known.\",\n \"Thus, a need exists for G protein-coupled receptors that have been identified and show promise as targets for therapeutic intervention in a variety of animals, including humans.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>The present invention relates to an isolated nucleic acid molecule that comprises a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence homologous to even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a fragment thereof.\",\n \"The nucleic acid molecule encodes at least a portion of nGPCR-x.\",\n \"In some embodiments, the nucleic acid molecule comprises a sequence that encodes a polypeptide comprising even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a fragment thereof.\",\n \"In some embodiments, the nucleic acid molecule comprises a sequence homologous to odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or a fragment thereof.\",\n \"In some embodiments, the nucleic acid molecule comprises a sequence selected from the group consisting of odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, and fragments thereof.\",\n \"According to some embodiments, the present invention provides vectors which comprise the nucleic acid molecule of the invention.\",\n \"In some embodiments, the vector is an expression vector.\",\n \"According to some embodiments, the present invention provides host cells which comprise the vectors of the invention.\",\n \"In some embodiments, the host cells comprise expression vectors.\",\n \"The present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence complementary to at least a portion of a sequence from an odd numbered sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, said portion comprising at least 10 nucleotides.\",\n \"The present invention provides a method of producing a polypeptide comprising a sequence from an even numbered sequence ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a homolog or fragment thereof.\",\n \"The method comprising the steps of introducing a recombinant expression vector that includes a nucleotide sequence that encodes the polypeptide into a compatible host cell, growing the host cell under conditions for expression of the polypeptide and recovering the polypeptide.\",\n \"The present invention provides an isolated antibody which binds to an epitope on a polypeptide comprising a sequence from an even numbered sequence ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a homolog or fragment thereof.\",\n \"The present invention provides an method of inducing an immune response in a mammal against a polypeptide comprising a sequence from an even numbered sequence ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a homolog or fragment thereof.\",\n \"The method comprises administering to a mammal an amount of the polypeptide sufficient to induce said immune response.\",\n \"The present invention provides a method for identifying a compound which binds nGPCR-x.\",\n \"The method comprises the steps of: contacting nGPCR-x with a compound and determining whether the compound binds nGPCR-x.\",\n \"The present invention provides a method for identifying a compound which binds a nucleic acid molecule encoding nGPCR-x.\",\n \"The method comprises the steps of contacting said nucleic acid molecule encoding nGPCR-x with a compound and determining whether said compound binds said nucleic acid molecule.\",\n \"The present invention provides a method for identifying a compound which modulates the activity of nGPCR-x.\",\n \"The method comprises the steps of contacting nGPCR-x with a compound and determining whether nGPCR-x activity has been modulated.\",\n \"The present invention provides a method of identifying an animal homolog of nGPCR-x.\",\n \"The method comprises the steps screening a nucleic acid database of the animal with an odd numbered sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or a portion thereof and determining whether a portion of said library or database is homologous to said odd numbered sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or portion thereof.\",\n \"The present invention provides a method of identifying an animal homolog of nGPCR-x.\",\n \"The methods comprises the steps screening a nucleic acid library of the animal with a nucleic acid molecule having an odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or a portion thereof; and determining whether a portion of said library or database is homologous to said odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or a portion thereof.\",\n \"Another aspect of the present invention relates to methods of screening a human subject to diagnose a disorder affecting the brain or genetic predisposition therefor.\",\n \"The methods comprise the steps of assaying nucleic acid of a human subject to determine a presence or an absence of a mutation altering an amino acid sequence, expression, or biological activity of at least one nGPCR that is expressed in the brain.\",\n \"The nGPCR comprise an amino acid sequence selected from the group consisting of: SEQ ID NO:74, SEQ ID NO: 186, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:90, and SEQ ID NO:94, and allelic variants thereof.\",\n \"A diagnosis of the disorder or predisposition is made from the presence or absence of the mutation.\",\n \"The presence of a mutation altering the amino acid sequence, expression, or biological activity of the nGPCR in the nucleic acid correlates with an increased risk of developing the disorder.\",\n \"The present invention further relates to methods of screening for a nGPCR-40 or nGPCR-54 hereditary schizophrenia genotype in a human patient.\",\n \"The methods comprise the steps of providing a biological sample comprising nucleic acid from the patient, in which the nucleic acid includes sequences corresponding to alleles of nGPCR-40 or nGPCR-54.The presence of one or more mutations in the nGPCR-40 allele or the nGPCR-54 allele is detected indicative of a hereditary schizophrenia genotype.\",\n \"The present invention provides kits for screening a human subject to diagnose schizophrenia or a genetic predisposition therefor.\",\n \"The kits include an oligonucleotide useful as a probe for identifying polymorphisms in a human nGPCR-40 gene or a human nGPCR-54 gene.\",\n \"The oligonucleotide comprises 6-50 nucleotides in a sequence that is identical or complementary to a sequence of a wild type human nGPCR-40 or nGPCR-54 gene sequence or nGPCR-40 or nGPCR-54 coding sequence, except for one sequence difference selected from the group consisting of a nucleotide addition, a nucleotide deletion, or nucleotide substitution.\",\n \"The kit also includes a media packaged with the oligonucleotide.\",\n \"The media contains information for identifying polymorphisms that correlate with schizophrenia or a genetic predisposition therefor, the polymorphisms being identifiable using the oligonucleotide as a probe.\",\n \"The present invention further relates to methods of identifying nGPCR allelic variants that correlates with mental disorders.\",\n \"The methods comprise the steps of providing biological samples that comprise nucleic acid from a human patient diagnosed with a mental disorder, or from the patient's genetic progenitors or progeny, and detecting in the nucleic acid the presence of one or more mutations in an nGPCR that is expressed in the brain.\",\n \"The nGPCR comprises an amino acid sequence selected from the group consisting of SEQ ID NO:74, SEQ ID NO: 186, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:90, and SEQ ID NO:94, and allelic variants thereof.\",\n \"The nucleic acid includes sequences corresponding to the gene or genes encoding nGPCR.\",\n \"The one or more mutations detected indicate an allelic variant that correlates with a mental disorder.\",\n \"The present invention further relates to purified polynucleotides comprising nucleotide sequences encoding alleles of nGPCR-40 or nGPCR-54 from a human with schizophrenia.\",\n \"The polynucleotide hybridizes to the complement of SEQ ID NO:83 or of SEQ ID NO:85 under the following hybridization conditions: (a) hybridization for 16 hours at 42° C. in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% dextran sulfate and (b) washing 2 times for 30 minutes at 60° C. in a wash solution comprising 0.1×SSC and 1% SDS.\",\n \"The polynucleotide that encodes nGPCR-40 or nGPCR-54 amino acid sequence of the human differs from SEQ ID NO:84 or SEQ ID NO:86 by at least one residue.\",\n \"The present invention also provides methods for identifying a modulator of biological activity of nGPCR-40 or nGPCR-54 comprising the steps of contacting a cell that expresses nGPCR-40 or nGPCR-54 in the presence and in the absence of a putative modulator compound and measuring nGPCR-40 or nGPCR-54 biological activity in the cell.\",\n \"The decreased or increased nGPCR-40 or nGPCR-54 biological activity in the presence versus absence of the putative modulator is indicative of a modulator of biological activity.\",\n \"The present invention further provides methods to identify compounds useful for the treatment of schizophrenia.\",\n \"The methods comprise the steps of contacting a composition comprising nGPCR-40 with a compound suspected of binding nGPCR-40 or contacting a composition comprising nGPCR-54 with a compound suspected of binding nGPCR-54.The binding between nGPCR-40 and the compound suspected of binding nGPCR-40 or between nGPCR-54 and the compound suspected of binding nGPCR-54 is detected.\",\n \"Compounds identified as binding nGPCR-40 or nGPCR-54 are candidate compounds useful for the treatment of schizophrenia.\",\n \"The present invention further provides methods for identifying a compound useful as a modulator of binding between nGPCR-40 and a binding partner of nGPCR-40 or between nGPCR-54 and a binding partner of nGPCR-54.The methods comprise the steps of contacting the binding partner and a composition comprising nGPCR-40 or nGPCR-54 in the presence and in the absence of a putative modulator compound and detecting binding between the binding partner and nGPCR-40 or nGPCR-54.Decreased or increased binding between the binding partner and nGPCR-40 or nGPCR-54 in the presence of the putative modulator, as compared to binding in the absence of the putative modulator is indicative a modulator compound useful for the treatment of schizophrenia.\",\n \"Another aspect of the present invention relates to methods of purifying a G protein from a sample containing a G protein.\",\n \"The methods comprise the steps of contacting the sample with an nGPCR for a time sufficient to allow the G protein to form a complex with the nGPCR; isolating the complex from remaining components of the sample; maintaining the complex under conditions which result in dissociation of the G protein from the nGPCR; and isolating said G protein from the nGPCR.\",\n \"detailed-description description=\\\"Detailed Description\\\" end=\\\"lead\\\"?\"\n ],\n [\n \"FIELD OF THE INVENTION The present invention relates generally to the fields of genetics and cellular and molecular biology.\",\n \"More particularly, the invention relates to novel G protein coupled receptors, to polynucleotides that encode such novel receptors, to reagents such as antibodies, probes, primers and kits comprising such antibodies, probes, primers related to the same, and to methods which use the novel G protein coupled receptors, polynucleotides or reagents.\",\n \"BACKGROUND OF THE INVENTION The G protein-coupled receptors (GPCRs) form a vast superfamily of cell surface receptors which are characterized by an amino-terminal extracellular domain, a carboxyl terminal intracellular domain, and a serpentine structure that passes through the cell membrane seven times.\",\n \"Hence, such receptors are sometimes also referred to as seven transmembrane (7TM) receptors.\",\n \"These seven transmembrane domains define three extracellular loops and three intracellular loops, in addition to the amino- and carboxy-terminal domains.\",\n \"The extracellular portions of the receptor have a role in recognizing and binding one or more extracellular binding partners (e.g., ligands), whereas the intracellular portions have a role in recognizing and communicating with downstream molecules in the signal transduction cascade.\",\n \"The G protein-coupled receptors bind a variety of ligands including calcium ions, hormones, chemokines, neuropeptides, neurotransmitters, nucleotides, lipids, odorants, and even photons, and are important in the normal (and sometimes the aberrant) function of many cell types.\",\n \"[See generally Strosberg, Eur.\",\n \"J. Biochem.\",\n \"196:1-10 (1991) and Bohm et al., Biochem J.\",\n \"322:1-18 (1997).]\",\n \"When a specific ligand binds to its corresponding receptor, the ligand typically stimulates the receptor to activate a specific heterotrimeric guanine-nucleotide-binding regulatory protein (G-protein) that is coupled to the intracellular portion of the receptor.\",\n \"The G protein in turn transmits a signal to an effector molecule within the cell, by either stimulating or inhibiting the activity of that effector molecule.\",\n \"These effector molecules include adenylate cyclase, phospholipases and ion channels.\",\n \"Adenylate cyclase and phospholipases are enzymes that are involved in the production of the second messenger molecules cAMP, inositol triphosphate and diacyglycerol.\",\n \"It is through this sequence of events that an extracellular ligand stimuli exerts intracellular changes through a G protein-coupled receptor.\",\n \"Each such receptor has its own characteristic primary structure, expression pattern, ligand-binding profile, and intracellular effector system.\",\n \"Because of the vital role of G protein-coupled receptors in the communication between cells and their environment, such receptors are attractive targets for therapeutic intervention, for example by activating or antagonizing such receptors.\",\n \"For receptors having a known ligand, the identification of agonists or antagonists may be sought specifically to enhance or inhibit the action of the ligand.\",\n \"Some G protein-coupled receptors have roles in disease pathogenesis (e.g., certain chemokine receptors that act as HIV co-receptors may have a role in AIDS pathogenesis), and are attractive targets for therapeutic intervention even in the absence of knowledge of the natural ligand of the receptor.\",\n \"Other receptors are attractive targets for therapeutic intervention by virtue of their expression pattern in tissues or cell types that are themselves attractive targets for therapeutic intervention.\",\n \"Examples of this latter category of receptors include receptors expressed in immune cells, which can be targeted to either inhibit autoimmune responses or to enhance immune responses to fight pathogens or cancer; and receptors expressed in the brain or other neural organs and tissues, which are likely targets in the treatment of schizophrenia, depression, bipolar disease, or other neurological disorders.\",\n \"This latter category of receptor is also useful as a marker for identifying and/or purifying (e.g., via fluorescence-activated cell sorting) cellular subtypes that express the receptor.\",\n \"Unfortunately, only a limited number of G protein receptors from the central nervous system (CNS) are known.\",\n \"Thus, a need exists for G protein-coupled receptors that have been identified and show promise as targets for therapeutic intervention in a variety of animals, including humans.\",\n \"SUMMARY OF THE INVENTION The present invention relates to an isolated nucleic acid molecule that comprises a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence homologous to even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a fragment thereof.\",\n \"The nucleic acid molecule encodes at least a portion of nGPCR-x.\",\n \"In some embodiments, the nucleic acid molecule comprises a sequence that encodes a polypeptide comprising even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a fragment thereof.\",\n \"In some embodiments, the nucleic acid molecule comprises a sequence homologous to odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or a fragment thereof.\",\n \"In some embodiments, the nucleic acid molecule comprises a sequence selected from the group consisting of odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, and fragments thereof.\",\n \"According to some embodiments, the present invention provides vectors which comprise the nucleic acid molecule of the invention.\",\n \"In some embodiments, the vector is an expression vector.\",\n \"According to some embodiments, the present invention provides host cells which comprise the vectors of the invention.\",\n \"In some embodiments, the host cells comprise expression vectors.\",\n \"The present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence complementary to at least a portion of a sequence from an odd numbered sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, said portion comprising at least 10 nucleotides.\",\n \"The present invention provides a method of producing a polypeptide comprising a sequence from an even numbered sequence ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a homolog or fragment thereof.\",\n \"The method comprising the steps of introducing a recombinant expression vector that includes a nucleotide sequence that encodes the polypeptide into a compatible host cell, growing the host cell under conditions for expression of the polypeptide and recovering the polypeptide.\",\n \"The present invention provides an isolated antibody which binds to an epitope on a polypeptide comprising a sequence from an even numbered sequence ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a homolog or fragment thereof.\",\n \"The present invention provides an method of inducing an immune response in a mammal against a polypeptide comprising a sequence from an even numbered sequence ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a homolog or fragment thereof.\",\n \"The method comprises administering to a mammal an amount of the polypeptide sufficient to induce said immune response.\",\n \"The present invention provides a method for identifying a compound which binds nGPCR-x.\",\n \"The method comprises the steps of: contacting nGPCR-x with a compound and determining whether the compound binds nGPCR-x.\",\n \"The present invention provides a method for identifying a compound which binds a nucleic acid molecule encoding nGPCR-x.\",\n \"The method comprises the steps of contacting said nucleic acid molecule encoding nGPCR-x with a compound and determining whether said compound binds said nucleic acid molecule.\",\n \"The present invention provides a method for identifying a compound which modulates the activity of nGPCR-x.\",\n \"The method comprises the steps of contacting nGPCR-x with a compound and determining whether nGPCR-x activity has been modulated.\",\n \"The present invention provides a method of identifying an animal homolog of nGPCR-x.\",\n \"The method comprises the steps screening a nucleic acid database of the animal with an odd numbered sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or a portion thereof and determining whether a portion of said library or database is homologous to said odd numbered sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or portion thereof.\",\n \"The present invention provides a method of identifying an animal homolog of nGPCR-x.\",\n \"The methods comprises the steps screening a nucleic acid library of the animal with a nucleic acid molecule having an odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or a portion thereof; and determining whether a portion of said library or database is homologous to said odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or a portion thereof.\",\n \"Another aspect of the present invention relates to methods of screening a human subject to diagnose a disorder affecting the brain or genetic predisposition therefor.\",\n \"The methods comprise the steps of assaying nucleic acid of a human subject to determine a presence or an absence of a mutation altering an amino acid sequence, expression, or biological activity of at least one nGPCR that is expressed in the brain.\",\n \"The nGPCR comprise an amino acid sequence selected from the group consisting of: SEQ ID NO:74, SEQ ID NO: 186, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:90, and SEQ ID NO:94, and allelic variants thereof.\",\n \"A diagnosis of the disorder or predisposition is made from the presence or absence of the mutation.\",\n \"The presence of a mutation altering the amino acid sequence, expression, or biological activity of the nGPCR in the nucleic acid correlates with an increased risk of developing the disorder.\",\n \"The present invention further relates to methods of screening for a nGPCR-40 or nGPCR-54 hereditary schizophrenia genotype in a human patient.\",\n \"The methods comprise the steps of providing a biological sample comprising nucleic acid from the patient, in which the nucleic acid includes sequences corresponding to alleles of nGPCR-40 or nGPCR-54.The presence of one or more mutations in the nGPCR-40 allele or the nGPCR-54 allele is detected indicative of a hereditary schizophrenia genotype.\",\n \"The present invention provides kits for screening a human subject to diagnose schizophrenia or a genetic predisposition therefor.\",\n \"The kits include an oligonucleotide useful as a probe for identifying polymorphisms in a human nGPCR-40 gene or a human nGPCR-54 gene.\",\n \"The oligonucleotide comprises 6-50 nucleotides in a sequence that is identical or complementary to a sequence of a wild type human nGPCR-40 or nGPCR-54 gene sequence or nGPCR-40 or nGPCR-54 coding sequence, except for one sequence difference selected from the group consisting of a nucleotide addition, a nucleotide deletion, or nucleotide substitution.\",\n \"The kit also includes a media packaged with the oligonucleotide.\",\n \"The media contains information for identifying polymorphisms that correlate with schizophrenia or a genetic predisposition therefor, the polymorphisms being identifiable using the oligonucleotide as a probe.\",\n \"The present invention further relates to methods of identifying nGPCR allelic variants that correlates with mental disorders.\",\n \"The methods comprise the steps of providing biological samples that comprise nucleic acid from a human patient diagnosed with a mental disorder, or from the patient's genetic progenitors or progeny, and detecting in the nucleic acid the presence of one or more mutations in an nGPCR that is expressed in the brain.\",\n \"The nGPCR comprises an amino acid sequence selected from the group consisting of SEQ ID NO:74, SEQ ID NO: 186, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:90, and SEQ ID NO:94, and allelic variants thereof.\",\n \"The nucleic acid includes sequences corresponding to the gene or genes encoding nGPCR.\",\n \"The one or more mutations detected indicate an allelic variant that correlates with a mental disorder.\",\n \"The present invention further relates to purified polynucleotides comprising nucleotide sequences encoding alleles of nGPCR-40 or nGPCR-54 from a human with schizophrenia.\",\n \"The polynucleotide hybridizes to the complement of SEQ ID NO:83 or of SEQ ID NO:85 under the following hybridization conditions: (a) hybridization for 16 hours at 42° C. in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% dextran sulfate and (b) washing 2 times for 30 minutes at 60° C. in a wash solution comprising 0.1×SSC and 1% SDS.\",\n \"The polynucleotide that encodes nGPCR-40 or nGPCR-54 amino acid sequence of the human differs from SEQ ID NO:84 or SEQ ID NO:86 by at least one residue.\",\n \"The present invention also provides methods for identifying a modulator of biological activity of nGPCR-40 or nGPCR-54 comprising the steps of contacting a cell that expresses nGPCR-40 or nGPCR-54 in the presence and in the absence of a putative modulator compound and measuring nGPCR-40 or nGPCR-54 biological activity in the cell.\",\n \"The decreased or increased nGPCR-40 or nGPCR-54 biological activity in the presence versus absence of the putative modulator is indicative of a modulator of biological activity.\",\n \"The present invention further provides methods to identify compounds useful for the treatment of schizophrenia.\",\n \"The methods comprise the steps of contacting a composition comprising nGPCR-40 with a compound suspected of binding nGPCR-40 or contacting a composition comprising nGPCR-54 with a compound suspected of binding nGPCR-54.The binding between nGPCR-40 and the compound suspected of binding nGPCR-40 or between nGPCR-54 and the compound suspected of binding nGPCR-54 is detected.\",\n \"Compounds identified as binding nGPCR-40 or nGPCR-54 are candidate compounds useful for the treatment of schizophrenia.\",\n \"The present invention further provides methods for identifying a compound useful as a modulator of binding between nGPCR-40 and a binding partner of nGPCR-40 or between nGPCR-54 and a binding partner of nGPCR-54.The methods comprise the steps of contacting the binding partner and a composition comprising nGPCR-40 or nGPCR-54 in the presence and in the absence of a putative modulator compound and detecting binding between the binding partner and nGPCR-40 or nGPCR-54.Decreased or increased binding between the binding partner and nGPCR-40 or nGPCR-54 in the presence of the putative modulator, as compared to binding in the absence of the putative modulator is indicative a modulator compound useful for the treatment of schizophrenia.\",\n \"Another aspect of the present invention relates to methods of purifying a G protein from a sample containing a G protein.\",\n \"The methods comprise the steps of contacting the sample with an nGPCR for a time sufficient to allow the G protein to form a complex with the nGPCR; isolating the complex from remaining components of the sample; maintaining the complex under conditions which result in dissociation of the G protein from the nGPCR; and isolating said G protein from the nGPCR.\",\n \"DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Definitions Various definitions are made throughout this document.\",\n \"Most words have the meaning that would be attributed to those words by one skilled in the art.\",\n \"Words specifically defined either below or elsewhere in this document have the meaning provided in the context of the present invention as a whole and as are typically understood by those skilled in the art.\",\n \"“Synthesized” as used herein and understood in the art, refers to polynucleotides produced by purely chemical, as opposed to enzymatic, methods.\",\n \"“Wholly” synthesized DNA sequences are therefore produced entirely by chemical means, and “partially” synthesized DNAs embrace those wherein only portions of the resulting DNA were produced by chemical means.\",\n \"By the term “region” is meant a physically contiguous portion of the primary structure of a biomolecule.\",\n \"In the case of proteins, a region is defined by a contiguous portion of the amino acid sequence of that protein.\",\n \"The term “domain” is herein defined as referring to a structural part of a biomolecule that contributes to a known or suspected function of the biomolecule.\",\n \"Domains may be co-extensive with regions or portions thereof; domains may also incorporate a portion of a biomolecule that is distinct from a particular region, in addition to all or part of that region.\",\n \"Examples of GPCR protein domains include, but are not limited to, the extracellular (i.e., N-terminal), transmembrane and cytoplasmic (i.e., C-terminal) domains, which are co-extensive with like-named regions of GPCRS; each of the seven transmembrane segments of a GPCR; and each of the loop segments (both extracellular and intracellular loops) connecting adjacent transmembrane segments.\",\n \"As used herein, the term “activity” refers to a variety of measurable indicia suggesting or revealing binding, either direct or indirect; affecting a response, i.e.\",\n \"having a measurable affect in response to some exposure or stimulus, including, for example, the affinity of a compound for directly binding a polypeptide or polynucleotide of the invention, or, for example, measurement of amounts of upstream or downstream proteins or other similar functions after some stimulus or event.\",\n \"Unless indicated otherwise, as used herein, the abbreviation in lower case (gpcr) refers to a gene, cDNA, RNA or nucleic acid sequence, while the upper case version (GPCR) refers to a protein, polypeptide, peptide, oligopeptide, or amino acid sequence.\",\n \"The term “nGPCR-x” refers to any of the nGPCRs taught herein, while specific reference to a nGPCR (for example nGPCR-5) refers only to that specific nGPCR.\",\n \"As used herein, the term “antibody” is meant to refer to complete, intact antibodies, and Fab, Fab′, F(ab)2, and other fragments thereof.\",\n \"Complete, intact antibodies include monoclonal antibodies such as murine monoclonal antibodies, chimeric antibodies and humanized antibodies.\",\n \"As used herein, the term “binding” means the physical or chemical interaction between two proteins or compounds or associated proteins or compounds or combinations thereof.\",\n \"Binding includes ionic, nononic, Hydrogen bonds, Van der Waals, hydrophobic interactions, etc.\",\n \"The physical interaction, the binding, can be either direct or indirect, indirect being through or due to the effects of another protein or compound.\",\n \"Direct binding refers to interactions that do not take place through or due to the effect of another protein or compound but instead are without other substantial chemical intermediates.\",\n \"Binding may be detected in many different manners.\",\n \"As a non-limiting example, the physical binding interaction between a nGPCR-x of the invention and a compound can be detected using a labeled compound.\",\n \"Alternatively, functional evidence of binding can be detected using, for example, a cell transfected with and expressing a nGPCR-x of the invention.\",\n \"Binding of the transfected cell to a ligand of the nGPCR that was transfected into the cell provides functional evidence of binding.\",\n \"Other methods of detecting binding are well-known to those of skill in the art.\",\n \"As used herein, the term “compound” means any identifiable chemical or molecule, including, but not limited to, small molecule, peptide, protein, sugar, nucleotide, or nucleic acid, and such compound can be natural or synthetic.\",\n \"As used herein, the term “complementary” refers to Watson-Crick basepairing between nucleotide units of a nucleic acid molecule.\",\n \"As used herein, the term “contacting” means bringing together, either directly or indirectly, a compound into physical proximity to a polypeptide or polynucleotide of the invention.\",\n \"The polypeptide or polynucleotide can be in any number of buffers, salts, solutions etc.\",\n \"Contacting includes, for example, placing the compound into a beaker, microtiter plate, cell culture flask, or a microarray, such as a gene chip, or the like, which contains the nucleic acid molecule, or polypeptide encoding the nGPCR or fragment thereof.\",\n \"As used herein, the phrase “homologous nucleotide sequence,” or “homologous amino acid sequence,” or variations thereof, refers to sequences characterized by a homology, at the nucleotide level or amino acid level, of at least the specified percentage.\",\n \"Homologous nucleotide sequences include those sequences coding for isoforms of proteins.\",\n \"Such isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA.\",\n \"Alternatively, isoforms can be encoded by different genes.\",\n \"Homologous nucleotide sequences include nucleotide sequences encoding for a protein of a species other than humans, including, but not limited to, mammals.\",\n \"Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein.\",\n \"A homologous nucleotide sequence does not, however, include the nucleotide sequence encoding other known GPCRs.\",\n \"Homologous amino acid sequences include those amino acid sequences which contain conservative amino acid substitutions and which polypeptides have the same binding and/or activity.\",\n \"A homologous amino acid sequence does not, however, include the amino acid sequence encoding other known GPCRs.\",\n \"Percent homology can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using the default settings, which uses the algorithm of Smith and Waterman (Adv.\",\n \"Appl.\",\n \"Math., 1981, 2, 482 489, which is incorporated herein by reference in its entirety).\",\n \"As used herein, the term “isolated” nucleic acid molecule refers to a nucleic acid molecule (DNA or RNA) that has been removed from its native environment.\",\n \"Examples of isolated nucleic acid molecules include, but are not limited to, recombinant DNA molecules contained in a vector, recombinant DNA molecules maintained in a heterologous host cell, partially or substantially purified nucleic acid molecules, and synthetic DNA or RNA molecules.\",\n \"As used herein, the terms “modulates” or “modifies” means an increase or decrease in the amount, quality, or effect of a particular activity or protein.\",\n \"As used herein, the term “oligonucleotide” refers to a series of linked nucleotide residues which has a sufficient number of bases to be used in a polymerase chain reaction (PCR).\",\n \"This short sequence is based on (or designed from) a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.\",\n \"Oligonucleotides comprise portions of a DNA sequence having at least about 10 nucleotides and as many as about 50 nucleotides, preferably about 15 to 30 nucleotides.\",\n \"They are chemically synthesized and may be used as probes.\",\n \"As used herein, the term “probe” refers to nucleic acid sequences of variable length, preferably between at least about 10 and as many as about 6,000 nucleotides, depending on use.\",\n \"They are used in the detection of identical, similar, or complementary nucleic acid sequences.\",\n \"Longer length probes are usually obtained from a natural or recombinant source, are highly specific and much slower to hybridize than oligomers.\",\n \"They may be single- or double-stranded and carefully designed to have specificity in PCR, hybridization membrane based, or ELISA-like technologies.\",\n \"The term “preventing” refers to decreasing the probability that an organism contracts or develops an abnormal condition.\",\n \"The term “treating” refers to having a therapeutic effect and at least partially alleviating or abrogating an abnormal condition in the organism.\",\n \"The term “therapeutic effect” refers to the inhibition or activation factors causing or contributing to the abnormal condition.\",\n \"A therapeutic effect relieves to some extent one or more of the symptoms of the abnormal condition.\",\n \"In reference to the treatment of abnormal conditions, a therapeutic effect can refer to one or more of the following: (a) an increase in the proliferation, growth, and/or differentiation of cells; (b) inhibition (i.e., slowing or stopping) of cell death; (c) inhibition of degeneration; (d) relieving to some extent one or more of the symptoms associated with the abnormal condition; and (e) enhancing the function of the affected population of cells.\",\n \"Compounds demonstrating efficacy against abnormal conditions can be identified as described herein.\",\n \"The term “abnormal condition” refers to a function in the cells or tissues of an organism that deviates from their normal functions in that organism.\",\n \"An abnormal condition can relate to cell proliferation, cell differentiation, cell signaling, or cell survival.\",\n \"An abnormal condition may also include obesity, diabetic complications such as retinal degeneration, and irregularities in glucose uptake and metabolism, and fatty acid uptake and metabolism.\",\n \"Abnormal cell proliferative conditions include cancers such as fibrotic and mesangial disorders, abnormal angiogenesis and vasculogenesis, wound healing, psoriasis, diabetes mellitus, and inflammation.\",\n \"Abnormal differentiation conditions include, but are not limited to, neurodegenerative disorders, slow wound healing rates, and slow tissue grafting healing rates.\",\n \"Abnormal cell signaling conditions include, but are not limited to, psychiatric disorders involving excess neurotransmitter activity.\",\n \"Abnormal cell survival conditions may also relate to conditions in which programmed cell death (apoptosis) pathways are activated or abrogated.\",\n \"A number of protein kinases are associated with the apoptosis pathways.\",\n \"Aberrations in the function of any one of the protein kinases could lead to cell immortality or premature cell death.\",\n \"The term “administering” relates to a method of incorporating a compound into cells or tissues of an organism.\",\n \"The abnormal condition can be prevented or treated when the cells or tissues of the organism exist within the organism or outside of the organism.\",\n \"Cells existing outside the organism can be maintained or grown in cell culture dishes.\",\n \"For cells harbored within the organism, many techniques exist in the art to administer compounds, including (but not limited to) oral, parenteral, dermal, injection, and aerosol applications.\",\n \"For cells outside of the organism, multiple techniques exist in the art to administer the compounds, including (but not limited to) cell microinjection techniques, transformation techniques and carrier techniques.\",\n \"The abnormal condition can also be prevented or treated by administering a compound to a group of cells having an aberration in a signal transduction pathway to an organism.\",\n \"The effect of administering a compound on organism function can then be monitored.\",\n \"The organism is preferably a mouse, rat, rabbit, guinea pig or goat, more preferably a monkey or ape, and most preferably a human.\",\n \"By “amplification” it is meant increased numbers of DNA or RNA in a cell compared with normal cells.\",\n \"“Amplification” as it refers to RNA can be the detectable presence of RNA in cells, since in some normal cells there is no basal expression of RNA.\",\n \"In other normal cells, a basal level of expression exists, therefore in these cases amplification is the detection of at least 1 to 2-fold, and preferably more, compared to the basal level.\",\n \"As used herein, the phrase “stringent hybridization conditions” or “stringent conditions” refers to conditions under which a probe, primer, or oligonucleotide will hybridize to its target sequence, but to no other sequences.\",\n \"Stringent conditions are sequence-dependent and will be different in different circumstances.\",\n \"Longer sequences hybridize specifically at higher temperatures.\",\n \"Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.\",\n \"The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium.\",\n \"Since the target sequences are generally present in excess, at Tm, 50% of the probes are occupied at equilibrium.\",\n \"Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes, primers or oligonucleotides (e.g.\",\n \"10 to 50 nucleotides) and at least about 60° C. for longer probes, primers or oligonucleotides.\",\n \"Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.\",\n \"The amino acid sequences are presented in the amino to carboxy direction, from left to right.\",\n \"The amino and carboxy groups are not presented in the sequence.\",\n \"The nucleotide sequences are presented by single strand only, in the 5′ to 3′ direction, from left to right.\",\n \"Nucleotides and amino acids are represented in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission or (for amino acids) by three letters code.\",\n \"Polynucleotides The present invention provides purified and isolated polynucleotides (e.g., DNA sequences and RNA transcripts, both sense and complementary antisense strands, both single- and double-stranded, including splice variants thereof) that encode unknown G protein-coupled receptors heretofore termed novel GPCRs, or nGPCRs.\",\n \"These genes are described herein and designated herein collectively as nGPCR-x (where x is 1, 3, 4, 5, 9, 11, 12, 14, 15, 18, 16, 17, 20, 21, 22, 24, 27, 28, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 53, 54, 55, 56, 57, 58, 59, or 60).\",\n \"That is, these genes are described herein and designated herein as nGPCR-1 (also referred to as beGPCR-1), nGPCR-3 (also referred to as beGPCR-3), nGPCR-4 (also referred to as beGPCR-4), nGPCR-5 (also referred to as beGPCR-5 and TL-GPCR-5), nGPCR-9 (also referred to as beGPCR-9), nGPCR-11 (also referred to as beGPCR-11), nGPCR-12 (also referred to as beGPCR-12), nGPCR-14 (also referred to as beGPCR-14), nGPCR-15 (also referred to as beGPCR-15), nGPCR-18 (also referred to as beGPCR-18), nGPCR-16 (also referred to as beGPCR-16), nGPCR-17 (also referred to as beGPCR-17), nGPCR-20 (also referred to as beGPCR-20), nGPCR-21 (also referred to as beGPCR-21), nGPCR-22 (also referred to as beGPCR-22), nGPCR-24 (also referred to as beGPCR-24), nGPCR-27 (also referred to as beGPCR-27), nGPCR-28 (also referred to as beGPCR-28), nGPCR-31 (also referred to as beGPCR-31), nGPCR-32 (also referred to as beGPCR-32), nGPCR-33 (also referred to as beGPCR-33), nGPCR-34 (also referred to as beGPCR-34), nGPCR-35 (also referred to as beGPCR-35), nGPCR-36 (also referred to as beGPCR-36), nGPCR-37 (also referred to as beGPCR-37), nGPCR-38 (also referred to as beGPCR-38), nGPCR-40 (also referred to as beGPCR-40), nGPCR-1 (also referred to as beGPCR-41), nGPCR-53, nGPCR-54, nGPCR-55, nGPCR-56, nGPCR-57, nGPCR-58, nGPCR-59, and nGPCR-60.Table 1 below identifies the novel gene sequence nGPCR-x designation, the SEQ ID NO: of the gene sequence, the SEQ ID NO: of the polypeptide hereby, and the U.S.\",\n \"Provisional Application in which the gene sequence has been TABLE 1 Nucleotide Amino acid Sequence Sequence (SEQ ID (SEQ ID Originally nGPCR NO:) NO:) filed in: 1 1 2 A 1 73 74 E 3 3 4 A 3 185 186 P 4 5 6 A 5 7 8 A 5 75 76 F 9 9 10 A 9 77 78 G 11 11 12 A 11 79 80 H 12 13 14 A 14 15 16 A 14 191 192 herein 15 17 18 A 18 19 20 A 16 21 22 B 16 81 82 I 17 23 24 B 20 25 26 B 21 27 28 B 22 29 30 B 24 31 32 B 27 33 34 B 28 35 36 B 31 37 38 B 32 39 40 B 33 41 42 C 34 43 44 C 35 45 46 C 36 47 48 C 37 49 50 C 38 51 52 C 40 53 54 C 40 83 84 J 41 55 56 C 53 57 58 D 54 59 60 D 54 85 86 K 55 61 62 D 56 63 64 D 56 87 88 L 56 89 90 M 57 65 66 D 58 67 68 D 58 91 92 N 58 93 94 O 59 69 70 D 60 71 72 D Legend A = Ser.\",\n \"No.\",\n \"60/165,838 B = Ser.\",\n \"No.\",\n \"60/166,701 C = Ser.\",\n \"No.\",\n \"60/166,678 D = Ser.\",\n \"No.\",\n \"60/173,396 E = Ser.\",\n \"No.\",\n \"60/184,129 F = Ser.\",\n \"No.\",\n \"60/188,114 G = Ser.\",\n \"No.\",\n \"60/185,421 H = Ser.\",\n \"No.\",\n \"60/186,811 I = Ser.\",\n \"No.\",\n \"60/186,530 J = Ser.\",\n \"No.\",\n \"60/207,094 K = Ser.\",\n \"No.\",\n \"60/203,111 L = Ser.\",\n \"No.\",\n \"60/190,310 M = Ser.\",\n \"No.\",\n \"60/201,190 N = Ser.\",\n \"No.\",\n \"60/185554 O = Ser.\",\n \"No.\",\n \"60/190,800 P = Ser.\",\n \"No.\",\n \"60/198,568 When a specific nGPCR is identified (for example nGPCR-5), it is understood that only that specific nGPCR is being referred to.\",\n \"As described in Example 4 below, the genes encoding nGPCR-1 (nucleic acid sequence SEQ ID NO: 1, SEQ ID NO: 73, amino acid sequence SEQ ID NO: 2, SEQ ID NO:74), nGPCR-9 (nucleic acid sequence SEQ ID NO:9, SEQ ID NO:77, amino acid sequence SEQ ID NO:10, SEQ ID NO:78), nGPCR-11 (nucleic acid sequence SEQ ID NO:11, SEQ ID NO:79, amino acid sequence SEQ ID NO:12, SEQ ID NO:80), nGPCR-16 (nucleic acid sequence SEQ ID NO: 21, SEQ ID NO:81, amino acid sequence SEQ ID NO: 22, SEQ ID NO:82), nGPCR-40 (nucleic acids sequence SEQ ID NO:53, SEQ ID NO:83, amino acid sequence SEQ ID NO:54, SEQ ID NO:84), nGPCR-54 (nucleic acid sequence SEQ ID NO:59, SEQ ID NO:85, amino acid sequence SEQ ID NO:60, SEQ ID NO: 86), nGPCR-56 (nucleic acid sequence SEQ ID NO:63, SEQ ID NO:87, SEQ ID NO:89, amino acid sequence SEQ ID NO:64, SEQ ID NO: 88, SEQ ID NO:90), nGPCR-58 (nucleic acid sequence SEQ ID NO:67, SEQ ID NO:91, SEQ ID NO:93, amino acid sequence SEQ ID NO:68, SEQ ID NO: 92, SEQ ID NO:94) and nGPCR-3 (nucleic acid sequence SEQ ID NO:3, SEQ ID NO:185, amino acid sequence SEQ ID NO:4, SEQ ID NO: 186) have been detected in brain tissue indicating that these n-GPCR-x proteins are neuroreceptors.\",\n \"The invention provides purified and isolated polynucleotides (e.g., cDNA, genomic DNA, synthetic DNA, RNA, or combinations thereof, whether single or double-stranded) that comprise a nucleotide sequence encoding the amino acid sequence of the polypeptides of the invention.\",\n \"Such polynucleotides are useful for recombinantly expressing the receptor and also for detecting expression of the receptor in cells (e.g., using Northern hybridization and in situ hybridization assays).\",\n \"Such polynucleotides also are useful in the design of antisense and other molecules for the suppression of the expression of nGPCR-x in a cultured cell, a tissue, or an animal; for therapeutic purposes; or to provide a model for diseases or conditions characterized by aberrant nGPCR-x expression.\",\n \"Specifically excluded from the definition of polynucleotides of the invention are entire isolated, non-recombinant native chromosomes of host cells.\",\n \"A preferred polynucleotide has the sequence of the sequence set forth in odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191, which correspond to naturally occurring nGPCR-x sequences.\",\n \"It will be appreciated that numerous other polynucleotide sequences exist that also encode nGPCR-x having the sequence set forth in even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO:192, due to the well-known degeneracy of the universal genetic code.\",\n \"The invention also provides a purified and isolated polynucleotide comprising a nucleotide sequence that encodes a mammalian polypeptide, wherein the polynucleotide hybridizes to a polynucleotide having the sequence set forth in odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185, and SEQ ID NO:191, or the non-coding strand complementary thereto, under the following hybridization conditions: (a) hybridization for 16 hours at 42° C. in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% dextran sulfate; and (b) washing 2 times for 30 minutes each at 60° C. in a wash solution comprising 0.1% SSC, 1% SDS.\",\n \"Polynucleotides that encode a human allelic variant are highly preferred.\",\n \"The present invention relates to molecules which comprise the gene sequences that encode the nGPCRs; constructs and recombinant host cells incorporating the gene sequences; the novel GPCR polypeptides encoded by the gene sequences; antibodies to the polypeptides and homologs; kits employing the polynucleotides and polypeptides, and methods of making and using all of the foregoing.\",\n \"In addition, the present invention relates to homologs of the gene sequences and of the polypeptides and methods of making and using the same.\",\n \"Genomic DNA of the invention comprises the protein-coding region for a polypeptide of the invention and is also intended to include allelic variants thereof.\",\n \"It is widely understood that, for many genes, genomic DNA is transcribed into RNA transcripts that undergo one or more splicing events wherein intron (i.e., non-coding regions) of the transcripts are removed, or “spliced out.” RNA transcripts that can be spliced by alternative mechanisms, and therefore be subject to removal of different RNA sequences but still encode a nGPCR-x polypeptide, are referred to in the art as splice variants which are embraced by the invention.\",\n \"Splice variants comprehended by the invention therefore are encoded by the same original genomic DNA sequences but arise from distinct mRNA transcripts Allelic variants are modified forms of a wild-type gene sequence, the modification resulting from recombination during chromosomal segregation or exposure to conditions which give rise to genetic mutation.\",\n \"Allelic variants, like wild type genes, are naturally occurring sequences (as opposed to non-naturally occurring variants that arise from in vitro manipulation).\",\n \"The invention also comprehends cDNA that is obtained through reverse transcription of an RNA polynucleotide encoding nGPCR-x (conventionally followed by second strand synthesis of a complementary strand to provide a double-stranded DNA).\",\n \"Preferred DNA sequences encoding human nGPCR-x polypeptides are set out in odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191.A preferred DNA of the invention comprises a double stranded molecule along with the complementary molecule (the “non-coding strand” or “complement”) having a sequence unambiguously deducible from the coding strand according to Watson-Crick base-pairing rules for DNA.\",\n \"Also preferred are other polynucleotides encoding the nGPCR-x polypeptide of even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO:192 which differ in sequence from the polynucleotides of odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 192, by virtue of the well-known degeneracy of the universal nuclear genetic code.\",\n \"In a preferred embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence which encodes a fragment of polypeptide comprising a sequence of SEQ ID NO: 192.The fragment of the polypeptide comprising a sequence of SEQ ID NO: 192 comprises at least one or more amino acid residues from one or more of the following regions of SEQ ID NO: 192: amino acid residues 1 to 42 of SEQ ID NO: 192; amino acid residues 68 to 77 of SEQ ID NO: 192; amino acid residues 185 to 197 of SEQ ID NO: 192; or amino acid residues 293 to 513 of SEQ ID NO: 192.In a preferred embodiment, the isolated nucleic acid comprises a nucleotide sequence of SEQ ID NO: 191, and fragments thereof, that encode a polypeptide having a sequence of SEQ ID NO: 192, or fragments thereof.\",\n \"The fragment of the nucleotide sequence of SEQ ID NO: 191 comprises at least one or more nucleotides from one or more of the following regions of SEQ ID NO: 191: nucleotides 1 to 193 of SEQ ID NO: 191; nucleotides 612 to 644 of SEQ ID NO:191; nucleotides 697 to 706 of SEQ ID NO:191; nucleotides 1011 to 1049 of SEQ ID NO: 191; nucleotides 1051 to 1057 of SEQ ID NO:191; nucleotides 1090 to 1096 of SEQ ID NO:191; or nucleotides 1141 to 1642 of SEQ ID NO:191.The invention further embraces other species, preferably mammalian, homologs of the human nGPCR-x DNA.\",\n \"Species homologs, sometimes referred to as “orthologs,” in general, share at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homology with human DNA of the invention.\",\n \"Generally, percent sequence “homology” with respect to polynucleotides of the invention may be calculated as the percentage of nucleotide bases in the candidate sequence that are identical to nucleotides in the nGPCR-x sequence set forth in odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.\",\n \"Polynucleotides of the invention permit identification and isolation of polynucleotides encoding related nGPCR-x polypeptides, such as human allelic variants and species homologs, by well-known techniques including Southern and/or Northern hybridization, and polymerase chain reaction (PCR).\",\n \"Examples of related polynucleotides include human and non-human genomic sequences, including allelic variants, as well as polynucleotides encoding polypeptides homologous to nGPCR-x and structurally related polypeptides sharing one or more biological, immunological, and/or physical properties of nGPCR-x.\",\n \"Non-human species genes encoding proteins homologous to nGPCR-x can also be identified by Southern and/or PCR analysis and are useful in animal models for nGPCR-x disorders.\",\n \"Knowledge of the sequence of a human nGPCR-x DNA also makes possible through use of Southern hybridization or polymerase chain reaction (PCR) the identification of genomic DNA sequences encoding nGPCR-x expression control regulatory sequences such as promoters, operators, enhancers, repressors, and the like.\",\n \"Polynucleotides of the invention are also useful in hybridization assays to detect the capacity of cells to express nGPCR-x.\",\n \"Polynucleotides of the invention may also provide a basis for diagnostic methods useful for identifying a genetic alteration(s) in a nGPCR-x locus that underlies a disease state or states, which information is useful both for diagnosis and for selection of therapeutic strategies.\",\n \"According to the present invention, the nGPCR-x nucleotide sequences disclosed herein may be used to identify homologs of the nGPCR-x, in other animals, including but not limited to humans and other mammals, and invertebrates.\",\n \"Any of the nucleotide sequences disclosed herein, or any portion thereof, can be used, for example, as probes to screen databases or nucleic acid libraries, such as, for example, genomic or cDNA libraries, to identify homologs, using screening procedures well known to those skilled in the art.\",\n \"Accordingly, homologs having at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably at least 100% homology with nGPCR-x sequences can be identified.\",\n \"The disclosure herein of full-length polynucleotides encoding nGPCR-x polypeptides makes readily available to the worker of ordinary skill in the art every possible fragment of the full-length polynucleotide.\",\n \"One preferred embodiment of the present invention provides an isolated nucleic acid molecule comprising a sequence homologous to odd numbered sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:93, SEQ ID NO: 185 and SEQ ID NO: 191, and fragments thereof.\",\n \"Another preferred embodiment provides an isolated nucleic acid molecule comprising a sequence selected from the group of odd numbered sequences consisting of SEQ ID NO: 1 to SEQ ID NO:93, SEQ ID NO: 185 and SEQ ID NO: 191, and fragments thereof.\",\n \"As used in the present invention, fragments of nGPCR-x-encoding polynucleotides comprise at least 10, and preferably at least 12, 14, 16, 18, 20, 25, 50, or 75 consecutive nucleotides of a polynucleotide encoding nGPCR-x.\",\n \"Preferably, fragment polynucleotides of the invention comprise sequences unique to the nGPCR-x-encoding polynucleotide sequence, and therefore hybridize under highly stringent or moderately stringent conditions only (i.e., “specifically”) to polynucleotides encoding nGPCR-x (or fragments thereof).\",\n \"Polynucleotide fragments of genomic sequences of the invention comprise not only sequences unique to the coding region, but also include fragments of the full-length sequence derived from introns, regulatory regions, and/or other non-translated sequences.\",\n \"Sequences unique to polynucleotides of the invention are recognizable through sequence comparison to other known polynucleotides, and can be identified through use of alignment programs routinely utilized in the art, e.g., those made available in public sequence databases.\",\n \"Such sequences also are recognizable from Southern hybridization analyses to determine the number of fragments of genomic DNA to which a polynucleotide will hybridize.\",\n \"Polynucleotides of the invention can be labeled in a manner that permits their detection, including radioactive, fluorescent, and enzymatic labeling.\",\n \"Fragment polynucleotides are particularly useful as probes for detection of full-length or fragments of nGPCR-x polynucleotides.\",\n \"One or more polynucleotides can be included in kits that are used to detect the presence of a polynucleotide encoding nGPCR-x, or used to detect variations in a polynucleotide sequence encoding nGPCR-x.\",\n \"The invention also embraces DNAs encoding nGPCR-x polypeptides that hybridize under moderately stringent or high stringency conditions to the non-coding strand, or complement, of the polynucleotides set forth in odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:192.Exemplary highly stringent hybridization conditions are as follows: hybridization at 42° C. in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% Dextran sulfate, and washing twice for 30 minutes at 60° C. in a wash solution comprising 0.1×SSC and 1% SDS.\",\n \"It is understood in the art that conditions of equivalent stringency can be achieved through variation of temperature and buffer, or salt concentration as described Ausubel et al.\",\n \"(Eds.\",\n \"), Protocols in Molecular Biology, John Wiley & Sons (1994), pp.\",\n \"6.0.3 to 6.4.10.Modifications in hybridization conditions can be empirically determined or precisely calculated based on the length and the percentage of guanosine/cytosine (GC) base pairing of the probe.\",\n \"The hybridization conditions can be calculated as described in Sambrook, et al., (Eds.\",\n \"), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y. (1989), pp.\",\n \"9.47 to 9.51.With the knowledge of the nucleotide sequence information disclosed in the present invention, one skilled in the art can identify and obtain nucleotide sequences which encode nGPCR-x from different sources (i.e., different tissues or different organisms) through a variety of means well known to the skilled artisan and as disclosed by, for example, Sambrook et al., “Molecular cloning: a laboratory manual”, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (19.89), which is incorporated herein by reference in its entirety.\",\n \"For example, DNA that encodes nGPCR-x may be obtained by screening of mRNA, cDNA, or genomic DNA with oligonucleotide probes generated from the nGPCR-x gene sequence information provided herein.\",\n \"Probes may be labeled with a detectable group, such as a fluorescent group, a radioactive atom or a chemiluminescent group in accordance with procedures known to the skilled artisan and used in conventional hybridization assays, as described by, for example, Sambrook et al.\",\n \"A nucleic acid molecule comprising any of the nGPCR-x nucleotide sequences described above can alternatively be synthesized by use of the polymerase chain reaction (PCR) procedure, with the PCR oligonucleotide primers produced from the nucleotide sequences provided herein.\",\n \"See U.S. Pat.\",\n \"No.\",\n \"4,683,195 to Mulliset al.\",\n \"and U.S. Pat.\",\n \"No.\",\n \"4,683,202 to Mullis.\",\n \"The PCR reaction provides a method for selectively increasing the concentration of a particular nucleic acid sequence even when that sequence has not been previously purified and is present only in a single copy in a particular sample.\",\n \"The method can be used to amplify either single- or double-stranded DNA.\",\n \"The essence of the method involves the use of two oligonucleotide probes to serve as primers for the template dependent, polymerase mediated replication of a desired nucleic acid molecule.\",\n \"A wide variety of alternative cloning and in vitro amplification methodologies are well known to those skilled in the art Examples of these techniques are found in, for example, Berger et al., Guide to Molecular Cloning Techniques, Methods in Enzymology 152, Academic Press, Inc., San Diego, Calif. (Berger), which is incorporated herein by reference in its entirety.\",\n \"Automated sequencing methods can be used to obtain or verify the nucleotide sequence of nGPCR-x.\",\n \"The nGPCR-x nucleotide sequences of the present invention are believed to be 100% accurate.\",\n \"However, as is known in the art, nucleotide sequence obtained by automated methods may contain some errors.\",\n \"Nucleotide sequences determined by automation are typically at least about 90%, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of a given nucleic acid molecule.\",\n \"The actual sequence may be more precisely determined using manual sequencing methods, which are well known in the art.\",\n \"An error in a sequence which results in an insertion or deletion of one or more nucleotides may result in a frame shift in translation such that the predicted amino acid sequence will differ from that which would be predicted from the actual nucleotide sequence of the nucleic acid molecule, starting at the point of the mutation.\",\n \"The nucleic acid molecules of the present invention, and fragments derived therefrom, are useful for screening for restriction fragment length polymorphism (RFLP) associated with certain disorders, as well as for genetic mapping.\",\n \"The polynucleotide sequence information provided by the invention makes possible large-scale expression of the encoded polypeptide by techniques well known and routinely practiced in the art.\",\n \"Vectors Another aspect of the present invention is directed to vectors, or recombinant expression vectors, comprising any of the nucleic acid molecules described above.\",\n \"Vectors are used herein either to amplify DNA or RNA encoding nGPCR-x and/or to express DNA which encodes nGPCR-x.\",\n \"Preferred vectors include, but are not limited to, plasmids, phages, cosmids, episomes, viral particles or viruses, and integratable DNA fragments (i.e., fragments integratable into the host genome by homologous recombination).\",\n \"Preferred viral particles include, but are not limited to, adenoviruses, baculoviruses, parvoviruses, herpesviruses, poxviruses, adeno-associated viruses, Semliki Forest viruses, vaccinia viruses, and retroviruses.\",\n \"Preferred expression vectors include, but are not limited to, pcDNA3 (Invitrogen) and pSVL (Pharmacia Biotech).\",\n \"Other expression vectors include, but are not limited to, pSPORT™ vectors, pGEM™ vectors (Promega), pPROEX vectors™ (LTI, Bethesda, Md.\",\n \"), Bluescript™ vectors (Stratagene), pQE™ vectors (Qiagen), pSE420™ (Invitrogen), and pYES2™ (Invitrogen).\",\n \"Expression constructs preferably comprise GPCR-x-encoding polynucleotides operatively linked to an endogenous or exogenous expression control DNA sequence and a transcription terminator.\",\n \"Expression control DNA sequences include promoters, enhancers, operators, and regulatory element binding sites generally, and ore typically selected based on the expression systems in which the expression construct is to be utilized.\",\n \"Preferred promoter and enhancer sequences are generally selected for the ability to increase gene expression, while operator sequences are generally selected for the ability to regulate gene expression.\",\n \"Expression constructs of the invention may also include sequences encoding one or more selectable markers that permit identification of host cells bearing the construct.\",\n \"Expression constructs may also include sequences that facilitate, and preferably promote, homologous recombination in a host cell.\",\n \"Preferred constructs of the invention also include sequences necessary for replication in a host cell.\",\n \"Expression constructs are preferably utilized for production of an encoded protein, but may also be utilized simply to amplify a nGPCR-x-encoding polynucleotide sequence.\",\n \"In preferred embodiments, the vector is an expression vector wherein the polynucleotide of the invention is operatively linked to a polynucleotide comprising an expression control sequence.\",\n \"Autonomously replicating recombinant expression constructs such as plasmid and viral DNA vectors incorporating polynucleotides of the invention are also provided.\",\n \"Preferred expression vectors are replicable DNA constructs in which a DNA sequence encoding nGPCR-x is operably linked or connected to suitable control sequences capable of effecting the expression of the nGPCR-x in a suitable host DNA regions are operably linked or connected when they are functionally related to each other.\",\n \"For example, a promoter is operably linked or connected to a coding sequence if it controls the transcription of the sequence.\",\n \"Amplification vectors do not require expression control domains, but rather need only the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants.\",\n \"The need for control sequences in the expression vector will vary depending upon the host selected and the transformation method chosen.\",\n \"Generally, control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding and sequences which control the termination of transcription and translation.\",\n \"Preferred vectors preferably contain a promoter that is recognized by the host organism.\",\n \"The promoter sequences of the present invention may be prokaryotic, eukaryotic or viral.\",\n \"Examples of suitable prokaryotic sequences include the PR and PL promoters of bacteriophage lambda (The bacteriophage Lambda, Hershey, A. D., Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1973), which is incorporated herein by reference in its entirety; Lambda II, Hendrix, R. W., Ed., Cold Spring Habor Press, Cold Spring Harbor, N.Y. (1980), which is incorporated herein by reference in its entirety); the trp, recA, heat shock, and lacZ promoters of E. coli and the SV40 early promoter (Benoist et al.\",\n \"Nature, 1981, 290, 304-310, which is incorporated herein by reference in its entirety).\",\n \"Additional promoters include, but are not limited to, mouse mammary tumor virus, long terminal repeat of human immunodeficiency virus, maloney virus, cytomegalovirus immediate early promoter, Epstein Barr virus, Rous sarcoma virus, human actin, human myosin, human hemoglobin, human muscle creatine, and human metalothionein.\",\n \"Additional regulatory sequences can also be included in preferred vectors.\",\n \"Preferred examples of suitable regulatory sequences are represented by the Shine-Dalgarno of the replicase gene of the phage MS-2 and of the gene cII of bacteriophage lambda.\",\n \"The Shine-Dalgarno sequence may be directly followed by DNA encoding nGPCR-x and result in the expression of the mature nGPCR-x protein.\",\n \"Moreover, suitable expression vectors can include an appropriate marker that allows the screening of the transformed host cells.\",\n \"The transformation of the selected host is carried out using any one of the various techniques well known to the expert in the art and described in Sambrook et al., supra.\",\n \"An origin of replication can also be provided either by construction of the vector to include an exogenous origin or may be provided by the host cell chromosomal replication mechanism.\",\n \"If the vector is integrated into the host cell chromosome, the latter may be sufficient.\",\n \"Alternatively, rather than using vectors which contain viral origins of replication, one skilled in the art can transform mammalian cells by the method of co-transformation with a selectable marker and nGPCR-x DNA.\",\n \"An example of a suitable marker is dihydrofolate reductase (DHFR) or thymidine kinase (see, U.S. Pat.\",\n \"No.\",\n \"4,399,216).\",\n \"Nucleotide sequences encoding GPCR-x may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesiderable joining, and ligation with appropriate ligases.\",\n \"Techniques for such manipulation are disclosed by Sambrook et al., supra and are well known in the art Methods for construction of mammalian expression vectors are disclosed in, for example, Okayama et al, Mol.\",\n \"Cell.\",\n \"Biol., 1983, 3, 280, Cosman et al., Mol.\",\n \"Immutol., 1986, 23, 935, Cosman et al., Nature, 1984, 312, 768, EP-A-0367566, and WO 91/18982, each of which is incorporated herein by reference in its entirety.\",\n \"Host Cells According to another aspect of the invention, host cells are provided, including prokaryotic and eukaryotic cells, comprising a polynucleotide of the invention (or vector of the invention) in a manner that permits expression of the encoded nGPCR-x polypeptide.\",\n \"Polynucleotides of the invention may be introduced into the host cell as part of a circular plasmid, or as linear DNA comprising an isolated protein coding region or a viral vector.\",\n \"Methods for introducing DNA into the host cell that are well known and routinely practiced in the art include transformation, transfection, electroporation, nuclear injection, or fusion with carriers such as liposomes, micelles, ghost cells, and protoplasts.\",\n \"Expression systems of the invention include bacterial, yeast, fungal, plant, insect, invertebrate, vertebrate, and mammalian cells systems.\",\n \"The invention provides host cells that are transformed or transfected (stably or transiently) with polynucleotides of the invention or vectors of the invention.\",\n \"As stated above, such host cells are useful for amplifying the polynucleotides and also for expressing the nGPCR-x polypeptide or fragment thereof encoded by the polynucleotide.\",\n \"In still another related embodiment, the invention provides a method for producing a nGPCR-x polypeptide (or fragment thereof) comprising the steps of growing a host cell of the invention in a nutrient medium and isolating the polypeptide or variant thereof from the cell or the medium.\",\n \"Because nGPCR-x is a seven transmembrane receptor, it will be appreciated that, for some applications, such as certain activity assays, the preferable isolation may involve isolation of cell membranes containing the polypeptide embedded therein, whereas for other applications a more complete isolation may be preferable.\",\n \"According to some aspects of the present invention, transformed host cells having an expression vector comprising any of the nucleic acid molecules described above are provided.\",\n \"Expression of the nucleotide sequence occurs when the expression vector is introduced into an appropriate host cell.\",\n \"Suitable host cells for expression of the polypeptides of the invention include, but are not limited to, prokaryotes, yeast, and eukaryotes.\",\n \"If a prokaryotic expression vector is employed, then the appropriate host cell would be any prokaryotic cell capable of expressing the cloned sequences.\",\n \"Suitable prokaryotic cells include, but are not limited to, bacteria of the genera Escherichia, Bacillus, Salmonella, Pseudomonas, Streptomyces, and Staphylococcus.\",\n \"If an eukaryotic expression vector is employed, then the appropriate host cell would be any eukaryotic cell capable of expressing the cloned sequence.\",\n \"Preferably, eukaryotic cells are cells of higher eukaryotes.\",\n \"Suitable eukaryotic cells include, but are not limited to, non-human mammalian tissue culture cells and human tissue culture cells.\",\n \"Preferred host cells include, but are not limited to, insect cells, HeLa cells, Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (COS cells), human 293 cells, and murine 3T3 fibroblasts.\",\n \"Propagation of such cells in cell culture has become a routine procedure (see, Tissue Culture, Academic Press, Kruse and Patterson, eds.\",\n \"(1973), which is incorporated herein by reference in its entirety).\",\n \"In addition, a yeast host may be employed as a host cell.\",\n \"Preferred yeast cells include, but are not limited to, the genera Saccharomyces, Pichia, and Kluveromyces.\",\n \"Preferred yeast hosts are S. cerevisiae and P. pastoris.\",\n \"Preferred yeast vectors can contain an origin of replication sequence from a 2T yeast plasmid, an autonomously replication sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene.\",\n \"Shuttle vectors for replication in both yeast and E. coli are also included herein.\",\n \"Alternatively, insect cells may be used as host cells.\",\n \"In a preferred embodiment, the polypeptides of the invention are expressed using a baculovirus expression system (sea Luckow et al., Bio/Technology, 1988, 6, 47, Baculovirus Expression Vectors: A Laboratory Manual, O'Rielly et al (Eds.\",\n \"), W.H.\",\n \"Freeman and Company, New York, 1992, and U.S. Pat.\",\n \"No.\",\n \"4,879,236, each of which is incorporated herein by reference in its entirety).\",\n \"In addition, the MAXBAC™ complete baculovirus expression system (Invitrogen) can, for example, be used for production in insect cells.\",\n \"Host cells of the invention are a valuable source of immunogen for development of antibodies specifically immunoreactive with nGPCR-x.\",\n \"Host cells of the invention are also useful in methods for the large-scale production of nGPCR-x polypeptides wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated from the cells, or from the medium in which the cells are grown, by purification methods known in the art, e.g., conventional chromatographic methods including immunoaffinity chromatography, receptor affinity chromatography, hydrophobic interaction chromatography, lectin affinity chromatography, size exclusion filtration, cation or anion exchange chromatography, high pressure liquid chromatography (HPLC), reverse phase HPLC, and the like.\",\n \"Still other methods of purification include those methods wherein the desired protein is expressed and purified as a fusion protein having a specific tag, label, or chelating moiety that is recognized by a specific binding partner or agent.\",\n \"The purified protein can be cleaved to yield the desired protein, or can be left as an intact fusion protein.\",\n \"Cleavage of the fusion component may produce a form of the desired protein having additional amino acid residues as a result of the cleavage process.\",\n \"Knowledge of nGPCR-x DNA sequences allows for modification of cells to permit, or increase, expression of endogenous nGPCR-x.\",\n \"Cells can be modified (e.g., by homologous recombination) to provide increased expression by replacing, in whole or in part, the naturally occurring nGPCR-x promoter with all or part of a heterologous promoter so that the cells express nGPCR-x at higher levels.\",\n \"The heterologous promoter is inserted in such a manner that it is operatively linked to endogenous nGPCR-x encoding sequences.\",\n \"(See, for example, PCT International Publication No.\",\n \"WO 94/12650, PCT International Publication No.\",\n \"WO 92/20808, and PCT International Publication No.\",\n \"WO 91/09955.)\",\n \"It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamoyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA.\",\n \"If linked to the nGPCR-x coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the nGPCR-x coding sequences in the cells.\",\n \"Knockouts The DNA sequence information provided by the present invention also makes possible the development (e.g., by homologous recombination or “knock-out” strategies; see Capecchi, Science 244:1288-1292 (1989), which is incorporated herein by reference) of animals that fail to express functional nGPCR-x or that express a variant of nGPCR-x.\",\n \"Such animals (especially small laboratory animals such as rats, rabbits, and mice) are useful as models for studying the in vivo activities of nGPCR-x and modulators of nGPCR-x.\",\n \"Antisense Also made available by the invention are anti-sense polynucleotides that recognize and hybridize to polynucleotides encoding nGPCR-x.\",\n \"Full-length and fragment anti-sense polynucleotides are provided.\",\n \"Fragment antisense molecules of the invention include (i) those that specifically recognize and hybridize to nGPCR-x RNA (as determined by sequence comparison of DNA encoding nGPCR-x to DNA encoding other known molecules).\",\n \"Identification of sequences unique to nGPCR-x encoding polynucleotides can be deduced through use of any publicly available sequence database, and/or through use of commercially available sequence comparison programs.\",\n \"After identification of the desired sequences, isolation through restriction digestion or amplification using any of the various polymerase chain reaction techniques well known in the art can be performed.\",\n \"Antisense polynucleotides are particularly relevant to regulating expression of nGPCR-x by those cells expressing nGPCR-x mRNA.\",\n \"Antisense nucleic acids (preferably 10 to 30 base-pair oligonucleotides) capable of specifically binding to nGPCR-x expression control sequences or nGPCR-x RNA are introduced into cells (e.g., by a viral vector or colloidal dispersion system such as a liposome).\",\n \"The antisense nucleic acid binds to the nGPCR-x target nucleotide sequence in the cell and prevents transcription and/or translation of the target sequence.\",\n \"Phosphorothioate and methylphosphonate antisense oligonucleotides are specifically contemplated for therapeutic use by the invention.\",\n \"The antisense oligonucleotides may be further modified by adding poly-L-lysine, transferrin polylysine, or cholesterol moieties at their 5′ end.\",\n \"Suppression of nGPCR-x expression at either the transcriptional or translational level is useful to generate cellular or animal models for diseases/conditions characterized by aberrant nGPCR-x expression.\",\n \"Antisense oligonucleotides, or fragments of odd numbered nucleotide sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191, or sequences complementary or homologous thereto, derived from the nucleotide sequences of the present invention encoding nGPCR-x are useful as diagnostic tools for probing gene expression in various tissues.\",\n \"For example, tissue can be probed in situ with oligonucleotide probes carrying detectable groups by conventional autoradiography techniques to investigate native expression of this enzyme or pathological conditions relating thereto.\",\n \"Antisense oligonucleotides are preferably directed to regulatory regions of odd numbered nucleotide sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191, or mRNA corresponding thereto, including, but not limited to, the initiation codon, TATA box, enhancer sequences, and the like.\",\n \"Transcription Factors The nGPCR-x sequences taught in the present invention facilitate the design of novel transcription factors for modulating nGPCR-x expression in native cells and animals, and cells transformed or transfected with nGPCR-x polynucleotides.\",\n \"For example, the Cys2-His2 zinc finger proteins, which bind DNA via their zinc finger domains, have been shown to be amenable to structural changes that lead to the recognition of different target sequences.\",\n \"These artificial zinc finger proteins recognize specific target sites with high affinity and low dissociation constants, and are able to act as gene switches to modulate gene expression.\",\n \"Knowledge of the particular nGPCR-x target sequence of the present invention facilitates the engineering of zinc finger proteins specific for the target sequence using known methods such as a combination of structure-based modeling and screening of phage display libraries (Segal et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"(USA) 96:2758-2763 (1999); Liu et al, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"(USA) 94:5525-5530 (1997); Greisman et al., Science 275:657-661 (1997); Choo et al., J. Mol.\",\n \"Biol.\",\n \"273:525-532 (1997)).\",\n \"Each zinc finger domain usually recognizes three or more base pairs.\",\n \"Since a recognition sequence of 18 base pairs is generally sufficient in length to render it unique in any known genome, a zinc finger protein consisting of 6 tandem repeats of zinc fingers would be expected to ensure specificity for a particular sequence (Segal et al.)\",\n \"The artificial zinc finger repeats, designed based on nGPCR-x sequences, are fused to activation or repression domains to promote or suppress nGPCR-x expression (Liu et al.)\",\n \"Alternatively, the zinc finger domains can be fused to the TATA box-binding factor (TBP) with varying lengths of linker region between the zinc finger peptide and the TBP to create either transcriptional activators or repressors (Kim et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"(USA) 94:3616-3620 (1997).\",\n \"Such proteins and polynucleotides that encode them, have utility for modulating nGPCR-x expression in vivo in both native cells, animals and humans; and/or cells transfected with nGPCR-x-encoding sequences.\",\n \"The novel transcription factor can be delivered to the target cells by transfecting constructs that express the transcription factor (gene therapy), or by introducing the protein.\",\n \"Engineered zinc finger proteins can also be designed to bind RNA sequences for use in therapeutics as alternatives to antisense or catalytic RNA methods (McColl et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"(USA) 96:9521-9526 (1997); Wu et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"(USA) 92:344-348 (1995)).\",\n \"The present invention contemplates methods of designing such transcription factors based on the gene sequence of the invention, as well as customized zinc finger proteins, that are useful to modulate nGPCR-x expression in cells (native or transformed) whose genetic complement includes these sequences.\",\n \"Polypeptides The invention also provides purified and isolated mammalian nGPCR-x polypeptides encoded by a polynucleotide of the invention.\",\n \"Presently preferred is a human nGPCR-x polypeptide comprising the amino acid sequence set out in even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO:192, or fragments thereof comprising an epitope specific to the polypeptide.\",\n \"By “epitope specific to” is meant a portion of the nGPCR receptor that is recognizable by an antibody that is specific for the nGPCR, as defined in detail below.\",\n \"Although the sequences provided are particular human sequences, the invention is intended to include within its scope other human allelic variants; non-human mammalian forms of nGPCR-x, and other vertebrate forms of nGPCR-x.\",\n \"It will be appreciated that extracellular epitopes are particularly useful for generating and screening for antibodies and other binding compounds that bind to receptors such as nGPCR-x.\",\n \"Thus, in another preferred embodiment, the invention provides a purified and isolated polypeptide comprising at least one extracellular domain (e.g., the N-terminal extracellular domain or one of the three extracellular loops) of nGPCR-x.\",\n \"Purified and isolated polypeptides comprising the N-terminal extracellular domain of nGPCR-x are highly preferred.\",\n \"Also preferred is a purified and isolated polypeptide comprising a nGPCR-x fragment selected from the group consisting of the N-terminal extracellular domain of nGPCR-x, transmembrane domains of nGPCR-x, an extracellular loop connecting transmembrane domains of nGPCR-x, an intracellular loop connecting transmembrane domains of nGPCR-x, the C-terminal cytoplasmic region of nGPCR-x, and fusions thereof.\",\n \"Such fragments may be continuous portions of the native receptor.\",\n \"However, it will also be appreciated that knowledge of the nGPCR-x gene and protein sequences as provided herein permits recombining of various domains that are not contiguous in the native protein.\",\n \"Using a FORTRAN computer program called “tmtrest all” [Parodi et al., Comput.\",\n \"Appl.\",\n \"Biosci.\",\n \"5:527-535 (1994)], nGPCR-x was shown to contain transmembrane-spanning domains.\",\n \"The invention also embraces polypeptides that have at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55% or at least 50% identity and/or homology to the preferred polypeptide of the invention.\",\n \"Percent amino acid sequence “identity” with respect to the preferred polypeptide of the invention is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues in the nGPCR-x sequence after aligning both sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.\",\n \"Percent sequence “homology” with respect to the preferred polypeptide of the invention is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues in the nGPCR-x sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and also considering any conservative substitutions as part of the sequence identity.\",\n \"In one aspect, percent homology is calculated as the percentage of amino acid residues in the smaller of two sequences which align with identical amino acid residue in the sequence being compared, when four gaps in a length of 100 amino acids may be introduced to maximize alignment [Dayhoff, in Atlas of Protein Sequence and Structure, Vol.\",\n \"5, p. 124, National Biochemical Research Foundation, Washington, D.C. (1972), incorporated herein by reference].\",\n \"Polypeptides of the invention may be isolated from natural cell sources or may be chemically synthesized, but are preferably produced by recombinant procedures involving host cells of the invention.\",\n \"Use of mammalian host cells is expected to provide for such post-translational modifications (e.g., glycosylation, truncation, lipidation, and phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the invention.\",\n \"Glycosylated and non-glycosylated forms of nGPCR-x polypeptides are embraced by the invention.\",\n \"The invention also embraces variant (or analog) nGPCR-x polypeptides.\",\n \"In one example, insertion variants are provided wherein one or more amino acid residues supplement a nGPCR-x amino acid sequence.\",\n \"Insertions may be located at either or both termini of the protein, or may be positioned within internal regions of the nGPCR-x amino acid sequence.\",\n \"Insertional variants with additional residues at either or both termini can include, for example, fusion proteins and proteins including amino acid tags or labels.\",\n \"Insertion variants include nGPCR-x polypeptides wherein one or more amino acid residues are added to a nGPCR-x acid sequence or to a biologically active fragment thereof.\",\n \"Variant products of the invention also include mature nGPCR-x products, ie., nGPCR-x products wherein leader or signal sequences are removed, with additional amino terminal residues.\",\n \"The additional amino terminal residues may be derived from another protein, or may include one or more residues that are not identifiable as being derived from specific proteins.\",\n \"nGPCR-x products with an additional methionine residue at position −1 (Met−1-nGPCR-x) are contemplated, as are variants with additional methionine and lysine residues at positions −2 and −1 (Met−2-Lys−1-nGPCR-x).\",\n \"Variants of nGPCR-x with additional Met, Met-Lys, Lys residues (or, one or more basic residues in general) are particularly useful for enhanced recombinant protein production in bacterial host cells.\",\n \"The invention also embraces nGPCR-x variants having additional amino acid residues that result from use of specific expression systems.\",\n \"For example, use of commercially available vectors that express a desired polypeptide as part of a glutathione-S-transferase (GST) fusion product provides the desired polypeptide having an additional glycine residue at position −1 after cleavage of the GST component from the desired polypeptide.\",\n \"Variants that result from expression in other vector systems are also contemplated.\",\n \"Insertional variants also include fusion proteins wherein the amino terminus and/or the carboxy terminus of nGPCR-x is/are fused to another polypeptide.\",\n \"In another aspect, the invention provides deletion variants wherein one or more amino acid residues in a nGPCR-x polypeptide are removed.\",\n \"Deletions can be effected at one or both termini of the nGPCR-x polypeptide, or with removal of one or more non-terminal amino acid residues of nGPCR-x.\",\n \"Deletion variants, therefore, include all fragments of a nGPCR-x polypeptide.\",\n \"The invention also embraces polypeptide fragments of the even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, wherein the fragments maintain biological (e.g., ligand binding and/or intracellular signaling) immunological properties of a nGPCR-x polypeptide.\",\n \"In one preferred embodiment of the invention, an isolated nucleic acid molecule comprises a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence homologous to even numbered sequences selected from the group consisting of: SEQ ID NO:2 to SEQ ID NO:94, SEQ ID NO: 186 and SEQ ID NO:192, and fragments thereof, wherein the nucleic acid molecule encoding at least a portion of nGPCR-x.\",\n \"In a more preferred embodiment, the isolated nucleic acid molecule comprises a sequence that encodes a polypeptide comprising even numbered sequences selected from the group consisting of SEQ ID NO:2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, and fragments thereof.\",\n \"As used in the present invention, polypeptide fragments comprise at least 5, 10, 15, 20, 25, 30, 35, or 40 consecutive amino acids of the even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO:192.Preferred polypeptide fragments display antigenic properties unique to, or specific for, human nGPCR-x and its allelic and species homologs.\",\n \"Fragments of the invention having the desired biological and immunological properties can be prepared by any of the methods well known and routinely practiced in the art.\",\n \"In still another aspect, the invention provides substitution variants of nGPCR-x polypeptides.\",\n \"Substitution variants include those polypeptides wherein one or more amino acid residues of a nGPCR-x polypeptide are removed and replaced with alternative residues.\",\n \"In one aspect, the substitutions are conservative in nature; however, the invention embraces substitutions that are also non-conservative.\",\n \"Conservative substitutions for this purpose may be defined as set out in Tables 2, 3, or 4 below.\",\n \"Variant polypeptides include those wherein conservative substitutions have been introduced by modification of polynucleotides encoding polypeptides of the invention.\",\n \"Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure.\",\n \"A conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties.\",\n \"Exemplary conservative substitutions are set out in Table 2 (from WO 97/09433, page 10, published Mar.\",\n \"13, 1997 (PCT/GB96/02197, filed Sep. 6, 1996), immediately below.\",\n \"TABLE 2 Conservative Substitutions I SIDE CHAIN CHARACTERISTIC AMINO ACID Aliphatic Non-polar GAP ILV Polar - uncharged CSTM NQ Polar - charged DE KR Aromatic HFWY Other NQDE Alternatively, conservative amino acids can be grouped as described in Lehninger, [Biochemistry, Second Edition; Worth Publishers, Inc. NY, N.Y. (1975), pp.71-77] as set out in Table 3, below.\",\n \"Table 3 Conservative Substitutions II TABLE 3 Conservative Substitutions II SIDE CHAIN CHARACTERISTIC AMINO ACID Non-polar (hydrophobic) A. Aliphatic: ALIVP B. Aromatic: FW C. Sulfur-containing: M D. Borderline: G Uncharged-polar A. Hydroxyl: STY B. Amides: NQ C. Sulfhydryl: C D. Borderline: G Positively Charged (Basic): KRH Negatively Charged (Acidic): DE As still another alternative, exemplary conservative substitutions are set out in Table 4, below.\",\n \"TABLE 4 Conservative Substitutions III Original Residue Exemplary Substitution Ala (A) Val, Leu, Ile Arg (R) Lys, Gln, Asn Asn (N) Gln, His, Lys, Arg Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp His (H) Asn, Gln, Lys, Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu (L) Ile, Val, Met, Ala, Phe Lys (K) Arg, Gln, Asn Met (M) Leu, Phe, Ile Phe (F) Leu, Val, Ile, Ala Pro (P) Gly Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp, Phe, Thr, Ser Val (V) Ile, Leu, Met, Phe, Ala It should be understood that the definition of polypeptides of the invention is intended to include polypeptides bearing modifications other than insertion, deletion, or substitution of amino acid residues.\",\n \"By way of example, the modifications may be covalent in nature, and include for example, chemical bonding with polymers, lipids, other organic, and inorganic moieties.\",\n \"Such derivatives may be prepared to increase circulating half-life of a polypeptide, or may be designed to improve the targeting capacity of the polypeptide for desired cells, tissues, or organs.\",\n \"Similarly, the invention further embraces nGPCR-x polypeptides that have been covalently modified to include one or more water-soluble polymer attachments such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.\",\n \"Variants that display ligand binding properties of native nGPCR-x and are expressed at higher levels, as well as variants that provide for constitutively active receptors, are particularly useful in assays of the invention; the variants are also useful in providing cellular, tissue and animal models of diseases/conditions characterized by aberrant nGPCR-x activity.\",\n \"In a related embodiment, the present invention provides compositions comprising purified polypeptides of the invention.\",\n \"Preferred compositions comprise, in addition to the polypeptide of the invention, a pharmaceutically acceptable (i.e., sterile and non-toxic) liquid, semisolid, or solid diluent that serves as a pharmaceutical vehicle, excipient, or medium.\",\n \"Any diluent known in the art may be used.\",\n \"Exemplary diluents include, but are not limited to, water, saline solutions, polyoxyethylene sorbitan monolaurate, magnesium stearate, methyl- and propylhydroxybenzoate, talc, alginates, starches, lactose, sucrose, dextrose, sorbitol, mannitol, glycerol, calcium phosphate, mineral oil, and cocoa butter.\",\n \"Variants that display ligand binding properties of native nGPCR-x and are expressed at higher levels, as well as variants that provide for constitutively active receptors, are particularly useful in assays of the invention; the variants are also useful in assays of the invention and in providing cellular, tissue and animal models of diseases/conditions characterized by aberrant nGPCR-x activity.\",\n \"The G protein-coupled receptor functions through a specific heterotrimeric guanine-nucleotide-binding regulatory protein (G-protein) coupled to the intracellular portion of the G protein-coupled receptor molecule.\",\n \"Accordingly, the G protein-coupled receptor has a specific affinity to G protein.\",\n \"G proteins specifically bind to guanine nucleotides.\",\n \"Isolation of G proteins provides a means to isolate guanine nucleotides.\",\n \"G Proteins may be isolated using commercially available anti-G protein antibodies or isolated G protein-coupled receptors.\",\n \"Similarly, G proteins may be detected in a sample isolated using commercially available detectable anti-G protein antibodies or isolated G protein-coupled receptors.\",\n \"According to the present invention, the isolated n-GPCR-x proteins of the present invention are useful to isolate and purify G proteins from samples such as cell lysates.\",\n \"Example 15 below sets forth an example of isolation of G proteins using isolated nGPCR-x proteins.\",\n \"Such methodolgy may be used in place of the use of commercially available ant-G protein antibodies which are used to isolate G proteins.\",\n \"Moreover, G proteins may be detected using nGPCR-x proteins in place of commercially available detectable anti-G protein antibodies.\",\n \"Since nGPCR-x proteins specifically bind to G proteins, they can be employed in any specific use where G protein specific affinity is required, such as those uses where commercially available anti-G protein antibodies are employed.\",\n \"Antibodies Also comprehended by the present invention are antibodies (e.g.\",\n \"monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifunctional/bispecific antibodies, humanized antibodies, human antibodies, and complementary determining region (CDR)-grafted antibodies, including compounds which include CDR sequences which specifically recognize a polypeptide of the invention) specific for nGPCR-x or fragments thereof.\",\n \"Preferred antibodies of the invention are human antibodies that are produced and identified according to methods described in WO93/11236, published Jun.\",\n \"20, 1993, which is incorporated herein by reference in its entirety.\",\n \"Antibody fragments, including Fab, Fab′, F(ab′)2, and Fv, are also provided by the invention.\",\n \"The term “specific for,” when used to describe antibodies of the invention, indicates that the variable regions of the antibodies of the invention recognize and bind nGPCR-x polypeptides exclusively (i.e., are able to distinguish nGPCR-x polypeptides from other known GPCR polypeptides by virtue of measurable differences in binding affinity, despite the possible existence of localized sequence identity, homology, or similarity between nGPCR-x and such polypeptides).\",\n \"It will be understood that specific antibodies may also interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and, in particular, in the constant region of the molecule.\",\n \"Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art.\",\n \"For a comprehensive discussion of such assays, see Harlow et al.\",\n \"(Eds.\",\n \"), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988), Chapter 6.Antibodies that recognize and bind fragments of the nGPCR-x polypeptides of the invention are also contemplated, provided that the antibodies are specific for nGPCR-x polypeptides.\",\n \"Antibodies of the invention can be produced using any method well known and routinely practiced in the art.\",\n \"The invention provides an antibody that is specific for the nGPCR-x of the invention.\",\n \"Antibody specificity is described in greater detail below.\",\n \"However, it should be emphasized that antibodies that can be generated from polypeptides that have previously been described in the literature and that are capable of fortuitously cross-reacting with nGPCR-x (e.g., due to the fortuitous existence of a similar epitope in both polypeptides) are considered “cross-reactive” antibodies.\",\n \"Such cross-reactive antibodies are not antibodies that are “specific” for nGPCR-x.\",\n \"The determination of whether an antibody is specific for nGPCR-x or is cross-reactive with another known receptor is made using any of several assays, such as Western blotting assays, that are well known in the art.\",\n \"For identifying cells that express nGPCR-x and also for modulating nGPCR-x-ligand binding activity, antibodies that specifically bind to an extracellular epitope of the nGPCR-x are preferred.\",\n \"In one preferred variation, the invention provides monoclonal antibodies.\",\n \"Hybridomas that produce such antibodies also are intended as aspects of the invention.\",\n \"In yet another variation, the invention provides a humanized antibody.\",\n \"Humanized antibodies are useful for in vivo therapeutic indications.\",\n \"In another variation, the invention provides a cell-free composition comprising polyclonal antibodies, wherein at least one of the antibodies is an antibody of the invention specific for nGPCR-x.\",\n \"Antisera isolated from an animal is an exemplary composition, as is a composition comprising an antibody fraction of an antisera that has been resuspended in water or in another diluent, excipient, or carrier.\",\n \"In still another related embodiment, the invention provides an anti-idiotypic antibody specific for an antibody that is specific for nGPCR-x.\",\n \"It is well known that antibodies contain relatively small antigen binding domains that can be isolated chemically or by recombinant techniques.\",\n \"Such domains are useful nGPCR-x binding molecules themselves, and also may be reintroduced into human antibodies, or fused to toxins or other polypeptides.\",\n \"Thus, in still another embodiment, the invention provides a polypeptide comprising a fragment of a nGPCR-x-specific antibody, wherein the fragment and the polypeptide bind to the nGPCR-x.\",\n \"By way of non-limiting example, the invention provides polypeptides that are single chain antibodies and CDR-grafted antibodies.\",\n \"Non-human antibodies may be humanized by any of the methods known in the art.\",\n \"In one method, the non-human CDRs are inserted into a human antibody or consensus antibody framework sequence.\",\n \"Further changes can then be introduced into the antibody framework to modulate affinity or immunogenicity.\",\n \"Antibodies of the invention are useful for, e.g., therapeutic purposes (by modulating activity of nGPCR-x), diagnostic purposes to detect or quantitate nGPCR-x, and purification of nGPCR-x.\",\n \"Kits comprising an antibody of the invention for any of the purposes described herein are also comprehended.\",\n \"In general, a kit of the invention also includes a control antigen for which the antibody is immunospecific.\",\n \"Compositions Mutations in the nGPCR-x gene that result in loss of normal function of the nGPCR-x gene product underlie nGPCR-x-related human disease states.\",\n \"The invention comprehends gene therapy to restore nGPCR-x activity to treat those disease states.\",\n \"Delivery of a functional nGPCR-x gene to appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), or er vivo by use of physical DNA transfer methods (erg, liposomes or chemical treatments).\",\n \"See, for example, Anderson, Nature, supplement to vol.\",\n \"392, no.\",\n \"6679, pp.25-20 (1998).\",\n \"For additional reviews of gene therapy technology see Friedmann, Science, 244: 1275-1281 (1989); Verna, Scientific American: 68-84 (1990); and Miller, Nature, 357: 455-460 (1992).\",\n \"Alternatively, it is contemplated that in other human disease states, preventing the expression of, or inhibiting the activity of, nGPCR-x will be useful in treating disease states.\",\n \"It is contemplated that antisense therapy or gene therapy could be applied to negatively regulate the expression of nGPCR-x.\",\n \"Another aspect of the present invention is directed to compositions, including pharmaceutical compositions, comprising any of the nucleic acid molecules or recombinant expression vectors described above and an acceptable carrier or diluent.\",\n \"Preferably, the carrier or diluent is pharmaceutically acceptable.\",\n \"Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field, which is incorporated herein by reference in its entirety.\",\n \"Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin.\",\n \"Liposomes and nonaqueous vehicles such as fixed oils may also be used.\",\n \"The formulations are sterilized by commonly used techniques.\",\n \"Also within the scope of the invention are compositions comprising polypeptides, polynucleotides, or antibodies of the invention that have been formulated with, e.g., a pharmaceutically acceptable carrier.\",\n \"The invention also provides methods of using antibodies of the invention.\",\n \"For example, the invention provides a method for modulating ligand binding of a nGPCR-x comprising the step of contacting the nGPCR-x with an antibody specific for the nGPCR-x, under conditions wherein the antibody binds the receptor.\",\n \"GPCRs that may be expressed in the brain, such as nGPCR-x, provide an indication that aberrant nGPCR-x signaling activity may correlate with one or more neurological or psychological disorders.\",\n \"The invention also provides a method for treating a neurological or psychiatric disorder comprising the step of administering to a mammal in need of such treatment an amount of an antibody-like polypeptide of the invention that is sufficient to modulate ligand binding to a nGPCR-x in neurons of the mammal.\",\n \"nGPCR-x may also be expressed in other tissues, including but not limited to, peripheral blood lymphocytes, pancreas, ovary, uterus, testis, salivary gland, thyroid gland, kidney, adrenal gland, liver, bone marrow, prostate, fetal liver, colon, muscle, and fetal brain, and may be found in many other tissues.\",\n \"Within the brain, nGPCR-x mRNA transcripts may be found in many tissues, including, but not limited to, frontal lobe, hypothalamus, pons, cerebellum, caudate nucleus, and medulla.\",\n \"Tissues and brain regions where specific nGPCRs of the present invention are expressed are identified in the Examples below.\",\n \"Kits The present invention is also directed to kits, including pharmaceutical kits.\",\n \"The kits can comprise any of the nucleic acid molecules described above, any of the polypeptides described above, or any antibody which binds to a polypeptide of the invention as described above, as well as a negative control.\",\n \"The kit preferably comprises additional components, such as, for example, instructions, solid support, reagents helpful for quantification, and the like.\",\n \"In another aspect, the invention features methods for detection of a polypeptide in a sample as a diagnostic tool for diseases or disorders, wherein the method comprises the steps of: (a) contacting the sample with a nucleic acid probe which hybridizes under hybridization assay conditions to a nucleic acid target region of a polypeptide having the sequence of even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, said probe comprising the nucleic acid sequence encoding the polypeptide, fragments thereof, and the complements of the sequences and fragments; and (b) detecting the presence or amount of the probe:target region hybrid as an indication of the disease.\",\n \"In preferred embodiments of the invention, the disease is selected from the group consisting of thyroid disorders (e.g.\",\n \"thyreotoxicosis, myxoedema); renal failure; inflammatory conditions (e.g., Crohn's disease); diseases related to cell differentiation and homeostasis; rheumatoid arthritis; autoimmune disorders; movement disorders; CNS disorders (e.g., pain including migraine; stroke; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, anxiety, generalized anxiety disorder, post-traumatic-stress disorder, depression, bipolar disorder, delirium, dementia, severe mental retardation; dyskinesias, such as Huntington's disease or Tourette's Syndrome; attention disorders including ADD and ADHD, and degenerative disorders such as Parkinson's, Alzheimer's; movement disorders, including ataxias, supranuclear palsy, etc.\",\n \"); infections, such as viral infections caused by HIV-1 or HIV-2; metabolic and cardiovascular diseases and disorders (e.g., type 2 diabetes, obesity, anorexia, hypotension, hypertension, thrombosis, myocardial infarction, cardiomyopathies, atherosclerosis, etc.\",\n \"); proliferative diseases and cancers (e.g., different cancers such as breast, colon, lung, etc., and hyperproliferative disorders such as psoriasis, prostate hyperplasia, etc.\",\n \"); hormonal disorders (e.g., male/female hormonal replacement, polycystic ovarian syndrome, alopecia, etc); and sexual dysfunction, among others.\",\n \"As described above and in Example 4 below, the genes encoding nGPCR-1 (nucleic acid sequence SEQ ID NO: 1, SEQ ID NO: 73, amino acid sequence SEQ ID NO: 2, SEQ ID NO:74), nGPCR-9 (nucleic acid sequence SEQ ID NO:9, SEQ ID NO:77, amino acid sequence SEQ ID NO:10, SEQ ID NO:78), nGPCR-11 (nucleic acid sequence SEQ ID NO:11, SEQ ID NO:79, amino acid sequence SEQ ID NO:12, SEQ ID NO:80), nGPCR-16 (nucleic acid sequence SEQ ID NO: 21, SEQ ID NO:81, amino acid sequence SEQ ID NO: 22, SEQ ID NO:82), nGPCR-40 (nucleic acid sequence SEQ ID NO:53, SEQ ID NO:83, amino acid sequence SEQ ID NO:54, SEQ ID NO:84), nGPCR-54 (nucleic acid sequence SEQ ID NO:59, SEQ ID NO:85, amino acid sequence SEQ ID NO:60, SEQ ID NO: 86), nGPCR-56 (nucleic acid sequence SEQ ID NO:63, SEQ ID NO:87, SEQ ID NO:89, amino acid sequence SEQ ID NO:64, SEQ ID NO: 88, SEQ ID NO:90), nGPCR-58 (nucleic acid sequence SEQ ID NO:67, SEQ ID NO:91, SEQ ID NO:93, amino acid sequence SEQ ID NO:68, SEQ ID NO: 92, SEQ ID NO:94) and nGPCR-3 (nucleic acid sequence SEQ ID NO:3, SEQ ID NO:185, amino acid sequence SEQ ID NO:4, SEQ ID NO: 186) have been detected in brain tissue indicating that these n-GPCR-x proteins are neuroreceptors.\",\n \"Kits may be designed to detect either expression of polynucleotides encoding these proteins or the proteins themselves in order to identify tissue as being neurological.\",\n \"For example, oligonucleotide hybridization kits can be provided which include a container having an oligonucleotide probe specific for the n-GPCR-x-specific DNA and optionally, containers with positive and negative controls and/or instructions.\",\n \"Similarly, PCR kits can be provided which include a container having primers specific for the n-GPCR-x-specific sequences, DNA and optionally, containers with size markers, positive and negative controls and/or instructions.\",\n \"Hybridization conditions should be such that hybridization occurs only with the genes in the presence of other nucleic acid molecules.\",\n \"Under stringent hybridization conditions only highly complementary nucleic acid sequences hybridize.\",\n \"Preferably, such conditions prevent hybridization of nucleic acids having 1 or 2 mismatches out of 20 contiguous nucleotides.\",\n \"Such conditions are defined supra.\",\n \"The diseases for which detection of genes in a sample could be diagnostic include diseases in which nucleic acid (DNA and/or RNA) is amplified in comparison to normal cells.\",\n \"By “amplification” is meant increased numbers of DNA or RNA in a cell compared with normal cells.\",\n \"The diseases that could be diagnosed by detection of nucleic acid in a sample preferably include central nervous system and metabolic diseases.\",\n \"The test samples suitable for nucleic acid probing methods of the present invention include, for example, cells or nucleic acid extracts of cells, or biological fluids.\",\n \"The samples used in the above-described methods will vary based on the assay format, the detection method and the nature of the tissues, cells or extracts to be assayed.\",\n \"Methods for preparing nucleic acid extracts of cells are well known in the art and can be readily adapted in order to obtain a sample that is compatible with the method utilized.\",\n \"Alternatively, immunoassay kits can be provided which have containers container having antibodies specific for the n-GPCR-x-protein and optionally, containers with positive and negative controls and/or instructions.\",\n \"Kits may also be provided useful in the identification of GPCR binding partners such as natural ligands or modulators (agonists or antagonists).\",\n \"Substances useful for treatment of disorders or diseases preferably show positive results in one or more in vitro assays for an activity corresponding to treatment of the disease or disorder in question.\",\n \"Substances that modulate the activity of the polypeptides preferably include, but are not limited to, antisense oligonucleotides, agonists and antagonists, and inhibitors of protein kinases.\",\n \"Methods of Inducing Immune Response Another aspect of the present invention is directed to methods of inducing an immune response in a mammal against a polypeptide of the invention by administering to the mammal an amount of the polypeptide sufficient to induce an immune response.\",\n \"The amount will be dependent on the animal species, size of the animal, and the like but can be determined by those skilled in the art.\",\n \"Methods of Identifying Ligands The invention also provides assays to identify compounds that bind nGPCR-x.\",\n \"One such assay comprises the steps of: (a) contacting a composition comprising a nGPCR-x with a compound suspected of binding nGPCR-x; and (b) measuring binding between the compound and nGPCR-x.\",\n \"In one variation, the composition comprises a cell expressing nGPCR-x on its surface.\",\n \"In another variation, isolated nGPCR-x or cell membranes comprising nGPCR-x are employed.\",\n \"The binding may be measured directly, e.g., by using a labeled compound, or may be measured indirectly by several techniques, including measuring intracellular signaling of nGPCR-x induced by the compound (or measuring changes in the level of nGPCR-x signaling).\",\n \"Specific binding molecules, including natural ligands and synthetic compounds, can be identified or developed using isolated or recombinant nGPCR-x products, nGPCR-x variants, or preferably, cells expressing such products.\",\n \"Binding partners are useful for purifying nGPCR-x products and detection or quantification of nGPCR-x products in fluid and tissue samples using known immunological procedures.\",\n \"Binding molecules are also manifestly useful in modulating (i.e., blocking, inhibiting or stimulating) biological activities of nGPCR-x, especially those activities involved in signal transduction.\",\n \"The DNA and amino acid sequence information provided by the present invention also makes possible identification of binding partner compounds with which a nGPCR-x polypeptide or polynucleotide will interact.\",\n \"Methods to identify binding partner compounds include solution assays, in vitro assays wherein nGPCR-x polypeptides are immobilized, and cell-based assays.\",\n \"Identification of binding partner compounds of nGPCR-x polypeptides provides candidates for therapeutic or prophylactic intervention in pathologies associated with nGPCR-x normal and aberrant biological activity.\",\n \"The invention includes several assay systems for identifying nGPCR-x binding partners.\",\n \"In solution assays, methods of the invention comprise the steps of (a) contacting a nGPCR-x polypeptide with one or more candidate binding partner compounds and (b) identifying the compounds that bind to the nGPCR-x polypeptide.\",\n \"Identification of the compounds that bind the nGPCR-x polypeptide can be achieved by isolating the nGPCR-x polypeptide/binding partner complex, and separating the binding partner compound from the nGPCR-x polypeptide.\",\n \"An additional step of characterizing the physical, biological, and/or biochemical properties of the binding partner compound is also comprehended in another embodiment of the invention.\",\n \"In one aspect, the nGPCR-x polypeptide/binding partner complex is isolated using an antibody immunospecific for either the nGPCR-x polypeptide or the candidate binding partner compound.\",\n \"In still other embodiments, either the nGPCR-x polypeptide or the candidate binding partner compound comprises a label or tag that facilitates its isolation, and methods of the invention to identify binding partner compounds include a step of isolating the nGPCR-x polypeptide/binding partner complex through interaction with the label or tag.\",\n \"An exemplary tag of this type is a poly-histidine sequence, generally around six histidine residues, that permits isolation of a compound so labeled using nickel chelation.\",\n \"Other labels and tags, such as the FLAG® tag (Eastman Kodak, Rochester, N.Y.), well known and routinely used in the art, are embraced by the invention.\",\n \"In one variation of an in vitro assay, the invention provides a method comprising the steps of (a) contacting an immobilized nGPCR-x polypeptide with a candidate binding partner compound and (b) detecting binding of the candidate compound to the nGPCR-x polypeptide.\",\n \"In an alternative embodiment, the candidate binding partner compound is immobilized and binding of nGPCR-x is detected.\",\n \"Immobilization is accomplished using any of the methods well known in the art, including covalent bonding to a support, a bead, or a chromatographic resin, as well as non-covalent, high affinity interactions such as antibody binding, or use of streptavidin/biotin binding wherein the immobilized compound includes a biotin moiety.\",\n \"Detection of binding can be accomplished (i) using a radioactive label on the compound that is not immobilized, (ii) using of a fluorescent label on the non-immobilized compound, (iii) using an antibody immunospecific for the non-immobilized compound, (iv) using a label on the non-immobilized compound that excites a fluorescent support to which the immobilized compound is attached, as well as other techniques well known and routinely practiced in the art.\",\n \"The invention also provides cell-based assays to identify binding partner compounds of a nGPCR-x polypeptide.\",\n \"In one embodiment, the invention provides a method comprising the steps of contacting a nGPCR-x polypeptide expressed on the surface of a cell with a candidate binding partner compound and detecting binding of the candidate binding partner compound to the nGPCR-x polypeptide.\",\n \"In a preferred embodiment, the detection comprises detecting a calcium flux or other physiological event in the cell caused by the binding of the molecule.\",\n \"Another aspect of the present invention is directed to methods of identifying compounds that bind to either nGPCR-x or nucleic acid molecules encoding nGPCR-x, comprising contacting nGPCR-x, or a nucleic acid molecule encoding the same, with a compound, and determining whether the compound binds nGPCR-x or a nucleic acid molecule encoding the same.\",\n \"Binding can be determined by binding assays which are well known to the skilled artisan, including, but not limited to, gel-shift assays, Western blots, radiolabeled competition assay, phage-based expression cloning, co-fractionation by chromatography, co-precipitation, cross linking, interaction trap/two-hybrid analysis, southwestern analysis, ELISA, and the like, which are described in, for example, Current Protocols in Molecular Biology, 1999, John Wiley & Sons, NY, which is incorporated herein by reference in its entirety.\",\n \"The compounds to be screened include (which may include compounds which are suspected to bind nGPCR-x, or a nucleic acid molecule encoding the same), but are not limited to, extracellular, intracellular, biologic or chemical origin.\",\n \"The methods of the invention also embrace ligands, especially neuropeptides, that are attached to a label, such as a radiolabel (e.g., 125I, 35S, 32P, 33P, 3H), a fluorescence label, a chemiluminescent label, an enzymic label and an immunogenic label.\",\n \"Modulators falling within the scope of the invention include, but are not limited to, non-peptide molecules such as non-peptide mimetics, non-peptide allosteric effectors, and peptides.\",\n \"The nGPCR-x polypeptide or polynucleotide employed in such a test may either be free in solution, attached to a solid support, borne on a cell surface or located intracellularly or associated with a portion of a cell.\",\n \"One skilled in the art can, for example, measure the formation of complexes between nGPCR-x and the compound being tested.\",\n \"Alternatively, one skilled in the art can examine the diminution in complex formation between nGPCR-x and its substrate caused by the compound being tested.\",\n \"In another embodiment of the invention, high throughput screening for compounds having suitable binding affinity to nGPCR-x is employed.\",\n \"Briefly, large numbers of different small peptide test compounds are synthesized on a solid substrate.\",\n \"The peptide test compounds are contacted with nGPCR-x and washed.\",\n \"Bound nGPCR-x is then detected by methods well known in the art.\",\n \"Purified polypeptides of the invention can also be coated directly onto plates for use in the aforementioned drug screening techniques.\",\n \"In addition, non-neutralizing antibodies can be used to capture the protein and immobilize it on the solid support.\",\n \"Generally, an expressed nGPCR-x can be used for HTS binding assays in conjunction with its defined ligand, in this case the corresponding neuropeptide that activates it.\",\n \"The identified peptide is labeled with a suitable radioisotope, including, but not limited to, 125I, 3H, 35S or 32P, by methods that are well known to those skilled in the art.\",\n \"Alternatively, the peptides may be labeled by well-known methods with a suitable fluorescent derivative (Baindur et al., Drug Dev.\",\n \"Res., 1994, 33, 373-398; Rogers, Drug Discovery Today, 1997, 2, 156-160).\",\n \"Radioactive ligand specifically bound to the receptor in membrane preparations made from the cell line expressing the recombinant protein can be detected in HTS assays in one of several standard ways, including filtration of the receptor-ligand complex to separate bound ligand from unbound ligand (Williams, Med.\",\n \"Res.\",\n \"Rev., 1991, 11, 147-184; Sweetnam et al., J.\",\n \"Natural Products, 1993, 56, 441455).\",\n \"Alternative methods include a scintillation proximity assay (SPA) or a FlashPlate format in which such separation is unnecessary (Nakayama, Cur.\",\n \"Opinion Drug Disc.\",\n \"Dev., 1998,1,85-91 Bosse et al., J. Biomolecular Screening, 1998, 3, 285-292.).\",\n \"Binding of fluorescent ligands can be detected in various ways, including fluorescence energy transfer (FRET), direct spectrophotofluorometric analysis of bound ligand, or fluorescence polarization (Rogers, Drug Discovery Today, 1997, 2, 156-160; Hill, Cur.\",\n \"Opinion Drug Disc.\",\n \"Dev, 1998, 1, 92-97).\",\n \"Other assays may be used to identify specific ligands of a nGPCR-x receptor, including assays that identify ligands of the target protein through measuring direct binding of test ligands to the target protein, as well as assays that identify ligands of target proteins through affinity ultrafiltration with ion spray mass spectroscopy/HPLC methods or other physical and analytical methods.\",\n \"Alternatively, such binding interactions are evaluated indirectly using the yeast two-hybrid system described in Fields et al., Nature, 340:245-246 (1989), and Fields et al., Trends in Genetics, 10:286-292 (1994), both of which are incorporated herein by reference.\",\n \"The two-hybrid system is a genetic assay for detecting interactions between two proteins or polypeptides.\",\n \"It can be used to identify proteins that bind to a known protein of interest, or to delineate domains or residues critical for an interaction.\",\n \"Variations on this methodology have been developed to clone genes that encode DNA binding proteins, to identify peptides that bind to a protein, and to screen for drugs.\",\n \"The two-hybrid system exploits the ability of a pair of interacting proteins to bring a transcription activation domain into close proximity with a DNA binding domain that binds to an upstream activation sequence (UAS) of a reporter gene, and is generally performed in yeast.\",\n \"The assay requires the construction of two hybrid genes encoding (1) a DNA-binding domain that is fused to a first protein and (2) an activation domain fused to a second protein.\",\n \"The DNA-binding domain targets the first hybrid protein to the UAS of the reporter gene; however, because most proteins lack an activation domain, this DNA-binding hybrid protein does not activate transcription of the reporter gene.\",\n \"The second hybrid protein, which contains the activation domain, cannot by itself activate expression of the reporter gene because it does not bind the UAS.\",\n \"However, when both hybrid proteins are present, the noncovalent interaction of the first and second proteins tethers the activation domain to the UAS, activating transcription of the reporter gene.\",\n \"For example, when the first protein is a GPCR gene product, or fragment thereof, that is known to interact with another protein or nucleic acid, this assay can be used to detect agents that interfere with the binding interaction.\",\n \"Expression of the reporter gene is monitored as different test agents are added to the system.\",\n \"The presence of an inhibitory agent results in lack of a reporter signal.\",\n \"The function of nGPCR-x gene products is unclear and no ligands have yet been found which bind the gene product.\",\n \"The yeast two-hybrid assay can also be used to identify proteins that bind to the gene product.\",\n \"In an assay to identify proteins that bind to a nGPCR-x receptor, or fragment thereof, a fusion polynucleotide encoding both a nGPCR-x receptor (or fragment) and a UAS binding domain (i.e., a first protein) may be used.\",\n \"In addition, a large number of hybrid genes each encoding a different second protein fused to an activation domain are produced and screened in the assay.\",\n \"Typically, the second protein is encoded by one or more members of a total cDNA or genomic DNA fusion library, with each second protein-coding region being fused to the activation domain.\",\n \"This system is applicable to a wide variety of proteins, and it is not even necessary to know the identity or function of the second binding protein.\",\n \"The system is highly sensitive and can detect interactions not revealed by other methods; even transient interactions may trigger transcription to produce a stable mRNA that can be repeatedly translated to yield the reporter protein.\",\n \"Other assays may be used to search for agents that bind to the target protein.\",\n \"One such screening method to identify direct binding of test ligands to a target protein is described in U.S. Pat.\",\n \"No.\",\n \"5,585,277, incorporated herein by reference.\",\n \"This method relies on the principle that proteins generally exist as a mixture of folded and unfolded states, and continually alternate between the two states.\",\n \"When a test ligand binds to the folded form of a target protein (i.e., when the test ligand is a ligand of the target protein), the target protein molecule bound by the ligand remains in its folded state.\",\n \"Thus, the folded target protein is present to a greater extent in the presence of a test ligand which binds the target protein, than in the absence of a ligand.\",\n \"Binding of the ligand to the target protein can be determined by any method that distinguishes between the folded and unfolded states of the target protein.\",\n \"The function of the target protein need not be known in order for this assay to be performed.\",\n \"Virtually any agent can be assessed by this method as a test ligand, including, but not limited to, metals, polypeptides, proteins, lipids, polysaccharides, polynucleotides and small organic molecules.\",\n \"Another method for identifying ligands of a target protein is described in Wieboldt et al., Anal.\",\n \"Chem., 69:1683-1691 (1997), incorporated herein by reference.\",\n \"This technique screens combinatorial libraries of 20-30 agents at a time in solution phase for binding to the target protein.\",\n \"Agents that bind to the target protein are separated from other library components by simple membrane washing.\",\n \"The specifically selected molecules that are retained on the filter are subsequently liberated from the target protein and analyzed by HPLC and pneumatically assisted electrospray (ion spray) ionization mass spectroscopy.\",\n \"This procedure selects library components with the greatest affinity for the target protein, and is particularly useful for small molecule libraries.\",\n \"Other embodiments of the invention comprise using competitive screening assays in which neutralizing antibodies capable of binding a polypeptide of the invention specifically compete with a test compound for binding to the polypeptide.\",\n \"In this manner, the antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants with nGPCR-x.\",\n \"Radiolabeled competitive binding studies are described in A. H. Lin et al.\",\n \"Antimicrobial Agents and Chemotherapy, 1997, vol.\",\n \"41, no.\",\n \"10.pp.\",\n \"2127-2131, the disclosure of which is incorporated herein by reference in its entirety.\",\n \"As described above and in Example 4 below, the genes encoding nGPCR-1 (nucleic acid sequence SEQ ID NO: 1, SEQ ID NO: 73, amino acid sequence SEQ ID NO: 2, SEQ ID NO:74), nGPCR-9 (nucleic acid sequence SEQ ID NO:9, SEQ ID NO:77, amino acid sequence SEQ ID NO:10, SEQ ID NO:78), nGPCR-11 (nucleic acid sequence SEQ ID NO:11, SEQ ID NO:79, amino acid sequence SEQ ID NO:12, SEQ ID NO:80), nGPCR-16 (nucleic acid sequence SEQ ID NO: 21, SEQ ID NO:81, amino acid sequence SEQ ID NO: 22, SEQ ID NO:82), nGPCR-40 (nucleic acid sequence SEQ ID NO:53, SEQ ID NO:83, amino acid sequence SEQ ID NO:54, SEQ ID NO:84), nGPCR-54 (nucleic acid sequence SEQ ID NO:59, SEQ ID NO:85, amino acid sequence SEQ ID NO:60, SEQ ID NO: 86), nGPCR-56 (nucleic acid sequence SEQ ID NO:63, SEQ ID NO:87, SEQ ID NO:89, amino acid sequence SEQ ID NO:64, SEQ ID NO: 88, SEQ ID NO:90), nGPCR-58 (nucleic acid sequence SEQ ID NO:67, SEQ ID NO:91, SEQ ID NO:93, amino acid sequence SEQ ID NO:68, SEQ ID NO: 92, SEQ ID NO:94), and nGPCR-3 (nucleic acid sequence SEQ ID NO:3, SEQ ID NO:185, amino acid sequence SEQ ID NO:4, SEQ ID NO: 186) have been detected in brain tissue indicating that these nGPCR-x proteins are neuroreceptors.\",\n \"Accordingly, natural binding partners of these molecules include neurotransmitters.\",\n \"Identification of Modulating Agents The invention also provides methods for identifying a modulator of binding between a nGPCR-x and a nGPCR-x binding partner, comprising the steps of: (a) contacting a nGPCR-x binding partner and a composition comprising a nGPCR-x in the presence and in the absence of a putative modulator compound; (b) detecting binding between the binding partner and the nGPCR-x; and (c) identifying a putative modulator compound or a modulator compound in view of decreased or increased binding between the binding partner and the nGPCR-x in the presence of the putative modulator, as compared to binding in the absence of the putative modulator.\",\n \"nGPCR-x binding partners that stimulate nGPCR-x activity are useful as agonists in disease states or conditions characterized by insufficient nGPCR-x signaling (e.g., as a result of insufficient activity of a nGPCR-x ligand).\",\n \"nGPCR-x binding partners that block ligand-mediated nGPCR-x signaling are useful as nGPCR-x antagonists to treat disease states or conditions characterized by excessive nGPCR-x signaling.\",\n \"In addition nGPCR-x modulators in general, as well as nGPCR-x polynucleotides and polypeptides, are useful in diagnostic assays for such diseases or conditions.\",\n \"In another aspect, the invention provides methods for treating a disease or abnormal condition by administering to a patient in need of such treatment a substance that modulates the activity or expression of a polypeptide having the sequence of even numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192.Agents that modulate (i.e., increase, decrease, or block) nGPCR-x activity or expression may be identified by incubating a putative modulator with a cell containing a nGPCR-x polypeptide or polynucleotide and determining the effect of the putative modulator on nGPCR-x activity or expression.\",\n \"The selectivity of a compound that modulates the activity of nGPCR-x can be evaluated by comparing its effects on nGPCR-x to its effect on other GPCR compounds.\",\n \"Selective modulators may include, for example, antibodies and other proteins, peptides, or organic molecules that specifically bind to a nGPCR-x polypeptide or a nGPCR-x-encoding nucleic acid.\",\n \"Modulators of nGPCR-x activity will be therapeutically useful in treatment of diseases and physiological conditions in which normal or aberrant nGPCR-x activity is involved.\",\n \"nGPCR-x polynucleotides, polypeptides, and modulators may be used in the treatment of such diseases and conditions as infections, such as viral infections caused by HIV-1 or HIV-2; pain; cancers; Parkinson's disease; hypotension; hypertension; and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Tourette's Syndrome, among others.\",\n \"nGPCR-x polynucleotides and polypeptides, as well as nGPCR-x modulators, may also be used in diagnostic assays for such diseases or conditions.\",\n \"Methods of the invention to identify modulators include variations on any of the methods described above to identify binding partner compounds, the variations including techniques wherein a binding partner compound has been identified and the binding assay is carried out in the presence and absence of a candidate modulator.\",\n \"A modulator is identified in those instances where binding between the nGPCR-x polypeptide and the binding partner compound changes in the presence of the candidate modulator compared to binding in the absence of the candidate modulator compound.\",\n \"A modulator that increases binding between the nGPCR-x polypeptide and the binding partner compound is described as an enhancer or activator, and a modulator that decreases binding between the nGPCR-x polypeptide and the binding partner compound is described as an inhibitor.\",\n \"The invention also comprehends high-throughput screening (HTS) assays to identify compounds that interact with or inhibit biological activity (i.e., affect enzymatic activity, binding activity, etc.)\",\n \"of a nGPCR-x polypeptide.\",\n \"HTS assays permit screening of large numbers of compounds in an efficient manner.\",\n \"Cell-based HTS systems are contemplated to investigate nGPCR-x receptor-ligand interaction.\",\n \"HTS assays are designed to identify “hits” or “lead compounds” having the desired property, from which modifications can be designed to improve the desired property.\",\n \"Chemical modification of the “hit” or “lead compound” is often based on an identifiable structure/activity relationship between the “hit” and the nGPCR-x polypeptide.\",\n \"Another aspect of the present invention is directed to methods of identifying compounds which modulate (i.e., increase or decrease) activity of nGPCR-x comprising contacting nGPCR-x with a compound, and determining whether the compound modifies activity of nGPCR-x.\",\n \"The activity in the presence of the test compared is measured to the activity in the absence of the test compound.\",\n \"Where the activity of the sample containing the test compound is higher than the activity in the sample lacking the test compound, the compound will have increased activity.\",\n \"Similarly, where the activity of the sample containing the test compound is lower than the activity in the sample lacking the test compound, the compound will have inhibited activity.\",\n \"The present invention is particularly useful for screening compounds by using nGPCR-x in any of a variety of drug screening techniques.\",\n \"The compounds to be screened include (which may include compounds which are suspected to modulate nGPCR-x activity), but are not limited to, extracellular, intracellular, biologic or chemical origin.\",\n \"The nGPCR-x polypeptide employed in such a test may be in any form, preferably, free in solution, attached to a solid support, borne on a cell surface or located intracellularly.\",\n \"One skilled in the art can, for example, measure the formation of complexes between nGPCR-x and the compound being tested.\",\n \"Alternatively, one skilled in the art can examine the diminution in complex formation between nGPCR-x and its substrate caused by the compound being tested.\",\n \"The activity of nGPCR-x polypeptides of the invention can be determined by, for example, examining the ability to bind or be activated by chemically synthesized peptide ligands.\",\n \"Alternatively, the activity of nGPCR-x polypeptides can be assayed by examining their ability to bind calcium ions, hormones, chemokines, neuropeptides, neurotransmitters, nucleotides, lipids, odorants, and photons.\",\n \"Alternatively, the activity of the nGPCR-x polypeptides can be determined by examining the activity of effector molecules including, but not limited to, adenylate cyclase, phospholipases and ion channels.\",\n \"Thus, modulators of nGPCR-x polypeptide activity may alter a GPCR receptor function, such as a binding property of a receptor or an activity such as G protein-mediated signal transduction or membrane localization.\",\n \"In various embodiments of the method, the assay may take the form of an ion flux assay, a yeast growth assay, a non-hydrolyzable GTP assay such as a [35S]-GTP S assay, a cAMP assay, an inositol triphosphate assay, a diacylglycerol assay, an Aequorin assay, a Luciferase assay, a FLIPR assay for intracellular Ca2+ concentration, a mitogenesis assay, a MAP Kinase activity assay, an arachidonic acid release assay (e.g., using [3H]-arachidonic acid), and an assay for extracellular acidification rates, as well as other binding or function-based assays of nGPCR-x activity that are generally known in the art.\",\n \"In several of these embodiments, the invention comprehends the inclusion of any of the G proteins known in the art, such as G16, G15, or chimeric Gqd5, Gqs5, Gqo5, Gq25, and the like.\",\n \"nGPCR-x activity can be determined by methodologies that are used to assay for FaRP activity, which is well known to those skilled in the art.\",\n \"Biological activities of nGPCR-x receptors according to the invention include, but are not limited to, the binding of a natural or an unnatural ligand, as well as any one of the functional activities of GPCRs known in the art.\",\n \"Non-limiting examples of GPCR activities include transmembrane signaling of various forms, which may involve G protein association and/or the exertion of an influence over G protein binding of various guanidylate nucleotides; another exemplary activity of GPCRs is the binding of accessory proteins or polypeptides that differ from known G proteins.\",\n \"The modulators of the invention exhibit a variety of chemical structures, which can be generally grouped into non-peptide mimetics of natural GPCR receptor ligands, peptide and non-peptide allosteric effectors of GPCR receptors, and peptides that may function as activators or inhibitors (competitive, uncompetitive and non-competitive) (e.g., antibody products) of GPCR receptors.\",\n \"The invention does not restrict the sources for suitable modulators, which may be obtained from natural sources such as plant, animal or mineral extracts, or non-natural sources such as small molecule libraries, including the products of combinatorial chemical approaches to library construction, and peptide libraries.\",\n \"Examples of peptide modulators of GPCR receptors exhibit the following primary structures: GLGPRPLRFamide, GNSFLRFamide, GGPQGPLRFamide, GPSGPLRFamide, PDVDHVFLRFamide, and pyro-EDVDHVFLRFamide.\",\n \"Other assays can be used to examine enzymatic activity including, but not limited to, photometric, radiometric, HPLC, electrochemical, and the like, which are described in, for example, Enzyme Assays: A Practical Approach, eds.\",\n \"R. Eisenthal and M. J. Danson, 1992, Oxford University Press, which is incorporated herein by reference in its entirety.\",\n \"The use of cDNAs encoding GPCRs in drug discovery programs is well-known; assays capable of testing thousands of unknown compounds per day in high-throughput screens (HTSs) are thoroughly documented.\",\n \"The literature is replete with examples of the use of radiolabelled ligands in HTS binding assays for drug discovery (see Williams, Medicinal Research Reviews, 1991, 11, 147-184.; Sweetnam, et al., J.\",\n \"Natural Products, 1993, 56, 441-455 for review).\",\n \"Recombinant receptors are preferred for binding assay HTS because they allow for better specificity (higher relative purity), provide the ability to generate large amounts of receptor material, and can be used in a broad variety of formats (see Hodgson, Bio/Technology, 1992, 10, 973-980; each of which is incorporated herein by reference in its entirety).\",\n \"A variety of heterologous systems is available for functional expression of recombinant receptors that are well known to those skilled in the art.\",\n \"Such systems include bacteria (Strosberg, et al., Trends in Pharmacological Sciences, 1992, 13, 95-98), yeast (Pausch, Trends in Biotechnology, 1997, 15, 487-494), several kinds of insect cells (Vanden Broeck, Int.\",\n \"Rev.\",\n \"Cytology, 1996, 164, 189-268), amphibian cells (Jayawickreme et al., Current Opinion in Biotechnology, 1997, 8, 629634) and several mammalian cell lines (CHO, BEK293, COS, etc.\",\n \"; see Gerhardt, et al., Eur.\",\n \"J. Pharmacology, 1997, 334, 1-23).\",\n \"These examples do not preclude the use of other possible cell expression systems, including cell lines obtained from nematodes (PCT application WO 98/37177).\",\n \"In preferred embodiments of the invention, methods of screening for compounds that modulate nGPCR-x activity comprise contacting test compounds with nGPCR-x and assaying for the presence of a complex between the compound and nGPCR-x.\",\n \"In such assays, the ligand is typically labeled.\",\n \"After suitable incubation, free ligand is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of the particular compound to bind to nGPCR-x.\",\n \"It is well known that activation of heterologous receptors expressed in recombinant systems results in a variety of biological responses, which are mediated by G proteins expressed in the host cells.\",\n \"Occupation of a GPCR by an agonist results in exchange of bound GDP for GIT at a binding site on the Ga subunit; one can use a radioactive, non-hydrolyzable derivative of GTP, GTPγ[35S], to measure binding of an agonist to the receptor (Sim et al., Neuroreport, 1996, 7, 729-733).\",\n \"One can also use this binding to measure the ability of antagonists to bind to the receptor by decreasing binding of GTPγ[35S] in the presence of a known agonist.\",\n \"One could therefore construct a HTS based on GTPγ[35S] binding, though this is not the preferred method.\",\n \"The G proteins required for functional expression of heterologous GPCRs can be native constituents of the host cell or can be introduced through well-known recombinant technology.\",\n \"The G proteins can be intact or chimeric.\",\n \"Often, a nearly universally competent G protein (e.g., Gα16) is used to couple any given receptor to a detectable response pathway.\",\n \"G protein activation results in the stimulation or inhibition of other native proteins, events that can be linked to a measurable response.\",\n \"Examples of such biological responses include, but are not limited to, the following: the ability to survive in the absence of a limiting nutrient in specifically engineered yeast cells (Pausch, Trends in Biotechnology, 1997, 15, 487-494); changes in intracellular Ca2+ concentration as measured by fluorescent dyes (Murphy, et al, Cur.\",\n \"Opinion Drug Disc.\",\n \"Dev., 1998, 1, 192-199).\",\n \"Fluorescence changes can also be used to monitor ligand-induced changes in membrane potential or intracellular pH; an automated system suitable for HTS has been described for these purposes (Schroeder, et al., J. Biomolecular Screening, 1996, 1, 75-80).\",\n \"Melanophores prepared from Xenopus laevis show a ligand-dependent change in pigment organization in response to heterologous GPCR activation; this response is adaptable to HTS formats (Jayawickreme et al, Cur.\",\n \"Opinion Biotechnology, 1997, 8, 629634).\",\n \"Assays are also available for the measurement of common second messengers, including cAMP, phosphoinositides and arachidonic acid, but these are not generally preferred for HTS.\",\n \"Preferred methods of HTS employing these receptors include permanently transfected CHO cells, in which agonists and antagonists can be identified by the ability to specifically alter the binding of GTPγ[35S] in membranes prepared from these cells.\",\n \"In another embodiment of the invention, permanently transfected CHO cells could be used for the preparation of membranes which contain significant amounts of the recombinant receptor proteins; these membrane preparations would then be used in receptor binding assays, employing the radiolabelled ligand specific for the particular receptor.\",\n \"Alternatively, a functional assay, such as fluorescent monitoring of ligand-induced changes in internal Ca2+ concentration or membrane potential in permanently transfected CHO cells containing each of these receptors individually or in combination would be preferred for HTS.\",\n \"Equally preferred would be an alternative type of mammalian cell, such as HEK293 or COS cells, in similar formats.\",\n \"More preferred would be permanently transfected insect cell lines, such as Drosophila S2 cells.\",\n \"Even more preferred would be recombinant yeast cells expressing the Drosophila melanogaster receptors in HTS formats well known to those skilled in the art (e.g., Pausch, Trends in Biotechnology, 1997, 15, 487-494).\",\n \"The invention contemplates a multitude of assays to screen and identify inhibitors of ligand binding to nGPCR-x receptors.\",\n \"In one example, the nGPCR-x receptor is immobilized and interaction with a binding partner is assessed in the presence and absence of a candidate modulator such as an inhibitor compound.\",\n \"In another example, interaction between the nGPCR-x receptor and its binding partner is assessed in a solution assay, both in the presence and absence of a candidate inhibitor compound.\",\n \"In either assay, an inhibitor is identified as a compound that decreases binding between the nGPCR-x receptor and its binding partner.\",\n \"Another contemplated assay involves a variation of the dihybrid assay wherein an inhibitor of protein/protein interactions is identified by detection of a positive signal in a transformed or transfected host cell, as described in PCT publication number WO 95/20652, published Aug. 3, 1995.Candidate modulators contemplated by the invention include compounds selected from libraries of either potential activators or potential inhibitors.\",\n \"There are a number of different libraries used for the identification of small molecule modulators, including: (1) chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules.\",\n \"Chemical libraries consist of random chemical structures, some of which are analogs of known compounds or analogs of compounds that have been identified as “hits” or “leads” in other drug discovery screens, some of which are derived from natural products, and some of which arise from non-directed synthetic organic chemistry.\",\n \"Natural product libraries are collections of microorganisms, animals, plants, or marine organisms which are used to create mixtures for screening by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of plants or marine organisms.\",\n \"Natural product libraries include polyketides, non-ribosomal peptides, and variants (non-naturally occurring) thereof.\",\n \"For a review, see Science 282:63-68 (1998).\",\n \"Combinatorial libraries are composed of large numbers of peptides, oligonucleotides, or organic compounds as a mixture.\",\n \"These libraries are relatively easy to prepare by traditional automated synthesis methods, PCR, cloning, or proprietary synthetic methods.\",\n \"Of particular interest are non-peptide combinatorial libraries.\",\n \"Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries.\",\n \"For a review of combinatorial chemistry and libraries created therefrom, see Myers, Curr.\",\n \"Opin.\",\n \"Biotechnol.\",\n \"8:701-707 (1997).\",\n \"Identification of modulators through use of the various libraries described herein permits modification of the candidate “hit” (or “lead”) to optimize the capacity of the “hit” to modulate activity.\",\n \"Still other candidate inhibitors contemplated by the invention can be designed and include soluble forms of binding partners, as well as such binding partners as chimeric, or fusion, proteins.\",\n \"A “binding partner” as used herein broadly encompasses non-peptide modulators, as well as such peptide modulators as neuropeptides other than natural ligands, antibodies, antibody fragments, and modified compounds comprising antibody domains that are immunospecific for the expression product of the identified nGPCR-x gene.\",\n \"The polypeptides of the invention are employed as a research tool for identification, characterization and purification of interacting, regulatory proteins.\",\n \"Appropriate labels are incorporated into the polypeptides of the invention by various methods known in the art and the polypeptides are used to capture interacting molecules.\",\n \"For example, molecules are incubated with the labeled polypeptides, washed to remove unbound polypeptides, and the polypeptide complex is quantified.\",\n \"Data obtained using different concentrations of polypeptide are used to calculate values for the number, affinity, and association of polypeptide with the protein complex.\",\n \"Labeled polypeptides are also useful as reagents for the purification of molecules with which the polypeptide interacts including, but not limited to, inhibitors.\",\n \"In one embodiment of affinity purification, a polypeptide is covalently coupled to a chromatography column.\",\n \"Cells and their membranes are extracted, and various cellular subcomponents are passed over the column.\",\n \"Molecules bind to the column by virtue of their affinity to the polypeptide.\",\n \"The polypeptide-complex is recovered from the column, dissociated and the recovered molecule is subjected to protein sequencing.\",\n \"This amino acid sequence is then used to identify the captured molecule or to design degenerate oligonucleotides for cloning the corresponding gene from an appropriate cDNA library.\",\n \"Alternatively, compounds may be identified which exhibit similar properties to the ligand for the nGPCR-x of the invention, but which are smaller and exhibit a longer half time than the endogenous ligand in a human or animal body.\",\n \"When an organic compound is designed, a molecule according to the invention is used as a “lead” compound.\",\n \"The design of mimetics to known pharmaceutically active compounds is a well-known approach in the development of pharmaceuticals based on such “lead” compounds.\",\n \"Mimetic design, synthesis and testing are generally used to avoid randomly screening a large number of molecules for a target property.\",\n \"Furthermore, structural data deriving from the analysis of the deduced amino acid sequences encoded by the DNAs of the present invention are useful to design new drugs, more specific and therefore with a higher pharmacological potency.\",\n \"Comparison of the protein sequence of the present invention with the sequences present in all the available databases showed a significant homology with the transmembrane portion of G protein coupled receptors.\",\n \"Accordingly, computer modeling can be used to develop a putative tertiary structure of the proteins of the invention based on the available information of the transmembrane domain of other proteins.\",\n \"Thus, novel ligands based on the predicted structure of nGPCR-x can be designed.\",\n \"In a particular embodiment, the novel molecules identified by the screening methods according to the invention are low molecular weight organic molecules, in which case a composition or pharmaceutical composition can be prepared thereof for oral intake, such as in tablets.\",\n \"The compositions, or pharmaceutical compositions, comprising the nucleic acid molecules, vectors, polypeptides, antibodies and compounds identified by the screening methods described herein, can be prepared for any route of administration including, but not limited to, oral, intravenous, cutaneous, subcutaneous, nasal, intramuscular or intraperitoneal.\",\n \"The nature of the carrier or other ingredients will depend on the specific route of administration and particular embodiment of the invention to be administered.\",\n \"Examples of techniques and protocols that are useful in this context are, inter alia, found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A (ed.\",\n \"), 1980, which is incorporated herein by reference in its entirety.\",\n \"The dosage of these low molecular weight compounds will depend on the disease state or condition to be treated and other clinical factors such as weight and condition of the human or animal and the route of administration of the compound.\",\n \"For treating human or animals, between approximately 0.5 mg/kg of body weight to 500 mg/kg of body weight of the compound can be administered.\",\n \"Therapy is typically administered at lower dosages and is continued until the desired therapeutic outcome is observed.\",\n \"The present compounds and methods, including nucleic acid molecules, polypeptides, antibodies, compounds identified by the screening methods described herein, have a variety of pharmaceutical applications and may be used, for example, to treat or prevent unregulated cellular growth, such as cancer cell and tumor growth.\",\n \"In a particular embodiment, the present molecules are used in gene therapy.\",\n \"For a review of gene therapy procedures, see e.g.\",\n \"Anderson, Science, 1992, 256, 808-813, which is incorporated herein by reference in its entirety.\",\n \"The present invention also encompasses a method of agonizing (stimulating) or antagonizing a nGPCR-x natural binding partner associated activity in a mammal comprising administering to said mammal an agonist or antagonist to one of the above disclosed polypeptides in an amount sufficient to effect said agonism or antagonism.\",\n \"One embodiment of the present invention, then, is a method of treating diseases in a mammal with an agonist or antagonist of the protein of the present invention comprises administering the agonist or antagonist to a mammal in an amount sufficient to agonize or antagonize nGPCR-x-associated functions.\",\n \"In an effort to discover novel treatments for diseases, biomedical researchers and chemists have designed, synthesized, and tested molecules that inhibit the function of protein polypeptides.\",\n \"Some small organic molecules form a class of compounds that modulate the function of protein polypeptides.\",\n \"Examples of molecules that have been reported to inhibit the function of protein kinases include, but are not limited to, bis monocyclic, bicyclic or heterocyclic aryl compounds (PCT WO 92/20642, published Nov. 26, 1992 by Maguire et al.\",\n \"), vinylene-azaindole derivatives (PCT WO 94/14808, published Jul.\",\n \"7, 1994 by Ballinari et al.\",\n \"), 1-cyclopropyl-4-pyridyluinolones (U.S. Pat.\",\n \"No.\",\n \"5,330,992), styryl compounds (U.S. Pat.\",\n \"No.\",\n \"5,217,999), styryl-substituted pyridyl compounds (U.S. Pat.\",\n \"No.\",\n \"5,302,606), certain quinazoline derivatives (EP Application No.\",\n \"0 566 266 A1), seleoindoles and selenides (PCT WO 94/03427, published Feb. 17, 1994 by Denny et al.\",\n \"), tricyclic polyhydroxylic compounds (PCT WO 92/21660, published Dec. 10, 1992 by Dow), and benzylphosphonic acid compounds (PCT WO 91/15495, published Oct. 17, 1991 by Dow et al), all of which are incorporated by reference herein, including any drawings.\",\n \"Exemplary diseases and conditions amenable to treatment based on the present invention include, but are not limited to, thyroid disorders (e.g.\",\n \"thyreotoxicosis, myxoedema); renal failure; inflammatory conditions (e g., Chron's disease); diseases related to cell differentiation and homeostasis; rheumatoid arthritis; autoimmune disorders; movement disorders; CNS disorders (e.g., pain including migraine; stroke; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, anxiety, generalized anxiety disorder, post-traumatic-stress disorder, depression, bipolar disorder, delirium, dementia, severe mental retardation; dyskinesias, such as Huntington's disease or Tourette's Syndrome; attention disorders including ADD and ADHD, and degenerative disorders such as Parkinson's, Alzheimer's; movement disorders, including ataxias, supranuclear palsy, etc); infections, such as viral infections caused by HIV-1 or HIV-2; metabolic and cardiovascular diseases and disorders (e.g., type 2 diabetes, obesity, anorexia, hypotension, hypertension, thrombosis, myocardial infarction, cardiomyopathies, atherosclerosis, etc.\",\n \"); proliferative diseases and cancers (e.g., different cancers such as breast, colon, lung, etc., and hyperproliferative disorders such as psoriasis, prostate hyperplasia, etc); hormonal disorders (e.g., male/female hormonal replacement, polycystic ovarian syndrome, alopecia, etc.\",\n \"); sexual dysfunction, among others.\",\n \"Compounds that can traverse cell membranes and are resistant to acid hydrolysis are potentially advantageous as therapeutics as they can become highly bioavailable after being administered orally to patients.\",\n \"However, many of these protein inhibitors only weakly inhibit function.\",\n \"In addition, many inhibit a variety of protein kinases and will therefore cause multiple side effects as therapeutics for diseases.\",\n \"Some indolinone compounds, however, form classes of acid resistant and membrane permeable organic molecules.\",\n \"WO 96/22976 (published Aug. 1, 1996 by Ballinari et at) describes hydrosoluble indolinone compounds that harbor tetralin, naphthalene, quinoline, and indole substituents fused to the oxindole ring.\",\n \"These bicyclic substituents are in turn substituted with polar groups including hydroxylated alkyl, phosphate, and ether substituents.\",\n \"U.S. patent application Ser.\",\n \"No.\",\n \"08/702,232, filed Aug. 23, 1996, entitled “Indolinone Combinatorial Libraries and Related Products and Methods for the Treatment of Disease” by Tang et al.\",\n \"(Lyon & Lyon Docket No.\",\n \"221/187) and 08/485,323, filed Jun.\",\n \"7, 1995, entitled “Benzylidene-Z-Indoline Compounds for the Treatment of Disease” by Tang et al.\",\n \"(Lyon & Lyon Docket No.\",\n \"223/298) and International Patent Publication WO 96/22976, published Aug. 1, 1996 by Ballinari et al., all of which are incorporated herein by reference in their entirety, including any drawings, describe indolinone chemical libraries of indolinone compounds harboring other bicyclic moieties as well as monocyclic moieties fused to the oxindole ring.\",\n \"Application Ser.\",\n \"Nos.\",\n \"08/02,232, filed Aug. 23, 1996, entitled “Indolinone Combinatorial Libraries and Related Products and Methods for the Treatment of Disease” by Tang et al.\",\n \"(Lyon & Lyon Docket No.\",\n \"221/187), 08/485,323, filed Jun.\",\n \"7, 1995, entitled “Benzylidene-Z-Indoline Compounds for the Treatment of Disease” by Tang et al.\",\n \"(Lyon & Lyon Docket No.\",\n \"223/298), and WO 96/22976, published Aug. 1, 1996 by Ballinari et al.\",\n \"teach methods of indolinone synthesis, methods of testing the biological activity of indolinone compounds in cells, and inhibition patterns of indolinone derivatives, both of which are incorporated by reference herein, including any drawings.\",\n \"Other examples of substances capable of modulating kinase activity include, but are not limited to, tyrphostins, quinazolines, quinoxolines, and quinolines.\",\n \"The quinazolines, tyrphostins, quinolines, and quinoxolines referred to above include well-known compounds such as those described in the literature.\",\n \"For example, representative publications describing quinazolines include Barker et al., EPO Publication No.\",\n \"0 520 722 A1; Jones et al., U.S. Pat.\",\n \"No.\",\n \"4,447,608; Kabbe et al., U.S. Pat.\",\n \"No.\",\n \"4,757,072; Kaul and Vougioukas, U.S. Pat.\",\n \"No.\",\n \"5,316,553; Kreighbaum and Corner, U.S. Pat.\",\n \"No.\",\n \"4,343,940; Pegg and Wardleworth, EPO Publication No.\",\n \"0 562 734 A1; Barker et al., Proc.\",\n \"of Am.\",\n \"Assoc.\",\n \"for Cancer Research 32:327 (1991); Bertino, J. R., Cancer Research 3:293-304 (1979); Bertino, J. R., Cancer Research 9(2 part 1):293-304 (1979); Curtinet al., Br.\",\n \"J.\",\n \"Cancer 53:361-368 (1986); Fernandes et al., Cancer Research 43:1117-1123 (1983); Ferris et al.\",\n \"J. Org.\",\n \"Chem.\",\n \"44(2):173-178; Fry et al., Science 265:1093-1095 (1994); Jackman et al., Cancer Research 51:5579-5586 (1981); Jones et al.\",\n \"J. Med.\",\n \"Chem.\",\n \"29(6):1114-1118; Lee and Skibo, Biochemistry 26(23):7355-7362 (1987); Lemus et al., J. Org.\",\n \"Chem.\",\n \"54:3511-3518 (1989); Ley and Seng, Synthesis 1975:415-522 (1975); Maxwell et al., Magnetic Resonance in Medicine 17:189-196 (1991); Mini et al., Cancer Research 45:325-330 (1985); Phillips and Castle, J. Heterocyclic Chem.\",\n \"17(19):1489-1596 (1980); Reece et al., Cancer Research 47(11):2996-2999 (1977); Sculier et al., Cancer Immunol.\",\n \"and Immunother.\",\n \"23:A65 (1986); Sikora et al., Cancer Letters 23:289-295 (1984); and Sikora et al., Analytical Biochem.\",\n \"172:344-355 (1988), all of which are incorporated herein by reference in their entirety, including any drawings.\",\n \"Quinoxaline is described in Kaul and Vougioukas, U.S. Pat.\",\n \"No.\",\n \"5,316,553, incorporated herein by reference in its entirety, including any drawings.\",\n \"Quinolines are described in Dolle et al., J. Med.\",\n \"Chem.\",\n \"37:2627-2629 (1994); MaGuire, J. Med.\",\n \"Chem.\",\n \"37:2129-2131 (1994); Burke et al., J. Med.\",\n \"Chem.\",\n \"36:425-432 (1993); and Burke et al.\",\n \"BioOrganic Med.\",\n \"Chem.\",\n \"Letters 2:1771-1774 (1992), all of which are incorporated by reference in their entirety, including any drawings.\",\n \"Tyrphostins are described in Allen et al., Clin.\",\n \"Exp.\",\n \"Immunol.\",\n \"91:141-156 (1993); Anafi et al., Blood 82:12:3524-3529 (1993); Baker et al., J.\",\n \"Cell Sci.\",\n \"102:543-555 (1992); Bilder et al., Amer.\",\n \"Physiol.\",\n \"Soc.\",\n \"pp.\",\n \"6363-6143:C721-C730 (1991); Brunton et al., Proceedings of Amer.\",\n \"Assoc.\",\n \"Cancer Rsch.\",\n \"33:558 (1992); Bryckaert et al., Experimental Cell Research 199:255-261 (1992); Dong et al., J. Leukocyte Biology 53:53-60 (1993); Dong et al., J. Immunol.\",\n \"151(5):2717-2724 (1993); Gazit et al., J. Med.\",\n \"Chem.\",\n \"32:2344-2352 (1989); Gazit et al., J. Med.\",\n \"Chem.\",\n \"36:3556-3564 (1993); Kaur et al., Anti-Cancer Drugs 5:213-222 (1994); King et al., Biochem.\",\n \"J.\",\n \"275:413418 (1991); Kuo et al., Cancer Letters 74:197-202 (1993); Levitzkd, A., The FASEB J.\",\n \"6:3275-3282 (1992); Lyall et al., J. Biol.\",\n \"Chem.\",\n \"264:14503-14509 (1989); Peterson et al., The Prostate 22:335-345 (1993); Pillemer et al., Int.\",\n \"J.\",\n \"Cancer 50:80-85 (1992); Posner et al., Molecular Pharmacology 45:673-683 (1993); Rendu et al., Biol.\",\n \"Pharmacology 44(5):881-888 (1992); Sauro and Thomas, Life Sciences 53:371-376 (1993); Sauro and Thomas, J. Pharm.\",\n \"and Experimental Therapeutics 267(3):119-1125 (1993); Wolbring et al., J. Biol.\",\n \"Chem.\",\n \"269(36):22470-22472 (1994); and Yoneda et al., Cancer Research 51:44304435 (1991); all of which are incorporated herein by reference in their entirety, including any drawings.\",\n \"Other compounds that could be used as modulators include oxindolinones such as those described in U.S. patent application Ser.\",\n \"No.\",\n \"08/702,232 filed Aug. 23, 1996, incorporated herein by reference in its entirety, including any drawings.\",\n \"Methods of determining the dosages of compounds to be administered to a patient and modes of administering compounds to an organism are disclosed in U.S. application Ser.\",\n \"No.\",\n \"08/702,282, filed Aug. 23, 1996 and International patent publication number WO 96/22976, published Aug. 1, 1996, both of which are incorporated herein by reference in their entirety, including any drawings, figures or tables.\",\n \"Those skilled in the art will appreciate that such descriptions are applicable to the present invention and can be easily adapted to it.\",\n \"The proper dosage depends on various factors such as the type of disease being treated, the particular composition being used and the size and physiological condition of the patient.\",\n \"Therapeutically effective doses for the compounds described herein can be estimated initially from cell culture and animal models.\",\n \"For example, a dose can be formulated in animal models to achieve a circulating concentration range that initially takes into account the IC50 as determined in cell culture assays.\",\n \"The animal model data can be used to more accurately determine useful doses in humans.\",\n \"Plasma half-life and biodistribution of the drug and metabolites in the plasma, tumors and major organs can also be determined to facilitate the selection of drugs most appropriate to inhibit a disorder.\",\n \"Such measurements can be carried out.\",\n \"For example, HPLC analysis can be performed on the plasma of animals treated with the drug and the location of radiolabeled compounds can be determined using detection methods such as X-ray, CAT scan and MRI.\",\n \"Compounds that show potent inhibitory activity in the screening assays, but have poor pharmacokinetic characteristics, can be optimized by altering the chemical structure and retesting.\",\n \"In this regard, compounds displaying good pharmacokinetic characteristics can be used as a model.\",\n \"Toxicity studies can also be carried out by measuring the blood cell composition.\",\n \"For example, toxicity studies can be carried out in a suitable animal model as follows: 1) the compound is administered to mice (an untreated control mouse should also be used); 2) blood samples are periodically obtained via the tail vein from one mouse in each treatment group; and 3) the samples are analyzed for red and white blood cell counts, blood cell composition and the percent of lymphocytes versus polymorphonuclear cells.\",\n \"A comparison of results for each dosing regime with the controls indicates if toxicity is present.\",\n \"At the termination of each toxicity study, further studies can be carried out by sacrificing the animals (preferably, in accordance with the American Veterinary Medical Association guidelines Report of the American Veterinary Medical Assoc.\",\n \"Panel on Euthanasia, Journal of American Veterinary Medical Assoc., 202:229-249, 1993).\",\n \"Representative animals from each treatment group can then be examined by gross necropsy for immediate evidence of metastasis, unusual illness or toxicity.\",\n \"Gross abnormalities in tissue are noted and tissues are examined histologically.\",\n \"Compounds causing a reduction in body weight or blood components are less preferred, as are compounds having an adverse effect on major organs.\",\n \"In general, the greater the adverse effect the less preferred the compound.\",\n \"For the treatment of cancers the expected daily dose of a hydrophobic pharmaceutical agent is between 1 to 500 mg/day, preferably 1 to 250 mg/day, and most preferably 1 to 50 mg/day.\",\n \"Drugs can be delivered less frequently provided plasma levels of the active moiety are sufficient to maintain therapeutic effectiveness.\",\n \"Plasma levels should reflect the potency of the drug.\",\n \"Generally, the more potent the compound the lower the plasma levels necessary to achieve efficacy.\",\n \"nGPCR-x mRNA transcripts may found in many tissues, including, but not limited to, brain, peripheral blood lymphocytes, pancreas, ovary, uterus, testis, salivary gland, kidney, adrenal gland, liver, bone marrow, prostate, fetal liver, colon, muscle, and fetal brain, and may be found in many other tissues.\",\n \"Within the brain, nGPCR-x mRNA transcripts may be found in many tissues, including, but not limited to, frontal lobe, hypothalamus, pons, cerebellum, caudate nucleus, and medulla.\",\n \"Tissues and brain regions where specific nGPCR mRNA transcripts are expressed are identified in the Examples, below.\",\n \"Odd numbered nucleotide sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191 will, as detailed above, enable screening the endogenous neurotransmitters/hormones/ligands which activate, agonize, or antagonize nGPCR-x and for compounds with potential utility in treating disorders including, but not limited to, thyroid disorders (e.g.\",\n \"thyreotoxicosis, myxoedema); renal failure; inflammatory conditions (e.g., Chron's disease); diseases related to cell differentiation and homeostasis; rheumatoid arthritis; autoimmune disorders; movement disorders; CNS disorders (e.g., pain including migraine; stroke; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, anxiety, generalized anxiety disorder, post-traumatic-stress disorder, depression, bipolar disorder, delirium, dementia, severe mental retardation; dyskinesias, such as Huntington's disease or Tourette's Syndrome; attention disorders including ADD and ADHD, and degenerative disorders such as Parkinson's, Alzheimer's; movement disorders, including ataxias, supranuclear palsy, etc.\",\n \"); infections, such as viral infections caused by HIV-1 or HIV-2; metabolic and cardiovascular diseases and disorders (e.g., type 2 diabetes, obesity, anorexia, hypotension, hypertension, thrombosis, myocardial infarction, cardiomyopathies, atherosclerosis, etc.\",\n \"); proliferative diseases and cancers (e.g., different cancers such as breast, colon, lung, etc, and hyperproliferative disorders such as psoriasis, prostate hyperplasia, etc.\",\n \"); hormonal disorders (e.g., male/female hormonal replacement, polycystic ovarian syndrome, alopecia, etc.\",\n \"); sexual dysfunction, among others.\",\n \"For example, nGPCR-x may be useful in the treatment of respiratory ailments such as asthma, where T cells are implicated by the disease.\",\n \"Contraction of airway smooth muscle is stimulated by thrombin.\",\n \"Cicala et al (1999) Br J Pharmacol 126:478484.Additionally, in bronchiolitis obliterans, it has been noted that activation of thrombin receptors may be deleterious.\",\n \"Hauck et al.\",\n \"(1999) Am J Physiol 277:L22-L29.Furthermore, mast cells have also been shown to have thrombin receptors.\",\n \"Cirino et al (1996) J Exp Med 183:821-827.nGPCR-x may also be useful in remodeling of airway structure s in chronic pulmonary inflammation via stimulation of fibroblast procollagen synthesis.\",\n \"See, e.g., Chambers et al.\",\n \"(1998) Biochem J 333:121-127; Trejo et al.\",\n \"(1996) J Biol Chem 271:21536-21541.In another example, increased release of sCD40L and expression of CD40L by T cells after activation of thrombin receptors suggests that nGPCR-x may be useful in the treatment of unstable angina due to the role of T cells and inflammation.\",\n \"See Aukrust et al.\",\n \"(1999) Circulation 100:614-620.A further example is the treatment of inflammatory diseases, such as psoriasis, inflammatory bowel disease, multiple sclerosis, rheumatoid arthritis, and thyroiditis.\",\n \"Due to the tissue expression profile of nGPCR-x, inhibition of thrombin receptors may be beneficial for these diseases.\",\n \"See, e.g., Morris et al.\",\n \"(1996) Ann Rheum Dis 55:841-843.In addition to T cells, NK cells and monocytes are also critical cell types which contribute to the pathogenesis of these diseases.\",\n \"See, e.g., Naldini & Carney (1996) Cell Immunol 172:3542; Hoffman & Cooper (1995) Blood Cells Mol Dis 21:156-167; Colotta et al.\",\n \"(1994) Am J Pathol 144:975-985.Expression of nGPCR-x in bone marrow and spleen may suggest that it may play a role in the proliferation of hematopoietic progenitor cells.\",\n \"See DiCuccio et al.\",\n \"(1996) Exp Hematol 24:914-918.As another example, nGPCR-x may be useful in the treatment of acute and/or traumatic brain injury.\",\n \"Astrocytes have been demonstrated to express thrombin receptors.\",\n \"Activation of thrombin receptors may be involved in astrogliosis following brain injury.\",\n \"Therefore, inhibition of receptor activity may be beneficial for limiting neuroinflammation.\",\n \"Scar formation mediated by astrocytes may also be limited by inhibiting thrombin receptors.\",\n \"See, e.g., Pindon et al.\",\n \"(1998) Eur J Biochem 255:766-774; Ubl & Reiser.\",\n \"(1997) Glia 21:361-369; Grabham & Cunningham (1995) J Neurochem 64:583-591.nGPCR-x receptor activation may mediate neuronal and astrocyte apoptosis and prevention of neurite outgrowth.\",\n \"Inhibition would be beneficial in both chronic and acute brain injury.\",\n \"See, e.g., Donovan et al.\",\n \"(1997) J Neurosci 17:5316-5326; Turgeon et al (1998) J Neurosci 18:6882-6891; Smith-Swintosky et al.\",\n \"(1997) J Neurochem 69:1890-1896; Gill et al.\",\n \"(1998) Brain Res 797:321-327; Suidan et al.\",\n \"(1996) Semin Thromb Hemost 22:125-133.The attached Sequence Listing contains the sequences of the polynucleotides and polypeptides of the invention and is incorporated herein by reference in its entirety.\",\n \"As described above and in Example 4 below, the genes encoding nGPCR-1 (nucleic acid sequence SEQ ID NO: 1, SEQ ID NO: 73, amino acid sequence SEQ ID NO: 2, SEQ ID NO:74), nGPCR-9 (nucleic acid sequence SEQ ID NO:9, SEQ ID NO:77, amino acid sequence SEQ ID NO: 10, SEQ ID NO:78), nGPCR-11 (nucleic acid sequence SEQ ID NO:11, SEQ ID NO:79, amino acid sequence SEQ ID NO:12, SEQ ID NO:80), nGPCR-16 (nucleic acid sequence SEQ ID NO: 21, SEQ.\",\n \"ID NO:81, amino acid sequence SEQ ID NO: 22, SEQ ID NO:82), nGPCR-40 (nucleic acid sequence SEQ ID NO:53, SEQ ID NO:83, amino acid sequence SEQ ID NO: 54, SEQ ID NO:84), nGPCR-54 (nucleic acid sequence SEQ ID NO:59, SEQ ID NO:85, amino acid sequence SEQ ID NO:60, SEQ ID NO: 86), nGPCR-56 (nucleic acid sequence SEQ ID NO:63, SEQ ID NO:87, SEQ ID NO:89, amino acid sequence SEQ ID NO:64, SEQ ID NO: 88, SEQ ID NO:90), nGPCR-58 (nucleic acid sequence SEQ ID NO:3, SEQ ID NO: 185, amino acid sequence SEQ ID NO:4, SEQ ID NO: 186) have been detected in brain tissue indicating that these nGPCR-x proteins are neuroreceptors.\",\n \"The identification of modulators such as agonists and antagonists is therefore useful for the identification of compounds useful to treat neurological diseases and disorders.\",\n \"Such neurological diseases and disorders, including but are not limited to, schizophrenia, affective disorders, ADHD/ADD (i.e., Attention Deficit-Hyperactivity Disorder/Attention Deficit Disorder), and neural disorders such as Alzheimer's disease, Parkinson's disease, migraine, and senile dementia as well as depression, anxiety, bipolar disease, epilepsy, neuritis, neurasthenia, neuropathy, neuroses, and the like.\",\n \"Methods of Screening Human Subjects Thus in yet another embodiment, the invention provides genetic screening procedures that entail analyzing a person's genome—in particular their alleles for GPCRs of the invention—to determine whether the individual possesses a genetic characteristic found in other individuals that are considered to be afflicted with, or at risk for, developing a mental disorder or disease of the brain that is suspected of having a hereditary component.\",\n \"For example, in one embodiment, the invention provides a method for determining a potential for developing a disorder affecting the brain in a human subject comprising the steps of analyzing the coding sequence of one or more GPCR genes from the human subject; and determining development potential for the disorder in said human subject from the analyzing step.\",\n \"More particularly, the invention provides a method of screening a human subject to diagnose a disorder affecting the brain or genetic predisposition therefor, comprising the steps of: (a) assaying nucleic acid of a human subject to determine a presence or an absence of a mutation altering the amino acid sequence, expression, or biological activity of at least one seven transmembrane receptor that is expressed in the brain, wherein the seven transmembrane receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 74, 186, 78, 80, 82, 84, 86, 90, and 94, or an allelic variant thereof, and wherein the nucleic acid corresponds to the gene encoding the seven transmembrane receptor; and (b) diagnosing the disorder or predisposition from the presence or absence of said mutation, wherein the presence of a mutation altering the amino acid sequence, expression, or biological activity of allele in the nucleic acid correlates with an increased risk of developing the disorder.\",\n \"In preferred variations, the seven transmembrane receptor is nGPCR-40 or nGPCR-54 comprising amino acid sequences set forth in SEQ ID NO: 84 for nGPCR-40 and SEQ ID NO: 86 for nGPCR-54, or an allelic variant thereof, and the disease is schizophrenia.\",\n \"By “human subject” is meant any human being, human embryo, or human fetus.\",\n \"It will be apparent that methods of the present invention will be of particular interest to individuals that have themselves been diagnosed with a disorder affecting the brain or have relatives that have been diagnosed with a disorder affecting the brain.\",\n \"By “screening for an increased risk” is meant determination of whether a genetic variation exists in the human subject that correlates with a greater likelihood of developing a disorder affecting the brain than exists for the human population as a whole, or for a relevant racial or ethnic human sub-population to which the individual belongs.\",\n \"Both positive and negative determinations (i.e., determinations that a genetic predisposition marker is present or is absent) are intended to fall within the scope of screening methods of the invention.\",\n \"In preferred embodiments, the presence of a mutation altering the sequence or expression of at least one nGPCR-40 or nGPCR-54 seven transmembrane receptor allele in the nucleic acid is correlated with an increased risk of developing schizophrenia, whereas the absence of such a mutation is reported as a negative determination.\",\n \"The “assaying” step of the invention may involve any techniques available for analyzing nucleic acid to determine its characteristics, including but not limited to well-known techniques such as single-strand conformation polymorphism analysis (SSCP) [Orita et al., Proc Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA, 86: 2766-2770 (1989)]; heteroduplex analysis [White et al., Genomics, 12: 301-306 (1992)]; denaturing gradient gel electrophoresis analysis [Fischer et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA, 80: 1579-1583 (1983); and Riesner et al., Electrophoresis, 10: 377-389 (1989)]; DNA sequencing; RNase cleavage [Myers et al., Science, 230: 1242-1246 (1985)]; chemical cleavage of mismatch techniques [Rowley et al., Genomics, 30: 574-582 (1995); and Roberts et al., Nucl.\",\n \"Acids Res., 25: 3377-3378 (1997)]; restriction fragment length polymorphism analysis; single nucleotide primer extension analysis [Shumaker et al., Hum.\",\n \"Mutat., 7: 346-354 (1996); and Pastinen et al., Genome Res., 7: 606-614 (1997)]; 5′ nuclease assays [Pease et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA, 91:5022-5026 (1994)]; DNA Microchip analysis [Raisay, G., Nature Biotechnology, 16: 4048 (1999); and Chee et al., U.S. Pat.\",\n \"No.\",\n \"5,837,832]; and ligase chain reaction [Whiteley et al., U.S. Pat.\",\n \"No.\",\n \"5,521,065].\",\n \"[See generally, Schafer and Hawkins, Nature Biotechnology, 16: 33-39 (1998).]\",\n \"All of the foregoing documents are hereby incorporated by reference in their entirety.\",\n \"Thus, in one preferred embodiment involving screening nGPCR-40 or nGPCR-54 sequences, for example, the assaying step comprises at least one procedure selected from the group consisting of: (a) determining a nucleotide sequence of at least one codon of at least one nGPCR-40 or nGPCR-54 allele of the human subject; (b) performing a hybridization assay to determine whether nucleic acid from the human subject has a nucleotide sequence identical to or different from one or more reference sequences; (c) performing a polynucleotide migration assay to determine whether nucleic acid from the human subject has a nucleotide sequence identical to or different from one or more reference sequences; and (d) performing a restriction endonuclease digestion to determine whether nucleic acid from the human subject has a nucleotide sequence identical to or different from one or more reference sequences.\",\n \"In a highly preferred embodiment, the assaying involves sequencing of nucleic acid to determine nucleotide sequence thereof, using any available sequencing technique.\",\n \"[See, e.g., Sanger et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"(USA), 74: 5463-5467 (1977) (dideoxy chain termination method); Mirzabekov, TIBTECH, 12: 27-32 (1994) (sequencing by hybridization); Drmanac et al., Nature Biotechnology, 16: 54-58 (1998); U.S. Pat.\",\n \"No.\",\n \"5,202,231; and Science, 260: 1649-1652 (1993) (sequencing by hybridization); Kieleczawa et al., Science, 258: 1787-1791 (1992) (sequencing by primer walking); (Douglas et al., Biotechniques, 14: 824828 (1993) (Direct sequencing of PCR products); and Akane et al., Biotechniques 16: 238-241 (1994); Maxam and Gilbert, Meth.\",\n \"Enzymol., 65: 499-560 (1977) (chemical termination sequencing), all incorporated herein by reference.]\",\n \"The analysis may entail sequencing of the entire nGPCR gene genomic DNA sequence, or portions thereof, or sequencing of the entire seven transmembrane receptor coding sequence or portions thereof.\",\n \"In some circumstances, the analysis may involve a determination of whether an individual possesses a particular allelic variant, in which case sequencing of only a small portion of nucleic acid—enough to determine the sequence of a particular codon characterizing the allelic variant—is sufficient This approach is appropriate, for example, when assaying to determine whether one family member inherited the same allelic variant that has been previously characterized for another family member, or, more generally, whether a person's genome contains an allelic variant that has been previously characterized and correlated with a mental disorder having a heritable component.\",\n \"In another highly preferred embodiment, the assaying step comprises performing a hybridization assay to determine whether nucleic acid from the human subject has a nucleotide sequence identical to or different from one or more reference sequences.\",\n \"In a preferred embodiment, the hybridization involves a determination of whether nucleic acid derived from the human subject will hybridize with one or more oligonucleotides, wherein the oligonucleotides have nucleotide sequences that correspond identically to a portion of the GPCR gene sequence taught herein, such as the nGPCR-40 or nGPCR-54 coding sequence set forth in SEQ ID NOS: 83 for nGPCR-40 or 85 for nGPCR-54, or that correspond identically except for one mismatch.\",\n \"The hybridization conditions are selected to differentiate between perfect sequence complementarity and imperfect matches differing by one or more bases.\",\n \"Such hybridization experiments thereby can provide single nucleotide polymorphism sequence information about the nucleic acid from the human subject, by virtue of knowing the sequences of the oligonucleotides used in the experiments.\",\n \"Several of the techniques outlined above involve an analysis wherein one performs a polynucleotide migration assay, e.g., on a polyacrylamide electrophoresis gel (or in a capillary electrophoresis system), under denaturing or non-denaturing conditions.\",\n \"Nucleic acid derived from the human subject is subjected to gel electrophoresis, usually adjacent to (or co-loaded with) one or more reference nucleic acids, such as reference GPCR-encoding sequences having a coding sequence identical to all or a portion of SEQ ID NOS: 83 or 85 (or identical except for one known polymorphism).\",\n \"The nucleic acid from the human subject and the reference sequence(s) are subjected to similar chemical or enzymatic treatments and then electrophoresed under conditions whereby the polynucleotides will show a differential migration pattern, unless they contain identical sequences.\",\n \"[See generally Ausubel et al.\",\n \"(eds.\",\n \"), Current Protocols in Molecular Biology, New York: John Wiley & Sons, Inc. (1987-1999); and Sambrook et al., (eds.\",\n \"), Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press (1989), both incorporated herein by reference in their entirety.]\",\n \"In the context of assaying, the term “nucleic acid of a human subject” is intended to include nucleic acid obtained directly from the human subject (e.g., DNA or RNA obtained from a biological sample such as a blood, tissue, or other cell or fluid sample); and also nucleic acid derived from nucleic acid obtained directly from the human subject.\",\n \"By way of non-limiting examples, well known procedures exist for creating cDNA that is complementary to RNA derived from a biological sample from a human subject, and for amplifying (e.g., via polymerase chain reaction (PCR)) DNA or RNA derived from a biological sample obtained from a human subject.\",\n \"Any such derived polynucleotide which retains relevant nucleotide sequence information of the human subject's own DNA/RNA is intended to fall within the definition of “nucleic acid of a human subject” for the purposes of the present invention.\",\n \"In the context of assaying, the term “mutation” includes addition, deletion, and/or substitution of one or more nucleotides in the GPCR gene sequence (e.g., as compared to the seven transmembrane receptor-encoding sequences set forth of SEQ ID NOS: 74, 186, 78, 80, 82, 84, 86, 90, and 94) and other polymorphisms that occur in introns (where introns exist) and that are identifiable via sequencing, restriction fragment length polymorphism, or other techniques.\",\n \"The various activity examples provided herein permit determination of whether a mutation modulates activity of the relevant receptor in the presence or absence of various test substances.\",\n \"In a related embodiment, the invention provides methods of screening a person's genotype with respect to GPCR's of the invention, and correlating such genotypes with diagnoses for disease or with predisposition for disease (for genetic counseling).\",\n \"For example, the invention provides a method of screening for an nGPCR-40 or nGPCR-54 hereditary schizophrenia genotype in a human patient, comprising the steps of: (a) providing a biological sample comprising nucleic acid from the patient, the nucleic acid including sequences corresponding to said patients nGPCR-40 or nGPCR-54 alleles; (b) analyzing the nucleic acid for the presence of a mutation or mutations; (c) determining an nGPCR-40 or nGPCR-54 genotype from the analyzing step; and (d) correlating the presence of a mutation in an nGPCR-40 or nGPCR-54 allele with a hereditary schizophrenia genotype.\",\n \"In a preferred embodiment, the biological sample is a cell sample containing human cells that contain genomic DNA of the human subject.\",\n \"The analyzing can be performed analogously to the assaying described in preceding paragraphs.\",\n \"For example, the analyzing comprises sequencing a portion of the nucleic acid (e.g., DNA or RNA), the portion comprising at least one codon of the nGPCR-40 or nGPCR-54 alleles.\",\n \"Although more time consuming and expensive than methods involving nucleic acid analysis, the invention also may be practiced by assaying protein of a human subject to determine the presence or absence of an amino acid sequence variation in GPCR protein from the human subject.\",\n \"Such protein analyses may be performed, e.g., by fragmenting GPCR protein via chemical or enzymatic methods and sequencing the resultant peptides; or by Western analyses using an antibody having specificity for a particular allelic variant of the GPCR.\",\n \"The invention also provides materials that are useful for performing methods of the invention.\",\n \"For example, the present invention provides oligonucleotides useful as probes in the many analyzing techniques described above.\",\n \"In general, such oligonucleotide probes comprise 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides that have a sequence that is identical, or exactly complementary, to a portion of a human GPCR gene sequence taught herein (or allelic variant thereof), or that is identical or exactly complementary except for one nucleotide substitution.\",\n \"In a preferred embodiment, the oligonucleotides have a sequence that corresponds in the foregoing manner to a human GPCR coding sequence taught herein, and in particular, the coding sequences set forth in SEQ ID NO: 83 and 85.In one variation, an oligonucleotide probe of the invention is purified and isolated.\",\n \"In another variation, the oligonucleotide probe is labeled, e.g., with a radioisotope, chromophore, or fluorophore.\",\n \"In yet another variation, the probe is covalently attached to a solid support.\",\n \"[See generally Ausubel et al.\",\n \"And Sambrook et al., supra.]\",\n \"In a related embodiment, the invention provides kits comprising reagents that are useful for practicing methods of the invention.\",\n \"For example, the invention provides a kit for screening a human subject to diagnose schizophrenia or a genetic predisposition therefor, comprising, in association: (a) an oligonucleotide useful as a probe for identifying polymorphisms in a human nGPCR-40 or nGPCR-54 seven transmembrane receptor gene, the oligonucleotide comprising 6-50 nucleotides that have a sequence that is identical or exactly complementary to a portion of a human nGPCR-40 or nGPCR-54 gene sequence or nGPCR-40 or nGPCR-54 coding sequence, except for one sequence difference selected from the group consisting of a nucleotide addition, a nucleotide deletion, or nucleotide substitution; and (b) a media packaged with the oligonucleotide containing information identifying polymorphisms identifiable with the probe that correlate with schizophrenia or a genetic predisposition therefor.\",\n \"Exemplary information-containing media include printed paper package inserts or packaging labels; and magnetic and optical storage media that are readable by computers or machines used by practitioners who perform genetic screening and counseling services.\",\n \"The practitioner uses the information provided in the media to correlate the results of the analysis with the oligonucleotide with a diagnosis.\",\n \"In a preferred variation, the oligonucleotide is labeled.\",\n \"In still another embodiment, the invention provides methods of identifying those allelic variants of GPCRs of the invention that correlate with mental disorders.\",\n \"For example, the invention provides a method of identifying a seven transmembrane allelic variant that correlates with a mental disorder, comprising steps of: (a) providing a biological sample comprising nucleic acid from a human patient diagnosed with a mental disorder, or from the patient's genetic progenitors or progeny; (b) analyzing the nucleic acid for the presence of a mutation or mutations in at least one seven transmembrane receptor that is expressed in the brain, wherein the at least one seven transmembrane receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 74, 186, 78, 80, 82, 84, 86, 90, and 94 or an allelic variant thereof, and wherein the nucleic acid includes sequence corresponding to the gene or genes encoding the at least one seven transmembrane receptor, (c) determining a genotype for the patient for the at least one seven transmembrane receptor from said analyzing step; and (d) identifying an allelic variant that correlates with the mental disorder from the determining step.\",\n \"To expedite this process, it may be desirable to perform linkage studies in the patients (and possibly their families) to correlate chromosomal markers with disease states.\",\n \"The chromosomal localization data provided herein facilitates identifying an involved GPCR with a chromosomal marker.\",\n \"The foregoing method can be performed to correlate GPCR's of the invention to a number of disorders having hereditary components that are causative or that predispose persons to the disorder.\",\n \"For example, in one preferred variation, the disorder is schizophrenia, and the at least one seven transmembrane receptor comprises nGPCR-40 having an amino acid sequence set forth in SEQ ID NO: 84 or an allelic variant thereof.\",\n \"Also contemplated as part of the invention are polynucleotides that comprise the allelic variant sequences identified by such methods, and polypeptides encoded by the allelic variant sequences, and oligonucleotide and oligopeptide fragments thereof that embody the mutations that have been identified.\",\n \"Such materials are useful in in vitro cell-free and cell-based assays for identifying lead compounds and therapeutics for treatment of the disorders.\",\n \"For example, the variants are used in activity assays, binding assays, and assays to screen for activity modulators described herein.\",\n \"In one preferred embodiment, the invention provides a purified and isolated polynucleotide comprising a nucleotide sequence encoding a nGPCR-40 or nGPCR-54 receptor allelic variant identified according to the methods described above; and an oligonucleotide that comprises the sequences that differentiate the allelic variant from the nGPCR-40 or nGPCR-54 sequences set forth in SEQ ID NOS: 83 and 88.The invention also provides a vector comprising the polynucleotide (preferably an expression vector); and a host cell transformed or transfected with the polynucleotide or vector.\",\n \"The invention also provides an isolated cell line that is expressing the allelic variant GPCR polypeptide; purified cell membranes from such cells; purified polypeptide; and synthetic peptides that embody the allelic variation amino acid sequence.\",\n \"In one particular embodiment, the invention provides a purified polynucleotide comprising a nucleotide sequence encoding a nGPCR-40 seven transmembrane receptor protein of a human that is affected with schizophrenia; wherein said polynucleotide hybridizes to the complement of SEQ ID NO: 83 under the following hybridization conditions: (a) hybridization for 16 hours at 42° C. in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% dextran sulfate and (b) washing 2 times for 30 minutes at 60° C. in a wash solution comprising 0.1×SSC and 1% SDS; and wherein the polynucleotide encodes a nGPCR-40 amino acid sequence that differs from SEQ ID NO: 84 by at least one residue.\",\n \"An exemplary assay for using the allelic variants is a method for identifying a modulator of nGPCR-x biological activity, comprising the steps of: (a) contacting a cell expressing the allelic variant in the presence and in the absence of a putative modulator compound; (b) measuring nGPCR-x biological activity in the cell; and (c) identifying a putative modulator compound in view of decreased or increased nGPCR-x biological activity in the presence versus absence of the putative modulator.\",\n \"Additional features of the invention will be apparent from the following Examples.\",\n \"Examples 1, 2, 4, 11, 12, and 13 are actual, while the remaining Examples are prophetic.\",\n \"Additional features and variations of the invention will be apparent to those skilled in the art from the entirety of this application, including the detailed description, and all such features are intended as aspects of the invention.\",\n \"Likewise, features of the invention described herein can be re-combined into additional embodiments that also are intended as aspects of the invention, irrespective of whether the combination of features is specifically mentioned above as an aspect or embodiment of the invention.\",\n \"Also, only such limitations which are described herein as critical to the invention should be viewed as such; variations of the invention lacking limitations which have not been described herein as critical are intended as aspects of the invention.\",\n \"EXAMPLES Example 1 Identification of nGPCR-X A.\",\n \"Database Search The Celera database was searched using known GPCR receptors as query sequences to find patterns suggestive of novel G protein-coupled receptors.\",\n \"Positive hits were further analyzed with the GCG program BLAST to determine which ones were the most likely candidates to encode G protein-coupled receptors, using the standard (default) alignment produced by BLAST as a guide.\",\n \"Briefly, the BLAST algorithm, which stands for Basic Local Alignment Search Tool is suitable for determining sequence similarity (Altschul et al., J. Molec.\",\n \"Biol., 1990, 215, 403-410, which is incorporated herein by reference in its entirety).\",\n \"Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).\",\n \"This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence.\",\n \"T is referred to as the neighborhood word score threshold (Altschul et al., supra).\",\n \"These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing them.\",\n \"The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased.\",\n \"Extension for the word hits in each direction are halted when: 1) the cumulative alignment score falls off by the quantity X from its maximum achieved value; 2) the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or 3) the end of either sequence is reached.\",\n \"The Blast algorithm parameters W, T and X determine the sensitivity and speed of the alignment.\",\n \"The Blast program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA, 1992, 89, 10915-10919, which is incorporated herein by reference in its entirety) alignments (13) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.\",\n \"The BLAST algorithm (Karlin et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA, 1993, 90, 5873-5787, which is incorporated herein by reference in its entirety) and Gapped BLAST perform a statistical analysis of the similarity between two sequences.\",\n \"One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.\",\n \"For example, a nucleic acid is considered similar to a GPCR gene or cDNA if the smallest sum probability in comparison of the test nucleic acid to a GPCR nucleic acid is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.Homology searches were performed with the program BLAST version 2.08.A collection of 340 query amino acid sequences derived from GPCR's was used to search the genomic DNA sequence using TBLASTN and alignments with an E-value lower than 0.01 were collected from each BLAST search.\",\n \"The amino acid sequences have been edited to remove regions in the sequence that produce non-significant alignments with proteins that are not related to GPCR's.\",\n \"Multiple query sequences may have a significant alignment to the same genomic region, although each alignment may not cover exactly the same DNA region.\",\n \"A procedure is used to determine the region of maximum common overlap between the alignments from several query sequences.\",\n \"This region is called the consensus DNA region.\",\n \"The procedure for determining this consensus involves the automatic parsing of the BLAST output files using the program MSPcrunch to produce a tabular report.\",\n \"From this tabular report the start and end of each alignment in the genomic DNA is extracted.\",\n \"This information was used by a PERL script to derive the maximum common overlap.\",\n \"These regions were reported in the form of a unique sequence identifier, a start and the end position in the sequence.\",\n \"The sequences defined by these regions were extracted from the original genomic sequence file using the program fetchdb.\",\n \"The consensus regions were assembled into a non-redundant set by using the program phrap.\",\n \"After assembly with phrap a set of contigs and singletons was defined as candidate DNA regions coding for nGPCR-x.\",\n \"These sequences were then submitted for further sequence analysis.\",\n \"Further sequence analysis involved the removal of sequences previously isolated and removal of sequences related to olfactory GPCRs.\",\n \"The transmembrane regions for the sequences that remained were determined using a FORTRAN computer program called “tmtrest.all” [Parodi et al., Comput.\",\n \"Appl.\",\n \"Biosci.\",\n \"5:527-535(1994)].\",\n \"Only sequences that contained transmembrane regions in a pattern found in GPCRs were retained.\",\n \"cDNAs were sequenced directly using an ABI377 fluorescence-based sequencer (Perkin-Elmer/Applied Biosystems Division, PE/ABD, Foster City, Calif.) and the ABI PRISM™ Ready Dye-Deoxy Terminator kit with Taq FS™ polymerase.\",\n \"Each ABI cycle sequencing reaction contained about 0.5 μg of plasmid DNA.\",\n \"Cycle-sequencing was performed using an initial denaturation at 98° C. for 1 minute, followed by 50 cycles using the following parameters: 98° C. for 30 seconds, annealing at 50° C. for 30 seconds, and extension at 60° C. for 4 minutes.\",\n \"Temperature cycles and times were controlled by a Perkin-Elmer 9600 thermocycler.\",\n \"Extension products were purified using Centriflex™ gel filtration cartridges (Advanced Genetic Technologies Corp., Gaithersburg, Md.).\",\n \"Each reaction product was loaded by pipette onto the column, which is then centrifuged in a swinging bucket centrifuge (Sorvall model RT6000B tabletop centrifuge) at 1500×g for 4 minutes at room temperature.\",\n \"Column-purified samples were dried under vacuum for about 40 minutes and then dissolved in 5 μl of a DNA loading solution (83% deionized formamide, 8.3 mM EDTA, and 1.6 mg/ml Blue Dextran).\",\n \"The samples were then heated to 90° C. for three minutes and loaded into the gel sample wells for sequence analysis using the ABI377 sequencer.\",\n \"Sequence analysis was performed by importing ABI377 files into the Sequencer program (Gene Codes, Ann Arbor, Mich.).\",\n \"Generally, sequence reads of 700 bp were obtained.\",\n \"Potential sequencing errors were minimized by obtaining sequence information from both DNA strands and by re-sequencing difficult areas using primers annealing at different locations until all sequencing ambiguities were removed.\",\n \"The following Table 5 contains the sequences of the polynucleotides and polypeptides of the invention.\",\n \"Start and stop codons within the polynucleotide sequence are identified by boldface type.\",\n \"The transmembrane domains within the polypeptide sequence are identified by underlining.\",\n \"TABLE 5 The following DNA sequence beGPCR-seq1 <SEQ ID NO.\",\n \"1> was identified in H. sapiens: GTCTGGGGGTGGGGGATGCTGGGACAGGGGTCAATTGCCTGAAGCAAGTGCTCTCATCCCCCTAGCTCCTGC TGATCTAGTTGGGGCTCCAGAGTGGGGAGGAGAAAGGCACTTTGAAACTTCTCTGCCCTTACCGTCTTAGCC ATCAAACTCTGAGCTGGAGATAGTGACGATGTGACAGGAACTTTCCCTGGGCCTCTCTGGGCCACAATTCCT GGCCGAGAGAAAGAGGAGGAATGAGGTGAGCACCTTCTTCACTCCTAGGGCCATGTGGTAGAGCTGCAGTCG CACCTCCTTCTGCCAATAGGCATAGATGAGTGGGTTGAGCAGGGAGTTGCCCACGCCGAGCAGCCACAGGTA CCGTTCCAGCACTAGGTAGAGGTGACACTCCTGGCAGGCCACCTGCACAATGCCAGTGATAAGGAAGGGGGT CCAGGATAGAGCAAAGCTCCCAATGAGAACAGACACAGTACGGAGAGCTTTGAAGTCGCTGGGAGTCCGTGG GGATCGATAACCTCCAGCCATGGCTCCTGCATGTTCCATCTTTCGAATCTGCTGGCTGTGCATGGAGGCAAT TTGAGCATGTCGCAGTAGAAGAAGACAAAGAGGAGCATGGCTGGGAAGAAGCCAACGCAGGAGAGGGTCAGC ACGAAGTGAGGGTGAAATACAGCAAAGAAGCTGCACTGCCCTTTGTAGGCAGTCTGCTGGAACATGGGGATT CCGAGTGGGAGGAAGCCAATGAGGTAAGACACTAACCACAGCCCGGCAATGCAGGCCCCGGCCACGAACCCA CTCATGATCTTCAAGTAGCGGAAGGGCTGCTTGATGGCAAGGTACCTGTCAAAGGTGATCAGCATGACCGTG AGGACAGAGGCAGCTGCGGAGGAAGTGACAAATGCCATCCGCAGGCTGCACAGGGTCTTCTGTGTGGGCCGA GAAGGGCTGGAGAGCTGGTCTGTGAGTAGGCCAGAGATGGCCACACCAATCAAGGTGTCAGCCACAGCCAGA TTCAAGGTGAAGCAGAGACTGACACCATCATTCTTGTGGATCAACAGCAGCACAGCCACAGCCACTAGTGTG TTAGTAGCAATGATGAGGGAGGCCAGGACAGCAAGGATCACTCCAAATGAGAAAGATGATTCCATGTCTCGA AGTGGCAGGACTTCACTTACCAGGGCATG The following amino acid sequence <SEQ ID NO.\",\n \"2> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"1: MESSFSFGVILAVLASLIIATNTLVAVAVLLLIHKNDGVSLCFTLNLAVADTLIGVAISGLLTDQLSSPSRPT QKTLCSLRMAFVTSSAAASVLTVMLITFDRYLAIKQPFRYLKIMSGFVAGACIAGLWLVSYLIGFLPLGIPMF QQTAYKGQCSFFAVFHPHFVLTLSCVGFFPAMLLFVFFYCDMLKIASMHSQQIRKMEHAGAMAGGYRSPRTPS DFKALRTVSVLIGSFALSWTPFLITGIVQVACQECHLYLVLERYLWLLGVGNSLLNPLIYAYWQKEVRLQLYH MALGVKKVLTSFLLFLSARNCGPERPRESSCHIVTISSSEFDG The following DNA sequence beGPCR-seq3 <SEQ ID NO.\",\n \"3> was identified in H. sapiens: CAGCGCGAGCGCCTTCATGGTGACGGTGTCCATGCGCTGGCAGTGTCTGCGTGCCACCCGGTGCACCTGGAG CGAGGTGAGGCAGAGCACCGCCAGCGGCAGCACGAAGCCCACGGCATGGAGCGTGGCGGTGAAGGCTGCGAA GCGCGGACGCTCAGGCTCGGGCGGCAGGCGCAGCGAACAGGACGCGAAGGCGCTGCTGTAGCCAAGCCACGA GCAGCCAAGTGCAGCGCCTGAGAAGGCCAGCGACTGTCCCCAGGCACAGCCCAGCAGCAGGCCGGCATAGCG CGGTCGCAGGCGTCCGGCGTAGCGCAGTGGGAAGCCCACTGCCAGCCACTGGTCTGCGCTCAGCGCCGCCAC GCTCAGCGCCGCGTTGGACGCCAGGAAGGTGTCCAGGAAGCCAATGACTTGGCATGCGCCGGGCGCCGACGG TGTCCGCCCGCGCATCACACCGAGCAGCGTGAAGGGCATGTCCAGCGCCGCCAGCAGCAGGTGGCCCAGAGA CAGATTCACCAGGAGGACGCCTGAGGCTCGAGTGCGGAGCTCAGCGCTGTAGGCGCAACAAAGCAGCACCAG TGCGTTGGATAGCAGCGCCACGGCCAGTACCATCACCAGGAGACCCGCCAGCAGCGCCTCGCCGGGGCCCAT GGCGCTAGC The following amino acid sequence <SEQ ID NO.\",\n \"4> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"3: SAMGPGEALLAGLLVMVLAVALLSNALVLLCCAYSAELRTRASGVLLVNLSLGHLLLAALDMPFTLLGVMRGR TPSAPGACQVIGFLDTFLASNAALSVAALSADQWLAVGFPLRYAGRLRPRYAGLLLGCAWGQSLAFSGAALGC SWLGYSSAFASCSLRLPPEPERPRFAAFTATLHAVGFVLPLAVLCLTSLQVHRVARRHCQRMDTVTMKALA The following DNA sequence beGPCR-seq4 <SEQ ID NO.\",\n \"5> was identified in H. sapiens: TGTGCAGGTGTGATCTCCATTCCTTTGTACATCCCTCACACGCTGTTCGATGGGATTTTGGAAAGGAAATCT GTGTATTTTGGCTCACTACTGACTATCTGTTATGTACAGCATCTGTATATAACATTGTCCTCATCAGCTATG ATCGATACCTGTCAGTCTCAAATGCTGTAAGTCGAACACATTAATTTATCCCCCTTAGAAGATTATGTAAAT GTATA The following amino acid sequence <SEQ ID NO.\",\n \"6> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"5: CAGVISIPLYIPHTLFEWDFGKEICVFWLTTDYLLCTASVYNIVLISYDRYLSVSNAVSRTHFIPLR RLCKCI The following DNA sequence beGPCR-seq5 <SEQ ID NO.\",\n \"7> was identified in H. sapiens: GACGTCGAAGCAGGTGATGATGCCCAGGGCGTGCACCGGGTAGGTGAGATCGGTGCGCGCCAGCGGGGACAGG GCGGTCAGGAGCAGCAGCCAGGTCCCTGCACACGCGGCCACCGCGTAACGACGGCGGCGCCAGCGCTTGGAGC TGAGCGGGTACAGGATCCCCAGGAAGCGCTCCACGCTGATACAGGTCATGGTGAGGATGCTGGAATACATGTT TGCGTAAAAGGCCACGGTCACCACGTTGCAAAGCAGCACCCCGAATACCCAGTGGTGGCGGTTGCAATGGTAG TAGATTTGGAAAGGCAACACGCTGGCCAGCATCAGGTCCGTGACGCTCAGGTTGATCATGAAGATGACCGACG GGGATCTGGGCCCCATGCGCCGGCACAGCACCCACAGAGAGAAGAGGTTGCCCGGGATGCTGACCGCCGCCAC CAGCGAGTACACCACGGGCAGGGCCACCGCGATCGCCGGGTTCCGCAGCATCTGCAGCGTCGCGTTGTC The following amino acid sequence <SEQ ID NO.\",\n \"8> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"7: DNATLQMLRNPAIAVALPVVYSLVAAVSIPGNLFSLWVLCRRMGPRSPSVIFMINLSVTDLMLASVLPFQIYY HCNRHHWVFGVLCNLVVTVAFYANMYSSILTMTCISVERFLGILYPLSSKRWRRRRYAVAACAGTWLLLLTAL SPLARTDLTYPVHALGIITCFDV The following DNA sequence beGPCR-seq9 <SEQ ID NO.\",\n \"9> was identified in H. sapiens: CCCATGTTCCTGCTCCTGGGCAGCCTCACGTTGTCGGATCTGCTGGCAGGCGCCGCCTACGCCGCCAACAT CCTACTGTCGGGGCCGCTCACGCTGAAACTGTCCCCCGCGCTCTGGTTCGCACGGGAGGGAGGCGTCTTCG TGGCACTCACTGCGTCCGTGCTGAGCCTCCTGGGCATCGCGCTGGAGCGCAGCCTCACCATGGCGCGCAGG GGGCCCGCGCCCGTCTCCAGTCGGGGGCGCACGCTGGCGATGGCAGCCGCGGCCTGG The following amino acid sequence <SEQ ID NO.\",\n \"10> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"9: PMFLLLGSLTLSDLLAGAAYAANILLSGPLTLKLSPALWFAREGGVFVALTASVLSLLGIALERSLTMARRGP APVSSRGRTLAMAAAAW The following DNA sequence beGPCR-seq11 <SEQ ID NO.\",\n \"11> was identified in H. sapiens: CTGCTCATTGTGGCCTTTGTGCTGGGCGCACTAGGCAATGGGGTCGCCCTGTGTGGTTTCTGCTTCCACAT GAAGACCTGGAAGCCCAGCACTGTTTACCTTTTCAATTTGGCCGTGGCTGATTTCCTCCTTATGATCTGCC TGCCTTTTCGGACAGACTATTACCTCAGACGTAGACACTGGGCTTTTGGGGACATTCCCTGCCGAGTGGGG CTCTTCACGTTGGCCATGAACAGGGCCGGGAGCATCGTGTTCCTTACGGTGGTGGCTGCGGACAGGTATTT CAAAGTGGTCCACCCCCACCACGCGGTGAACACTATCTCCACCCGGGTGGCGGCTGGCATCGTCTGCACCC TGTGGGCCCTGGTCATCCTGGGAACAGTGTATCTTTTGCTGGAGAACCATCTCTGCGTGCAAGAGACGGCC GTCTCCTGTGAGAGCTTCATCATGGAGTCGGCCAATGGCTGGCATGACATCATGTTCCAGCTGGAGTTCTT TATGCCCCTCGGCATCATCTTATTTTGCTCCTTCAAGATTGTTTGGAGCCTGAGGCGGAGGCAGCAGCTGG CCAGACAGGCTCGGATGAAGAAGGCGACCCGGTTCATCATGGTGGTGGCAATTGTGTTCATCACATGCTAC CTGCCCAGCGTGTCTGCTAGACTCTATTTCCTCTGGACGGTGCCCTCGAGTGCCTGCGATCCCTCTGTCCA TGGGGCCCTGCACATAACCCTCAGCTTCACCTACATGAACAGCATGCTGGATCCCCTGGTGTATTATTTTT CAAGCCCCTCCTTTCCCAAATTCTACAACAAGCTCAAAATCTGCAGTCTGAAACCCAAGCAGCCAGGACAC TCAAAAACACAAAGGCCGGAAGAGATGCCAATTTCG The following amino acid sequence <SEQ ID NO.\",\n \"12> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"11: LLIVAFVLGALGNGVALCGFCFHMKTWKPSTVYLFNLAVADFLLMICLPFRTDYYLRRRHWAFGDIPCRVGLF TLAMNRAGSIVFLTVVAADRYFKVVHPHHAVNTISTRVAAGIVCTLWALVILGTVYLLLENHLCVQETAVSCE SFIMESANGWHDIMFQLEFFMPLGIILFCSFKIVWSLRRRQQLARQARMKKATRFIMVVAIVFITCYLPSVSA RLYFLWTVPSSACDPSVHGALHITLSFTYMNSMLDPLVYYFSSPSFPKFYNKLKICSLKPKQPGHSKTQRPEE MPIS The following DNA sequence beGPCR-seq12 <SEQ ID NO.\",\n \"13> was identified in H. sapiens: TGGAGCTGTGCCACCACCTATCTGGTGAACCTGATGGTGGCCGACCTGCTTTATGTGCTATTGCCCTTCCT CATCATCACCTACTCACTAGATGACAGGTGGCCCTTCGGGGAGCTGCTCTGCAAGCTGGTGCACTTCCTGT TCTATATCAACCTTTACGGCAGCATCCTGCTGCTGACCTGCATCTCTGTGCACCAGTTCCTAGGTGTGTGC CACCCACTGTGTTCGCTGCCCTACCGGACCCGCAGGCATGCCTGGCTGGGCACCAGCACCACCTGGGCCCT GGTGGTCCTCCAGCTGCTGCCCACACTGGCCTTCTCCCACACGGACTACATCAATGGCCAGATGATCTGGT ATGACATGACCAGCCAAGAGAATTTTGATCGGCTTTTTGCCTACGGCATAGTTCTGACATTGTCTGGCTTT CTTTCCCTCCTTGGTCATTTTGGTGTGCTATTCACTGATGGTCAGGAGCCTGATCAAGCCAGAGGAGAACC TCATGAGGACAGG The following amino acid sequence <SEQ ID NO.\",\n \"14> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"13: WSCATTYLVNLMVADLLYVLLPFLIITYSLDDRWPFGELLCKLVHFLFYINLYGSILLLTCISVHQFLGVCHP LCSLPYRTRRHAWLGTSTTWALVVLQLLPTLAFSHTDYINGQMIWYDMTSQENFDRLFAYGIVLTLSGFLSLL GHFGVLFTDGQEPDQARGEPHEDR The following DNA sequence beGPCR-seq14 <SEQ ID NO.\",\n \"15> was identified in H. sapiens: CCACCACGCGCAGCACGCCGACAGGGCCTCTCCCTCCCATTCTCCCGCAGGCCCGGACGACCACGCTGCCT CCAGCCGGTCGGCAAACTAGGGCAGCTCGCAGCCCACGAACAGCAGCCCCAGCAGCTGGCTCATCTTCAGG CTCTGCACCTTGGCGCGGGGCATCGCGCTGGGCGCACGGGCTCCACCTGGGCTCGCCGACCAGGCCGCTGC ACCCGCTGGGGCCTTCAGCCGGTGCCGCCACCAGACGGAGAGTAGGTGGCCACAAGCGACACCCATGATCT TAACAGGCGCGACGAAGCCCGCGACGGCCTCATAGAACGCGTACACCTGCACGTGCCAGCGCTGCAGGAGC GCGAAGATCCAGTGGCAGCGACGCATCCCCGGCCAGGCTCGGGCGGAGAGTGGCGCGCCTGGCTGCAGAGA CGTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAGTACTAGCGCACCACAAACCCCGACCCCCGCGCCA GCAGCAGTGCCAGCAGCCAGCCCAGGGCGGCGAGGGCACGCGCGGGCAGCGGCCGGCCGTGCGGAAGACGC ACCGCGCGCCGGCGCTCGAGGGCGATGAGCACCACGAGGTGGGCCGAGGCGCCCCGCCCGGATGCCTGCAG CAGCTGCAGGAAGCGGCACGCCAGGTCCCCCGTGGCCGCGCGGGGCTCGCCCAGCAGTTCCCAGGCCAGCT GTGACAGCGCCGTGCCCCCGCACGCGTACAGGTCCGCCAGGGCCAGCTGCACCAGCAGGAAGTCCATCTTG CGACGCTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNACAGGCGGCACAGCACTGTGGTGTTGCCTGCCAC CGCCACCACCAGGATGACCCCCAGGAACACCAGGCGGACGCG The following amino acid sequence <SEQ ID NO.\",\n \"16> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"15: RVRLVFLGVILVVAVAGNTTVLCRLXXXXXXXXXXKRRKMDFLLVQLALADLYACGGTALSQLAWELLGEPRA ATGDLACRFLQLLQASGRGASAHLVVLIALERRRAVRLPHGRPLPARALAALGWLLALLLARGSGFVVRYXXX XXXXXXXXTSLQPGAPLSARAWPGMRRCHWIFALLQRWHVQVYAFYEAVAGFVAPVKIMGVACGHLLSVWWRH RLKAPAGAAAWSASPGGARAPSAMPRAKVQSLKMSQLLGLLFVGCELPFADRLEAAWSSGPAGEWEGEALSAC CAWW The following DNA sequence beGPCR-seq15 <SEQ ID NO.\",\n \"17> was identified in H. sapiens: TCTAAGTTTTTCTCTGAACTTTGAGCCTGTGAAAAAAGAAGGGATGCTGCCTCAGGCCACCCCAGCCTAGA TACTCACTCTGAGTGCCATGAGGTAGTAGAGGACACTGATGACAGTCATGGGGAGGAGGTAGAATAGGAAG GAGGTGACCTGGATGATGAAATTGTAGATCCACATGGGCTTGATGACCGTACAGGTGGCCGAACCTGGGAC CAGGGACCCATTGGGGAAGTAGTGGAACTTGATGCCATGGATGCTGGTGTTGGGCAGGGAGAAGAGCACGG AGAAGCCCCAGACGATGCCGAGGATCCTGAGGGCCCGGCGCCGGGTGCTCTGCAGTTTGGCGCGGAACGGG TGTAGGATGGCCACGTAGCGCTCCACGCTGACGGTGGTGATGCTGAGGATGGAGGCGAAGCACACGGTCTC AAAGAGGGCCGTCTTGAAGTAGCAGCCCACGGGCCCGAACAAGAAAGGGTAGTTGCGCCACATCTCATAGA CCTCCAGGGGCATTCCAAGGAGCAGGACCAGGAGGTCAGAGACCGCCAGGCTGAAGAGGTAGTAGTTGGTG GGCGTCTTCATAGCCTGGTGCTGCAGAATCACCAGGCACACCAGGACATTGCCAATGACCCCCACCACAAA AATTGGCACATACACCACAGACACGGGGAGGAAGAAGTGGCTGCGCCGAGGTCCGCAGAGGAAGGCCAGAT ACTCCTCGGTGCTGTTCAGGTGTTTCTGGAATGGATCTTCTAGTTTCTGCTGGTAGATCCAGGAAGCATTC TGAAGTTTTTCCATCCCTGA The following amino acid sequence <SEQ ID NO.\",\n \"18> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"17: SGMEKLQNASWIYQQKLEDPFQKHLNSTEEYLAFLCGPRRSHFFLPVSVVYVPIFVVGVIGNVLVCLVILQHQ AMKTPNTYYLFSLAVSDLLVLLLGMPLEVYEMWRNYPFLFGPVGCYFKTALFETVCFASILSITTVSVERYVA ILHPFRAKLQSTRRRALRILGIVWGFSVLFSLPNTSIHGIKFHYFPNGSLVPGSATCTVIKPMWIYNFIIQVT SFLFYLLPMTVISVLYYLMALRVSIAGVAG The following DNA sequence beGPCR-seq18 <SEQ ID NO.\",\n \"19> was identified in H. sapiens: ATCAAGATGATTTTTGCTATCGTGCAAATTATTGGATTTTCCAACTCCATCTGTAATCCCATTGTCTATGC ATTTATGAATGAAAACTTCAAAAAAAATGTTTTGTCTGCAGTTTGTTATTGCATAGTAAATAAAACCTTCT CTCCAGCACAAAGGCATGGAAATTCAGGAATTACAATGATGCGGAAGAAAGCAAAGTTTTCCCTCAGAGAG AATCCAGTG The following amino acid sequence <SEQ ID NO.\",\n \"20> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"19: IKMIFAIVQIIGFSNSICNPIVYAFMNENFKKNVLSAVCYCIVNKTFSPAQRHGNSGITMMRKKAKFSLRENP The following DNA sequence beGPCR-seq16 <SEQ ID NO.\",\n \"21> was identified in H. sapiens: GCCACAGCATGCAGTTTTCTGTAGAATTCCACTTTGTCTTTGCACTTGAAGAAGATGAGGTATCTGGTGAC CAGGATCACCACATAGAATAGGAACCGTGAGGTACATGTGGATGTGCAGCATGGCACTCACAAATTTGCAG AAGGGCAGCCCAAACATCCAAGTCTTCTTGATGAGGTAGGTCAAGCGAAATGGCACTGTCAGCAGAAAAAC GCTGTGGACCACCACCAAGTTAATGACCGCCATGGTGGTCACTGACCGGGTGTTCATTTTCACCAGGAGGA AAAGAATGGAAATGACACCCACCAGCCCGCCAATAAGCACTATGAAGTAGAGGCTGATTAAGTGGGGTGTC ACTATAGGATCGCAAGAGGAATTCCTGGAGGTATTGTGGCCAGGCATACTTGGGAAGTCACCTGGAGGAGA AAAAGCACCAGAGTAACTGAC The following amino acid sequence <SEQ ID NO.\",\n \"22> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"21: VSYSGAFSPPGDFPSMPGHNTSRNSSCDPIVTPHLISLYFIVLIGGLVGVISILFLLVKMNTRSVTTMAVINL VVVHSVFLLTVPFRLTYLIKKTWMFGLPFCKFVSAMLHIHMYLTVPILCGDPGHQIPHLLQVQRQSGILQKTA CCG The following DNA sequence beGPCR-seq17 <SEQ ID NO.\",\n \"23> was identified in H. sapiens: ACTGACCAAGGTCAGGGCATCGACTGAGGCTAGAAGGCCACAGGAAATGCCAGTCAAGGTGTTGGCGCCTG CAATCGCACCTACCACAAACTTGACCGGGGGCAGGGGGGCAGGCCCGCCAGCGAACACGGTCAGCAGCACC AGTCCATTGCAGAGCACGGAGAGCAACACGATGGCCCACACGGCCAGGCGGATGCCCCAGCTTTCAAAGAG GTACTCACA The following amino acid sequence <SEQ ID NO.\",\n \"24> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"23: CEYLFESWGIRLAVWAIVLLSVLCNGLVLLTVFAGGPAPLPPVKFVVGAIAGANTLTGISCGLLASVDALTLV S The following DNA sequence beGPCR-seq20 <SEQ ID NO.\",\n \"25> was identified in H. sapiens: AACCCCATCATCTACACGCTCACCAACCGCGACCTGCGCCACGCGCTCCTGCGCCTGGTCTGCTGCGGACG CCACTCCTGCGGCAGAGACCCGAGTGGCTCCCAGCAGTCGGCGAGCGCGGCTGAGGCTTCCGGGGGCCTGC GCCGCTGCCTGCCCCCGGGCCTTGATGGGAGCTTCAGCGGCTCGGAGCGCTCATCGCCCCAGCGCGACGGG CTGGACACCAGCGGCTCCACAGGCAGCCCCGGT The following amino acid sequence <SEQ ID NO.\",\n \"26> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"25: NPIIYTLTNRDLRHALLRLVCCGRHSCGRDPSGSQQSASAAEASGGLRRCLPPGLDGSFSGSERSSPQRDGLD TSGSTGSPG The following DNA sequence beGPCR-seq21 <SEQ ID NO.\",\n \"27> was identified in H. sapiens: CGTGAAGAACAGCGCCACCATGACCAGCATGTGCACCACGCGCGCTCTGCGCCGCGATGCTCGCGGGTCCG CAGCCTCCTNNNNNNNNNNNNNNNNNNNNNNNNNNTGGCAGAGCTTGCGCGCGATGCGGGCGTACATGACC ACGATGAGCGCCAGCGGCGCCAGGTAGATGTGCGAGAAGAGCACAGTGGTGTAGACCCTGCGCATGCCCTT CTCGGGCCAGGCCTCCCAGCAGGAGTAGAGAGGGTAGGAGCGGTTGCGGGCGTCCACCATGAAGTGGTGCT CCTCACGGGTGACGGTCAGCGTGACGGCCGAGGGACACATGATGAGCAGCGCCAGGGCCCAGATGACGGCG ATGGTGACGAGCGCCTTCCGCAGGGTCAGCTTCTCGCGGAAAGGGTGCACGATGCAGCGGAACCT The following amino acid sequence <SEQ ID NO.\",\n \"28> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"27: FRCIVHPFREKLTLRKALVTIAVIWALALLIMCPSAVTLTVTREEHHFMVDARNRSYPLYSCWEAWPEKGM RRVYTTVLFSHIYLAPLALIVVMYARIARKLCXXXXXXXXXXEAADPRASRRRARVVHMLVMVALFFT The following DNA sequence beGPCR-seq22 <SEQ ID NO.\",\n \"29> was identified in H. sapiens: GCAGGGGGCGTGAGTCCTCAGGCACTTCTTGAGGTCCTTGTTGAGCAGGAAGCAGACAATTGGGTTGACGG CAGCCTGGGCGAAGCTCATCCAAACAGCATGGCCAGGTAGCGGTGGGGCACAGCACAGGCTTTCACAAACA CTCGCCAGTAGCAGGCCACGATGTAGGGTGACCAGAGGAGCAGAAAGAGCAGTGTGATCGCGTAGAACATG CGGCCCAGCTGCTTTTCACCCTTGACCTCGTCCATGCCCAGTAGCCGCCGGCTGGCTGCATGCCCATTCTG CCGGATACCCAGCAGGGTTGGTGGCATGGGCCC The following amino acid sequence <SEQ ID NO.\",\n \"30> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"29: GPMPPTLLGIRQNGHASRRLLGMDEVKGEKQLGRMFYAITLLFLLLWSPYIVACYWRVFVKACAVPHRYLAT AVWMSFAQAAVNPIVCFLLNKDLKKCLRTHAPC The following DNA sequence beGPCR-seq24 <SEQ ID NO.\",\n \"31> was identified in H. sapiens: TATTCTGTAATGAAGAATGTCATTCACACTGCCATTGGCACATCCAGTGGCCTCACCTAGCATTGTGAAAG CCCTTCGGTTGGTGTATTGCCACTTCATTTTAAAAGGATGCACAAGTCCCTGGTGCCTTTCCACAGCAATG CAGGTCATAGTGAGGATTTCTGTCACAACAGCGGTAGACTGGACAAATGGCACCATCTTGCAAATGAAAGC ACCTGCAGTAAGGAAATAGGATAAATCATACATCAAAACAAAAAGAATAAAGGTTTCATCTGTGTCTTTGT AATTATCACTATCAGTCCATTCTGAGCCTCTGCCAAAAAGTTTGATAATTGTAATTACTCTGTAGACACA The following amino acid sequence <SEQ ID NO.\",\n \"32> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"31: VYRVITIIKLFGRGSEWTDSDNYKDTDETFILFVLMYDLSYFLTAGAFICKMVPFVQSTAVVTEILTMTCIAV ERHQGLVHPFKMKWQYTNRRAFTMLGEATGCANGSVNDILHYRI The following DNA sequence beGPCR-seq27 <SEQ ID NO.\",\n \"33> was identified in H. sapiens: GAGCAACATGATCTTTTTGAAGTACTTGACGGTGTCGTTCTTGACGGTCACGAAGCACAGAGTGTTGATCA TGCTGTTGCTCATGGCGATGCACTCGACGATGTAGAAGGCAGTGAGGTAGTGCTTCTCCTTCACAAACACG GTGGGGAAGAAGTCGCGCACGATGGTGAAGCCGTAGAAGGGCGCCCAGCATAGCACGTAGGCGGTGAGGAT GCACATGAGCACCAGGACCGTCTTCCTGCGGCAGCGCAGCCTCTTGCGGATCTGCTCTGTCTGGAATCCAG GGACCGCCTTGAACCAGAGCTCCCGGGAGATCCTGGCATAGCACAGGGTCATGGTGACCACGGGGCCCACG AATTCTATGCCAAAGATAAAGAGGAAGTAGGACTTGTAGTAGAGCTGCTGGTCCACAGGCCAGATCTGGCC GCAGAAGATCTTTTCCTGGCTCTTGACAATGACGAGGACCGTCTCGGTGGTGAAGTAGGCGGAAGGGATGG CGATCAGGATGGACACCGTCCACACCAAGGCAATCAGGCCAGTGGCTGTTTGGCACTTCATTCGTGGTCTC AGCGGATGGACAATAGCCAGATACCTAGGGCAAGAACACAAGTGGAGGCAGCC The following amino acid sequence <SEQ ID NO.\",\n \"34> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"33: GCLHLCSCPRYLAIVHPLRPRMKCQTATGLIALVWTVSILIAIPSAYFTTETVLVIVKSQEKIFCGQIWPVDQ QLYYKSYFLFIFGIEFVGPVVTMTLCYARISRELWFKAVPGFQTEQIRKRLRCRRKTVLVLMCILTAYVLCWA PFYGFTIVRDFFPTVFVKEKHYLTAFYIVECIAMSNSMINTLCFVTVKNDTVKYFKKIMLL The following DNA sequence beGPCR-seq28 <SEQ ID NO.\",\n \"35> was identified in H. sapiens: CAGCCACACTGCAGTGATGAAATCAAATGTCCAACACCAACCATAGTCACCATTACTAACTAAGAAGCCAC AAAACTTCCCTTCCAGGGTGTTCAGCAGCAGGGACAGGGCCCAGGGCAGGGCACACATGACAGTTGACAGG TTTCTTGGGCAGCAGCAGCAGTACCAGATAGGCCGCAGGACAGACAGGCAGCACTCAGTACTGATGGCACT CAGCATGCTCAGGCCTACAAGGTAGGCAAAGGTCATCACGCTGGTGAAGAAGCTAGGGAAATTGATGGAGA TGGAACAGAAGAAGTTACTGAGGTACACCAGGCAATTTATAATCTGGAAGCAGAGGAAGAGGAAGTCGGCC CCGGCCAGGCTGAGGACGTAGACAGAGAAGGCGTTCCTGCGCATGCGGAAGCCCAGGAGCCAGAGCACAAA CCCGTTTCCTACCAGCCCGACCAGGGCAATGAAAAGGATCAGGAAGACCGGGATCAG The following amino acid sequence <SEQ ID NO.\",\n \"36> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"35: LIPVFLILFIALVGLVGNGFVLWLLGFRMRRNAFSVYVLSLAGADFLFLCFQIINCLVYLSNFFCSISINFPS FFTSVMTFAYLVGLSMLSAISTECCLSVLRPIWYCCCCPRNLSTVMCALPWALSLLLNTLEGKFCGFLVSNGD YGWCWTFDFITAVWL The following DNA sequence beGPCR-seq31 <SEQ ID NO.\",\n \"37> was identified in H. sapiens: GAGAGTCTGATTCTGACTTACATCACATATGTAGGCCTGGGCATTTCTATTTGCAGCCTGATCCTTTGCTTGT CCGTTGAGGTCCTAGTCTGGAGCCAAGTGACAAAGACAGAGATCACCTATTTACGCCATGTGTGCATTGTTAA CATTGCAGCCACTTTGCTGATGGCAGATGTGTGGTTCATTGTGGCTTCCTTTCTTAGTGGCCCAATAACACAC CACAAGGGATGTGTGGCAGCCACATTTTTTGGTCATTTCTTTTACCTTTCTGTATTTTTCTGGATGCTTGCCA AGGCACTCCTTATCCTCTATGGAATCATGATTGTTTTC The following amino acid sequence <SEQ ID NO.\",\n \"38> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"37: ESLILTYITYVGLGISICSLILCLSVEVLVWSQVTKTEITYLRHVCIVNIAATLLMADVWFIVASFLSGPITH HKGCVAATFFGHFFYLSVFFWMLAKALLILYGIMIVF The following DNA sequence beGPCR-seq32 <SEQ ID NO.\",\n \"39> was identified in H. sapiens: TTGTGTGGCAGTAGAGAGATGTCAGGCTTCAGAGTCAACAAGAACTGGATTTCAAACTGGATTTGAGGACCCC CACCTTTGGTAAGTGACTTATTATCTGCGAGCCTCTGTTTCTCTCTTCTTTAAATGAGGACAGTAAATCCCAT ACGGCAGGGTGGTGGGGAGAATCAGAGATGATACAGCTGGTGATCACATCTGGTTTGTGTTCCCAGGGGCACC AGACTAGGGTTTCTGAGCATGGATCCAACCGTCCCAGTCTTCGGTACAAAACTGACACCAATCAACGGACGTG AGGAGACTCCTTGCTACAATCAGACCCTGAGCTTCACGGTGCTGACGTGCATCATTTCCCTTGTCGGACTGAC AGGAAACGCGGTAGTGCTCTGGCTCCTGGGCTACCGCATGCGCAGGAACGCTGTCTCCATCTACATCCTCAAC CTGGCCGCAGCAGACTTCCTCTTCCTCAGCTTCCAGATTATACGTTCGCCATTACGCCTCATCAATATCAGCC ATCTCATCCGCAAAATCCTCGTTTCTGTGATGACCTTTCCCTACTTTACAGGCCTGAGTATGCTGAGCGCCAT CAGCACCGAGCGCTGCCTGTCTGTTCTGTGGCCCATCTGGTACC The following amino acid sequence <SEQ ID NO.\",\n \"40> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"39: LCGSREMSGFRVNKNWISNWIGPPPLVSDLLSASLCFSLLMRTVNPIRQGGGENQRYSWSHLVCVPRGTRLGF LSMDPTVPVFGTKLTPINGREETPCYNQTLSFTVLTCIISLVGLTGNAVVLWLLGYRMRRNAVSIYILNLAAA DFLFLSFQIIRSPLRLINISHLIRKILVSVMTFPYFTGLSMLSAISTERCLSVLWPIWY The following DNA sequence beGPCR-seq33 <SEQ ID NO.\",\n \"41> was identified in H. sapiens: ACAGAAAGCAAGGCCACCAGGACCTTAGGCATAGTCATGGGAGTGTTTGTGTTGTGCTGGCTGCCCTTCTTTG TCTTGACGATCACAGATCCTTTCATTAATTTTACAACCCTTGAAGATCTGTACAATGTCTTCCTCTGGCTAGG CTATTTCAACTCTGCTTTCAATCCCATTTTATATGGCATGCTTTATCCTTGGTTTCGCAAGGCATTGAGGATG ATTGTCACAGGCATGATCTTCCACCCTGACTCTTCCACCCTAAGCCTGTTTTCTGCCCATGCTTAGGCTGTGT TCATCATTCAATAGGACTCTTTTCTGG The following amino acid sequence <SEQ ID NO.\",\n \"42> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"41: TESKATRTLGIVMGVFVLCWLPFFVLTITDPFINFTTLEDLYNVFLWLGYFNSAFNPILYGMLYPWFRKALRM IVTGMIFHPDSSTLSLFSAHAAVFIIQDSF The following DNA sequence beGPCR-seq34 <SEQ ID NO.\",\n \"43> was identified in H. sapiens: TAGGAATCTCAGAGAAGAAAGTAAGGAACCAGAAAACCATAAAAGAATGTAAATGGAAAAGAATCAGCAAATC TTATTCACTTATCACTAAATCTAAAATATGTCAAAATACATGAAGACAACAAATGCTTTAGAACAACTGTTGA ATGTATTGTCCTACAACTTGGCATATGATCATGCTTGCCTCTCTATGTCCAAGTGTTTATTTTTGCAGTTGAC CTTAATTTCAAGTTAGTTTTGAGGTCTCTACAGTAATGTTTTTAATCTGTCTCTACTTCTTCAGAAAATAAAT TAGTTGTTGACGAATCAGTCCTTAAGACCTTGCCGCTTACAATAAGTTTTATTGCCTTCCCAAACCATTGGTA AAAGAAAGCATAAATCAAGGGGTTCATAGCTGAATTATAATAAACACACCAAACTAAAATCTCATAAACATAA GGAGGAGTTATAAAATTCATATAAGCATCAATCACTGCATCAACGAGGTATGGTAGCCAAGAGACAAGAAATG CTGC The following amino acid sequence <SEQ ID NO.\",\n \"44> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"43: LHQRGMVAKRQEMLAAFLVSWLPYLVDAVIDAYMNFITPPYVYEILVWCVYYNSAMNPLIYAFFYQWFGKAIK LIVSGKVLRTDSSTTNLFSEEVETDKHYCRDLKTNLKLRSTAKINTWTRGKHDHMPSCRTIHSTVVLKHLLSS CI The following DNA sequence beGPCR-seq35 <SEQ ID NO.\",\n \"45> was identified in H. sapiens: CTGGAAAGAGGTCCTCGATCTATCCTCTACGCCGTCCTTGGTTTTGGGGCTGTGCTGGCAGCGTTTGGAAACT TACTGGTCATGATTGCTATCCTTCACTTCTAACAACTGCACACACCTACAAACTTTCTGATTGCGTCGCTGGC CTGTGCTGACTTCTTGGTGGGAGTCACTGTGATGCCCTTCAGCACAGTGAGGTCTGTGGAGAGCTGTTGGTAC TTTGGGGACAGTTACTGTAAATTCCATACATGTTTTGACACATCTTTCTGTTTTGCTTCTTTATTTCATTTAT GCTGTATCTCTGTTGATAGATACATTGCTGTTACTGATCCTCTGACCTATCCAACCAAGTTTACTGTGTCAGT TTCAGGGATATGCATTGTTCTTTCCTGGTTCTTTTCTGTCACATACAGCTTTTCGATCTTTTACACGGGAGCC AACGAAGAAGGAATTGAGGAATTAGTAGTTGCTCTAACCTGTGTAGGAGGCTGCCAGGCTCCACTGAATCAAA ACTGGGTCCTACTTTGTTTTCTTCTATTCTTTATACCCAATGTCGCCATGGTGTTTATATACAGTAAGATATT TTTGGTGGCCAAGCATCAGGCTAGGAAGATAGAAAGTACAGCCAGCCAAGCTCAGTCCTTCTCAGAGAGTTAC AAGGAAAGAGTAGCAAAAAGAGAGAGAAAGGCTGCCAAAACCTTGGGAATTGCTATGGCAGCATTTCTT The following amino acid sequence <SEQ ID NO.\",\n \"46> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"45: LERGPRSILYAVLGFGAVLAAFGNLLVMIAILHFQLHTPTNFLIASLACADFLVGVTVMPFSTVRSVESCWYF GDSYCKFHTCFDTSFCFASLFHLCCISVDRYIAVTDPLTYPTKFTVSVSGICIVLSWFFSVTYSFSIFYTGAN EEGIEELVVALTCVGGCQAPLNQNWVLLCFLLFFIPNVAMVFIYSKIFLVAKHQARKIESTASQAQSFSESYK ERVAKRERKAAKTLGIAMAAFL The following DNA sequence beGPCR-seq36 <SEQ ID NO.\",\n \"47> was identified in H. sapiens: AACCAGGTGGCCTTACTCCTAAGACCCCTGGCCTTGTCTATGGCCTTTATCAACAGCTGTCTCAATCCAGTTC TCTATGTCTTCATTGGGCATGACTTCTGGGAGCACTTGCTCCACTCCCTGCTAGCTGCCTTAGAACGGGCACT TAGCGAGGAGCCAGATAGTGCCTGAATCCCAGCTCCCAGGCAGATGAGTCCTTTATAACATGACCCAATTTCC TACTCCATTTTCCCACCACTCAATCCTCTTCCCAAACAGCTCTACCATAATCCAACATCCAACAGAATTTAAG AGAATAAACCACAACTTTTAAGTGAGCTCTATGTGCTAGGTCATGTTTTAGAATACAACCTTAAGTGCCTGGA AGATGGAGGCAAGAAACAAACAAGGTCTCATTCTTTAGAGGAAGACAGTTCACCAAGACTCAAACAGAAAAAA AGATAGTTATCTTGTGACAAAACAAGTCATAAAATTGGGTCAGGACCTGCAGCAATGACTTTATGCTAGAATC CAGAGCACTAGCAGGAAACTGCTTAAATTTTACTTAATCAAAGTCAAGTTTGGACATACATGTCAGGTAAAAC CTAGCAGAGATGAGCTACCTTGATTTTAAAACTTCAAGGGATAGCTCAATGTCATCAAGATCCTTTTGATGAC TTG The following amino acid sequence <SEQ ID NO.\",\n \"48> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"47: NQVALLLRPLALSMAFINSCLNPVLYVFIGHDFWEHLLHSLLAALERALSEEPDSAIPAPRQMSPLHDPISYS IFPPLNPLPKQLYHNPTSNRIENKPQLLSELYVLGHVLEYNLKCLEDGGKKQTRSHSLEEDSSPRLKQKKRLS CDKTSHKIGSGPAAMTLCNPEHQETAILLNQSQVWTYMSGKTQRATLILKLQGIAQCHQDPFDDL The following DNA sequence beGPCR-seq37 <SEQ ID NO.\",\n \"49> was identified in H. sapiens: GCTTGTTCACGGCCACCATCCTCAAGCTGTTGCGCACGGAGGAGGCGCACGGCCGGGAGCAGCGGAGGCGCGC GGTGGGCCTGGCCGCGGTGGTCTTGCTGGCCTTTGTCACCTGCTTCGCCCCCAACAACTTCGTGCTCCTGGCG CACATCGTGAGCCGCCTGTTCTACGGCAAGAGCTACTACCACGTGTACAAGCTCACGCTGTGTCTCAGCTGCC TCAACAACTGTCTGGACCCGTTTGTTTATTACTTTGCGTCCCGGGAATTCCAGCTGCGCCTGCGGGAATATTT GGGCTGCCGCCGGGTGCCCAGAGACACCCTGGACACGCGCCGCGAGAGCCTCTTCTCCGCCAGGACCACGTCC GTGCGCTCCGAGGCCGGTGCGCACCCTGAAGGGATGGAGGGAGCCACCAGGCCCGGCCTCCAGAGGCAGGAGA GTGTGTTCTGAGTCCCGGGGGCGCAGC The following amino acid sequence <SEQ ID NO.\",\n \"50> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"49: LFTATILKLLRTEEAHGREQRRRAVGLAAVVLLAFVTCFAPNNFVLLAHIVSRLFYGKSYYHVYKLTLCLSCL NNCLDPFVYYFASREFQLRLREYLGCRRVPRDTLDTRRESLFSARTTSVRSEAGAHPEGMEGATRPGLQRQES VFVPGAQAAPPGLR The following DNA sequence beGPCR-seq38 <SEQ ID NO.\",\n \"51> was identified in H. sapiens: TTACTTATTCTGCCCTTTATCCAACTTTTAATTCCCTTTGCTATTCTCCTGCCTCATTTTCTGGCCTCATTTT CCCTATTATCCTGCCTCACATTGATCAAGGGATGAGGCTGGCAGGATCCGGAACCCACAGGGCCCCGTGGGCC ATGAGAGGCTCCTGGACTTGAACCTCAGGACACTCCCACTCTGGCTGCCGGCAGGGATGGAAGCTGGATGAGC AGGCAGGAGCTGGCAGTGGGGGTGGAGAGCCATAGGCTATTGGGGTGGACAGGCTTGGGTGCCTCATGGGAGC TCCCCATGGGAGCTGTGGCCCCTTGGGGCCTCTTATTTCTCACCCCAGGCTTTCCCGGGAGAGGTTCAAGTCA GAAGATGCCCCAAAGATCCACGTGGCCCTGGGTGGCAGCCTGTTCCTCCTGAATCTGGCCTTCTTGGTCAATG TGGGGAGTGGCTCAAAGGGGTCTGATGCTGCCTGCTGGGCCCGGGGGGCTGTCTTCCACTACTTCCTGCTCTG TGCCTTCACCTGGATGGGCCTTGAAGCCTTCCACCTCTACCTGCTCGCTGTCAGGGTCTTCAACACCTACTTC GGGCACTACTTCCTGAAGC The following amino acid sequence <SEQ ID NO.\",\n \"52> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"51: ETYSALYPTFNSLCYSPASFSGLIFPIILPHIDQGMRLAGSGTHRAPWAMRGSWTTSGHSHSGCRQGWKLDEQ AGAGSGGGEPAIGVDRLGCLMGAPHGSCGPLGPLISHPRLSRERFKSEDAPKIHVALGGSLFLLNLAFLVNVG SGSKGSDAACWARGAVFHYFLLCAFTWMGLEAFHLYLLAVRVFNTYFGHYFL The following DNA sequence beGPCR-seq40 <SEQ ID NO.\",\n \"53> was identified in H. sapiens: AATTGGTCGGAGAGTGCAGCTGCTTGAAATGGAGGATTGAAATCATCACCAGGAGGTTTCCAAACACAGCCAG CACAGCCCCAAAGCCAAACACTATGTACAGAATCACCCGGGATCCCGGCGAGAAGGGGATTTTCACACAGGAC CCATTCACGTTCGCGTAGCACAGCTGCACAGCCACCAGCAGGGATGAATTGCTGCTCATAACGCTGGTATTTA CATATGGAGAAATTTTGTCCTTGTTGATTATCACAAAAAATACAGGATTGTTCCTGATTTTCATTGCTCCTGC GGAAAAAAACACATATTCACCAGGATGCCAGAGGAAATGATCA The following amino acid sequence <SEQ ID NO.\",\n \"54> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"53: DHFLWHPGEYVFFSAGAMKIRNNPVFFVIINKDKISPYVNTSVMSSNSSLLVAVQLCYANVNGSCVKIPFSPG SRVILYIVFGFGAVLAVFGNLLVMISILHFKQLHSPTN The following DNA sequence beGPCR-seq41 <SEQ ID NO.\",\n \"55> was identified in H. sapiens: CACATCTTAACAAGACTGAAAAACATTGATTTGTTTTTAATTTGAAGAGCAATTTATTTGCTATTCATTCATA GTCTTACTTGATTTTTAAAAACTCATTTCGCTTGGTAATTTTAAAGGTATCCTGAACTTCGTCTATCCAACTG CTTATATATGTTCAGAAAACAAATTCATGGTTGCTGAACTGTTCTTTAAAACCTGACCAGTTACAATAACTTT TATTGCTTTCCTAAACCATGGGTAAAATAAAGCATAAATCAAAGGATTCATGGCTGAGTTATAATAAGCACAC CAACAGCATCATAAATACAGGCAGGGGTTATAAAGCCCATAAAGGCATCAATTAATGAATCAATGCTATATGG TAACCATGAAATCATAAATGCTACCACTGTGACCCCCAGGGTTTTAGCTGCTTTTCTCTCTCTCCTGGCCACT CTGGCTTTGTAACTCTCTGAGGATGATTCTGTCTTGCTACCAGTATTTTCTATCTTTTTCGCCTGTCGTCTAG CCACAAGAAATATGTTACCATACAGAATTATCATAATAAAGGTAGGTATAAAGAAGGATAGAAAATCTGTCAA CA The following amino acid sequence <SEQ ID NO.\",\n \"56> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"55: LTDFLSFFIPTFIMIILYGNIFLVARRQAKKIENTGSKTESSSESYKARVARRERKAAKTLGVTVVAFMISWL PYSIDSLIDAFMGFITPACIYEICCWCAYYNSAMNPLIYALFYPWFRKAIKVIVTGQVLKNSSATMNLFSEHI AVGTKFRIPLKLPSEMSFKSSKTMNEQINCSSNKQINVFQSCDV The following DNA sequence nGPCR-seq53 <SEQ ID NO.\",\n \"57> was identified in H. sapiens: TTTGTGGCAAGGAGACCCTGATCCCGGTCTTCCTGATCCTTTTCATTGCCCTGGTCGGGCTGGTAGGAAACGG GTTTGTGCTCTGGCTCCTGGGCTTCCGCATGCGCAGGAACGCCTTCTCTGTCTACGTCCTCAGCCTGGCCGGG GCCGACTTCCTCTTCCTCTGCTTCCAGATTATAAATTGCCTGGTGTACCTCAGTAACTTCTTCTGTTCCATCT CCATCAATTTCCCTAGCTTCTTCACCACTGTGATGACCTGTGCCTACCTTGCAGGCCTGAGCATGCTGAGCAC CGTCAGCACCGAGCGCTGCCTGTCCGTCCTGTGGCCCATCTGGTATCGCTGCCGCCGCCCCAGACACCTGTCA GCGGTCGTGTGTGTCCTGCTCTGGGCCCTGTCCCTACTGCTGAGCATCTTGGAAGGGAAGTTCTGTGGCTTCT TATTTAGTGATGGTGACTCTGGTTGGTGTCAGACATTTGATTTCATCACTGCAGCGTGGCTGATTTTTTTATT CATGGTTCTCTGTGGGTCCAGTCTGGCCCTGCTGGTCAGGATCCTCTGTGGCTCCAGGGGTCTGCCACTGACC AGGCTGTACCTGACCATCCTGCTCACAGTGCTGGTGTCCCTCCTCTGCGGCCTGCCCTTTGGCATTCAGTGGT TCCTAATATTATGGATCTGGAAGGATTCTGATGTCTTATTTTGTCATATTCATCCAGTTTCAGTTGTCCTGTC ATCTCTTAACAGCAGTGCCAACCCCATCATTTACTTCTTCGTGGGCTCTTTTAGGAAGCAGTGGCGGSTGCAG CACCCGATCCTCAAGCTGGCTCTCCAGAGGGCTCTGCAGGACATTGCTGAGGTGGATCACAGTGAAGGATGCT TCCGTCAGGGCACCCGGAGATTCAAAGAAGCATTCTGGTGTAGGGATGGACCCCTCTACTTCCATCATATATA TGTGGCTTTGAGAGGCAACTTTGCCCC The following amino acid sequence <SEQ ID NO.\",\n \"58> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"57: CGKETLIPVFLILFIALVGLVGNGFVLWLLGFRMRRNAFSVYVLSLAGADFLFLCFQIINCLVYLSNFFCSIS INFPSFFTTVMTCAYLAGLSMLSTVSTERCLSVLWPIWYRCRRPRHLSAVVCVLLWALSLLLSILEGKFCGFL FSDGDSGWCQTFDFITAAWLIFLFMVLCGSSLALLVRILCGSRGLPLTRLYLTILLTVLVSLLCGLPFGIQWF LILWIWKDSDVLFCHIHPVSVVLSSLNSSANPIIYFFVGSFRKQWRXQHPILKLALQRALQDIAEVDHSEGCF RQGTRRFKEAFWCRDGPLYFHHIYVALRGNFA The following DNA sequence nGPCR-seq54 <SEQ ID NO.\",\n \"59> was identified in H. sapiens: CTTTGCATCTCACTGTTGAGCAGACAGCCTGCTGAAAGTTGTCGCTGACCACCACATATAGTAACAGGTTACC AAAGGTGTTCAGAGCAGCATAATGGTCTAGAAACGATGTAAGCTTCATGGATCTGATTCTCAATGGAACAACT GATTGAAAGCAGGCTGAGATTCGATCCTGAATGACCCTCAAGATATGGAAGGGTAAAAAACATACGTAAAATG CAAGGAGTAGCAGAATGGTTAGCCTTCGTGCTTTCTGCTTAAGGCAGCTGTCAGTTTGCAGTCCATGGGTCAA AGTGTGGATAATCGTGGTATAGCAAAGTGTCACTATCACCAAGGGGAGGCAGAAAGTACTTGCAGTCAAAATC AGGTTGTACCACTTAATAGTATTGAGTTCATCCGAACTGGTGAGGTCGAGACAGGCTGATCTGTTGGTCCTGT TGGTTGATGTGATCAAGAAGGTCATCGGAATGACAGCTACCAGTGAAATGATCCACACCACAGCACAGGCTAC AACTGCACATCGAGTTTTGTGAATGGAAAAGCAGCTCATTGGGTGAATGATCACACAGTAGCGGAAG The following amino acid sequence <SEQ ID NO.\",\n \"60> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"59: FRYCVIIHPMSCFSIHKTRCAVVACAVVWIISLVAVIPMTFLITSTNRTNRSACLDLTSSDELNTIKWYNLIL TASTFCLPLVIVTLCYTTIIHTLTHGLQTDSCLKQKARRLTILLLLAFYVCFLPFHILRVIQDRISACFQSVV PLRIRSMKLTSFLDHYAALNTFGNLLLYVVVSDNFQQAVCSTVRCK The following DNA sequence nGPCR-seq55 <SEQ ID NO.\",\n \"61> was identified in H. sapiens, where the underlined ATG identifies a probable start codon: GGGAGGGCTCGTAGACACACTAACCCTACCCTTTCTGTTTCTTCCTCATCTTTCCTTTCCATCTGTTTCTCAT GGTCTCCTGTCTGTCTCTCTCTCTCTCCCCTCTTTCTCTCTCCTCGCTCTTTCTCATCCCCTCCATTTCTGTG TCAATCTCAATCCATTTATATCGGTGGCCACTTTTCTATCTCTTTGTTCTATCTCTCTCTCTCTCTCTTTCCC ACTTTGTCTCTGCACGCCTGTTGTGTTTTTCTGCCTGTCTCTCTCTTGCCCTCATCTCTCTGTCTCTCTCTTG CCCTCATCTCTCTGTCTCTCTGTGTCTGTGTCTCCCCCGCTCATTCCCATTTGCAGGTGCAATGTAGCAGGAC AACTCATGGAGCCCCCCCGGGCCCATCGAGTACCGGACTGGCTGACCCCCTAGGGTTGGCAGTAGCCCCTGAC CCTCAGTATGGCCAACACTACCGGAGAGCCTGAGGAGGTGAGCGGCGCTCTGTCCCCACCGTCCGCATCAGCT TATGTGAAGCTGGTACTGCTGGGACTGATTATGTGCGTGAGCCTGGCGGGTAACGCCATCTTGTCCCTGCTGG TGCTCAAGGAGCGGGCCCTGCACAAGGCTCCTTACTACTTCCTGCTGGACCTGTGCCTGGCCGATGGCATACG CTCTGCCGTCTGCTTCCCCTTTGTGCTGGCTTCTGTGCGCCACGGCTCTTCATGGACCTTCAGTGCACTCAGC TGCAAGATTGTGGCCTTTATGGCCGTGCTCTTTTGCTTCCATGCGGCCTTCATGCTGTTCTGCATCAGCGTCA CCCGCTACATGGCCATCGCCCACCACCGCTTCTACGCCAAGCGCATGACACTCTGGACATGCGCGGCTG The following amino acid sequence <SEQ ID NO.\",\n \"62> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"61: MANTTGEPEEVSGALSPPSASAYVKLVLLGLIMCVSLAGNAILSLLVLKERALHKAPYYFLLDLCLADGIRSA VCFPFVLASVRHGSSWTFSALSCKIVAFMAVLFCFHAAFMLFCISVTRYMAIAHHRFYAKRMTLWTCAAE The following DNA sequence nGPCR-seq56 <SEQ ID NO.\",\n \"63> was identified in H. sapiens: AAAAATTGCTGTACTGAACTATTGAATGGAACTTGGAAATAAAGTCCCTTCCAAAATAACTATTCTTCAACAG AGAGTAATAGGTAAATGTTTTAGAAGTGAGAGGACTCAAATTGCCAATGATTTACTCTTTTATTTTTCCTCCT AGGTTTCTGGGATAAGTATGTGCAAATAAAAAATAAACATGAGAAGGAACTGTAACCTGATTATGGATTTGGG AAAAAGATAAATCAACACACAAAGGGAAAAGTAAACTGATTGACAGCCCTCAGGAATGATGCCCTTTTGCCAC AATATAATTAATATTTCCTGTGTGAAAAACAACTGGTCAAATGATGTCCGTGCTTCCCTGTACAGTTTAATGG TGCTCATAATTCTGACCACACTCGTTGGCAATCTGATAGTTATTGTTTCTATATCACACTTCAAACAACTTCA TACCCCAACAAATTGGCTCATTCATTCCATGGCCACTGTGGACTTTCTTCTGGGGTGTCTGGTCATGCCTTAC AGTATGGTGAGATCTGCTGAGCACTGTTGGTATTTTGGAGAAGTCTTCTGTAAAATTCACACAAGCACCGACA TTATGCTGAGCTCAGCCTCCATTTTCCATTTGTCTTTCATCTCCATTGACCGCTACTATGCTGTGTGTGATCC ACTGAGATATAAAGCCAAGATGAATATCTTGGTTATTTGTGTGATGATCTTCATTAGTTGGAGTGTCCCTGCT GTTTTTGCATTTGGAATGATCTTTCTGGAGCTAAACTTCAAAGGCGCTGAAGAGATATATTACAAACATGTTC ACTGCAGAGGAGGTTGCTCTGTCTTCTTTAGCAAAATATCTGGGGTACTGACCTTTATGACTTCTTTTTATAT ACCTGGATCTATTATGTTATGTGTCTATTACAGAATATATCTTATCGCTAAAGAACAGGCAAGATTAATTAGT GATGCCAATCAGA The following amino acid sequence <SEQ ID NO.\",\n \"64> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"63: REKTDQPSGMMPFCRNIINISCVKNNWSNDVRASLYSLMVLIILTTLVGNLIVIVSISHFKQLHTPTNWLIHS MATVDFLLGCLVMPYSMVRSAEHCWYFGEVFCKIHTSTDIMLSSASIFHLSFISIDRYYAVCDPLRYKAKMNI LVICVMIFISWSVPAVFAFGMIFLELNFKGAEEIYYKHVHCRGGCSVFFSKISGVLTFMTSFYIPGSIMLCVY YRIYLIAKEQARLISDANQ The following DNA sequence nGPCR-seq57 <SEQ ID NO.\",\n \"65> was identified in H. sapiens: AACAGTCCCGGGTGGAACCTGGGCATGTATATTTTGATTGTTTTATGCATACTCCTAGTGAAGAACCAATGTC TTGCTCAGATAGAAGCAAGATACTCAGACTTAGTTTCTCTGTAGCTCCTGCTTTTTATTATTCCTGGTTGGAT TGCACCACTACTCAGTTTCTATTTTATAATACTGATTATAAAACATGGGAGGGAAATAACTTTGTATTGGTTT TTATGGATAATTTATTATGTGTCCTAGACTCTGGCCTTGTCAAAAGAAGGACGTAAGAAGGCACGATGTATTA TACTTGGGAATGATAGAAGAGACTGACCTGGTATTTCCACCCGGAAGAGGGAAAGGATTTTAACTACAAATAC AGGAATCCAGCAGATGGCATCAGAGAACACTATAAAAAAGAAACGATTTGCAACAGCCACCTCTCTTCCAAAA CAATTCCTTACTTCTGTGGTCTGCAAGGCGGTTTTTTGAATGGAACAGAACATAGTAATATAGGAAAACACAA TGATGAGAAAAGCCAGCAAGTTCACACCTGTTGGGGAAAAGCACACTTTTAACATCTCAGGCGTAAAAGTCAA CAGTAAAATTACTGTGGTACAGGTTGAGTATCCCTTACCCAAAATGTTTGAAACCAGAAATGTTTTGGATTTC GGATTTCGGAATATTTACACATTCATAATGATATATCTTGGAAATGGTTCCCAAGTCTAAACACAAAATTTAT TTATGTTTCATATACACCTTATACACATAGTCTGAAAGTAATTTTGTACAATATTTTAAATAATTTTGGGCAT GAAACAAAGTTTGCATACATTGAACCATCAGACAGCAAAAGCTTCAGGTGTGGAATTTTCCACTTGTGGCATC ATGTTGATGCTCAAAAAGTTCCATATTTTAGAGCATTTCAAATTTTGGATTTTCAAATTACAAATGCTTAACC TGTACTTAGATGTTAAATACAGTGCCTCTTCCACGGGCACTTTCAGGAAGCATTCTTTTATATAAGCCC The following amino acid sequence <SEQ ID NO.\",\n \"66> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"65: YIKECFLKVPVEEALYLTSKYRLSICNLKIQNLKCSKIWNFLSINMMPQVENSTPEAFAVWFNVCKLCFMPKI INIVQNYFQTMCIRCININKFCVTWEPFPRYIIMNVIFRNPKSKTFLVSNILGKGYSTCTTVILLLTFTPEML KVCFSPTGVNLLAFLIIVFSYITMFCSIQKTALQTTEVRNCFGREVAVANRFFFIVFSDAICWIPVFVVKILS LFRVEIPGQSLLSFPSIIHRAFLRPSFDKARVDTIIHKNQYKVISLPCFIISIIKKLSSGAIQPGIIKSRSYR ETKSEYLASIARHWFFTRSMHKTIKIYMPRFHPGL The following DNA sequence nGPCR-seq58 <SEQ ID NO.\",\n \"67> was identified in H. sapiens: ACTACCATGGAAGCTGACCTGGGTGCCACTGGCCACAGGCCCCGCACAGAGCTTGATGATGAGGACTCCTACC CCCAAGGTGGCTGGGACACGGTCTTCCTGGTGGCCCTGCTGCTCCTTGGGCTGCCAGCCAATGGGTTGATGGC GTGGCTGGCCGGCTCCCAGGCCCGGCATGGAGCTGGCACGCGTCTGGCGCTGCTCCTGCTCAGCCTGGCCCTC TCTGACTTCTTGTTCCTGGCAGCAGCGGCCTTCCAGATCCTAGAGATCCGGCATGGGGGACACTGGCCGCTGG GGACAGCTGCCTGCCGCTTCTACTACTTCCTATGGGGCGTGTCCTACTCCTCCGGCCTCTTCCTGCTGGCCGC CCTCAGCCTCGACCGCTGCCTGCTGGCGCTGTGCCCACACTGGTACCCTGGGCACCGCCCAGTCCGCCTGCCC CTCTGGGTCTGCGCCGGTGTCTGGGTGCTGGCCACACTCTTCAGCGTGCCCTGGCTGGTCTTCCCCGAGGCTG CCGTCTGGTGGTACGACCTGGTCATCTGCCTGGACTTCTGGGACAGCGAGGAGCTGTCGCTGAGGATGCTGGA GGTCCTGGGGGGCTTCCTGCCTTTCCTCCTGCTGCTCGTCTGCCACGTGCTCACCCAGGCCACAGCCTGTCGC ACCTGCCACCGCCAACAGCAGCCCGCAGCCTGCCGGGGCTTCGCCCGTGTGGCCAGGACCATTCTGTCAGCCT ATGTGGTCCTGAGGCTGCCCTACCAGCTGGCCCAGCTGCTCTACCTGGCCTTCCTGTGGGACGTCTACTCTGG CTACCTGCTCTGGGAGGCCCTGGTCTACTCCGACTACCTGATCCTACTCAACAGCTGCCTCAGCCCCTTCCTC TGCCTCATGGCCAGTGCCGACCTCCGGACCCTGCTGCGCTCCGTGCTCTCGTCCTTCGCGGCAGCTCTCTGCG AGGAGCGGCCGGGCAGCTTCACGCCCACTGAGCCACAGACCCAGCTAGATTCTGAGGGTCCAACTCTGCCAGA GCCGATGGCAGAGGCCCAGTCACAGATGGATCCTGTGGCCCAGCCTCAGGTGAACCCCACACTCCAGCCACGA TCGGATCCCACAGCTCAGCCACAGCTGAACCCTACGGCCCAGCCACAGTCGGATCCCACAGCCCAGCCACAGC TGAACCTCATGGCCCAGCCACAGTCAGATTCTGTGGCCCAGCCACAGGCAGACACTAACGTCCAGACCCCTGC ACCTGCTGCC The following amino acid sequence <SEQ ID NO.\",\n \"68> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"67: TTMEADLGATGHRPRTELDDEDSYPQGGWDTVFLVALLLLGLPANGLMAWLAGSQARHGAGTRLALLL LSLALSDFLFLAAAAFQILEIRHGGHWPLGTAACRFYYFLWGVSYSSGLFLLAALSLDRCLLALCPHW YPGHRPVRLPLWVCAGVWVLATLFSVPWLVFPEAAVWWYDLVICLDFWDSEELSLRMLEVLGGFLPFL LLLVCHVLTQATACRTCHRQQQPAACRGFARVARTILSAYVVLRLPYQLAQLLYLAFLWDVYSGYLLW EALVYSDYLILLNSCLSPFLCLMASADLRTLLRSVLSSFAAALCEERPGSFTPTEPQTQLDSEGPTLP EPMAEAQSQMDPVAQPQVNPTLQPRSDPTAQPQLNPTAQPQSDPTAQPQLNLMAQPQSDSVAQPQADT NVQTPAPAA The following DNA sequence nGPCR-seq59 <SEQ ID NO.\",\n \"69> was identified in H. sapiens: TACAGGCCTGAGCATGCTGGGCTCCATCAGCACCAAGCACTGCCTGTCCATCCTGTGGCCCATCTAGTACCGC TGCCACCACCCCACACACCTGTCAGCAGTCGTGTGTCCTGCTCTGGGCCCTGTCCCTGCTGCAGAGCATCCTG GAATGGATGTTCTGTGGCTTCCTGTCTAGTGGTGCTGATTCTGTTTGGTGTGAAACATCAGATTTCATCACAG TCACATGGCTGATTTTTTTATGTGTGGTTCTCTGCGGGTCCAGCCCGGTTCTGCTGGTCAGGATCCTTTGTGG ATCCCGGAAGATGCCCTTGACCAGGCTGTACATGACCATCCTGCTCAGAGTGCTGGTCTTCCTCCTCTGTGAC CTGCCCTTTGGCATTCAGTGATTCCTATTTTTCTGGATCCACGTGGATTTGTCACGTTCGTCTAGTTTCCATT TTCCTGTCCACTCTTAACAGCAGTGCCAACCCCATTATTTACTTCTTCATGGGCTCCTTTAGGCAGCTTCAAA ACAGGAAGACTCTCTAGCTGGTTCTCCAGAGGGCTCTGCAGGACACGCCTGAGGTGGAAGAAGGCAGATGGCG GCTTTCTGAGGAAACCCTGGAGCTGTCATGAAGCAGATTGGGGCCATGAGGAAGAGCCTCTGCCCTGTCAGTC AG The following amino acid sequence <SEQ ID NO.\",\n \"70> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"69: YRPEHAGLHQHQALPVHPVAHLVPLPPPHTPVSSRVSCSGPCPCCRASWNGCSVASCLVVLILFGVKHQISSQ SHGFFYVWFSAGPARFCWSGSFVDPGRCPPGCTPSCSECWSSSSVTCPLAFSDSYFSGSTWICHVRLVSIFLS TLNSSANPIIYFFMGSFRQLQNRKTLLVLQRALQDTPEVEEGRWRLSEETLELSSRLGPGRASALSV The following DNA sequence nGPCR-seq60 <SEQ ID NO.\",\n \"71> was identified in H. sapiens: ATGCCGAAGGCAGGCCGCAGAAGAGAAGAGGAGGACGGTGAGGAGGATGAGCCCAGGGAAGCCCCGGGGTGGG GGCCGCTGGGGGCCTCGCTCCACCCGCAGCAGCAGCATAAGGCTGGCCCCACACATGGTGCAACACAGCAGAG CCAGCAGCACCGCTGCCACCAGCCACAGCGTCCGGCACAAGTGGCGGCTGGGCTCCCCGAAGAACTGGGTGCA GGCGCCGCTGAGCAGCAGGTGCAGCAGCAGGCAGAGGGCCCAGGTGAGGGCGCACACACAGGTGGTCAGGTGG CGTGGGCGGCGGCACGAGTACCAGGCTGGGAAGAGGGCGGCCAGGCACTGCTCCACGCTGACGGCCGCCAGGA GACTCAGGCCCACGATGTAGCAGAAGAAGCGCAGCGTTGCCAGGCTGGTCTGCACGAAGCCCGGGAAGTCCAG CCGGCCTTGCAGCAAGTCGGGGACGATGGCCACCATGTGGCAGCCAAGGAAGATGAGATCCGCGCAGGCCACG TCCAGGAGGTAGATGGCGAAAGGGTTTCTGTAGACATTGGAGCTGAGC The following amino acid sequence <SEQ ID NO.\",\n \"72> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"71: LSSNVYRNPFAIYLLDVACADLIFLGCHMVAIVPDLLQGRLDFPGFVQTSLATLRFFCYIVGLSLLA AVSVEQCLAALFPAWYSCRRPRHLTTCVCALTWALCLLLHLTTCVCALTWALCLLLHLLLSGACTLL LSGACTQFFGEPSRHLCRTLWLVAAVLLALLCCTMCGASLMLLLRVERGPQRPPPRGFPGLILLTVL LFSSAACLRH The following DNA sequence nGPCR-1 <SEQ ID NO.\",\n \"73> was identified in H. sapiens: ATGGAATCATCTTTCTCATTTGGAGTGATCCTTGCTGTCCTGGCCTCCCTCATCATTGCTACTAACACACTAG TGGCTGTGGCTGTGCTGCTGTTGATCCACAAGAATGATGGTGTCAGTCTCTGCTTCACCTTGAATCTGGCTGT GGCTGACACCTTGATTGGTGTGGCCATCTCTGGCCTACTCACAGACCAGCTCTCCAGCCCTTCTCGGCCCACA CAGAAGACCCTGTGCAGCCTGCGGATGGCATTTGTCACTTCCTCCGCAGCTGCCTCTGTCCTCACGGTCATGC TGATCACCTTTGACAGGTACCTTGCCATCAAGCAGCCCTTCCGCTACTTGAAGATCATGAGTGGGTTCGTGGC CGGGGCCTGCATTGCCGGGCTGTGGTTAGTGTCTTACCTCATTGGCTTCCTCCCACTCGGAATCCCCATGTTC CAGCAGACTGCCTACAAAGGGCAGTGCAGCTTCTTTGCTGTATTTCACCCTCACTTCGTGCTGACCCTCTCCT GCGTTGGCTTCTTCCCAGCCATGCTCCTCTTTGTCTTCTTCTACTGCGACATGCTCAAGATTGCCTCCATGCA CAGCCAGCAGATTCGAAAGATGGAACATGCAGGAGCCATGGCTGGAGGTTATCGATCCCCACGGACTCCCAGC GACTTCAAAGCTCTCCGTACTGTGTCTGTTCTCATTGGGAGCTTTGCTCTATCCTGGACCCCCTTCCTTATCA CTGGCATTGTGCAGGTGGCCTGCCAGGAGTGTCACCTCTACCTAGTGCTGGAACGGTACCTGTGGCTGCTCGG CGTGGGCAACTCCCTGCTCAACCCACTCATCTATGCCTATTGGCAGAAGGAGGTGCGACTGCAGCTCTACCAC ATGGCCCTAGGAGTGAAGAAGGTGCTCACCTCATTCCTCCTCTTTCTCTCGGCCAGGAATTGTGGCCCAGAGA GGCCCAGGGAAAGTTCCTGTCACATCGTCACTATCTCCAGCTCAGAGTTTGATGGCTAA The following amino acid sequence <SEQ ID NO.\",\n \"74> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"73: MESSFSFGVILAVLASLIIATNTLVAVAVLLLIHKNDGVSLCFTLNLAVADTLIGVAISGLLTDQLSSPSRPT QKTLCSLRMAFVTSSAAASVLTVMLITFDRYLAIKQPFRYLKIMSGFVAGACIAGLWLVSYLIGFLPLGIPMF QQTAYKGQCSFFAVFHPHFVLTLSCVGFFPAMLLFVFFYCDMLKIASMHSQQIRKMEHAGAMAGGYRSPRTPS DFKALRTVSVLIGSFALSWTPFLITGIVQVACQECHLYLVLERYLWLLGVGNSLLNPLIYAYWQKEVRLQLY HMALGVKKVLTSFLLFLSARNCGPERPRESSCHIVTISSSEFDG The following DNA sequence TL-GPCR-seq5 <SEQ ID NO.\",\n \"75> was identified in H. sapiens.\",\n \"AACTGGAAGGGCAGCCGTCTGCCGCCCACGAACACCTTCTCAAGCACTTTGAGTGACCACGGCTTGCAAGCTG GTGGCTGGCCCCCCGAGTCCCGGGCTCTGAGGCACGGCCGTCGACTTAAGCGTTGCATCCTGTTACCTGGAGA CCCTCTGAGCTCTCACCTGCTACTTCTGCCGCTGCTTCTGCACAGAGCCCGGGCGAGGACCCCTCCAGGATGC AGGTCCCGAACAGCACCGGCCCGGACAACGCGACGCTGCAGATGCTGCGGAACCCGGCGATCGCGGTGGCCCT GCCCGTGGTGTACTCGCTGGTGGCGGCGGTCAGCATCCCGGGCAACCTCTTCTCTCTGTGGGTGCTGTGCCGG CGCATGGGGCCCAGATCCCCGTCGGTCATCTTCATGATCAACCTGAGCGTCACGGACCTGATGCTGGCCAGCG TGTTGCCTTTCCAAATCTACTACCATTGCAACCGCCACCACTGGGTATTCGGGGTGCTGCTTTGCAACGTGGT GACCGTGGCCTTTTACGCAAACATGTATTCCAGCATCCTCACCATGACCTGTATCAGCGTGGAGCGCTTCCTG GGGGTCCTGTACCCGCTCAGCTCCAAGCGCTGGCGCCGCCGTCGTTACGCGGTGGCCGCGTGTGCAGGGACCT GGCTGCTGCTCCTGACCGCCCTGTCCCCGCTGGCGCGCACCGATCTCACCTACCCGGTGCACGCCCTGGGCAT CATCACCTGCTTCGACGTCCTCAAGTGGACGATGCTCCCCAGCGTGGCCATGTGGGCCGTGTTCCTCTTCACC ATCTTCATCCTGCTGTTCCTCATCCCGTTCGTGATCACCGTGGCTTGTTACACGGCCACCATCCTCAAGCTGT TGCGCACGGAGGAGGCGCACGGCCGGGAGCAGCGGAGGCGCGCGGTGGGCCTGGCCGCGGTGGTCTTGCTGGC CTTTGTCACCTGCTTCGCCCCCAACAACTTCGTGCTCCTGGCGCACATCGTGAGCCGCCTGTTCTACGGCAAG AGCTACTACCACGTGTACAAGCTCACGCTGTGTCTCAGCTGCCTCAACAACTGTCTGGACCCGTTTGTTTATT ACTTTGCGTCCCGGGAATTCCAGCTGCGCCTGCGGGAATATTTGGGCTGCCGCCGGGTGCCCAGAGACACCCT GGACACGCGCCGCGAGAGCCTCTTCTCCGCCAGGACCACGTCCGTGCGCTCCGAGGCCGGTGCGCACCCTGAA GGGATGGAGGGAGCCACCAGGCCCGGCCTCCAGAGGCAGGAGAGTGTGTTCTGAGTCCCGGGGGCGCAGCTTG GAGAGCCGGGGGCGCAGCTTGGAGGATCCAGGGGCGCATGGAGAGGCCACGGTGCCAGAGGTTCAGGGAGAAC AGCTGCGTTGCTCCCAGGCACTGCAGAGGCCCGGTGGGGAAGGGTCTCCAGGCTTTATTCCTCCCAGGCACTG CAGAGGCACCGGTGAGGAAGGGTCTCCAGGCTTCACTCAGGGTAGAGAAACAAGCAAAGCCCAGCAGCGCACA GGGTGCTTGTTATCCTGCAGAGGGTGCCTCTGCCTCTCTGTGTCAGGGGACAGCTTGTGTCACCACGCCCGGC TAATTTTTGTATTTTTTTTAGTAGAGCTGGGCTGTCACCCCCGAGCTCCTTAGACACTCCTCACACCTGTCCA TACCCGAGGATGGATATTCAACCAGCCCCACCGCCTACCCGACTCGGTTTCTGGATATCCTCTGTGGGCGAAC TGCGAGCCCCATTCCCAGCTCTTCTCCCTGCTGACATCGTCCCTTAGCACACCTGTCCATACCCGAGGATGGA TATTCAACCAGCCCCACCGCCTACCCGACTCGGTTTCTGGATATCCTCTGTGGGCGAACTGCGAGCCCCATTC CCAGCTCTTCTCCCTGCTGACATCGTCCCTTAGTTGTGGTTCTGGCCTTCTCCATTCTCCTCCAGGGGTTCTG GTCTCCGTAGCCCGGTGCACGCCGAAATTTCTGTTTATTTCACTCAGGGGCACTGTGGTTGCTGTGGTTGGAA TTCTTCTTTCAGAGGAGCGCCTGGGGCTCCTGCAAGTCAGCTACTCTCCGTGCCCACTTCCCCTCACACACAC ACCCCCCTCGTGCCGAATTC The following amino acid sequence <SEQ ID NO.\",\n \"76> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"75.MQVPNSTGPDNATLQMLRNPAIAVALPVVYSLVAAVSIPGNLFSLWVLCRRMGPRSPSVIFMINLSVTDLMLA SVLPFQIYYHCNRHHWVFGVLLCNVVTVAFYANMYSSILTMTCISVERFLGVLYPLSSKRWRRRRYAVAACAG TWLLLLTALSPLARTDLTYPVHALGIITCFDVLKWTMLPSVAMWAVFLFTIFILLFLIPFVITVACYTATILK LLRTEEAHGREQRRRAVGLAAVVLLAFVTCFAPNNFVLLAHIVSRLFYGKSYYHVYKLTLCLSCLNNCLDPFV YYFASREFQLRLREYLGCRRVPRDTLDTRRESLFSARTTSVRSEAGAHPEGMEGATRPGLQRQESVF The following DNA sequence nGPCR-9 <SEQ ID NO.\",\n \"77> was identified in H. sapiens: ATGGAGTCGGGGCTGCTGCGGCCGGCGCCGGTGAGCGAGGTCATCGTCCTGCATTACAACTACACCGGCAAGC TCCGCGGTGCGCGCTACCAGCCGGGTGCCGGCCTGCGCGCCGACGCCGTGGTGTGCCTGGCGGTGTGCGCCTT CATCGTGCTAGAGAATCTAGCCGTGTTGTTGGTGCTCGGACGCCACCCGCGCTTCCACGCTCCCATGTTCCTG CTCCTGGGCAGCCTCACGTTGTCGGATCTGCTGGCAGGCGCCGCCTACGCCGCCAACATCCTACTGTCGGGGC CGCTCACGCTGAAACTGTCCCCCGCGCTCTGGTTCGCACGGGAGGGAGGCGTCTTCGTGGCACTCACTGCGTC CGTGCTGAGCCTCCTGGCCATCGCGCTGGAGCGCAGCCTCACCATGGCGCGCAGGGGGCCCGCGCCCGTCTCC AGTCGGGGGCGCACGCTGGCGATGGCAGCCGCGGCCTGGGGCGTGTCGCTGCTCCTCGGGCTCCTGCCAGCGC TGGGCTGGAATTGCCTGGGTCGCCTGGACGCTTGCTCCACTGTCTTGCCGCTCTACGCCAAGGCCTACGTGCT CTTCTGCGTGCTCGCCTTCGTGGGCATCCTGGCCGCTATCTGTGCACTCTACGCGCGCATCTACTGCCAGGTA CGCGCCAACGCGCGGCGCCTGCCGGCACGGCCCGGGACTGCGGGGACCACCTCGACCCGGGCGCGTCGCAAGC CGCGCTCGCTGGCCTTGCTGCGCACGCTCAGCGTGGTGCTCCTGGCCTTTGTGGCATGTTGGGGCCCCCTCTT CCTGCTGCTGTTGCTCGACGTGGCGTGCCCGGCGCGCACCTGTCCTGTACTCCTGCAGGCCGATCCCTTCCTG GGACTGGCCATGGCCAACTCACTTCTGAACCCCATCATCTACACGCTCACCAACCGCGACCTGCGCCACGCGC TCCTGCGCCTGGTCTGCTGCGGACGCCACTCCTGCGGCAGAGACCCGAGTGGCTCCCAGCAGTCGGCGAGCGC GGCTGAGGCTTCCGGGGGCCTGCGCCGCTGCCTGCCCCCGGGCCTTGATGGGAGCTTCAGCGGCTCGGAGCGC TCATCGCCCCAGCGCGACGGGCTGGACACCAGCGGCTCCACAGGCAGCCCCGGTGCACCCACAGCCGCCCGGA CTCTGGTATCAGAACCGGCTGCAGACTGA The following amino acid sequence <SEQ ID NO.\",\n \"78> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"77: MESGLLRPAPVSEVIVLHYNYTGKLRGARYQPGAGLRADAVVCLAVCAFIVLENLAVLLVLGRHPRFHAPMFL LLGSLTLSDLLAGAAYAANILLSGPLTLKLSPALWFAREGGVFVALTASVLSLLAIALERSLTMARRGPAPVS SRGRTLAMAAAAWGVSLLLGLLPALGWNCLGRLDACSTVLPLYAKAYVLFCVLAFVGILAAICALYARIYCQV RANARRLPARPGTAGTTSTRARRKPRSLALLRTLSVVLLAFVACWGPLFLLLLLDVACPARTCPVLLQADPFL GLAMANSLLNPIIYTLTNRDLRHALLRLVCCGRHSCGRDPSGSQQSASAAEASGGLRRCLPPGLDGSFSGSER SSPQRDGLDTSGSTGSPGAPTAARTLVSEPAAD The following DNA sequence nGPcR-11 <SEQ ID NO.\",\n \"79> was identified in H. sapiens: ATGTACAACGGGTCGTGCTGCCGCATCGAGGGGGACACCATCTCCCAGGTGATGCCGCCGCTGCTCATTGTGG CCTTTGTGCTGGGCGCACTAGGCAATGGGGTCGCCCTGTGTGGTTTCTGCTTCCACATGAAGACCTGGAAGCC CAGCACTGTTTACCTTTTCAATTTGGCCGTGGCTGATTTCCTCCTTATGATCTGCCTGCCTTTTCGGACAGAC TATTACCTCAGACGTAGACACTGGGCTTTTGGGGACATTCCCTGCCGAGTGGGGCTCTTCACGTTGGCCATGA ACAGGGCCGGGAGCATCGTGTTCCTTACGGTGGTGGCTGCGGACAGGTATTTCAAAGTGGTCCACCCCCACCA CGCGGTGAACACTATCTCCACCCGGGTGGCGGCTGGCATCGTCTGCACCCTGTGGGCCCTGGTCATCCTGGGA ACAGTGTATCTTTTGCTGGAGAACCATCTCTGCGTGCAAGAGACGGCCGTCTCCTGTGAGAGCTTCATCATGG AGTCGGCCAATGGCTGGCATGACATCATGTTCCAGCTGGAGTTCTTTATGCCCCTCGGCATCATCTTATTTTG CTCCTTCAAGATTGTTTGGAGCCTGAGGCGGAGGCAGCAGCTGGCCAGACAGGCTCGGATGAAGAAGGCGACC CGGTTCATCATGGTGGTGGCAATTGTGTTCATCACATGCTACCTGCCCAGCGTGTCTGCTAGACTCTATTTCC TCTGGACGGTGCCCTCGAGTGCCTGCGATCCCTCTGTCCATGGGGCCCTGCACATAACCCTCAGCTTCACCTA CATGAACAGCATGCTGGATCCCCTGGTGTATTATTTTTCAAGCCCCTCCTTTCCCAAATTCTACAACAAGCTC AAAATCTGCAGTCTGAAACCCAAGCAGCCAGGACACTCAAAAACACAAAGGCCGGAAGAGATGCCAATTTCGA ACCTCGGTCGCAGGAGTTGCATCAGTGTGGCAAATAGTTTCCAAAGCCAGTCTGATGGGCAATGGGATCCCCA CATTGTTGAGTGGCACTGA The following amino acid sequence <SEQ ID NO.\",\n \"80> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"79: MYNGSCCRIEGDTISQVMPPLLIVAFVLGALGNGVALCGFCFHMKTWKPSTVYLFNLAVADFLLMICLPFRTD YYLRRRHWAFGDIPCRVGLFTLAMNRAGSIVFLTVVAADRYFKVVHPHHAVNTISTRVAAGIVCTLWALVILG TVYLLLENHLCVQETAVSCESFIMESANGWHDIMFQLEFFMPLGIILFCSFKIVWSLRRRQQLARQARMKKAT RFIMVVAIVFITCYLPSVSARLYFLWTVPSSACDPSVHGALHITLSFTYMNSMLDPLVYYFSSPSFPKFYNKL KICSLKPKQPGHSKTQRPEEMPISNLGRRSCISVANSFQSQSDGQWDPHIVEWH The following DNA sequence nGPCR-16 <SEQ ID NO.\",\n \"81> was identified in H. sapiens: ATGACAGGTGACTTCCCAAGTATGCCTGGCCACAATACCTCCAGGAATTCCTCTTGCGATCCTATAGACACCC CACTTAATCAGCCTCTACTTCATAGTGCTTATTGGCGGGCTGGTGGGTGTCATTTCCATTCTTTTCCTCCTGG TGAAAATGAACACCCGGTCAGTGACCACCATGGCGGTCATTAACTTGGTGGTGGTCCACAGCGTTTTTCTGCT GACAGTGCCATTTCGCTTGACCTACCTCATCAAGAAGACTTGGATGTTTGGGCTGCCCTTCTGCAAATTTGTG AGTGCCATGCTGCACATCCACATGTACCTCACGTTCCTATTCTATGTGGTGATCCTGGTCACCAGATACCTCA TCTTCTTCAAGTGCAAAGACAAAGTGGAATTCTACAGAAAACTGCATGCTGTGGCTGCCAGTGCTGGCATGTG GACGCTGGTGATTGTCATTGTGGTACCCCTGGTTGTCTCCCGGTATGGAATCCATGAGGAATACAATGAGGAG CACTGTTTTAAATTTCACAAAGAGCTTGCTTACACATATGTGAAAATCATCAACTATATGATAGTCATTTTTG TCATAGCCGTTGCTGTGATTCTGTTGGTCTTCCAGGTCTTCATCATTATGTTGATGGTGCAGAAGCTACGCCA CTCTTTACTATCCCACCAGGAGTTCTGGGCTCAGCTGAAAAACCTATTTTTTATAGGGGTCATCCTTGTTTGT TTCCTTCCCTACCAGTTCTTTAGGATCTATTACTTGAATGTTGTGACGCATTCCAATGCCTGTAACAGCAAGG TTGCATTTTATAACGAAATCTTCTTGAGTGTAACAGCAATTAGCTGCTATGATTTGCTTCTCTTTGTCTTTGG GGGAAGCCATTGGTTTAAGCAAAAGATAATTGGCTTATGGAATTGTGTTTTGTGCCGTTAGCCACAAACTACA GTATTCATATTTGCTTCCTTTATATTGGGAATAAAAATGGGTATAGGGGAGGTAAGAATGGTATTTCATTACT TGATCAAAACCATGCCTTGATGTACCCAAAACAAAAGGACTATAAAATGCAAGAGCCCTCATTGTAGTCCTTA TGGGATCCCTCCCATCTCTGAGTGATGGCCGTACAAAGACCAGTGTTGTTGAATCCACCTGGAGTTGCAATAT TACATTATTTTCCAGTACAGAATGTCTGTGTGGCCCATGAAAGCAACATAGGTTTTAAGAGTTTTAGAGTTTC ATTAGCTCATTCTAAGTTCCTCTGTTTGAAGCATGGTCTCTTAGGTTTTGGACTGAACTCAGACCTTTAGTTC TTTTCATCCCACTTCACCTTAGGTAAGTAAATTCTGGCCACCACCCAGCTCCAAAGACACAAACTCTCCTTCG CTAACCAGGTTAGATGTCCCATTCATCTCATGCCCTGATAAAAACTGATAAGGGGAGAGAATAGTTAAAAATT TTTCTAGGGTATCATAACTCTGGTAGGAAGTCATCTGTCTAGAAATCAAGAGAAAAAGAACGTGTGGCCTCCT GTTATAACAAGGGTTTCTAGATTTGTCCTGTGAAAGGTCGTTTAAGGACTTGGGGATCAACTTCCTCAATTAT CACCAATTGCACTGTTGCTCCAAAAATCATTTAAAAGCTTACTGGACATATCTACATAATGGTGAAACTGTAA TTTAGAGACTATCCCTGACTAATGTGCTGGTAGGCATTAAAATGAGTTCCCAAGGGAAGTGATTAAAATTTTT TTCTCTTCTGTTTTTTGAGAGAATTTCTAGATGTCCTGGGCCACAGTTAATTAAGATTTTTAGGGGGGACAGA AAGTTATACTGAAATCTTTAGAGCTCCCTTCCGCCGTTAAAATTATATATATATATATTTAAATTATACCTTA AGTTCTGGGGTACATGTGCAGAATGTGCAGGTTTGTTACATAGGTATACACGTGCCATGGTGGTTTGCGGCAC CTGTCAACCCATCTACATTAGGTATTTCTCCTAATGCTCTCCCTCCCCTAGCCCCCCACCCCTGGACAGGCCC CATTGTGTGATGTTCCCCTCCCTGTGTCCATGTGTTTTCATTGTTCAACTCCCACTTCTAAGTGAGAACATGC GGTGTTTGGTTTTCTGTTCCTGTGTTAGTTTGCTGAGAATGATGGTTTCCAGGTTAAAATTATATATTTTTAA ATAAATGAAAACTGTGTTTTTAAAAGAGGACTTTTGAGAAGTATATAGAAAAACCATTAATTTAGACTCTGTG AGATTAGGTTGCATGAAGAAGGTTTTCTGAATATTTGAAGAGTGGATAAATAAATGTCCCCCAAAGCAATAAA ATCATAATCCTTTAAAATATAGGAAAAATAACTAATGGGAACTAGGCTTAATACTCGGGATGAAATAATCTGT ACAACAAACTCCCATGACACATGTTTACCTATGTAACAAACCTGCACATGTACCCCTGAACTTAAAATAAAAT TTAAAGTATAATAATAAAATAATATGGATTTTCTTT The following amino acid sequence <SEQ ID NO.\",\n \"82> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"81: MTGDFPSMPGHNTSRNSSCDPIVTPHLISLYFIVLIGGLVGVISILFLLVKMNTRSVTTMAVINLVVVHSVFL LTVPFRLTYLIKKTWMFGLPFCKFVSAMLHIHMYLTFLFYVVILVTRYLIFFKCKDKVEFYRKLHAVAASAGM WTLVIVIVVPLVVSRYGIHEEYNEEHCFKFHKELAYTYVKIINYMIVIFVIAVAVILLVFQVFIIMLMVQKLR HSLLSHQEFWAQLKNLFFIGVILVCFLPYQFFRIYYLNVVTHSNACNSKVAFYNEIFLSVTAISCYDLLLFVF GGSHWFKQKIIGLWNCVLCR The following DNA sequence nGPcR-40 <SEQ ID NO.\",\n \"83> was identified in H. sapiens: GCAGGAGCACTGAAAATCAGGAACAATCCTGTATTTTTTGTGATAATCAACAAGGACAAAACTTCTCCATATG TAAATAACAGCGTTATGAGCAGCAATTCATCCCTGCTGGTGGCTGTGCAGCTGTGCTACGCGAACGTGAATGG GTCCTGTGTGAAAATCCCCTTCTCGCCGGGATCCCGGGTGATTCTGTACATAGTGTTTGGCTTTGGGGCTGTG CTGGCTGTGTTTGGAAACCTCCTGGTGATGATTTCAATCCTCCATTTCAAGCAGCTGCACTCTCCGACCAATT TTCTCGTTGCCTCTCTGGCCTGCGCTGATTTCTTGGTGGGTGTGACTGTGATGCCCTTCAGCATGGTCAGGAC GGTGGAGAGCTGCTGGTATTTTGGGAGGAGTTTTTGTACTTTCCACACCTGCTGTGATGTGGCATTTTGTTAC TCTTCTCTCTTTCACTTGTGCTTCATCTCCATCGACAGGTACATTGCGGTTACTGACCCCCTGGTCTATCCTA CCAAGTTCACCGTATCTGTGTCAGGAATTTGCATCAGCGTGTCCTGGATCCTGCCCCTCATGTACAGCGGTGC TGTGTTCTACACAGGTGTCTATGACGATGGGCTGGAGGAATTATCTGATGCCCTAAACTGTATAGGAGGTTGT CAGACCGTTGTAAATCAAAACTGGGTGTTGACAGATTTTCTATCCTTCTTTATACCTACCTTTATTATGATAA TTCTGTATGGTAACATATTTCTTGTGGCTAGACGACAGGCGAAAAAGATAGAAAATACTGGTAGCAAGACAGA ATCATCCTCAGAGAGTTACAAAGCCAGAGTGGCCAGGAGAGAGAGAAAAGCAGCTAAAACCCTGGGGGTCACA GTGGTAGCATTTATGATTTCATGGTTACCATATAGCATTGATTCATTAATTGATGCCTTTATGGGCTTTATAA CCCCTGCCTGTATTTATGAGATTTGCTGTTGGTGTGCTTATTATAACTCAGCCATGAATCCTTTGATTTATGC TTTATTTTACCCATGGTTTAGGAAAGCAATAAAAGTTATTGTAACTGGTCAGGTTTTAAAGAACAGTTCAGCA ACCATGAATTTGTTTTCTGAACATATATAA The following amino acid sequence <SEQ ID NO.\",\n \"84> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"83: MSSNSSLLVAVQLCYANVNGSCVKIPFSPGSRVILYIVFGFGAVLAVFGNLLVMISILHFKQLHSPTNFLVAS LACADFLVGVTVMPFSMVRTVESCWYFGRSFCTFHTCCDVAFCYSSLFHLCFISIDRYIAVTDPLVYPTKFTV SVSGICISVSWILPLMYSGAVFYTGVYDDGLEELSDALNCIGGCQTVVNQNWVLTDFLSFFIPTFIMIILYGN IFLVARRQAKKIENTGSKTESSSESYKARVARRERKAAKTLGVTVVAFMISWLPYSIDSLIDAFMGFITPACI YEICCWCAYYNSAMNPLIYALFYPWFRKAIKVIVTGQVLKNSSATMNLFSEHI The following DNA sequence nGPCR-54 <SEQ ID NO.\",\n \"85> was identified in H. sapiens: ACCATGAATGAGCCACTAGACTATTTAGCAAATGCTTCTGATTTCCCCGATTATGCAGCTGCTTTTGGAAATT GCACTGATGAAAACATCCCACTCAAGATGCACTACCTCCCTGTTATTTATGGCATTATCTTCCTCGTGGGATT TCCAGGCAATGCAGTAGTGATATCCACTTACATTTTCAAAATGAGACCTTGGAAGAGCAGCACCATCATTATG CTGAACCTGGCCTGCACAGATCTGCTGTATCTGACCAGCCTCCCCTTCCTGATTCACTACTATGCCAGTGGCG AAAACTGGATCTTTGGAGATTTCATGTGTAAGTTTATCCGCTTCAGCTTCCATTTCAACCTGTATAGCAGCAT CCTCTTCCTCACCTGTTTCAGCATCTTCCGCTACTGTGTGATCATTCACCCAATGAGCTGCTTTTCCATTCAC AAAACTCGATGTGCAGTTGTAGCCTGTGCTGTGGTGTGGATCATTTCACTGGTAGCTGTCATTCCGATGACCT TCTTGATCACATCAACCAACAGGACCAACAGATCAGCCTGTCTCGACCTCACCAGTTCGGATGAACTCAATAC TATTAAGTGGTACAACCTGATTTTGACTGCAAGTACTTTCTGCCTCCCCTTGGTGATAGTGACACTTTGCTAT ACCACGATTATCCACACTTTGACCCATGGACTGCAAACTGACAGCTGCCTTAAGCAGAAAGCACGAAGGCTAA CCATTCTGCTACTCCTTGCATTTTACGTATGTTTTTTACCCTTCCATATCTTGAGGGTCATTCAGGATCGAAT CTCAGCCTGCTTTCAATCAGTTGTTCCATTGAGAATCAGATCCATGAAGCTTACATCGTTTCTAGACCATTAT GCTGCTCTGAACACCTTTGGTAACCTGTTACTATATGTGGTGGTCAGCGACAACTTTCAGCAGGCTGTCTGCT CAACAGTGAGATGCAAAGTAAGCGGGAACCTTGAGCAAGCAAAGAAAATTAGTTACTCAAACAACCCTTGA The following amino acid sequence <SEQ ID NO.\",\n \"86> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"85: MNEPLDYLANASDFPDYAAAFGNCTDENIPLKMHYLPVIYGIIFLVGFPGNAVVISTYIFKMRPWKSSTIIML NLACTDLLYLTSLPFLIHYYASGENWIFGDFMCKFIRFSFHFNLYSSILFLTCFSIFRYCVIIHPMSCFSIHK TRCAVVACAVVWIISLVAVIPMTFLITSTNRTNRSACLDLTSSDELNTIKWYNLILTASTFCLPLVIVTLCYT TIIHTLTHGLQTDSCLKQKARRLTILLLLAFYVCFLPFHILRVIQDRISACFQSVVPLRIRSMKLTSFLDHYA ALNTFGNLLLYVVVSDNFQQAVCSTVRCKVSGNLEQAKKISYSN The following DNA sequence nGPCR-56 <SEQ ID NO.\",\n \"87> was identified in H. sapiens: AAAAATTGCTGTACTGAACTATTGAATGGAACTTGGAAATAAAGTCCCTTCCAAAATAACTATTCTTCAACAG AGAGTAATAGGTAAATGTTTTAGAAGTGAGAGGACTCAAATTGCCAATGATTTACTCTTTTATTTTTCCTCCT AGGTTTCTGGGATAAGTATGTGCAAATAAAAAATAAACATGAGAAGGAACTGTAACCTGATTATGGATTTGGG AAAAAGATAAATCAACACACAAAGGGAAAAGTAAACTGATTGACAGCCCTCAGGAATGATGCCCTTTTGCCAC AATATAATTAATATTTCCTGTGTGAAAAACAACTGGTCAAATGATGTCCGTGCTTCCCTGTACAGTTTAATGG TGCTCATAATTCTGACCACACTCGTTGGCAATCTGATAGTTATTGTTTCTATATCACACTTCAAACAACTTCA TACCCCAACAAATTGGCTCATTCATTCCATGGCCACTGTGGACTTTCTTCTGGGGTGTCTGGTCATGCCTTAC AGTATGGTGAGATCTGCTGAGCACTGTTGGTATTTTGGAGAAGTCTTCTGTAAAATTCACACAAGCACCGACA TTATGCTGAGCTCAGCCTCCATTTTCCATTTGTCTTTCATCTCCATTGACCGCTACTATGCTGTGTGTGATCC ACTGAGATATAAAGCCAAGATGAATATCTTGGTTATTTGTGTGATGATCTTCATTAGTTGGAGTGTCCCTGCT GTTTTTGCATTTGGAATGATCTTTCTGGAGCTAAACTTCAAAGGCGCTGAAGAGATATATTACAAACATGTTC ACTGCAGAGGAGGTTGCTCTGTCTTCTTTAGCAAAATATCTGGGGTACTGACCTTTATGACTTCTTTTTATAT ACCTGGATCTATTATGTTATGTGTCTATTACAGAATATATCTTATCGCTAAAGAACAGGCAAGATTAATTAGT GATGCCAATCAGAAGCTCCAAATTGGATTGGAAATGAAAAATGGAATTTCACAAAGCAAAGAAAGGAAAGCTG TGAAGACATTGGGGATTGTGATGGGAGTTTTCCTAATATGCTGGTGCCCTTTCTTTATCTGTACAGTCATGGA CCCTTTTCTTCACTACATTATTCCACCTACTTTGAATGATGTA The following amino acid sequence <SEQ ID NO.\",\n \"88> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"87: MMPFCHNIINISCVKNNWSNDVRASLYSLMVLIILTTLVGNLIVIVSISHFKQLHTPTNWLIHSMATVDFLLG CLVMPYSMVRSAEHCWYFGEVFCKIHTSTDIMLSSASIFHLSFISIDRYYAVCDPLRYKAKMNILVICVMIFI SWSVPAVFAFGMIFLELNFKGAEEIYYKHVHCRGGCSVFFSKISGVLTFMTSFYIPGSIMLCVYYRIYLIAKE QARLISDANQKLQIGLEMKNGISQSKERKAVKTLGIVMGVFLICWCPFFICTVMDPFLHYIIPPTLNDARGSR ANSA The following DNA sequence nGPCR-56 <SEQ ID NO.\",\n \"89> was identified in H. sapiens: GGAATGATGCCCTTTTGCCACAATATAATTAATATTTCCTGTGTGAAAAACAACTGGTCAAATGATGTCCGTG CTTCCCTGTACAGTTTAATGGTGCTCATAATTCTGACCACACTCGTTGGCAATCTGATAGTTATTGTTTCTAT ATCACACTTCAAACAACTTCATACCCCAACAAATTGGCTCATTCATTCCATGGCCACTGTGGACTTTCTTCTG GGGTGTCTGGTCATGCCTTACAGTATGGTGAGATCTGCTGAGCACTGTTGGTATTTTGGAGAAGTCTTCTGTA AAATTCACACAAGCACCGACATTATGCTGAGCTCAGCCTCCATTTTCCATTTGTCTTTCATCTCCATTGACCG CTACTATGCTGTGTGTGATCCACTGAGATATAAAGCCAAGATGAATATCTTGGTTATTTGTGTGATGATCTTC ATTAGTTGGAGTGTCCCTGCTGTTTTTGCATTTGGAATGATCTTTCTGGAGCTAAACTTCAAAGGCGCTGAAG AGATATATTACAAACATGTTCACTGCAGAGGAGGTTGCTCTGTCTTCTTTAGCAAAATATCTGGGGTACTGAC CTTTATGACTTCTTTTTATATACCTGGATCTATTATGTTATGTGTCTATTACAGAATATATCTTATCGCTAAA GAACAGGCAAGATTAATTAGTGATGCCAATCAGAAGCTCCAAATTGGATTGGAAATGAAAAATGGAATTTCAC AAAGCAAAGAAAGGAAAGCTGTGAAGACATTGGGGATTGTGATGGGAGTTTTCCTAATATGCTGGTGCCCTTT CTTTATCTGTACAGTCATGGACCCTTTTCTTCACTACATTATTCCACCTACTTTGAATGATGTATTGATTTGG TTTGGCTACTTGAACTCTACATTTAATCCAATGGTTTATGCATTTTTCTATCCTTGGTTTAGAAAAGCACTGA AGATGATGCTGTTTGGTAAAATTTTCCAAAAAGATTCATCCAGGTGTAAATTATTTTTGGAATTGAGTTCATA G The following amino acid sequence <SEQ ID NO.\",\n \"90> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"89: MMPFCHNIINISCVKNNWSNDVRASLYSLMVLIILTTLVGNLIVIVSISHFKQLHTPTNWLIHSMATVDFLLG CLVMPYSMVRSAEHCWYFGEVFCKIHTSTDIMLSSASIFHLSFISIDRYYAVCDPLRYKAKMNILVICVMIFI SWSVPAVFAFGMIFLELNFKGAEEIYYKHVHCRGGCSVFFSKISGVLTFMTSFYIPGSIMLCVYYRIYLIAKE QARLISDANQKLQIGLEMKNGISQSKERKAVKTLGIVMGVFLICWCPFFICTVMDPFLHYIIPPTLNDVLIWF GYLNSTFNPMVYAFFYPWFRKALKMMLFGKIFQKDSSRCKLFLELSS The following DNA sequence nGPCR-58 <SEQ ID NO.\",\n \"91> was identified in H. sapiens: CTGTAAAGTAGATTGTATGAGGACTCCATGAGGTCATCCACTTCAAGTCCTTGGCATAGGATAATTACTCAAA AGGTGATGACAATGGCGCAGGGAGGGATGGTGACTTGCCTGGAGATGCACAGCACCGTCTCTCCCATACTCGG TCATTCACACCATCATTGATTCACCAGGCACCACTCCGTGTCCAGCAGGACTCTGGGGACCCCAAATGGACAC TACCATGGAAGCTGACCTGGGTGCCACTGGCCACAGGCCCCGCACAGAGCTTGATGATGAGGACTCCTACCCC CAAGGTGGCTGGGACACGGTCTTCCTGGTGGCCCTGCTGCTCCTTGGGCTGCCAGCCAATGGGTTGATGGCGT GGCTGGCCGGCTCCCAGGCCCGGCATGGAGCTGGCACGCGTCTGGCGCTGCTCCTGCTCAGCCTGGCCCTCTC TGACTTCTTGTTCCTGGCAGCAGCGGCCTTCCAGATCCTAGAGATCCGGCATGGGGGACACTGGCCGCTGGGG ACAGCTGCCTGCCGCTTCTACTACTTCCTATGGGGCGTGTCCTACTCCTCCGGCCTCTTCCTGCTGGCCGCCC TCAGCCTCGACCGCTGCCTGCTGGCGCTGTGCCCACACTGGTACCCTGGGCACCGCCCAGTCCGCCTGCCCCT CTGGGTCTGCGCCGGTGTCTGGGTGCTGGCCACACTCTTCAGCGTGCCCTGGCTGGTCTTCCCCGAGGCTGCC GTCTGGTGGTACGACCTGGTCATCTGCCTGGACTTCTGGGACAGCGAGGAGCTGTCGCTGAGGATGCTGGAGG TCCTGGGGGGCTTCCTGCCTTTCCTCCTGCTGCTCGTCTGCCACGTGCTCACCCAGGCCACAGCCTGTCGCAC CTGCCACCGCCAACAGCAGCCCGCAGCCTGCCGGGGCTTCGCCCGTGTGGCCAGGACCATTCTGTCAGCCTAT GTGGTCCTGAGGCTGCCCTACCAGCTGGCCCAGCTGCTCTACCTGGCCTTCCTGTGGGACGTCTACTCTGGCT ACCTGCTCTGGGAGGCCCTGGTCTACTCCGACTACCTGATCCTACTCAACAGCTGCCTCAGCCCCTTCCTCTG CCTCATGGCCAGTGCCGACCTCCGGACCCTGCTGCGCTCCGTGCTCTCGTCCTTCGCGGCAGCTCTCTGCGAG GAGCGGCCGGGCAGCTTCACGCCCACTGAGCCACAGACCCAGCTAGATTCTGAGGGTCCAACTCTGCCAGAGC CGATGGCAGAGGCCCAGTCACAGATGGATCCTGTGGCCCAGCCTCAGGTGAACCCCACACTCCAGCCACGATC GGATCCCACAGCTCAGCCACAGCTGAACCCTACGGCCCAGCCACAGTCGGATCCCACAGCCCAGCCACAGCTG AACCTCATGGCCCAGCCACAGTCAGATTCTGTGGCCCAGCCACAGGCAGACACTAACGTCCAGACCCCTGCAC CTGCTGCCAGTTCTGTGCCCAGTCCCTGTGATGAAGCTTCCCCAACCCCATCCTCGCATCCTACCCCAGGGGC CCTTGAGGACCCAGCCACACCTCCTGCCTCTGAAGGAGAAAGCCCCAGCAGCACCCCGCCAGAGGCGGCCCCG GGCGCAGGCCCCACGTGAGGGTCCAGGAACACGCAGGCCCACCAGAGCAGTGAAAGAGCCCAGGGCAGACAGA GGAACCAGCCAGTCAGA The following amino acid seqence <SEQ ID NO.\",\n \"92> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"91: LAWRCTAPSLPYSVIHTIIDSPGTTPCPAGLWGPQMDTTMEADLGATGHRPRTELDDEDSYPQGGWDTVFLVA LLLLGLPANGLMAWLAGSQARHGAGTRLALLLLSLALSDFLFLAAAAFQILEIRHGGHWPLGTAACRFYYFLW GVSYSSGLFLLAALSLDRCLLALCPHWYPGHRPVRLPLWVCAGVWVLATLFSVPWLVFPEAAVWWYDLVICLD FWDSEELSLRMLEVLGGFLPFLLLLVCHVLTQATACRTCHRQQQPAACRGFARVARTILSAYVVLRLPYQLAQ LLYLAFIMDVYSGYLLWEALVYSDYLILLNSCLSPFLCLMASADLRTLLRSVLSSFAAALCEERPGSFTPTEP QTQLDSEGPTLPEPMAEAQSQMDPVAQPQVNPTLQPRSDPTAQPQLNPTAQPQSDPTAQPQLNLMAQPQSDSV AQPQADTNVQTPAPAASSVPSPCDEASPTPSSHPTPGALEDPATPPASEGESPSSTPPEAAPGAGPT The following DNA sequence nGPCR-58 <SEQ ID NO.\",\n \"93> was identified in H. sapiens: ATGGACACTACCATGGAAGCTGACCTGGGTGCCACTGGCCACAGGCCCCGCACAGAGCTTGATGATGAGGACT CCTACCCCCAAGGTGGCTGGGACACGGTCTTCCTGGTGGCCCTGCTGCTCCTTGGGCTGCCAGCCA ATGGGTTGATGGCGTGGCTGGCCGGCTCCCAGGCCCGGCATGGAGCTGGCACGCGTCTGGCGCTGCTCCTGCT CAGCCTGGCCCTCTCTGACTTCTTGTTCCTGGCAGCAGCGGCCTTCCAGATCCTAGAGATCCGGCATGGGGGA CACTGGCCGCTGGGGACAGCTGCCTGCCGCTTCTACTACTTCCTATGGGGCGTGTCCTACTCCTCCGGCCTCT TCCTGCTGGCCGCCCTCAGCCTCGACCGCTGCCTGCTGGCGCTGTGCCCACACTGGTACCCTGGGCACCGCCC AGTCCGCCTGCCCCTCTGGGTCTGCGCCGGTGTCTGGGTGCTGGCCACACTCTTCAGCGTGCCCTGGCTGGTC TTCCCCGAGGCTGCCGTCTGGTGGTACGACCTGGTCATCTGCCTGGACTTCTGGGACAGCGAGGAGCTGTCGC TGAGGATGCTGGAGGTCCTGGGGGGCTTCCTGCCTTTCCTCCTGCTGCTCGTCTGCCACGTGCTCACCCAGGC CACAGCCTGTCGCACCTGCCACCGCCAACAGCAGCCCGCAGCCTGCCGGGGCTTCGCCCGTGTGGCCAGGACC ATTCTGTCAGCCTATGTGGTCCTGAGGCTGCCCTACCAGCTGGCCCAGCTGCTCTACCTGGCCTTCCTGTGGG ACGTCTACTCTGGCTACCTGCTCTGGGAGGCCCTGGTCTACTCCGACTACCTGATCCTACTCAACAGCTGCCT CAGCCCCTTCCTCTGCCTCATGGCCAGTGCCGACCTCCGGACCCTGCTGCGCTCCGTGCTCTCGTCCTTCGCG GCAGCTCTCTGCGAGGAGCGGCCGGGCAGCTTCACGCCCACTGAGCCACAGACCCAGCTAGATTCTGAGGGTC CAACTCTGCCAGAGCCGATGGCAGAGGCCCAGTCACAGATGGATCCTGTGGCCCAGCCTCAGGTGAACCCCAC ACTCCAGCCACGATCGGATCCCACAGCTCAGCCACAGCTGAACCCTACGGCCCAGCCACAGTCGGATCCCACA GCCCAGCCACAGCTGAACCTCATGGCCCAGCCACAGTCAGACTCTGTGGCCCAGCCACAGGCAGACACTAACG TCCAGACCCCTGCACCTGCTGCCAGTTCTGTGCCCAGTCCCTGTGATGAAGCTTCCCCAACCCCATCCTCGCA TCCTACCCCAGGGGCCCTTGAGGACCCAGCCACACCTCCTGCCTCTGAAGGAGAAAGCCCCAGCAGCACCCCG CCAGAGGCGGCCCCGGGCGCAGGCCCCACGTGA The following amino acid sequence <SEQ ID NO.\",\n \"94> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"93: MDTTMEADLGATGHRPRTELDDEDSYPQGGWDTVFLVALLLLGLPANGLMAWLAGSQARHGAGTRLALLLLSL ALSDFLFLAAAAFQILEIRHGGHWPLGTAACRFYYFLWGVSYSSGLFLLAALSLDRCLLALCPHWYPGHRPVR LPLWVCAGVWVLATLFSVPWLVFPEAAVWWYDLVICLDFWDSEELSLRMLEVLGGFLPFLLLLVCHVLTQATA CRTCHRQQQPAACRGFARVARTILSAYVVLRLPYQLAQLLYLAFLWDVYSGYLLWEALVYSDYLILLNSCLSP FLCLMASADLRTLLRSVLSSFAAALCEERPGSFTPTEPQTQLDSEGPTLPEPMAEAQSQMDPVAQPQVNPTLQ PRSDPTAQPQLNPTAQPQSDPTAQPQLNLMAQPQSDSVAQPQADTNVQTPAPAA The following DNA sequence nGPCR-3 <SEQ ID NO.\",\n \"185> was identified in H. sapiens: AGGCTCGCGCCCGAAGCAGAGCCATGAGAACCCCAGGGTGCCTGGCGAGCCGCTAGCGCCATGGGCCCCGGCG AGGCGCTGCTGGCGGGTCTCCTGGTGATGGTACTGGCCGTGGCGCTGCTATCCAACGCACTGGTGCTGCTTTG TTGCGCCTACAGCGCTGAGCTCCGCACTCGAGCCTCAGGCGTCCTCCTGGTGAATCTGTCTCTGGGCCACCTG CTGCTGGCGGCGCTGGACATGCCCTTCACGCTGCTCGGTGTGATGCGCGGGCGGACACCGTCGGCGCCCGGCG CATGCCAAGTCATTGGCTTCCTGGACACCTTCCTGGCGTCCAACGCGGCGCTGAGCGTGGCGGCGCTGAGCGC AGACCAGTGGCTGGCAGTGGGCTTCCCACTGCGCTACGCCGGACGCCTGCGACCGCGCTATGCCGGCCTGCTG CTGGGCTGTGCCTGGGGACAGTCGCTGGCCTTCTCAGGCGCTGCACTTGGCTGCTCGTGGCTTGGCTACAGCA GCGCCTTCGCGTCCTGTTCGCTGCGCCTGCCGCCCGAGCCTGAGCGTCCGCGCTTCGCAGCCTTCACCGCCAC GCTCCATGCCGTGGGCTTCGTGCTGCCGCTGGCGGTGCTCTGCCTCACCTCGCTCCAGGTGCACCGGGTGGCA CGCAGACACTGCCAGCGCATGGACACCGTCACCATGAAGGCGCTCGCGCTGCTCGCCGACCTGCACCCCAGTG TGCGGCAGCGCTGCCTCATCCAGCAGAAGCGGCGCCGCCACCGCGCCACCAGGAAGATTGGCATTGCTATTGC GACCTTCCTCATCTGCTTTGCCCCGTATGTCATGACCAGGCTGGCGGAGCTCGTGCCCTTCGTCACCGTGAAC GCCCAGTGGGGCATCCTCAGCAAGTGCCTGACCTACAGCAAGGCGGTGGCCGACCCGTTCACGTACTCTCTGC TCCGCCGGCCGTTCCGCCAAGTCCTGGCCGGCATGGTGCACCGGCTGCTGAAGAGAACCCCGCGCCCAGCATC CACCCATGACAGCTCTCTGGATGTGGCCGGCATGGTGCACCAGCTGCTGAAGAGAACCCCGCGCCCAGCGTCC ACCCACAACGGCTCTGTGGACACAGAGAATGATTCCTGCCTGCAGCAGACACACTGAGGGCCTGGCAGGGCTC ATCGCCCCCACCTTCTAAGA The following amino acid sequence <SEQ ID NO.\",\n \"186> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"185: MGPGEALLAGLLVMVLAVALLSNALVLLCCAYSAELRTRASGVLLVNLSLGHLLLAALDMPFTLLGVMRGRTP SAPGACQVIGFLDTFLASNAALSVAALSADQWLAVGFPLRYAGRLRPRYAGLLLGCAWGQSLAFSGAALGCSW LGYSSAFASCSLRLPPEPERPRFAAFTATLHAVGFVLPLAVLCLTSLQVHRVARRHCQRMDTVTMKALALLAD LHPSVRQRCLIQQKRRRHRATRKIGIAIATFLICFAPYVMTRLAELVPFVTVNAQWGILSKCLTYSKAVADPF TYSLLRRPFRQVLAGMVHRLLKRTPRPASTHDSSLDVAGMVHQLLKRTPRPASTHNGSVDTENDSCLQQTH The following DNA sequence nGPCR-58 <SEQ ID NO.\",\n \"93> was identified in H. sapiens: ATGGACACTACCATGGAAGCTGACCTGGGTGCCACTGGCCACAGGCCCCGCACAGAGCTTGATGATGAGGACT CCTACCCCCAAGGTGGCTGGGACACGGTCTTCCTGGTGGCCCTGCTGCTCCTTGGGCTGCCAGCCA ATGGGTTGATGGCGTGGCTGGCCGGCTCCCAGGCCCGGCATGGAGCTGGCACGCGTCTGGCGCTGCTCCTGCT CAGCCTGGCCCTCTCTGACTTCTTGTTCCTGGCAGCAGCGGCCTTCCAGATCCTAGAGATCCGGCATGGGGGA CACTGGCCGCTGGGGACAGCTGCCTGCCGCTTCTACTACTTCCTATGGGGCGTGTCCTACTCCTCCGGCCTCT TCCTGCTGGCCGCCCTCAGCCTCGACCGCTGCCTGCTGGCGCTGTGCCCACACTGGTACCCTGGGCACCGCCC AGTCCGCCTGCCCCTCTGGGTCTGCGCCGGTGTCTGGGTGCTGGCCACACTCTTCAGCGTGCCCTGGCTGGTC TTCCCCGAGGCTGCCGTCTGGTGGTACGACCTGGTCATCTGCCTGGACTTCTGGGACAGCGAGGAGCTGTCGC TGAGGATGCTGGAGGTCCTGGGGGGCTTCCTGCCTTTCCTCCTGCTGCTCGTCTGCCACGTGCTCACCCAGGC CACAGCCTGTCGCACCTGCCACCGCCAACAGCAGCCCGCAGCCTGCCGGGGCTTCGCCCGTGTGGCCAGGACC ATTCTGTCAGCCTATGTGGTCCTGAGGCTGCCCTACCAGCTGGCCCAGCTGCTCTACCTGGCCTTCCTGTGGG ACGTCTACTCTGGCTACCTGCTCTGGGAGGCCCTGGTCTACTCCGACTACCTGATCCTACTCAACAGCTGCCT CAGCCCCTTCCTCTGCCTCATGGCCAGTGCCGACCTCCGGACCCTGCTGCGCTCCGTGCTCTCGTCCTTCGCG GCAGCTCTCTGCGAGGAGCGGCCGGGCAGCTTCACGCCCACTGAGCCACAGACCCAGCTAGATTCTGAGGGTC CAACTCTGCCAGAGCCGATGGCAGAGGCCCAGTCACAGATGGATCCTGTGGCCCAGCCTCAGGTGAACCCCAC ACTCCAGCCACGATCGGATCCCACAGCTCAGCCACAGCTGAACCCTACGGCCCAGCCACAGTCGGATCCCACA GCCCAGCCACAGCTGAACCTCATGGCCCAGCCACAGTCAGACTCTGTGGCCCAGCCACAGGCAGACACTAACG TCCAGACCCCTGCACCTGCTGCCAGTTCTGTGCCCAGTCCCTGTGATGAAGCTTCCCCAACCCCATCCTCGCA TCCTACCCCAGGGGCCCTTGAGGACCCAGCCACACCTCCTGCCTCTGAAGGAGAAAGCCCCAGCAGCACCCCG CCAGAGGCGGCCCCGGGCGCAGGCCCCACGTGA The following amino acid sequence <SEQ ID NO.\",\n \"94> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"93: MDTTMEADLGATGHRPRTELDDEDSYPQGGWDTVFLVALLLLGLPANGLMAWLAGSQARHGAGTRLALLLLSL ALSDFLFLAAAAFQILEIRHGGHWPLGTAACRFYYFLWGVSYSSGLFLLAALSLDRCLLALCPHWYPGHRPVR LPLWVCAGVWVLATLFSVPWLVFPEAAVWWYDLVICLDFWDSEELSLRMLEVLGGFLPFLLLLVCHVLTQATA CRTCHRQQQPAACRGFARVARTILSAYVVLRLPYQLAQLLYLAFLWDVYSGYLLWEALVYSDYLILLNSCLSP FLCLMASADLRTLLRSVLSSFAAALCEERPGSFTPTEPQTQLDSEGPTLPEPMAEAQSQMDPVAQPQVNPTLQ PRSDPTAQPQLNPTAQPQSDPTAQPQLNLMAQPQSDSVAQPQADTNVQTPAPAA The following DNA sequence nGPCR-14 <SEQ ID NO.\",\n \"191> was identified in H. sapiens: ACTAACTTTGGGAACTCGTATAGACCCAGCGTCGCTCCCCGCGCCGCCTCGCCTCCACTTTGGTTTCCCGCGT CCTGCCCGCCCTCTTCGGTGCCTCCTCTTCCTCCGGGACAAGGATGGAGGATCTCTTTAGCCCCTCAATTCTG CCGCCGGCGCCCAACATTTCCGTGCCCATCTTGCTGGGCTGGGGTCTCAACCTGACCTTGGGGCAAGGACCCC CTGCCTCTGGGCCGCCCAGCCCGCGTGCGGGGGCACGGCGCTGTCACAGCTGGCCTGGGAACTGCTGGGCGAG CCCCGCGCGGCCACGGGGGACCTGGCGTGCCGCTTCCTGCAGCTGCTGCAGGCATCCGGGCGGGGCGCCTCGG CCCACCTAGTGGTGCTCATCGCCCTCGAGCGCCGGCGCGCGGTGCGTCTTCCGCACGGCCGGCCGCTGCCCGC GCGTGCCCTCGCCGCCCTGGGCTGGCTGCTGGCACTGCTGCTGGCGCTGCCCCCGGCCTTCGTGGTGCGCGGG GACTCCCCCTCGCCGCTGCCGCCGCCGCCGCCGCCAACGTCCCTGCAGCCAGGCGCGCCCCCGGCCGCCCGCG CCTGGCCGGGGGAGCGTCGCTGCCACGGGATCTTCGCGCCCCTGCCGCGCTGGCACCTGCAGGTCTACGCGTT CTACGAGGCCGTCGCGGGCTTCGTCGCGCCTGTTACGGTCCTGGGCGTCGCTTGCGGCCACCTACTCTCCGTC TGGTGGCGGCACCGGCCGCAGGCCCCCGCGGCTGCAGCGCCCTGGTCGGCGAGCCCAGGTCGAGCCCCTGCGC CCAGCGCGCTGCCCCGCGCCAAGGTGCAGAGCCTGAAGATGAGCCTGCTGCTGGCGCTGCTGTTCGTGGGCTG CGAGCTGCCCTACTTTGCCGCCCGGCTGGCGGCCGCGTGGTCGTCCGGGCCCGCGGGAGACTGGGAGGGAGAG GGCCTGTCGGCGGCGCTGCGCGTGgTGGCGATGGCCAACAGCGCTCTCAATCCCTTCGTCTACCTCTTCTTCC AGGCGGGCGACTGCTGGCTCCGGCGACAGCTGCGGAAGCGGCTGGGCTCTCTGTGCTGCGCGCCGCAGGGAGG CGCGGAGGACGAGGAGGGGCCCCGGGGCCACCAGGCGCTCTACCGCCAACGCTGGCCCCACCCTCATTATCAC CATGCTCGGCGGGAACCCGCTGGACGAGGGCGGCTTGCGCCCACCCCCTCCGCGCCCCAGACCCCTGCCTTGC TCCTGCGAAAGTGCCTTCTAGGTGCTTGGTGGTCAGAGACGGGTCATCTGTCGCTAAGGCGCAACCTCCAGGG AACTCGAGGCCTGCCAGGGTCTGTCCAGATCACAAGGGGCAGGAGAGTCTGTGAGAGAGTGACACTGAAGTTG TCCCCTTCCTCCACTCTCCTATTCCCTTCTCATGTTTACATTTCCCTATGCTCTTCCAGTTTCTCTTCTTCCC TACAGTTCCTCTCATATCTCCCCATTTGGAGACAGTGAGCCACTGGAAAGTTGTAAAAACAAAAACAGTTATT TTTGCAGTTTTCTTTCACGCATTTATAGTGCTCTGGATAATGCCATTTATTTTTGCTGATTACCCAACTTTCA GTATTTGCTGTGTTATCATCTGTATTTACTTATTTTGA The following amino acid sequence <SEQ ID NO.\",\n \"192> is the predicted amino acid sequence derived from the DNA sequence of SEQ ID NO.\",\n \"191: MEDLFSPSILPPAPNISVPILLGWGLNLTLGQGAPASGPPSRRVRLVFLGVILVVAVAGNTTVLCRLCGGGG PWAGPKRRKMDFLLVQLALADLYACGGTALSQLAWELLGEPRAATGDLACRFLQLLQASGRGASAHLVVLIA LERRRAVRLPHGRPLPARALAALGWLLALLLALPPAFVVRGDSPSPLPPPPPPTSLQPGAPPAARAWPGERR CHGIFAPLPRWHLQVYAFYEAVAGFVAPVTVLGVACGHLLSVWWRHRPQAPAAAAPWSASPGRAPAPSALPR AKVQSLKMSLLLALLFVGCELPYFAARLAAAWSSGPAGDWEGEGLSAALRVVAMANSALNPFVYLFFQAGDC WLRRQLRKRLGSLCCAPQGGAEDEEGPRGHQALYRQRWPHPHYHHARREPAGRGRLAPTPSAPQTPALLLRK CLLGAWWSETGHLSLRRNLQGTRGLPGSVQITRGRRVCERVTLKLSPSSTLLFPSHVYISLCSSSFSSSLQF LSYLPIWRQ Example 2 Cloning of nGPCR-x To isolate a cDNA clone encoding full length nGPCR-x, a DNA fragment corresponding to a nucleotide sequence set forth in odd numbered nucleotide sequences ranging from SEQ ID NO: 1-93, or a portion thereof, can be used as a probe for hybridization screening of a phage cDNA library.\",\n \"The DNA fragment is amplified by the polymerase chain reaction (PCR) method.\",\n \"The PCR reaction mixture of 50 μl contains polymerase mixture (0.2 mM dNTPs, 1×PCR Buffer and 0.75 μl Expand High Fidelity Polymerase (Roche Biochemicals)), 1 μg of plasmid, and 50 pmoles of forward primer and 50 pmoles of reverse primer.\",\n \"The primers are preferably 10 to 25 nucleotides in length and are determined by procedures well known to those skilled in the art.\",\n \"Amplification is performed in an Applied Biosystems PE2400 thermocycler, using the following program: 95° C. for 15 seconds, 52° C. for 30 seconds and 72° C. for 90 seconds; repeated for 25 cycles.\",\n \"The amplified product is separated from the plasmid by agarose gel electrophoresis, and purified by Qiaquick™ gel extraction kit (Qiagen).\",\n \"A lambda phage library containing cDNAs cloned into lambda ZAPII phagovector is plated with E. coli XL-1 blue host, on 15 cm LB-agar plates at a density of 50,000 pfu per plate, and grown overnight at 37° C.; (plated as described by Sambrook et al., supra).\",\n \"Phage plaques are transferred to nylon membranes (Amersham Hybond NJ), denatured for 2 minutes in denaturation solution (0.5 M NaOH, 1.5 M NaCl), renatured for 5 minutes in renaturation solution (1 M Tris pH 7.5, 1.5 M NaCl), and washed briefly in 2×SSC (20×SSC: 3 M NaCl, 0.3 M Na-citrate).\",\n \"Filter membranes are dried and incubated at 80° C. for 120 minutes to cross-link the phage DNA to the membranes.\",\n \"The membranes are hybridized with a DNA probe prepared as described above.\",\n \"A DNA fragment (25 ng) is labeled with α-32P-dCTP (NEN) using Rediprime™ random priming (Amersham Pharmacia Biotech), according to manufacturers instructions.\",\n \"Labeled DNA is separated from unincorporated nucleotides by S200 spin columns (Amersham Pharmacia Biotech), denatured at 95° C. for 5 minutes and kept on ice.\",\n \"The DNA-containing membranes (above) are pre-hybridized in 50 ml ExpressHyb™ (Clontech) solution at 68° C. for 90 minutes.\",\n \"Subsequently, the labeled DNA probe is added to the hybridization solution, and the probe is left to hybridize to the membranes at 68° C. for 70 minutes.\",\n \"The membranes are washed five times in 2×SSC, 0.1% SDS at 42° C. for 5 minutes each, and finally washed 30 minutes in 0.1×SSC, 0.2% SDS.\",\n \"Filters are exposed to Kodak XAR™ film (Eastman Kodak Company, Rochester, N.Y., USA) with an intensifying screen at −80° C. for 16 hours.\",\n \"One positive colony is isolated from the plates, and replated with about 1000 pfu on a 15 cm LB plate.\",\n \"Plating, plaque lift to filters and hybridization are performed as described above.\",\n \"About four positive phage plaques are isolated form this secondary screening.\",\n \"cDNA containing plasmids (pBluescript SK−) are rescued from the isolated phages by in vivo excision by culturing XL-1 blue cells co-infected with the isolated phages and with the Excision helper phage, as described by manufacturer (Stratagene).\",\n \"XL-blue cells containing the plasmids are plated on LB plates and grown at 37° C. for 16 hours.\",\n \"Colonies (18) from each plate are replated on LB plates and grown.\",\n \"One colony from each plate is stricken onto a nylon filter in an ordered array, and the filter is placed on a LB plate to raise the colonies.\",\n \"The filter is then hybridized with a labeled probe as described above.\",\n \"About three positive colonies are selected and grown up in LB medium.\",\n \"Plasmid DNA is isolated from the three clones by Qiagen Midi Kit™ (Qiagen) according to the manufacturer's instructions.\",\n \"The size of the insert is determined by digesting the plasmid with the restriction enzymes NotI and SalI, which establishes an insert size.\",\n \"The sequence of the entire insert is determined by automated sequencing on both strands of the plasmids.\",\n \"nGPCR-1: PCR and Subcloning cDNAs were sequenced directly using an AB1377 fluorescence-based sequencer (Perkin Elmer/Applied Biosystems Division, PE/ABD, Foster City, Calif.) and the ABI PRISM Ready Dye-Deoxy Terminator kit with Taq FS polymerase.\",\n \"Each ABI cycle sequencing reaction contained about 0.5 μg of plasmid DNA.\",\n \"Cycle-sequencing was performed using an initial denaturation at 98° C. for 1 min, followed by 50 cycles: 99° C. for 30 sec, annealing at 50° C. for 30 sec, and extension at 60° C. for 4 min.\",\n \"Temperature cycles and times were controlled by a Perkin-Elmer 9600 thermocycler.\",\n \"Extension products were purified using AGTC® gel filtration block (Edge BiosSystems, Gaithersburg, Md.).\",\n \"Each reaction product was loaded by pipette onto the column, which was then centrifuged in a swinging bucket centrifuge (Sorvall model RT6000B table top centrifuge) at 1500×g for 4 min at room temperature.\",\n \"Column-purified samples were dried under vacuum for about 40 min and then dissolved in 5 μl of a DNA loading solution (83% deionized formamide, 8.3 mM EDTA, and 1.6 mg/ml Blue Dextran).\",\n \"The samples were then heated to 90° C. for three min and loaded into the gel sample wells for sequence analysis by the ABI377 sequencer.\",\n \"Sequence analysis was performed by importing ABI373A files into the Sequencher program (Gene Codes, Ann Arbor, Mich.).\",\n \"The PCR reaction was performed in 50 μL samples containing 41.9 μL H2O, 5 μL 10× Buffer containing 15 mM MgCl2 (Boehringer Mannheim Expand High Fidelity PCR System), 0.5 μL 10 mM dNTP mix, 1.5 μL human genomic DNA (Clontech #6550-1, 0.1 μg/μL), 0.3 μL primer VR1A (1 μg/μL), 0.3 μL primer VR1B (1 μg/μL), and 0.5 μL High Fidelity Taq polymerase (Boehringer Mannheim, 3.5U/μl).\",\n \"The primer sequences for and, respectively were: 5′TCAAAGCTTATGGAATCATCTTTCTCATTTGGAGTGATCCTTGCTGTC, (VR1A) (SEQ ID NO: 95) corresponding to the 5′ end of the coding region and containing a HindIII restriction site, and: 5′ TTCACTCGAGTTAGCCATCAAACTCTGAGCTGGAGATAGTGACGATGTG (VR1B) (SEQ ID NO: 96) corresponding to the 3′ end of the coding region and containing an XhoI restriction site (Genosys).\",\n \"The PCR reaction was carried out using a GeneAmp PCR9700 thermocycler (Perkin Elmer Applied Biosystems) and started with 1 cycle of 94° C. for 2 min followed by 5 cycles at 94° C. for 30 sec, 60° C. for 2 min, 72° C. for 1.5 min, followed by 20 cycles at 94° C. for 30 sec, 60° C. for 30 sec, 72° C. for 1.5 min.\",\n \"The PCR reaction was loaded onto a 0.75% agarose gel.\",\n \"The DNA band was excised from the gel and the DNA eluted from the agarose using a QIAquick gel extraction kit (Qiagen).\",\n \"The eluted DNA was ethanol-precipitated and resuspended in 4 μL H2O for ligation.\",\n \"The ligation reaction consisted of 4 μL of fresh ethanol-precipitated PCR product and 1 μL of pCRII-TOPO vector (Invitrogen).\",\n \"The reaction was gently mixed and allowed to incubate for 5 min.\",\n \"at room temperature followed by the addition of 1 μL of 6×TOPO cloning stop solution and mixing for 10 sec.\",\n \"at room temperature.\",\n \"The sample was then placed on ice and 2 μL was transformed in 50 μL of One Shot cells (Invitrogen) and plated onto ampicillin plates.\",\n \"Four white colonies were chosen and the presence of an insert was verified by PCR in the following manner.\",\n \"Each colony was resuspended in 2 ml LB broth for 2 hrs.\",\n \"A 500 μL aliquot was spun down in the microfuge, the supernatant discarded, and the pellet resuspended in 25 μL of H2O.\",\n \"A 16 μL aliquot was removed and boiled for 5 min and the sample was placed on ice.\",\n \"The sample was microfuged briefly to pellet any bacterial debris and PCR was carried out with 15 μL sample using primers VR1A and VR1B, described above.\",\n \"Colonies from positive clones identified by PCR were used to inoculate a 4 ml culture of LB medium containing 100 μg/ml ampicillin.\",\n \"Plasmid DNA was purified using the Wizard Plus Minipreps DNA purification system (Promega).\",\n \"Since the primers used to amplify the fragment of nGPCR-1 from genomic DNA were engineered to have HindIII and XhoI sites, the cDNA obtained from the minipreps was digested with these restriction enzymes.\",\n \"One clone was verified by gel electrophoresis to give a DNA band of the correct size.\",\n \"cDNA from this clone was then sequenced, yielding the sequence of SEQ ID NO: 73.nGPCR-3: PCR and Subcloning First-strand cDNA synthesis was performed following the directions for 3′-RACE ready cDNA from the SMART™ RACE cDNA Amplification Kit (Clontech).\",\n \"First 3 μl of H2O, 1 μl human whole brain poly A+ RNA (1 μg/μl) (Clontech, 6516-1) and 1 μl 3′-CDS primer were mixed together, incubated at 70° C. for 2 minutes, then placed on ice for 2 minutes.\",\n \"Added to the tube was 2 μl 5× First-Strand buffer, 1 μl 20 mM DTT, 1 μl dNTP mix (10 mM) and 1 μl Superscript II RT (200 units/μl) (GIBCO/BRL).\",\n \"The tube was incubated at 42° C. for 1.5 hours then the reaction was diluted with 250 μl of Tricine-EDTA buffer.\",\n \"PCR was performed in a 50 μl reaction using components that come with the Advantage®-GC cDNA PCR Kit.\",\n \"The PCR reaction contained 22.4 μl H2O, 10 μl 5×GC cDNA PCR Reaction buffer, 10 μl 5M GC Melt, 1 μl 50×dNTP mix (10 mM each), 5 μl human brain cDNA, 0.3 μl of LW1649 (SEQ ID NO: 187) (1 μg/μl), 0.3 μl of LW1650 (SEQ ID NO: 188) (1 μg/μl), 1 μl 50× Advantage-GC cDNA polymerase mix.\",\n \"The PCR reaction was performed in a Perkin-Elmer 9600 GeneAmp PCR System starting with 1 cycle of 94° C. for 2 min then 8 cycles at 94° C. for 15 sec, 72° C. for 2 min (decreasing 1° C. with each cycle), 72° C. for 3 min, followed by 30 cycles of 94° C. for 15 sec, 68° C. for 3 min.\",\n \"The PCR reaction was loaded onto a 1.2% agarose gel.\",\n \"The DNA band was excised from the gel, placed in GenElute Agarose spin column (Supelco) and spun for 10 min at maximum speed in a microcentrifuge.\",\n \"The eluted DNA was EtOH precipitated and resuspended in 4H2O for ligation.\",\n \"The PCR primer sequence for LW1649 was: GCATAAGCTTGCCATGGGCCCCGGCGAGG (SEQ ID NO: 187) and for LW1650 was: GCATTCTAGACCTCAGTGTGTCTGCTGC (SEQ ID NO: 188).\",\n \"The underlined portion of the primers matches the 5′ and 3′ areas, respectively, of the coding region.\",\n \"The ligation reaction used solutions from the TOPO TA Cloning Kit (Invitrogen) which consisted of 4 μl PCR product DNA, 1 μl Salt Solution and 1 μl pCRII-TOPO vector that was incubated for 5 minutes at room temperature and then placed on ice.\",\n \"Two microliters of the ligation reaction was transformed in One-Shot TOP10 cells (Invitrogen), and placed on ice for 30 minutes.\",\n \"The cells were heat-shocked for 30 seconds at 42° C., placed on ice for two minutes, 250 μl of SOC was added, then incubated at 37° C. with shaking for one hour and then plated onto ampicillin plates.\",\n \"A single colony containing an insert was used to inoculate a 5 ml culture of LB medium.\",\n \"Plasmid DNA was purified using a Concert Rapid Plasmid Miniprep System (GibcoBRL) and then sequenced.\",\n \"The DNA subcloned into pCRII-TOPO was sequenced using the ABI PRISM™ 310 Genetic Analyzer (PE Applied Biosystems) which uses advanced capillary electrophoresis technology and the ABI PRISM™ BigDye™ Terminator Cycle Sequencing Ready Reaction Kit.\",\n \"Each cycle-sequencing reaction contained 6 μl of H2O, 8 μl of BigDye Terminator mix, 5 μl mini-prep DNA (0.1 μg/μl), and 1 μl primer (25 ng/μl) and was performed in a Perkin-Elmer 9600 thermocycler with 25 cycles of 96° C. for 10 sec, 50° C. for 10 sec, and 60° C. for 4 min.\",\n \"The product was purified using a Centriflex™ gel filtration cartridge, dried under vacuum, then dissolved in 16 μl of Template Suppression Reagent (PE Applied Biosystems).\",\n \"The samples were heated at 95° C. for 5 min then placed in the 310 Genetic Analyzer, yielding the sequence of SEQ ID NO: 95.nGPCR-9: PCR and Subcloning The PCR reaction was performed in 50 μl containing 34.5 μl H2O, 5 μl Buffer II (PE Applied Biosystems AmpliTaq Gold system), 6 μl 25 mM MgCl2, 2 μl 10 mM dNTP mix, 1.5 μl human genomic DNA (Clontech #6550-1, 0.1 μg/μl), 0.3 μl primer VR9A (1 μg/μl), 0.3 μl primer VR9B (1 μg/μl), and 0.4 μl AmpliTaq Gold™ DNA Polymerase.\",\n \"The primer sequences for VR9A and VR9B were as follows: VR9A 5′TTCAAAGCTTATGGAGTCGGGGCTGCTG 3′ (SEQ ID NO: 101), corresponding to the 5′ end of the coding region and containing a HindIII restriction site, and the reverse primer was: VR9B 5′ TTCACTCGAGTCAGTCTGCAGCCGGTTCTG 3′, (SEQ ID NO: 102), corresponding to the 3′ end of the coding region and containing an XhoI restriction site (Genosys).\",\n \"The PCR reaction was carried out using a GeneAmp PCR 9700 thermocycler (Perkin Elmer Applied Biosystems) and started with 1 cycle of 95° C. for 10 min, then 10 cycles at 95° C. for 30 sec, 72° C. for 2 min decreasing 1° C. each cycle, 72° C. for 1 min.\",\n \"followed by 30 cycles at 95° C. for 30 sec, 60° C. for 30 sec, 72° C. for 1 min.\",\n \"The PCR reaction was loaded on a 0.75% gel.\",\n \"The DNA band was excised from the gel and the DNA was eluted from the agarose using a QIAquick gel extraction kit (Qiagen).\",\n \"The eluted DNA was ethanol-precipitated and resuspended in 4 μl H2O for ligation.\",\n \"The ligation reaction consisted of 4 μl of fresh ethanol-precipitated PCR product and 1 μl of pCRII-TOPO vector (Invitrogen).\",\n \"The reaction was gently mixed and allowed to incubate for 5 min at room temperature followed by the addition of 1 μl of 6×TOPO cloning stop solution and mixing for 10 sec at room temperature.\",\n \"The sample was then placed on ice and 2 μl was transformed in 50 μl of One Shot cells (Invitrogen) and plated onto ampicillin plates.\",\n \"Five white colonies were chosen and were used to inoculate a 4 ml culture of LB medium containing 100 μg/ml ampicillin.\",\n \"Plasmid DNA was purified using the Wizard Plus Minipreps DNA purification system (Promega).\",\n \"Since the primers used to PCR SEQ-9 from genomic DNA were engineered to have HindIII and XhoI sites, the cDNA obtained from the minipreps was digested with these restriction enzymes.\",\n \"One clone was verified by gel electrophoresis to give a DNA band of the correct size.\",\n \"cDNA from this clone was then submitted for sequencing.\",\n \"One mutation was found (bp 621 T→G) and repaired as described as below.\",\n \"The mutation in the identified clone was repaired using the QuikChange Site-Directed Mutagenesis Kit (Stratagene).\",\n \"The PCR reaction contained 39.3 μl H2O, 5 μl 10× reaction buffer, 50 ng mini-prep cDNA, 1.25 μl primer VR9E (100 ng/μl), 1.25 μl primer VR9F (100 ng/μl), 1 μl 20 mM dNTP mix, 1 μl Pfu DNA polymerase.\",\n \"The cycle conditions were 95° C. for 30 sec, then 12 cycles at 95° C. for 30 sec, 55° C. for 1 min, 68° C. for 10 min.\",\n \"One μl of DpnI was added and the tube incubated at 37° C. for 1 hr.\",\n \"One μl of the DpnI-treated DNA was transformed into 50 μl Epicurian coli XL1-Blue supercompetent cells and the entire insert was re-sequenced.\",\n \"The primer sequences used were: VR9E: 5′ GCATCCTGGCCGCTATCTGTGCACTCTACG 3′ (SEQ ID NO: 103) and VR9F: 5′ CGTAGAGTGCACAGATAGCGGCCAGGATGC 3′ (SEQ ID NO: 104) where the base underlined was the base being corrected.\",\n \"The clone described above was sequenced directly using an ABI377 fluorescence-based sequencer (Perkin Elmer/Applied Biosystems Division, PE/ABD, Foster City, Calif.) and the ABI BigDye™ Terminator Cycle Sequencing Ready Reaction kit with Taq FS™ polymerase.\",\n \"Each ABI cycle sequencing reaction contained 0.5 μg of plasmid DNA.\",\n \"Cycle-sequencing was performed using an initial denaturation at 98° C. for 1 min, followed by 50 cycles: 96° C. for 30 sec, annealing at 50° C. for 30 sec, and extension at 60° C. for 4 min.\",\n \"Temperature cycles and times were controlled by a Perkin-Elmer 9600 thermocycler.\",\n \"Extension products were purified using AGTC (R) gel filtration block (Edge BiosSystems, Gaithersburg, Md.).\",\n \"Each reaction product was loaded by pipette onto the column, which was then centrifuged in a swinging bucket centrifuge (Sorvall model RT6000B tabletop centrifuge) at 1500×g for 4 min at room temperature.\",\n \"Column-purified samples were dried under vacuum for about 40 min and then dissolved in 3 μl of a DNA loading solution (83% deionized formamide, 8.3 mM EDTA, and 1.6 mg/ml Blue Dextran).\",\n \"The samples were then heated to 90° C. for 3.5 min and loaded into the gel sample wells for sequence analysis by the ABI377 sequencer.\",\n \"Sequence analysis was performed by importing ABI377 files into the 310 Genetic Analyzer, yielding the sequence of SEQ ID NO: 77.nGPCR-11: PCR and Subcloning PCR was performed in a 50 g reaction containing 32 μl H2O, 5 A 10×TT buffer (140 mM Ammonium Sulfate, 0.1% gelatin, 0.6 M Tris-tricine pH 8.4), 5 μl 15 mM MgSO4, 2 μl 10 mM dNTP, 5 μl human genomic DNA (0.3 μg/μL) (Clontech), 0.3 μl of LW1564 (1 μg/μl), 0.3 μl of LW1565 (1 μg/μl), 0.4 μl High Fidelity Taq polymerase (Boehringer Mannheim).\",\n \"The PCR reaction was performed in a GeneAmp 9600 PCR thermocycler (PE Applied Biosystems) starting with 1 cycle of 94° C. for 2 min followed by 17 cycles at 94° C. for 30 sec, 72° C. for 2 min decreasing 1° C. each cycle, 68° C. for 2 min, then 25 cycles of 94° C. for 30 sec, 55° C. for 30 sec, 68° C. for 2 min.\",\n \"The PCR reaction was loaded onto a 1.2% agarose gel.\",\n \"The DNA band was excised from the gel, placed in GenElute Agarose spin column (Supelco) and spun for 10 min at maximum speed in a microcentrifuge.\",\n \"The eluted DNA was EtOH precipitated and resuspended in 4 μl H2O for ligation.\",\n \"The forward PCR primer sequence was: LW1564: GCATAAGCTTCCATGTACAACGGGTCGTGCTGC (SEQ ID NO: 107), and the reverse PCR primer was: LW1565: GCATTCTAGATCAGTGCCACTCAACAATGTGGG (SEQ ID NO: 108).\",\n \"The ligation reaction used solutions from the TOPO TA Cloning Kit (vitrogen) which consisted of 4 μl PCR product DNA and 1 μl pCkII-TOPO vector that was incubated for 5 minutes at room temperature.\",\n \"To the ligation reaction one microliter of 6×TOPO Cloning Stop Solution was added then the reaction was placed on ice.\",\n \"Two microliters of the ligation reaction was transformed in One Shot TOP10 cells (Invitrogen), and placed on ice for 30 minutes.\",\n \"The cells were heat-shocked for 30 seconds at 42° C., placed on ice for two minutes, 250 Tl of SOC was added, then incubated at 37° C. with shaking for one hour and then plated onto ampicillin plates.\",\n \"A single colony containing an insert was used to inoculate a 5 ml culture of LB medium.\",\n \"Plasmid DNA was purified using a Concert Rapid Plasmid Miniprep System (GibcoBRL) and then sequenced.\",\n \"The DNA subcloned into pCRII was sequenced using the ABI PRISM™ 310 Genetic Analyzer (PE Applied Biosystems) which uses advanced capillary electrophoresis technology and the ABI PRISM™ BigDye™ Terminator Cycle Sequencing Ready Reaction Kit.\",\n \"Each cycle-sequencing reaction contained 6 μl of H2O, 8 μl of BigDye Terminator mix, 5 μl mini-prep DNA (0.1 μg/μl), and 1 μl primer (25 ng/11) and was performed in a Perkin-Elmer 9600 thermocycler with 25 cycles of 96° C. for 10 sec, 50° C. for 10 sec, and 60° C. for 4 min.\",\n \"The product was purified using a Centriflex™ gel filtration cartridge, dried under vacuum, then dissolved in 16 μl of Template Suppression Reagent (PE Applied Biosystems).\",\n \"The samples were heated at 95° C. for 5 min then placed in the 310 Genetic Analyzer, yielding the sequence of SEQ ID NO: 79.nGPCR-16: PCR and Subcloning PCR was performed in a 50 μl reaction containing 32 μl H2O, 5 μl 10×TT buffer (140 mM Ammonium Sulfate, 0.1% gelatin, 0.6 M Tris-tricine pH 8.4), 5 μl 15 mM MgSO4, 2 μl 10 mM dNTP, 5 μl 2445704H1 DNA (0.17 Tg/Tl), 0.3 μl of LW1587 (1 μg/μl), 0.3 μl of LW1588 (1 μg/μl), 0.4 μl High Fidelity Taq polymerase (Boehringer Mannheim).\",\n \"The PCR reaction was performed on a Robocycler thermocycler (Stratagene) starting with 1 cycle of 94° C. for 2 min followed by 15 cycles of 94° C. for 30 sec, 55° C. for 1.3 min, 68° C. for 2 min.\",\n \"The PCR reaction was loaded onto a 1.2% agarose gel.\",\n \"The DNA band was excised from the gel, placed in GenElute Agarose spin column (Supelco) and spun for 10 min at maximum speed in a microcentrifuge.\",\n \"The eluted DNA was EtOH precipitated and resuspended in 12 μl H2O for ligation.\",\n \"The PCR primer sequence for the forward primer was: LW1587: GATCAAGCTTATGACAGGTGACTTCCCAAGTATGC (SEQ ID NO: 111), and the sequence for the reverse primer was: LW1588: GATCCTCGAGGCTAACGGCACAAAACACAATTCC (SEQ ID NO: 112).\",\n \"The ligation reaction used solutions from the TOPO TA Cloning Kit (Invitrogen) which consisted of 4 μl PCR product DNA and 1 μl pCRII-TOPO vector that was incubated for 5 minutes at room temperature.\",\n \"To the ligation reaction one microliter of 6×TOPO Cloning Stop Solution was added then the reaction was placed on ice.\",\n \"Two microliters of the ligation reaction was transformed in One-Shot TOP10 cells (Invitrogen), and placed on ice for 30 minutes.\",\n \"The cells were heat-shocked for 30 seconds at 42° C., placed on ice for two minutes, 250 μl of SOC was added, then incubated at 37° C. with shaking for one hour and then plated onto ampicillin plates.\",\n \"A single colony containing an insert was used to inoculate a 5 ml culture of LB medium.\",\n \"Plasmid DNA was purified using a Concert Rapid Plasmid Miniprep System (GibcoBRL) and then sequenced.\",\n \"The DNA subcloned into pCRII was sequenced using the ABI PRISM™ 310 Genetic Analyzer (PE Applied Biosystems) which uses advanced capillary electrophoresis technology and the ABI PRISM™ BigDye™ Terminator Cycle Sequencing Ready Reaction Kit.\",\n \"Each cycle-sequencing reaction contained 6 μl of H2O, 8 μl of BigDye Terminator mix, 5 μl mini-prep DNA (0.1 μg/μl), and 1 μl primer (25 ng/μl) and was performed in a Perkin-Elmer 9600 thermocycler with 25 cycles of 96° C. for 10 sec, 50° C. for 10 sec, and 60° C. for 4 min.\",\n \"The product was purified using a Centriflex™ gel filtration cartridge, dried under vacuum, then dissolved in 16 μl of Template Suppression Reagent (PE Applied Biosystems).\",\n \"The samples were heated at 95° C. for 5 min then placed in the 310 Genetic Analyzer, yielding the sequence of SEQ ID NO: 81.nGPCR-40: PCR and Subcloning PCR was performed in a 50 μl reaction containing utilizing Herculase DNA Polymerase blend (Stratagene), using the buffer recommendations provided by the manufacturer, 200 ng each of primers PSK 18 and 19 (SEQ ID NOS: 115 and 116), 150 ng of human genomic DNA (Clontech), and 2% DMSO.\",\n \"The PCR reaction was performed on a Robocycler thermocycler (Stratagene) starting with 1 cycle of 94° C. for 2 min followed by 35 cycles of 94° C. for 30 sec, 65° C. for.\",\n \"30 sec, 72° C. for 2 min.\",\n \"The PCR reaction was purified using the QiaQuick PCR Purification Kit (Qiagen), and then eluted in TE.\",\n \"The PCR primer sequences were: PSK 18 GATC GAATTCGCAGGAGCAATG AAAATCAGGAAC (SEQ ID NO: 115), and: PSK19: GATCGAATTCTTATATATGTTCAGAAAACAAATTCATGG (SEQ ID NO: 116)).\",\n \"The underlined portion of the primer matches the 5′ and 3′ areas, respectively, of a portion of the 5′ untranslated region and coding region.\",\n \"Initiation and termination codons are shown above in bold.\",\n \"The PCR product was ligated into the pCR-BluntII-TOPO vector (Invitrogen) using the Zero Blunt Topo PCR TA cloning kit as follow: 3 μl PCR product DNA, 1 μl pCRII-TOPO vector, and 1 μl TOPOII salt solution (1.2M NaCl, 0.06M MgCl2).\",\n \"The mixture was incubated for 5 minutes at room temperature.\",\n \"To the ligation reaction one microliter of 6×TOPO Cloning Stop Solution was added, and then the reaction was placed on ice.\",\n \"Two microliters of the ligation reaction was transformed in One-Shot TOP10 cells (Invitrogen), and placed on ice for 30 minutes.\",\n \"The cells were heat-shocked for 30 seconds at 42° C., placed on ice for two minutes, 250 μl of SOC was added, then incubated at 37° C. with shaking for one hour and then plated onto ampicillin plates supplemented with Xgal and IPTG.\",\n \"Single colonies were screened by PCR for the presence of the insert, and a plasmid DNA from colony 58 was purified using a Qiagen Endo-Free plasmid purification kit.\",\n \"nGPCR-40 was sequenced directly using an ABI377 fluorescence-based sequencer (Perkin Elmer/Applied Biosystems Division, PE/ABD, Foster City, Calif.) and the ABI BigDye™ Terminator Cycle Sequencing Ready Reaction kit with Taq FS™ polymerase.\",\n \"Each ABI cycle sequencing reaction contained about 0.5 μg of plasmid DNA.\",\n \"Cycle-sequencing was performed using an initial denaturation at 98° C. for 1 min, followed by 50 cycles: 96° C. for 30 sec, annealing at 50° C. for 30 sec, and extension at 60° C. for 4 min.\",\n \"Temperature cycles and times were controlled by a Perkin-Elmer 9600 thermocycler.\",\n \"Extension products were purified using AGTC® gel filtration block (Edge BiosSystems, Gaithersburg, Md.).\",\n \"Each reaction product was loaded by pipette onto the column, which was then centrifuged in a swinging bucket centrifuge (Sorvall model RT6000B tabletop centrifuge) at 1500×g for 4 min at room temperature.\",\n \"Column-purified samples were dried under vacuum for about 40 min and then dissolved in 3 μl of DNA loading solution (83% deionized formamide, 8.3 mM EDTA, and 1.6 mg/ml Blue Dextran).\",\n \"The samples were then heated to 90° C. for 3.5 min and loaded into the gel sample wells for sequence analysis by the ABI377 sequencer.\",\n \"Sequence analysis was performed by importing ABI377 files into the Sequencher program (Gene Codes, Aim Arbor, M), which yielded a sequence identical to SEQ ID NO:83 with the exception that the nucleotide at position 10 was identified as an “A” which incorrectly indicated the presence of an initiation codon at that position.\",\n \"Subsequent analysis of genomic DNA samples indicated that this position was incorrectly assigned and that the correct nucleotide at that position was a “C”.\",\n \"The sequence reported at SEQ ID NO.\",\n \"83 correctly identifies the nucleotide at position 10 and indicates that the first initiation codon occurs at position 88-90.nGPCR-54: PCR and Subcloning Two microliters of a human genomic library (˜108 PFU/ml) (Clontech) was added to 6 ml of an overnight culture of K802 cells (Clontech), then distributed as 250 μl aliquots into each of 24 tubes.\",\n \"The tubes were incubated at 37° C. for 15 min.\",\n \"Seven milliliters of 0.8% agarose was added to each tube, mixed, then poured onto LB agar+10 mM MgSO4 plates and incubated overnight at 37° C. To each plate 5 ml of SM (0.1M NaCl, 8.1 mM MgSO4-7H2O, 50 mM Tris-Cl (pH 7.5), 0.0001% gelatin) phage buffer was added and the top agarose was removed with a microscope slide and placed in a 50 ml centrifuge tube.\",\n \"A drop of chloroform was added and the tube was place in a 37° C. shaker for 15 min, then centrifuged for 20 min at 4000 RPM (Sorvall RT6000 table top centrifuge) and the supernatant stored at 4° C. as a stock solution.\",\n \"Two μl of phage from each tube was heated to 99° C. for 4 min then cooled to 10° C. Added to the phage was a PCR mix containing 8.8 μl H2O, 4 μl 5× Rapid-load Buffer (Origene), 2 μl 10×PCR buffer II (Perkin-Elmer), 2 μl 25 mM MgCl2, 0.8 μl 10 mM dNTP, 0.12 μl LW1634 (1 μg/μl) (SEQ ID NO: 119), 0.12 μl LW1635 (1 μg/μl) (SEQ ID NO: 120), 0.2 μl AmpliTaq Gold polymerase (Perlin Elmer).\",\n \"The PCR reaction involved 1 cycle at 95° C. for 10 min followed by 35 cycles at 95° C. for 45 sec, 53.5° C. for 2 min, 72° C. for 45 sec.\",\n \"The reaction was loaded onto a 2% agarose gel.\",\n \"From the tube that gave a PCR product of the correct size, 10 μl was used to set up five 1:10 dilutions that were plated onto LB agar+10 mM MgSO4 plates and incubated overnight.\",\n \"A BA85 nitrocellulose filter (Schleicher & Schuell) was placed on top of each plate for 1 hour.\",\n \"The filter was removed, placed phage side up in a petri dish, and covered with 4 ml of SM for 15 min to elute the phage.\",\n \"One milliliter of SM was removed from each plate and used to set up a PCR reaction as above.\",\n \"The plate of the lowest dilution to give a PCR product was subdivided, filter-lifted and the PCR reaction was repeated.\",\n \"The series of dilutions and subdividing of the plate was continued until a single plaque was isolated that gave a positive PCR band.\",\n \"Once a single plaque was isolated, 10 μl phage supernatant was added to 100 μl SM and 200 μl of K802 cells per plate with a total of 8 plates set up.\",\n \"The plates were incubated overnight at 37° C. The top agarose was removed by adding 8 ml of SM then scrapping off the agarose with a microscope slide and collected in a centrifuge tube.\",\n \"To the tube, 3 drops of chloroform was added, vortexed, incubated at 37° C. for 15 min then centrifuged for 20 min at 4000 RPM (Sorvall RT6000 table top centrifuge) to recover the phage, which was used to isolate genomic phage DNA using the Qiagen Lambda Midi Kit.\",\n \"The sequence for primer LW1634 was: CTGAAAGTTGTCGCTGACC (SEQ ID NO: 119), and for primer LW1635 was: CGATTATCCACACTTTGACCC (SEQ ID NO: 120).\",\n \"The PCR reaction for the coding region was performed in a 50 μl reaction containing 33 μl H2O, 5 μl 10×TT buffer (140 mM Ammonium Sulfate, 0.1% gelatin, 0.6 M Tris tricine pH 8.4), 5 μl 15 mM MgSO4, 2 μl 10 mM DNTP, 4 μl genomic phage DNA (0.25 μg/μl), 0.3 μl LW1698 (1 μg/μl) (SEQ ID NO: 121), 0.3 μl LW1699 (1 μg/μl) (SEQ ID NO: 122), 0.4 μl High Fidelity Taq polymerase (Boehringer Mannheim).\",\n \"The PCR reaction was started with 1 cycle of 94° C. for 2 min followed by 30 cycles at 94° C. for 30 sec, 55° C. for 30 sec., 68° C. for 2 min.\",\n \"The PCR reaction was loaded onto a 2% agarose gel.\",\n \"The DNA band was excised from the gel, placed in GenElute Agarose spin column (Supelco) and spun for 10 min at maximum speed.\",\n \"The eluted DNA was EtOH precipitated and resuspended in 8 μl H2O.\",\n \"The PCR primer sequence for primer LW1698 was: GCATACCATGAATGAGCCACTAGAC (SEQ ID NO: 121), and for primer LW1699 was: GCATCTCGAGTCAAGGGTTGTTTGAGTAAC (SEQ ID NO: 122).\",\n \"The underlined portion of the primer matches the 5′ and 3′ areas, respectively, of the coding region of nGPCR-54.The ligation reaction used solutions from the TOPO TA Cloning Kit (Invitrogen) which consisted of 4 μl PCR product DNA, 1 μl of salt solution and 1 μl pCRII-TOPO vector that was incubated for 5 minutes at room temperature then the reaction was placed on ice.\",\n \"Two microliters of the ligation reaction was transformed in One-Shot TOP10 cells (Invitrogen), and placed on ice for 30 minutes.\",\n \"The cells were heat-shocked for 30 seconds at 42° C., placed on ice for two minutes, 250 μl of SOC was added, then incubated at 37° C. with shaking for one hour and then plated onto ampicillin plates.\",\n \"A single colony containing an insert was used to inoculate a 5 ml culture of LB medium.\",\n \"Plasmid DNA was purified using a Concert Rapid Plasmid Miniprep System (GibcoBRL) and then sequenced.\",\n \"nGPCR-54 genomic phage DNA was sequenced using the ABI PRISM™ 310 Genetic Analyzer (PE Applied Biosystems) which uses advanced capillary electrophoresis technology and the ABI PRISM™ BigDye™ Terminator Cycle Sequencing Ready Reaction Kit.\",\n \"The cycle-sequencing reaction contained 14 μl of H2O, 16 μl of BigDye Terminator mix, 7 μl genomic phage DNA (0.1 μg/μl), and 3 μl primer (25 ng/μl).\",\n \"The reaction was performed in a Perkin-Elmer 9600 thermocycler at 95° C. for 5 min, followed by 99 cycles of 95° C. for 30 sec, 55° C. for 20 sec, and 60° C. for 4 min.\",\n \"The product was purified using a Centriflex™ gel filtration cartridges, dried under vacuum, then dissolved in 16 μl of Template Suppression Reagent.\",\n \"The samples were heated at 95° C. for 5 min then placed in the 310 Genetic Analyzer.\",\n \"The DNA subcloned into pCRII was sequenced using the ABI PRISM™ 310 Genetic Analyzer (PE Applied Biosystems) which uses advanced capillary electrophoresis technology and the ABI PRISM™ BigDye™ Terminator Cycle Sequencing Ready Reaction Kit.\",\n \"Each cycle-sequencing reaction contained 6 μl of H2O, 8 μl of BigDye Terminator mix, 5 μl mini-prep DNA (0.1 μg/μl), and 1 μl primer (25 ng/μl) and was performed in a Perkin-Elmer 9600 thermocycler with 25 cycles of 96° C. for 10 sec, 50° C. for 10 sec, and 60° C. for 4 min.\",\n \"The product was purified using a Centriflex™ gel filtration cartridge, dried under vacuum, then dissolved in 16 μl of Template Suppression Reagent (PE Applied Biosystems).\",\n \"The samples were heated at 95° C. for 5 min then placed in the 310 Genetic Analyzer, yielding the sequence of SEQ ID NO: 85.nGPCR-56: PCR and Subcloning The PCR reaction for the coding region of nGPCR-56 used components that come with PLATINUM® Pfx DNA Polymerase (GibcoBRL) containing 35.5 μl H2O, 5 μl 10×Pfx Amplification buffer, 1.5 μl 50 mM MgSO4, 2 μl 10 mM &M, 5 μl human genomic DNA (0.3 μg/μl) (Clontech), 0.3 μl of LW1603 (1 μg/μl) (SEQ ID NO: 152), 0.3 μl of LW1604 (1 μg/μl) (SEQ ID NO: 153), 0.4 μl PLATINUM® Pfa DNA Polymerase (2.5 U/Tl).\",\n \"The PCR reaction was performed in a Robocycler Gradient 96 (Stratagene) starting with 1 cycle of 94° C. for 5 min followed by 30 cycles at 94° C. for 40 sec, 55° C. for 2 min, 68° C. for 3 min.\",\n \"Following the final cycle, 0.5 μl of AmpliTaq DNA Polymerase (5 U/μl) was added and the tube was incubated at 72° C. for 5 min.\",\n \"The sequence of LW1603 is: GATCAAGCTTGGAATGATGCCCTITTGCCAC (SEQ ID NO: 152), and for LW1604 is: GATCCTCGAGCATCATTCAAAGTAGGTGG.\",\n \"(SEQ ID NO: 153).\",\n \"The underlined portion of the primer matches the 5′ and 3′ areas, respectively, of a portion of the coding region of nGPCR-56.The PCR reaction for the coding region was performed in a 50 μl reaction containing 32 μl H2O, 5 μl 10×TT buffer (140 mM Ammonium Sulfate, 0.1% gelatin, 0.6 M Tris tricine pH 8.4), 5 μl 15 mM MgSO4, 2 μl 10 mM DNTP, 5 μl human genomic DNA (0.3 μg/μl) (Clontech), 0.3 μl LW1603 (1 μg/μl) (SEQ ID NO: 152), 0.3 μl LW1696 (1 μg/μl) (SEQ ID NO: 154), 0.4 μl High Fidelity Taq polymerase (Boehringer Mannheim).\",\n \"The PCR reaction was started with 1 cycle of 94° C. for 2 min followed by 25 cycles at 94° C. for 40 sec, 55° C. for 60 sec., 68° C. for 2 min.\",\n \"The PCR reaction was loaded onto a 2% agarose gel.\",\n \"The DNA band was excised from the gel, placed in GenElute Agarose spin column (Supelco) and spun for 10 min at maximum speed.\",\n \"The eluted DNA was EtOH precipitated and resuspended in 12 μl H2O for ligation.\",\n \"The PCR primer sequence for LW1603 is: GATCAAGCTTGGAATGATGCCCTTTTGCCAC (SEQ ID NO: 152), and LW1696: GATCCTCGAGCTATGAACTCAATTCCAAAAATAATTTACACC (SEQ ID NO: 154).\",\n \"The underlined portion of the primer matches the 5′ and 3′ areas, respectively, of a portion of the coding region.\",\n \"The ligation reaction used solutions from the TOPO TA Cloning Kit (Invitrogen) which consisted of 4 μl PCR product DNA, 1 μl of salt solution and 1 μl pCRH-TOPO vector that was incubated for 5 minutes at room temperature then the reaction was placed on ice.\",\n \"Two microliters of the ligation reaction was transformed in One-Shot TOP10 cells (Invitrogen), and placed on ice for 30 minutes.\",\n \"The cells were heat-shocked for 30 seconds at 42° C., placed on ice for two minutes, 250 μl of SOC was added, then incubated at 37° C. with shaking for one hour and then plated onto ampicillin plates.\",\n \"A single colony containing an insert was used to inoculate a 5 ml culture of LB medium.\",\n \"Plasmid DNA was purified using a Concert Rapid Plasmid Miniprep System (GibcoBRL) and then sequenced.\",\n \"The mutation in nGPCR-56 was repaired using the QuikChange Site-Directed Mutagenesis Kit (Stratagene).\",\n \"The PCR reaction contained 40 μl H2O, 5 μl 10× Reaction buffer, 1 μl mini-prep DNA, 1 μl LW1700 (125 ng/μl) (SEQ ID NO: 155), 1 μl LW1701 (125 ng/μl) (SEQ ID NO: 156), 1 μl 10 mM dNTP, 1 μl Pfu DNA polymerase.\",\n \"The cycle conditions were 95° C. for 30 sec then 14 cycles at 95° C. for 30 sec, 55° C. for 1 min, 68° C. for 12 min.\",\n \"The tube was placed on ice for 2 min, then 1 μl of DpnI was added and the tube incubated at 37° C. for one hour.\",\n \"One microliter of the DpnI-treated DNA was transformed into Epicurian coli XL1-Blue supercompetent cells and the entire insert was re sequenced.\",\n \"The primer sequences are: GCTACTTGAACTCTACATTTAATCCAATGGTTTATGCATITTTCTATCC (LW1700) (SEQ ID NO: 155), and: GGATAGAAAAATGCATAAACCATTGGATTAAATGTAGAGTTCAAGTAGC (LW1701) (SEQ ID NO: 156).\",\n \"The DNA subcloned into pCRII was sequenced using the ABI PRISM™ 310 Genetic Analyzer (PE Applied Biosystems) which uses advanced capillary electrophoresis technology and the ABI PRISM™ BigDye™ Terminator Cycle Sequencing Ready Reaction Kit.\",\n \"Each cycle-sequencing reaction contained 6 μl of H2O, 8 μl of BigDye Terminator mix, 5 μl mini-prep DNA (0.1 μg/μl), and 1 μl primer (25 ng/μl) and was performed in a Perkin-Elmer 9600 thermocycler with 25 cycles of 96° C. for 10 sec, 50° C. for 10 sec, and 60° C. for 4 min.\",\n \"The product was purified using a Centriflex™ gel filtration cartridge, dried under vacuum, then dissolved in 16 μl of Template Suppression Reagent (PE Applied Biosystems).\",\n \"The samples were heated at 95° C. for 5 min then placed in the 310 Genetic Analyzer, yielding the sequence of SEQ ID NO: 89.nGPCR-58: PCR and Subcloning Isolation of a clone for nGPCR-58 from genomic DNA was performed by PCR in a 50 μl reaction containing Herculase DNA Polymerse blend (Stratagene), with buffer recommendations as supplied by the manufacturer, 200 ng each primers PSK14 (SEQ ID NO: 157) and PSK15 (SEQ ID NO: 158), 150 ng of human genomic DNA (Clontech) and 6% DMSO.\",\n \"The PCR reaction was performed on a Robocycler thermocycler (Stratagene) starting with 1 cycle of 94° C. for 2 min followed by 35 cycles of 94° C. for 30 sec, 65° C. for 30 sec, 72° C. for 2 min.\",\n \"The PCR reaction was purified by the QiaQuick PCR Purification Kit (Qiagen) and eluted in Th.\",\n \"The PCR primer sequences were: PSK14: 5′GATCGAATTCATGGACACTACCATGGAAGCTGACC (SEQ ID NO: 157), and: PSK15: 5′GATCCTCGAGTCACGTGGGGCCTGCGCCCGG (SEQ ID NO: 158).\",\n \"The underlined portion of the primers match the 5′ and 3′ areas, respectively, of a portion of the 5′ untranslated region and coding region.\",\n \"Translation initiation and termination codons are shown above in bold.\",\n \"The blunt ended PCR product was prepared for cloning by the addition of a single base “A” residue by AmpliTaq Gold (Perkin Elmer) in a reaction with 1×PCR Buffer II, 1 mM MgCl2, 200 uM each dATP, dGTP, dCTP, and dTTP.\",\n \"The reaction was incubated at 94° C. for 10 minutes followed by 72° C. for 10 minutes.\",\n \"The products were cloned into the pCRII-TOPO vector (Invitrogen) using the TOPO TA cloning kit as follows: 3 μl PCR product DNA, 1 μl pCRII-TOPO vector, and 1 μl TOPOII salt solution (1.2M NaCl, 0.06M MgCl2) was incubated for 5 minutes at room temperature.\",\n \"To the ligation reaction one microliter of 6×TOPO Cloning Stop Solution was added then the reaction was placed on ice.\",\n \"Two microliters of the ligation reaction was transformed in One-Shot TOP10 cells (Invitrogen), and placed on ice for 30 minutes.\",\n \"The cells were heat-shocked for 30 seconds at 42° C., placed on ice for two minutes, 250 μl of SOC was added, then incubated at 37° C. with shaking for one hour and then plated onto ampicillin plates supplemented with X-gal and IPTG.\",\n \"Single colonies were screened by PCR for the presence of the insert, and a plasmid DNA from colony 58-6 was purified using a Qiagen Endo-Free plasmid purification kit and deposited as nGPCR-58.nGPCR-58 was sequenced directly using an ABI377 fluorescence-based sequencer (Perkin Elmer/Applied Biosystems Division, PE/ABD, Foster City, Calif.) and the ABI BigDye™ Terminator Cycle Sequencing Ready Reaction kit with Taq FS™ polymerase.\",\n \"Each ABI cycle sequencing reaction contained about 0.5 μg of plasmid DNA.\",\n \"Cycle-sequencing was performed using an initial denaturation at 98° C. for 1 min, followed by 50 cycles: 96° C. for 30 sec, annealing at 50° C. for 30 sec, and extension at 60° C. for 4 min.\",\n \"Temperature cycles and times were controlled by a Perkin-Elmer 9600 thermocycler.\",\n \"Extension products were purified using AGTC (R) gel filtration block (Edge RiosSystems, Gaithersburg, Md.).\",\n \"Each reaction product was loaded by pipette onto the column, which was then centrifuged in a swinging bucket centrifuge (Sorvall model RT6000B tabletop centrifuge) at 1500×g for 4 min at room temperature.\",\n \"Column-purified samples were dried under vacuum for about 40 min and then dissolved in 3 μl of a DNA loading solution (83% deionized formamide, 8.3 mM EDTA, and 1.6 mg/ml Blue Dextran).\",\n \"The samples were then heated to 90° C. for 3.5 min and loaded into the gel sample wells for sequence analysis by the ABI377 sequencer.\",\n \"Sequence analysis was performed by importing ABI377 files into the Sequencer program (Gene Codes, Ann Arbor, Mich.), yielding the sequence of SEQ ID NO: 93.Example 3 Hybridization Analysis to Demonstrate nGPCR-X Expression in Brain The expression of nGPCR-x in mammals, such as the rat, may be investigated by in situ hybridization histochemistry.\",\n \"To investigate expression in the brain, for example, coronal and sagittal rat brain cryosections (20 pun thick) are prepared using a Reichert-Jung cryostat.\",\n \"Individual sections are thaw-mounted onto silanized, nuclease-free slides (CEL Associates, Inc., Houston, Tex.\",\n \"), and stored at −80° C. Sections are processed starting with post-fixation in cold 4% paraformaldehyde, rinsed in cold phosphate-buffered saline (PBS), acetylated using acetic anhydride in triethanolamine buffer, and dehydrated through a series of alcohol washes in 70%, 95%, and 100% alcohol at room temperature.\",\n \"Subsequently, sections are delipidated in chloroform, followed by rehydration through successive exposure to 100% and 95% alcohol at room temperature.\",\n \"Microscope slides containing processed cryosections are allowed to air dry prior to hybridization.\",\n \"Other tissues may be assayed in a similar fashion.\",\n \"A nGPCR-x-specific probe is generated using PCR.\",\n \"Following PCR amplification, the fragment is digested with restriction enzymes and cloned into pBluescript II cleaved with the same enzymes.\",\n \"For production of a probe specific for the sense strand of nGPCR-x, the nGPCR-x clone in pBluescript II is linearized with a suitable restriction enzyme, which provides a substrate for labeled run-off transcripts (i.e., cRNA riboprobes) using the vector-borne T7 promoter and commercially available T7 RNA polymerase.\",\n \"A probe specific for the antisense strand of nGPCR-x is also readily prepared using the nGPCR-x clone in pBluescript II by cleaving the recombinant plasmid with a suitable restriction enzyme to generate a linearized substrate for the production of labeled run-off cRNA transcripts using the T3 promoter and cognate polymerase.\",\n \"The riboprobes are labeled with [35S]-UTP to yield a specific activity of about 0.40×106 cpm/pmol for antisense riboprobes and about 0.65×106 cpm/pmol for sense-strand riboprobes.\",\n \"Each riboprobe is subsequently denatured and added (2 pmol/ml) to hybridization buffer which contained 50% formamide, 10% dextran, 0.3 M NaCl, 10 mM Tris (pH 8.0), 1 mM EDTA, 1× Denhardt's Solution, and 10 mM dithiothreitol.\",\n \"Microscope slides containing sequential brain cryosections are independently exposed to 45 μl of hybridization solution per slide and silanized cover slips are placed over the sections being exposed to hybridization solution.\",\n \"Sections are incubated overnight (15-18 hours) at 52° C. to allow hybridization to occur.\",\n \"Equivalent series of cryosections are exposed to sense or antisense nGPCR-40-specific cRNA riboprobes.\",\n \"Following the hybridization period, coverslips are washed off the slides in 1×SSC, followed by RNase A treatment involving the exposure of slides to 20 μg/ml RNase A in a buffer containing 10 mM Tris-HCl (pH 7.4), 0.5 M EDTA, and 0.5 M NaCl for 45 minutes at 37° C. The cryosections are then subjected to three high-stringency washes in 0.1×SSC at 52° C. for 20 minutes each.\",\n \"Following the series of washes, cryosections are dehydrated by consecutive exposure to 70%, 95%, and 100% ammonium acetate in alcohol, followed by air drying and exposure to Kodak BioMax™ MR-1 film.\",\n \"After 13 days of exposure, the film is developed.\",\n \"Based on these results, slides containing tissue that hybridized, as shown by film autoradiograms, are coated with Kodak NTB-2 nuclear track emulsion and the slides are stored in the dark for 32 days.\",\n \"The slides are then developed and counterstained with hematoxylin.\",\n \"Emulsion-coated sections are analyzed microscopically to determine the specificity of labeling.\",\n \"The signal is determined to be specific if autoradiographic grains (generated by antisense probe hybridization) are clearly associated with cresyl violate-stained cell bodies.\",\n \"Autoradiographic grains found between cell bodies indicates non-specific binding of the probe.\",\n \"Expression of nGPCR-x in the brain provides an indication that modulators of nGPCR-x activity have utility for treating neurological disorders, including but not limited to, schizophrenia, affective disorders, ADHD/ADD (i.e., Attention Deficit-Hyperactivity Disorder/Attention Deficit Disorder), and neural disorders such as Alzheimer's disease, Parkinson's disease, migraine, and senile dementia.\",\n \"Some other diseases for which modulators of nGPCR-x may have utility include depression, anxiety, bipolar disease, epilepsy, neuritis, neurasthenia, neuropathy, neuroses, and the like.\",\n \"Use of nGPCR-x modulators, including nGPCR-x ligands and anti-nGPCR-x antibodies, to treat individuals having such disease states is intended as an aspect of the invention.\",\n \"Example 4 Tissue Expression Profiling Tissue specific expression of the cDNAs encoding nGPCR-1, nGPCR-3, nGPCR-9, nGPCR-11, nGPCR-16, nGPCR-40, nGPCR-54, nGPCR-56, and nGPCR-58 was detected using a PCR-based system.\",\n \"Tissue specific expression of cDNAs encoding nGPCR-x may be accomplished using similar methods.\",\n \"Primers were synthesized by Genosys Corp., The Woodlands, Tex.\",\n \"PCR reactions were assembled using the components of the Expand Hi-Fi PCR System™ (Roche Molecular Biochemicals, Indianapolis, Ind.).\",\n \"nGPCR-1 The RapidScan™ Gene Expression Panel was used to generate a comprehensive expression profile of the putative GPCR in human tissues.\",\n \"Human tissues in the array may include: brain, heart, kidney, spleen, liver, colon, lung, small intestine, muscle, stomach, testis, placenta, salivary gland, thyroid, adrenal gland, pancreas, ovary, uterus, prostate, skin, PBL, bone marrow, fetal brain, fetal liver.\",\n \"Human brain regions in the array may include: frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.\",\n \"Expression of the nGPCR-1 in the various tissues was detected by using PCR primers designed based on the available sequence of the receptor that will prime the synthesis of a 212 bp fragment in the presence of the appropriate cDNA.\",\n \"The forward primer was: GCTCAACCCACTCATCTATGCC (SEQ ID NO: 97), and the reverse primer was: AAACTTCTCTGCCCTTACCGTC (SEQ ID NO: 98) The PCR reaction mixture was added to each well of the PCR plate.\",\n \"The plate was placed in a GeneAmp PCR9700 PCR thermocycler (Perkin Elmer Applied Biosystems).\",\n \"The plate was then exposed to the following cycling parameters: Pre-soak 94° C. for 3 min; denaturation at 94° C. for 30 seconds; annealing at primer Tm for 45 seconds; extension 72° C. for 2 minutes; for 35 cycles.\",\n \"PCR products were then separated and analyzed by electrophoresis on a 1.5-% agarose gel.\",\n \"The 4-log dilution range of cDNA deposited on the plate ensured that the amplification reaction is within the linear range and, hence, facilitated the semi-quantitative determination of relative mRNA accumulation in the various tissues or brain regions examined.\",\n \"Expression of nGPCR-1 was found to be highest in the testis, adrenal gland and heart.\",\n \"Significant levels of expression were also found in the brain, kidney, spleen ovary, prostate, muscle, PBL, stomach and bone marrow.\",\n \"Within the brain, expression levels were highest in the cerebellum, amygdala, thalamus and spinal cord, with significant levels of expression in the frontal lobe, hippocampus, substantia nigra, hypothalamus and pons.\",\n \"Expression of nGPCR-1 in the brain provided an indication that modulators of nGPCR-1 activity have utility for treating neurological disorders, including but not limited to, schizophrenia, affective disorders, ADHD/ADD (i.e., Attention Deficit-Hyperactivity Disorder/Attention Deficit Disorder), and neural disorders such as Alzheimer's disease, Parkinson's disease, migraine, and senile dementia.\",\n \"Some other diseases for which modulators of nGPCR-1 may have utility include depression, anxiety, bipolar disease, epilepsy, neuritis, neurasthenia, neuropathy, neuroses, and the like.\",\n \"Use of nGPCR-1 modulators, including nGPCR-1 ligands and ant-nGPCR-1 antibodies, to treat individuals having such disease states is intended as an aspect of the invention.\",\n \"nGPCR-3 Tissue specific expression of the cDNA encoding nGPCR-3 was detected using a PCR-based method.\",\n \"Multiple Choice™ first strand cDNAs (OriGene Technologies, Rockville, Md.)\",\n \"from 6 human tissues were serially diluted over a 3-log range and arrayed into a multi-well PCR plate.\",\n \"This array was used to generate a comprehensive expression profile of the putative GPCR in human tissues.\",\n \"Human tissues arrayed included: brain, heart, kidney, peripheral blood leukocytes, lung and testis.\",\n \"PCR primers were designed based on the available sequence of the putative GPCR.\",\n \"The sequence of the forward primer used was: 5′TGCTGCTTTGTTGCGCCTAC3′ (SEQ ID NO: 189), corresponding to base pairs 77 through 96 of the predicted coding sequence of nGPCR-3.The sequence of the reverse primer used was: 5′TTGGACGCCAGGAAGGTG3′ (SEQ ID NO: 190), corresponding to base pairs 258 through 285 of the predicted coding sequence of nGPCR-3.This primer set primes the synthesis of a 298 base pair fragment in the presence of the appropriate cDNA.\",\n \"For detection of expression within brain regions, the same primer set was used with the Human Brain Rapid Scan™ Panel (OriGene Technologies, Rockville, Md.).\",\n \"This panel represents serial dilutions over a 3 log range of first strand cDNA from the following brain regions arrayed in a 96 well format: frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.\",\n \"Primers were synthesized by Genosys Corp., The Woodlands, Tex.\",\n \"PCR reactions were assembled using the components of the Expand Hi-Fi PCR System (Roche Molecular Biochemicals, Indianapolis, Ind.).\",\n \"Twenty-five microliters of the PCR reaction mixture was added to each well of the RapidScan PCR plate.\",\n \"The plate was placed in a GeneAmp 9700 PCR thermocycler (Perkin Elmer Applied Biosystems).\",\n \"The following cycling program was executed: Pre-soak at (94° C. for 3 min.)\",\n \"followed by 35 cycles of [(94° C. for 45 sec.\",\n \"), (53° C. for 2 min.\",\n \"), and (72° C. for 45 sec.)].\",\n \"PCR reaction products were then separated and analyzed by electrophoresis on a 2.0% agarose gel stained with ethidium bromide.\",\n \"The results indicated that nGPCR-3 was expressed in the brain, heart, kidney, peripheral blood lymphocytes, lung, and testis.\",\n \"In the brain, nGPCR-3 was expressed in frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla, as well as in the spinal cord.\",\n \"nGPCR-9 The RapidScan™ Gene Expression Panel was used to generate a comprehensive expression profile of the putative GPCR in human tissues.\",\n \"Human tissues arrayed include: brain, heart, kidney, spleen, liver, colon, lung, small intestine, muscle, stomach, testis, placenta, salivary gland, thyroid, adrenal gland, pancreas, ovary, uterus, prostate, skin, PBL, bone marrow, fetal brain, fetal liver.\",\n \"The forward primer used was to detect expression of nGPCR-9 was: 5′ AACCCCATCATCTACACGC 3′(SEQ ID NO: 105), and, the reverse primer was: 5′ TGCCTGTGGAGCCGCTGG 3′(SEQ ID NO: 106).\",\n \"This primer set will prime the synthesis of a 238 base pair fragment in the presence of the appropriate cDNA.\",\n \"For detection of expression within brain regions, the same primer set was used with the Human Brain Rapid Scan™ Panel (OriGene Technologies, Rockville, Md.).\",\n \"This panel represents serial dilutions over a 2-log range of first strand cDNA from the following brain regions arrayed in a 96 well format: frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.\",\n \"Twenty-five microliters of the PCR reaction mixture was added to each well of the PCR plate.\",\n \"The plate was placed in a GeneAmp 9700 PCR thermocycler (Perkin Elmer Applied Biosystems).\",\n \"The following cycling program was executed: Pre-soak at (94° C. for 3 min.)\",\n \"followed by 35 cycles of [(94° C. for 45 sec.)\",\n \"(52° C. for 2 min.)\",\n \"(72° C. for 45 sec.)].\",\n \"PCR reaction products were then separated and analyzed by electrophoresis on a 2.0% agarose gel and stained with ethidium bromide.\",\n \"nGPCR-9 was expressed in the brain, peripheral blood leukocytes, heart, kidney, adrenal gland, spleen, pancreas, liver, lung, skin, bone marrow, testis, placenta, salivary gland, uterus, small intestine, muscle, stomach, and fetal liver.\",\n \"Within the brain, nGPCR-9 was expressed in all areas examined including the frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.\",\n \"Expression of nGPCR-9 in the brain provided an indication that modulators of nGPCR-9 activity have utility for treating disorders, including but not limited to, schizophrenia, affective disorders, movement disorders, metabolic disorders, inflammatory disorders, cancers, ADHD/ADD (i.e., Attention Deficit-Hyperactivity Disorder/Attention Deficit Disorder), and neural disorders such as Alzheimer's disease, Parkinson's disease, migraine, and senile dementia.\",\n \"Use of nGPCR-9 modulators, including nGPCR-9 ligands and anti-nGPCR-9 antibodies, to treat individuals having such disease states is intended as an aspect of the invention.\",\n \"nGPCR-41 The RapidScan™ Gene Expression Panel was used to generate a comprehensive expression profile of the putative GPCR in human tissues.\",\n \"Human tissues in the array included, inter alia: brain, heart, kidney, spleen, liver, colon, lung, small intestine, muscle, stomach, testis, placenta, salivary gland, thyroid, adrenal gland, pancreas, ovary, uterus, prostate, skin, PBL, bone marrow, fetal brain, fetal liver.\",\n \"Human brain regions in the array included, inter alia: frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.\",\n \"Expression of nGPCR-11 in the various tissues was detected by using PCR primers designed based on the available sequence of the receptor that will prime the synthesis of a 206 bp fragment in the presence of the appropriate cDNA.\",\n \"The forward primer used to detect expression of nGPCR-11 was: 5′-GAAGCCCAGCACTGTTTACC-3′ (SEQ ID NO: 109), and the reverse primer was: 5′-TGAAATACCTGTCCGCAGCC-3 (SEQ ID NO: 10).\",\n \"Twenty-five microliters of the PCR reaction mixture was added to each well of the RapidScan PCR plate.\",\n \"The plate was placed in a GeneAmp 9700 PCR thermocycler (PE Applied Biosystems).\",\n \"The following cycling program was executed: Pre-soak 94° C. for 3 min; denaturation at 94° C. for 30 seconds; annealing at primer Tm for 45 seconds; extension at 72° C. for 2 minutes; for 35 cycles.\",\n \"PCR reaction products were then separated and analyzed by electrophoresis on a 2.0% agarose gel stained with ethidium bromide.\",\n \"The 4-log dilution range of cDNA deposited on the plate ensured that the amplification reaction was within the linear range and, facilitated semi-quantitative determination of relative mRNA accumulation in the various tissues or brain regions examined.\",\n \"nGPCR-11 was expressed in the thyroid gland, brain, heart, kidney, adrenal gland, spleen, liver, ovary, muscle, testis, salivary gland, colon, prostate, small intestine, skin stomach, bone marrow, fetal brain and placenta.\",\n \"Within the brain, nGPCR-11 was expressed in the temporal lobe, amygdala, substantia nigra, pons, spinal cord, frontal lobe, and cerebellum.\",\n \"Expression of the nGPCR-11 in the brain provided an indication that modulators of nGPCR-11 activity have utility for treating disorders, including but not limited to, schizophrenia, affective disorders, metabolic disorders, inflammatory disorders, cancers, ADHD/ADD (i.e., Attention Deficit-Hyperactivity Disorder/Attention Deficit Disorder), and neural disorders such as Alzheimer's disease, Parkinson's disease, migraine, and senile dementia.\",\n \"Some other diseases for which modulators of nGPCR-11 may have utility include depression, anxiety, bipolar disease, epilepsy, neuritis, neurasthenia, neuropathy, neuroses, and the lice.\",\n \"Use of nGPCR-11 modulators, including nGPCR1-11 ligands and anti-nGPCR-11 antibodies, to treat individuals having such disease states is intended as an aspect of the invention.\",\n \"Expression of nGPCR-11 in the thyroid gland, indicates that agonists or antagonists could be of use in the treatment of thyroid dysfunction such as thyreotoxicosis and myxoedema.\",\n \"They could also be of use in the stimulation of thyroid hormone release leading to overall increase in metabolic rate and weight reduction.\",\n \"The expression of nGPCR-11 in liver and muscle indicate a use for agonists or antagonists in regulation of glucose metabolism applicable in diabetes type II.\",\n \"nGCPR-16 The RapidScan™ Gene Expression Panel was used to generate a comprehensive expression profile of the putative GPCR in human tissues.\",\n \"Human tissues in the array included, inter alia: brain, heart, kidney, spleen, liver, colon, lung, small intestine, muscle, stomach, testis, placenta, salivary gland, thyroid, adrenal gland, pancreas, ovary, uterus, prostate, skin, PBL, bone marrow, fetal brain, fetal liver.\",\n \"Human brain regions in the array included, inter alia: frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.\",\n \"Expression of nGPCR-16 in the various tissues was detected by using PCR primers designed based on the available sequence of the receptor that will prime the synthesis of a 205 bp fragment in the presence of the appropriate cDNA.\",\n \"The forward primer used to detect expression of nGPCR-16 was: 5′ CAGCCCAAACATCCAAGTC 3′.\",\n \"(SEQ ID NO: 113).\",\n \"The reverse primer used to detect expression of nGPCR-16 was: 5′ ACCCCACTTAATCAGCCTC 3′ (SEQ ID NO: 114).\",\n \"For detection of expression within brain regions, the same primer set was used with the Human Brain Rapid Scan™ Panel (OriGene Technologies, Rockville, Md.).\",\n \"This panel represents serial dilutions over a 2 log range of first strand cDNA from the following brain regions arrayed in a 96 well format: frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.\",\n \"Twenty-five microliters of the PCR reaction mixture was added to each well of the RapidScan PCR plate.\",\n \"The plate was placed in a GeneAmp 9700 PCR thermocycler (Perkin Elmer Applied Biosystems).\",\n \"The following cycling program was executed: Pre-soak at (94° for 3 min.)\",\n \"followed by 35 cycles of [(94° C. for 45 sec.)\",\n \"(53° C. for 2 min.)\",\n \"(72° C. for 45 sec.)].\",\n \"PCR reaction products were then separated and analyzed by electrophoresis on a 2.0% agarose gel, and stained with ethidium bromide.\",\n \"The 4-log dilution range of cDNA deposited on the plate ensured that the amplification reaction was within the linear range and, facilitated semi-quantitative determination of relative mRNA accumulation in the various tissues or brain regions examined.\",\n \"nGPCR-16 was expressed in the ovary, lung, prostate, bone marrow, salivary gland, heart, adrenal gland, spleen, liver, small intestine, skin, muscle, peripheral blood leukocytes, testis, placenta, fetal liver, brain, thyroid gland, kidney, pancreas, colon, uterus, and stomach.\",\n \"Within the brain, nGPCR-16 was expressed in all areas examined including the frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.\",\n \"Expression of nGPCR-16 in the brain provides an indication that modulators of nGPCR-16 activity have utility for treating neurological disorders, including but not limited to, schizophrenia, affective disorders, ADHD/ADD (i.e., Attention Deficit-Hyperactivity Disorder/Attention Deficit Disorder), and neural disorders such as Alzheimer's disease, Parkinson's disease, migraine, and senile dementia.\",\n \"Some other diseases for which modulators of nGPCR-16 may have utility include depression, anxiety, bipolar disease, epilepsy, neuritis, neurasthenia, neuropathy, neuroses, and the like.\",\n \"Use of nGPCR-16 modulators, including nGPCR-16 ligands and anti-nGPCR-16 antibodies, to treat individuals having such disease states is intended as an aspect of the invention.\",\n \"nGPCR-40 The RapidScan™ Gene Expression Panel (OriGene Technologies, Rockville, Md.)\",\n \"was used to generate a comprehensive expression profile of the putative GPCR in human tissues.\",\n \"Human tissues arrayed include: brain, heart, kidney, spleen, liver, colon, lung, small intestine, muscle, stomach, testis, placenta, salivary gland, thyroid, adrenal gland, pancreas, ovary, uterus, prostate, skin, PBL, bone marrow, fetal brain, fetal liver.\",\n \"The forward primer used was: 5′ACAGCCCCAAAGCCAAACAC3′, (SEQ ID NO: 117), and the reverse primer was: 5′CCGCAGGAGCAATGAAAATCAG3′, (SEQ ID NO: 118).\",\n \"This primer set primed the synthesis of a 220 base pair fragment in the presence of the appropriate cDNA.\",\n \"For detection of expression within brain regions, the same primer set was used with the Human Brain RapidScan™ Panel (OriGene Technologies, Rockville, Md.).\",\n \"This panel represents serial dilutions over a 2 log range of first strand cDNA from the following brain regions arrayed in a 96 well format: frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.\",\n \"Twenty-five microliters of the PCR reaction mixture was added to each well of the RapidScan PCR plate.\",\n \"The plate was placed in a GeneAmp 9700 PCR thermocycler (Perkin Elmer Applied Biosystems).\",\n \"The following cycling program was executed: Pre-soak at (94° C. for 3 min.)\",\n \"followed by 35 cycles of [(94° for 45 sec.)\",\n \"(54° C. for 2 min.)\",\n \"(72° for 45 sec.)].\",\n \"PCR reaction products were then separated and analyzed by electrophoresis on a 2.0% agarose gel stained with ethidium bromide.\",\n \"The dilution range of cDNA deposited on the plates ensured that the amplification reaction was within the linear range and, hence, facilitated semi-quantitative determination of relative mRNA accumulation in the various tissues or brain regions examined.\",\n \"nGPCR-40 was expressed in the brain, peripheral blood lymphocytes, pancreas, ovary, uterus, testis, salivary gland, kidney, adrenal gland, liver, bone marrow, prostate, fetal liver, colon, muscle, and fetal brain, may be found in many other tissues, including, but not limited to, lung, small intestine, fetal brain cord, and bone.\",\n \"Within the brain, nGPCR-40 was expressed in the frontal lobe, hypothalamus, pons, cerebellum, caudate nucleus, and medulla.\",\n \"Expression of nGPCR-40 in the brain provides an indication that modulators of nGPCR-40 activity have utility for treating neurological disorders, including but not limited to, movement disorders, affective disorders, metabolic disorders, inflammatory disorders and cancers.\",\n \"Use of nGPCR-40 modulators, including nGPCR-40 ligands and anti-nGPCR-40 antibodies, to treat individuals having such disease states is intended as an aspect of the invention.\",\n \"nGPCR-54 Multiple Choice™ first strand cDNAs (OriGene Technologies, Rockville, Md.)\",\n \"from 12 human tissues were serially diluted over a 3-log range and arrayed into a multi-well PCR plate.\",\n \"Human tissues arrayed include: brain, heart, kidney, peripheral blood leukocytes, liver, lung, muscle, ovary, prostate, small intestine, spleen and testis.\",\n \"PCR primers were designed based on the sequence of nGPCR-54 provided herein.\",\n \"The forward primer used was: 5′CTGTCTCTCTGTCCTCTTCC3′, (SEQ ID NO: 123).\",\n \"The reverse primer used was: 5′GCACCGATCTTCATTGAATITC3′, (SEQ ID NO: 124).\",\n \"This primer set primes the synthesis of a 145 base pair fragment in the presence of the appropriate cDNA.\",\n \"For detection of expression within brain regions, the same primer set was used with the Human Brain Rapid Scan™ Panel (OriGene Technologies, Rockville, Md.).\",\n \"This panel represents serial dilutions over a 3 log range of first strand cDNA from the following brain regions arrayed in a 96 well format: frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.\",\n \"Twenty-five microliters of the PCR reaction mixture was added to each well of the RapidScan PCR plate.\",\n \"The plate was placed in a GeneAmp 9700 PCR thermocycler (Perkin Elmer Applied Biosystems).\",\n \"The following cycling program was executed: Pre-soak at (94° C. for 3 min.)\",\n \"followed by 35 cycles of [(94° C. for 45 sec.)\",\n \"(52.5° C. for 2 min.)\",\n \"(72° C. for 45 sec.)].\",\n \"PCR reaction products were then separated and analyzed by electrophoresis on a 2.0% agarose gel stained with ethidium bromide.\",\n \"nGPCR-54 was expressed in the brain, kidney, lung, muscle, testis, heart, liver, ovary, prostate, small intestine, spleen, and peripheral blood leukocytes.\",\n \"Within the brain, nGPCR-54 was expressed in the cerebellum, hippocampus, substantia nigra, thalamus, hypothalamus, pons, frontal lobe, temporal lobe, caudate nucleus, medulla, spinal cord, and amygdala.\",\n \"Expression of the nGPCR-54 in the brain provides an indication that modulators of nGPCR-54 activity have utility for treating neurological disorders, including but not limited to, movement disorders, affective disorders, metabolic disorders, inflammatory disorders and cancers.\",\n \"Use of nGPCR-54 modulators, including nGPCR-54 ligands and anti-nGPCR-54 antibodies, to treat individuals having such disease states is intended as an aspect of the invention.\",\n \"nGPCR-56 The RapidScan™ Gene Expression Panel was used to generate a comprehensive expression profile of the putative GPCR in human tissues.\",\n \"Human tissues arrayed include: brain, heart, kidney, spleen, liver, colon, lung, small intestine, muscle, stomach, testis, placenta, salivary gland, thyroid, adrenal gland, pancreas, ovary, uterus, prostate, skin, PBL, bone marrow, fetal brain, fetal liver.\",\n \"The forward primer used was: 5′ ACTRCAAACAACTTCATACCCC 3′ (SEQ ID NO: 125), and the reverse primer used was: 5′ACACACAGCATAGTAGCG 3′ (SEQ ID NO: 126).\",\n \"This primer set will prime the synthesis of a 231 base pair fragment in the presence of the appropriate cDNA.\",\n \"For detection of expression within brain regions, the same primer set was used with the Human Brain Rapid Scan™ Panel (OriGene Technologies, Rockville, Md.).\",\n \"This panel represents serial dilutions over a 2 log range of first strand cDNA from the following brain regions arrayed in a 96 well format: frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.\",\n \"Twenty-five microliters of the PCR reaction mixture was added to each well of the RapidScan PCR plate.\",\n \"The plate was placed in a GeneAmp 9700 PCR thermocycler (Perkin Elmer Applied Biosystems).\",\n \"The following cycling program was executed: Pre-soak at (94° C. for 3 min.)\",\n \"followed by 35 cycles of [(94° C. for 45 sec.)\",\n \"(53° C. for 2 min.)\",\n \"(72° C. for 45 sec.)].\",\n \"PCR reaction products were then separated and analyzed by electrophoresis on a 2.0% agarose gel stained with ethidium bromide.\",\n \"nGPCR-56 was expressed in peripheral blood lymphocytes, testis, salivary gland, kidney, spleen, skin, stomach, placenta, ovary, bone marrow, fetal liver, small intestine, and fetal brain.\",\n \"Expression of nGPCR-56 in the brain provides an indication that modulators of nGPCR-56 activity have utility for treating neurological disorders, including but not limited to, movement disorders, affective disorders, metabolic disorders, inflammatory disorders and cancers.\",\n \"Use of nGPCR-56 modulators, including nGPCR-56 ligands and anti-nGPCR-56 antibodies, to treat individuals having such disease states is intended as an aspect of the invention.\",\n \"nGPCR-58 The RapidScan™ Gene Expression Panel was used to generate a comprehensive expression profile of the putative GPCR in human tissues.\",\n \"Human tissues in the array included: brain, heart, kidney, spleen, liver, lung, small intestine, muscle, testis, ovary, prostate, and PBL.\",\n \"Human brain regions in the array included: frontal lobe, temporal lobe, cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinal cord.\",\n \"Expression of the nGPCR-58 in the various tissues was detected by using PCR primers designed based on the available sequence of the receptor that will prime the synthesis of a 282 bp fragment in the presence of the appropriate cDNA.\",\n \"The forward primer was: CAGAGCTTGATGATGAGGAC (SEQ ID NO: 127), and the reverse primer was: CCCATAGGAAGTAGTAGAAG (SEQ ID NO: 128).\",\n \"The PCR reaction mixture was added to each well of the PCR plate.\",\n \"The plate was placed in a GeneAmp PCR9700 PCR thermocycler (Perkin Elmer Applied Biosystems).\",\n \"The plate was then exposed to the following cycling parameters: Pre-soak 94° for 3 min; denaturation at 94° for 30 seconds; annealing at primer Tm for 45 seconds; extension at 72° for 2 minutes; for 35 cycles.\",\n \"PCR productions were then separated and analyzed by electrophoresis on a 1.5-% agarose gel.\",\n \"The 4-log dilution range of cDNA deposited on the plate ensured that the amplification reaction was within the linear range and, hence, facilitated semi-quantitative determination of relative mRNA accumulation in the various tissues or brain regions examined.\",\n \"nGPCR-58 was expressed in all tissues included on the array, including brain, muscle, prostate, kidney, peripheral blood lymphocytes, liver, lung, small intestine, spleen, testis, heart, and ovary.\",\n \"Within the brain, nGPCR-58 was expressed in many regions including, but not limited to cerebellum, substantia nigra, thalamus, pons, spinal cord, frontal lobe, temporal lobe, hippocampus, caudate nucleus, amygdala, hypothalamus, and medulla.\",\n \"Expression of the nGPCR-58 in the brain provided an indication that modulators of nGPCR-58 activity have utility for treating disorders, including but not limited to, schizophrenia, affective disorders, ADHD/ADD (i.e., Attention Deficit-Hyperactivity Disorder/Attention Deficit Disorder), neural disorders such as Alzheimer's disease, Parkinson's disease, migraine, senile dementia, depression, anxiety, bipolar disease, epilepsy, neuritis, neurasthenia, neuropathy, neuroses, metabolic disorders, inflammatory disorders, cancers and the like.\",\n \"Use of nGPCR-58 modulators, including nGPCR-58 ligands and anti-nGPCR-58 antibodies, to treat individuals having such disease states is intended as an aspect of the invention.\",\n \"Example 5 Northern Blot Analysis Northern blots are performed to examine the expression of nGPCR-x mRNA.\",\n \"The sense orientation oligonucleotide and the antisense-orientation oligonucleotide, described above, are used as primers to amplify a portion of the GPCR-x cDNA sequence of an odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191.Multiple human tissue northern blots from Clontech (Human II # 7767-1) are hybridized with the probe.\",\n \"Pre-hybridization is carried out at 42 C for 4 hours in 5×SSC, 1× Denhardt's reagent, 0.1% SDS, 50% formamide, 250 mg/ml salmon sperm DNA.\",\n \"Hybridization is performed overnight at 42° C. in the same mixture with the addition of about 1.5×106 cpm/ml of labeled probe.\",\n \"The probe is labeled with α-32P-DCTP by Rediprime™ DNA labeling system (Amersham Pharmacia), purified on Nick Column (Amersham Pharmacia) and added to the hybridization solution.\",\n \"The filters are washed several times at 42 C in 0.2×SSC, 0.1% SDS.\",\n \"Filters are exposed to Kodak XAR film (Eastman Kodak Company, Rochester, N.Y., USA) with intensifying screen at −80° C. Example 6 Recombinant Expression of nGPCR-x in Eukaryotic Host Cells A.\",\n \"Expression of nGPCR-x in Mammalian Cells To produce nGPCR-x protein, a nGPCR-x-encoding polynucleotide is expressed in a suitable host cell using a suitable expression vector and standard genetic engineering techniques.\",\n \"For example, the nGPCR-x-encoding sequence described in Example 1 is subcloned into the commercial expression vector pzeoSV2 (Invitrogen, San Diego, Calif.) and transfected into Chinese Hamster Ovary (CHO) cells using the transfection reagent FuGENE6™ (Boehringer-Mannheim) and the transfection protocol provided in the product insert.\",\n \"Other eukaryotic cell lines, including human embryonic kidney (HEK 293) and COS cells, are suitable as well.\",\n \"Cells stably expressing nGPCR-x are selected by growth in the presence of 100 μg/ml zeocin (Stratagene, LaJolla, Calif.).\",\n \"Optionally, nGPCR-x may be purified from the cells using standard chromatographic techniques.\",\n \"To facilitate purification, antisera is raised against one or more synthetic peptide sequences that correspond to portions of the nGPCR-x amino acid sequence, and the antisera is used to affinity purify nGPCR-x.\",\n \"The nGPCR-x also may be expressed in-frame with a tag sequence (e.g.\",\n \"polyhistidine, hemagluttinin, FLAG) to facilitate purification.\",\n \"Moreover, it will be appreciated that many of the uses for nGPCR-x polypeptides, such as assays described below, do not require purification of nGPCR-x from the host cell.\",\n \"B.\",\n \"Expression of nGPCR-x in 293 Cells For expression of nGPCR-x in mammalian cells 293 (transformed human, primary embryonic kidney cells), a plasmid bearing the relevant nGPCR-x coding sequence is prepared, using vector pSecTag2A (Invitrogen).\",\n \"Vector pSecTag2A contains the murine IgK chain leader sequence for secretion, the c-myc epitope for detection of the recombinant protein with the anti-myc antibody, a C-terminal polyhistidine for purification with nickel chelate chromatography, and a Zeocin resistant gene for selection of stable transfectants.\",\n \"The forward primer for amplification of this GPCR cDNA is determined by routine procedures and preferably contains a 5′ extension of nucleotides to introduce the HindIII cloning site and nucleotides matching the GPCR sequence.\",\n \"The reverse primer is also determined by routine procedures and preferably contains a 5′ extension of nucleotides to introduce an XhoI restriction site for cloning and nucleotides corresponding to the reverse complement of the nGPCR-x sequence.\",\n \"The PCR conditions are 55° C. as the annealing temperature.\",\n \"The PCR product is gel purified and cloned into the HindIII-XhoI sites of the vector.\",\n \"The DNA is purified using Qiagen chromatography columns and transfected into 293 cells using DOTAP™ transfection media (Boehringer Mannheim, Indianapolis, Ind.).\",\n \"Transiently transfected cells are tested for expression after 24 hours of transfection, using western blots probed with anti-His and anti-nGPCR-x peptide antibodies.\",\n \"Permanently transfected cells are selected with Zeocin and propagated.\",\n \"Production of the recombinant protein is detected from both cells and media by western blots probed with anti-His, anti-Myc or anti-GPCR peptide antibodies.\",\n \"C. Expression of nGPCR-x in COS Cells For expression of the nGPCR-x in COS7 cells, a polynucleotide molecule having an odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191 can be cloned into vector p3-CI.\",\n \"This vector is a pUC18-derived plasmid that contains the HCMV (human cytomegalovirus) promoter-intron located upstream from the bGH (bovine growth hormone) polyadenylation sequence and a multiple cloning site.\",\n \"In addition, the plasmid contains the dhrf (dihydrofolate reductase) gene which provides selection in the presence of the drug methotrexane (WIX) for selection of stable transformants.\",\n \"The forward primer is determined by routine procedures and preferably contains a 5′ extension which introduces an XbaI restriction site for cloning, followed by nucleotides which correspond to an odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191.The reverse primer is also determined by routine procedures and preferably contains 5′-extension of nucleotides which introduces a SalI cloning site followed by nucleotides which correspond to the reverse complement of an odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191.The PCR consists of an initial denaturation step of 5 min at 95° C. 30 cycles of 30 sec denaturation at 95° C., 30 sec annealing at 58° C. and 30 sec extension at 72° C., followed by 5 min extension at 72° C. The PCR product is gel purified and ligated into the XbaI and SalI sites of vector p3-CI.\",\n \"This construct is transformed into E. coli cells for amplification and DNA purification.\",\n \"The DNA is purified with Qiagen chromatography columns and transfected into COS 7 cells using Lipofectamine™ reagent from BRL, following the manufacturer's protocols.\",\n \"Forty-eight and 72 hours after transfection, the media and the cells are tested for recombinant protein expression.\",\n \"nGPCR-x expressed from a COS cell culture can be purified by concentrating the cell-growth media to about 10 mg of protein/ml, and purifying the protein by, for example, chromatography.\",\n \"Purified nGPCR-x is concentrated to 0.5 mg/ml in an Amicon concentrator fitted with a YM-10 membrane and stored at −80° C. D. Expression of nGPCR-x in Insect Cells For expression of nGPCR-x in a baculovirus system, a polynucleotide molecule having an odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191 can be amplified by PCR.\",\n \"The forward primer is determined by routine procedures and preferably contains a 5′ extension which adds the NdeI cloning site, followed by nucleotides which correspond to an odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191.The reverse primer is also determined by routine procedures and preferably contains a 5′ extension which introduces the KpnI cloning site, followed by nucleotides which correspond to the reverse complement of an odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191.The PCR product is gel purified, digested with NdeI and KpnI, and cloned into the corresponding sites of vector pACHTL-A (Pharmingen, San Diego, Calif.).\",\n \"The pAcHTL expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV), and a 6×His tag upstream from the multiple cloning site.\",\n \"A protein kinase site for phosphorylation and a thrombin site for excision of the recombinant protein precede the multiple cloning site is also present.\",\n \"Of course, many other baculovirus vectors could be used in place of pAcHTL-A, such as pAc373, pVL941 and pAcIM1.Other suitable vectors for the expression of GPCR polypeptides can be used, provided that the vector construct includes appropriately located signals for transcription, translation, and trafficking, such as an in-frame AUG and a signal peptide, as required.\",\n \"Such vectors are described in Luckow et al., Virology 170:31-39, among others.\",\n \"The virus is grown and isolated using standard baculovirus expression methods, such as those described in Summers et al.\",\n \"(A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experimental Station Bulletin No.\",\n \"1555 (1987)).\",\n \"In a preferred embodiment, pAcHLT-A containing nGPCR-x gene is introduced into baculovirus using the “BaculoGold™” transfection kit (Pharmingen, San Diego, Calif.) using methods established by the manufacturer.\",\n \"Individual virus isolates are analyzed for protein production by radiolabeling infected cells with 35S-methionine at 24 hours post infection.\",\n \"Infected cells are harvested at 48 hours post infection, and the labeled proteins are visualized by SDS-PAGE.\",\n \"Viruses exhibiting high expression levels can be isolated and used for scaled up expression.\",\n \"For expression of a nGPCR-x polypeptide in a Sf9 cells, a polynucleotide molecule having the sequence of an odd numbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191 can be amplified by PCR using the primers and methods described above for baculovirus expression.\",\n \"The nGPCR-x cDNA is cloned into vector pAcHLT-A (Pharmingen) for expression in Sf9 insect.\",\n \"The insert is cloned into the NdeI and KpnI sites, after elimination of an internal NdeI site (using the same primers described above for expression in baculovirus).\",\n \"DNA is purified with Qiagen chromatography columns and expressed in Sf9 cells.\",\n \"Preliminary Western blot experiments from non-purified plaques are tested for the presence of the recombinant protein of the expected size which reacted with the GPCR-specific antibody.\",\n \"These results are confirmed after further purification and expression optimization in HiG5 cells.\",\n \"Example 7 Interaction Trap/Two-Hybrid System In order to assay for nGPCR-x-interacting proteins, the interaction trap/two-hybrid library screening method can be used.\",\n \"This assay was first described in Fields et al., Nature, 1989, 340, 245, which is incorporated herein by reference in its entirety.\",\n \"A protocol is published in Current Protocols in Molecular Biology 1999, John Wiley & Sons, NY, and Ausubel, F. M et al.\",\n \"1992, Short protocols in molecular biology, Fourth edition, Greene and Wiley-interscience, NY, each of which is incorporated herein by reference in its entirety.\",\n \"Kits are available from Clontech, Palo Alto, Calif. (Matchmaker Two-Hybrid System 3).\",\n \"A fusion of the nucleotide sequences encoding all or partial nGPCR-x and the yeast transcription factor GAL4 DNA-binding domain (DNA-BD) is constructed in an appropriate plasmid (ie., pGBKT7) using standard subcloning techniques.\",\n \"Similarly, a GALA active domain (AD) fusion library is constructed in a second plasmid (i.e., pGADT7) from cDNA of potential GPCR-binding proteins (for protocols on forming cDNA libraries, see Sambrook et al.\",\n \"1989, Molecular cloning: a laboratory manual, second edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.), which is incorporated herein by reference in its entirety.\",\n \"The DNA-BD/nGPCR-x fusion construct is verified by sequencing, and tested for autonomous reporter gene activation and cell toxicity, both of which would prevent a successful two-hybrid analysis.\",\n \"Similar controls are performed with the AD/library fusion construct to ensure expression in host cells and lack of transcriptional activity.\",\n \"Yeast cells are transformed (ca.\",\n \"105 transformants/mg DNA) with both the nGPCR-x and library fusion plasmids according to standard procedures (Ausubel et al., 1992, Short protocols in molecular biology, fourth edition, Greene and Wiley-interscience, NY, which is incorporated herein by reference in its entirety).\",\n \"In vivo binding of DNA-BD/nGPCR-x with AD/library proteins results in transcription of specific yeast plasmid reporter genes (i.e., lacZ, HIS3, ADE2, LEU2).\",\n \"Yeast cells are plated on nutrient-deficient media to screen for expression of reporter genes.\",\n \"Colonies are dually assayed for β-galactosidase activity upon growth in Xgal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) supplemented media (filter assay for β-galactosidase activity is described in Breeden et al., Cold Spring Harb.\",\n \"Symp.\",\n \"Quant.\",\n \"Biol., 1985, 50, 643, which is incorporated herein by reference in its entirety).\",\n \"Positive AD-library plasmids are rescued from transformants and reintroduced into the original yeast strain as well as other strains containing unrelated DNA-BD fusion proteins to confirm specific nGPCR-x/library protein interactions.\",\n \"Insert DNA is sequenced to verify the presence of an open reading frame fused to GAL4 AD and to determine the identity of the nGPCR-x-binding protein.\",\n \"Example 8 Mobility Shift DNA-Binding Assay Using Gel Electrophoresis A gel electrophoresis mobility shift assay can rapidly detect specific protein-DNA interactions.\",\n \"Protocols are widely available in such manuals as Sambrook et al.\",\n \"1989, Molecular cloning: a laboratory manual, second edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. and Ausubel, F. M. et al., 1992, Short Protocols in Molecular Biology, fourth edition, Greene and Wiley-interscience, NY, each of which is incorporated herein by reference in its entirety.\",\n \"Probe DNA (<300 bp) is obtained from synthetic oligonucleotides, restriction endonuclease fragments, or PCR fragments and end-labeled with 32P.\",\n \"An aliquot of purified nGPCR-x (ca.\",\n \"15 μg) or crude nGPCR-x extract (ca.\",\n \"15 ng) is incubated at constant temperature (in the range 22-37 C) for at least 30 minutes in 10-15 μl of buffer (i.e.\",\n \"TAE or TBE, pH 8.0-8.5) containing radiolabeled probe DNA, nonspecific carrier DNA (ca 1 μg), BSA (300 μg/ml), and 10% (v/v) glycerol.\",\n \"The reaction mixture is then loaded onto a polyacrylamide gel and run at 30-35 mA until good separation of free probe DNA from protein-DNA complexes occurs.\",\n \"The gel is then dried and bands corresponding to free DNA and protein-DNA complexes are detected by autoradiography.\",\n \"Example 9 Antibodies to nGPCR-X Standard techniques are employed to generate polyclonal or monoclonal antibodies to the nGPCR-x receptor, and to generate useful antigen-binding fragments thereof or variants thereof, including “humanized” variants.\",\n \"Such protocols can be found, for example, in Sambrook et al.\",\n \"(1989) and Harlow et al.\",\n \"(Eds.\",\n \"), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988).\",\n \"In one embodiment, recombinant nGPCR-x polypeptides (or cells or cell membranes containing such polypeptides) are used as antigen to generate the antibodies.\",\n \"In another embodiment, one or more peptides having amino acid sequences corresponding to an immunogenic portion of nGPCR-x (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids) are used as antigen.\",\n \"Peptides corresponding to extracellular portions of nGPCR-x, especially hydrophilic extracellular portions, are preferred.\",\n \"The antigen may be mixed with an adjuvant or linked to a hapten to increase antibody production.\",\n \"A. Polyclonal or Monoclonal Antibodies As one exemplary protocol, recombinant nGPCR-x or a synthetic fragment thereof is used to immunize a mouse for generation of monoclonal antibodies (or larger mammal, such as a rabbit, for polyclonal antibodies).\",\n \"To increase antigenicity, peptides are conjugated to Keyhole Lympet Hemocyanin (Pierce), according to the manufacturer's recommendations.\",\n \"For an initial injection, the antigen is emulsified with Freund's Complete Adjuvant and injected subcutaneously.\",\n \"At intervals of two to three weeks, additional aliquots of nGPCR-x antigen are emulsified with Freund's Incomplete Adjuvant and injected subcutaneously.\",\n \"Prior to the final booster injection, a serum sample is taken from the immunized mice and assayed by western blot to confirm the presence of antibodies that immunoreact with nGPCR-x.\",\n \"Serum from the immunized animals may be used as polyclonal antisera or used to isolate polyclonal antibodies that recognize nGPCR-x.\",\n \"Alternatively, the mice are sacrificed and their spleen removed for generation of monoclonal antibodies.\",\n \"To generate monoclonal antibodies, the spleens are placed in 10 ml serum-free RPMI 1640, and single cell suspensions are formed by grinding the spleens in serum-free RPMI 1640, supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, and 100 μg/ml streptomycin (RPMI) (Gibco, Canada).\",\n \"The cell suspensions are filtered and washed by centrifugation and resuspended in serum-free RPMI.\",\n \"Thymocytes taken from three naive Balb/c mice are prepared in a similar manner and used as a Feeder Layer.\",\n \"NS-1 myeloma cells, kept in log phase in RPMI with 10% fetal bovine serum (FBS) (Hyclone Laboratories, Inc., Logan, Utah) for three days prior to fusion, are centrifuged and washed as well.\",\n \"To produce hybridoma fusions, spleen cells from the immunized mice are combined with NS-1 cells and centrifuged, and the supernatant is aspirated.\",\n \"The cell pellet is dislodged by tapping the tube, and 2 ml of 37° C. PEG 1500 (50% in 75 mM HEPES, pH 8.0) (Boehringer-Mannheim) is stirred into the pellet, followed by the addition of serum-free RPMI.\",\n \"Thereafter, the cells are centrifuged, resuspended in RPMI containing 15% FBS, 100 μM sodium hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine (HAT) (Gibco), 25 units/ml IL-6 (Boehringer-Mannheim) and 1.5×106 thymocytes/ml, and plated into 10 Corning flat-bottom 96-well tissue culture plates (Corning, Corning N.Y.).\",\n \"On days 2, 4, and 6 after the fusion, 100 μl of medium is removed from the wells of the fusion plates and replaced with fresh medium.\",\n \"On day 8, the fusions are screened by ELISA, testing for the presence of mouse IgG that binds to nGPCR-x.\",\n \"Selected fusion wells are further cloned by dilution until monoclonal cultures producing anti-nGPCR-x antibodies are obtained.\",\n \"B. Humanization of Anti-nGPCR-x Monoclonal Antibodies The expression pattern of nGPCR-x as reported herein and the proven track record of GPCRs as targets for therapeutic intervention suggest therapeutic indications for nGPCR-x inhibitors (antagonists).\",\n \"nGPCR-x-neutralizing antibodies comprise one class of therapeutics useful as nGPCR-x antagonists.\",\n \"Following are protocols to improve the utility of anti-nGPCR-x monoclonal antibodies as therapeutics in humans by “humanizing” the monoclonal antibodies to improve their serum half-life and render them less immunogenic in human hosts (i.e., to prevent human antibody response to non-human anti-nGPCR-x antibodies).\",\n \"The principles of humanization have been described in the literature and are facilitated by the modular arrangement of antibody proteins.\",\n \"To minimize the possibility of binding complement, a humanized antibody of the IgG4 isotype is preferred.\",\n \"For example, a level of humanization is achieved by generating chimeric antibodies comprising the variable domains of non-human antibody proteins of interest with the constant domains of human antibody molecules.\",\n \"(See, e.g., Morrison et al., Adv.\",\n \"Immunol., 44:65-92 (1989)).\",\n \"The variable domains of nGPCR-x-neutralizing anti-nGPCR-x antibodies are cloned from the genomic DNA of a B-cell hybridoma or from cDNA generated from mRNA isolated from the hybridoma of interest.\",\n \"The V region gene fragments are linked to exons encoding human antibody constant domains, and the resultant construct is expressed in suitable mammalian host cells (e.g., myeloma or CHO cells).\",\n \"To achieve an even greater level of humanization, only those portions of the variable region gene fragments that encode antigen-binding complementarity determining regions (“CDR”) of the non-human monoclonal antibody genes are cloned into human antibody sequences.\",\n \"(See, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-36 (1988); and Tempest et al., Bio/Technology 9: 266-71 (1991)).\",\n \"If necessary, the P-sheet framework of the human antibody surrounding the CDR3 regions also is modified to more closely mirror the three dimensional structure of the antigen-binding domain of the original monoclonal antibody.\",\n \"(See Kettleborough et al., Protein Engin., 4:773-783 (1991); and Foote et al., J. Mol.\",\n \"Biol., 224:487-499 (1992)).\",\n \"In an alternative approach, the surface of a non-human monoclonal antibody of interest is humanized by altering selected surface residues of the non-human antibody, e.g., by site-directed mutagenesis, while retaining all of the interior and contacting residues of the non-human antibody.\",\n \"See Padlan, Molecular Immunol., 28(4/5):489-98 (1991).\",\n \"The foregoing approaches are employed using nGPCR-x-neutralizing anti-nGPCR-x monoclonal antibodies and the hybridomas that produce them to generate humanized nGPCR-x-neutralizing antibodies useful as therapeutics to treat or palliate conditions wherein nGPCR-x expression or ligand-mediated nGPCR-x signaling is detrimental.\",\n \"C. Human nGPCR-x-Neutralizing Antibodies from Phage Display Human nGPCR-x-neutralizing antibodies are generated by phage display techniques such as those described in Aujame et al., Human Antibodies 8(4):155-168 (1997); Hoogenboom, TIBTECH 15:62-70 (1997); and Rader et al., Curr.\",\n \"Opin.\",\n \"Biotechnol.\",\n \"8:503-508 (1997), all of which are incorporated by reference.\",\n \"For example, antibody variable regions in the form of Fab fragments or linked single chain Fv fragments are fused to the amino terminus of filamentous phage minor coat protein pIII.\",\n \"Expression of the fusion protein and incorporation thereof into the mature phage coat results in phage particles that present an antibody on their surface and contain the genetic material encoding the antibody.\",\n \"A phage library comprising such constructs is expressed in bacteria, and the library is screened for nGPCR-x-specific phage-antibodies using labeled or immobilized nGPCR-x as antigen-probe.\",\n \"D. Human nGPCR-x-Neutralizing Antibodies from Transgenic Mice Human nGPCR-x-neutralizing antibodies are generated in transgenic mice essentially as described in Bruggemann et al., Immunol.\",\n \"Today 17(8):391-97 (1996) and Bruggemann et al., Curr.\",\n \"Opin.\",\n \"Biotechnol.\",\n \"8:455-58 (1997).\",\n \"Transgenic mice carrying human V-gene segments in germline configuration and that express these transgenes in their lymphoid tissue are immunized with a nGPCR-x composition using conventional immunization protocols.\",\n \"Hybridomas are generated using B cells from the immunized mice using conventional protocols and screened to identify hybridomas secreting anti-nGPCR-x human antibodies (e.g., as described above).\",\n \"Example 10 Assays to Identify Modulators of nGPCR-x Activity Set forth below are several nonlimiting assays for identifying modulators (agonists and antagonists) of nGPCR-x activity.\",\n \"Among the modulators that can be identified by these assays are natural ligand compounds of the receptor, synthetic analogs and derivatives of natural ligands; antibodies, antibody fragments, and/or antibody-like compounds derived from natural antibodies or from antibody-like combinatorial libraries; and/or synthetic compounds identified by high-throughput screening of libraries; and the like.\",\n \"All modulators that bind nGPCR-x are useful for identifying nGPCR-x in tissue samples (e.g., for diagnostic purposes, pathological purposes, and the like).\",\n \"Agonist and antagonist modulators are useful for up-regulating and down-regulating nGPCR-x activity, respectively, to treat disease states characterized by abnormal levels of nGPCR-x activity.\",\n \"The assays may be performed using single putative modulators, and/or may be performed using a known agonist in combination with candidate antagonists (or visa versa).\",\n \"A. cAMP Assays In one type of assay, levels of cyclic adenosine monophosphate (cAMP) are measured in nGPCR-x-transfected cells that have been exposed to candidate modulator compounds.\",\n \"Protocols for cAMP assays have been described in the literature.\",\n \"(See, e.g., Sutherland et al., Circulation 37: 279 (1968); Frandsen et al., Life Sciences 18: 529-541 (1976); Dooley et al., Journal of Pharmacology and Experimental Therapeutics 283 (2): 735-41 (1997); and George et al., Journal of Biomolecular Screening 2 (4): 23540 (1997)).\",\n \"An exemplary protocol for such an assay, using an Adenylyl Cyclase Activation FlashPlate® Assay from NEN™ Life Science Products, is set forth below.\",\n \"Briefly, the nGPCR-x coding sequence (e.g., a cDNA or intronless genomic DNA) is subcloned into a commercial expression vector, such as pzeoSV2 (Invitrogen), and transiently transfected into Chinese Hamster Ovary (CHO) cells using known methods, such as the transfection protocol provided by Boehringer-Mannheim when supplying the FuGENE 6 transfection reagent.\",\n \"Transfected CHO cells are seeded into 96-well microplates from the FlashPlate® assay kit, which are coated with solid scintillant to which antisera to cAMP has been bound.\",\n \"For a control, some wells are seeded with wild type (untransfected) CHO cells.\",\n \"Other wells in the plate receive various amounts of acAMP standard solution for use in creating a standard curve.\",\n \"One or more test compounds (i.e., candidate modulators) are added to the cells in each well, with water and/or compound-free medium/diluent serving as a control or controls.\",\n \"After treatment, cAMP is allowed to accumulate in the cells for exactly 15 minutes at room temperature.\",\n \"The assay is terminated by the addition of lysis buffer containing [125I]-labeled cAMP, and the plate is counted using a Packard Topcount™ 96-well microplate scintillation counter.\",\n \"Unlabeled cAMP from the lysed cells (or from standards) and fixed amounts of [125I]-cAMP compete for antibody bound to the plate.\",\n \"A standard curve is constructed, and cAMP values for the unknowns are obtained by interpolation.\",\n \"Changes in intracellular cAMP levels of cells in response to exposure to a test compound are indicative of nGPCR-x modulating activity.\",\n \"Modulators that act as agonists of receptors which couple to the Gs subtype of G proteins will stimulate production of cAMP, leading to a measurable 3-10 fold increase in cAMP levels.\",\n \"Agonists of receptors which couple to the Gi/o subtype of G proteins will inhibit forskolin-stimulated cAMP production, leading to a measurable decrease in cAMP levels of 50-100%.\",\n \"Modulators that act as inverse agonists will reverse these effects at receptors that are either constitutively active or activated by known agonists.\",\n \"B. Aequorin Assays In another assay, cells (e.g., CHO cells) are transiently co-transfected with both a nGPCR-x expression construct and a construct that encodes the photoprotein apoaquorin.\",\n \"In the presence of the cofactor coelenterazine, apoaquorin will emit a measurable luminescence that is proportional to the amount of intracellular (cytoplasmic) free calcium.\",\n \"(See generally, Cobbold, et al.\",\n \"“Aequorin measurements of cytoplasmic free calcium,” In: McCormack J. G. and Cobbold P. H., eds., Cellular Calcium: A Practical Approach.\",\n \"Oxford:IRL Press (1991); Stables et al., Analytical Biochemistry 252: 115-26 (1997); and Haugland, Handbook of Fluorescent Probes and Research Chemicals.\",\n \"Sixth edition.\",\n \"Eugene Oreg.\",\n \": Molecular Probes (1996).)\",\n \"In one exemplary assay, nGPCR-x is subcloned into the commercial expression vector pzeoSV2 (Invitrogen) and transiently co-transfected along with a construct that encodes the photoprotein apoaquorin (Molecular Probes, Eugene, Oreg.)\",\n \"into CHO cells using the transfection reagent FuGENE 6 (Boehringer-Mannheim) and the transfection protocol provided in the product insert.\",\n \"The cells are cultured for 24 hours at 37° C. in MEM (Gibco/BRL, Gaithersburg, Md.)\",\n \"supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin and 10 μg/ml streptomycin, at which time the medium is changed to serum-free MEM containing 5 μM coelenterazine (Molecular Probes, Eugene, Oreg.).\",\n \"Culturing is then continued for two additional hours at 37° C. Subsequently, cells are detached from the plate using VERSEN (Gibco/BRL), washed, and resuspended at 200,000 cells/ml in serum-free MEM.\",\n \"Dilutions of candidate nGPCR-x modulator compounds are prepared in serum-free MEM and dispensed into wells of an opaque 96-well assay plate at 50 μl/well.\",\n \"Plates are then loaded onto an MLX microtiter plate luminometer (Dynex Technologies, Inc., Chantilly, Va.).\",\n \"The instrument is programmed to dispense 50 μl cell suspensions into each well, one well at a time, and immediately read luminescence for 15 seconds.\",\n \"Dos response curves for the candidate modulators are constructed using the area under the curve for each light signal peak.\",\n \"Data are analyzed with SlideWrite, using the equation for a one-site ligand, and EC50 values are obtained.\",\n \"Changes in luminescence caused by the compounds are considered indicative of modulatory activity.\",\n \"Modulators that act as agonists at receptors which couple to the Gq subtype of G proteins give an increase in luminescence of up to 100 fold.\",\n \"Modulators that act as inverse agonists will reverse this effect at receptors that are either constitutively active or activated by known agonists.\",\n \"C. Luciferase Reporter Gene Assay The photoprotein luciferase provides another useful tool for assaying for modulators of nGPCR-x activity.\",\n \"Cells (e.g., CHO cells or COS 7 cells) are transiently co-transfected with both a nGPCR-x expression construct (e.g., nGPCR-x in pzeoSV2) and a reporter construct which includes a gene for the luciferase protein downstream from a transcription factor binding site, such as the cAMP-response element (CRE), AP-1, or NF-kappa B. Agonist binding to receptors coupled to the G. subtype of G proteins leads to increases in cAMP, thereby activating the CRE transcription factor and resulting in expression of the luciferase gene.\",\n \"Agonist binding to receptors coupled to the Gq subtype of G protein leads to production of diacylglycerol that activates protein kinase C, which activates the AP-1 or NF-kappa B transcription factors, in turn resulting in expression of the luciferase gene.\",\n \"Expression levels of luciferase reflect the activation status of the signaling events.\",\n \"(See generally, George et al, Journal of Biomolecular Screening 2(4): 235-240 (1997); and Stratowa et al, Current Opinion in Biotechnology 6: 574-581 (1995)).\",\n \"Luciferase activity may be quantitatively measured using, e.g., luciferase assay reagents that are commercially available from Promega (Madison, Wis.).\",\n \"In one exemplary assay, CHO cells are plated in 24-well culture dishes at a density of 100,000 cells/well one day prior to transfection and cultured at 37° C. in MEM (Gibco/BRL) supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin and 10 μg/ml streptomycin.\",\n \"Cells are transiently co-transfected with both a nGPCR-x expression construct and a reporter construct containing the luciferase gene.\",\n \"The reporter plasmids CRE-luciferase, AP-1-luciferase and NF-kappaB-luciferase may be purchased from Stratagene (LaJolla, Calif.).\",\n \"Transfections are performed using the FuGENE 6 transfection reagent (Boehringer-Mannheim) according to the supplier's instructions.\",\n \"Cells transfected with the reporter construct alone are used as a control.\",\n \"Twenty-four hours after transfection, cells are washed once with PBS pre-warmed to 37° C. Serum-free MEM is then added to the cells either alone (control) or with one or more candidate modulators and the cells are incubated at 37° C. for five hours.\",\n \"Thereafter, cells are washed once with ice-cold PBS and lysed by the addition of 100 μl of lysis buffer per well from the luciferase assay kit supplied by Promega.\",\n \"After incubation for 15 minutes at room temperature, 15 μl of the lysate is mixed with 50 μl of substrate solution (Promega) in an opaque-white, 96-well plate, and the luminescence is read immediately on a Wallace model 1450 MicroBeta scintillation and luminescence counter (Wallace Instruments, Gaithersburg, Md.).\",\n \"Differences in luminescence in the presence versus the absence of a candidate modulator compound are indicative of modulatory activity.\",\n \"Receptors that are either constitutively active or activated by agonists typically give a 3 to 20-fold stimulation of luminescence compared to cells transfected with the reporter gene alone.\",\n \"Modulators that act as inverse agonists will reverse this effect.\",\n \"D. Intracellular Calcium Measurement Using FLIPR Changes in intracellular calcium levels are another recognized indicator of G protein-coupled receptor activity, and such assays can be employed to screen for modulators of nGPCR-x activity.\",\n \"For example, CHO cells stably transfected with a nGPCR-x expression vector are plated at a density of 4×104 cells/well in Packard black-walled, 96-well plates specially designed to discriminate fluorescence signals emanating from the various wells on the plate.\",\n \"The cells are incubated for 60 minutes at 37° C. in modified Dulbecco's PBS (D-PBS) containing 36 mg/L pyruvate and 1 g/L glucose with the addition of 1% fetal bovine serum and one of four calcium indicator dyes (Fluo-3™ AM, Fluo-4™ AM, Calcium Green™-1 AM, or Oregon Green™ 488 BAPTA-1 AM), each at a concentration of 4 μM.\",\n \"Plates are washed once with modified D-PBS without 1% fetal bovine serum and incubated for 10 minutes at 37° C. to remove residual dye from the cellular membrane.\",\n \"In addition, a series of washes with modified D-PBS without 1% fetal bovine serum is performed immediately prior to activation of the calcium response.\",\n \"A calcium response is initiated by the addition of one or more candidate receptor agonist compounds, calcium ionophore A23187 (10 μM; positive control), or ATP (4 μM; positive control).\",\n \"Fluorescence is measured by Molecular Device's FLIPR with an argon laser (excitation at 488 nm).\",\n \"(See, e.g., Kuntzweiler et al., Drug Development Research, 44(1): 14-20 (1998)).\",\n \"The F-stop for the detector camera was set at 2.5 and the length of exposure was 0.4 milliseconds.\",\n \"Basal fluorescence of cells was measured for 20 seconds prior to addition of candidate agonist, ATP, or A23187, and the basal fluorescence level was subtracted from the response signal.\",\n \"The calcium signal is measured for approximately 200 seconds, taking readings every two seconds.\",\n \"Calcium ionophore A23187 and ATP increase the calcium signal 200% above baseline levels.\",\n \"In general, activated GPCRs increase the calcium signal approximately 10-15% above baseline signal.\",\n \"E. Mitogenesis Assay In a mitogenesis assay, the ability of candidate modulators to induce or inhibit nGPCR-x-mediated cell division is determined.\",\n \"(See, e.g., Lajiness et al., Journal of Pharmacology and Experimental Therapeutics 267(3): 1573-1581 (1993)).\",\n \"For example, CHO cells stably expressing nGPCR-x are seeded into 96-well plates at a density of 5000 cells/well and grown at 37° C. in MEM with 10% fetal calf serum for 48 hours, at which time the cells are rinsed twice with serum-free MEM.\",\n \"After rinsing, 80 μl of fresh MEM, or MEM containing a known mitogen, is added along with 20 μl MEM containing varying concentrations of one or more candidate modulators or test compounds diluted in serum-free medium.\",\n \"As controls, some wells on each plate receive serum-free medium alone, and some receive medium containing 10% fetal bovine serum.\",\n \"Untransfected cells or cells transfected with vector alone also may serve as controls.\",\n \"After culture for 16-18 hours, 1 μCi of [3H]-thymidine (2 Ci/mmol) is added to the wells and cells are incubated for an additional 2 hours at 37° C. The cells are trypsinized and collected on filter mats with a cell harvester (Tomtec); the filters are then counted in a Betaplate counter.\",\n \"The incorporation of [3H]-thymidine in serum-free test wells is compared to the results achieved in cells stimulated with serum (positive control).\",\n \"Use of multiple concentrations of test compounds permits creation and analysis of dose-response curves using the non-linear, least squares fit equation: A=B×[C/(D+C)]+G where A is the percent of serum stimulation; B is the maximal effect minus baseline; C is the EC50; D is the concentration of the compound; and G is the maximal effect.\",\n \"Parameters B, C and G are determined by Simplex optimization.\",\n \"Agonists that bind to the receptor are expected to increase [3H]-thymidine incorporation into cells, showing up to 80% of the response to serum.\",\n \"Antagonists that bind to the receptor will inhibit the stimulation seen with a known agonist by up to 100%.\",\n \"F. [35S]GTPγS Binding Assay Because G protein-coupled receptors signal through intracellular G proteins whose activity involves GTP binding and hydrolysis to yield bound GDP, measurement of binding of the non-hydrolyzable GTP analog [35S]GTPγS in the presence and absence of candidate modulators provides another assay for modulator activity.\",\n \"(See, e.g., Kowal et al., Neuropharmacology 37:179-187 (1998).)\",\n \"In one exemplary assay, cells stably transfected with a nGPCR-x expression vector are grown in 10 cm tissue culture dishes to subconfluence, rinsed once with 5 ml of ice-cold Ca2+/Mg2+-free phosphate-buffered saline, and scraped into 5 ml of the same buffer.\",\n \"Cells are pelleted by centrifugation (500×g, 5 minutes), resuspended in TEE buffer (25 mM Tris, pH 7.5, 5 in M EDTA, 5 mM EGTA), and frozen in liquid nitrogen.\",\n \"After thawing, the cells are homogenized using a Dounce homogenizer (one ml TEE per plate of cells), and centrifuged at 1,000×g for 5 minutes to remove nuclei and unbroken cells.\",\n \"The homogenate supernatant is centrifuged at 20,000×g for 20 minutes to isolate the membrane fraction, and the membrane pellet is washed once with TEE and resuspended in binding buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 1 mM EDTA).\",\n \"The resuspended membranes can be frozen in liquid nitrogen and stored at −70° C. until use.\",\n \"Aliquots of cell membranes prepared as described above and stored at −70° C. are thawed, homogenized, and diluted into buffer containing 20 mM HEPES, 10 mM MgCl, 1 mM EDTA, 120 mM NaCl, 10 μM GDP, and 0.2 mM ascorbate, at a concentration of 10-50 μg/ml.\",\n \"In a final volume of 90 μl, homogenates are incubated with varying concentrations of candidate modulator compounds or 100 μM GTP for 30 minutes at 30° C. and then placed on ice.\",\n \"To each sample, 10 μl guanosine 5′-O-(3[35S]thio) triphosphate (NEN, 1200 Ci/mmol; [35S]-GTPγS), was added to a final concentration of 100-200 pM.\",\n \"Samples are incubated at 30° C. for an additional 30 minutes, 1 ml of 10 mM HEPES, pH 7.4, 10 mM MgCl2, at 4° C. is added and the reaction is stopped by filtration.\",\n \"Samples are filtered over Whatman GF/B filters and the filters are washed with 20 ml ice-cold 10 mM HEPES, pH 7.4, 10 mM MgCl2.Filters are counted by liquid scintillation spectroscopy.\",\n \"Nonspecific binding of [35S]-GTPγS is measured in the presence of 100 μM GTP and subtracted from the total.\",\n \"Compounds are selected that modulate the amount of [35]-GTPγS binding in the cells, compared to untransfected control cells.\",\n \"Activation of receptors by agonists gives up to a five-fold increase in [35S]GTPγS binding.\",\n \"This response is blocked by antagonists.\",\n \"G. MAP Kinase Activity Assay Evaluation of MAP kinase activity in cells expressing a GPCR provides another assay to identify modulators of GPCR activity.\",\n \"(See, e.g.\",\n \"Lajiness et al., Journal of Pharmacology and Experimental Therapeutics 267(3):1573-1581 (1993) and Boulton et al., Cell 65:663-675 (1991).)\",\n \"In one embodiment, CHO cells stably transfected with nGPCR-x are seeded into 6-well plates at a density of 70,000 cells/well 48 hours prior to the assay.\",\n \"During this 48-hour period, the cells are cultured at 37° C. in MEM medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin and 10 μg/ml streptomycin.\",\n \"The cells are serum-starved for 1-2 hours prior to the addition of stimulants.\",\n \"For the assay, the cells are treated with medium alone or medium containing either a candidate agonist or 200 nM Phorbol ester-myristoyl acetate (i.e., PMA, a positive control), and the cells are incubated at 37° C. for varying times.\",\n \"To stop the reaction, the plates are placed on ice, the medium is aspirated, and the cells are rinsed with 1 ml of ice-cold PBS containing 1 mM EDTA.\",\n \"Thereafter, 200 μl of cell lysis buffer (12.5 mM MOPS, pH 7.3, 12.5 mM glycerophosphate, 7.5 mM MgCl2, 0.5 MM EGTA, 0.5 mM sodium vanadate, 1 mM benzamidine, 1 mM dithiothreitol, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 2 μg/ml pepstatin A, and 1 μM okadaic acid) is added to the cells.\",\n \"The cells are scraped from the plates and homogenized by 10 passages through a 23 3/4 G needle, and the cytosol fraction is prepared by centrifugation at 20,000×g for 15 minutes.\",\n \"Aliquots (5-10 μl containing 1-5 μg protein) of cytosol are mixed with 1 mM MAPK Substrate Peptide (APRTPGGRR (SEQ ID NO: 129), Upstate Biotechnology, Inc., N.Y.) and 50 μM [γ-32P]ATP (NEN, 3000 Ci/mmol), diluted to a final specific activity of ˜2000 cpm/pmol, in a total volume of 25 μl.\",\n \"The samples are incubated for 5 minutes at 30° C., and reactions are stopped by spotting 20 μl on 2 cm2 squares of Whatman P81 phosphocellulose paper.\",\n \"The filter squares are washed in 4 changes of 1% H3PO4, and the squares are subjected to liquid scintillation spectroscopy to quantitate bound label.\",\n \"Equivalent cytosolic extracts are incubated without MAPK substrate peptide, and the bound label from these samples are subtracted from the matched samples with the substrate peptide.\",\n \"The cytosolic extract from each well is used as a separate point.\",\n \"Protein concentrations are determined by a dye binding protein assay (Bio-Rad Laboratories).\",\n \"Agonist activation of the receptor is expected to result in up to a five-fold increase in MAPK enzyme activity.\",\n \"This increase is blocked by antagonists.\",\n \"H. [3H]Anachidonic Acid Release The activation of GPCRs also has been observed to potentiate arachidonic acid release in cells, providing yet another useful assay for modulators of GPCR activity.\",\n \"(See, e.g., Kanterman et al., Molecular Pharmacology 39:364-369 (1991).)\",\n \"For example, CHO cells that are stably transfected with a nGPCR-x expression vector are plated in 24-well plates at a density of 15,000 cells/well and grown in MEM medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin and 10 μg/ml streptomycin for 48 hours at 37° C. before use.\",\n \"Cells of each well are labeled by incubation with [3H]-arachidonic acid (Amersham Corp., 210 Ci/mmol) at 0.5 μCi/ml in 1 ml MEM supplemented with 10 mM HEPES, pH 7.5, and 0.5% fatty-acid-free bovine serum albumin for 2 hours at 37° C. The cells are then washed twice with 1 ml of the same buffer.\",\n \"Candidate modulator compounds are added in 1 ml of the same buffer, either alone or with 10 μM ATP and the cells are incubated at 37° C. for 30 minutes.\",\n \"Buffer alone and mock-transfected cells are used as controls.\",\n \"Samples (0.5 ml) from each well are counted by liquid scintillation spectroscopy.\",\n \"Agonists which activate the receptor will lead to potentiation of the ATP-stimulated release of [3H]-arachidonic acid.\",\n \"This potentiation is blocked by antagonists.\",\n \"I. Extracellular Acidification Rate In yet another assay, the effects of candidate modulators of nGFCR-x activity are assayed by monitoring extracellular changes in pH induced by the test compounds.\",\n \"(See, e.g., Dunlop et al, Journal of Pharmacological and Toxicological Methods 40(1):47-55 (1998).)\",\n \"In one embodiment, CHO cells transfected with a nGPCR-x expression vector are seeded into 12 mm capsule cups (Molecular Devices Corp.) at 4×105 cells/cup in MEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 10 U/ml penicillin, and 10 μg/ml streptomycin.\",\n \"The cells are incubated in this medium at 37° C. in 5% CO2 for 24 hours.\",\n \"Extracellular acidification rates are measured using a Cytosensor microphysiometer (Molecular Devices Corp.).\",\n \"The capsule cups are loaded into the sensor chambers of the microphysiometer and the chambers are perfused with running buffer (bicarbonate-free MEM supplemented with 4 mM L-glutamine, 10 units/ml penicillin, 10 μg/ml streptomycin, 26 mM NaCl) at a flow rate of 100 μl/minute.\",\n \"Candidate agonists or other agents are diluted into the running buffer and perfused through a second fluid path.\",\n \"During each 60-second pump cycle, the pump is run for 38 seconds and is off for the remaining 22 seconds.\",\n \"The pH of the running buffer in the sensor chamber is recorded during the cycle from 43-58 seconds, and the pump is re-started at 60 seconds to start the next cycle.\",\n \"The rate of acidification of the running buffer during the recording time is calculated by the Cytosoft program.\",\n \"Changes in the rate of acidification are calculated by subtracting the baseline value (the average of 4 rate measurements immediately before addition of a modulator candidate) from the highest rate measurement obtained after addition of a modulator candidate.\",\n \"The selected instrument detects 61 mV/pH unit.\",\n \"Modulators that act as agonists of the receptor result in an increase in the rate of extracellular acidification compared to the rate in the absence of agonist.\",\n \"This response is blocked by modulators which act as antagonists of the receptor.\",\n \"Example 11 In Situ Hybridization DNA Probe Preparation for nGPCR-11, -16, 40, -54, and -56 DNA probes for in situ hybridization were prepared as follows.\",\n \"Two sets of primer pairs were prepared.\",\n \"The first set has the sequence for T7 polymerase promoter on the 5′ primer to make the sense RNA, and the second set has the T7 polymerase promoter sequence on the 3′ primer to make the antisense RNA.\",\n \"PCR was performed in a 50 μl reaction containing 36.5 μl H2O, 5 μl 10×TT buffer (140 mM Ammonium Sulfate, 0.1% gelatine, 0.6 M Tris-tricine pH 8.4), 5 μl 25 mM MgCl2, 2 μl 10 mM dNTP, 0.4 μl Incyte clone 1722192 DNA, 0.5 μl AmpliTaq (PE Applied Biosystems), and 0.3 μl oligo1 (1 mg/ml) and 0.3 μl oligo2 (1 mg/ml) [to make the sense RNA], or 0.3 μl oligo3 (1 mg/ml) and 0.3 μl oligo4 (1 mg/ml) [to make the antisense RNA].\",\n \"The PCR reaction involved one cycle at 94° C. for 2 min followed by 35 cycles at 94° C. for 30 sec, 60° C. for 30 sec, 72° C. for 30 sec.\",\n \"The two PCR reactions were loaded onto a 1.2% agarose gel.\",\n \"The DNA band was excised from the gel, placed in a GenElute Agarose spin column (Supelco) and spun for 10 min at maximum speed.\",\n \"The eluted DNA was EtOH precipitated and resuspended in transcription buffer.\",\n \"The primer sequences for each nGPCR tested are listed below.\",\n \"For nGPCR-11, the sense primers were: GCGTAATACGACTCACTATAGGGAGACCGCGTGTCTGCTAGACTCTATTTCC 3′(LW1658) (SEQ ID NO: 159), and: 5′ TGCCACACTGATGCAACTCC 3′ (LW661) (SEQ ID NO: 160).\",\n \"The antisense primers were: GCGTAATACGACTCACTATAGGGAGACCTGCCACACTGATGCAACTCC (LW1659) SEQ ID NO: 161) and: 5′GCGTGTCTGCTAGACTCTATITCC 3′ (LW1660) (SEQ ID NO: 162).\",\n \"The primer pairs yielded a product of 275 bp.\",\n \"For nGPCR-16, the sense primers were: 5′GCGTAATACGACTCACTATAGGGAGACCGCACGCCACTCTTTACTATCCC (LW1645) (SEQ ID NO: 163), and: 5′ GCACAAAACACAATFCCATAAGCC 3′ (LW1648) (SEQ ID NO: 164).\",\n \"The antisense primers were: 5′GCGTAATACGACTCACTATAGGGAGACCGCACAAAACACAATTCCATAAGCC 3′ (LW1646) (SEQ ID NO: 165), and: 5′ GCTACGCCACTCTTrACTATCCC 3′ (LW1647) (SEQ ID NO: 166).\",\n \"The primer pairs yielded a product of 283 bp.\",\n \"For nGPCR-40, the sense primers were: 5′GCGTAATACGACTCACTATAGGGAGACCTTATGAGCAGCAATTCATCCC 3′(LW1704) (SEQ ID NO: 167), and: 5′CACACCCACCAAGAAATCAG 3′(LW1707) (SEQ ID NO: 168).\",\n \"The antisense primers were: 5′GCGTAATACGACTCACTATAGGGAGACCCACACCCACCAAGAAATCAG 3′(LW1705) (SEQ ID NO: 169), and: 5′ TTATGAGCAGCAATTCATCCC 3′ (LW1706) (SEQ ID NO: 170).\",\n \"The primer pairs yielded a product of 25 lbp.\",\n \"For nGPCR-54, the sense primers were: 5′GCGTAATACGACTCACTATAGGGAGACCCGATTATCCACACTTTGACCC 3′ (LW1803) (SEQ ID NO: 171), and: 5′CTGAAAGTrGTCGCTGACC 3′ (LW1634) (SEQ ID NO: 172).\",\n \"The anti-sense primers were: GCGTAATACGACTCACTATAGGGAGACCCTGCTGAAAGTTGTCGCTGACC 3′ (LW1804) (SEQ ID NO: 173), and: 5′CGATTATCCACACTTTGACGC 3′ (LW1635) (SEQ ID NO: 174).\",\n \"The primer pairs yielded a product of 286 bp.\",\n \"For nGPCR-56, the sense primers were: GCGTAATACGACTCACTATAGGGAGACCCTGTAAAATTCACACAAGCACC 3′ (LW1763) (SEQ ID NO: 175), and: 5′AGAAGACAGAGCAACCTCC 3′ (L[W1766) (SEQ ID NO: 176).\",\n \"The anti-sense primers were: GCGTAATACGACTCACTATAGGGAGACCAGAAGACAGAGCAACCTCC (LW1764) (SEQ ID NO: 177) and: CTGTAAAATTCACACAAGCACC (LW1765) (SEQ ID NO: 178).\",\n \"The primer pairs yielded a product of 272 bp.\",\n \"DNA Probe Preparation For nGPCR-1 Probes for nGPCR-1 were prepared as above with the following modifications.\",\n \"Using a sense primer: GCATGGATCCTCTTTTGCTGTATTTCACCCTC) (LW1595) (SEQ ID NO: 179) and an antisense primer: 5′GCATGAATTCACAATGCCAGTGATAAGGAAG 3′ (LW1596) (SEQ ID NO: 180), a 271 bp fragment was generated by PCR.\",\n \"The fragment was digested with BamHI and EcoRI and ligated into a BluescriptII vector that had been cut with BamHI and EcoRI.\",\n \"The orientation of the insert was such that T7 polymerase generates the anti-sense strand and T3 polymerase generates the sense strand.\",\n \"Histochemistry Coronal and sagittal oriented rat brain sections were cryosectioned (20 μm thick) using a Reichert-Jung cryostat.\",\n \"The individual sections were thaw-mounted onto silanated, nuclease-free slides (CEL Associates, Inc., Houston, Tex.\",\n \"), and stored at −80° C. The sections were processed starting with post-fixation in cold 4% paraformaldehyde, rinsed in cold PBS, acetylated using acetic anhydride in triethanolamine buffer and dehydrated through 70%, 95%, and 100% alcohols at room temperature (RT).\",\n \"This was followed with delipidation in chloroform then rehydration in 100% and 95% alcohol at room temperature.\",\n \"Sections were air-dried prior to hybridization.\",\n \"Two PCR fragments (˜250 bp) were generated, one that contained T7 polymerase on the 5′ end (sense) and the other with T7 polymerase on the 3′ end (antisense).\",\n \"The PCR fragments were labeled with 35S-UTP to yield a specific activity of 0.655×106 cpm/pmol for antisense and 0.675×106 cpm/pmol for sense probe.\",\n \"Both riboprobes were denatured and added to hybridization buffer containing 50% formamide, 10% dextran, 0.3M NaCl, 10 mM Tris, 1 mM EDTA, 1× Denhardts, and 10 mM DTT.\",\n \"Sequential brain cryosections were hybridized with 45 μl/slide of the sense and antisense riboprobe hybridization mixture, then covered with silanized glass coverslips.\",\n \"The sections were hybridized overnight (15-18 hrs) at 42° C. in an incubator.\",\n \"Coverslips were washed off the slides in 1×SSC, followed by RNase A treatment, and high temperature stringency washes (3×, 20 mins at 41° C.) in 0.1×SSC.\",\n \"Slides were dehydrated with 70%, 95% NH4OAc, and 100% NH4OAc alcohols, air-dried and exposed to Kodak BioMax MR-1 film.\",\n \"After 9 days of exposure, the film was developed.\",\n \"This was followed with coating selected tissue slides with Kodak NTB-2 nuclear track emulsion and storing the slides in the dark for 23 days.\",\n \"The slides were then developed and counterstained with hematoxylin.\",\n \"Emulsion-coated sections were analyzed microscopically to determine the specificity of labeling.\",\n \"Presence of autoradiographic grains (generated by antisense probe hybridization) over cell bodies (versus between cell bodies) was used as an index of specific hybridization.\",\n \"Results In Situ Hybridization Results Indicated Localization in the Following Brain Areas: nGPCR-1 was localized to the dentate gyrus of hippocampus, piriform cortex, and red nucleus.\",\n \"nGPCR-11 was localized to the piriform cortex, hippocampus, red nucleus, subthalamic nuclei, dorsal raphe, interpeduncular nucleus, and habenula.\",\n \"nGPCR-16 was localized to the cortex, piriform cortex, hippocampus, thalamus, subthalamic nuclei, hypothalamus, bed nucleus stria terminals and posterior striatum.\",\n \"nGPCR-40 was localized to the cortex, piriform cortex, hippocampus, substantia nigra compacta, hypothalamus, laterial septus, bed nucleus stria terminalis, thalamus, ventral tegmental area, interpeduncular nucleus, dorsal raphe, medical geniculate, islands of Calleja, subthamalmic nuclei, choroid plexus.\",\n \"nGPCR-54 was localized to the piriform cortex and hippocampus, including the dentate gyms, CA1 and CA3.nGPCR-56 was localized to the piriform cortex, cortex, interpeduncular nuceus, red nucleus, hippocampus, habenula, substantia nigra pars compacta, mamillary body stria terminalis, hypothalamus, subthamalmic nuclei, corsal raphe, and ventral tegmental area.\",\n \"Example 12 Chromosomal Localization Methods Chromosomal location of the genes encoding nGPCRs was determined using the Stanford G3 Radiation Hybrid Panel (Research Genetics, Inc., Huntsville, Ala.).\",\n \"This panel contains 83 radiation hybrid clones of the entire human genome created by the Stanford Human Genome Center.\",\n \"PCR reactions were assembled containing 25 ng of DNA from each clone and the components of the Expand Hi-Fi PCR System™ (Roche Molecular Biochemicals, Indianapolis, Ind.)\",\n \"in a final reaction volume of 15 μl.\",\n \"PCR primers were synthesized by Genosys Corp., The Woodlands, Tex.\",\n \"PCR reactions were incubated in a GeneAmp 9700 PCR thermocycler (Perkin Elmer Applied Biosystems).\",\n \"The following cycling program was executed: Pre-soak at (940 for 3 min.)\",\n \"(94° for 30 sec.)\",\n \"(52° C. for 60 sec.)\",\n \"(72′ for 2 min.)]\",\n \"for 35 cycles.\",\n \"PCR reaction products were then separated and analyzed by electrophoresis on a 2.0% agarose gel, and stained with ethidium bromide.\",\n \"Lanes were scored for the presence or absence of the expected PCR product and the results submitted to the Stanford Human Genome Center via e-mail for analysis (http://wwwshgc.stanford.edu./RH/rhserverformnew.html).\",\n \"nGPCR-40 PCR primers were designed based on the available sequence of the Celera sequence HUM_IDS|Contig|11000258115466.The forward primer used was: 5′ACAGCCCCAAAGCCAAACAC3′ (SEQ ID NO: 181).\",\n \"The reverse primer was: 5′CCGCAGGAGCAATG-AAAATCAG3′ (SEQ ID NO: 182).\",\n \"This prier set will prime the synthesis of a 220 base pair fragment in the presence of the appropriate genomic DNA.\",\n \"G3 Radiation Hybrid Panel Analysis places nGPCR-40 on chromosome 6, most nearly linked to Stanford marker SHGC-1836 with a LOD score of 11.84.This marker lies at position 6q21.In a genome scanning data set, Cao et al.\",\n \"(Genomics 1997 Jul.\",\n \"1: 43(1): 1-8) found excess allele sharing for markers on 6q13-q26.Greatest allele sharing was at interval 6q21-q22.3 with a maximum multipoint MLS value of 3.06 close to marker D6S278.Replication data from a second data set found maximum multipoint MLS at the interval D6S424-D6S275.These results provide suggestive evidence for a susceptibility locus for schizophrenia in chromosome 6q from two independent data sets.\",\n \"nGPCR-54 PCR primers were designed based on the available sequence of the Celera sequence GA—11824020.The forward primer used was: 5′CTGTCTCTCTGTCCTCTTCC3′, (SEQ ID NO: 183).\",\n \"The reverse primer used was: 5′GCACCGATCTTCATTGAATTTC3′, (SEQ ID NO: 184).\",\n \"This primer set will prime the synthesis of a 145 base pair fragment in the presence of the appropriate genomic DNA.\",\n \"G3 Radiation Hybrid Panel Analysis places nGPCR-54 on chromosome 13, most nearly linked to Stanford marker SHGC-68276 with a LOD score of 6.31.This marker lies at position 13q32.Numerous investigations have found significant suggestion of linkage of schizophrenia to this region of chromosome 13q32.See, for example, Brzustowicz et al., Am J Hum Genet 1999 October; 65(4): 1096-1103; Blouinet al., Nat Genet 1998 September; 20(1): 70-3; Shaw et al., Am J Med Genet.\",\n \"1998 Sep. 7; 81(5): 364-76; Lin et al., Hum Genet 1997 March, 99(3): 417-20; Pulver et al., Cold Spring Harb Symp Quant Biol 1996; 61:797-814.Genes localized to chromosomal regions in linkage with schizophrenia are candidate genes for disease susceptibility.\",\n \"Genes in these regions with the potential to play a biochemical/functional role in the disease process (like G protein coupled receptors) have a high probability of being a disease-modifying locus.\",\n \"nGPCR-40 and -54, because of their chromosomal location, are attractive targets therefore for screening ligands useful in modulating cellular processes involved in schizophrenia.\",\n \"Example 13 Clone Deposit Information In accordance with the Budapest Treaty, clones of the present invention have been deposited at the Agricultural Research Culture Collection (NRRL) International Depository Authority, 1815 N. University Street, Peoria, Ill. 61604, U.S.A. Accession numbers and deposit dates are provided below in Table 6.TABLE 6 DEPOSIT INFORMATION Accession Number Nudapest Treaty Clone NRRL Deposit Date nGPCR-1 (SEQ ID NO:73) B-30243 Jan. 18, 2000 nGPCR-5 (SEQ ID NO: 75) B-30244 Jan. 18, 2000 nGPCR-16 (SEQ ID NO: 81) B-30245 Jan. 18, 2000 nGPCR-11 (SEQ ID NO: 79) B-30258 Feb. 2, 2000 nGPCR-17 (SEQ ID NO: 23) B-30259 Feb. 3, 2000 nGPCR-9 (SEQ ID NO: 77) B-30262 Feb. 22, 2000 nGPCR-58 (SEQ ID NO: 91) B-30274 Mar.\",\n \"23, 2000 nGPCR-56 (SEQ ID NO: 89) B-30288 May 5, 2000 nGPCR-3 (SEQ ID NO: 185) B-30290 May 5, 2000 nGPCR-54 (SEQ ID NO: 85) B-30291 May 5, 2000 nGPCR-40 (SEQ ID NO: 83*) B-30299N Jun.\",\n \"2, 2000 *The clone deposited with NRLL Accession Number N30299N comprises a sequence identical to SEQ ID NO: 83 but with the substitution of an “A” at neucleotide position 10.Example 14 Using nGPCR-x Proteins to Isolate Neurotransmitters The isolated nGPCR-x proteins can be used to isolate novel or known neurotransmitters (Saito et al., Nature 400: 265-269, 1999).\",\n \"The cDNAs that encode the isolated nGPCR-x can be cloned into mammalian expression vectors and used to stably or transiently transfect mammalian cells including CHO, Cos or HEK293 cells.\",\n \"Receptor expression can be determined by Northern blot analysis of transfected cells and identification of an appropriately sized mRNA band (predicted size from the cDNA).\",\n \"Brain regions shown by mRNA analysis to express each of the nGPCR-x proteins could be processed for peptide extraction using any of several protocols ((Reinsheidk R. K. et al., Science 270: 243-247, 1996; Sakurai, T., et al., Cell 92; 573-585, 1998; Hinuma, S., et al., Nature 398: 272-276, 1998).\",\n \"Chromotographic fractions of brain extracts could be tested for ability to activate nGPCR-x proteins by measuring second messenger production such as changes in cAMP production in the presence or absence of forskolin, changes in inositol 3-phosphate levels, changes in intracellular calcium levels or by indirect measures of receptor activation including receptor stimulated mitogenesis, receptor mediated changes in extracellular acidification or receptor mediated changes in reporter gene activation in response to cAMP or calcium (these methods should all be referenced in other sections of the patent).\",\n \"Receptor activation could also be monitored by co-transfecting cells with a chimeric GIq/i3 to force receptor coupling to a calcium stimulating pathway (Conklin et al., Nature 363; 274-276, 1993).\",\n \"Neurotransmitter mediated activation of receptors could also be monitored by measuring changes in [35S]-GTPKS binding in membrane fractions prepared from transfected mammalian cells.\",\n \"This assay could also be performed using baculoviruses containing nGPCR-x proteins infected into SF9 insect cells.\",\n \"The neurotransmitter which activates nGPCR-x proteins can be purified to homogeneity through successive rounds of purification using nGPCR-x proteins activation as a measurement of neurotransmitter activity.\",\n \"The composition of the neurotransmitter can be determined by mass spectrometry and Edman degradation if peptidergic.\",\n \"Neurotramitters isolated in this manner will be bioactive materials which will alter neurotransmission in the central nervous system and will produce behavioral and biochemical changes.\",\n \"Example 15 Using nGPCR-x Proteins to Isolate and Purify G Proteins cDNAs encoding nGPCR-x proteins are epitope-tagged at the amino terminus end of the cDNA with the cleavable influenza-hemagglutinin signal sequence followed by the FLAG epitope (IBI, New Haven, Conn.).\",\n \"Additionally, these sequences are tagged at the carboxyl terminus with DNA encoding six histidine residues.\",\n \"(Amino and Carboxyl Terminal Modifications to Facilitate the Production and Purification of a G Protein-Coupled Receptor, B. K. Kobilka, Analytical Biochemistry, Vol.\",\n \"231, No.\",\n \"1, October 1995, pp.\",\n \"269-271).\",\n \"The resulting sequences are cloned into a baculovirus expression vector such as pVL1392 (Invitrogen).\",\n \"The baculovirus expression vectors are used to infect SF-9 insect cells as described (Guan, X. M., Kobilka, T. S., and Kobilka, B. K. (1992) J. Biol.\",\n \"Chem.\",\n \"267, 21995-21998).\",\n \"Infected SF-9 cells could be grown in 1000-ml cultures in SF900 II medium (Life Technologies, Inc.) containing 5% fetal calf serum (Gemini, Calabasas, Calif.) and 0.1 mg/ml gentamicin (Life Technologies, Inc.) for 48 hours at which time the cells could be harvested.\",\n \"Cell membrane preparations could be separated from soluble proteins following cell lysis.\",\n \"nGPCR-x protein purification is carried out as described for purification of the θ2 receptor (Kobilka, Anal.\",\n \"Biochem., 231 (1): 269-271, 1995) including solubilization of the membranes in 0.8-1.0% n-dodecyl-D-maltoside (DM) (CalBiochem, La Jolla, Calif.) in buffer containing protease inhibitors followed by Ni-column chromatography using chelating Sepharose™ (Pharmacia, Uppsala, Sweden).\",\n \"The eluate from the Ni-column is further purified on an M1 anti-FLAG antibody column (IBI).\",\n \"Receptor containing fractions are monitored by using receptor specific antibodies following western blot analysis or by SDS-PAGE analysis to look for an appropriate sized protein band (appropriate size would be the predicted molecular weight of the protein).\",\n \"This method of purifying G protein is particularly useful to isolate G proteins that bind to the nGPCR-x proteins in the absence of an activating ligand.\",\n \"Some of the preferred embodiments of the invention described above are outlined below and include, but are not limited to, the following embodiments.\",\n \"As those skilled in the art will appreciate, numerous changes and modifications may be made to the preferred embodiments of the invention without departing from the spirit of the invention.\",\n \"It is intended that all such variations fall within the scope of the invention.\",\n \"The entire disclosure of each publication cited herein is hereby incorporated by reference.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10467492"}}},{"rowIdx":369,"cells":{"text":{"kind":"list like","value":[["Pharmaceutical formulation","The present invention provides a pharmaceutical formulation which comprises a core comprising a first active ingredient, a coating comprising a second active ingredient which is incompatible with the first active ingredient and a barrier between the core and the coating which prevents physical contact between the core and the coating, characterised in that the barrier is formed on the core by film-coating and the coating is formed on the barrier by press-coating."],["1.A pharmaceutical formulation which comprises a core comprising a first active ingredient, a coating comprising a second active ingredient which is incompatible with the first active ingredient and a barrier between the core and the coating which prevents physical contact between the core and the coating, characterised in that the barrier is formed on the core by film-coating and the coating is formed on the barrier by press-coating.","2.A pharmaceutical formulation as claimed in claim 1 wherein the core further comprises one or more pharmaceutical excipients, disintegrants, lubricants, anti-adhesion agents, flow agents or diluents.","3.A pharmaceutical formulation as claimed in claim 1 wherein the coating further comprises one or more pharmaceutical excipients, disintegrants, lubricants, anti-adhesion agents, flow agents or diluents.","4.A pharmaceutical formulation as claimed in claim 1 wherein the core comprises ranitidine or a physiologically acceptable salt thereof and the coating comprises acetylsalicylic acid or a physiologically acceptable salt thereof.","5.A pharmaceutical formulation as claimed in claim 4 wherein the ranitidine is present in the core in an amount of from 10 to 200 mg or a physiologically acceptable salt of ranitidine is present in the core in an amount which is equivalent to from 10 to 200 mg of ranitidine.","6.A pharmaceutical formulation as claimed in claim 4 wherein the ranitidine is present in the form of ranitidine hydrochloride.","7.A pharmaceutical formulation as claimed in claim 4 wherein acetylsalicylic acid is present in the coating in an amount of from 50 to 1000 mg or a physiologically acceptable salt of acetylsalicylic acid is present in the core in an amount which is equivalent to from 50 to 1000 mg of acetylsalicylic acid.","8.A pharmaceutical formulation as claimed in claim 4 wherein the acetylsalicylic acid or the physiologically acceptable salt of acetylsalicylic acid is microencapsulated with ethyl cellulose.","9.A pharmaceutical formulation as claimed in claim 1 wherein the barrier also reduces the transmission of moisture between the core and the coating.","10.A pharmaceutical formulation as claimed in claim 1 wherein the barrier is present in an amount of from 1 to 20% by weight based on the total weight of the core."],["The present invention relates to improvements in the formulation of pharmaceutical compositions.","In particular, it relates to improvements in the formulation of pharmaceutical compositions wherein two or more active ingredients are present in the composition and wherein at least two of the active ingredients are incompatible with one another.","In the context of the present application, the term “incompatible”, when applied to active ingredients, means that the active ingredients cannot normally be formulated into a pharmaceutical composition wherein the active ingredients are in physical contact.","The incompatibility is usually, but not necessarily, the result of a chemical reaction between the active ingredients which results in a reduction of the therapeutic activity of at least one of the active ingredients.","It will be appreciated by a person skilled in the art that many pharmaceutical compositions which comprise incompatible active ingredients may be formulated as described herein.","However, the present invention is particularly directed to compositions which comprise acetylsalicylic acid or a physiologically acceptable salt of acetylsalicylic acid and ranitidine or a physiologically acceptable salt of ranitidine, such as ranitidine hydrochloride.","Systemic non-steroidal anti-inflammatory drugs such as acetylsalicylic acid are known to give undesirable side effects.","In particular they are known to be ulcerogenic and can therefore give rise to gastric ulceration when administered orally over a period of time to a patient.","Ranitidine is the approved name for N-[2-[[[5-(dimethylamino)methyl]-2-furanyl]methyl]thio]ethyl-N′-methyl-2-nitro-1,1-ethanediamine which is described and claimed in British Patent No 1,565,966.It is known to be a potent histamine H2-antagonist which may be used in the treatment of conditions where there is an advantage in lowering gastric acidity, particularly in gastric and peptic ulceration.","It is also known from British Patent No 2,105,193 that mucosal lesions of the gastrointestinal tract caused by systemic non-steroidal anti-inflammatory drugs can be significantly reduced by co-administering ranitidine.","British Patent No 2,105,193 discloses pharmaceutical compositions comprising a systemic non-steroidal anti-inflammatory drug and ranitidine or a physiologically acceptable salt thereof.","However, experiments show that there is a clear chemical incompatibility between ranitidine hydrochloride (the most preferred physiologically acceptable salt of ranitidine) and acetylsalicylic acid.","These two active ingredients react chemically with each other to produce degradation products.","The reaction becomes manifest in a matter of days and implies a serious stability problem for pharmaceutical compositions containing both active ingredients.","The present invention addresses the problems outlined above and provides a pharmaceutical formulation which comprises a core comprising a first active ingredient, a coating comprising a second active ingredient which is incompatible with the first active ingredient and a barrier between the core and the coating which prevents physical contact between the core and the coating, characterised in that the barrier is formed on the core by film-coating and the coating is formed on the barrier by press-coating.","It will be appreciated by those skilled in the art that further barriers and coatings may be formed on the composition if so desired.","This may be necessary if, for example, more than two active ingredients are present and each active ingredient is incompatible with all or some of the other active ingredients.","In one embodiment of the present invention the core comprises ranitidine or a physiologically acceptable salt thereof and the coating comprises acetylsalicylic acid or a physiologically acceptable salt thereof.","In a preferred embodiment, ranitidine or a physiologically acceptable salt thereof is present in the core in an amount sufficient to reduce gastrointestinal distress caused by acetylsalicylic acid.","In a more preferred embodiment, ranitidine is present in the core in an amount of from 10 to 200 mg or a physiologically acceptable salt of ranitidine is present in the core in an amount which is equivalent to from 10 to 200 mg of ranitidine.","In a more preferred embodiment, ranitidine is present in the core in an amount of from 50 to 100 mg or a physiologically acceptable salt of ranitidine is present in the core in an amount which is equivalent to from 50 to 100 mg of ranitidine.","In a more preferred embodiment, ranitidine is present in the core in an amount of from 70 to 80 mg or a physiologically acceptable salt of ranitidine is present in the core in an amount which is equivalent to from 70 to 80 mg of ranitidine.","It is preferable that the ranitidine be present in the form of a physiologically acceptable salt.","Such salts include salts of inorganic or organic acids such as the hydrochloride, hydrobromide, sulphate, acetate, maleate, succinate and fumarate salts.","In a particularly preferred embodiment, ranitidine is present in the form of ranitidine hydrochloride.","As well as the first active ingredient, the core may also comprise one or more pharmaceutical excipients, disintegrants, lubricants, anti-adhesion agents, flow agents, diluents or taste-masking agents.","In a preferred embodiment, the one or more lubricants are present in the core in an amount of from 0.1 to 5% by weight based on the total weight of the core.","In a more preferred embodiment, the one or more lubricants are present in the core in an amount of from 0.1 to 3% by weight based on the total weight of the core.","In a still more preferred embodiment, the one or more lubricants are present in the core in an amount of from 0.5 to 1.0% by weight based on the total weight of the core.","Examples of suitable lubricants include magnesium stearate, calcium stearate, zinc stearate, stearic acid, palmitic acid and sodium stearyl fumarate.","A preferred lubricant is magnesium stearate.","In a preferred embodiment, the one or more diluents are present in the core in an amount of from 10 to 90% by weight based on the total weight of the core.","In a more preferred embodiment, the one or more diluents are present in the core in an amount of from 30 to 70% by weight based on the total weight of the core.","In a still more preferred embodiment, the one or more diluents are present in the core in an amount of from 40 to 60% by weight based on the total weight of the core.","Examples of suitable diluents include microcrystalline cellulose, powdered cellulose, lactose, mannitol, sucrose and calcium phosphate.","A preferred diluent is microcrystalline cellulose.","In a preferred embodiment, the one or more disintegrants are present in the core in an amount of from 0.1 to 10% by weight based on the total weight of the core.","In a more preferred embodiment, the one or more disintegrants are present in the core in an amount of from 0.1 to 5% by weight based on the total weight of the core.","In a still more preferred embodiment, the one or more disintegrants are present in the core in an amount of from 1 to 3% by weight based on the total weight of the core.","An example of a suitable disintegrant is sodium croscaramelose.","In a preferred embodiment, acetylsalicylic acid is present in the coating in an amount of from 50 to 1000 mg or a physiologically acceptable salt of acetylsalicylic acid is present in the core in an amount which is equivalent to from 50 to 1000 mg of acetylsalicylic acid.","In a more preferred embodiment, acetylsalicylic acid is present in the coating in an amount of from 300 to 700 mg or a physiologically acceptable salt of acetylsalicylic acid is present in the coating in an amount which is equivalent to from 300 to 700 mg of acetylsalicylic acid.","In a more preferred embodiment, acetylsalicylic acid is present in the coating in an amount of from 450 to 550 mg or a physiologically acceptable salt of acetylsalicylic acid is present in the coating in an amount which is equivalent to from 450 to 550 mg of acetylsalicylic acid.","In an embodiment the acetylsalicylic acid or the physiologically acceptable salt of acetylsalicylic acid may be microencapsulated with ethyl cellulose.","An example of a commercially available microencapsulated acetylsalicylic acid product is Rhodine NCR-P™.","As well as the second active ingredient, the coating may also comprise one or more pharmaceutical excipients, disintegrants, lubricants, anti-adhesion agents, flow agents, diluents or taste-masking agents.","In a preferred embodiment, the one or more diluents are present in the coating in an amount of from 5 to 90% by weight based on the total weight of the coating.","In a more preferred embodiment, the one or more diluents are present in the coating in an amount of from 5 to 50% by weight based on the total weight of the coating.","In a still more preferred embodiment, the one or more diluents are present In the coating in an amount of from 10 to 20% by weight based on the total weight of the coating.","Examples of suitable diluents include copovidone, microcrystalline cellulose, powdered cellulose, maize starch, lactose, Cellactose™80 (a compound formed from 25% powdered cellulose and 75% lactose monohydrate) and Ludipress™ (a compound formed from 93% lactose monohydrate, 3.5% povidone and 3.5% crospovidone).","A preferred diluent is Cellactose™80.In a preferred embodiment, the one or more disintegrants are present in the coating in an amount of from 0.1 to 10% by weight based on the total weight of the coating.","In a more preferred embodiment, the one or more disintegrants are present in the coating in an amount of from 0.1 to 5% by weight based on the total weight of the coating.","In a still more preferred embodiment, the one or more disintegrants are present in the coating in an amount of from 1 to 3% by weight based on the total weight of the coating.","Examples of suitable disintegrants include maize starch, crospovidone, sodium starch glycolate, bentonite, aluminium magnesium silicate and sodium croscaramelose.","A preferred disintegrant is sodium croscaramelose.","In a preferred embodiment, the one or more lubricants are present in the coating in an amount of from 0.1 to 10% by weight based on the total weight of the coating.","In a more preferred embodiment, the one or more lubricants are present in the coating in an amount of from 0.1 to 5% by weight based on the total weight of the coating.","In a still more preferred embodiment, the one or more lubricants are present in the coating in an amount of from 0.1 to 1.0% by weight based on the total weight of the coating.","Examples of suitable lubricants include hydrogenated vegetable oil, stearic acid, palmitic acid and sodium stearyl fumarate.","A preferred lubricant is sodium stearyl fumarate.","In a preferred embodiment, the one or more anti-adhesion agents are present in the coating in an amount of from 0.1 to 10% by weight based on the total weight of the coating.","Examples of suitable anti-adhesion agents are talc and hydrogenated vegetable oil.","In a preferred embodiment, the one or more flow agents are present in the coating in an amount of from 0.01 to 5% by weight based on the total weight of the coating.","In a more preferred embodiment, the one or more flow agents are present in the coating in an amount of from 0.01 to 1% by weight based on the total weight of the coating.","In a still more preferred embodiment, the one or more flow agents are present in the coating in an amount of from 0.01 to 0.5% by weight based on the total weight of the coating.","An example of a suitable flow agent is colloidal anhydrous silica.","The barrier is present in order to prevent physical contact between the core and the coating and therefore prevent reaction between the first and second active ingredients which are present in the core and the coating respectively.","Preferably the barrier also reduces the transmission of moisture between the core and the coating.","More preferably the barrier is substantially impermeable to the transmission of moisture between the core and the coating.","In a preferred embodiment the barrier is present in an amount of from 1 to 20% by weight based on the total weight of the core.","In a more preferred embodiment the barrier is present in an amount of from 3 to 15% by weight based on the total weight of the core.","In a still more preferred embodiment the barrier is present in an amount of from 5 to 10% by weight based on the total weight of the core.","In general terms any material that reduces water transmission and is compatible with the active ingredients contained in the core and the coating can be used to physically separate them.","Thus, a barrier material which comprises one or more of stearic acid, polyvinyl alcohol, ethyl cellulose, microcrystalline cellulose, hydroxypropyl cellulose and methacrylic acid copolymer type C, can be used.","An example of a suitable barrier material is OPADRY™ Aqueous Moisture Barrier (AMB) produced by the Colorcon Company (Reference OY-B-28920).","OPADRY™ AMB is made up of polyvinyl alcohol, titanium dioxide, purified talc, lecithin and xanthan gum.","The formula is supplied as a powder which is reconstituted using purified cold water before it is sprayed on the core.","The actual solids content of the coating suspensions can be varied according to the atomising capabilities of the spraying equipment.","Preferably, the solids content is approximately 20% w/w.","Another example of a suitable barrier material is SEPIFILM™ Low Permeability (LP) produced by Seppic.","SEPIFILM™ LP is made up of hydroxypropylmethyl cellulose, microcrystalline cellulose, stearic acid and pigments and/or lakes (if required).","Another example of a suitable barrier material is LUSTRECLEAR™ produced by FMC Corporation.","LUSTRECLEAR™ is made up of microcrystalline cellulose, carrageenan, polyethylene glycol, hydroxyethyl cellulose, maltodextrin and pigments and/or lakes (if required).","Another example of a suitable barrier material is EUDRAGIT™ L 30D-55 produced by Degussa.","If this barrier material is chosen, it should not be applied in an amount greater than approximately 1 mg/cm2 of barrier material on the tablet surface.","If more is used there is a danger of forming an enteric coating around the core which could reduce the bioavailability of the active ingredient in the core.","EUDRAGIT™ L 30D-55 is made up of methacrylic acid—ethyl acrylate copolymer as a 30% dispersion in water.","The mean relative molecular mass of the copolymer is approximately 250 000 and the ratio of carboxylic groups to ester groups is approximately 1:1.The material may also comprise surface-active agents such as sodium dodecyl sulphate and polysorbate 80.It contains not less than 46.0% m/m and not more than 50.6% m/m of methacrylic acid units, calculated with reference to the residue on evaporation.","Methods of manufacturing by press coating, also known as dry coating or compression coating are well known in the pharmaceutical industry.","See for example; Pharmaceutics, the Science of Dosage Form Design, edited by M E Aulton, Published by Churchill Livingstone (part of the Longman Group) 1988, ISBN 0-443-03643-8.In particular pages 675 to 676 explain the method of press coating as follows.","Press coating involves the compaction of granular material around an already preformed core using compressing equipment similar to that used for the core itself, e.g.","Manesty Drycota.","It is used mainly to separate chemically incompatible materials, one or more being placed in the core and the other(s) in the coating layer.","However, there is still an interface of contact left between the two layers.","In cases where even this is important then the process of press coating can be taken one step further.","It is possible to apply two press coatings to a tablet core using suitable equipment, e.g.","Manesty Bicota.","This equipment produces press coated tablets with perfect separation between the active core and the coating as these two can be separated by an inert coating.","The technique is also described in Reminton: The Science and Practice of Pharmacy 19th Edition 1995, published by Mack Publishing Company, ISBN 0-912734-04-3.See in particular pages 1616 and 1631 which explain the method of manufacture by press coating as follows.","Press coated tablets, also referred to as dry-coated, are prepared by feeding previously compressed tablets into a special tableting machine and compressing another granulation layer around the preformed tablets.","They have all the advantages of compressed tablets, i.e.","slotting, monogramming, speed of disintegration, etc, while retaining the attributes of sugar-coated tablets in masking the taste of the taste drug substance in the core tablets.","An example of a press-coated tablet press is the Manesty Drycota.","Press coated tablets can also be used to separate incompatible drug substances; in addition, they can provide a means to give an enteric coating to the core tablets.","The following journal articles also provide some discussion of the technique of press-coating: Press-coated tablets for the sequential pulsed administration of two different drugs, International Journal of Pharmaceutics, Volume 99, Issues 2-3, 1993, pages 173-179, Maggi L, et al.","Press-coated tablets for time-programmed released of drug, Biomaterials, Volume 14, Issue 13,1993 1017-1023, Conte U, et al.","The technique of film-coating is also well known in the pharmaceutical industry and is described for example in Pharmaceutics, the Science of Dosage Form Design, edited by M E Aulton, Published by Churchill Livingstone (part of the Longman Group) 1988, ISBN 0-443-03643-8.In particular pages 672 to 675 explain the method of film coating as follows.","Film coating involves the deposition, usually by a spray method, of a thin film of polymer surrounding the tablet core.","It is possible to utilize conventional panning equipment but more usually specialized equipment is employed to take advantage of fast coating times and a high degree of automation is possible.","The coating solution contains a polymer in a suitable liquid medium together with other ingredients such as pigments and plasticizers.","This solution is sprayed on to a rotated mixed tablet bed.","The drying conditions permit the removal of the solvent so as to leave a thin deposition of coating material around each tablet core.","Typically the coating solution formulation comprises: polymer, solvent, plasticizer and colorants.","The technique is also described in Reminton: The Science and Practice of Pharmacy 19th Edition 1995, published by Mack Publishing Company, ISBN 0-912734-04-3.See in particular pages 1652 to 1659 which explain the method of film coating as follows.","Film coating involves the deposition of a thin, but uniform, film onto the surface of the substrate.","Unlike sugar coating, the flexibility afforded in film coating allows additional substrates other than just compressed tablets, to be considered (eg, powders, granules, nonpareils, capsules).","Coatings are essentially applied continuously to a moving bed of material by means of a spray technique, although manual application procedures have also been used.","The following are examples of formulations which are representative of the present invention.","They are intended to illustrate the invention but are not intended to be limiting on the scope of the invention which is defined by the claims.","EXAMPLE 1 A double tablet having the following specifications for the core, barrier and coating was manufactured.","The first active ingredient is ranitidine hydrochloride and the second active ingredient is acetylsalicylic acid: Core Weight per % w/w (based on Core Ingredient tablet (mg) total core weight) Function Granulated 84.000 56.00 Active Ingredient Ranitidine Hydrochloride Microcrystalline 64.875 43.25 Diluent Cellulose Magnesium 1.125 0.75 Lubricant Stearate Barrier Barrier Weight per % w/w (based on Ingredient tablet (mg) total core weight) Function Opadry AMB ™ 12.000 8.00 Moisture Barrier Coating % w/w (based on Coating Weight per total coating Ingredient tablet (mg) weight) Function Acetylsalicylic 500.000 82.40 Active Ingredient acid Cellactose ™ 80 91.020 15.00 Diluent Sodium 12.140 2.00 Disintegrant Croscaramelose Sodium Stearyl 3.030 0.50 Lubricant Fumarate Colloidal 0.610 0.10 Flow Promoter Anhydrous Silica The manufacturing method was as follows, the steps being numbered sequentially: A) Manufacture of the Ranitidine Tablet Cores: Mixture of the Ranitidine Tablet Core Ingredients 1) The selected amount of granulated ranitidine hydrochloride, microcrystalline cellulose and magnesium stearate were weighed out according to the batch size to be manufactured.","2) The granulated ranitidine hydrochloride and the microcrystalline cellulose were added to a drum blender or equivalent mixing equipment.","3) The mixture was blended for 10 minutes at a speed of rotation of 15 rpm to provide a homogenous mixture.","Note that the mixing parameters may be varied according to the equipment used and batch size intended for manufacture.","4) To the homogenous mixture of ranitidine hydrochloride and microcrystalline cellulose was added the magnesium stearate.","Mixing was continued in the same drum blender or equivalent mixing equipment for 5 minutes at a speed of rotation of 15 rpm.","Again, the mixing parameters may be varied according to the equipment used and batch size intended for manufacture.","Compression of the Powder Mixture to Produce the Ranitidine Tablet Core 5) A tableting machine was used to compress the previous powder mixture into tablet cores in compliance with the following specifications: Weight: 150 mg Uniformity of Mass: In compliance with European Pharmacopeia Hardness: greater than 8 Kp Friability: less than 1% w/w Disintegration time: less than 15 minutes The punches used were standard concave, 8 mm diameter.","B) Film-Coating the Ranitidine Tablet Cores: 6) The coating suspension of Opadry AMB was prepared as follows.","The required amount of Opadry AMB and purified water to prepare a 20% w/w dispersion of Opadry AMB in purified water were measured out.","Note that the recommended solids level is 20% w/w, but the actual solids level in the suspension can be changed to allow for the atomising capabilities of the spraying equipment.","Opadry AMB was reconstituted as follows.","The total quantity of cold water was poured into a suitable container.","A propeller or similar type of stirrer was placed in the water, and rotated so that a vortex was produced, but without drawing air into the liquid.","The Opadry AMB powder was added to the vortex as quickly as possible so that undispersed powder did not float on the surface of the liquid.","During the addition step, the suspension viscosity rose, thus it was necessary to increase the stirrer speed in order to maintain the vortex.","After all the Opadry AMB powder had been added, the stirrer speed was reduced until the vortex was nearly eliminated and stirring continued for a further 45 minutes, after which time the coating suspension was ready for use.","It was preferable to provide gentle agitation to the coating suspension while it was being sprayed.","7) The selected amount of ranitidine tablet cores were placed into a suitable film coating equipment e.g.","ACCELA-COTA or similar equipment.","12 mg of Opadry AMB film was coated on the ranitidine tablet cores using the following film coating parameters: Inlet air temperature: ≧90° C. Product temperature before spraying: ≧70° C. Drum speed: 10 rpm Spray equipment: Manesty spray gun Atomising air pressure: 3.5 Bar The spraying parameters may be varied according to the equipment used and batch size intended for manufacture.","C) Press Coating the Ranitidine Film Coated Cores: Mixture of the Acetyl Salicylic Acid (ASA) Tablet Coat Ingredients 8) The selected amounts of ASA, Cellactose™ 80, sodium croscaramelose, sodium stearyl fumarate and colloidal anhydrous silica were weighed out.","9) The ASA, Cellactose™ 80, sodium croscaramelose, sodium stearyl fumarate and colloidal anhydrous silica were added to a drum blender or equivalent mixing equipment.","10) The mixture was blended for 10 minutes at 15 rpm to obtain a homogenous mixture.","Note that these parameters are Intended as a guide only.","The proper parameters will depend upon the equipment and the batch size intended for manufacture.","Press Coating the ASA Powder Mixture onto the Film Coated Ranitidine Cores 11) A tableting machine which is capable of producing double compressed tablets e.g.","Manesty Drycota or similar equipment was used to provide a tablet in compliance with the following specifications: Weight: 768.8 mg Uniformity of Mass: compliant with European Pharmacopeia Hardness: greater than 10 Kp Friability: less than 1% w/w The punches used were standard concave, 12 mm diameter The press coating process can be summarised in the following steps.","Approximately half of the ASA powder mixture was filled into a tablet die.","Then the filmed-coated ranitidine tablet was fed into the tablet die.","Afterwards, the rest of the ASA mixture was filled into the die.","The punches compressed the ASA coat onto the filmed-coated ranitidine tablet, producing a double compressed tablet where the tablet core is the filmed-coated ranitidine tablet and the external coat is the ASA.","This cycle was repeated by the tableting machine as more film coated ranitidine tablets and ASA mixture (dry coating material) were fed into the dies from the hoppers.","This formulation was subjected to a stability study under the following conditions: 25° C./60% relative humidity and 40° C./75% relative humidity for 1, 2, 3 and 6 months.","It was found that the ranitidine remains stable for 6 months at 25° C./60% relative humidity and at 40° C./75% relative humidity.","The acetylsalicylic acid is stable for 6 months at 25° C./60% relative humidity and is stable for 2 months at 40° C./75% relative humidity.","EXAMPLE 2 A double tablet having the following specifications for the core, barrier and coating was manufactured: Core Weight per % w/w (based on Core Ingredient tablet (mg) total core weight) Function Granulated 84.000 56.00 Active Ingredient Ranitidine Hydrochloride Microcrystalline 64.875 43.25 Diluent Cellulose Magnesium 1.125 0.75 Lubricant Stearate Barrier Barrier Weight per % w/w (based on Ingredient tablet (mg) total core weight) Function Opadry AMB ™ 12.000 8.00 Moisture Barrier Coating % w/w (based on Coating Weight per total coating Ingredient tablet (mg) weight) Function Acetylsalicylic 507.6-526.3(1) 82.7-85.7 Active Ingredient Acid micro- encapsulated with Ethyl Cellulose (Rhodine NCR- P ™) Cellactose ™ 80 73.7-92.4(2) 12.0-15.0 Diluent Sodium 12.3 2.0 Disintegrant Croscaramelose Sodium Stearyl 1.2 0.2 Lubricant Fumarate Colloidal 0.6 0.1 Flow Promoter Anhydrous Silica (1) The purity of acetylsalicylic acid micro-encapsulated with ethyl cellulose varies between 95% and 98.5% w/w.","Therefore the quantity of acetylsalicylic acid micro-encapsulated with ethyl cellulose that has to be included in each tablet can vary between 507.6 and 526.3 mg in order to provide a dose of 500 mg of acetylsalicylic acid.","(2) The amount of Cellactose™ 80 added can vary between 73.7 and 92.4 mg so as to provide a total of 600 mg for the combined weight of Cellactose™ 80 and acetylsalicylic acid micro-encapsulated with ethyl cellulose.","The manufacturing method was the same as for Example 1 above except that the weight of acetylsalicylic acid micro-encapsulated with ethyl cellulose has to be adjusted to provide 500 mg of acetylsalicylic acid and the difference in the final tablet weight is compensated with the amount of Cellactose™ 80.This formulation was subjected to a stability study under the following conditions: 25° C./60% relative humidity and 40° C./75% relative humidity for 1, 2, 3 and 6 months.","It was found that the ranitidine remains stable for 6 months at 25° C./60% relative humidity and at 40° C./75% relative humidity.","The acetylsalicylic acid is stable for 6 months at 25° C./60% relative humidity and is stable for 2 months at 40° C./75% relative humidity."]],"string":"[\n [\n \"Pharmaceutical formulation\",\n \"The present invention provides a pharmaceutical formulation which comprises a core comprising a first active ingredient, a coating comprising a second active ingredient which is incompatible with the first active ingredient and a barrier between the core and the coating which prevents physical contact between the core and the coating, characterised in that the barrier is formed on the core by film-coating and the coating is formed on the barrier by press-coating.\"\n ],\n [\n \"1.A pharmaceutical formulation which comprises a core comprising a first active ingredient, a coating comprising a second active ingredient which is incompatible with the first active ingredient and a barrier between the core and the coating which prevents physical contact between the core and the coating, characterised in that the barrier is formed on the core by film-coating and the coating is formed on the barrier by press-coating.\",\n \"2.A pharmaceutical formulation as claimed in claim 1 wherein the core further comprises one or more pharmaceutical excipients, disintegrants, lubricants, anti-adhesion agents, flow agents or diluents.\",\n \"3.A pharmaceutical formulation as claimed in claim 1 wherein the coating further comprises one or more pharmaceutical excipients, disintegrants, lubricants, anti-adhesion agents, flow agents or diluents.\",\n \"4.A pharmaceutical formulation as claimed in claim 1 wherein the core comprises ranitidine or a physiologically acceptable salt thereof and the coating comprises acetylsalicylic acid or a physiologically acceptable salt thereof.\",\n \"5.A pharmaceutical formulation as claimed in claim 4 wherein the ranitidine is present in the core in an amount of from 10 to 200 mg or a physiologically acceptable salt of ranitidine is present in the core in an amount which is equivalent to from 10 to 200 mg of ranitidine.\",\n \"6.A pharmaceutical formulation as claimed in claim 4 wherein the ranitidine is present in the form of ranitidine hydrochloride.\",\n \"7.A pharmaceutical formulation as claimed in claim 4 wherein acetylsalicylic acid is present in the coating in an amount of from 50 to 1000 mg or a physiologically acceptable salt of acetylsalicylic acid is present in the core in an amount which is equivalent to from 50 to 1000 mg of acetylsalicylic acid.\",\n \"8.A pharmaceutical formulation as claimed in claim 4 wherein the acetylsalicylic acid or the physiologically acceptable salt of acetylsalicylic acid is microencapsulated with ethyl cellulose.\",\n \"9.A pharmaceutical formulation as claimed in claim 1 wherein the barrier also reduces the transmission of moisture between the core and the coating.\",\n \"10.A pharmaceutical formulation as claimed in claim 1 wherein the barrier is present in an amount of from 1 to 20% by weight based on the total weight of the core.\"\n ],\n [\n \"The present invention relates to improvements in the formulation of pharmaceutical compositions.\",\n \"In particular, it relates to improvements in the formulation of pharmaceutical compositions wherein two or more active ingredients are present in the composition and wherein at least two of the active ingredients are incompatible with one another.\",\n \"In the context of the present application, the term “incompatible”, when applied to active ingredients, means that the active ingredients cannot normally be formulated into a pharmaceutical composition wherein the active ingredients are in physical contact.\",\n \"The incompatibility is usually, but not necessarily, the result of a chemical reaction between the active ingredients which results in a reduction of the therapeutic activity of at least one of the active ingredients.\",\n \"It will be appreciated by a person skilled in the art that many pharmaceutical compositions which comprise incompatible active ingredients may be formulated as described herein.\",\n \"However, the present invention is particularly directed to compositions which comprise acetylsalicylic acid or a physiologically acceptable salt of acetylsalicylic acid and ranitidine or a physiologically acceptable salt of ranitidine, such as ranitidine hydrochloride.\",\n \"Systemic non-steroidal anti-inflammatory drugs such as acetylsalicylic acid are known to give undesirable side effects.\",\n \"In particular they are known to be ulcerogenic and can therefore give rise to gastric ulceration when administered orally over a period of time to a patient.\",\n \"Ranitidine is the approved name for N-[2-[[[5-(dimethylamino)methyl]-2-furanyl]methyl]thio]ethyl-N′-methyl-2-nitro-1,1-ethanediamine which is described and claimed in British Patent No 1,565,966.It is known to be a potent histamine H2-antagonist which may be used in the treatment of conditions where there is an advantage in lowering gastric acidity, particularly in gastric and peptic ulceration.\",\n \"It is also known from British Patent No 2,105,193 that mucosal lesions of the gastrointestinal tract caused by systemic non-steroidal anti-inflammatory drugs can be significantly reduced by co-administering ranitidine.\",\n \"British Patent No 2,105,193 discloses pharmaceutical compositions comprising a systemic non-steroidal anti-inflammatory drug and ranitidine or a physiologically acceptable salt thereof.\",\n \"However, experiments show that there is a clear chemical incompatibility between ranitidine hydrochloride (the most preferred physiologically acceptable salt of ranitidine) and acetylsalicylic acid.\",\n \"These two active ingredients react chemically with each other to produce degradation products.\",\n \"The reaction becomes manifest in a matter of days and implies a serious stability problem for pharmaceutical compositions containing both active ingredients.\",\n \"The present invention addresses the problems outlined above and provides a pharmaceutical formulation which comprises a core comprising a first active ingredient, a coating comprising a second active ingredient which is incompatible with the first active ingredient and a barrier between the core and the coating which prevents physical contact between the core and the coating, characterised in that the barrier is formed on the core by film-coating and the coating is formed on the barrier by press-coating.\",\n \"It will be appreciated by those skilled in the art that further barriers and coatings may be formed on the composition if so desired.\",\n \"This may be necessary if, for example, more than two active ingredients are present and each active ingredient is incompatible with all or some of the other active ingredients.\",\n \"In one embodiment of the present invention the core comprises ranitidine or a physiologically acceptable salt thereof and the coating comprises acetylsalicylic acid or a physiologically acceptable salt thereof.\",\n \"In a preferred embodiment, ranitidine or a physiologically acceptable salt thereof is present in the core in an amount sufficient to reduce gastrointestinal distress caused by acetylsalicylic acid.\",\n \"In a more preferred embodiment, ranitidine is present in the core in an amount of from 10 to 200 mg or a physiologically acceptable salt of ranitidine is present in the core in an amount which is equivalent to from 10 to 200 mg of ranitidine.\",\n \"In a more preferred embodiment, ranitidine is present in the core in an amount of from 50 to 100 mg or a physiologically acceptable salt of ranitidine is present in the core in an amount which is equivalent to from 50 to 100 mg of ranitidine.\",\n \"In a more preferred embodiment, ranitidine is present in the core in an amount of from 70 to 80 mg or a physiologically acceptable salt of ranitidine is present in the core in an amount which is equivalent to from 70 to 80 mg of ranitidine.\",\n \"It is preferable that the ranitidine be present in the form of a physiologically acceptable salt.\",\n \"Such salts include salts of inorganic or organic acids such as the hydrochloride, hydrobromide, sulphate, acetate, maleate, succinate and fumarate salts.\",\n \"In a particularly preferred embodiment, ranitidine is present in the form of ranitidine hydrochloride.\",\n \"As well as the first active ingredient, the core may also comprise one or more pharmaceutical excipients, disintegrants, lubricants, anti-adhesion agents, flow agents, diluents or taste-masking agents.\",\n \"In a preferred embodiment, the one or more lubricants are present in the core in an amount of from 0.1 to 5% by weight based on the total weight of the core.\",\n \"In a more preferred embodiment, the one or more lubricants are present in the core in an amount of from 0.1 to 3% by weight based on the total weight of the core.\",\n \"In a still more preferred embodiment, the one or more lubricants are present in the core in an amount of from 0.5 to 1.0% by weight based on the total weight of the core.\",\n \"Examples of suitable lubricants include magnesium stearate, calcium stearate, zinc stearate, stearic acid, palmitic acid and sodium stearyl fumarate.\",\n \"A preferred lubricant is magnesium stearate.\",\n \"In a preferred embodiment, the one or more diluents are present in the core in an amount of from 10 to 90% by weight based on the total weight of the core.\",\n \"In a more preferred embodiment, the one or more diluents are present in the core in an amount of from 30 to 70% by weight based on the total weight of the core.\",\n \"In a still more preferred embodiment, the one or more diluents are present in the core in an amount of from 40 to 60% by weight based on the total weight of the core.\",\n \"Examples of suitable diluents include microcrystalline cellulose, powdered cellulose, lactose, mannitol, sucrose and calcium phosphate.\",\n \"A preferred diluent is microcrystalline cellulose.\",\n \"In a preferred embodiment, the one or more disintegrants are present in the core in an amount of from 0.1 to 10% by weight based on the total weight of the core.\",\n \"In a more preferred embodiment, the one or more disintegrants are present in the core in an amount of from 0.1 to 5% by weight based on the total weight of the core.\",\n \"In a still more preferred embodiment, the one or more disintegrants are present in the core in an amount of from 1 to 3% by weight based on the total weight of the core.\",\n \"An example of a suitable disintegrant is sodium croscaramelose.\",\n \"In a preferred embodiment, acetylsalicylic acid is present in the coating in an amount of from 50 to 1000 mg or a physiologically acceptable salt of acetylsalicylic acid is present in the core in an amount which is equivalent to from 50 to 1000 mg of acetylsalicylic acid.\",\n \"In a more preferred embodiment, acetylsalicylic acid is present in the coating in an amount of from 300 to 700 mg or a physiologically acceptable salt of acetylsalicylic acid is present in the coating in an amount which is equivalent to from 300 to 700 mg of acetylsalicylic acid.\",\n \"In a more preferred embodiment, acetylsalicylic acid is present in the coating in an amount of from 450 to 550 mg or a physiologically acceptable salt of acetylsalicylic acid is present in the coating in an amount which is equivalent to from 450 to 550 mg of acetylsalicylic acid.\",\n \"In an embodiment the acetylsalicylic acid or the physiologically acceptable salt of acetylsalicylic acid may be microencapsulated with ethyl cellulose.\",\n \"An example of a commercially available microencapsulated acetylsalicylic acid product is Rhodine NCR-P™.\",\n \"As well as the second active ingredient, the coating may also comprise one or more pharmaceutical excipients, disintegrants, lubricants, anti-adhesion agents, flow agents, diluents or taste-masking agents.\",\n \"In a preferred embodiment, the one or more diluents are present in the coating in an amount of from 5 to 90% by weight based on the total weight of the coating.\",\n \"In a more preferred embodiment, the one or more diluents are present in the coating in an amount of from 5 to 50% by weight based on the total weight of the coating.\",\n \"In a still more preferred embodiment, the one or more diluents are present In the coating in an amount of from 10 to 20% by weight based on the total weight of the coating.\",\n \"Examples of suitable diluents include copovidone, microcrystalline cellulose, powdered cellulose, maize starch, lactose, Cellactose™80 (a compound formed from 25% powdered cellulose and 75% lactose monohydrate) and Ludipress™ (a compound formed from 93% lactose monohydrate, 3.5% povidone and 3.5% crospovidone).\",\n \"A preferred diluent is Cellactose™80.In a preferred embodiment, the one or more disintegrants are present in the coating in an amount of from 0.1 to 10% by weight based on the total weight of the coating.\",\n \"In a more preferred embodiment, the one or more disintegrants are present in the coating in an amount of from 0.1 to 5% by weight based on the total weight of the coating.\",\n \"In a still more preferred embodiment, the one or more disintegrants are present in the coating in an amount of from 1 to 3% by weight based on the total weight of the coating.\",\n \"Examples of suitable disintegrants include maize starch, crospovidone, sodium starch glycolate, bentonite, aluminium magnesium silicate and sodium croscaramelose.\",\n \"A preferred disintegrant is sodium croscaramelose.\",\n \"In a preferred embodiment, the one or more lubricants are present in the coating in an amount of from 0.1 to 10% by weight based on the total weight of the coating.\",\n \"In a more preferred embodiment, the one or more lubricants are present in the coating in an amount of from 0.1 to 5% by weight based on the total weight of the coating.\",\n \"In a still more preferred embodiment, the one or more lubricants are present in the coating in an amount of from 0.1 to 1.0% by weight based on the total weight of the coating.\",\n \"Examples of suitable lubricants include hydrogenated vegetable oil, stearic acid, palmitic acid and sodium stearyl fumarate.\",\n \"A preferred lubricant is sodium stearyl fumarate.\",\n \"In a preferred embodiment, the one or more anti-adhesion agents are present in the coating in an amount of from 0.1 to 10% by weight based on the total weight of the coating.\",\n \"Examples of suitable anti-adhesion agents are talc and hydrogenated vegetable oil.\",\n \"In a preferred embodiment, the one or more flow agents are present in the coating in an amount of from 0.01 to 5% by weight based on the total weight of the coating.\",\n \"In a more preferred embodiment, the one or more flow agents are present in the coating in an amount of from 0.01 to 1% by weight based on the total weight of the coating.\",\n \"In a still more preferred embodiment, the one or more flow agents are present in the coating in an amount of from 0.01 to 0.5% by weight based on the total weight of the coating.\",\n \"An example of a suitable flow agent is colloidal anhydrous silica.\",\n \"The barrier is present in order to prevent physical contact between the core and the coating and therefore prevent reaction between the first and second active ingredients which are present in the core and the coating respectively.\",\n \"Preferably the barrier also reduces the transmission of moisture between the core and the coating.\",\n \"More preferably the barrier is substantially impermeable to the transmission of moisture between the core and the coating.\",\n \"In a preferred embodiment the barrier is present in an amount of from 1 to 20% by weight based on the total weight of the core.\",\n \"In a more preferred embodiment the barrier is present in an amount of from 3 to 15% by weight based on the total weight of the core.\",\n \"In a still more preferred embodiment the barrier is present in an amount of from 5 to 10% by weight based on the total weight of the core.\",\n \"In general terms any material that reduces water transmission and is compatible with the active ingredients contained in the core and the coating can be used to physically separate them.\",\n \"Thus, a barrier material which comprises one or more of stearic acid, polyvinyl alcohol, ethyl cellulose, microcrystalline cellulose, hydroxypropyl cellulose and methacrylic acid copolymer type C, can be used.\",\n \"An example of a suitable barrier material is OPADRY™ Aqueous Moisture Barrier (AMB) produced by the Colorcon Company (Reference OY-B-28920).\",\n \"OPADRY™ AMB is made up of polyvinyl alcohol, titanium dioxide, purified talc, lecithin and xanthan gum.\",\n \"The formula is supplied as a powder which is reconstituted using purified cold water before it is sprayed on the core.\",\n \"The actual solids content of the coating suspensions can be varied according to the atomising capabilities of the spraying equipment.\",\n \"Preferably, the solids content is approximately 20% w/w.\",\n \"Another example of a suitable barrier material is SEPIFILM™ Low Permeability (LP) produced by Seppic.\",\n \"SEPIFILM™ LP is made up of hydroxypropylmethyl cellulose, microcrystalline cellulose, stearic acid and pigments and/or lakes (if required).\",\n \"Another example of a suitable barrier material is LUSTRECLEAR™ produced by FMC Corporation.\",\n \"LUSTRECLEAR™ is made up of microcrystalline cellulose, carrageenan, polyethylene glycol, hydroxyethyl cellulose, maltodextrin and pigments and/or lakes (if required).\",\n \"Another example of a suitable barrier material is EUDRAGIT™ L 30D-55 produced by Degussa.\",\n \"If this barrier material is chosen, it should not be applied in an amount greater than approximately 1 mg/cm2 of barrier material on the tablet surface.\",\n \"If more is used there is a danger of forming an enteric coating around the core which could reduce the bioavailability of the active ingredient in the core.\",\n \"EUDRAGIT™ L 30D-55 is made up of methacrylic acid—ethyl acrylate copolymer as a 30% dispersion in water.\",\n \"The mean relative molecular mass of the copolymer is approximately 250 000 and the ratio of carboxylic groups to ester groups is approximately 1:1.The material may also comprise surface-active agents such as sodium dodecyl sulphate and polysorbate 80.It contains not less than 46.0% m/m and not more than 50.6% m/m of methacrylic acid units, calculated with reference to the residue on evaporation.\",\n \"Methods of manufacturing by press coating, also known as dry coating or compression coating are well known in the pharmaceutical industry.\",\n \"See for example; Pharmaceutics, the Science of Dosage Form Design, edited by M E Aulton, Published by Churchill Livingstone (part of the Longman Group) 1988, ISBN 0-443-03643-8.In particular pages 675 to 676 explain the method of press coating as follows.\",\n \"Press coating involves the compaction of granular material around an already preformed core using compressing equipment similar to that used for the core itself, e.g.\",\n \"Manesty Drycota.\",\n \"It is used mainly to separate chemically incompatible materials, one or more being placed in the core and the other(s) in the coating layer.\",\n \"However, there is still an interface of contact left between the two layers.\",\n \"In cases where even this is important then the process of press coating can be taken one step further.\",\n \"It is possible to apply two press coatings to a tablet core using suitable equipment, e.g.\",\n \"Manesty Bicota.\",\n \"This equipment produces press coated tablets with perfect separation between the active core and the coating as these two can be separated by an inert coating.\",\n \"The technique is also described in Reminton: The Science and Practice of Pharmacy 19th Edition 1995, published by Mack Publishing Company, ISBN 0-912734-04-3.See in particular pages 1616 and 1631 which explain the method of manufacture by press coating as follows.\",\n \"Press coated tablets, also referred to as dry-coated, are prepared by feeding previously compressed tablets into a special tableting machine and compressing another granulation layer around the preformed tablets.\",\n \"They have all the advantages of compressed tablets, i.e.\",\n \"slotting, monogramming, speed of disintegration, etc, while retaining the attributes of sugar-coated tablets in masking the taste of the taste drug substance in the core tablets.\",\n \"An example of a press-coated tablet press is the Manesty Drycota.\",\n \"Press coated tablets can also be used to separate incompatible drug substances; in addition, they can provide a means to give an enteric coating to the core tablets.\",\n \"The following journal articles also provide some discussion of the technique of press-coating: Press-coated tablets for the sequential pulsed administration of two different drugs, International Journal of Pharmaceutics, Volume 99, Issues 2-3, 1993, pages 173-179, Maggi L, et al.\",\n \"Press-coated tablets for time-programmed released of drug, Biomaterials, Volume 14, Issue 13,1993 1017-1023, Conte U, et al.\",\n \"The technique of film-coating is also well known in the pharmaceutical industry and is described for example in Pharmaceutics, the Science of Dosage Form Design, edited by M E Aulton, Published by Churchill Livingstone (part of the Longman Group) 1988, ISBN 0-443-03643-8.In particular pages 672 to 675 explain the method of film coating as follows.\",\n \"Film coating involves the deposition, usually by a spray method, of a thin film of polymer surrounding the tablet core.\",\n \"It is possible to utilize conventional panning equipment but more usually specialized equipment is employed to take advantage of fast coating times and a high degree of automation is possible.\",\n \"The coating solution contains a polymer in a suitable liquid medium together with other ingredients such as pigments and plasticizers.\",\n \"This solution is sprayed on to a rotated mixed tablet bed.\",\n \"The drying conditions permit the removal of the solvent so as to leave a thin deposition of coating material around each tablet core.\",\n \"Typically the coating solution formulation comprises: polymer, solvent, plasticizer and colorants.\",\n \"The technique is also described in Reminton: The Science and Practice of Pharmacy 19th Edition 1995, published by Mack Publishing Company, ISBN 0-912734-04-3.See in particular pages 1652 to 1659 which explain the method of film coating as follows.\",\n \"Film coating involves the deposition of a thin, but uniform, film onto the surface of the substrate.\",\n \"Unlike sugar coating, the flexibility afforded in film coating allows additional substrates other than just compressed tablets, to be considered (eg, powders, granules, nonpareils, capsules).\",\n \"Coatings are essentially applied continuously to a moving bed of material by means of a spray technique, although manual application procedures have also been used.\",\n \"The following are examples of formulations which are representative of the present invention.\",\n \"They are intended to illustrate the invention but are not intended to be limiting on the scope of the invention which is defined by the claims.\",\n \"EXAMPLE 1 A double tablet having the following specifications for the core, barrier and coating was manufactured.\",\n \"The first active ingredient is ranitidine hydrochloride and the second active ingredient is acetylsalicylic acid: Core Weight per % w/w (based on Core Ingredient tablet (mg) total core weight) Function Granulated 84.000 56.00 Active Ingredient Ranitidine Hydrochloride Microcrystalline 64.875 43.25 Diluent Cellulose Magnesium 1.125 0.75 Lubricant Stearate Barrier Barrier Weight per % w/w (based on Ingredient tablet (mg) total core weight) Function Opadry AMB ™ 12.000 8.00 Moisture Barrier Coating % w/w (based on Coating Weight per total coating Ingredient tablet (mg) weight) Function Acetylsalicylic 500.000 82.40 Active Ingredient acid Cellactose ™ 80 91.020 15.00 Diluent Sodium 12.140 2.00 Disintegrant Croscaramelose Sodium Stearyl 3.030 0.50 Lubricant Fumarate Colloidal 0.610 0.10 Flow Promoter Anhydrous Silica The manufacturing method was as follows, the steps being numbered sequentially: A) Manufacture of the Ranitidine Tablet Cores: Mixture of the Ranitidine Tablet Core Ingredients 1) The selected amount of granulated ranitidine hydrochloride, microcrystalline cellulose and magnesium stearate were weighed out according to the batch size to be manufactured.\",\n \"2) The granulated ranitidine hydrochloride and the microcrystalline cellulose were added to a drum blender or equivalent mixing equipment.\",\n \"3) The mixture was blended for 10 minutes at a speed of rotation of 15 rpm to provide a homogenous mixture.\",\n \"Note that the mixing parameters may be varied according to the equipment used and batch size intended for manufacture.\",\n \"4) To the homogenous mixture of ranitidine hydrochloride and microcrystalline cellulose was added the magnesium stearate.\",\n \"Mixing was continued in the same drum blender or equivalent mixing equipment for 5 minutes at a speed of rotation of 15 rpm.\",\n \"Again, the mixing parameters may be varied according to the equipment used and batch size intended for manufacture.\",\n \"Compression of the Powder Mixture to Produce the Ranitidine Tablet Core 5) A tableting machine was used to compress the previous powder mixture into tablet cores in compliance with the following specifications: Weight: 150 mg Uniformity of Mass: In compliance with European Pharmacopeia Hardness: greater than 8 Kp Friability: less than 1% w/w Disintegration time: less than 15 minutes The punches used were standard concave, 8 mm diameter.\",\n \"B) Film-Coating the Ranitidine Tablet Cores: 6) The coating suspension of Opadry AMB was prepared as follows.\",\n \"The required amount of Opadry AMB and purified water to prepare a 20% w/w dispersion of Opadry AMB in purified water were measured out.\",\n \"Note that the recommended solids level is 20% w/w, but the actual solids level in the suspension can be changed to allow for the atomising capabilities of the spraying equipment.\",\n \"Opadry AMB was reconstituted as follows.\",\n \"The total quantity of cold water was poured into a suitable container.\",\n \"A propeller or similar type of stirrer was placed in the water, and rotated so that a vortex was produced, but without drawing air into the liquid.\",\n \"The Opadry AMB powder was added to the vortex as quickly as possible so that undispersed powder did not float on the surface of the liquid.\",\n \"During the addition step, the suspension viscosity rose, thus it was necessary to increase the stirrer speed in order to maintain the vortex.\",\n \"After all the Opadry AMB powder had been added, the stirrer speed was reduced until the vortex was nearly eliminated and stirring continued for a further 45 minutes, after which time the coating suspension was ready for use.\",\n \"It was preferable to provide gentle agitation to the coating suspension while it was being sprayed.\",\n \"7) The selected amount of ranitidine tablet cores were placed into a suitable film coating equipment e.g.\",\n \"ACCELA-COTA or similar equipment.\",\n \"12 mg of Opadry AMB film was coated on the ranitidine tablet cores using the following film coating parameters: Inlet air temperature: ≧90° C. Product temperature before spraying: ≧70° C. Drum speed: 10 rpm Spray equipment: Manesty spray gun Atomising air pressure: 3.5 Bar The spraying parameters may be varied according to the equipment used and batch size intended for manufacture.\",\n \"C) Press Coating the Ranitidine Film Coated Cores: Mixture of the Acetyl Salicylic Acid (ASA) Tablet Coat Ingredients 8) The selected amounts of ASA, Cellactose™ 80, sodium croscaramelose, sodium stearyl fumarate and colloidal anhydrous silica were weighed out.\",\n \"9) The ASA, Cellactose™ 80, sodium croscaramelose, sodium stearyl fumarate and colloidal anhydrous silica were added to a drum blender or equivalent mixing equipment.\",\n \"10) The mixture was blended for 10 minutes at 15 rpm to obtain a homogenous mixture.\",\n \"Note that these parameters are Intended as a guide only.\",\n \"The proper parameters will depend upon the equipment and the batch size intended for manufacture.\",\n \"Press Coating the ASA Powder Mixture onto the Film Coated Ranitidine Cores 11) A tableting machine which is capable of producing double compressed tablets e.g.\",\n \"Manesty Drycota or similar equipment was used to provide a tablet in compliance with the following specifications: Weight: 768.8 mg Uniformity of Mass: compliant with European Pharmacopeia Hardness: greater than 10 Kp Friability: less than 1% w/w The punches used were standard concave, 12 mm diameter The press coating process can be summarised in the following steps.\",\n \"Approximately half of the ASA powder mixture was filled into a tablet die.\",\n \"Then the filmed-coated ranitidine tablet was fed into the tablet die.\",\n \"Afterwards, the rest of the ASA mixture was filled into the die.\",\n \"The punches compressed the ASA coat onto the filmed-coated ranitidine tablet, producing a double compressed tablet where the tablet core is the filmed-coated ranitidine tablet and the external coat is the ASA.\",\n \"This cycle was repeated by the tableting machine as more film coated ranitidine tablets and ASA mixture (dry coating material) were fed into the dies from the hoppers.\",\n \"This formulation was subjected to a stability study under the following conditions: 25° C./60% relative humidity and 40° C./75% relative humidity for 1, 2, 3 and 6 months.\",\n \"It was found that the ranitidine remains stable for 6 months at 25° C./60% relative humidity and at 40° C./75% relative humidity.\",\n \"The acetylsalicylic acid is stable for 6 months at 25° C./60% relative humidity and is stable for 2 months at 40° C./75% relative humidity.\",\n \"EXAMPLE 2 A double tablet having the following specifications for the core, barrier and coating was manufactured: Core Weight per % w/w (based on Core Ingredient tablet (mg) total core weight) Function Granulated 84.000 56.00 Active Ingredient Ranitidine Hydrochloride Microcrystalline 64.875 43.25 Diluent Cellulose Magnesium 1.125 0.75 Lubricant Stearate Barrier Barrier Weight per % w/w (based on Ingredient tablet (mg) total core weight) Function Opadry AMB ™ 12.000 8.00 Moisture Barrier Coating % w/w (based on Coating Weight per total coating Ingredient tablet (mg) weight) Function Acetylsalicylic 507.6-526.3(1) 82.7-85.7 Active Ingredient Acid micro- encapsulated with Ethyl Cellulose (Rhodine NCR- P ™) Cellactose ™ 80 73.7-92.4(2) 12.0-15.0 Diluent Sodium 12.3 2.0 Disintegrant Croscaramelose Sodium Stearyl 1.2 0.2 Lubricant Fumarate Colloidal 0.6 0.1 Flow Promoter Anhydrous Silica (1) The purity of acetylsalicylic acid micro-encapsulated with ethyl cellulose varies between 95% and 98.5% w/w.\",\n \"Therefore the quantity of acetylsalicylic acid micro-encapsulated with ethyl cellulose that has to be included in each tablet can vary between 507.6 and 526.3 mg in order to provide a dose of 500 mg of acetylsalicylic acid.\",\n \"(2) The amount of Cellactose™ 80 added can vary between 73.7 and 92.4 mg so as to provide a total of 600 mg for the combined weight of Cellactose™ 80 and acetylsalicylic acid micro-encapsulated with ethyl cellulose.\",\n \"The manufacturing method was the same as for Example 1 above except that the weight of acetylsalicylic acid micro-encapsulated with ethyl cellulose has to be adjusted to provide 500 mg of acetylsalicylic acid and the difference in the final tablet weight is compensated with the amount of Cellactose™ 80.This formulation was subjected to a stability study under the following conditions: 25° C./60% relative humidity and 40° C./75% relative humidity for 1, 2, 3 and 6 months.\",\n \"It was found that the ranitidine remains stable for 6 months at 25° C./60% relative humidity and at 40° C./75% relative humidity.\",\n \"The acetylsalicylic acid is stable for 6 months at 25° C./60% relative humidity and is stable for 2 months at 40° C./75% relative humidity.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10467504"}}},{"rowIdx":370,"cells":{"text":{"kind":"list like","value":[["Method of education and scholastic management for cyber education system utilizing internet","This invention relates to the method of cyber education through internet capable to check the true attendance of a cyber class by a fingerprint and calculate the accumulative learning time of each registered lectures by the host computer for the scholastic management of the cyber education system.","In addition, utilizing this invention, it provides a pre-school system by using a game and animation created for pre-school curriculums capable to evaluate the education development of each children with a method for calculating the accumulative learning time of each subject by individual ID, and a software system for studying subjects at a stage of character formation for kids and evaluating the mental development of human being by means of playing game."],["1.The method for checking the attendance of the cyber class through internet, comprising the step of, (a) registering the fingerprint of registrant for completing a course of study and being issued ID number.","(b) confirming the attendance of the cyber class by inputting the registrant's fingerprint through internet; (c) confirming the time to depart from the class by inputting the fingerprint when the registrant finishes attending a lecture; (d) verifying the true attendance of class by the fingerprint data inputted from the registrant by comparing the fingerprint data with the fingerprint data memorized in the host computer in accordance with ID data of each registrant.","2.The method of claim 1, further comprising the step of alarming with a sound signal every set of time to the student for inputting the fingerprint, and verifying the true attendance of the cyber class by checking the fingerprint for a certain interval of the time.","3.The method of claim 1 or claim 2, further comprising the step of giving credits for the completion of the course by calculating the accumulated time of each student's log in time checked by means of fingerprint.","4.The method of claim 1 and 2, further comprising the step of creating database for each subject and lessons, and divide total running time of the program to small quantity, and when student logs-in to database with fingerprint and only views a few parts of the lesson then host computer records partial time spent and accumulates and giving credits when partial lessons are accumulating to complete lesson time which host computer recognizes as attendance.","5.The method of claim 1, wherein checking the attendance of the class through the verification of the iris of the eye, the voice signal or a passwords of the students instead of fingerprint.","6.The method of the scholastic management for granting academic degree at cyber school comprising of; (a) calculating by the host computer the starting time and ending time of each lesson by student's fingerprint data inputted and accumulate the total logging time in the memory device of the host computer; (b) granting credits when the host computer recognizes individual's accumulative time spent of each lesson is qualified according to the school's guide line for completion of the course; (c) giving a qualification to the student to submit a thesis through on-line or off-line when student finishes academic requirements according to the records of credits by the host computer; (d) granting a degree to the student when student passes all examinations concerning a thesis and complete academic cyber courses.","7.The method of claim 6, further comprising the step of giving an examination by the host computer and receiving the answer through internet to earn enough credits to graduate.","8.The method of claim 6, wherein further comprising the step of examining the thesis by attending the student on the thesis examination committee of the institute and giving the degree after the said examination.","9.The method of the scholastic management for operating the universalized university such as a cyber university or a cyber graduate school for worldwide, comprising the step of; (a) constructing the database of the lectures in different languages comprising at least one or more different languages of the each lecture of the each professor; (b) registering the fingerprint of the student for completing a course of study and being issued ID code; (c) logging into the system with registered ID and selecting the lecture by the language and the professor of the database; (d) attending the cyber class by confirming the starting time and the ending time of each lecture by inputting the student's fingerprint to check the true attendance of the cyber class; (e) granting credits when the host computer recognizes the student's accumulative time spent of each lesson is qualified according to the school's guide line for completion of the course; (f) giving a qualification to the student to submit a thesis through on-line or off-line when student finishes academic requirements according to the records of credits calculated by the host computer; (g) granting a degree to the student when student passes all examinations concerning a thesis and complete academic cyber courses.","10.The educational system for operating the cyber pre-school system utilizing internet comprising the step of; (a) a database comprising animation and/or game of each subject suitable for constructing the pre-school curriculums for the kids for studying by playing the game and/or animation; (b) a host computer for, (i) registering the cyber pre-school for completing a course of study by the parents and being issued ID code of the kids, (ii) calculating the time of log-in and log-out of each subject by the ID code for recording the accumulated playing time of each subject, (iii) qualifying the kids for completion of the program when individual's accumulative time spent of the each subject according to school's guide line, and (iv) recognizing the kids as completion of the academic programs when the kids complete all the courses, according to the records of the host computer.","11.The educational system of claim 10, further comprising a means for registering kid's fingerprint to indicate starting time and ending time for accessing DB to check an accurate attendance.","12.The educational system of claim 10, further comprising a database of the educational curriculums such as a language course, a music course or mathematics course for issuing a certificate for completion of the said educational curriculums by recognizing the kids as completion of the course when kid's accumulative time spent of the course is qualified by the host computer.","13.The educational system of claim 10, further comprising a database of internet on-line games for providing internet on-line game service to kids for interchanging among other kids.","14.The educational system of claim 10, further comprising a database for providing internet on-line game service and a means for a visual chatting service for kids to interchange among other kids.","15.The educational system of claim 10, further registering the kids of off-line pre-school to cyber pre-school for logging into the database through internet and recognizing the completion of courses with data calculated by the host computer.","16.The business method for operating the cyber pre-school utilizing internet comprising the step of; (a) creating a DB (database) for studying the each subject divided into session according to the pre-school curriculums; (b) issuing ID to log in to the DB by paying the tuition; (c) calculating the accumulated time of studying by the kids according to the time of log-in and log-out of each subject with the host computer; (d) advising the accumulated studying time of each subject through internet to the parents to select the subject for their kids to study more according to the said accumulated learning time data of each subject; (e) qualifying the kids for completion of the pre-school when individual's accumulative time spent of the each subject is qualified according to the guide line of the school by the host computer.","17.The business method of claim 16, further comprising a step for evaluating the development of study by calculating the signal from the kids for selecting answer among the questionnaires comprising animations or games with explanation voice.","18.The business method of claim 17, wherein the step for calculating further comprising a step for calculating the accumulated time of student's study by the subject and the number of the complete study of the subject.","19.The business method of claim 17, wherein the database is comprising educational game created to learn the subject of the pre-school curriculums for studying through playing the games.","20.The business method of claim 17, wherein the database is comprising educational animation and/or animated game created to study according to the curriculums for studying through playing the game and/or viewing animation.","21.The business method of claim 20, further comprising a cyber class created to study the subject according to the curriculums for studying through the explanation of the cyber teacher and the class activities of the characters in the cyber class.","22.The method for evaluating the mental development of a human being through internet comprising the step of, (a) making a DB (database) of games created to evaluate the mental development such as a character, a disposition and/or a morality of a human being in a category of each checking subject; (b) registering ID to log in the database with/without the fingerprint of the player; (c) accessing the database with said ID with/without the fingerprint of the player and playing the games through internet; (d) calculating the each action of the cyber characters played by the player and giving different points according to the favorable action and the non-favorable action based on the mental development evaluating standard in a category of each subject by the host computer of the DB system; (e) advising the result of the test in a category of each checking subject, calculated by the host computer for evaluating the mental developments of the player.","23.The method of claim 22, wherein the database comprising games to check the mental development and/or knowledge of pre-school kids and evaluating the mental development and/or knowledge of the kids according to the action of the cyber characters played by kids.","24.The method of claim 22 or claim 23, wherein the DB contents comprising animated questionnaires with/without a sound explanation to be selected by the player for evaluating the mental development of the player.","25.The software system for evaluating the mental development of human being with the PC comprising, (a) an animated game created to evaluate the player's intention according to the action of the game character in the categories of each situation through playing the game by the player; (b) a calculating means for calculating the each action of the game characters played by the player by giving different points according to the favorable action and the non-favorable action based on the mental development evaluation standard in a category of each subject to check; (c) a displaying means for displaying the result of the calculation by the point in a category of each subject to evaluate the mental development of the player.","26.The software system of claim 25, wherein the calculating means calculating the accumulated points in category of each subject and the number of the completion of the game to display according to the player.","27.The software system for studying the educational subjects with PC comprising, an animated game created to learn the subject through playing the game by the kids; a means for calculating the playing time of the each subject of the software by the each subject; and a means for displaying the accumulated playing time according to the subject to check the studying intensive of the kids.","28.The software system of claim 27 further comprising a cyber class created to study the subject to learn through the activities of the kids and the guidance of the teacher in the cyber class.","29.The software system of claim 27 or claim 28, further comprising a animated questionnaires for the kids to select the answer for calculating points to display on the monitor of the PC to evaluate the kid's understanding."],["<SOH> FIELD OF INVENTION <EOH>The present invention relates to the method of cyber education and scholastic management system related to the cyber education through Internet capable to check the true attendance of the cyber class by a fingerprint and calculate the accumulative learning time of each registered lectures by the host computer for determining automatically the completion of program for cyber education's scholastic management.","In addition, utilizing the system that calculates accumulative learning time of each registered lectures by the host computer, a children's educational software program can be created for the pre-school children at the stage of character formation where they start learning language, morality and human nature by imitation on the model of parents and home environment, the system can accumulate how much they absorb knowledge on each subject and how many subjects are covered according to the accumulated learning time data of the each subject by the child's individual ID, to provide the data for evaluating each child's progress in data by the category of each subject, so that the parents may know what kind of the subjects to be covered more for their kids."],["<SOH> BRIEF DESCRIPTION OF THE DRAWING <EOH>The figures in the drawings are briefly described as follows: FIG.","1 is a block diagram of the registering fingerprint data into education management system.","FIG.","2 is a block diagram of the system that calculates logging time on educational institution's database detailed-description description=\"Detailed Description\" end=\"lead\"?"],["FIELD OF INVENTION The present invention relates to the method of cyber education and scholastic management system related to the cyber education through Internet capable to check the true attendance of the cyber class by a fingerprint and calculate the accumulative learning time of each registered lectures by the host computer for determining automatically the completion of program for cyber education's scholastic management.","In addition, utilizing the system that calculates accumulative learning time of each registered lectures by the host computer, a children's educational software program can be created for the pre-school children at the stage of character formation where they start learning language, morality and human nature by imitation on the model of parents and home environment, the system can accumulate how much they absorb knowledge on each subject and how many subjects are covered according to the accumulated learning time data of the each subject by the child's individual ID, to provide the data for evaluating each child's progress in data by the category of each subject, so that the parents may know what kind of the subjects to be covered more for their kids.","DESCRIPTION OF THE RELATED ART The conventional way of Internet education is when a student registers for a program, the student receives an ID, attends class with that given ID and takes a form of tests to get grades or get completion of courses.","However, this method can not distinguish the true attendance of the cyber class from the wrong access by the substitutes.","Also, when student does not want to be presented at a complete lecture at the same time, attending a part of lecture, the conventional system have a problem with not enough data base information to accumulate actual time spent.","Further, taking advantage of world wide internet, cyber lecture course can be used on a world wide for many different people using different language, and it needs more developed technology rather than administering by human force in order to administer, control and provide the multi language lecture on the same subject by the same professor.","In addition, conventional curriculums for pre-school children courses on Internet have a problem with not providing enough information to evaluate the learning time of each subject according to the curriculums and how much children understand the program by the individual child's ID.","Especially, when a child is registered on a off-line pre-school program, parents do not know how many hours the child spent on what subject, and what area needs to be covered more for the children's human mental development.","Just by completing the program, parents assume that their child obtain more knowledge from the school.","Children are imitative by nature and begin to build up their character and temper by recognizing objects, and learning from daily activity of their parents and home environment from the age of 2-3 years old.","It is difficult for the parents to be a standard model for every aspect and subject for providing enough learning experiences for their children, therefore, it needs to develop a software for human brains that provide a progressive learning effect and memorizing the knowledge just like a software for the computer.","Learning experience on the model of parents from childhood helps to develop personality and morality of that person for lifetime so it is crucial that children's learning experience should be from very early stage of their lives.","The conventional ordinary off-line course or on-line course does not provide an accurate information such as what kind of learning experience children absorb and what needs to cover more in order to form a good character and a good disposition.","SUMMERY OF THE INVENTION A primary object of the present invention is to resolve prior mentioned problems by when a student registers for Internet course, the student inputs his/her own fingerprint data or iris of the eyes data in school's host computer.","When the student begins the class and ends the class, the student is required to put personal data such as fingerprints or an iris of the eyes, then host computer from school compares the data information from the memory mean of host computer to the student's data when he/she inputted to recognize if true person took the lesson.","When a student is taking a course, the host computer calculates data information containing how many hours on each subject the student spent and progress to determine grades and manage educational matters.","Further, regarding the pre-school curriculums that are made by an educational institution, the pre-school curriculums should construct games and animations data to help student to learn by himself by playing the PC, create a ID to log into the system and calculate number of logging time and hours spent on each subject to provide accurate data for parents to understand progress.","Therefore, parents can determine shortage on certain subject and what more needs to be done according to the progressive report data of the host computer.","Also, by given ID for students, the host computer accumulates hours spent on each subject and determines qualification for completion of the program to give out graduation certificates to the educational institution to manage its business.","BRIEF DESCRIPTION OF THE DRAWING The figures in the drawings are briefly described as follows: FIG.","1 is a block diagram of the registering fingerprint data into education management system.","FIG.","2 is a block diagram of the system that calculates logging time on educational institution's database DETAILED DESCRIPTION OF THE INVENTION To overcome the limitations in the prior art describe above, base on understanding and predicting current situation and certain limitation that will occur in the future.","In managing cyber institution, the present invention provides a method to verify an accurate attendance and registration by comparing fingerprints or iris of the eyes, and provides a method of scholastic management that cyber institution calculates automatically the true attendance of the cyber class by student's individual ID by a host computer to prevent waste of human source and operating cost by the more accurate and economical managing system.","In addition, utilizing the technology that calculates an accumulative learning time of each registered lectures by the host computer, the present invention is providing a pre-school education system through internet.","The pre-school management can construct a data base program on variety of subjects for pre-school children, and under the guidance of parents, children can log into an access to the program and complete courses.","Furthermore, it provides a progressive data by children's ID, and parents can determine what programs are covered and how many hours spend to measure the children's learning stage.","It also provides a technology to construct a software to evaluate the mental development of the children's memory from early stage of learning to form a good character and temper.","It is understood that many modifications and variations can be made therein, without departing from the spirit of the invention and from the scope of the appended claims.","In the following description of the exemplary embodiment, reference is made to the accompanying drawings, which is shown by way of illustrations of specific embodiment in the invention.","It is made without departing from the scope of the present invention.","FIG.","1 illustrates a block diagram of the registering fingerprint data into education management system.","According to the illustration of FIG.","1, the invention comprises a cyber educational institution (110); an institution's host computer (115); a data base (111) that stores each subject's lessons as in a form of digital data and controlled by host computer, when students (114-1 to 114-n) log into the data base through internet (113); an access control mean (112) to control fraud by checking ID and fingerprint data For checking attendance, when students (114-l-114-n) registers for the program, student inputs fingerprint to the host computer (115) and receives an ID to log into the data base (111).","The host computer then stores registered ID and fingerprint as in a form of digital data to memory.","When a student tries to log into institution's database (111) through the Internet (113) and put the given ID as same as conventional way.","When student wants to log in and try to view a lecture for a subject, the student needs to input fingerprint data through personal computer.","Then, the host computer compares the inputted data to the data from the memory.","If both of data matches, then, the computer recognizes ID and allows student to access the lecture and stores the data such as the log-in time, the learning time by the subject of the lectures.","When the student wishes to end the lecture, with ending alert signal, the student needs to input fingerprint data through personal computer.","Then, host computer (115) compares the inputted data to the data from the memory.","If both data matches, then, computer calculates accumulative logging time and store to the memory with ID.","In case, if both fingerprint data does not match, the host computer indicates as “ERROR” to the student and asks to re-enter fingerprint If the lecture session is ended or log-off without verifying fingerprint, then host the computer considers as fraud and stores the learning time as for the session.","Also, if the cyber educational institution tries to check constantly for more accurate attendance, set the program for certain set time such as in every 30 min for host computer (115) to transmit a signal to input fingerprint through student's PC monitor and compare the data in the same way.","Further more, if the institution creates a program for 2 hour lecture session and divides lecture into 30 min session or 1 hour session, the student can stop or continue each session for convenient and host computer stores and accumulates session time for each ID automatically and when all the sessions are complete then the host computer can give the grades.","According to the program mentioned above, when a student is complete with all the courses, then host computer accumulates all the credits and notify the student to do finals step or any kind of graduation process such as an examination, or a presentation of a thesis for granting a degree to the student.","If the student finishes the final step through on-line or off-line according to the institution's guideline, then the student is qualified for the completion of courses and granted for a degree.","These steps can be used for public school system, private schools or any kind of educational institutions such as a college, an university and a graduate school.","Also, instead of the fingerprint recognition system, when an iris recognition system or a voice recognition system become popular and economical, the system can be replaced and gets same effects.","Internet is the worldwide source without any borders among countries, when the educational institution want to manage worldwide, construct data base for each subject in different languages according to the professor, when students register and log in with choice of language, subject and professor then institution can check attendance with the prior mentioned systems to manage the institution worldwide.","FIG.","2 illustrates the block diagram for a pre school educational system utilizing the technology of the present invention.","According to FIG.","2, the present invention comprises a school (216) for creating and managing a cyber pre-school system; an educational program data base (214) created suitable for constructing the pre-school curriculums by subjects; a host computer (213-1, 213-2) for controlling access to the database by the non-authorized user, and calculating accumulative the learning time and number of log in times and controlling the system; an on-line game server (215) for storing the data of games for students; user's personal computers(211-1, 211-2, - - - 211-n) to log in the data base (214) and the game server (215) through Internet (212).","The database (214) includes a pre-school or a kindergarten's educational curriculums on each subject as a form of games or animations.","Each subject is divided to cover all the details on educational program.","In addition to the basic programs, a dance program, a music program, a language program, a mathematics program and an honor program can be created and added for the additional education in addition to the pre-school curriculums.","Game server (215) provides children to play game, stores all different kinds of pre-school game programs and provides multi media chatting services.","When student registers to the school (216) then student receives an ID code issue by school to log in to the system including program database (214) and games server (215).","Student pays tuition to the school and the tuition is main source of the cyber school's income.","When the student registers a fingerprint for checking attendance the scholastic management of the cyber pre-school can be used for same effects as described prior.","Student's parents can link to school's educational program database (214) through internet (212) using their personal computers (211-2, 211-2, - - - 211,n) to view and select each subject and help student to log in.","Educational program database (214) includes each subject's lessons through games, animations, and videos to help understand and teach children who are just starting to learn.","For example, “Christmas” can be put as search word, then children can learn about Christmas, and or “food” can be main search word to explain relationship between human body and food and all related topics.","Parents can select certain program if they feel more lessons should be provided certain area for the kids.","If parents select daily curriculums or lessons for children, children can join programs by simply pushing few buttons to play games, watch cartoons and videos and can repeat the programs for greater effects by the progressive learning effect.","After finishing with internet lessons, parents can discuss daily subject which children learned and add more explanation to help understand better and this process is reaching the goal of educational program and help children learn step by step.","The host computer (213-1, 213-2) then make data and stores information containing each subject logging time, time spent with user's ID to calculate accumulative time spent and indicate learning progress.","Each completed subject and time spent is stored as data then those stored data can be used for providing and checking completion of the entire program to give out graduation certificate and base for children's progress in learning.","Those stored data can provide and use for checking individual's progress, if parents think there are more area which needs to be covered, it is simple to recognize that information, and from stored data, the school as a service provider can make individual's progress report data for parents for convenience.","Addition to the basic curriculums, an honor program, a dance lessons, a music lessons, a language lessons or a mathematics can be added to the data base (215) for the extra curriculums and more professional lessons.","Calculating and checking progress is same as a basic system.","For extra curriculums, the school can provide special certificate for completion of those courses in addition to basic graduation certificate.","Off-line or conventional pre-school or kindergarten can join cyber program and instead of parents providing and selecting each subject or course for individual child, off-line school's teacher or guardian can do same part.","All data and progress report can provide base for off-line schools to help issue certificates.","Also, when students are log into games server (215) through internet (212), the students can be able to chat online with system setting, also when proper equipment is installed for video chatting, a child can join with other friends to talk online with monitor showing actual friends image.","Children can develop friendship and teamwork to solve problems for the games and children would want to stay with the program for social gathering.","This advantage can be helpful for increasing number of membership of the cyber pre-school service provider and increasing profit for the school.","The present invention provides a progressive structured learning on each curriculum for children by a software through Internet.","It also provides a scientific accurate accumulative time records from the beginning to the end of each software program.","Computer organizes all the progressive learning records, so it becomes simple to look at each individual's progress and check area for more supplement learning.","Children begin to recognize and learn about objects and everything around daily life when they are two or three years old.","From parent's behavior, children also learn and adopt a way of life and value of life.","Parents become teachers in early stage in every aspect and children develop their own personality from that stage.","So it is very crucial to the childhood to help to learn, and to help to memorize all kinds of knowledge from the very beginning stage, and accordingly, the present invention provides teaching methods and steps.","Just like a brand new computer needs to be installed with all different kinds of software, it is important for human being to learn proper knowledge and moral standard from beginning because it will be effect for rest of their lives.","Therefore, to provide a good knowledge and a good character for very young children, “learning software” can be created.","The leaning software can be made when top of professionals design curriculums for each subject which the kids have to know and divide them into small lessons and each small sessions are created in games and animations for the children who do not know letters or text yet.","On the way to help children to learn and memorize the learning software, since the software is designed with games and animations when children start to play the games, the children will try to understand the subjects and will begin to memorize by playing.","When subjects are described or explained in forms of animation, the effects become greater.","Furthermore, the method to check the progress for individuals can be done with a calculator means of the computer when children's curriculums are divided into each subject and more divided into small sessions.","For example, when 100 is perfect score to get when all the sessions are completed, computer records gives certain grades for each child's ID with the number of sessions play, the play time, and the number of sessions that are completed.","Each of these gets certain value for grades and calculator mean accumulates all the scores to check the progress.","Also, when there are animations containing visual and audio effects to test children with the multiple questionnaires by a form of animation or game then computer the combines the answers, more accurate information about progress can be made and let parents to figure out what area needs to cover more.","In addition, to overcome current problems of the on-line pre-school system, the present invention improves the quality by providing online games that can be played with multiple users with visual chatting to help social life of the kids.","For the business, since the program is designed with grading system with a proper graduation certificate, make it more interesting with online games to reach out to more customers.","Eventually number of the customers will be increased with the customers who want to stay with the programs for longer time and these will become profit to the business of the pre-school service provider.","This invention also can be used for evaluating the character, the disposition and the moral standard of the human being not only kids through internet or CD with personal computer.","The database or CD can be created with a games designed to check the mental development such as the character, the disposition and morality of the player by giving different points according to the favorable action and the non-favorable action based on the mental development evaluating standard in a category of each subject for checking those points by the action of the cyber characters played in the cyber situation by the player.","The method of this invention for evaluating the mental development, and the moral standard of a human being with a game is also providing the technology for making diagnoses of a mental diseases/disorder, and for evaluating the character, the disposition and the moral standards of human being for the assignment of a position for the right man in the right post."]],"string":"[\n [\n \"Method of education and scholastic management for cyber education system utilizing internet\",\n \"This invention relates to the method of cyber education through internet capable to check the true attendance of a cyber class by a fingerprint and calculate the accumulative learning time of each registered lectures by the host computer for the scholastic management of the cyber education system.\",\n \"In addition, utilizing this invention, it provides a pre-school system by using a game and animation created for pre-school curriculums capable to evaluate the education development of each children with a method for calculating the accumulative learning time of each subject by individual ID, and a software system for studying subjects at a stage of character formation for kids and evaluating the mental development of human being by means of playing game.\"\n ],\n [\n \"1.The method for checking the attendance of the cyber class through internet, comprising the step of, (a) registering the fingerprint of registrant for completing a course of study and being issued ID number.\",\n \"(b) confirming the attendance of the cyber class by inputting the registrant's fingerprint through internet; (c) confirming the time to depart from the class by inputting the fingerprint when the registrant finishes attending a lecture; (d) verifying the true attendance of class by the fingerprint data inputted from the registrant by comparing the fingerprint data with the fingerprint data memorized in the host computer in accordance with ID data of each registrant.\",\n \"2.The method of claim 1, further comprising the step of alarming with a sound signal every set of time to the student for inputting the fingerprint, and verifying the true attendance of the cyber class by checking the fingerprint for a certain interval of the time.\",\n \"3.The method of claim 1 or claim 2, further comprising the step of giving credits for the completion of the course by calculating the accumulated time of each student's log in time checked by means of fingerprint.\",\n \"4.The method of claim 1 and 2, further comprising the step of creating database for each subject and lessons, and divide total running time of the program to small quantity, and when student logs-in to database with fingerprint and only views a few parts of the lesson then host computer records partial time spent and accumulates and giving credits when partial lessons are accumulating to complete lesson time which host computer recognizes as attendance.\",\n \"5.The method of claim 1, wherein checking the attendance of the class through the verification of the iris of the eye, the voice signal or a passwords of the students instead of fingerprint.\",\n \"6.The method of the scholastic management for granting academic degree at cyber school comprising of; (a) calculating by the host computer the starting time and ending time of each lesson by student's fingerprint data inputted and accumulate the total logging time in the memory device of the host computer; (b) granting credits when the host computer recognizes individual's accumulative time spent of each lesson is qualified according to the school's guide line for completion of the course; (c) giving a qualification to the student to submit a thesis through on-line or off-line when student finishes academic requirements according to the records of credits by the host computer; (d) granting a degree to the student when student passes all examinations concerning a thesis and complete academic cyber courses.\",\n \"7.The method of claim 6, further comprising the step of giving an examination by the host computer and receiving the answer through internet to earn enough credits to graduate.\",\n \"8.The method of claim 6, wherein further comprising the step of examining the thesis by attending the student on the thesis examination committee of the institute and giving the degree after the said examination.\",\n \"9.The method of the scholastic management for operating the universalized university such as a cyber university or a cyber graduate school for worldwide, comprising the step of; (a) constructing the database of the lectures in different languages comprising at least one or more different languages of the each lecture of the each professor; (b) registering the fingerprint of the student for completing a course of study and being issued ID code; (c) logging into the system with registered ID and selecting the lecture by the language and the professor of the database; (d) attending the cyber class by confirming the starting time and the ending time of each lecture by inputting the student's fingerprint to check the true attendance of the cyber class; (e) granting credits when the host computer recognizes the student's accumulative time spent of each lesson is qualified according to the school's guide line for completion of the course; (f) giving a qualification to the student to submit a thesis through on-line or off-line when student finishes academic requirements according to the records of credits calculated by the host computer; (g) granting a degree to the student when student passes all examinations concerning a thesis and complete academic cyber courses.\",\n \"10.The educational system for operating the cyber pre-school system utilizing internet comprising the step of; (a) a database comprising animation and/or game of each subject suitable for constructing the pre-school curriculums for the kids for studying by playing the game and/or animation; (b) a host computer for, (i) registering the cyber pre-school for completing a course of study by the parents and being issued ID code of the kids, (ii) calculating the time of log-in and log-out of each subject by the ID code for recording the accumulated playing time of each subject, (iii) qualifying the kids for completion of the program when individual's accumulative time spent of the each subject according to school's guide line, and (iv) recognizing the kids as completion of the academic programs when the kids complete all the courses, according to the records of the host computer.\",\n \"11.The educational system of claim 10, further comprising a means for registering kid's fingerprint to indicate starting time and ending time for accessing DB to check an accurate attendance.\",\n \"12.The educational system of claim 10, further comprising a database of the educational curriculums such as a language course, a music course or mathematics course for issuing a certificate for completion of the said educational curriculums by recognizing the kids as completion of the course when kid's accumulative time spent of the course is qualified by the host computer.\",\n \"13.The educational system of claim 10, further comprising a database of internet on-line games for providing internet on-line game service to kids for interchanging among other kids.\",\n \"14.The educational system of claim 10, further comprising a database for providing internet on-line game service and a means for a visual chatting service for kids to interchange among other kids.\",\n \"15.The educational system of claim 10, further registering the kids of off-line pre-school to cyber pre-school for logging into the database through internet and recognizing the completion of courses with data calculated by the host computer.\",\n \"16.The business method for operating the cyber pre-school utilizing internet comprising the step of; (a) creating a DB (database) for studying the each subject divided into session according to the pre-school curriculums; (b) issuing ID to log in to the DB by paying the tuition; (c) calculating the accumulated time of studying by the kids according to the time of log-in and log-out of each subject with the host computer; (d) advising the accumulated studying time of each subject through internet to the parents to select the subject for their kids to study more according to the said accumulated learning time data of each subject; (e) qualifying the kids for completion of the pre-school when individual's accumulative time spent of the each subject is qualified according to the guide line of the school by the host computer.\",\n \"17.The business method of claim 16, further comprising a step for evaluating the development of study by calculating the signal from the kids for selecting answer among the questionnaires comprising animations or games with explanation voice.\",\n \"18.The business method of claim 17, wherein the step for calculating further comprising a step for calculating the accumulated time of student's study by the subject and the number of the complete study of the subject.\",\n \"19.The business method of claim 17, wherein the database is comprising educational game created to learn the subject of the pre-school curriculums for studying through playing the games.\",\n \"20.The business method of claim 17, wherein the database is comprising educational animation and/or animated game created to study according to the curriculums for studying through playing the game and/or viewing animation.\",\n \"21.The business method of claim 20, further comprising a cyber class created to study the subject according to the curriculums for studying through the explanation of the cyber teacher and the class activities of the characters in the cyber class.\",\n \"22.The method for evaluating the mental development of a human being through internet comprising the step of, (a) making a DB (database) of games created to evaluate the mental development such as a character, a disposition and/or a morality of a human being in a category of each checking subject; (b) registering ID to log in the database with/without the fingerprint of the player; (c) accessing the database with said ID with/without the fingerprint of the player and playing the games through internet; (d) calculating the each action of the cyber characters played by the player and giving different points according to the favorable action and the non-favorable action based on the mental development evaluating standard in a category of each subject by the host computer of the DB system; (e) advising the result of the test in a category of each checking subject, calculated by the host computer for evaluating the mental developments of the player.\",\n \"23.The method of claim 22, wherein the database comprising games to check the mental development and/or knowledge of pre-school kids and evaluating the mental development and/or knowledge of the kids according to the action of the cyber characters played by kids.\",\n \"24.The method of claim 22 or claim 23, wherein the DB contents comprising animated questionnaires with/without a sound explanation to be selected by the player for evaluating the mental development of the player.\",\n \"25.The software system for evaluating the mental development of human being with the PC comprising, (a) an animated game created to evaluate the player's intention according to the action of the game character in the categories of each situation through playing the game by the player; (b) a calculating means for calculating the each action of the game characters played by the player by giving different points according to the favorable action and the non-favorable action based on the mental development evaluation standard in a category of each subject to check; (c) a displaying means for displaying the result of the calculation by the point in a category of each subject to evaluate the mental development of the player.\",\n \"26.The software system of claim 25, wherein the calculating means calculating the accumulated points in category of each subject and the number of the completion of the game to display according to the player.\",\n \"27.The software system for studying the educational subjects with PC comprising, an animated game created to learn the subject through playing the game by the kids; a means for calculating the playing time of the each subject of the software by the each subject; and a means for displaying the accumulated playing time according to the subject to check the studying intensive of the kids.\",\n \"28.The software system of claim 27 further comprising a cyber class created to study the subject to learn through the activities of the kids and the guidance of the teacher in the cyber class.\",\n \"29.The software system of claim 27 or claim 28, further comprising a animated questionnaires for the kids to select the answer for calculating points to display on the monitor of the PC to evaluate the kid's understanding.\"\n ],\n [\n \"<SOH> FIELD OF INVENTION <EOH>The present invention relates to the method of cyber education and scholastic management system related to the cyber education through Internet capable to check the true attendance of the cyber class by a fingerprint and calculate the accumulative learning time of each registered lectures by the host computer for determining automatically the completion of program for cyber education's scholastic management.\",\n \"In addition, utilizing the system that calculates accumulative learning time of each registered lectures by the host computer, a children's educational software program can be created for the pre-school children at the stage of character formation where they start learning language, morality and human nature by imitation on the model of parents and home environment, the system can accumulate how much they absorb knowledge on each subject and how many subjects are covered according to the accumulated learning time data of the each subject by the child's individual ID, to provide the data for evaluating each child's progress in data by the category of each subject, so that the parents may know what kind of the subjects to be covered more for their kids.\"\n ],\n [\n \"<SOH> BRIEF DESCRIPTION OF THE DRAWING <EOH>The figures in the drawings are briefly described as follows: FIG.\",\n \"1 is a block diagram of the registering fingerprint data into education management system.\",\n \"FIG.\",\n \"2 is a block diagram of the system that calculates logging time on educational institution's database detailed-description description=\\\"Detailed Description\\\" end=\\\"lead\\\"?\"\n ],\n [\n \"FIELD OF INVENTION The present invention relates to the method of cyber education and scholastic management system related to the cyber education through Internet capable to check the true attendance of the cyber class by a fingerprint and calculate the accumulative learning time of each registered lectures by the host computer for determining automatically the completion of program for cyber education's scholastic management.\",\n \"In addition, utilizing the system that calculates accumulative learning time of each registered lectures by the host computer, a children's educational software program can be created for the pre-school children at the stage of character formation where they start learning language, morality and human nature by imitation on the model of parents and home environment, the system can accumulate how much they absorb knowledge on each subject and how many subjects are covered according to the accumulated learning time data of the each subject by the child's individual ID, to provide the data for evaluating each child's progress in data by the category of each subject, so that the parents may know what kind of the subjects to be covered more for their kids.\",\n \"DESCRIPTION OF THE RELATED ART The conventional way of Internet education is when a student registers for a program, the student receives an ID, attends class with that given ID and takes a form of tests to get grades or get completion of courses.\",\n \"However, this method can not distinguish the true attendance of the cyber class from the wrong access by the substitutes.\",\n \"Also, when student does not want to be presented at a complete lecture at the same time, attending a part of lecture, the conventional system have a problem with not enough data base information to accumulate actual time spent.\",\n \"Further, taking advantage of world wide internet, cyber lecture course can be used on a world wide for many different people using different language, and it needs more developed technology rather than administering by human force in order to administer, control and provide the multi language lecture on the same subject by the same professor.\",\n \"In addition, conventional curriculums for pre-school children courses on Internet have a problem with not providing enough information to evaluate the learning time of each subject according to the curriculums and how much children understand the program by the individual child's ID.\",\n \"Especially, when a child is registered on a off-line pre-school program, parents do not know how many hours the child spent on what subject, and what area needs to be covered more for the children's human mental development.\",\n \"Just by completing the program, parents assume that their child obtain more knowledge from the school.\",\n \"Children are imitative by nature and begin to build up their character and temper by recognizing objects, and learning from daily activity of their parents and home environment from the age of 2-3 years old.\",\n \"It is difficult for the parents to be a standard model for every aspect and subject for providing enough learning experiences for their children, therefore, it needs to develop a software for human brains that provide a progressive learning effect and memorizing the knowledge just like a software for the computer.\",\n \"Learning experience on the model of parents from childhood helps to develop personality and morality of that person for lifetime so it is crucial that children's learning experience should be from very early stage of their lives.\",\n \"The conventional ordinary off-line course or on-line course does not provide an accurate information such as what kind of learning experience children absorb and what needs to cover more in order to form a good character and a good disposition.\",\n \"SUMMERY OF THE INVENTION A primary object of the present invention is to resolve prior mentioned problems by when a student registers for Internet course, the student inputs his/her own fingerprint data or iris of the eyes data in school's host computer.\",\n \"When the student begins the class and ends the class, the student is required to put personal data such as fingerprints or an iris of the eyes, then host computer from school compares the data information from the memory mean of host computer to the student's data when he/she inputted to recognize if true person took the lesson.\",\n \"When a student is taking a course, the host computer calculates data information containing how many hours on each subject the student spent and progress to determine grades and manage educational matters.\",\n \"Further, regarding the pre-school curriculums that are made by an educational institution, the pre-school curriculums should construct games and animations data to help student to learn by himself by playing the PC, create a ID to log into the system and calculate number of logging time and hours spent on each subject to provide accurate data for parents to understand progress.\",\n \"Therefore, parents can determine shortage on certain subject and what more needs to be done according to the progressive report data of the host computer.\",\n \"Also, by given ID for students, the host computer accumulates hours spent on each subject and determines qualification for completion of the program to give out graduation certificates to the educational institution to manage its business.\",\n \"BRIEF DESCRIPTION OF THE DRAWING The figures in the drawings are briefly described as follows: FIG.\",\n \"1 is a block diagram of the registering fingerprint data into education management system.\",\n \"FIG.\",\n \"2 is a block diagram of the system that calculates logging time on educational institution's database DETAILED DESCRIPTION OF THE INVENTION To overcome the limitations in the prior art describe above, base on understanding and predicting current situation and certain limitation that will occur in the future.\",\n \"In managing cyber institution, the present invention provides a method to verify an accurate attendance and registration by comparing fingerprints or iris of the eyes, and provides a method of scholastic management that cyber institution calculates automatically the true attendance of the cyber class by student's individual ID by a host computer to prevent waste of human source and operating cost by the more accurate and economical managing system.\",\n \"In addition, utilizing the technology that calculates an accumulative learning time of each registered lectures by the host computer, the present invention is providing a pre-school education system through internet.\",\n \"The pre-school management can construct a data base program on variety of subjects for pre-school children, and under the guidance of parents, children can log into an access to the program and complete courses.\",\n \"Furthermore, it provides a progressive data by children's ID, and parents can determine what programs are covered and how many hours spend to measure the children's learning stage.\",\n \"It also provides a technology to construct a software to evaluate the mental development of the children's memory from early stage of learning to form a good character and temper.\",\n \"It is understood that many modifications and variations can be made therein, without departing from the spirit of the invention and from the scope of the appended claims.\",\n \"In the following description of the exemplary embodiment, reference is made to the accompanying drawings, which is shown by way of illustrations of specific embodiment in the invention.\",\n \"It is made without departing from the scope of the present invention.\",\n \"FIG.\",\n \"1 illustrates a block diagram of the registering fingerprint data into education management system.\",\n \"According to the illustration of FIG.\",\n \"1, the invention comprises a cyber educational institution (110); an institution's host computer (115); a data base (111) that stores each subject's lessons as in a form of digital data and controlled by host computer, when students (114-1 to 114-n) log into the data base through internet (113); an access control mean (112) to control fraud by checking ID and fingerprint data For checking attendance, when students (114-l-114-n) registers for the program, student inputs fingerprint to the host computer (115) and receives an ID to log into the data base (111).\",\n \"The host computer then stores registered ID and fingerprint as in a form of digital data to memory.\",\n \"When a student tries to log into institution's database (111) through the Internet (113) and put the given ID as same as conventional way.\",\n \"When student wants to log in and try to view a lecture for a subject, the student needs to input fingerprint data through personal computer.\",\n \"Then, the host computer compares the inputted data to the data from the memory.\",\n \"If both of data matches, then, the computer recognizes ID and allows student to access the lecture and stores the data such as the log-in time, the learning time by the subject of the lectures.\",\n \"When the student wishes to end the lecture, with ending alert signal, the student needs to input fingerprint data through personal computer.\",\n \"Then, host computer (115) compares the inputted data to the data from the memory.\",\n \"If both data matches, then, computer calculates accumulative logging time and store to the memory with ID.\",\n \"In case, if both fingerprint data does not match, the host computer indicates as “ERROR” to the student and asks to re-enter fingerprint If the lecture session is ended or log-off without verifying fingerprint, then host the computer considers as fraud and stores the learning time as for the session.\",\n \"Also, if the cyber educational institution tries to check constantly for more accurate attendance, set the program for certain set time such as in every 30 min for host computer (115) to transmit a signal to input fingerprint through student's PC monitor and compare the data in the same way.\",\n \"Further more, if the institution creates a program for 2 hour lecture session and divides lecture into 30 min session or 1 hour session, the student can stop or continue each session for convenient and host computer stores and accumulates session time for each ID automatically and when all the sessions are complete then the host computer can give the grades.\",\n \"According to the program mentioned above, when a student is complete with all the courses, then host computer accumulates all the credits and notify the student to do finals step or any kind of graduation process such as an examination, or a presentation of a thesis for granting a degree to the student.\",\n \"If the student finishes the final step through on-line or off-line according to the institution's guideline, then the student is qualified for the completion of courses and granted for a degree.\",\n \"These steps can be used for public school system, private schools or any kind of educational institutions such as a college, an university and a graduate school.\",\n \"Also, instead of the fingerprint recognition system, when an iris recognition system or a voice recognition system become popular and economical, the system can be replaced and gets same effects.\",\n \"Internet is the worldwide source without any borders among countries, when the educational institution want to manage worldwide, construct data base for each subject in different languages according to the professor, when students register and log in with choice of language, subject and professor then institution can check attendance with the prior mentioned systems to manage the institution worldwide.\",\n \"FIG.\",\n \"2 illustrates the block diagram for a pre school educational system utilizing the technology of the present invention.\",\n \"According to FIG.\",\n \"2, the present invention comprises a school (216) for creating and managing a cyber pre-school system; an educational program data base (214) created suitable for constructing the pre-school curriculums by subjects; a host computer (213-1, 213-2) for controlling access to the database by the non-authorized user, and calculating accumulative the learning time and number of log in times and controlling the system; an on-line game server (215) for storing the data of games for students; user's personal computers(211-1, 211-2, - - - 211-n) to log in the data base (214) and the game server (215) through Internet (212).\",\n \"The database (214) includes a pre-school or a kindergarten's educational curriculums on each subject as a form of games or animations.\",\n \"Each subject is divided to cover all the details on educational program.\",\n \"In addition to the basic programs, a dance program, a music program, a language program, a mathematics program and an honor program can be created and added for the additional education in addition to the pre-school curriculums.\",\n \"Game server (215) provides children to play game, stores all different kinds of pre-school game programs and provides multi media chatting services.\",\n \"When student registers to the school (216) then student receives an ID code issue by school to log in to the system including program database (214) and games server (215).\",\n \"Student pays tuition to the school and the tuition is main source of the cyber school's income.\",\n \"When the student registers a fingerprint for checking attendance the scholastic management of the cyber pre-school can be used for same effects as described prior.\",\n \"Student's parents can link to school's educational program database (214) through internet (212) using their personal computers (211-2, 211-2, - - - 211,n) to view and select each subject and help student to log in.\",\n \"Educational program database (214) includes each subject's lessons through games, animations, and videos to help understand and teach children who are just starting to learn.\",\n \"For example, “Christmas” can be put as search word, then children can learn about Christmas, and or “food” can be main search word to explain relationship between human body and food and all related topics.\",\n \"Parents can select certain program if they feel more lessons should be provided certain area for the kids.\",\n \"If parents select daily curriculums or lessons for children, children can join programs by simply pushing few buttons to play games, watch cartoons and videos and can repeat the programs for greater effects by the progressive learning effect.\",\n \"After finishing with internet lessons, parents can discuss daily subject which children learned and add more explanation to help understand better and this process is reaching the goal of educational program and help children learn step by step.\",\n \"The host computer (213-1, 213-2) then make data and stores information containing each subject logging time, time spent with user's ID to calculate accumulative time spent and indicate learning progress.\",\n \"Each completed subject and time spent is stored as data then those stored data can be used for providing and checking completion of the entire program to give out graduation certificate and base for children's progress in learning.\",\n \"Those stored data can provide and use for checking individual's progress, if parents think there are more area which needs to be covered, it is simple to recognize that information, and from stored data, the school as a service provider can make individual's progress report data for parents for convenience.\",\n \"Addition to the basic curriculums, an honor program, a dance lessons, a music lessons, a language lessons or a mathematics can be added to the data base (215) for the extra curriculums and more professional lessons.\",\n \"Calculating and checking progress is same as a basic system.\",\n \"For extra curriculums, the school can provide special certificate for completion of those courses in addition to basic graduation certificate.\",\n \"Off-line or conventional pre-school or kindergarten can join cyber program and instead of parents providing and selecting each subject or course for individual child, off-line school's teacher or guardian can do same part.\",\n \"All data and progress report can provide base for off-line schools to help issue certificates.\",\n \"Also, when students are log into games server (215) through internet (212), the students can be able to chat online with system setting, also when proper equipment is installed for video chatting, a child can join with other friends to talk online with monitor showing actual friends image.\",\n \"Children can develop friendship and teamwork to solve problems for the games and children would want to stay with the program for social gathering.\",\n \"This advantage can be helpful for increasing number of membership of the cyber pre-school service provider and increasing profit for the school.\",\n \"The present invention provides a progressive structured learning on each curriculum for children by a software through Internet.\",\n \"It also provides a scientific accurate accumulative time records from the beginning to the end of each software program.\",\n \"Computer organizes all the progressive learning records, so it becomes simple to look at each individual's progress and check area for more supplement learning.\",\n \"Children begin to recognize and learn about objects and everything around daily life when they are two or three years old.\",\n \"From parent's behavior, children also learn and adopt a way of life and value of life.\",\n \"Parents become teachers in early stage in every aspect and children develop their own personality from that stage.\",\n \"So it is very crucial to the childhood to help to learn, and to help to memorize all kinds of knowledge from the very beginning stage, and accordingly, the present invention provides teaching methods and steps.\",\n \"Just like a brand new computer needs to be installed with all different kinds of software, it is important for human being to learn proper knowledge and moral standard from beginning because it will be effect for rest of their lives.\",\n \"Therefore, to provide a good knowledge and a good character for very young children, “learning software” can be created.\",\n \"The leaning software can be made when top of professionals design curriculums for each subject which the kids have to know and divide them into small lessons and each small sessions are created in games and animations for the children who do not know letters or text yet.\",\n \"On the way to help children to learn and memorize the learning software, since the software is designed with games and animations when children start to play the games, the children will try to understand the subjects and will begin to memorize by playing.\",\n \"When subjects are described or explained in forms of animation, the effects become greater.\",\n \"Furthermore, the method to check the progress for individuals can be done with a calculator means of the computer when children's curriculums are divided into each subject and more divided into small sessions.\",\n \"For example, when 100 is perfect score to get when all the sessions are completed, computer records gives certain grades for each child's ID with the number of sessions play, the play time, and the number of sessions that are completed.\",\n \"Each of these gets certain value for grades and calculator mean accumulates all the scores to check the progress.\",\n \"Also, when there are animations containing visual and audio effects to test children with the multiple questionnaires by a form of animation or game then computer the combines the answers, more accurate information about progress can be made and let parents to figure out what area needs to cover more.\",\n \"In addition, to overcome current problems of the on-line pre-school system, the present invention improves the quality by providing online games that can be played with multiple users with visual chatting to help social life of the kids.\",\n \"For the business, since the program is designed with grading system with a proper graduation certificate, make it more interesting with online games to reach out to more customers.\",\n \"Eventually number of the customers will be increased with the customers who want to stay with the programs for longer time and these will become profit to the business of the pre-school service provider.\",\n \"This invention also can be used for evaluating the character, the disposition and the moral standard of the human being not only kids through internet or CD with personal computer.\",\n \"The database or CD can be created with a games designed to check the mental development such as the character, the disposition and morality of the player by giving different points according to the favorable action and the non-favorable action based on the mental development evaluating standard in a category of each subject for checking those points by the action of the cyber characters played in the cyber situation by the player.\",\n \"The method of this invention for evaluating the mental development, and the moral standard of a human being with a game is also providing the technology for making diagnoses of a mental diseases/disorder, and for evaluating the character, the disposition and the moral standards of human being for the assignment of a position for the right man in the right post.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10467520"}}},{"rowIdx":371,"cells":{"text":{"kind":"list like","value":[["Fuel cell arrangement and method for operating a fuel cell arrangement","A fuel cell arrangement (10) and its operation are described.","This fuel cell arrangement (10) comprises one or more fuel cells (1) to which a combustible gas or a cathode gas is fed and which supply an electric power Pext to an external consuming device (2).","One or more internal electrical consuming devices (3) can optionally be switched on and off by means of a control device (5) as a function of the operating condition of the fuel cell arrangement (10).","Excessive heat is removed from the fuel cell arrangement by means of a cooling device (4).","According to the invention, a portion of the electric power PBZ generated by the fuel cells (1) is converted at the internal electrical consuming devices (3) during the operation of the fuel cell arrangement (10).","When the electric power Pext supplied to the external electrical consuming device (2) increases or decreases, the electric power Pint converted at the internal electrical consuming devices (3) is reduced or increased inversely in the sense of making the electric power PBZ generated by the fuel cells (1) uniform.","This is carried out by the control device (5) of the fuel cell arrangement (10)."],["1-40.","(Cancelled) 41.A method of operating a fuel cell arrangement having at least one fuel cell comprising: feeding a combustible gas and a cathode gas to said fuel cell arrangement; supplying an electric power Pext to an external consuming device; optionally switching on and off at least one internal electrical consuming device; and removing excessive heat from said fuel cell arrangement by a cooling device; wherein an electric power PBZ is generated by said at least one fuel cell, and an electric power Pint is consumed by said at least one internal electrical consuming device, said power Pint being a portion of said power PBZ.","42.The method of claim 41, wherein an increase or decrease of said power Pext causes a decrease or increase in said power Pint, respectively.","43.The method of claim 41, wherein said power Pext and said power Pint are inversely related.","44.The method of claim 42, wherein said power PBZ is uniform.","45.The method of claim 43, wherein, for a load-following operation, an increase of said power Pext supplied to said external electrical consuming device is compensated by a reduction of said power Pint, and a reduction of said power Pext is compensated by an increase of said power Pint.","46.The method of claim 45, wherein, for a standby operation, a switching-off of said external electrical consuming device is compensated by an increase in said power Pint.","47.The method of claim 46, wherein a change of said power Pint takes place essentially immediately with a change of the power Pext.","48.The method of claim 47, wherein said power PBZ is kept essentially constant.","49.The method of claim 48, wherein said power Pint maximally amounts to approximately one fourth to approximately one half of a nominal electric power of said at least one fuel cell.","50.The method of claim 48, wherein said power Pint is removed from said fuel cell arrangement by an increased use of said cooling device.","51.A fuel cell arrangement having at least one fuel cell comprising: at least one internal electrical consuming device, said at least one internal electrical consuming device being optionally switched on and off; a cooling device for removing excess heat from said fuel cell arrangement; and a control device, said control device controlling said at least one internal electrical consuming device as a function of an operating condition of said fuel cell arrangement; wherein an electric power PBZ is generated by said fuel cell arrangement, an electric power Pint is consumed by said at least one internal electrical consuming device, said power Pint being a portion of said power PBZ, and an electric power Pext is consumed by an external consuming device.","52.The fuel cell arrangement of claim 51, wherein an increase or decrease of said power Pext causes a decrease or increase in said power Pint, respectively.","53.The fuel cell arrangement of claim 51, wherein said power Pext and said power Pint are inversely related.","54.The fuel cell arrangement of claim 52, wherein said power PBZ is uniform.","55.The fuel cell arrangement of claim 53, wherein, for a load-following operation, said control device controlling said at least one internal consuming device such that an increase of said power Pext is compensated by a reduction of said power Pint, and a reduction of said power Pext is compensated by an increase of said power Pint.","56.The fuel cell arrangement of claim 55, wherein, for a standby operation, said control device controlling said at least one internal consuming device such that a switching-off of said external electrical consuming device is compensated by an increase in said power Pint.","57.The fuel cell arrangement of claim 56, wherein said control device controlling said at least one internal consuming device such that a change of said power Pint takes place essentially immediately with a change of the power Pext.","58.The fuel cell arrangement of claim 57, wherein said control device controlling said at least one internal consuming device such that said power PBZ is kept essentially constant.","59.The fuel cell arrangement of claim 58, wherein said control device controlling said at least one internal consuming device such that said power Pint maximally amounts to approximately one fourth to approximately one half of a nominal electric power of said fuel cell arrangement.","60.The fuel cell arrangement of claim 58, wherein said cooling device is constructed such that it removes said power Pint from said fuel cell arrangement.","61.The fuel cell arrangement of claim 60, said at least one internal electrical consuming device further comprising at least one of a start heating device, a ventilator device, and another aggregate.","62.The fuel cell arrangement of claim 61, further comprising an electrical inverter for converting a direct voltage supplied by said at least one fuel cell into an alternating voltage, said alternating voltage being supplied to said external consuming device.","63.The fuel cell arrangement of claim 62, wherein said at least one internal electrical consuming device being connected, via said control device, to said external electrical consuming device in parallel with an output of said inverter."],["<SOH> BACKGROUND OF THE INVENTION <EOH>Fuel cell arrangements are known which comprise one or more fuel cells to which a combustible gas and a cathode gas are fed and which supply electric power to an external consuming device.","One or more internal electrical consuming devices of the fuel cell arrangement can optionally be switched on and off.","A cooling device is provided for eliminating excessive heat from the fuel cell arrangement.","One difficulty during the operation of fuel cell arrangements of the above-mentioned type is the fact that, with respect to their performance, they cannot arbitrarily rapidly follow a change of the electric load of the connected external consuming device.","The reason is based less on the dynamics of the electrochemical events taking place in the fuel cells than in the slowness of supplying the fuel cells with their reaction gases: the combustible gas and the cathode gas.","The load-following operation therefore requires electric power at the expense of the efficiency in order to be able to immediately react in the event of a load demand.","In the case of a power supply system failure, i.e., in the event of a sudden failure of the load demand of the connected external consuming device, the fuel cell arrangement, if possible, should not switch off but change to a standby operation, in which the current requirement for the internal consuming devices of the fuel cell arrangement is covered.","The intrinsic consumption of the fuel cell arrangement is minimized for economical reasons, so that the system generates little electric power.","In the load-following operation, the amount of the current production of a fuel cell arrangement is defined by the external consuming devices connected to it.","Such a condition, which is also called an “island operation”, particularly in the case of small system sizes, results in load demands, which fluctuate considerably with respect to their percentages as a result of the connecting or disconnecting of individual consumption points.","Rapid load changes occur during failures of the external consuming device, for example, in the event of a power supply system failure.","The fuel cell arrangement will then supply no electric power to the external consuming device.","In this case, the fuel cell arrangement is to change to a standby operation in which it generates just enough electric power in order to satisfy the intrinsic consumption."],["<SOH> SUMMARY OF THE INVENTION <EOH>It is an object of the invention to provide a method of operating a fuel cell arrangement of the initially mentioned type by means of which the fuel cell arrangement can be operated in the sense of a fast response to changing load requirements.","Furthermore, by means of the invention, a fuel cell arrangement may be created which is capable of rapidly responding to changing load requirements.","With respect to the method, the object is achieved by means of the method of operating a fuel cell arrangement indicated herein.","With respect to the device, the object is achieved by means of the fuel cell arrangement indicated herein.","The invention creates a method of operating a fuel cell arrangement having one or more fuel cells, to which a combustible gas and a cathode gas are fed and which supply electric power to an external consuming device, and having one or more internal electrical consuming devices which can optionally be switched on and off, as well as having a cooling device by which excessive heat is eliminated from the fuel cell arrangement.","According to the invention, it is provided that, in the operation of the fuel cell arrangement, a portion of the electric power generated by the fuel cells is converted at the internal electrical consuming devices, and that, in the case of an increase or decrease of the electric power supplied to the external electrical consuming device, the electric power converted at the internal electrical consuming devices is reduced or increased inversely in the sense of making the electric power generated by the fuel cells uniform.","It is an advantage of the method according to the invention that, while the generating of the electric power is uniform, the fuel cells can rapidly respond to a change of the load demand by the external electrical consuming device.","According to a preferred embodiment of the invention, it is provided that, for a load-following operation, an increase of the power supplied to the external electrical consuming device is compensated by a reduction of the electric power converted at the internal electrical consuming devices, and a reduction of the power supplied to the external electrical consuming device is compensated by an increase of the power converted at the internal electrical consuming devices.","According to another aspect of the invention, it is provided that, for a standby operation, a switching-off of the external electrical consuming device is compensated by an increase of the electric power converted at the internal electrical consuming devices.","It is preferably provided that the change of the electric power inverted at the internal electrical consuming devices takes place essentially immediately with the change of the power supplied to the external electrical consuming device.","Here, it is particularly advantageous for the electric power generated by the fuel cells to be kept essentially constant.","According to an advantageous embodiment of the invention, it is provided that maximally an electric power is converted at the internal electrical consuming devices which amounts to approximately one fourth to approximately half of the nominal electric power of the fuel cells.","Preferably, the electric power converted at the internal electrical consuming devices is removed from the fuel cell arrangement by an increased use of the cooling device.","Furthermore, by means of the invention, a fuel cell arrangement is created which has one or more fuel cells to which a combustible gas and a cathode gas are fed and which supply an electric power to an external consuming device, and which has one or more internal electrical consuming devices which can optionally be switched on and off, and which has a cooling device by means of which the excessive heat is removed from the fuel cell arrangement, and which has a control device for controlling the operation of the internal consuming devices as a function of the operating condition of the fuel cell arrangement.","According to the invention, it is provided that the control device controls the internal electrical consuming devices such that a portion of the electric power generated by the fuel cells is converted at the internal electrical consuming devices, and that, in the case of an increase or decrease of the electric power supplied to the external electrical consuming device, the electric power converted at the internal electrical consuming devices is reduced or increased inversely in the sense of making the electric power generated by the fuel cells uniform.","It is an advantage of the fuel cell arrangement according to the invention that, while the generating of the electric power is essentially uniform, this fuel cell arrangement can rapidly respond to a change of the load demand of the connected electrical consuming device.","According to an aspect of the invention, it is provided that the control device controls the internal electrical consuming devices such that, for a load-following operation, an increase of the power supplied to the external electrical consuming device is compensated by a reduction of the electric power converted at the internal electrical consuming devices, and a reduction of the power supplied to the external electrical consuming device is compensated by an increase of the electric power converted at the internal electrical consuming devices.","According to another aspect of the invention, it is provided that the control device controls the internal electrical consuming devices such that, for a standby operation, a switching-off of the external electrical consuming device is compensated by an increase of the electric power converted at the internal electrical consuming devices.","Preferably, it is provided that the control device controls the internal electrical consuming devices such that the change of the electric power converted at the internal electrical consuming devices takes place essentially immediately with the change of the power supplied to the external electrical consuming device.","Furthermore, it is an advantage for the control device to control the internal electrical consuming devices such that the electric power generated by the fuel cells is essentially kept constant.","According to an advantageous embodiment of the invention, it is provided that maximally an electric power is converted at the internal electrical consuming devices which amounts to approximately one fourth to approximately half of the nominal electric power of the fuel cells.","Furthermore, it is advantageous for the cooling device to be constructed such that it removes the electric power converted at the internal electrical consuming devices from the fuel cell arrangement.","Advantageously, the internal electrical consuming devices comprise a start heating device and/or a ventilating device and/or other aggregates of the fuel cell arrangement.","According to a preferred embodiment of the invention, it is provided that the fuel cell arrangement comprises an electrical inverter for converting the direct voltage supplied by the fuel cells into an alternating voltage which is fed to the external consuming device.","Finally, according to an advantageous embodiment of the invention, it is provided that the internal electrical consuming devices can be connected by means of the control device optionally parallel to the external electric consuming device with the output of the inverter."],["CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to German Patent Application No.","101 06 219.2, which was filed Feb. 10, 2001.FIELD OF THE INVENTION The invention relates to a method of operating a fuel cell arrangement and further to a fuel cell arrangement.","BACKGROUND OF THE INVENTION Fuel cell arrangements are known which comprise one or more fuel cells to which a combustible gas and a cathode gas are fed and which supply electric power to an external consuming device.","One or more internal electrical consuming devices of the fuel cell arrangement can optionally be switched on and off.","A cooling device is provided for eliminating excessive heat from the fuel cell arrangement.","One difficulty during the operation of fuel cell arrangements of the above-mentioned type is the fact that, with respect to their performance, they cannot arbitrarily rapidly follow a change of the electric load of the connected external consuming device.","The reason is based less on the dynamics of the electrochemical events taking place in the fuel cells than in the slowness of supplying the fuel cells with their reaction gases: the combustible gas and the cathode gas.","The load-following operation therefore requires electric power at the expense of the efficiency in order to be able to immediately react in the event of a load demand.","In the case of a power supply system failure, i.e., in the event of a sudden failure of the load demand of the connected external consuming device, the fuel cell arrangement, if possible, should not switch off but change to a standby operation, in which the current requirement for the internal consuming devices of the fuel cell arrangement is covered.","The intrinsic consumption of the fuel cell arrangement is minimized for economical reasons, so that the system generates little electric power.","In the load-following operation, the amount of the current production of a fuel cell arrangement is defined by the external consuming devices connected to it.","Such a condition, which is also called an “island operation”, particularly in the case of small system sizes, results in load demands, which fluctuate considerably with respect to their percentages as a result of the connecting or disconnecting of individual consumption points.","Rapid load changes occur during failures of the external consuming device, for example, in the event of a power supply system failure.","The fuel cell arrangement will then supply no electric power to the external consuming device.","In this case, the fuel cell arrangement is to change to a standby operation in which it generates just enough electric power in order to satisfy the intrinsic consumption.","SUMMARY OF THE INVENTION It is an object of the invention to provide a method of operating a fuel cell arrangement of the initially mentioned type by means of which the fuel cell arrangement can be operated in the sense of a fast response to changing load requirements.","Furthermore, by means of the invention, a fuel cell arrangement may be created which is capable of rapidly responding to changing load requirements.","With respect to the method, the object is achieved by means of the method of operating a fuel cell arrangement indicated herein.","With respect to the device, the object is achieved by means of the fuel cell arrangement indicated herein.","The invention creates a method of operating a fuel cell arrangement having one or more fuel cells, to which a combustible gas and a cathode gas are fed and which supply electric power to an external consuming device, and having one or more internal electrical consuming devices which can optionally be switched on and off, as well as having a cooling device by which excessive heat is eliminated from the fuel cell arrangement.","According to the invention, it is provided that, in the operation of the fuel cell arrangement, a portion of the electric power generated by the fuel cells is converted at the internal electrical consuming devices, and that, in the case of an increase or decrease of the electric power supplied to the external electrical consuming device, the electric power converted at the internal electrical consuming devices is reduced or increased inversely in the sense of making the electric power generated by the fuel cells uniform.","It is an advantage of the method according to the invention that, while the generating of the electric power is uniform, the fuel cells can rapidly respond to a change of the load demand by the external electrical consuming device.","According to a preferred embodiment of the invention, it is provided that, for a load-following operation, an increase of the power supplied to the external electrical consuming device is compensated by a reduction of the electric power converted at the internal electrical consuming devices, and a reduction of the power supplied to the external electrical consuming device is compensated by an increase of the power converted at the internal electrical consuming devices.","According to another aspect of the invention, it is provided that, for a standby operation, a switching-off of the external electrical consuming device is compensated by an increase of the electric power converted at the internal electrical consuming devices.","It is preferably provided that the change of the electric power inverted at the internal electrical consuming devices takes place essentially immediately with the change of the power supplied to the external electrical consuming device.","Here, it is particularly advantageous for the electric power generated by the fuel cells to be kept essentially constant.","According to an advantageous embodiment of the invention, it is provided that maximally an electric power is converted at the internal electrical consuming devices which amounts to approximately one fourth to approximately half of the nominal electric power of the fuel cells.","Preferably, the electric power converted at the internal electrical consuming devices is removed from the fuel cell arrangement by an increased use of the cooling device.","Furthermore, by means of the invention, a fuel cell arrangement is created which has one or more fuel cells to which a combustible gas and a cathode gas are fed and which supply an electric power to an external consuming device, and which has one or more internal electrical consuming devices which can optionally be switched on and off, and which has a cooling device by means of which the excessive heat is removed from the fuel cell arrangement, and which has a control device for controlling the operation of the internal consuming devices as a function of the operating condition of the fuel cell arrangement.","According to the invention, it is provided that the control device controls the internal electrical consuming devices such that a portion of the electric power generated by the fuel cells is converted at the internal electrical consuming devices, and that, in the case of an increase or decrease of the electric power supplied to the external electrical consuming device, the electric power converted at the internal electrical consuming devices is reduced or increased inversely in the sense of making the electric power generated by the fuel cells uniform.","It is an advantage of the fuel cell arrangement according to the invention that, while the generating of the electric power is essentially uniform, this fuel cell arrangement can rapidly respond to a change of the load demand of the connected electrical consuming device.","According to an aspect of the invention, it is provided that the control device controls the internal electrical consuming devices such that, for a load-following operation, an increase of the power supplied to the external electrical consuming device is compensated by a reduction of the electric power converted at the internal electrical consuming devices, and a reduction of the power supplied to the external electrical consuming device is compensated by an increase of the electric power converted at the internal electrical consuming devices.","According to another aspect of the invention, it is provided that the control device controls the internal electrical consuming devices such that, for a standby operation, a switching-off of the external electrical consuming device is compensated by an increase of the electric power converted at the internal electrical consuming devices.","Preferably, it is provided that the control device controls the internal electrical consuming devices such that the change of the electric power converted at the internal electrical consuming devices takes place essentially immediately with the change of the power supplied to the external electrical consuming device.","Furthermore, it is an advantage for the control device to control the internal electrical consuming devices such that the electric power generated by the fuel cells is essentially kept constant.","According to an advantageous embodiment of the invention, it is provided that maximally an electric power is converted at the internal electrical consuming devices which amounts to approximately one fourth to approximately half of the nominal electric power of the fuel cells.","Furthermore, it is advantageous for the cooling device to be constructed such that it removes the electric power converted at the internal electrical consuming devices from the fuel cell arrangement.","Advantageously, the internal electrical consuming devices comprise a start heating device and/or a ventilating device and/or other aggregates of the fuel cell arrangement.","According to a preferred embodiment of the invention, it is provided that the fuel cell arrangement comprises an electrical inverter for converting the direct voltage supplied by the fuel cells into an alternating voltage which is fed to the external consuming device.","Finally, according to an advantageous embodiment of the invention, it is provided that the internal electrical consuming devices can be connected by means of the control device optionally parallel to the external electric consuming device with the output of the inverter.","BRIEF DESCRIPTION OF THE DRAWINGS FIG.","1 illustrates a block diagram of a fuel cell arrangement according to an embodiment of the invention.","DETAILED DESCRIPTION OF THE INVENTION FIG.","1 illustrates a fuel cell arrangement which, as a whole, has the reference number 10, and comprises one or more fuel cells 1.A combustible gas and a cathode gas are fed (not shown in the figure) to the fuel cells 1, from which electric energy is obtained in an electrochemical manner.","This electric energy is supplied by the fuel cells 1 to an external consuming device 2 as electric power Pext.","The external consuming device 2 is illustrated in FIG.","1 as an electrical line network, which supplies several consuming points that are not separately shown in the figure.","The fuel cell arrangement 10 comprises one or more internal electrical consuming devices 3 which are used for the operation of the fuel cell arrangement 10 and which can optionally be switched on and off.","Such internal electrical consuming devices 3 may, for example, comprise a start heating device 7 required for the starting operation and/or a ventilator device 8 required for the circulation of reaction gases, such as the combustible gas and the cathode gas or a cooling gas, and/or other aggregates 9 of the fuel cell arrangement.","The fuel cell arrangement 10 also has a cooling device 4 by means of which excessive heat generated during the operation of the fuel cell arrangement 10 is removed from the latter.","A control device 5 is used for controlling the operation of the internal consuming devices 3 as a function of the operating condition of the fuel cell arrangement 10.During the operation of the fuel cell arrangement 10, a portion of the electric power PBZ generated by the fuel cells 1 is converted at the internal electrical consuming devices 3.When the electric power Pext supplied to the external electrical consuming device 2 increases or decreases, the electric power Pint converted at the internal electrical consuming devices 3 is inversely reduced or increased in the sense of making the electric power PBZ generated by the fuel cells uniform.","The control device 5 controls the internal electrical consuming devices 3 in this sense.","For a load-following operation, thus an operation in which the fuel cell arrangement adapts the electric power provided by it to the load requirement of the connected external consuming device 2, that is, of the connected electrical power supply system, an increase of the power Pext supplied to the external electrical consuming device 2 is compensated by a reduction of the electric power Pint converted at the internal electrical consuming devices 3.In a corresponding manner, a reduction of the power Pext supplied to the external electrical consuming device 2 is compensated by an increase of the electric power Pint converted at the internal electrical consuming devices 3.For a standby operation, thus an operation in which, for example, in the event of the failure of the connected power supply system, or if its load requirement should be zero, no electric power Pext can be supplied anymore to the outside, a switching-off of the external electrical consuming device 2, for example, the above-mentioned power supply system failure, is compensated by an increase of the electric power Pint converted at the internal electrical consuming device 3.As a result, the fuel cell arrangement does not have to be moved to a minimal operating point but can still generate electric power in the amount of the electric power Pint converted at the internal electrical consuming devices 3.As required, thus when the external electrical consuming device 2, that is the connected power supply system, is restored, this power will immediately be available again.","The change of the electric power Pint converted at the internal electrical consuming devices 3 essentially takes place immediately with the change of the power Pint supplied to the external electrical consuming device 2.This is carried out by the control device 5.The electric power PBZ generated by the fuel cells 1 is kept essentially constant; that is, PBZ−Pint=Pext.","In the embodiment described here, the internal electrical consuming devices 3 are dimensioned such that maximally an electric power Pint is converted which amounts approximately to one quarter to approximately half of the nominal electric power of the fuel cells 1.The electric power Pint converted at the internal electrical consuming devices 3 is removed from the fuel cell arrangement 10 by an increased use of the cooling device 4.The electric power supplied by the fuel cells 1 to the external consuming device 2 is converted by an inverter 6 provided in the fuel cell arrangement 10 from the direct voltage supplied by the fuel cells 1 to an alternating voltage that is supplied to the external consuming device 2.The internal electrical consuming devices 3 can be connected by means of the control device 5 optionally parallel to the external electrical consuming device 2 with the output of the inverter 6.The adaptation of the electric power Pint converted at the internal electrical consuming devices 3 to the power Pext supplied to the external electrical consuming device 2 in the sense of making the electric power PBZ uniform which is generated by the fuel cells 1, can take place in steps or continuously, depending on the type and construction of the control device 5 by means of which this adaptation is carried out."]],"string":"[\n [\n \"Fuel cell arrangement and method for operating a fuel cell arrangement\",\n \"A fuel cell arrangement (10) and its operation are described.\",\n \"This fuel cell arrangement (10) comprises one or more fuel cells (1) to which a combustible gas or a cathode gas is fed and which supply an electric power Pext to an external consuming device (2).\",\n \"One or more internal electrical consuming devices (3) can optionally be switched on and off by means of a control device (5) as a function of the operating condition of the fuel cell arrangement (10).\",\n \"Excessive heat is removed from the fuel cell arrangement by means of a cooling device (4).\",\n \"According to the invention, a portion of the electric power PBZ generated by the fuel cells (1) is converted at the internal electrical consuming devices (3) during the operation of the fuel cell arrangement (10).\",\n \"When the electric power Pext supplied to the external electrical consuming device (2) increases or decreases, the electric power Pint converted at the internal electrical consuming devices (3) is reduced or increased inversely in the sense of making the electric power PBZ generated by the fuel cells (1) uniform.\",\n \"This is carried out by the control device (5) of the fuel cell arrangement (10).\"\n ],\n [\n \"1-40.\",\n \"(Cancelled) 41.A method of operating a fuel cell arrangement having at least one fuel cell comprising: feeding a combustible gas and a cathode gas to said fuel cell arrangement; supplying an electric power Pext to an external consuming device; optionally switching on and off at least one internal electrical consuming device; and removing excessive heat from said fuel cell arrangement by a cooling device; wherein an electric power PBZ is generated by said at least one fuel cell, and an electric power Pint is consumed by said at least one internal electrical consuming device, said power Pint being a portion of said power PBZ.\",\n \"42.The method of claim 41, wherein an increase or decrease of said power Pext causes a decrease or increase in said power Pint, respectively.\",\n \"43.The method of claim 41, wherein said power Pext and said power Pint are inversely related.\",\n \"44.The method of claim 42, wherein said power PBZ is uniform.\",\n \"45.The method of claim 43, wherein, for a load-following operation, an increase of said power Pext supplied to said external electrical consuming device is compensated by a reduction of said power Pint, and a reduction of said power Pext is compensated by an increase of said power Pint.\",\n \"46.The method of claim 45, wherein, for a standby operation, a switching-off of said external electrical consuming device is compensated by an increase in said power Pint.\",\n \"47.The method of claim 46, wherein a change of said power Pint takes place essentially immediately with a change of the power Pext.\",\n \"48.The method of claim 47, wherein said power PBZ is kept essentially constant.\",\n \"49.The method of claim 48, wherein said power Pint maximally amounts to approximately one fourth to approximately one half of a nominal electric power of said at least one fuel cell.\",\n \"50.The method of claim 48, wherein said power Pint is removed from said fuel cell arrangement by an increased use of said cooling device.\",\n \"51.A fuel cell arrangement having at least one fuel cell comprising: at least one internal electrical consuming device, said at least one internal electrical consuming device being optionally switched on and off; a cooling device for removing excess heat from said fuel cell arrangement; and a control device, said control device controlling said at least one internal electrical consuming device as a function of an operating condition of said fuel cell arrangement; wherein an electric power PBZ is generated by said fuel cell arrangement, an electric power Pint is consumed by said at least one internal electrical consuming device, said power Pint being a portion of said power PBZ, and an electric power Pext is consumed by an external consuming device.\",\n \"52.The fuel cell arrangement of claim 51, wherein an increase or decrease of said power Pext causes a decrease or increase in said power Pint, respectively.\",\n \"53.The fuel cell arrangement of claim 51, wherein said power Pext and said power Pint are inversely related.\",\n \"54.The fuel cell arrangement of claim 52, wherein said power PBZ is uniform.\",\n \"55.The fuel cell arrangement of claim 53, wherein, for a load-following operation, said control device controlling said at least one internal consuming device such that an increase of said power Pext is compensated by a reduction of said power Pint, and a reduction of said power Pext is compensated by an increase of said power Pint.\",\n \"56.The fuel cell arrangement of claim 55, wherein, for a standby operation, said control device controlling said at least one internal consuming device such that a switching-off of said external electrical consuming device is compensated by an increase in said power Pint.\",\n \"57.The fuel cell arrangement of claim 56, wherein said control device controlling said at least one internal consuming device such that a change of said power Pint takes place essentially immediately with a change of the power Pext.\",\n \"58.The fuel cell arrangement of claim 57, wherein said control device controlling said at least one internal consuming device such that said power PBZ is kept essentially constant.\",\n \"59.The fuel cell arrangement of claim 58, wherein said control device controlling said at least one internal consuming device such that said power Pint maximally amounts to approximately one fourth to approximately one half of a nominal electric power of said fuel cell arrangement.\",\n \"60.The fuel cell arrangement of claim 58, wherein said cooling device is constructed such that it removes said power Pint from said fuel cell arrangement.\",\n \"61.The fuel cell arrangement of claim 60, said at least one internal electrical consuming device further comprising at least one of a start heating device, a ventilator device, and another aggregate.\",\n \"62.The fuel cell arrangement of claim 61, further comprising an electrical inverter for converting a direct voltage supplied by said at least one fuel cell into an alternating voltage, said alternating voltage being supplied to said external consuming device.\",\n \"63.The fuel cell arrangement of claim 62, wherein said at least one internal electrical consuming device being connected, via said control device, to said external electrical consuming device in parallel with an output of said inverter.\"\n ],\n [\n \"<SOH> BACKGROUND OF THE INVENTION <EOH>Fuel cell arrangements are known which comprise one or more fuel cells to which a combustible gas and a cathode gas are fed and which supply electric power to an external consuming device.\",\n \"One or more internal electrical consuming devices of the fuel cell arrangement can optionally be switched on and off.\",\n \"A cooling device is provided for eliminating excessive heat from the fuel cell arrangement.\",\n \"One difficulty during the operation of fuel cell arrangements of the above-mentioned type is the fact that, with respect to their performance, they cannot arbitrarily rapidly follow a change of the electric load of the connected external consuming device.\",\n \"The reason is based less on the dynamics of the electrochemical events taking place in the fuel cells than in the slowness of supplying the fuel cells with their reaction gases: the combustible gas and the cathode gas.\",\n \"The load-following operation therefore requires electric power at the expense of the efficiency in order to be able to immediately react in the event of a load demand.\",\n \"In the case of a power supply system failure, i.e., in the event of a sudden failure of the load demand of the connected external consuming device, the fuel cell arrangement, if possible, should not switch off but change to a standby operation, in which the current requirement for the internal consuming devices of the fuel cell arrangement is covered.\",\n \"The intrinsic consumption of the fuel cell arrangement is minimized for economical reasons, so that the system generates little electric power.\",\n \"In the load-following operation, the amount of the current production of a fuel cell arrangement is defined by the external consuming devices connected to it.\",\n \"Such a condition, which is also called an “island operation”, particularly in the case of small system sizes, results in load demands, which fluctuate considerably with respect to their percentages as a result of the connecting or disconnecting of individual consumption points.\",\n \"Rapid load changes occur during failures of the external consuming device, for example, in the event of a power supply system failure.\",\n \"The fuel cell arrangement will then supply no electric power to the external consuming device.\",\n \"In this case, the fuel cell arrangement is to change to a standby operation in which it generates just enough electric power in order to satisfy the intrinsic consumption.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>It is an object of the invention to provide a method of operating a fuel cell arrangement of the initially mentioned type by means of which the fuel cell arrangement can be operated in the sense of a fast response to changing load requirements.\",\n \"Furthermore, by means of the invention, a fuel cell arrangement may be created which is capable of rapidly responding to changing load requirements.\",\n \"With respect to the method, the object is achieved by means of the method of operating a fuel cell arrangement indicated herein.\",\n \"With respect to the device, the object is achieved by means of the fuel cell arrangement indicated herein.\",\n \"The invention creates a method of operating a fuel cell arrangement having one or more fuel cells, to which a combustible gas and a cathode gas are fed and which supply electric power to an external consuming device, and having one or more internal electrical consuming devices which can optionally be switched on and off, as well as having a cooling device by which excessive heat is eliminated from the fuel cell arrangement.\",\n \"According to the invention, it is provided that, in the operation of the fuel cell arrangement, a portion of the electric power generated by the fuel cells is converted at the internal electrical consuming devices, and that, in the case of an increase or decrease of the electric power supplied to the external electrical consuming device, the electric power converted at the internal electrical consuming devices is reduced or increased inversely in the sense of making the electric power generated by the fuel cells uniform.\",\n \"It is an advantage of the method according to the invention that, while the generating of the electric power is uniform, the fuel cells can rapidly respond to a change of the load demand by the external electrical consuming device.\",\n \"According to a preferred embodiment of the invention, it is provided that, for a load-following operation, an increase of the power supplied to the external electrical consuming device is compensated by a reduction of the electric power converted at the internal electrical consuming devices, and a reduction of the power supplied to the external electrical consuming device is compensated by an increase of the power converted at the internal electrical consuming devices.\",\n \"According to another aspect of the invention, it is provided that, for a standby operation, a switching-off of the external electrical consuming device is compensated by an increase of the electric power converted at the internal electrical consuming devices.\",\n \"It is preferably provided that the change of the electric power inverted at the internal electrical consuming devices takes place essentially immediately with the change of the power supplied to the external electrical consuming device.\",\n \"Here, it is particularly advantageous for the electric power generated by the fuel cells to be kept essentially constant.\",\n \"According to an advantageous embodiment of the invention, it is provided that maximally an electric power is converted at the internal electrical consuming devices which amounts to approximately one fourth to approximately half of the nominal electric power of the fuel cells.\",\n \"Preferably, the electric power converted at the internal electrical consuming devices is removed from the fuel cell arrangement by an increased use of the cooling device.\",\n \"Furthermore, by means of the invention, a fuel cell arrangement is created which has one or more fuel cells to which a combustible gas and a cathode gas are fed and which supply an electric power to an external consuming device, and which has one or more internal electrical consuming devices which can optionally be switched on and off, and which has a cooling device by means of which the excessive heat is removed from the fuel cell arrangement, and which has a control device for controlling the operation of the internal consuming devices as a function of the operating condition of the fuel cell arrangement.\",\n \"According to the invention, it is provided that the control device controls the internal electrical consuming devices such that a portion of the electric power generated by the fuel cells is converted at the internal electrical consuming devices, and that, in the case of an increase or decrease of the electric power supplied to the external electrical consuming device, the electric power converted at the internal electrical consuming devices is reduced or increased inversely in the sense of making the electric power generated by the fuel cells uniform.\",\n \"It is an advantage of the fuel cell arrangement according to the invention that, while the generating of the electric power is essentially uniform, this fuel cell arrangement can rapidly respond to a change of the load demand of the connected electrical consuming device.\",\n \"According to an aspect of the invention, it is provided that the control device controls the internal electrical consuming devices such that, for a load-following operation, an increase of the power supplied to the external electrical consuming device is compensated by a reduction of the electric power converted at the internal electrical consuming devices, and a reduction of the power supplied to the external electrical consuming device is compensated by an increase of the electric power converted at the internal electrical consuming devices.\",\n \"According to another aspect of the invention, it is provided that the control device controls the internal electrical consuming devices such that, for a standby operation, a switching-off of the external electrical consuming device is compensated by an increase of the electric power converted at the internal electrical consuming devices.\",\n \"Preferably, it is provided that the control device controls the internal electrical consuming devices such that the change of the electric power converted at the internal electrical consuming devices takes place essentially immediately with the change of the power supplied to the external electrical consuming device.\",\n \"Furthermore, it is an advantage for the control device to control the internal electrical consuming devices such that the electric power generated by the fuel cells is essentially kept constant.\",\n \"According to an advantageous embodiment of the invention, it is provided that maximally an electric power is converted at the internal electrical consuming devices which amounts to approximately one fourth to approximately half of the nominal electric power of the fuel cells.\",\n \"Furthermore, it is advantageous for the cooling device to be constructed such that it removes the electric power converted at the internal electrical consuming devices from the fuel cell arrangement.\",\n \"Advantageously, the internal electrical consuming devices comprise a start heating device and/or a ventilating device and/or other aggregates of the fuel cell arrangement.\",\n \"According to a preferred embodiment of the invention, it is provided that the fuel cell arrangement comprises an electrical inverter for converting the direct voltage supplied by the fuel cells into an alternating voltage which is fed to the external consuming device.\",\n \"Finally, according to an advantageous embodiment of the invention, it is provided that the internal electrical consuming devices can be connected by means of the control device optionally parallel to the external electric consuming device with the output of the inverter.\"\n ],\n [\n \"CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to German Patent Application No.\",\n \"101 06 219.2, which was filed Feb. 10, 2001.FIELD OF THE INVENTION The invention relates to a method of operating a fuel cell arrangement and further to a fuel cell arrangement.\",\n \"BACKGROUND OF THE INVENTION Fuel cell arrangements are known which comprise one or more fuel cells to which a combustible gas and a cathode gas are fed and which supply electric power to an external consuming device.\",\n \"One or more internal electrical consuming devices of the fuel cell arrangement can optionally be switched on and off.\",\n \"A cooling device is provided for eliminating excessive heat from the fuel cell arrangement.\",\n \"One difficulty during the operation of fuel cell arrangements of the above-mentioned type is the fact that, with respect to their performance, they cannot arbitrarily rapidly follow a change of the electric load of the connected external consuming device.\",\n \"The reason is based less on the dynamics of the electrochemical events taking place in the fuel cells than in the slowness of supplying the fuel cells with their reaction gases: the combustible gas and the cathode gas.\",\n \"The load-following operation therefore requires electric power at the expense of the efficiency in order to be able to immediately react in the event of a load demand.\",\n \"In the case of a power supply system failure, i.e., in the event of a sudden failure of the load demand of the connected external consuming device, the fuel cell arrangement, if possible, should not switch off but change to a standby operation, in which the current requirement for the internal consuming devices of the fuel cell arrangement is covered.\",\n \"The intrinsic consumption of the fuel cell arrangement is minimized for economical reasons, so that the system generates little electric power.\",\n \"In the load-following operation, the amount of the current production of a fuel cell arrangement is defined by the external consuming devices connected to it.\",\n \"Such a condition, which is also called an “island operation”, particularly in the case of small system sizes, results in load demands, which fluctuate considerably with respect to their percentages as a result of the connecting or disconnecting of individual consumption points.\",\n \"Rapid load changes occur during failures of the external consuming device, for example, in the event of a power supply system failure.\",\n \"The fuel cell arrangement will then supply no electric power to the external consuming device.\",\n \"In this case, the fuel cell arrangement is to change to a standby operation in which it generates just enough electric power in order to satisfy the intrinsic consumption.\",\n \"SUMMARY OF THE INVENTION It is an object of the invention to provide a method of operating a fuel cell arrangement of the initially mentioned type by means of which the fuel cell arrangement can be operated in the sense of a fast response to changing load requirements.\",\n \"Furthermore, by means of the invention, a fuel cell arrangement may be created which is capable of rapidly responding to changing load requirements.\",\n \"With respect to the method, the object is achieved by means of the method of operating a fuel cell arrangement indicated herein.\",\n \"With respect to the device, the object is achieved by means of the fuel cell arrangement indicated herein.\",\n \"The invention creates a method of operating a fuel cell arrangement having one or more fuel cells, to which a combustible gas and a cathode gas are fed and which supply electric power to an external consuming device, and having one or more internal electrical consuming devices which can optionally be switched on and off, as well as having a cooling device by which excessive heat is eliminated from the fuel cell arrangement.\",\n \"According to the invention, it is provided that, in the operation of the fuel cell arrangement, a portion of the electric power generated by the fuel cells is converted at the internal electrical consuming devices, and that, in the case of an increase or decrease of the electric power supplied to the external electrical consuming device, the electric power converted at the internal electrical consuming devices is reduced or increased inversely in the sense of making the electric power generated by the fuel cells uniform.\",\n \"It is an advantage of the method according to the invention that, while the generating of the electric power is uniform, the fuel cells can rapidly respond to a change of the load demand by the external electrical consuming device.\",\n \"According to a preferred embodiment of the invention, it is provided that, for a load-following operation, an increase of the power supplied to the external electrical consuming device is compensated by a reduction of the electric power converted at the internal electrical consuming devices, and a reduction of the power supplied to the external electrical consuming device is compensated by an increase of the power converted at the internal electrical consuming devices.\",\n \"According to another aspect of the invention, it is provided that, for a standby operation, a switching-off of the external electrical consuming device is compensated by an increase of the electric power converted at the internal electrical consuming devices.\",\n \"It is preferably provided that the change of the electric power inverted at the internal electrical consuming devices takes place essentially immediately with the change of the power supplied to the external electrical consuming device.\",\n \"Here, it is particularly advantageous for the electric power generated by the fuel cells to be kept essentially constant.\",\n \"According to an advantageous embodiment of the invention, it is provided that maximally an electric power is converted at the internal electrical consuming devices which amounts to approximately one fourth to approximately half of the nominal electric power of the fuel cells.\",\n \"Preferably, the electric power converted at the internal electrical consuming devices is removed from the fuel cell arrangement by an increased use of the cooling device.\",\n \"Furthermore, by means of the invention, a fuel cell arrangement is created which has one or more fuel cells to which a combustible gas and a cathode gas are fed and which supply an electric power to an external consuming device, and which has one or more internal electrical consuming devices which can optionally be switched on and off, and which has a cooling device by means of which the excessive heat is removed from the fuel cell arrangement, and which has a control device for controlling the operation of the internal consuming devices as a function of the operating condition of the fuel cell arrangement.\",\n \"According to the invention, it is provided that the control device controls the internal electrical consuming devices such that a portion of the electric power generated by the fuel cells is converted at the internal electrical consuming devices, and that, in the case of an increase or decrease of the electric power supplied to the external electrical consuming device, the electric power converted at the internal electrical consuming devices is reduced or increased inversely in the sense of making the electric power generated by the fuel cells uniform.\",\n \"It is an advantage of the fuel cell arrangement according to the invention that, while the generating of the electric power is essentially uniform, this fuel cell arrangement can rapidly respond to a change of the load demand of the connected electrical consuming device.\",\n \"According to an aspect of the invention, it is provided that the control device controls the internal electrical consuming devices such that, for a load-following operation, an increase of the power supplied to the external electrical consuming device is compensated by a reduction of the electric power converted at the internal electrical consuming devices, and a reduction of the power supplied to the external electrical consuming device is compensated by an increase of the electric power converted at the internal electrical consuming devices.\",\n \"According to another aspect of the invention, it is provided that the control device controls the internal electrical consuming devices such that, for a standby operation, a switching-off of the external electrical consuming device is compensated by an increase of the electric power converted at the internal electrical consuming devices.\",\n \"Preferably, it is provided that the control device controls the internal electrical consuming devices such that the change of the electric power converted at the internal electrical consuming devices takes place essentially immediately with the change of the power supplied to the external electrical consuming device.\",\n \"Furthermore, it is an advantage for the control device to control the internal electrical consuming devices such that the electric power generated by the fuel cells is essentially kept constant.\",\n \"According to an advantageous embodiment of the invention, it is provided that maximally an electric power is converted at the internal electrical consuming devices which amounts to approximately one fourth to approximately half of the nominal electric power of the fuel cells.\",\n \"Furthermore, it is advantageous for the cooling device to be constructed such that it removes the electric power converted at the internal electrical consuming devices from the fuel cell arrangement.\",\n \"Advantageously, the internal electrical consuming devices comprise a start heating device and/or a ventilating device and/or other aggregates of the fuel cell arrangement.\",\n \"According to a preferred embodiment of the invention, it is provided that the fuel cell arrangement comprises an electrical inverter for converting the direct voltage supplied by the fuel cells into an alternating voltage which is fed to the external consuming device.\",\n \"Finally, according to an advantageous embodiment of the invention, it is provided that the internal electrical consuming devices can be connected by means of the control device optionally parallel to the external electric consuming device with the output of the inverter.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS FIG.\",\n \"1 illustrates a block diagram of a fuel cell arrangement according to an embodiment of the invention.\",\n \"DETAILED DESCRIPTION OF THE INVENTION FIG.\",\n \"1 illustrates a fuel cell arrangement which, as a whole, has the reference number 10, and comprises one or more fuel cells 1.A combustible gas and a cathode gas are fed (not shown in the figure) to the fuel cells 1, from which electric energy is obtained in an electrochemical manner.\",\n \"This electric energy is supplied by the fuel cells 1 to an external consuming device 2 as electric power Pext.\",\n \"The external consuming device 2 is illustrated in FIG.\",\n \"1 as an electrical line network, which supplies several consuming points that are not separately shown in the figure.\",\n \"The fuel cell arrangement 10 comprises one or more internal electrical consuming devices 3 which are used for the operation of the fuel cell arrangement 10 and which can optionally be switched on and off.\",\n \"Such internal electrical consuming devices 3 may, for example, comprise a start heating device 7 required for the starting operation and/or a ventilator device 8 required for the circulation of reaction gases, such as the combustible gas and the cathode gas or a cooling gas, and/or other aggregates 9 of the fuel cell arrangement.\",\n \"The fuel cell arrangement 10 also has a cooling device 4 by means of which excessive heat generated during the operation of the fuel cell arrangement 10 is removed from the latter.\",\n \"A control device 5 is used for controlling the operation of the internal consuming devices 3 as a function of the operating condition of the fuel cell arrangement 10.During the operation of the fuel cell arrangement 10, a portion of the electric power PBZ generated by the fuel cells 1 is converted at the internal electrical consuming devices 3.When the electric power Pext supplied to the external electrical consuming device 2 increases or decreases, the electric power Pint converted at the internal electrical consuming devices 3 is inversely reduced or increased in the sense of making the electric power PBZ generated by the fuel cells uniform.\",\n \"The control device 5 controls the internal electrical consuming devices 3 in this sense.\",\n \"For a load-following operation, thus an operation in which the fuel cell arrangement adapts the electric power provided by it to the load requirement of the connected external consuming device 2, that is, of the connected electrical power supply system, an increase of the power Pext supplied to the external electrical consuming device 2 is compensated by a reduction of the electric power Pint converted at the internal electrical consuming devices 3.In a corresponding manner, a reduction of the power Pext supplied to the external electrical consuming device 2 is compensated by an increase of the electric power Pint converted at the internal electrical consuming devices 3.For a standby operation, thus an operation in which, for example, in the event of the failure of the connected power supply system, or if its load requirement should be zero, no electric power Pext can be supplied anymore to the outside, a switching-off of the external electrical consuming device 2, for example, the above-mentioned power supply system failure, is compensated by an increase of the electric power Pint converted at the internal electrical consuming device 3.As a result, the fuel cell arrangement does not have to be moved to a minimal operating point but can still generate electric power in the amount of the electric power Pint converted at the internal electrical consuming devices 3.As required, thus when the external electrical consuming device 2, that is the connected power supply system, is restored, this power will immediately be available again.\",\n \"The change of the electric power Pint converted at the internal electrical consuming devices 3 essentially takes place immediately with the change of the power Pint supplied to the external electrical consuming device 2.This is carried out by the control device 5.The electric power PBZ generated by the fuel cells 1 is kept essentially constant; that is, PBZ−Pint=Pext.\",\n \"In the embodiment described here, the internal electrical consuming devices 3 are dimensioned such that maximally an electric power Pint is converted which amounts approximately to one quarter to approximately half of the nominal electric power of the fuel cells 1.The electric power Pint converted at the internal electrical consuming devices 3 is removed from the fuel cell arrangement 10 by an increased use of the cooling device 4.The electric power supplied by the fuel cells 1 to the external consuming device 2 is converted by an inverter 6 provided in the fuel cell arrangement 10 from the direct voltage supplied by the fuel cells 1 to an alternating voltage that is supplied to the external consuming device 2.The internal electrical consuming devices 3 can be connected by means of the control device 5 optionally parallel to the external electrical consuming device 2 with the output of the inverter 6.The adaptation of the electric power Pint converted at the internal electrical consuming devices 3 to the power Pext supplied to the external electrical consuming device 2 in the sense of making the electric power PBZ uniform which is generated by the fuel cells 1, can take place in steps or continuously, depending on the type and construction of the control device 5 by means of which this adaptation is carried out.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10467525"}}},{"rowIdx":372,"cells":{"text":{"kind":"list like","value":[["Recombinant proteinase k","The invention concerns recombinant proteinase K. Furthermore a method for producing recombinant proteinase K is disclosed, which is characterized in that a) a host cell is transformed with a recombinant nucleic acid which codes for the zymogenic precursor of proteinase K, b) the host cell is cultured in such a manner that the zymogenic precursor of proteinase K is formed in the form of inclusion bodies in the host cell, c) the inclusion bodies are isolated and natured under conditions which result in the formation of the protease part of the zymogenic precursor in its natural conformation, d) the natured proteinase K is activated and purified."],["1-22.","(canceled) 23.A method for the naturation of denatured zymogenic proteinase K comprising transferring the denatured zymogenic proteinase K to a folding buffer, the buffer comprising low molecular weight substances which aid folding, a redox shuffling system, and a complexing agent at a substoichiometric concentration relative to any Ca2+ ions that are present, the buffer having a pH of 7.5 to 10.5 and the method being carried out at a temperature between 0° C. and 37° C. 24.The method of claim 23 wherein the redox shuffling system comprises mixed disulfides or thiosulfonates.","25.The method of claim 23 wherein the pH range is 8 to 9.26.The method of claim 23 wherein the temperature is between 0° C. and 25° C. 27.The method of claim 23 wherein the buffer further comprises denaturing agents at a concentration of less than 50 mM.","28.The method of claim 23 wherein the low molecular weight substances are selected from the group consisting of L-arginine at a concentration of 0.5 to 2.0 M, Tris at a concentration of 0.5 M to 2.0 M, triethanolamine at a concentration of 0.5 M to 2.0 M, and α-cyclodextrin at a concentration of 60 mM to 120 mM.","29.The method of claim 23 wherein the Ca2+ ion concentration is 1 to 20 mM.","30.The method of claim 23 wherein the denatured zymogenic proteinase K is transferred to the folding buffer while reducing the concentration of denaturing agents that may be present.","31.A folding buffer comprising low molecular weight substances which aid folding, a redox shuffling system, and a complexing agent at a substoichiometric concentration relative to any Ca2+ ions that are present, the buffer having a pH value in the range of 7.5 to 10.5.32.The buffer of claim 31 wherein the pH value is 8 to 9 and the redox shuffling system comprises mixed disulfides or thiosulfonates.","33.A method for activating a natured zymogenic precursor of active proteinase K comprising adding a detergent to an inactive complex comprising a native proteinase K and an inhibitory propeptide of the active proteinase K, thereby releasing the active proteinase K from the inactive complex.","34.The method of claim 33 wherein the detergent is SDS at a concentration of 0.1 to 2% (w/v).","35.A method for producing active recombinant proteinase K comprising (a) producing an inactive zymogenic proform of proteinase K in an inclusion body, (b) naturing in vitro the zymogenic proform of proteinase K, and (c) activating the zymogenic proform by autocatalytic cleavage, thereby converting it to the active proteinase K. 36.The method of claim 35 wherein the naturing step comprises transferring the denatured zymogenic proteinase K to a folding buffer, the buffer comprising low molecular weight substances which aid folding, a redox shuffling system, and a complexing agent at a substoichiometric concentration relative to any Ca2+ ions that are present, the buffer having a pH of 7.5 to 10.5 and the naturing step being carried out at a temperature between 0° C. and 37° C. 37.The method of claim 35 wherein the inclusion body is solubilized by a denaturing agent and a reducing agent.","38.The method of claim 37 wherein the denaturing agent is guanidinium hydrochloride at a concentration of 6-8 M or urea at a concentration of 8-10 M and the reducing agent is DTT or DTE at a concentration of 50-200 mM.","39.A method for producing active recombinant proteinase K comprising (a) transforming a host cell with a vector containing a DNA sequence coding for a zymogenic precursor of proteinase K (b) expressing the zymogenic precursor in inclusion bodies, (c) isolating the inclusion bodies and solubilizing the zymogenic precursor, (d) naturing the zymogenic precursor with a folding buffer comprising low molecular weight substances which aid folding, a redox shuffling system, and a complexing agent at a substoichiometric concentration relative to any Ca2+ ions that are present, the buffer having a pH of 7.5 to 10.5 and the naturing step being carried out at a temperature between 0° C. and 37° C., and (e) activating the zymogenic precursor by autocatalytic cleavage, thereby converting it to the active proteinase K 40.The method of claim 39 wherein the host cell is a prokaryotic cell.","41.The method of claim 39 wherein the host cell is Escherichia coli.","42.A codon-optimized recombinant nucleic acid comprising DNA coding for a recombinant zymogenic proteinase K which has been optimized for expression in Escherichia coli.","43.A vector containing the recombinant nucleic acid of claim 42.44.A host cell transformed with the vector of claim 43."],["The present invention concerns the preparation of recombinant proteinase K from Tritirachium album Limber and corresponding methods for the expression, in vitro naturation and activation of the recombinant proteinase K. Proteinase K (E.C.","3.4.21.64, also known as endopeptidase K) is an extracellular endopeptidase which is synthesized by the fungus Tritirachium album Limber.","It is a member of the class of serine proteases with the typical catalytic triad Asp39-His69-Ser224 (Jany, K. D. et al.","(1986) FEBS Letters Vol.","199(2), 139-144).","Since the sequence of the polypeptide chain of 279 amino acids in length (Gunkel, F. A. and Gassen, H. G. (1989) Eur.","J. Biochem.","Vol.","179(1), 185-194) and the three dimensional structure (Betzel, C. et al.","(1988) Eur.","J. Biochem.","Vol.","178(1), 155-71) has a high degree of homology to bacterial subtilisins, proteinase K is classified as a member of the subtilisin family (Pahler, A. et al.","(1984) EMBO J. Vol.","3(6), 1311-1314; Jany, K. D. and Mayer, B.","(1985), Biol.","Chem.","Hoppe-Seyler, Vol.","366(5), 485-492).","Proteinase K was named on the basis of its ability to hydrolyse native keratin and thus allows the fungus to grow on keratin as the only source of carbon and nitrogen (Ebeling, W. et al.","(1974) Eur.","J. Biochem.","Vol.","47(1), 91-97) Roelcke and Uhlenbruch, 1069, Z. Med.","Mikrobiol.","Immunol.","Vol.","155(2), 156-170).","Proteinase K has a specific activity of more than 30 U/mg and is thus one of the most active of the known endopeptidases (Betzel, C. et al.","(1986) FEBS Lett.","Vol.","197(1-2), 105-110) and unspecifically hydrolyses native and denatured proteins (Kraus, E. and Femfert, U, (1976) Hoppe Seylers Z. Physiol.","Chem.","Vol.","357(7):937-947).","Proteinase K from Tritirachium album Limber is translated in its natural host as a preproprotein.","The sequence of the cDNA of the gene which codes for proteinase K was decoded in 1989 by Gunkel, F. A. and Gassen, H. G. (1989) Eur.","J. Biochem.","Vol.","179(1), 185-194.According to this the gene for prepro-proteinase K is composed of two exons and codes for a signal sequence of 15 amino acids in length, a prosequence of 90 amino acids in length and a mature proteinase K of 279 amino acids in length.","A 63 bp intron is located in the region of the prosequence.","The prepeptide is cleaved off during translocation into the endoplasmatic reticulum (ER).","At present very little is known about the subsequent processing to form mature proteinase K with cleavage of the propeptide.","Consequently mature proteinase K consists of 279 amino acids.","The compact structure is stabilized by two disulfide bridges and two bound calcium ions.","This explains why proteinase K compared to other subtilisins has a considerably higher stability towards extreme pH values, high temperatures, chaotropic substances and detergents (Dolashka, P. et al.","(1992) Int.","J. Pept.","Protein.","Res.","Vol.","40(5), 465-471).","Proteinase K is characterized by a high thermostability (up to 65° C., Bajorath et al.","(1988), Eur.","J. Biochem.","Vol.","176, 441-447) and a wide pH range (pH 7.5-12.0, Ebeling, W. et al.","(1974) Eur.","J. Biochem.","Vol.","47(1), 91-97).","Its activity is increased in the presence of denaturing substances such as urea or SDS (Hilz, H. et al.","(1975) J. Biochem.","Vol.","56(1), 103-108; Jany, K. D. and Mayer, B.","(1985) Biol.","Chem.","Hoppe-Seyler, Vol.","366(5), 485-492).","The above-mentioned properties make proteinase K of particular interest for biotechnological applications in which an unspecific protein degradation is required.","Special examples are nucleic acid isolation (DNA or RNA) from crude extracts and sample preparation in DNA analysis (Goldenberger, D. et al.","(1995) PCR Methods Appl.","Vol.","4(6), 368-370; U.S. Pat.","No.","5,187,083; U.S. Pat.","No.","5,346,999).","Other applications are in the field of protein analysis such as structure elucidation.","Proteinase K is obtained commercially in large amounts by fermentation of the fungus Tritirachium album Limber (e.g.","CBS 348.55, Merck strain No.","2429 or the strain ATCC 22563).","The production of proteinase K is suppressed by glucose or free amino acids.","Since protein-containing media also induce the expression of proteases, it is necessary to use proteins such as BSA, milk powder or soybean flour as the only nitrogen source.","The secretion of the protease starts as soon as the stationary phase of growth is reached (Ebeling, W. et al.","(1974) Eur.","J. Biochem.","Vol.","47(1), 91-97).","Since Tritirachium album Limber is consequently unsuitable for fermentation on a large scale and moreover is difficult to genetically manipulate, various attempts have been made to overexpress recombinant proteinase K in other host cells.","However, no significant activity was detected in these experiments due to lack of expression, formation of inactive inclusion bodies or problems with the naturation (Gunkel, F. A. and Gassen, H. G. (1989) Eur.","J. Biochem.","Vol.","179(1), 185-194; Samal, B.","B. et al.","(1996) Adv.","Exp.","Med.","Biol.","Vol.","379, 95-104).","Moreover, Tritirachium album Limber is a slowly growing fungus which only secretes small amounts of proteases into the medium.","The long fermentation period of one to two weeks is disadvantageous.","In addition it is known that T. album also produces other proteases apart from proteinase K which can contaminate the preparation (Samal, B.","B. et al.","(1991) Enzyme Microb.","Technol.","Vol.","13, 66-70).","The object of the present invention is to provide a method for the economical production of recombinant proteinase K and of inactive zymogenic precursors of proteinase K that can be autocatalytically activated.","The object was achieved by providing a method for producing recombinant proteinase K in which the inactive zymogenic proform of proteinase K is produced in an insoluble form in inclusion bodies, and the zymogenic proform of proteinase K is natured and the zymogenic proform processed i.e.","activated in subsequent steps.","The methods for the naturation and activation of proteinase K are also a subject matter of the present invention.","The present invention concerns a method for producing recombinant proteinase K characterized in that the zymogenic proform is folded by in vitro naturation and is converted by autocatalytic cleavage into the active form.","The present invention concerns in particular a method for producing a recombinant proteinase K in which a zymogenic precursor of proteinase K is converted by oxidative folding from isolated and solubilized inclusion bodies into the native structure i.e.","it is natured and subsequently the active proteinase K is obtained from the natively folded zymogen by autocatalytic cleavage by adding detergents.","Hence the present invention concerns a method for obtaining recombinant proteinase K by transforming a host cell with a DNA coding for the zymogenic proform of proteinase K characterized by the following process steps: a) Culturing the said host cell under conditions which result in an expression of the DNA coding for the zymogenic proform of proteinase K such that a zymogenic precursor of proteinase K is formed in the host cell in the form of insoluble inclusion bodies.","b) Isolating the inclusion bodies, solubilizing the enzyme and naturing of the zymogenic precursor of proteinase K under conditions in which the protease part of the zymogenic precursor of proteinase K is formed.","c) Activating the proteinase K by removing the propeptide and further purification.","The DNA coding for the zymogenic proform of proteinase K corresponds to the DNA shown in SEQ ID NO: 2 or a DNA corresponding thereto within the scope of the degeneracy of the genetic code.","SEQ ID NO: 2 includes the DNA sequence which codes for proteinase K and the propeptide.","Furthermore the DNA can be codon-optimized for expression in a particular host.","Method for codon-optimization are known to a person skilled in the art and are described in example 1.Hence the present invention concerns methods in which the host cell is transformed by a DNA which is selected from the above-mentioned group.","A proteinase K is obtained by the method according to the invention which is homogeneous and hence particularly suitable for analytical and diagnostic applications.","The zymogenic proform of proteinase K according to the invention can optionally contain additional N-terminal modifications and in particular sequences which facilitate purification of the target protein (affinity tags), sequences which increase the efficiency of translation, sequences which increase the folding efficiency or sequences which result in a secretion of the target protein into the culture medium (natural presequence and other signal peptides).","Proteinase K in the sense of the invention means the sequence according to Gassen et al.","(1989) shown in SEQ ID NO: 1 as well as other variants of proteinase K from Tritirachium album Limber like the amino acid sequence disclosed by Ch.","Betzel et al.","(Biochemistry 40 (2001), 3080-3088) and in particular proteinase T (Samal, B.","B. et al.","(1989) Gene Vol.","85(2), 329-333; Samal, B.","B. et al.","(1996) Adv.","Exp.","Med.","Biol.","Vol.","379, 95-104) and proteinase R (Samal, B.","B. et al.","(1990) Mol.","Microbiol.","Vol.","4(10), 1789-1792; U.S. Pat.","No.","5,278,062) and in addition variants produced by recombinant means (as described for example in WO 96/28556).","The sequence shown in SEQ ID NO: 1 comprises the signal sequence (1-15, 15 amino acids), the prosequence (16-105; 90 amino acids) and the sequence of the mature proteinase K (106-384; 279 amino acids).","The amino acid sequence described by Betzel et al.","(Biochemistry 40 (2001), 3080-3088) has in particular aspartate instead of a serine residue at position 207 of the active protease.","Pro-proteinase K in the sense of the invention means in particular a proteinase K whose N-terminus is linked to its prosequence.","In the case of the closely related subtilisin E and other variants it is known that the prosequence has an important influence on the folding and formation of active protease (Ikemura, H. et al.","(1987) Biol.","Chem.","Vol.","262(16), 7859-7864).","In particular it is presumed that the prosequence acts as an intramolecular chaperone (Inouye, M. (1991) Enzyme Vol.","45, 314-321).","After the folding it is processed to form the mature subtilisin protease by autocatalytically cleaving the propeptide (Ikemura, H. and Inouye, M. (1988) J. Biol.","Chem.","Vol.","263(26), 12959-12963).","This process occurs in the case of subtilisin E (Samal, B.","B. et al.","(1989) Gene vol.","85(2), 329-333; Volkov, A. and Jordan, F. (1996) J. Mol.","Biol.","Vol.","262, 595-599), subtilisin BPN′ (Eder, J. et al.","(1993) Biochemistry Vol.","32, 18-26), papain (Vernet, T. et al.","(1991) J. Biol.","Chem.","Vol.","266(32), 21451-21457) and thermolysin (Marie-Claire, C. (1998) J. Biol.","Chem.","Vol.","273(10), 5697-5701).","If added exogenously the propeptide can also act intermolecularly in trans as a chaperone on the folding of denatured mature subtilisin protease (Ohta, Y. et al.","(1991) Mol.","Microbiol.","Vol.","5(6), 1507-1510; Hu, Z. et al.","(1996) J. Biol.","Chem.","Vol.","271(7), 3375-3384).","The propeptide binds to the active centre of subtilisin (Jain, S. C. et al.","(1998) J. Mol.","Biol.","Vol.","284, 137-144) and acts as a specific inhibitor (Kojima, S. et al.","(1998) J. Mol.","Biol.","Vol.","277, 1007-1013; Li, Y. et al.","(1995) J. Biol.","Chem.","Vol.","270, 25127-25132; Ohta, Y.","(1991) Mol.","Microbiol.","Vol.","5(6), 1507-1510).","This effect is used in the sense of the invention in order to prevent autoproteolysis of proteolysis-sensitive folding intermediates by already folded, active proteinase K during the naturation.","Only certain, usually hydrophobic core regions of the prosequence appear to be necessary for the chaperone function since mutations in wide areas have no influence on the activity (Kobayashi, T. and Inouye, M. (1992) J. Mol.","Biol.","Vol.","226, 931-933).","In addition it is known that propeptides can be exchanged between various subtilisin variants.","Thus for example subtilisin BPN′ also recognizes the prosequence of subtilisin E (Hu, Z. et al.","(1996) J. Biol.","Chem.","Vol.","271(7), 3375-3384).","Inclusion bodies are microscopically visible particles consisting of insoluble and inactive protein aggregates which are often formed in the cytoplasm of the host cell when heterologous proteins are overexpressed and they contain very pure target protein.","Methods for producing and purifying such inclusion bodies are described for example in Creighton, T. E. (1978) Prog.","Biophys.","Mol.","Biol.","Vol.","33(3), 231-297; Marston, F. A.","(1986) Biochem.","J. Vol.","240(1), 1-12; Rudolph, R. (1997).","Folding proteins in: Creighton, T. E.","(ed.)","Protein Function: A practical approach.","Oxford University Press, 57-99; Fink, A. L. (1998) Fold.","Des.","Vol.","3(1), R9-23; and EP 0 114 506.In order to isolate inclusion bodies the host cells are lysed after fermentation by conventional methods e.g.","by ultrasound, high pressure dispersion or lysozyme.","The lysis preferably takes place in an aqueous neutral to slightly acid buffer.","The insoluble inclusion bodies can be separated and purified by various methods, preferably by centrifugation or filtration with several washing steps (Rudolph, R. (1997).","Folding Proteins In: Creighton, T. E.","(ed.)","Protein Function: A practical Approach.","Oxford University Press, 57-99).","The inclusion bodies obtained in this manner are then solubilized in a known manner.","Denaturing agents are advantageously used for this at a concentration which is suitable for dissolving the inclusion bodies, in particular guanidinium hydro-chloride and other guanidinium salts and/or urea.","In order to completely monomerize the inclusion body proteins it is also advantageous to add reducing agents such as dithiothreitol (DTT), dithioerythritol (DTE) or 2-mercaptoethanol during the solubilization in order to break possible disulfide bridges by reduction.","The invention also concerns a proteinase K in which the cysteines are not reduced but are derivatized in particular with GSSG to form mixed disulfides or thiocyanates (EP 0 393 725).","Hence according to the invention the inclusion bodies are solubilized by denaturing agents and reducing agents.","6-8 M guanidinium hydrochloride or 8-10 M urea are preferred as denaturing agents and 50-200 mM DTT (dithiothreitol) or DTE (dithioerythritol) are preferred as reducing agents.","Hence the present invention concerns the prosequence according to SEQ ID NO: 1 of 90 amino acids in length (amino acids 16-105) as well as other variants which facilitate folding.","It also concerns a propeptide which is added exogenously for the folding of mature proteinase K and has the functions described above.","A further subject matter of the invention is a recombinant vector which contains one or more copies of the recombinant DNA defined above.","The basic vector is advantageously a plasmid preferably containing a multi-copy origin of replication, but is also possible to use viral vectors.","The choice of expression vector depends on the selected host cell.","Methods are used to construct the expression vector and to transform the host cell with this vector that are familiar to a person skilled in the art and are described for example in Sambrook et al.","(1989), Molecular Cloning (see below).","A suitable vector for expression in E. coli is for example the pKKT5 expression vector or pKK177, pKK223, pUC, pET vectors (Novagen) as well as pQE vectors (Qiagen).","The expression plasmid pKKT5 is formed from pKK177-3 (Kopetzki et al., 1989, Mol.","Gen. Genet.","216:149-155) by exchanging the tac promoter for the T5 promoter from pDS (Bujard et al., 1987, Methods Enzymol.","155:416-433).","The EcoRI restriction endonuclease cleavage site in the sequence of the T5 promoter was removed by two point mutations.","In addition the coding DNA in the vector according to the invention is under the control of a preferably strong, regulatable promoter.","A promoter that can be induced by IPTG is preferred such as the lac, lacUV5, tac or T5 promoter.","The T5 promoter is especially preferred.","A host cell in the sense of the invention means any host cell in which proteins can form as inclusion bodies.","It is usually a microorganism e.g.","prokaryotes.","Prokaryotic cells are preferred and in particular Escherichia coli.","Particular preference is given to the following strains: E. coli K12 strains JM83, JM105, UT5600, RR1Δ15, DH5α, C600, TG1, NM522, M15 or the E. coli B derivatives BL21, HB101, E. coli M15 is particularly preferred.","The corresponding host cells are transformed according to the invention with a recombinant nucleic acid which encodes a recombinant zymogenic proteinase K according to SEQ ID NO:2 or with a nucleic acid which is derived from the said DNA by codon-optimization or with a DNA which is derived from the said DNA within the scope of the degeneracy of the genetic code.","The E. coli host cells are preferably transformed with a codon-optimized recombinant nucleic acid coding for a recombinant zymogenic proteinase K which has been optimized for expression in Escherichia coli.","Hence the present invention also concerns a suitable vector which is for example selected from the above-mentioned vectors and contains a recombinant nucleic acid that is codon-optimized for E. coli and codes for a recombinant proteinase K or a recombinant zymogenic proteinase K. Another subject matter of the invention is a host cell which is for example selected from the above-mentioned host cells which has been transformed by the above-mentioned vector.","A further subject matter of the present invention is a method for the naturation of denatured zymogenic proteinase K in which the denatured zymogenic proteinase K is transferred to a folding buffer which is characterized in that the folding buffer has the following features: A) pH value of the buffer is in the range of 7.5 to 10.5 B) presence of low-molecular weight substances which aid folding C) presence of a redox shuffling system D) presence of a complexing agent at a substoichiometric concentration relative to the Ca2+ ions and wherein the method is carried out at a temperature between 0° C. and 37° C. A low concentration of denaturing agents is preferably present during the naturation.","Denaturing agents may for example be present because they are still in the reaction solution due to the prior solubilization of the inclusion bodies.","The concentration of denaturing agents such as guanidine hydrochloride should be less than 50 mM.","Naturation in the sense of the invention is understood as a method in which denatured, essentially inactive protein is converted into a conformation in which the protein has the desired activity after autocatalytic cleavage and activation.","This is achieved by transferring the solubilized inclusion bodies to a folding buffer while reducing the concentration of the denaturing agent.","The conditions must be selected such that the protein remains in solution in this process.","This can be expediently carried out by rapid dilution or dialysis against the folding buffer.","It is preferred that the folding buffer has a pH of 8 to 9.Particularly preferred buffer substances are Tris/HCl buffer and bicine buffer.","The naturation method according to the invention is preferably carried out at a temperature between 0° C. and 25° C. The low molecular weight folding agents in the folding buffer are preferably selected from the following group of low molecular weight compounds.","They can be added alone as well as in mixtures, and other substances that aid folding may be present: L-arginine at a concentration of 0.5 to 2.0 M Tris at a concentration of 0.5 M to 2.0 M triethanolamine at a concentration of 0.5 M to 2.0 M α-cyclodextrin at a concentration of 60 mM to 120 mM Low molecular weight substances that aid folding are described for example in U.S. Pat.","No.","5,593,865; Rudolph, R. (1997) Folding Proteins.","In: Creighton, T. E.","(ed.)","Protein Function: A practical Approach.","Oxford University Press, 57-99 or De Bernardez Clark, E. et al.","(1999) Methods.","Enzymol.","Vol.","309,217-236.The above-mentioned redox shuffling system is preferably a mixed disulfide or thiosulfonate.","Systems are for example suitable as a redox shuffling system which consist of a thiol component in an oxidized and reduced form.","This allows the formation of disulfide bridges within the folding polypeptide chain during naturation by controlling the reduction potential, and on the other hand, enables the reshuffling of incorrect disulfide bridges within or between the folding polypeptide chains (Rudolph, R. (1997), see above).","Preferred thiol components are for example: glutathione in a reduced (GSH) and oxidized form (GSSH) cysteine and cystine cysteamine and cystamine 2-mercaptoethanol and 2-hydroxyethyldisulfide In the naturation method according to the invention the Ca2+ ions are preferably present at a concentration of 1 to 20 mM.","For example CaCl2 can be added in amounts of 1 to 20 mM.","The Ca2+ ions can bind to the calcium binding sites of the folding proteinase K. The presence of a complexing agent preferably EDTA, in a substoichiometric concentration relative to Ca2+ prevents the oxidation of the reducing agent by atmospheric oxygen and protects free SH groups.","The naturation is preferably carried out at a low temperature i.e.","below 20° C., preferably 10° C. to 20° C. In the method according to the invention the naturation is usually completed after a period of about 24 h to 48 h. The present invention also concerns a folding buffer which is characterized by the following features: A) pH value of the buffer is in the range of 7.5 to 10.5 B) presence of low-molecular weight substances which aid folding C) presence of a redox shuffling system D) presence of a complexing agent at a substoichiometric concentration relative to the Ca2+ ions.","It is especially preferred when the folding buffer has a pH of 8 to 9 and/or when the redox shuffling system is a mixed disulfide or thiosulfonate.","Another subject matter of the invention is a method for activating the natured zymogenic precursor of proteinase K. After the folding process according to the invention an inactive complex is formed from native proteinase K and the inhibitory propeptide.","The active proteinase K can be released from this complex.","Addition of detergents is preferred, SDS is particularly preferred at a concentration of 0.1 to 2% (w/v).","The advantages of the method disclosed here for producing recombinant proteinase K are: 1.The ability to utilize the high expression potential and the rapid and simple culture of Escherichia coli or other suitable microorganisms.","2.The possibility to genetically manipulate the recombinant DNA.","3.The uncomplicated purification after naturation.","4.The absence of eukaryotic impurities when a prokaryote is selected as a host cell.","A method would also be conceivable in which the nucleic acids which code for mature proteinase K and nucleic acids which code for the propeptide or pro-proteinase K are expressed separately in host cells and are then commonly transferred to a folding buffer for the naturation of mature proteinase K. DESCRIPTION OF THE FIGURES FIG.","1: Schematic representation of the PCR reaction to produce proteinase K fragments having an N-terminal BamHI cleavage site and an alternative enterokinase cleavage site for fusion with an N-terminal affinity tag.","FIG.","2: Dependency of the yield of naturation on temperature.","FIG.","3: Dependency of the yield of naturation on pH.","FIG.","4: Dependency of the yield of naturation on redox potential.","FIG.","5: Dependency of the yield of naturation on the arginine concentration.","FIG.","6: Dependency of the yield of naturation on the Tris concentration.","FIG.","7: Dependency of the yield of naturation on the α-cyclodextrin concentration.","FIG.","8: Dependency of the yield of naturation on the triethanolamine concentration.","FIG.","9: Dependency of the yield of naturation on the urea concentration.","FIG.","10: SDS polyacrylamide gel of the naturation of pro-proteinase K. FIG.","11: Reverse phase chromatography of natured proteinase K. FIG.","12: Renatured and processed proteinase K was analysed by analytical ultracentrifugation.","The centrifugation was carried out at 12000 rpm, 20° C. for 63 h. The data (o) could be fitted to a homogeneous species having an apparent molecular weight of 29-490 Da.","No systematic deviation was observed between the fitted and measured data (lower graph).","FIG.","13: Determination of the Km value of natured proteinase K. FIG.","14: Degradation pattern of blood serum proteins by natured proteinase K. FIG.","15: Purification of natured proteinase K by gel filtration.","EXAMPLE 1 Synthesis of the Gene which Codes for the Mature Form of Proteinase K. The gene for the mature proteinase K from Tritirachium album Limber without a signal sequence and without an intron was generated by means of gene synthesis.","The sequence of Gunkel, F. A. and Gassen, H. G. (1989) Eur.","J. Biochem.","Vol.","179(1), 185-194 of 837 bp in length (amino acids 106-384 from Swiss Prot P06873) was used as the template.","A codon usage optimized for Escherichia coli was used as the basis for retranslating the amino acid sequence to optimize the expression (Andersson, S. G. E. and Kurland, C. G. (1990) Microbiol.","Rev.","Vol.","54(2), 198-210, Kane, J. F. Curr.","Opin.","Biotechnol., Vol.","6, pp.","494-500).","The amino acid sequence is shown in SEQ ID NO: 1 and the nucleotide sequence is shown in SEQ ID NO: 2.For the gene synthesis the gene was divided into 18 fragments of sense and reverse, complementary counterstrand oligonucleotides in alternating sequence (SEQ ID NO:3-20).","An at least 15 bp region was attached to the 5′ end and to the 3′ end which in each case overlapped the neighbouring oligonucleotides.","Recognition sites for restriction endonucleases were attached to the 5′ and 3′ ends of the synthetic gene outside the coding region for subsequent cloning into expression vectors.","The oligonucleotide shown in SEQ ID NO:3 which contains an EcoRI cleavage site was used as a 5′ primer for cloning the pro-protein X gene without an N-terminal affinity tag.","SEQ ID NO: 20 shows the 3′ primer containing a HindIII cleavage site.","The 3′ primer contains an additional stop codon after the natural stop codon to ensure termination of the translation.","The oligonucleotide with a BamHI cleavage site shown in SEQ ID NO: 23 or the oligonucleotide with a BamHI cleavage site and enterokinase cleavage site shown in SEQ ID NO: 24 was used as a 5′ primer to clone the proprotein X gene with N-terminal affinity tags and an alternative enterokinase cleavage site as described in example 3.The oligonucleotides were linked together by means of a PCR reaction and the resulting gene was amplified.","For this the gene was firstly divided into three fragments of 6 oligonucleotides each and the three fragments were linked together in a second PCR cycle.","Fragment 1 is composed of the oligonucleotides shown in SEQ ID NO: 3-8, fragment 2 is composed of the oligonucleotides shown in SEQ ID NO: 9-14 and fragment 3 is composed of the oligonucleotides shown in SEQ ID NO: 15-20.The following PCR parameters were employed PCR reaction 1 (generation of three fragments) 5 min 95° C. hot start 2 min 95° C. 2 min 42° C. 1.5 min 72° C. {close oversize brace} 30 cycles 7 min 72° C. final extension PCR reaction 2 (linkage of the fragments to form the total gene) 5 min 95° C. hot start 1.5 min 95° C. 2 min 48° C. {close oversize brace} 6 cycles (without terminal primers) 2 min 72° C. addition of terminal primers 1.5 min 95° C. 1.5 min 60° C. {close oversize brace} 25 cycles (with terminal primers) 2 min 72° C. 7 min 72° C. final extension EXAMPLE 2 Cloning of the Synthetic Proteinase K Fragment from the Gene Synthesis The PCR mixture was applied to an agarose gel and the ca.","1130 bp PCR fragment was isolated from the agarose gel (Geneclean II Kit from Bio 101, Inc. CA USA).","The fragment was cleaved for 1 hour at 37° C. with EcoRI and HindIII restriction endonucleases (Roche Diagnostics GmbH, Germany).","At the same time the pUC18 plasmid (Roche Diagnostics GmbH, Germany) was cleaved for 1 hour at 37° C. with EcoRI and HindIII restriction endonucleases, the mixture was separated by agarose gel electrophoresis and the 2635 bp vector fragment was isolated.","Subsequently the PCR fragment and the vector fragment were ligated together using T4 DNA ligase.","For this 1 μl (20 ng) vector fragment and 3 μl (100 ng) PCR fragment, 1 μl 10× ligase buffer (Maniatis, T., Fritsch, E. F. and Sambrook, T. (1989).","Molecular Cloning: A laboratory manual.","2nd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y.), 1 μl T4 DNA ligase, 4 μl sterile redistilled H2O were pipetted, carefully mixed and incubated overnight at 16° C. The cloned gene was examined by restriction analysis and by multiple sequencing of both strands.","The sequence is shown in SEQ ID NO: 2.a) Construction of the pPK-1 Expression Plasmid In order to express proteinase K, the structural gene was cloned into the pKKT5 expression vector in such a manner that the structural gene is inserted in the correct orientation under the control of a suitable promoter, preferably a promoter that can be induced by IPTG such as the lac, lacUV5, tac or T5 promoter, particularly preferably the T5 promoter.","For this purpose the structural gene for proteinase K was cleaved from the plasmid pUC18 by EcoRI and HindIII, the restriction mixture was separated by agarose gel electrophoresis and the ca.","1130 bp fragment was isolated from the agarose gel.","At the same time the expression plasmid pKKT5 was cleaved with EcoRI and HindIII, the restriction mixture was separated by agarose gel electrophoresis and the ca.","2.5 kbp vector fragment was isolated from the agarose gel.","The fragments obtained in this manner were ligated together as described above.","The correct insertion of the gene was checked by sequencing.","b) Transformation of the Expression Plasmid pPK-1 in Various E. coli Expression Strains The expression vector was transformed in various expression strains that had been previously transformed with the plasmid pREP4 and/or pUBS520.The plasmid pREP4 contains a gene for the lacI repressor that should ensure a complete suppression of the expression before induction.","The plasmid pUBS520 (Brinkmann, U. et al.","(1989) Gene Vol.","85(1), 109-114) also contains the lacI repressor and additionally the dnaY gene which codes for the tRNA which is necessary to translate the rare arginine codons AGA and AGG in E. coli.","Competent cells of various E. coli strains were prepared according to the method of Hanahan, D. (1983) J. Mol.","Biol.","Vol.","166, 557-580.100 μl cells prepared in this manner was admixed with 20 ng isolated pPK-1 plasmid DNA.","After 30 min incubation on ice, they were heat-shocked (90 sec at 42° C.) and then incubated for 2 min on ice.","Subsequently the cells were transferred to 1 ml SOC medium and incubated for 1 hour at 37° C. while shaking for the phenotypic expression.","Aliquots of this transformation mixture were plated out on LB plates containing ampicillin as a selection marker and incubated for 15 hours at 37° C. Preferred strains are E. coli K12-strains JM83, JM105, UT5600, RR1Δ15, DH5α, C600, TG1, NM522, M15 or the E. coli B derivatives BL21, HB101; E. coli M15 is particularly preferred.","EXAMPLE 3 Cloning of an N-Terminal Affinity Tag In order to insert an N-terminal affinity tag, a BamHI cleavage site was inserted before the 5′ end of the gene for pro-proteinase K. This was achieved by PCR using the product obtained in example 1 as a template and the oligonucleotides described in SEQ ID NO:20, 23 and 24 as primers.","The primer described in SEQ ID NO:23 contains a BamHI cleavage site upstream of the 5′ region of pro-proteinase K, the primer described in SEQ ID NO:24 additionally contains an enterokinase cleavage site directly in front of the first codon of the prosequence.","SEQ ID NO:20 shows the 3′ primer that was also used in example 1 with a HindIII cleavage site.","The resulting PCR products were isolated as described above, digested with BamHI and HindIII and purified by agarose electrophoresis.","The affinity tag was inserted by means of a synthetic linker composed of two complementary oligonucleotides in such a manner that an EcoRI cleavage site was formed at the 5′ end and a BamHI cleavage site was formed at the 3′ end without further restriction digestion.","For a His tag the sense strand had the sequence shown in SEQ ID NO:21 and the antisense strand had the sequence shown in SEQ ID NO.22.The linker coded for a hexa-His tag with an N-terminal RGS motif.","The BamHI cleavage site between the linker and pro-proteinase K is translated into a Gly-Ser linker.","In order to anneal the linker, the two oligonucleotides (SEQ ID NO:21 and 22) were heated for 5 min to 95° C. in equimolar amounts (50 pmol/μl each) and subsequently cooled at 2° C. per min to room temperature.","As a result the annealing of the complementary oligonucleotides should be as complete as possible.","The linker was ligated with the BamHI/HindIII-digested PCR product.","(Rapid Ligation Kit from Roche Diagnostics GmbH, Germany) and purified by agarose gel electrophoresis (QIAquick gel extraction Kit from Qiagen, Germany).","The resulting ligation product was ligated into an expression vector analogously to example 2b via the EcoRI and HindIII overhangs and transformed correspondingly in expression strains.","This module system enables various affinity tags that are coded by the synthetic linker to be fused to the structural gene for pro-proteinase K. An enterokinase cleavage site can be alternatively inserted between the tag and propeptide by suitable selection of the corresponding 5′ primer if a subsequent removal of the tag is desired.","Furthermore a certain region of the proteinase K gene such as the mature proteinase K or the propeptide can be amplified by suitable selection of the overlapping regions of the PCR primers (FIG.","1).","EXAMPLE 4 Expression of Proteinase K in Escherichia coli Since proteinase K is a very active unspecific protease, it is preferable to express it in an inactive form preferably as inclusion bodies.","In order to express the gene which codes for proteinase K, 3 ml Lbamp medium was inoculated with plasmid-containing clones and incubated at 37° C. in a shaker.","LB medium: 10 g tryptone 10 yeast extract 5 g NaCl make up to a final volume of 1 l with distilled H2O, adjust to pH 7.0 with NaOH addition of antibiotics (100 μg/ml ampicillin) directly before inoculation The cells were induced with 1 mM IPTG at an optical density of 0.5 at 550 nm and incubated for 4 h at 37° C. in a shaker.","Subsequently the optical density of the individual expression clones was determined, an aliquot corresponding to an OD550 of 3/ml was removed and the cells were centrifuged (10 min 6000 rpm, 4° C.).","The cells were resuspended in 400 μl TE buffer, lysed by ultrasound and the soluble protein fraction was separated from the insoluble protein fraction by centrifugation (10 min, 14,000 rpm, 4° C.).","TE buffer: 50 mM Tris/HCl 50 mM EDTA pH 8.0 (at RT) Application buffer containing SDS and β-mercaptoethanol was added to all fractions and the proteins were denatured by heating (5 min 95° C.).","Subsequently 10 μl aliquots were analysed by means of a 12.5% analytical SDS gel (Laemmli, U.K. (1970) Nature Vol.","227(259), 680-685).","A very strong expression in the form of insoluble protein aggregates (inclusion bodies) was observed for the clones of mature proteinase K as well as for the clones of pro-proteinase K. Accordingly no proteinase K activity was measured.","EXAMPLE 5 Isolation of the Inclusion Bodies The inclusion bodies were prepared by known methods (Rudolph, R. (1997) see above).","For the cell lysis, 10 g wet biomass was in each case resuspended in 50 ml 100 mM Tris/HCl pH 7.0, 1 mM EDTA.","Afterwards 15 mg lysozyme was added, incubated for 60 min at 4° C. and the cells were subsequently lysed by high pressure (Gaulin cell lysis apparatus).","The DNA was digested for 30 min at RT by adding 3 mM MgCl2 and 10 μg/ml DNase to the crude extract.","The insoluble cell components which contain the inclusion bodies were separated by centrifugation (30 min 20,000 g) and washed once with washing buffer 1 and three times with washing buffer 2.washing buffer 1: 100 mM Tris/HCl 20 mM EDTA 2% (v/v) Triton X-100 0.5 M NaCl pH 7.0 (RT) washing buffer 2: 100 mM Tris/HCl 1 mM EDTA pH 7.0 (RT) The pellet of the last washing step constitutes the crude inclusion bodies which already contain highly pure target protein.","EXAMPLE 6 Solubilization of Inclusion Bodies a) Solubilization While Reducing with Cysteines 1 g crude inclusion bodies was suspended in 10 ml solubilization buffer and incubated for 2 h at RT while stirring gently.","Solubilization buffer: 100 mM Tris/HCl 6.0 M guanidinium hydrochloride 100 mM DTT pH 8.0 (RT) The solubilisate was titrated to pH 3 with 25% HCl and dialysed twice for 4 h at RT against 500 ml 6 M guanidine hydrochloride pH 3 and then overnight at 4° C. against 1000 ml guanidine hydrochloride pH 7.The protein concentration was determined by the Bradford method (Bradford, 1976) using a calculated extinction coefficient at 280 nm and was between 10 and 20 mg/ml.","The number of free cysteines was determined according to the Ellman method.","In accordance with the sequence 5 mol free cysteines per mol proteinase K were found.","The purity of the solubilized inclusion bodies was determined by 12.5% SDS PAGE and quantification of the bands after Coomassie staining.","b) Solubilization with Derivatization of the Cysteines to Form Mixed Disulfides Using Glutathione.","1 g Crude Inclusion Bodies Were Suspended in 10 ml Solubilization Buffer.","Solubilization buffer: 100 mM Tris/HCl 6.0 M guanidine hydrochloride 1 mM DTT pH 8.0 (RT) After 15 min incubation at RT while stirring gently, during which a catalytic amount of reduced cysteines was formed due to the small amounts of DTT, 100 mM GSSG was added, the pH was adjusted to 8.0 and it was incubated for a further 2 h at RT while stirring gently.","Further treatment as described under a).","EXAMPLE 7 Optimization of the Naturation of Pro-Proteinase K Various parameters were varied in order to optimize the yield in the folding and processing of pro-proteinase K from the solubilisates prepared in example 6a).","For all preparations the stated folding buffer was filtered, degassed, gassed with N2 and incubated at the desired temperature.","The redox shuffling system was not added until shortly before the start of the folding reaction and the pH was readjusted.","The folding was initiated by adding the solubilized inclusion bodies while rapidly mixing.","The volume of the folding mixtures was 1.8 ml in 2 ml glass tubes with a screw cap.","The yield was analysed by an activity test using the chromogenic substrate Suc-Ala-Ala-Pro-Phe-pNA from the Bachem Company (Heidelberg).","100 mM Tris/HCl, 5 mM CaCl2, pH 8.5 at 25° C. was used as the test buffer.","The concentration of the peptide in the test was 2 mM from a 200 mM stock solution in DMSO.","In order to activate the renaturate, 0.1% SDS was added to the sample (see example 8).","The absorbance at 410 nm was measured over a period of 20 min and the activity was calculated from the slope.","The following parameters were varied: a) Temperature and Time The folding buffer containing 100 mM Tris, 1.0 mM L-arginine, 10 mM CaCl2 was equilibrated at various temperatures.","After adding 3 mM GSH and 1 mM GSSG the pH was readjusted at the corresponding temperature.","The reaction was started by adding 50 μg/ml pro-proteinase K. After 12 h, 36 h and 60 h, aliquots were removed and tested for activity.","The results are shown in FIG.","2.b) pH Value A universal buffer containing 50 mM citrate, 50 mM MES, 50 mM bicine, 500 mM arginine, 2 mM CaCl2 and 1 mM EDTA was incubated at 15° C. and 3 mM GSH and 1 mM GSSG were added.","The pH was readjusted in a range between pH 4.0 and pH 12.0 and the folding reaction was started by adding 50 μg/ml pro-proteinase K inclusion bodies.","The activity measured after 18 h, 3 d and 5 d is shown in FIG.","3.c) Redox Potential Various redox potentials were set in a renaturation buffer containing 1.0 M L-arginine, 100 mM bicine, 2 mM CaCl2 and 10 mM CaCl2 by mixing various ratios of oxidized and reducing glutathione.","The protein concentration in the folding mixture was 50 μg/ml.","The folding was carried out at 15° C. The concentrations of GSH and GSSG are shown in table 1, the measurements are shown in FIG.","4.TABLE 1 concentrations of GSH and GSSG at the various redox potentials.","Redox potential (log(cGSH2/cGSSG) [M] c(GSH) [mM] c(GSSG) [mM] −00 0 2.500 −6.000 0.050 2.475 −5.500 0.088 2.456 −5.000 0.156 2.422 −4.500 0.273 2.363 −4.000 0.476 2.262 −3.500 0.814 2.093 −3.000 1.351 1.825 −2.500 2.130 1.435 −2.000 3.090 0.955 −1.500 3.992 0.504 −1.000 4.580 0.210 −0.500 4.851 0.074 0.000 4.951 0.025 +0.500 4.984 7.856e−3 +1.000 4.995 2.495e−3 +00 5.000 0 d) Solvent Additives that Promote Folding Various substances were examined for their ability to increase the folding yield of proteinase K. For this purpose solutions containing the substances at various concentrations were prepared and admixed with 2 mM CaCl2, 1 mM EDTA and 100 mM bicine.","The pH was adjusted to pH 8.75 at the folding temperature of 15° C. The protein concentration was 50 μg/ml.","FIG.","5 shows the relative yields of active proteinase K in relation to the concentration of the selected buffer additive.","EXAMPLE 8 Activation of the Natured Pro-Proteinase K After naturation of pro-proteinase K by the method according to the invention it was found to have no activity or only a very slight activity.","Chromatographic methods and SDS-PAGE showed that mature proteinase K is already present but is still associated in a complex with the propeptide.","This can be separated in a method which is referred to here as activation and is also a subject matter of the invention.","In this example SDS is added at a concentration of 2% (v/v) to the folding mixture and subsequently the folding additive and the SDS are removed by dialysis.","Alternatively SDS could also be added after removing the additives by dialysis.","In all cases full activity of proteinase K was detected.","EXAMPLE 9 Characterization of the Folding Product The proteinase K natured and activated by the method according to the invention was further characterized by various methods.","a) Analysis of Purity and Molecular Weight Determination by SDS Polyacrylamide Gel Electrophoresis Aliquots from various steps in the maturation process and the final product, the folded and activated recombinant proteinase K were applied to a 12.5% SDS polyacrylamide gel.","The samples each contained 10 mM DTT or 1% (v/v) 2-mercaptoethanol.","The recombinant proteinase K prepared by the method according to the invention had no significant contamination and runs identically with the authentic proteinase K at an apparent molecular weight of ca.","30 kDa (see FIG.","11).","b) Analysis of Purity Using RP-HPLC The folded and activated proteinase K and the authentic proteinase K from T. album and the pro-proteinase K inclusion bodies were analysed by means of reversed phase HPLC.","A Vydac C4 column having the dimensions 15 cm×4.6 cm diameter was used.","The samples were eluted with an acetonitrile gradient of 0% to 80% in 0.1% TFA.","The folding product exhibits mobility properties that are identical to the authentic proteinase K used as a standard (see FIG.","12).","c) Analytical Ultracentrifugation In order to analyse whether the renatured and processed proteinase K is present in a monomeric form without propeptide, the protein was examined by means of analytical ultracentrifugation.","The molecular weight was determined to be 29490 Da and corresponds to the mass of the monomeric mature proteinase K within the limits of error of this method (see FIG.","13).","Hence this showed that the propeptide was quantitatively cleaved by activation of the proteinase K. d) N-Terminal Sequence Analysis In order to examine whether the propeptide was cleaved at the correct cleavage site the natured and activated recombinant proteinase K was subjected to a sequence analysis.","For this the folding product was desalted by RP-HPLC as described in example 9b) and the first 6 residues were examined by N-terminal sequencing.","The result (AAQTNA) agrees with the authentic N-terminus of mature proteinase K. e) Activity and Km Value the Km value of the folded and activated proteinase K was compared with that of the authentic proteinase K. The tetrapeptide Suc-Ala-Ala-Pro-Phe-pNA was used as a substrate.","The test was carried out in 2.0 ml 50 mM Tris, pH 8.5 containing 1 mM CaCl2 at 25° C. The hydrolysis of the peptide was monitored spectroscopically at 410 nm.","A Km value of 0.16 mM was found for the recombinant proteinase K which corresponded very well with the Km value of authentic proteinase K (see FIG.","14).","f) Degradation Pattern of Blood Serum Proteins In an additional test to characterize the activity, the cleavage pattern of blood serum proteins was examined.","For this a defined amount of blood serum proteins was digested with 1 μg recombinant proteinase K or the same amount of authentic proteinase K. The cleavage pattern was analysed by means of RP-HPLC under identical conditions as described in example 9b).","FIG.","15 shows that the recombinant and the authentic proteinase K result in an identical degradation pattern.","EXAMPLE 10 Purification of the Folding Product The recombinant pro-proteinase K natured by the method according to the invention was purified by gel filtration.","As described in FIG.","11 the concentrated naturation solution was separated on a Superdex 75 pg after naturation in a first run without prior activation and in a second run with prior activation using 0.15% (w/v) SDS (30 min, 4° C.).","100 mM Tris/HCl, 150 mM NaCl pH 8.75 (4° C.) was used as the mobile buffer.","The application volume was 10 ml at a column volume of 1200 ml and a flow rate of 5 ml/min.","After completion of the application, 14 ml fractions were collected.","Aliquots of the fractions were precipitated with trichloroacetic acid, washed and taken up in Laemmli sample buffer containing 10 mM DTT.","The samples were applied to a 12.5% SDS polyacrylamide gel which was stained after the run with Coomassie blue R250.In the first run without activation a non-processed recombinant pro-proteinase K is seen in a first peak which probably runs in the form of microaggregates in the exclusion volume.","In a second peak one observes processed recombinant proteinase K which co-elutes with the propeptide which is non-covalently bound and acts as an inhibitor.","As a result no activity is found without prior activation.","Only after adding SDS to the fractions did the second peak exhibit significant proteinase K activity (not shown).","The second run in which the folded recombinant proteinase K was previously activated with SDS only shows one peak which elutes after an identical volume like proteinase K under the same conditions (not shown).","On the SDS gel one sees clean mature recombinant proteinase K without propeptide in this peak.","All impurities and the propeptide appear to have already been digested in the applied mixture by the activated recombinant proteinase K. As expected the fractions of the proteinase K peak exhibited activity without further activation with SDS.","The recombinant proteinase K purified in this manner appears to be almost 100% pure on the SDS gel and shows an identical migration behaviour to the authentic proteinase K (FIG.","16)."]],"string":"[\n [\n \"Recombinant proteinase k\",\n \"The invention concerns recombinant proteinase K. Furthermore a method for producing recombinant proteinase K is disclosed, which is characterized in that a) a host cell is transformed with a recombinant nucleic acid which codes for the zymogenic precursor of proteinase K, b) the host cell is cultured in such a manner that the zymogenic precursor of proteinase K is formed in the form of inclusion bodies in the host cell, c) the inclusion bodies are isolated and natured under conditions which result in the formation of the protease part of the zymogenic precursor in its natural conformation, d) the natured proteinase K is activated and purified.\"\n ],\n [\n \"1-22.\",\n \"(canceled) 23.A method for the naturation of denatured zymogenic proteinase K comprising transferring the denatured zymogenic proteinase K to a folding buffer, the buffer comprising low molecular weight substances which aid folding, a redox shuffling system, and a complexing agent at a substoichiometric concentration relative to any Ca2+ ions that are present, the buffer having a pH of 7.5 to 10.5 and the method being carried out at a temperature between 0° C. and 37° C. 24.The method of claim 23 wherein the redox shuffling system comprises mixed disulfides or thiosulfonates.\",\n \"25.The method of claim 23 wherein the pH range is 8 to 9.26.The method of claim 23 wherein the temperature is between 0° C. and 25° C. 27.The method of claim 23 wherein the buffer further comprises denaturing agents at a concentration of less than 50 mM.\",\n \"28.The method of claim 23 wherein the low molecular weight substances are selected from the group consisting of L-arginine at a concentration of 0.5 to 2.0 M, Tris at a concentration of 0.5 M to 2.0 M, triethanolamine at a concentration of 0.5 M to 2.0 M, and α-cyclodextrin at a concentration of 60 mM to 120 mM.\",\n \"29.The method of claim 23 wherein the Ca2+ ion concentration is 1 to 20 mM.\",\n \"30.The method of claim 23 wherein the denatured zymogenic proteinase K is transferred to the folding buffer while reducing the concentration of denaturing agents that may be present.\",\n \"31.A folding buffer comprising low molecular weight substances which aid folding, a redox shuffling system, and a complexing agent at a substoichiometric concentration relative to any Ca2+ ions that are present, the buffer having a pH value in the range of 7.5 to 10.5.32.The buffer of claim 31 wherein the pH value is 8 to 9 and the redox shuffling system comprises mixed disulfides or thiosulfonates.\",\n \"33.A method for activating a natured zymogenic precursor of active proteinase K comprising adding a detergent to an inactive complex comprising a native proteinase K and an inhibitory propeptide of the active proteinase K, thereby releasing the active proteinase K from the inactive complex.\",\n \"34.The method of claim 33 wherein the detergent is SDS at a concentration of 0.1 to 2% (w/v).\",\n \"35.A method for producing active recombinant proteinase K comprising (a) producing an inactive zymogenic proform of proteinase K in an inclusion body, (b) naturing in vitro the zymogenic proform of proteinase K, and (c) activating the zymogenic proform by autocatalytic cleavage, thereby converting it to the active proteinase K. 36.The method of claim 35 wherein the naturing step comprises transferring the denatured zymogenic proteinase K to a folding buffer, the buffer comprising low molecular weight substances which aid folding, a redox shuffling system, and a complexing agent at a substoichiometric concentration relative to any Ca2+ ions that are present, the buffer having a pH of 7.5 to 10.5 and the naturing step being carried out at a temperature between 0° C. and 37° C. 37.The method of claim 35 wherein the inclusion body is solubilized by a denaturing agent and a reducing agent.\",\n \"38.The method of claim 37 wherein the denaturing agent is guanidinium hydrochloride at a concentration of 6-8 M or urea at a concentration of 8-10 M and the reducing agent is DTT or DTE at a concentration of 50-200 mM.\",\n \"39.A method for producing active recombinant proteinase K comprising (a) transforming a host cell with a vector containing a DNA sequence coding for a zymogenic precursor of proteinase K (b) expressing the zymogenic precursor in inclusion bodies, (c) isolating the inclusion bodies and solubilizing the zymogenic precursor, (d) naturing the zymogenic precursor with a folding buffer comprising low molecular weight substances which aid folding, a redox shuffling system, and a complexing agent at a substoichiometric concentration relative to any Ca2+ ions that are present, the buffer having a pH of 7.5 to 10.5 and the naturing step being carried out at a temperature between 0° C. and 37° C., and (e) activating the zymogenic precursor by autocatalytic cleavage, thereby converting it to the active proteinase K 40.The method of claim 39 wherein the host cell is a prokaryotic cell.\",\n \"41.The method of claim 39 wherein the host cell is Escherichia coli.\",\n \"42.A codon-optimized recombinant nucleic acid comprising DNA coding for a recombinant zymogenic proteinase K which has been optimized for expression in Escherichia coli.\",\n \"43.A vector containing the recombinant nucleic acid of claim 42.44.A host cell transformed with the vector of claim 43.\"\n ],\n [\n \"The present invention concerns the preparation of recombinant proteinase K from Tritirachium album Limber and corresponding methods for the expression, in vitro naturation and activation of the recombinant proteinase K. Proteinase K (E.C.\",\n \"3.4.21.64, also known as endopeptidase K) is an extracellular endopeptidase which is synthesized by the fungus Tritirachium album Limber.\",\n \"It is a member of the class of serine proteases with the typical catalytic triad Asp39-His69-Ser224 (Jany, K. D. et al.\",\n \"(1986) FEBS Letters Vol.\",\n \"199(2), 139-144).\",\n \"Since the sequence of the polypeptide chain of 279 amino acids in length (Gunkel, F. A. and Gassen, H. G. (1989) Eur.\",\n \"J. Biochem.\",\n \"Vol.\",\n \"179(1), 185-194) and the three dimensional structure (Betzel, C. et al.\",\n \"(1988) Eur.\",\n \"J. Biochem.\",\n \"Vol.\",\n \"178(1), 155-71) has a high degree of homology to bacterial subtilisins, proteinase K is classified as a member of the subtilisin family (Pahler, A. et al.\",\n \"(1984) EMBO J. Vol.\",\n \"3(6), 1311-1314; Jany, K. D. and Mayer, B.\",\n \"(1985), Biol.\",\n \"Chem.\",\n \"Hoppe-Seyler, Vol.\",\n \"366(5), 485-492).\",\n \"Proteinase K was named on the basis of its ability to hydrolyse native keratin and thus allows the fungus to grow on keratin as the only source of carbon and nitrogen (Ebeling, W. et al.\",\n \"(1974) Eur.\",\n \"J. Biochem.\",\n \"Vol.\",\n \"47(1), 91-97) Roelcke and Uhlenbruch, 1069, Z. Med.\",\n \"Mikrobiol.\",\n \"Immunol.\",\n \"Vol.\",\n \"155(2), 156-170).\",\n \"Proteinase K has a specific activity of more than 30 U/mg and is thus one of the most active of the known endopeptidases (Betzel, C. et al.\",\n \"(1986) FEBS Lett.\",\n \"Vol.\",\n \"197(1-2), 105-110) and unspecifically hydrolyses native and denatured proteins (Kraus, E. and Femfert, U, (1976) Hoppe Seylers Z. Physiol.\",\n \"Chem.\",\n \"Vol.\",\n \"357(7):937-947).\",\n \"Proteinase K from Tritirachium album Limber is translated in its natural host as a preproprotein.\",\n \"The sequence of the cDNA of the gene which codes for proteinase K was decoded in 1989 by Gunkel, F. A. and Gassen, H. G. (1989) Eur.\",\n \"J. Biochem.\",\n \"Vol.\",\n \"179(1), 185-194.According to this the gene for prepro-proteinase K is composed of two exons and codes for a signal sequence of 15 amino acids in length, a prosequence of 90 amino acids in length and a mature proteinase K of 279 amino acids in length.\",\n \"A 63 bp intron is located in the region of the prosequence.\",\n \"The prepeptide is cleaved off during translocation into the endoplasmatic reticulum (ER).\",\n \"At present very little is known about the subsequent processing to form mature proteinase K with cleavage of the propeptide.\",\n \"Consequently mature proteinase K consists of 279 amino acids.\",\n \"The compact structure is stabilized by two disulfide bridges and two bound calcium ions.\",\n \"This explains why proteinase K compared to other subtilisins has a considerably higher stability towards extreme pH values, high temperatures, chaotropic substances and detergents (Dolashka, P. et al.\",\n \"(1992) Int.\",\n \"J. Pept.\",\n \"Protein.\",\n \"Res.\",\n \"Vol.\",\n \"40(5), 465-471).\",\n \"Proteinase K is characterized by a high thermostability (up to 65° C., Bajorath et al.\",\n \"(1988), Eur.\",\n \"J. Biochem.\",\n \"Vol.\",\n \"176, 441-447) and a wide pH range (pH 7.5-12.0, Ebeling, W. et al.\",\n \"(1974) Eur.\",\n \"J. Biochem.\",\n \"Vol.\",\n \"47(1), 91-97).\",\n \"Its activity is increased in the presence of denaturing substances such as urea or SDS (Hilz, H. et al.\",\n \"(1975) J. Biochem.\",\n \"Vol.\",\n \"56(1), 103-108; Jany, K. D. and Mayer, B.\",\n \"(1985) Biol.\",\n \"Chem.\",\n \"Hoppe-Seyler, Vol.\",\n \"366(5), 485-492).\",\n \"The above-mentioned properties make proteinase K of particular interest for biotechnological applications in which an unspecific protein degradation is required.\",\n \"Special examples are nucleic acid isolation (DNA or RNA) from crude extracts and sample preparation in DNA analysis (Goldenberger, D. et al.\",\n \"(1995) PCR Methods Appl.\",\n \"Vol.\",\n \"4(6), 368-370; U.S. Pat.\",\n \"No.\",\n \"5,187,083; U.S. Pat.\",\n \"No.\",\n \"5,346,999).\",\n \"Other applications are in the field of protein analysis such as structure elucidation.\",\n \"Proteinase K is obtained commercially in large amounts by fermentation of the fungus Tritirachium album Limber (e.g.\",\n \"CBS 348.55, Merck strain No.\",\n \"2429 or the strain ATCC 22563).\",\n \"The production of proteinase K is suppressed by glucose or free amino acids.\",\n \"Since protein-containing media also induce the expression of proteases, it is necessary to use proteins such as BSA, milk powder or soybean flour as the only nitrogen source.\",\n \"The secretion of the protease starts as soon as the stationary phase of growth is reached (Ebeling, W. et al.\",\n \"(1974) Eur.\",\n \"J. Biochem.\",\n \"Vol.\",\n \"47(1), 91-97).\",\n \"Since Tritirachium album Limber is consequently unsuitable for fermentation on a large scale and moreover is difficult to genetically manipulate, various attempts have been made to overexpress recombinant proteinase K in other host cells.\",\n \"However, no significant activity was detected in these experiments due to lack of expression, formation of inactive inclusion bodies or problems with the naturation (Gunkel, F. A. and Gassen, H. G. (1989) Eur.\",\n \"J. Biochem.\",\n \"Vol.\",\n \"179(1), 185-194; Samal, B.\",\n \"B. et al.\",\n \"(1996) Adv.\",\n \"Exp.\",\n \"Med.\",\n \"Biol.\",\n \"Vol.\",\n \"379, 95-104).\",\n \"Moreover, Tritirachium album Limber is a slowly growing fungus which only secretes small amounts of proteases into the medium.\",\n \"The long fermentation period of one to two weeks is disadvantageous.\",\n \"In addition it is known that T. album also produces other proteases apart from proteinase K which can contaminate the preparation (Samal, B.\",\n \"B. et al.\",\n \"(1991) Enzyme Microb.\",\n \"Technol.\",\n \"Vol.\",\n \"13, 66-70).\",\n \"The object of the present invention is to provide a method for the economical production of recombinant proteinase K and of inactive zymogenic precursors of proteinase K that can be autocatalytically activated.\",\n \"The object was achieved by providing a method for producing recombinant proteinase K in which the inactive zymogenic proform of proteinase K is produced in an insoluble form in inclusion bodies, and the zymogenic proform of proteinase K is natured and the zymogenic proform processed i.e.\",\n \"activated in subsequent steps.\",\n \"The methods for the naturation and activation of proteinase K are also a subject matter of the present invention.\",\n \"The present invention concerns a method for producing recombinant proteinase K characterized in that the zymogenic proform is folded by in vitro naturation and is converted by autocatalytic cleavage into the active form.\",\n \"The present invention concerns in particular a method for producing a recombinant proteinase K in which a zymogenic precursor of proteinase K is converted by oxidative folding from isolated and solubilized inclusion bodies into the native structure i.e.\",\n \"it is natured and subsequently the active proteinase K is obtained from the natively folded zymogen by autocatalytic cleavage by adding detergents.\",\n \"Hence the present invention concerns a method for obtaining recombinant proteinase K by transforming a host cell with a DNA coding for the zymogenic proform of proteinase K characterized by the following process steps: a) Culturing the said host cell under conditions which result in an expression of the DNA coding for the zymogenic proform of proteinase K such that a zymogenic precursor of proteinase K is formed in the host cell in the form of insoluble inclusion bodies.\",\n \"b) Isolating the inclusion bodies, solubilizing the enzyme and naturing of the zymogenic precursor of proteinase K under conditions in which the protease part of the zymogenic precursor of proteinase K is formed.\",\n \"c) Activating the proteinase K by removing the propeptide and further purification.\",\n \"The DNA coding for the zymogenic proform of proteinase K corresponds to the DNA shown in SEQ ID NO: 2 or a DNA corresponding thereto within the scope of the degeneracy of the genetic code.\",\n \"SEQ ID NO: 2 includes the DNA sequence which codes for proteinase K and the propeptide.\",\n \"Furthermore the DNA can be codon-optimized for expression in a particular host.\",\n \"Method for codon-optimization are known to a person skilled in the art and are described in example 1.Hence the present invention concerns methods in which the host cell is transformed by a DNA which is selected from the above-mentioned group.\",\n \"A proteinase K is obtained by the method according to the invention which is homogeneous and hence particularly suitable for analytical and diagnostic applications.\",\n \"The zymogenic proform of proteinase K according to the invention can optionally contain additional N-terminal modifications and in particular sequences which facilitate purification of the target protein (affinity tags), sequences which increase the efficiency of translation, sequences which increase the folding efficiency or sequences which result in a secretion of the target protein into the culture medium (natural presequence and other signal peptides).\",\n \"Proteinase K in the sense of the invention means the sequence according to Gassen et al.\",\n \"(1989) shown in SEQ ID NO: 1 as well as other variants of proteinase K from Tritirachium album Limber like the amino acid sequence disclosed by Ch.\",\n \"Betzel et al.\",\n \"(Biochemistry 40 (2001), 3080-3088) and in particular proteinase T (Samal, B.\",\n \"B. et al.\",\n \"(1989) Gene Vol.\",\n \"85(2), 329-333; Samal, B.\",\n \"B. et al.\",\n \"(1996) Adv.\",\n \"Exp.\",\n \"Med.\",\n \"Biol.\",\n \"Vol.\",\n \"379, 95-104) and proteinase R (Samal, B.\",\n \"B. et al.\",\n \"(1990) Mol.\",\n \"Microbiol.\",\n \"Vol.\",\n \"4(10), 1789-1792; U.S. Pat.\",\n \"No.\",\n \"5,278,062) and in addition variants produced by recombinant means (as described for example in WO 96/28556).\",\n \"The sequence shown in SEQ ID NO: 1 comprises the signal sequence (1-15, 15 amino acids), the prosequence (16-105; 90 amino acids) and the sequence of the mature proteinase K (106-384; 279 amino acids).\",\n \"The amino acid sequence described by Betzel et al.\",\n \"(Biochemistry 40 (2001), 3080-3088) has in particular aspartate instead of a serine residue at position 207 of the active protease.\",\n \"Pro-proteinase K in the sense of the invention means in particular a proteinase K whose N-terminus is linked to its prosequence.\",\n \"In the case of the closely related subtilisin E and other variants it is known that the prosequence has an important influence on the folding and formation of active protease (Ikemura, H. et al.\",\n \"(1987) Biol.\",\n \"Chem.\",\n \"Vol.\",\n \"262(16), 7859-7864).\",\n \"In particular it is presumed that the prosequence acts as an intramolecular chaperone (Inouye, M. (1991) Enzyme Vol.\",\n \"45, 314-321).\",\n \"After the folding it is processed to form the mature subtilisin protease by autocatalytically cleaving the propeptide (Ikemura, H. and Inouye, M. (1988) J. Biol.\",\n \"Chem.\",\n \"Vol.\",\n \"263(26), 12959-12963).\",\n \"This process occurs in the case of subtilisin E (Samal, B.\",\n \"B. et al.\",\n \"(1989) Gene vol.\",\n \"85(2), 329-333; Volkov, A. and Jordan, F. (1996) J. Mol.\",\n \"Biol.\",\n \"Vol.\",\n \"262, 595-599), subtilisin BPN′ (Eder, J. et al.\",\n \"(1993) Biochemistry Vol.\",\n \"32, 18-26), papain (Vernet, T. et al.\",\n \"(1991) J. Biol.\",\n \"Chem.\",\n \"Vol.\",\n \"266(32), 21451-21457) and thermolysin (Marie-Claire, C. (1998) J. Biol.\",\n \"Chem.\",\n \"Vol.\",\n \"273(10), 5697-5701).\",\n \"If added exogenously the propeptide can also act intermolecularly in trans as a chaperone on the folding of denatured mature subtilisin protease (Ohta, Y. et al.\",\n \"(1991) Mol.\",\n \"Microbiol.\",\n \"Vol.\",\n \"5(6), 1507-1510; Hu, Z. et al.\",\n \"(1996) J. Biol.\",\n \"Chem.\",\n \"Vol.\",\n \"271(7), 3375-3384).\",\n \"The propeptide binds to the active centre of subtilisin (Jain, S. C. et al.\",\n \"(1998) J. Mol.\",\n \"Biol.\",\n \"Vol.\",\n \"284, 137-144) and acts as a specific inhibitor (Kojima, S. et al.\",\n \"(1998) J. Mol.\",\n \"Biol.\",\n \"Vol.\",\n \"277, 1007-1013; Li, Y. et al.\",\n \"(1995) J. Biol.\",\n \"Chem.\",\n \"Vol.\",\n \"270, 25127-25132; Ohta, Y.\",\n \"(1991) Mol.\",\n \"Microbiol.\",\n \"Vol.\",\n \"5(6), 1507-1510).\",\n \"This effect is used in the sense of the invention in order to prevent autoproteolysis of proteolysis-sensitive folding intermediates by already folded, active proteinase K during the naturation.\",\n \"Only certain, usually hydrophobic core regions of the prosequence appear to be necessary for the chaperone function since mutations in wide areas have no influence on the activity (Kobayashi, T. and Inouye, M. (1992) J. Mol.\",\n \"Biol.\",\n \"Vol.\",\n \"226, 931-933).\",\n \"In addition it is known that propeptides can be exchanged between various subtilisin variants.\",\n \"Thus for example subtilisin BPN′ also recognizes the prosequence of subtilisin E (Hu, Z. et al.\",\n \"(1996) J. Biol.\",\n \"Chem.\",\n \"Vol.\",\n \"271(7), 3375-3384).\",\n \"Inclusion bodies are microscopically visible particles consisting of insoluble and inactive protein aggregates which are often formed in the cytoplasm of the host cell when heterologous proteins are overexpressed and they contain very pure target protein.\",\n \"Methods for producing and purifying such inclusion bodies are described for example in Creighton, T. E. (1978) Prog.\",\n \"Biophys.\",\n \"Mol.\",\n \"Biol.\",\n \"Vol.\",\n \"33(3), 231-297; Marston, F. A.\",\n \"(1986) Biochem.\",\n \"J. Vol.\",\n \"240(1), 1-12; Rudolph, R. (1997).\",\n \"Folding proteins in: Creighton, T. E.\",\n \"(ed.)\",\n \"Protein Function: A practical approach.\",\n \"Oxford University Press, 57-99; Fink, A. L. (1998) Fold.\",\n \"Des.\",\n \"Vol.\",\n \"3(1), R9-23; and EP 0 114 506.In order to isolate inclusion bodies the host cells are lysed after fermentation by conventional methods e.g.\",\n \"by ultrasound, high pressure dispersion or lysozyme.\",\n \"The lysis preferably takes place in an aqueous neutral to slightly acid buffer.\",\n \"The insoluble inclusion bodies can be separated and purified by various methods, preferably by centrifugation or filtration with several washing steps (Rudolph, R. (1997).\",\n \"Folding Proteins In: Creighton, T. E.\",\n \"(ed.)\",\n \"Protein Function: A practical Approach.\",\n \"Oxford University Press, 57-99).\",\n \"The inclusion bodies obtained in this manner are then solubilized in a known manner.\",\n \"Denaturing agents are advantageously used for this at a concentration which is suitable for dissolving the inclusion bodies, in particular guanidinium hydro-chloride and other guanidinium salts and/or urea.\",\n \"In order to completely monomerize the inclusion body proteins it is also advantageous to add reducing agents such as dithiothreitol (DTT), dithioerythritol (DTE) or 2-mercaptoethanol during the solubilization in order to break possible disulfide bridges by reduction.\",\n \"The invention also concerns a proteinase K in which the cysteines are not reduced but are derivatized in particular with GSSG to form mixed disulfides or thiocyanates (EP 0 393 725).\",\n \"Hence according to the invention the inclusion bodies are solubilized by denaturing agents and reducing agents.\",\n \"6-8 M guanidinium hydrochloride or 8-10 M urea are preferred as denaturing agents and 50-200 mM DTT (dithiothreitol) or DTE (dithioerythritol) are preferred as reducing agents.\",\n \"Hence the present invention concerns the prosequence according to SEQ ID NO: 1 of 90 amino acids in length (amino acids 16-105) as well as other variants which facilitate folding.\",\n \"It also concerns a propeptide which is added exogenously for the folding of mature proteinase K and has the functions described above.\",\n \"A further subject matter of the invention is a recombinant vector which contains one or more copies of the recombinant DNA defined above.\",\n \"The basic vector is advantageously a plasmid preferably containing a multi-copy origin of replication, but is also possible to use viral vectors.\",\n \"The choice of expression vector depends on the selected host cell.\",\n \"Methods are used to construct the expression vector and to transform the host cell with this vector that are familiar to a person skilled in the art and are described for example in Sambrook et al.\",\n \"(1989), Molecular Cloning (see below).\",\n \"A suitable vector for expression in E. coli is for example the pKKT5 expression vector or pKK177, pKK223, pUC, pET vectors (Novagen) as well as pQE vectors (Qiagen).\",\n \"The expression plasmid pKKT5 is formed from pKK177-3 (Kopetzki et al., 1989, Mol.\",\n \"Gen. Genet.\",\n \"216:149-155) by exchanging the tac promoter for the T5 promoter from pDS (Bujard et al., 1987, Methods Enzymol.\",\n \"155:416-433).\",\n \"The EcoRI restriction endonuclease cleavage site in the sequence of the T5 promoter was removed by two point mutations.\",\n \"In addition the coding DNA in the vector according to the invention is under the control of a preferably strong, regulatable promoter.\",\n \"A promoter that can be induced by IPTG is preferred such as the lac, lacUV5, tac or T5 promoter.\",\n \"The T5 promoter is especially preferred.\",\n \"A host cell in the sense of the invention means any host cell in which proteins can form as inclusion bodies.\",\n \"It is usually a microorganism e.g.\",\n \"prokaryotes.\",\n \"Prokaryotic cells are preferred and in particular Escherichia coli.\",\n \"Particular preference is given to the following strains: E. coli K12 strains JM83, JM105, UT5600, RR1Δ15, DH5α, C600, TG1, NM522, M15 or the E. coli B derivatives BL21, HB101, E. coli M15 is particularly preferred.\",\n \"The corresponding host cells are transformed according to the invention with a recombinant nucleic acid which encodes a recombinant zymogenic proteinase K according to SEQ ID NO:2 or with a nucleic acid which is derived from the said DNA by codon-optimization or with a DNA which is derived from the said DNA within the scope of the degeneracy of the genetic code.\",\n \"The E. coli host cells are preferably transformed with a codon-optimized recombinant nucleic acid coding for a recombinant zymogenic proteinase K which has been optimized for expression in Escherichia coli.\",\n \"Hence the present invention also concerns a suitable vector which is for example selected from the above-mentioned vectors and contains a recombinant nucleic acid that is codon-optimized for E. coli and codes for a recombinant proteinase K or a recombinant zymogenic proteinase K. Another subject matter of the invention is a host cell which is for example selected from the above-mentioned host cells which has been transformed by the above-mentioned vector.\",\n \"A further subject matter of the present invention is a method for the naturation of denatured zymogenic proteinase K in which the denatured zymogenic proteinase K is transferred to a folding buffer which is characterized in that the folding buffer has the following features: A) pH value of the buffer is in the range of 7.5 to 10.5 B) presence of low-molecular weight substances which aid folding C) presence of a redox shuffling system D) presence of a complexing agent at a substoichiometric concentration relative to the Ca2+ ions and wherein the method is carried out at a temperature between 0° C. and 37° C. A low concentration of denaturing agents is preferably present during the naturation.\",\n \"Denaturing agents may for example be present because they are still in the reaction solution due to the prior solubilization of the inclusion bodies.\",\n \"The concentration of denaturing agents such as guanidine hydrochloride should be less than 50 mM.\",\n \"Naturation in the sense of the invention is understood as a method in which denatured, essentially inactive protein is converted into a conformation in which the protein has the desired activity after autocatalytic cleavage and activation.\",\n \"This is achieved by transferring the solubilized inclusion bodies to a folding buffer while reducing the concentration of the denaturing agent.\",\n \"The conditions must be selected such that the protein remains in solution in this process.\",\n \"This can be expediently carried out by rapid dilution or dialysis against the folding buffer.\",\n \"It is preferred that the folding buffer has a pH of 8 to 9.Particularly preferred buffer substances are Tris/HCl buffer and bicine buffer.\",\n \"The naturation method according to the invention is preferably carried out at a temperature between 0° C. and 25° C. The low molecular weight folding agents in the folding buffer are preferably selected from the following group of low molecular weight compounds.\",\n \"They can be added alone as well as in mixtures, and other substances that aid folding may be present: L-arginine at a concentration of 0.5 to 2.0 M Tris at a concentration of 0.5 M to 2.0 M triethanolamine at a concentration of 0.5 M to 2.0 M α-cyclodextrin at a concentration of 60 mM to 120 mM Low molecular weight substances that aid folding are described for example in U.S. Pat.\",\n \"No.\",\n \"5,593,865; Rudolph, R. (1997) Folding Proteins.\",\n \"In: Creighton, T. E.\",\n \"(ed.)\",\n \"Protein Function: A practical Approach.\",\n \"Oxford University Press, 57-99 or De Bernardez Clark, E. et al.\",\n \"(1999) Methods.\",\n \"Enzymol.\",\n \"Vol.\",\n \"309,217-236.The above-mentioned redox shuffling system is preferably a mixed disulfide or thiosulfonate.\",\n \"Systems are for example suitable as a redox shuffling system which consist of a thiol component in an oxidized and reduced form.\",\n \"This allows the formation of disulfide bridges within the folding polypeptide chain during naturation by controlling the reduction potential, and on the other hand, enables the reshuffling of incorrect disulfide bridges within or between the folding polypeptide chains (Rudolph, R. (1997), see above).\",\n \"Preferred thiol components are for example: glutathione in a reduced (GSH) and oxidized form (GSSH) cysteine and cystine cysteamine and cystamine 2-mercaptoethanol and 2-hydroxyethyldisulfide In the naturation method according to the invention the Ca2+ ions are preferably present at a concentration of 1 to 20 mM.\",\n \"For example CaCl2 can be added in amounts of 1 to 20 mM.\",\n \"The Ca2+ ions can bind to the calcium binding sites of the folding proteinase K. The presence of a complexing agent preferably EDTA, in a substoichiometric concentration relative to Ca2+ prevents the oxidation of the reducing agent by atmospheric oxygen and protects free SH groups.\",\n \"The naturation is preferably carried out at a low temperature i.e.\",\n \"below 20° C., preferably 10° C. to 20° C. In the method according to the invention the naturation is usually completed after a period of about 24 h to 48 h. The present invention also concerns a folding buffer which is characterized by the following features: A) pH value of the buffer is in the range of 7.5 to 10.5 B) presence of low-molecular weight substances which aid folding C) presence of a redox shuffling system D) presence of a complexing agent at a substoichiometric concentration relative to the Ca2+ ions.\",\n \"It is especially preferred when the folding buffer has a pH of 8 to 9 and/or when the redox shuffling system is a mixed disulfide or thiosulfonate.\",\n \"Another subject matter of the invention is a method for activating the natured zymogenic precursor of proteinase K. After the folding process according to the invention an inactive complex is formed from native proteinase K and the inhibitory propeptide.\",\n \"The active proteinase K can be released from this complex.\",\n \"Addition of detergents is preferred, SDS is particularly preferred at a concentration of 0.1 to 2% (w/v).\",\n \"The advantages of the method disclosed here for producing recombinant proteinase K are: 1.The ability to utilize the high expression potential and the rapid and simple culture of Escherichia coli or other suitable microorganisms.\",\n \"2.The possibility to genetically manipulate the recombinant DNA.\",\n \"3.The uncomplicated purification after naturation.\",\n \"4.The absence of eukaryotic impurities when a prokaryote is selected as a host cell.\",\n \"A method would also be conceivable in which the nucleic acids which code for mature proteinase K and nucleic acids which code for the propeptide or pro-proteinase K are expressed separately in host cells and are then commonly transferred to a folding buffer for the naturation of mature proteinase K. DESCRIPTION OF THE FIGURES FIG.\",\n \"1: Schematic representation of the PCR reaction to produce proteinase K fragments having an N-terminal BamHI cleavage site and an alternative enterokinase cleavage site for fusion with an N-terminal affinity tag.\",\n \"FIG.\",\n \"2: Dependency of the yield of naturation on temperature.\",\n \"FIG.\",\n \"3: Dependency of the yield of naturation on pH.\",\n \"FIG.\",\n \"4: Dependency of the yield of naturation on redox potential.\",\n \"FIG.\",\n \"5: Dependency of the yield of naturation on the arginine concentration.\",\n \"FIG.\",\n \"6: Dependency of the yield of naturation on the Tris concentration.\",\n \"FIG.\",\n \"7: Dependency of the yield of naturation on the α-cyclodextrin concentration.\",\n \"FIG.\",\n \"8: Dependency of the yield of naturation on the triethanolamine concentration.\",\n \"FIG.\",\n \"9: Dependency of the yield of naturation on the urea concentration.\",\n \"FIG.\",\n \"10: SDS polyacrylamide gel of the naturation of pro-proteinase K. FIG.\",\n \"11: Reverse phase chromatography of natured proteinase K. FIG.\",\n \"12: Renatured and processed proteinase K was analysed by analytical ultracentrifugation.\",\n \"The centrifugation was carried out at 12000 rpm, 20° C. for 63 h. The data (o) could be fitted to a homogeneous species having an apparent molecular weight of 29-490 Da.\",\n \"No systematic deviation was observed between the fitted and measured data (lower graph).\",\n \"FIG.\",\n \"13: Determination of the Km value of natured proteinase K. FIG.\",\n \"14: Degradation pattern of blood serum proteins by natured proteinase K. FIG.\",\n \"15: Purification of natured proteinase K by gel filtration.\",\n \"EXAMPLE 1 Synthesis of the Gene which Codes for the Mature Form of Proteinase K. The gene for the mature proteinase K from Tritirachium album Limber without a signal sequence and without an intron was generated by means of gene synthesis.\",\n \"The sequence of Gunkel, F. A. and Gassen, H. G. (1989) Eur.\",\n \"J. Biochem.\",\n \"Vol.\",\n \"179(1), 185-194 of 837 bp in length (amino acids 106-384 from Swiss Prot P06873) was used as the template.\",\n \"A codon usage optimized for Escherichia coli was used as the basis for retranslating the amino acid sequence to optimize the expression (Andersson, S. G. E. and Kurland, C. G. (1990) Microbiol.\",\n \"Rev.\",\n \"Vol.\",\n \"54(2), 198-210, Kane, J. F. Curr.\",\n \"Opin.\",\n \"Biotechnol., Vol.\",\n \"6, pp.\",\n \"494-500).\",\n \"The amino acid sequence is shown in SEQ ID NO: 1 and the nucleotide sequence is shown in SEQ ID NO: 2.For the gene synthesis the gene was divided into 18 fragments of sense and reverse, complementary counterstrand oligonucleotides in alternating sequence (SEQ ID NO:3-20).\",\n \"An at least 15 bp region was attached to the 5′ end and to the 3′ end which in each case overlapped the neighbouring oligonucleotides.\",\n \"Recognition sites for restriction endonucleases were attached to the 5′ and 3′ ends of the synthetic gene outside the coding region for subsequent cloning into expression vectors.\",\n \"The oligonucleotide shown in SEQ ID NO:3 which contains an EcoRI cleavage site was used as a 5′ primer for cloning the pro-protein X gene without an N-terminal affinity tag.\",\n \"SEQ ID NO: 20 shows the 3′ primer containing a HindIII cleavage site.\",\n \"The 3′ primer contains an additional stop codon after the natural stop codon to ensure termination of the translation.\",\n \"The oligonucleotide with a BamHI cleavage site shown in SEQ ID NO: 23 or the oligonucleotide with a BamHI cleavage site and enterokinase cleavage site shown in SEQ ID NO: 24 was used as a 5′ primer to clone the proprotein X gene with N-terminal affinity tags and an alternative enterokinase cleavage site as described in example 3.The oligonucleotides were linked together by means of a PCR reaction and the resulting gene was amplified.\",\n \"For this the gene was firstly divided into three fragments of 6 oligonucleotides each and the three fragments were linked together in a second PCR cycle.\",\n \"Fragment 1 is composed of the oligonucleotides shown in SEQ ID NO: 3-8, fragment 2 is composed of the oligonucleotides shown in SEQ ID NO: 9-14 and fragment 3 is composed of the oligonucleotides shown in SEQ ID NO: 15-20.The following PCR parameters were employed PCR reaction 1 (generation of three fragments) 5 min 95° C. hot start 2 min 95° C. 2 min 42° C. 1.5 min 72° C. {close oversize brace} 30 cycles 7 min 72° C. final extension PCR reaction 2 (linkage of the fragments to form the total gene) 5 min 95° C. hot start 1.5 min 95° C. 2 min 48° C. {close oversize brace} 6 cycles (without terminal primers) 2 min 72° C. addition of terminal primers 1.5 min 95° C. 1.5 min 60° C. {close oversize brace} 25 cycles (with terminal primers) 2 min 72° C. 7 min 72° C. final extension EXAMPLE 2 Cloning of the Synthetic Proteinase K Fragment from the Gene Synthesis The PCR mixture was applied to an agarose gel and the ca.\",\n \"1130 bp PCR fragment was isolated from the agarose gel (Geneclean II Kit from Bio 101, Inc. CA USA).\",\n \"The fragment was cleaved for 1 hour at 37° C. with EcoRI and HindIII restriction endonucleases (Roche Diagnostics GmbH, Germany).\",\n \"At the same time the pUC18 plasmid (Roche Diagnostics GmbH, Germany) was cleaved for 1 hour at 37° C. with EcoRI and HindIII restriction endonucleases, the mixture was separated by agarose gel electrophoresis and the 2635 bp vector fragment was isolated.\",\n \"Subsequently the PCR fragment and the vector fragment were ligated together using T4 DNA ligase.\",\n \"For this 1 μl (20 ng) vector fragment and 3 μl (100 ng) PCR fragment, 1 μl 10× ligase buffer (Maniatis, T., Fritsch, E. F. and Sambrook, T. (1989).\",\n \"Molecular Cloning: A laboratory manual.\",\n \"2nd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y.), 1 μl T4 DNA ligase, 4 μl sterile redistilled H2O were pipetted, carefully mixed and incubated overnight at 16° C. The cloned gene was examined by restriction analysis and by multiple sequencing of both strands.\",\n \"The sequence is shown in SEQ ID NO: 2.a) Construction of the pPK-1 Expression Plasmid In order to express proteinase K, the structural gene was cloned into the pKKT5 expression vector in such a manner that the structural gene is inserted in the correct orientation under the control of a suitable promoter, preferably a promoter that can be induced by IPTG such as the lac, lacUV5, tac or T5 promoter, particularly preferably the T5 promoter.\",\n \"For this purpose the structural gene for proteinase K was cleaved from the plasmid pUC18 by EcoRI and HindIII, the restriction mixture was separated by agarose gel electrophoresis and the ca.\",\n \"1130 bp fragment was isolated from the agarose gel.\",\n \"At the same time the expression plasmid pKKT5 was cleaved with EcoRI and HindIII, the restriction mixture was separated by agarose gel electrophoresis and the ca.\",\n \"2.5 kbp vector fragment was isolated from the agarose gel.\",\n \"The fragments obtained in this manner were ligated together as described above.\",\n \"The correct insertion of the gene was checked by sequencing.\",\n \"b) Transformation of the Expression Plasmid pPK-1 in Various E. coli Expression Strains The expression vector was transformed in various expression strains that had been previously transformed with the plasmid pREP4 and/or pUBS520.The plasmid pREP4 contains a gene for the lacI repressor that should ensure a complete suppression of the expression before induction.\",\n \"The plasmid pUBS520 (Brinkmann, U. et al.\",\n \"(1989) Gene Vol.\",\n \"85(1), 109-114) also contains the lacI repressor and additionally the dnaY gene which codes for the tRNA which is necessary to translate the rare arginine codons AGA and AGG in E. coli.\",\n \"Competent cells of various E. coli strains were prepared according to the method of Hanahan, D. (1983) J. Mol.\",\n \"Biol.\",\n \"Vol.\",\n \"166, 557-580.100 μl cells prepared in this manner was admixed with 20 ng isolated pPK-1 plasmid DNA.\",\n \"After 30 min incubation on ice, they were heat-shocked (90 sec at 42° C.) and then incubated for 2 min on ice.\",\n \"Subsequently the cells were transferred to 1 ml SOC medium and incubated for 1 hour at 37° C. while shaking for the phenotypic expression.\",\n \"Aliquots of this transformation mixture were plated out on LB plates containing ampicillin as a selection marker and incubated for 15 hours at 37° C. Preferred strains are E. coli K12-strains JM83, JM105, UT5600, RR1Δ15, DH5α, C600, TG1, NM522, M15 or the E. coli B derivatives BL21, HB101; E. coli M15 is particularly preferred.\",\n \"EXAMPLE 3 Cloning of an N-Terminal Affinity Tag In order to insert an N-terminal affinity tag, a BamHI cleavage site was inserted before the 5′ end of the gene for pro-proteinase K. This was achieved by PCR using the product obtained in example 1 as a template and the oligonucleotides described in SEQ ID NO:20, 23 and 24 as primers.\",\n \"The primer described in SEQ ID NO:23 contains a BamHI cleavage site upstream of the 5′ region of pro-proteinase K, the primer described in SEQ ID NO:24 additionally contains an enterokinase cleavage site directly in front of the first codon of the prosequence.\",\n \"SEQ ID NO:20 shows the 3′ primer that was also used in example 1 with a HindIII cleavage site.\",\n \"The resulting PCR products were isolated as described above, digested with BamHI and HindIII and purified by agarose electrophoresis.\",\n \"The affinity tag was inserted by means of a synthetic linker composed of two complementary oligonucleotides in such a manner that an EcoRI cleavage site was formed at the 5′ end and a BamHI cleavage site was formed at the 3′ end without further restriction digestion.\",\n \"For a His tag the sense strand had the sequence shown in SEQ ID NO:21 and the antisense strand had the sequence shown in SEQ ID NO.22.The linker coded for a hexa-His tag with an N-terminal RGS motif.\",\n \"The BamHI cleavage site between the linker and pro-proteinase K is translated into a Gly-Ser linker.\",\n \"In order to anneal the linker, the two oligonucleotides (SEQ ID NO:21 and 22) were heated for 5 min to 95° C. in equimolar amounts (50 pmol/μl each) and subsequently cooled at 2° C. per min to room temperature.\",\n \"As a result the annealing of the complementary oligonucleotides should be as complete as possible.\",\n \"The linker was ligated with the BamHI/HindIII-digested PCR product.\",\n \"(Rapid Ligation Kit from Roche Diagnostics GmbH, Germany) and purified by agarose gel electrophoresis (QIAquick gel extraction Kit from Qiagen, Germany).\",\n \"The resulting ligation product was ligated into an expression vector analogously to example 2b via the EcoRI and HindIII overhangs and transformed correspondingly in expression strains.\",\n \"This module system enables various affinity tags that are coded by the synthetic linker to be fused to the structural gene for pro-proteinase K. An enterokinase cleavage site can be alternatively inserted between the tag and propeptide by suitable selection of the corresponding 5′ primer if a subsequent removal of the tag is desired.\",\n \"Furthermore a certain region of the proteinase K gene such as the mature proteinase K or the propeptide can be amplified by suitable selection of the overlapping regions of the PCR primers (FIG.\",\n \"1).\",\n \"EXAMPLE 4 Expression of Proteinase K in Escherichia coli Since proteinase K is a very active unspecific protease, it is preferable to express it in an inactive form preferably as inclusion bodies.\",\n \"In order to express the gene which codes for proteinase K, 3 ml Lbamp medium was inoculated with plasmid-containing clones and incubated at 37° C. in a shaker.\",\n \"LB medium: 10 g tryptone 10 yeast extract 5 g NaCl make up to a final volume of 1 l with distilled H2O, adjust to pH 7.0 with NaOH addition of antibiotics (100 μg/ml ampicillin) directly before inoculation The cells were induced with 1 mM IPTG at an optical density of 0.5 at 550 nm and incubated for 4 h at 37° C. in a shaker.\",\n \"Subsequently the optical density of the individual expression clones was determined, an aliquot corresponding to an OD550 of 3/ml was removed and the cells were centrifuged (10 min 6000 rpm, 4° C.).\",\n \"The cells were resuspended in 400 μl TE buffer, lysed by ultrasound and the soluble protein fraction was separated from the insoluble protein fraction by centrifugation (10 min, 14,000 rpm, 4° C.).\",\n \"TE buffer: 50 mM Tris/HCl 50 mM EDTA pH 8.0 (at RT) Application buffer containing SDS and β-mercaptoethanol was added to all fractions and the proteins were denatured by heating (5 min 95° C.).\",\n \"Subsequently 10 μl aliquots were analysed by means of a 12.5% analytical SDS gel (Laemmli, U.K. (1970) Nature Vol.\",\n \"227(259), 680-685).\",\n \"A very strong expression in the form of insoluble protein aggregates (inclusion bodies) was observed for the clones of mature proteinase K as well as for the clones of pro-proteinase K. Accordingly no proteinase K activity was measured.\",\n \"EXAMPLE 5 Isolation of the Inclusion Bodies The inclusion bodies were prepared by known methods (Rudolph, R. (1997) see above).\",\n \"For the cell lysis, 10 g wet biomass was in each case resuspended in 50 ml 100 mM Tris/HCl pH 7.0, 1 mM EDTA.\",\n \"Afterwards 15 mg lysozyme was added, incubated for 60 min at 4° C. and the cells were subsequently lysed by high pressure (Gaulin cell lysis apparatus).\",\n \"The DNA was digested for 30 min at RT by adding 3 mM MgCl2 and 10 μg/ml DNase to the crude extract.\",\n \"The insoluble cell components which contain the inclusion bodies were separated by centrifugation (30 min 20,000 g) and washed once with washing buffer 1 and three times with washing buffer 2.washing buffer 1: 100 mM Tris/HCl 20 mM EDTA 2% (v/v) Triton X-100 0.5 M NaCl pH 7.0 (RT) washing buffer 2: 100 mM Tris/HCl 1 mM EDTA pH 7.0 (RT) The pellet of the last washing step constitutes the crude inclusion bodies which already contain highly pure target protein.\",\n \"EXAMPLE 6 Solubilization of Inclusion Bodies a) Solubilization While Reducing with Cysteines 1 g crude inclusion bodies was suspended in 10 ml solubilization buffer and incubated for 2 h at RT while stirring gently.\",\n \"Solubilization buffer: 100 mM Tris/HCl 6.0 M guanidinium hydrochloride 100 mM DTT pH 8.0 (RT) The solubilisate was titrated to pH 3 with 25% HCl and dialysed twice for 4 h at RT against 500 ml 6 M guanidine hydrochloride pH 3 and then overnight at 4° C. against 1000 ml guanidine hydrochloride pH 7.The protein concentration was determined by the Bradford method (Bradford, 1976) using a calculated extinction coefficient at 280 nm and was between 10 and 20 mg/ml.\",\n \"The number of free cysteines was determined according to the Ellman method.\",\n \"In accordance with the sequence 5 mol free cysteines per mol proteinase K were found.\",\n \"The purity of the solubilized inclusion bodies was determined by 12.5% SDS PAGE and quantification of the bands after Coomassie staining.\",\n \"b) Solubilization with Derivatization of the Cysteines to Form Mixed Disulfides Using Glutathione.\",\n \"1 g Crude Inclusion Bodies Were Suspended in 10 ml Solubilization Buffer.\",\n \"Solubilization buffer: 100 mM Tris/HCl 6.0 M guanidine hydrochloride 1 mM DTT pH 8.0 (RT) After 15 min incubation at RT while stirring gently, during which a catalytic amount of reduced cysteines was formed due to the small amounts of DTT, 100 mM GSSG was added, the pH was adjusted to 8.0 and it was incubated for a further 2 h at RT while stirring gently.\",\n \"Further treatment as described under a).\",\n \"EXAMPLE 7 Optimization of the Naturation of Pro-Proteinase K Various parameters were varied in order to optimize the yield in the folding and processing of pro-proteinase K from the solubilisates prepared in example 6a).\",\n \"For all preparations the stated folding buffer was filtered, degassed, gassed with N2 and incubated at the desired temperature.\",\n \"The redox shuffling system was not added until shortly before the start of the folding reaction and the pH was readjusted.\",\n \"The folding was initiated by adding the solubilized inclusion bodies while rapidly mixing.\",\n \"The volume of the folding mixtures was 1.8 ml in 2 ml glass tubes with a screw cap.\",\n \"The yield was analysed by an activity test using the chromogenic substrate Suc-Ala-Ala-Pro-Phe-pNA from the Bachem Company (Heidelberg).\",\n \"100 mM Tris/HCl, 5 mM CaCl2, pH 8.5 at 25° C. was used as the test buffer.\",\n \"The concentration of the peptide in the test was 2 mM from a 200 mM stock solution in DMSO.\",\n \"In order to activate the renaturate, 0.1% SDS was added to the sample (see example 8).\",\n \"The absorbance at 410 nm was measured over a period of 20 min and the activity was calculated from the slope.\",\n \"The following parameters were varied: a) Temperature and Time The folding buffer containing 100 mM Tris, 1.0 mM L-arginine, 10 mM CaCl2 was equilibrated at various temperatures.\",\n \"After adding 3 mM GSH and 1 mM GSSG the pH was readjusted at the corresponding temperature.\",\n \"The reaction was started by adding 50 μg/ml pro-proteinase K. After 12 h, 36 h and 60 h, aliquots were removed and tested for activity.\",\n \"The results are shown in FIG.\",\n \"2.b) pH Value A universal buffer containing 50 mM citrate, 50 mM MES, 50 mM bicine, 500 mM arginine, 2 mM CaCl2 and 1 mM EDTA was incubated at 15° C. and 3 mM GSH and 1 mM GSSG were added.\",\n \"The pH was readjusted in a range between pH 4.0 and pH 12.0 and the folding reaction was started by adding 50 μg/ml pro-proteinase K inclusion bodies.\",\n \"The activity measured after 18 h, 3 d and 5 d is shown in FIG.\",\n \"3.c) Redox Potential Various redox potentials were set in a renaturation buffer containing 1.0 M L-arginine, 100 mM bicine, 2 mM CaCl2 and 10 mM CaCl2 by mixing various ratios of oxidized and reducing glutathione.\",\n \"The protein concentration in the folding mixture was 50 μg/ml.\",\n \"The folding was carried out at 15° C. The concentrations of GSH and GSSG are shown in table 1, the measurements are shown in FIG.\",\n \"4.TABLE 1 concentrations of GSH and GSSG at the various redox potentials.\",\n \"Redox potential (log(cGSH2/cGSSG) [M] c(GSH) [mM] c(GSSG) [mM] −00 0 2.500 −6.000 0.050 2.475 −5.500 0.088 2.456 −5.000 0.156 2.422 −4.500 0.273 2.363 −4.000 0.476 2.262 −3.500 0.814 2.093 −3.000 1.351 1.825 −2.500 2.130 1.435 −2.000 3.090 0.955 −1.500 3.992 0.504 −1.000 4.580 0.210 −0.500 4.851 0.074 0.000 4.951 0.025 +0.500 4.984 7.856e−3 +1.000 4.995 2.495e−3 +00 5.000 0 d) Solvent Additives that Promote Folding Various substances were examined for their ability to increase the folding yield of proteinase K. For this purpose solutions containing the substances at various concentrations were prepared and admixed with 2 mM CaCl2, 1 mM EDTA and 100 mM bicine.\",\n \"The pH was adjusted to pH 8.75 at the folding temperature of 15° C. The protein concentration was 50 μg/ml.\",\n \"FIG.\",\n \"5 shows the relative yields of active proteinase K in relation to the concentration of the selected buffer additive.\",\n \"EXAMPLE 8 Activation of the Natured Pro-Proteinase K After naturation of pro-proteinase K by the method according to the invention it was found to have no activity or only a very slight activity.\",\n \"Chromatographic methods and SDS-PAGE showed that mature proteinase K is already present but is still associated in a complex with the propeptide.\",\n \"This can be separated in a method which is referred to here as activation and is also a subject matter of the invention.\",\n \"In this example SDS is added at a concentration of 2% (v/v) to the folding mixture and subsequently the folding additive and the SDS are removed by dialysis.\",\n \"Alternatively SDS could also be added after removing the additives by dialysis.\",\n \"In all cases full activity of proteinase K was detected.\",\n \"EXAMPLE 9 Characterization of the Folding Product The proteinase K natured and activated by the method according to the invention was further characterized by various methods.\",\n \"a) Analysis of Purity and Molecular Weight Determination by SDS Polyacrylamide Gel Electrophoresis Aliquots from various steps in the maturation process and the final product, the folded and activated recombinant proteinase K were applied to a 12.5% SDS polyacrylamide gel.\",\n \"The samples each contained 10 mM DTT or 1% (v/v) 2-mercaptoethanol.\",\n \"The recombinant proteinase K prepared by the method according to the invention had no significant contamination and runs identically with the authentic proteinase K at an apparent molecular weight of ca.\",\n \"30 kDa (see FIG.\",\n \"11).\",\n \"b) Analysis of Purity Using RP-HPLC The folded and activated proteinase K and the authentic proteinase K from T. album and the pro-proteinase K inclusion bodies were analysed by means of reversed phase HPLC.\",\n \"A Vydac C4 column having the dimensions 15 cm×4.6 cm diameter was used.\",\n \"The samples were eluted with an acetonitrile gradient of 0% to 80% in 0.1% TFA.\",\n \"The folding product exhibits mobility properties that are identical to the authentic proteinase K used as a standard (see FIG.\",\n \"12).\",\n \"c) Analytical Ultracentrifugation In order to analyse whether the renatured and processed proteinase K is present in a monomeric form without propeptide, the protein was examined by means of analytical ultracentrifugation.\",\n \"The molecular weight was determined to be 29490 Da and corresponds to the mass of the monomeric mature proteinase K within the limits of error of this method (see FIG.\",\n \"13).\",\n \"Hence this showed that the propeptide was quantitatively cleaved by activation of the proteinase K. d) N-Terminal Sequence Analysis In order to examine whether the propeptide was cleaved at the correct cleavage site the natured and activated recombinant proteinase K was subjected to a sequence analysis.\",\n \"For this the folding product was desalted by RP-HPLC as described in example 9b) and the first 6 residues were examined by N-terminal sequencing.\",\n \"The result (AAQTNA) agrees with the authentic N-terminus of mature proteinase K. e) Activity and Km Value the Km value of the folded and activated proteinase K was compared with that of the authentic proteinase K. The tetrapeptide Suc-Ala-Ala-Pro-Phe-pNA was used as a substrate.\",\n \"The test was carried out in 2.0 ml 50 mM Tris, pH 8.5 containing 1 mM CaCl2 at 25° C. The hydrolysis of the peptide was monitored spectroscopically at 410 nm.\",\n \"A Km value of 0.16 mM was found for the recombinant proteinase K which corresponded very well with the Km value of authentic proteinase K (see FIG.\",\n \"14).\",\n \"f) Degradation Pattern of Blood Serum Proteins In an additional test to characterize the activity, the cleavage pattern of blood serum proteins was examined.\",\n \"For this a defined amount of blood serum proteins was digested with 1 μg recombinant proteinase K or the same amount of authentic proteinase K. The cleavage pattern was analysed by means of RP-HPLC under identical conditions as described in example 9b).\",\n \"FIG.\",\n \"15 shows that the recombinant and the authentic proteinase K result in an identical degradation pattern.\",\n \"EXAMPLE 10 Purification of the Folding Product The recombinant pro-proteinase K natured by the method according to the invention was purified by gel filtration.\",\n \"As described in FIG.\",\n \"11 the concentrated naturation solution was separated on a Superdex 75 pg after naturation in a first run without prior activation and in a second run with prior activation using 0.15% (w/v) SDS (30 min, 4° C.).\",\n \"100 mM Tris/HCl, 150 mM NaCl pH 8.75 (4° C.) was used as the mobile buffer.\",\n \"The application volume was 10 ml at a column volume of 1200 ml and a flow rate of 5 ml/min.\",\n \"After completion of the application, 14 ml fractions were collected.\",\n \"Aliquots of the fractions were precipitated with trichloroacetic acid, washed and taken up in Laemmli sample buffer containing 10 mM DTT.\",\n \"The samples were applied to a 12.5% SDS polyacrylamide gel which was stained after the run with Coomassie blue R250.In the first run without activation a non-processed recombinant pro-proteinase K is seen in a first peak which probably runs in the form of microaggregates in the exclusion volume.\",\n \"In a second peak one observes processed recombinant proteinase K which co-elutes with the propeptide which is non-covalently bound and acts as an inhibitor.\",\n \"As a result no activity is found without prior activation.\",\n \"Only after adding SDS to the fractions did the second peak exhibit significant proteinase K activity (not shown).\",\n \"The second run in which the folded recombinant proteinase K was previously activated with SDS only shows one peak which elutes after an identical volume like proteinase K under the same conditions (not shown).\",\n \"On the SDS gel one sees clean mature recombinant proteinase K without propeptide in this peak.\",\n \"All impurities and the propeptide appear to have already been digested in the applied mixture by the activated recombinant proteinase K. As expected the fractions of the proteinase K peak exhibited activity without further activation with SDS.\",\n \"The recombinant proteinase K purified in this manner appears to be almost 100% pure on the SDS gel and shows an identical migration behaviour to the authentic proteinase K (FIG.\",\n \"16).\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10467532"}}},{"rowIdx":373,"cells":{"text":{"kind":"list like","value":[["Antibody complexes and methods for immunolabeling","The present invention provides labeling reagents and methods for labeling primary antibodies and for detecting a target in a sample using an immuno-labeled complex that comprises a target-binding antibody and one or more labeling reagents.","The labeling reagents comprise monovalent antibody fragments or non-antibody monomeric proteins whereby the labeling proteins have affinity for a specific region of the target-binding antibody and are covalently attached to a label.","Typically, the labeling reagent is an anti-Fc Fab or Fab′ fragment that was generated by immunizing a goat or rabbit with the Fc fragment of an antibody.","The present invention provides for discrete subsets of labeling reagent and immuno-labeled complexes that facilitate the simultaneous detection of multiple targets in a sample wherein the immuno-labeled complexes are distinguished by i) a ratio of label to labeling reagent, or ii) a physical property of said label, or iii) a ratio of labeling reagent to said target-binding antibody, or iv) by said target-binding antibody.","This is particularly useful for fluorophore labels that can be attached to labeling reagents and subsequently immuno-labeled complexes in ratios for the detection of multiple targets."],["1.A method of forming an immuno-labeled complex, wherein said method comprises the steps of: b) contacting a solution of target-binding antibodies with a discreet labeling reagent subset, wherein said labeling reagent subsets are distinguished by i) ratio of label to labeling reagent or ii) a physical properties of said label; b) incubating said target-binding antibodies and said labeling reagent subset for a time period sufficient for one or more labeling reagents to form an immuno-labeled complex with a target-binding antibody wherein a region of said target binding antibody is selectively bound by labeling reagent whereby a discreet immuno-labeled complex subset is formed; c) optionally removing unbound labeling reagent by adding a capture reagent comprising immunoglobulin proteins or fragments thereof; and, c) optionally repeating said steps a), b), and c) to form a panel of immuno-labeled complex subsets wherein each subset is distinguished from another subset by i) a ratio of label to labeling reagent, or ii) a physical property of said label, or iii) a ratio of labeling reagent to said target-binding antibody, or iv) by said target-binding antibody.","2.The method according to claim 1, wherein said target binding antibody is a murine monoclonal antibody, a rabbit polyclonal antibody or a goat polyclonal antibody.","3.The method according to claim 2, wherein said target-binding antibodies are in a solution comprising serum proteins or ascites proteins.","4.The method according to claim 1, wherein said labeling reagent is a Fab or Fab′fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is a non-antibody protein selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.","5.The method according to claim 4, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme and a radioisotope.","6.The method according to claim 5, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine, and derivatives thereof.","7.The method according to claim 5, wherein said fluorescent protein is a phycobiliprotein.","8.The method according to claim 5, wherein said tandem dye is selected from the group consisting of a cyanine-phycobiliprotein derivative and xanthene-phycobiliprotein derivative.","9.The method according to claim 5, wherein said enzyme is selected from the group consisting of a peroxidase, a phosphatase, a glycosidase, and a luciferase.","10.A method for detecting a target in a sample, wherein said method comprises the steps of: a) contacting said sample with an immuno-labeled complex comprising a target-binding antibody and a labeling reagent; b) incubating said sample of step a) for a time sufficient to permit said immuno-labeled complex to selectively bind to said target; and, c) illuminating said immuno-labeled complex whereby said target is detected.","11.The method according to claim 11, wherein said sample comprises a population of cells, cellular extract, subcellular component, proteins, peptides, tissue culture, tissue, a bodily fluid, or a portion or combination thereof.","12.The method according to claim 11, wherein said sample is immobilized on a solid or semi-solid matrix.","13.The method according to claim 12, wherein said matrix is a gel, a membrane, an array, a glass surface or a microparticle.","14.The method according to claim 10, wherein said labeling reagent is a Fab or Fab′fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is a non-antibody protein selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.","15.The method according to claim 14, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme and a radioisotope.","16.The method according to claim 15, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine, and derivatives thereof.","17.The method according to claim 15, wherein said fluorescent protein is a phycobiliprotein.","18.The method according to claim 15, wherein said tandem dye is selected from the group consisting of a cyanine-phycobiliprotein derivative and xanthene-phycobiliprotein derivative.","19.The method according to claim 15, wherein said enzyme selected from the group consisting of a peroxidase, a phosphatase, a glycosidase, and a luciferase.","20.The method according to claim 19, wherein said method further comprises adding a colorimetric, fluorescent or chemiluminescent enzyme substrate.","21.The method according to claim 10, wherein said immuno-labeled complex comprises a labeling reagent that is a Fab or Fab′ anti-Fc fragment wherein said fragment is independently covalently bonded to one or more labels that are selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a phycobiliprotein, a borapolyazaindacene, a peroxidase, a phosphatase, a tandem dye and derivatives thereof.","22.A method for detecting multiple targets in a sample, said method comprising: a) contacting said sample with a solution comprising A) a pooled subset of immuno-labeled complexes wherein each said complex comprises a target-binding antibody and a labeling reagent, wherein each said subset is distinguished from another subset by i) a ratio of label to labeling reagent, or ii) a physical property of said label, or iii) a ratio of labeling reagent to said target-binding antibody, or iv) by said target-binding antibody or B) an individual immuno-labeled complex subset wherein multiple individual subsets are added to said sample; b) incubating said sample for a time sufficient to allow said immuno-labeled complex to selectively bind to said target; and, c) illuminating said immuno-labeled complex whereby said multiple targets are detected.","23.The method according to claim 22, wherein said sample comprises a population of cells, cellular extract, subcellular component, tissue culture, tissue, a bodily fluid, or a portion or combination thereof.","24.The method according to claim 23, wherein said sample is immobilized on a solid or semi-solid matrix.","25.The method according to claim 24, wherein said matrix is a gel, a membrane, an array, a glass surface or a microparticle.","26.The method according to claim 23, wherein said labeling reagent is a Fab or Fab′fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is a non-antibody protein selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.","27.The method according to claim 26, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, an electron transfer agent, a hapten, an enzyme and a radioisotope.","28.The method according to claim 27, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.","29.The method according to claim 27, wherein said fluorescent protein is a phycobiliprotein.","30.The method according to claim 27, wherein said tandem dye is selected from the group consisting of a cyanine-phycobiliprotein derivative and xanthene-phycobiliprotein derivative.","31.The method according to claim 27, wherein said enzyme is selected from the group consisting of a peroxidase, a phosphatase, a glycosidase, and a luciferase.","32.The method according to claim 31, wherein said method further comprises adding a calorimetric, fluorescent or chemiluminescent enzyme substrate.","33.A method for identifying and quantitating multiple targets in a sample, wherein said method comprises the steps of: a) contacting a population of cells or fragments thereof in a sample with A) a solution comprising a pooled subset of immuno-labeled complexes wherein said complex comprises a target-binding antibody and a labeling reagent, wherein each subset is.","distinguished from another subset i) a ratio of label to labeling reagent, or ii) a physical property of said label, or iii) a ratio of labeling reagent to said target-binding antibody, or iv) by said target-binding antibody or B) an individual subset of immuno-labeled complexes wherein multiple individual subsets are added to said sample; b) incubating said cells or fragments thereof for a time period sufficient to allow said immuno-labeled complex to bind said targets; c) passing said incubated population of cells or fragments thereof through an examination zone; and, d) collecting data from said cells or fragments thereof passed through said examination zone whereby said identification and quantitation of multiple targets is determined.","34.The method according to claim 33, wherein said labeling reagent is a Fab or Fab′fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is a non-antibody protein selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.","35.The method according to claim 34, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a radioisotope, and a hapten.","36.The method according to claim 35, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.","37.The method according to claim 35, wherein said fluorescent protein is a phycobiliprotein.","38.The method according to claim 35, wherein said tandem dye is selected from the group consisting of cyanine-phycobiliprotein and xanthene-phycobiliprotein.","39.A method of manufacturing an isolated labeling reagent, wherein said method comprises the steps of: a) cleaving an intact anti-region antibody with an enzyme to generate Fab fragments; b) isolating said anti-region Fab fragments of step a); c) contacting a matrix comprising intact immunoglobulin proteins or fragments that selectively bind anti-region Fab fragments with a solution comprising said anti-region fragments of step b) wherein said anti-region Fab fragments are immobilized on said matrix; d) contacting said matrix of step c) with a solution comprising a fluorophore label that contains a reactive group; e) washing said matrix of step d) to remove unbound label, and; f) eluting said labeling reagent from said matrix whereby said isolated labeling reagent is manufactured.","40.The method according to claim 39, wherein said anti-region Fab fragment are selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment.","41.The method according to claim 40, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.","42.A method of manufacturing an isolated labeling reagent, wherein said method comprises the steps of: a) contacting a matrix comprising intact immunoglobulin proteins or fragments thereof that selectively bind non-antibody proteins with a solution comprising said non-antibody proteins wherein said non-antibody proteins are immobilized on said matrix; b) contacting said matrix of step a) with a solution comprising a fluorophore label that contains a reactive group; c) washing said matrix to remove unbound label, and; d) eluting said labeling reagent from said matrix whereby said isolated labeling reagent is manufactured that comprises a fluorophore label.","43.The method according to claim 42, wherein said non-antibody protein is selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.","44.The method according to claim 43, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.","45.A method of manufacturing an isolated labeling reagent, wherein said method comprises the steps of: a) cleaving an intact anti-region antibody with an enzyme resulting in Fab or Fab′fragments; a) contacting said anti-region Fab or Fab′ fragments of step a) with a solution comprising a label that contains a reactive group; and, c) isolating labeled anti-region Fab or Fab′ fragments by size exclusion or affinity chromatography whereby an isolated labeling reagent is manufactured.","46.The method according to claim 45, wherein said anti-region Fab or Fab′ fragment is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment.","47.The method according to claim 46, wherein said label is a fluorescent protein or a tandem dye.","48.The method according to claim 47, wherein said fluorescent protein is a phycobiliprotein.","49.The method according to claim 47, wherein said tandem dye is selected from the group consisting of cyanine-phycobiliprotein and xanthene-phycobiliprotein.","50.An isolated labeling reagent made by a process comprising: a) cleaving an intact anti-region antibody with an enzyme resulting in Fab or Fab′fragments; b) contacting said Fab or Fab′ fragments of step a) with a solution comprising a label that contains a reactive group; and, c) isolating labeled anti-region Fab or Fab′ fragments by size exclusion or affinity chromatography.","51.The labeling reagent according to claim 50, wherein said anti-region Fab or Fab′fragment is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment.","52.The labeling reagent according to claim 51, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a radioisotope, and a hapten.","53.The labeling reagent according to claim 52, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.","54.An isolated labeling reagent made by a process comprising: a) cleaving an intact anti-region antibody with an enzyme to generate F(ab′)2 fragments; b) contacting said anti-region F(ab′)2 with a reducing agent to produce anti-region Fab′ fragments containing a thiol group; c) contacting said Fab′ fragments with a solution comprising a label that contains a reactive group; and, d) isolating said Fab′ fragments of step d) that are covalently attached to a label by size exclusion or affinity chromatography.","55.The labeling reagent according to claim 54, wherein said anti-region Fab′ fragment is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment.","56.The method according to claim 55, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a radioisotope, and a hapten.","57.An isolated labeling reagent made by a process comprising: a) cleaving an intact anti-region antibody with an enzyme to generate Fab fragments; b) isolating said anti-region Fab fragments of step a); c) contacting a matrix comprising intact immunoglobulin proteins or fragments thereof that specifically bind anti-region Fab fragments with a solution comprising said anti-region Fab fragments of step b) wherein said anti-region Fab fragments are immobilized; d) contacting said matrix of step c) with a solution comprising a fluorophore label that contains a reactive group; e) washing said matrix to remove unbound label, and; f) eluting said labeling reagent from said matrix whereby said labeling reagent is manufactured comprising a label and being isolated from other proteins and fragments thereof.","58.The labeling reagent according to claim 57, wherein said anti-region Fab fragment is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment.","59.The labeling reagent according to claim 58, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.","60.An isolated labeling reagent made by a process comprising: a) contacting a matrix comprising intact immunoglobulin proteins or fragments that selectively bind non-antibody proteins with a solution comprising said non-antibody proteins wherein said non-antibody proteins are immobilized; b) contacting said matrix of step c) with a solution comprising a fluorophore label that contains a reactive group; c) washing said matrix to remove unbound label, and; d) eluting said labeling reagent from said matrix whereby said labeling reagent is manufactured comprising a label.","61.The labeling reagent according to claim 60, wherein said non-antibody protein is selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.","62.The labeling reagent according to claim 61, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.","63.An immuno-labeled complex generated by a process comprising: a) contacting a solution of target-binding antibodies with a labeling reagent subset, wherein said labeling reagent subset is distinguished from another labeling reagent subset by i) ratio of label to labeling reagent or ii) a physical properties of said label; b) incubating said target-binding antibodies and said labeling reagent subset for a time period sufficient for one or more labeling reagents to form an immuno-labeled complex with a target-binding antibody wherein a region of said target binding antibody is selectively bound by labeling reagent; and, c) optionally removing unbound labeling reagent by adding a capture reagent comprising immunoglobulin proteins or fragments thereof whereby an immuno-labeled complex is produced.","64.The immuno-labeled complex according to claim 63, wherein said process further comprises repeating said steps a), b), and c) to form subsets of immuno-labeled complexes wherein each subset is distinguished from another subset by i) ratio of label to labeling reagent or ii) a physical properties of said label, or iii) a ratio of labeling reagent to said target-binding antibody or iv) by said target-binding antibody.","65.The immuno-labeled complex according to claim 63, wherein said target-binding antibody is a murine monoclonal antibody, a rabbit polyclonal antibody or a goat polyclonal antibody.","66.The immuno-labeled complex according to claim 65, wherein said labeling reagent is a Fab or Fab′ fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is a non-antibody protein selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.","67.The immuno-labeled complex according to claim 66, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme and a radioisotope.","68.The immuno-labeled complex according to claim 67, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine, and derivatives thereof.","69.A labeling solution comprising a buffer and either an individual labeling reagent subset or a pooled subset of labeling reagents, wherein each subset is distinguished from another subset by i) a ratio of label to labeling reagent, or ii) by a physical property of said label.","70.The solution according to claim 69, wherein said labeling reagent is a Fab or Fab′fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is non-antibody protein is selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.","71.The solution according to claim 70, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, an enzyme, a hapten and a radioisotope.","72.The solution according to claim 71, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine, and derivatives thereof.","73.A kit for preparing an immuno-labeling complex, said kit comprising: a) a labeling solution comprising a labeling reagent that is independently covalently bonded to one or more labels; or b) a panel of labeling solutions wherein each labeling solution comprises a subset of a labeling reagent and each subset is distinguished from another subset by i) a ratio of label to labeling reagent, or ii) by a physical property of said label; and, c) a solution comprising a capture reagent.","74.The kit according to claim 73, wherein said labeling reagent is a Fab or Fab′fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is a non-antibody protein selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.","75.The kit according to claim 74, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, an enzyme, a hapten and a radioisotope.","76.The kit according to claim 75, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.","77.The kit according to claim 76, wherein the kit further comprises an addition component selected from the group consisting of (a) stains for characterization of cellular organelles, cell viability, or cell proliferation state, (b) enzyme substrates and (c) enzyme conjugates."],["<SOH> BACKGROUND OF THE INVENTION <EOH>Immunolabeling is a method for qualitative or quantitative determination of the presence of a target in a sample, wherein antibodies are utilized for their specific binding capacity.","The antibodies form a complex with the target (antigen), wherein a detectable label is present on the antibody or on a secondary antibody.","The detectable label is a key feature of immunolabeling, which can be detected directly or indirectly.","The label provides a measurable signal by which the binding reaction is monitored providing a qualitative and/or quantitative measure of the degree of binding.","The relative quantity and location of signal generated by the labeled antibodies can serve to indicate the location and/or concentration of the target.","The label can also be used to select and isolate labeled targets, such as by flow sorting or using magnetic separation media.","Examples of labels include but are not limited to radioactive nucleotides ( 125 I, 3 H, 14 C, 32 P), chemiluminescent, fluorescent, or phosphorescent compounds (e.g., dioxetanes, xanthene, or carbocyanine dyes, lanthanide chelates), particles (e.g., gold clusters, colloidal gold, microspheres, quantum dots), and enzymes (e.g., peroxidases, glycosidases, phosphatases, kinases).","Ideally, the label is attached to the antibody in a manner that does not perturb the antibody's binding characteristics but enables the label to be measured by an appropriate detection technology.","The choice of labels is influenced by factors such as ease and sensitivity of detection, equipment availability, background in the sample (including other labels) and the degree to which such labels are readily attached to the particular antibody.","Both direct and indirect labeling of antibodies is utilized for immunolabeling.","Direct labeling utilizes only a primary antibody, i.e.","the antibody specific for the target, bound to the label.","In contrast, indirect labeling utilizes a secondary antibody bound to the label, which is specific for the primary antibody, e.g.","a goat anti-rabbit antibody.","The principal differences in immunolabeling methods and materials reside in the way that the label is attached to the antibody-antigen complex, the type of label that is used, and the means by which the antibody-antigen complex is detected.","Limitations for direct labeling primary antibodies include the need for buffers free of primary amines, or carrier proteins such as bovine serum albumin (BSA), and other compounds such as tris-(hydroxymethyl)aminomethane (TRIS), glycine, and ammonium ions.","These materials are, however, common components in antibody buffers and purification methods, and it may not be possible or feasible to remove them prior to the coupling reaction.","In particular, many monoclonal antibodies are available only as ascites fluid or in hybridoma culture supernatants, or diluted with carrier proteins, such as albumins.","Thus, direct labeling of antibodies in ascites fluid or other medias containing interfering compounds is not attainable.","The indirect immunolabeling method typically involves a multi-step process in which an unlabeled first antibody (typically a primary antibody) is directly added to the sample to form a complex with the antigen in the sample.","Subsequently, a labeled secondary antibody, specific for the primary antibody, is added to the sample, where it attaches noncovalently to the primary antibody-antigen complex.","Alternatively, a detectable label is covalently attached to an immunoglobulin-binding protein such as protein A and protein G to detect the antibody-antigen complex that has previously been formed with the target in the sample.","Using ligands, such as streptavidin, that are meant to amplify the detectable signal also expands this cascade binding.","Indirect immunolabeling often results in false positives and high background.","This is due to the fact that secondary antibodies, even when purified by adsorption against related species, nevertheless can exhibit significant residual cross-reactivity when used in the same sample.","For example, when mouse tissue is probed with a mouse monoclonal antibody, the secondary antibody must necessarily be a labeled anti-mouse antibody.","This anti-mouse antibody will detect the antibody of interest but will inevitably and additionally detect irrelevant, endogenous mouse immunoglobulins inherent in mouse tissue.","This causes a significant background problem, especially in diseased tissues, which reduces the usefulness and sensitivity of the assay.","Thus, the simultaneous detection of more than one primary antibody in a sample without this significant background interference depends on the availability of secondary antibodies that 1) do not cross-react with proteins intrinsic to the sample being examined, 2) recognize only one of the primary antibodies, and 3) do not recognize each other (Brelje, et al., METHODS IN CELL BIOLOGY 38, 97-181, especially 111-118 (1993)).","To address the background problem in indirect labeling, a number of strategies have been developed to block access of the anti-mouse secondary antibodies to the endogenous mouse immunoglobulins.","One such strategy for blocking involves complexing the primary antibody with a selected biotinylated secondary antibody to produce a complex of the primary and secondary antibodies, which is then mixed with diluted normal murine serum (Trojanowski et al., U.S. Pat No.","5,281,521 (1994)).","This method is limited by the necessity to utilize an appropriate ratio of primary-secondary complex.","Too low a ratio of primary-secondary complex will cause a decrease in specific staining and increased background levels due to the uncomplexed secondary anti-mouse antibody binding to endogenous mouse antibodies.","However, the ability of a whole IgG antibody (as was used in the referenced method) to simultaneously bind and cross-link two antigens results in too high a ratio, causing the complex to precipitate or form complexes that are too large to penetrate into the cell or tissue.","Another strategy for blocking access to endogenous immunoglobulins in the sample involves pre-incubating the sample with a monovalent antibody, such as Fab′ fragments, from an irrelevant species that recognize endogenous immunoglobulins.","This approach requires large quantities of expensive Fab′ fragments and gives mixed results and adds at least two steps (block and wash) to the overall staining procedure.","The addition of a cross-linking reagent has resulted in improved reduction of background levels (Tsao, et al., U.S. Pat.","No.","5,869,274 (1997)) but this is problematic when used with fluorophore-labeled antibodies.","The cross-linking causes an increase in the levels of autofluorescence and thus the background (J. Neurosci.","Meth.","83, 97 (1998); Mosiman et al., Methods 77, 191 (1997); Commun.","Clin.","Cytometry 30,151 (1997); Beisker et al., Cytometry 8, 235 (1987)).","In addition, pre-incubation with a cross-linking reagent often masks or prevents the antibody from binding to its antigen (J. Histochem.","Cytochem.","45, 327 (1997); J. Histochem.","Cytochem.","39, 741 (1991); J. Histochem.","Cytochem.","43,193 (1995); Appl.","Immunohistochem.","Molecul.","Morphol.","9,176 (2001)).","In a variation of this blocking strategy, a multi-step sequential-labeling procedure is used to overcome the problems of cross-reactivity.","The sample is incubated with a first antibody to form a complex with the first antigen, followed by incubation of the sample with a fluorophore-labeled goat Fab anti-mouse IgG to label the first antibody and block it from subsequently complexing when the second antibody is added.","In the third step, a second mouse antibody forms a complex with the second antigen.","Because the second antibody is blocked from cross-reacting with the first antibody, the second mouse antibody is detected with a standard indirect-labeling method using a goat anti-mouse antibody conjugated to a different fluorescent dye (J. Histochem.","Cytochem.","34, 703 (1986)).","This process requires multiple incubation steps and washing steps and it still cannot be used with mouse antibodies to probe mouse tissue.","Another blocking method is disclosed in the animal research kit (ARK) developed by DAKO.","In this kit, a primary antibody is complexed with biotin-labeled goat Fab anti-mouse IgG and excess free Fab is blocked with normal mouse serum.","However, since the Fab used in this process is generated from the intact IgG (rather than a selected region) there is a potential for the formation of anti-paratope or anti-idiotype antibodies that will block the antigen-binding site and prevent immunolabeling.","The biotinylated antibody also requires subsequent addition of a labeled avidin or streptavidin conjugate for its subsequent visualization.","The present invention is advantageous over previously described methods and compositions in that it provides the benefits of indirect labeling with the easy and flexibility of direct labeling for determination of a desired target in a biological sample.","The present invention provides labeled monovalent proteins specific for a target-binding antibody, which are complexed prior to addition with a biological sample.","Because these monovalent proteins are not bivalent antibodies, precipitation and cross-linking are not a problem.","Therefore the compositions of the present invention can be used with immunologically similar monoclonal or polyclonal antibodies of either an identical isotype or different isotypes.","The monovalent labeling reagents are specific for the Fc region of target-binding antibodies, these reagents will not interfere with the binding region of the primary antibody.","In addition, the monovalent labeling proteins are not negatively affected by the presence of primary amines like BSA, gelatin, hybridoma culture supernatants or ascites fluid, thus primary antibodies present in these media can be effectively labeled with the labeling reagents of the present invention.","Thus, the present invention provides numerous advantages over the conventional methods of immunolabeling."],["<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides labeling reagents and methods for labeling primary antibodies and for detecting a target in a sample using an immuno-labeled complex that comprises a target-binding antibody and one or more labeling reagents.","The labeling reagents comprise monovalent antibody fragments or non-antibody monomeric proteins whereby the labeling proteins have affinity for a specific region of the target-binding antibody and are covalently attached to a label.","Typically, the labeling reagent is an anti-Fc Fab or Fab′ fragment that was generated by immunizing a goat or rabbit with the Fc fragment of an antibody.","The methods for labeling a target-binding antibody with a labeling reagent comprise a) contacting a solution of target-binding antibodies with a labeling reagent, b) incubating said target-binding antibodies and said labeling reagent wherein a region of said target binding antibody is selectively bound by labeling reagent, and c) optionally removing unbound labeling reagent by adding a capture reagent comprising immunoglobulin proteins or fragments thereof that are optionally immobilized on a matrix.","The labeling of the target-binding antibody can be performed irrespective of the solution that the antibody is present in and includes proteins that are normally present in serum or ascites.","This feature of the labeling process of the target-binding antibody eliminates the need to purify and concentrate the target-binding antibody.","The time required for the labeling reagent to selectively bind to the target-binding antibody is typically very short, often less than 10 minutes.","Often the labeling reagent binds the target-binding antibody in the amount of time it takes to add and mix the labeling reagent with the target-binding antibody.","This formation of an immuno-labeled complex—a target-binding antibody and a labeling reagent—results in the formation of an target detection solution that is used to detect a target in a sample.","The labeling steps of the target-binding antibody are optionally repeated to form a panel of subsets, these immuno-labeled complex subsets may be used individually or pooled wherein each subset is distinguished from another subset by i) the target-binding antibody, or ii) a ratio of label to labeling reagent, or iii) a ratio of labeling reagent to the target-binding antibody or iv) by a physical property of the label.","Thus, it is appreciated that a wide range of subsets can be formed wherein the subsets can be used individually to detect a target in a sample or pooled to simultaneously detect multiple targets in a sample.","The simultaneous detection of multiple targets in a sample is especially useful in methods that utilize flow cytometry or methods that immobilize a population of cells or tissue on a surface.","The methods for determining a target in a sample using immuno-labeled subsets comprises forming a subset of immuno-labeled complexes, as described above, contacting a sample with said immuno-labeled complexes, incubating the sample for a time sufficient to allow the immuno-labeled complex to selectively bind to a desired target, and illuminating the immuno-labeled complex whereby the target is detected.","The sample is any material that may contain a target and typically comprises a population of cells, cellular extract, subcellular component, proteins, peptides, tissue culture, tissue, a bodily fluid, or a portion or combination thereof.","When multiple targets are detected a pooled subset of immuno-labeled complexes are formed and incubated with the sample or individual subsets are add sequentially to a sample.","For methods using flow cytometry the population of cells is illuminated when they pass through an optical examination zone and the data collected about the label determines the identity and quantity of the targets."],["CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Ser.","No.","60/329,068, filed Oct. 12, 2001; U.S. Ser.","No.","60/369,418 filed Apr.","1, 2002, U.S. Ser.","No.10/118,204 filed Apr.","5, 2002, and PCT/US02/31416 filed Oct. 2, 2002, which disclosures are herein incorporated by reference.","FIELD OF THE INVENTION The present invention relates to immuno-labeled complexes and methods for use in the detection and measurement of one or more targets in a biological sample.","The invention has applications in the fields of molecular biology, cell biology, immunohistochemistry, diagnostics, and therapeutics.","BACKGROUND OF THE INVENTION Immunolabeling is a method for qualitative or quantitative determination of the presence of a target in a sample, wherein antibodies are utilized for their specific binding capacity.","The antibodies form a complex with the target (antigen), wherein a detectable label is present on the antibody or on a secondary antibody.","The detectable label is a key feature of immunolabeling, which can be detected directly or indirectly.","The label provides a measurable signal by which the binding reaction is monitored providing a qualitative and/or quantitative measure of the degree of binding.","The relative quantity and location of signal generated by the labeled antibodies can serve to indicate the location and/or concentration of the target.","The label can also be used to select and isolate labeled targets, such as by flow sorting or using magnetic separation media.","Examples of labels include but are not limited to radioactive nucleotides (125I, 3H, 14C, 32P), chemiluminescent, fluorescent, or phosphorescent compounds (e.g., dioxetanes, xanthene, or carbocyanine dyes, lanthanide chelates), particles (e.g., gold clusters, colloidal gold, microspheres, quantum dots), and enzymes (e.g., peroxidases, glycosidases, phosphatases, kinases).","Ideally, the label is attached to the antibody in a manner that does not perturb the antibody's binding characteristics but enables the label to be measured by an appropriate detection technology.","The choice of labels is influenced by factors such as ease and sensitivity of detection, equipment availability, background in the sample (including other labels) and the degree to which such labels are readily attached to the particular antibody.","Both direct and indirect labeling of antibodies is utilized for immunolabeling.","Direct labeling utilizes only a primary antibody, i.e.","the antibody specific for the target, bound to the label.","In contrast, indirect labeling utilizes a secondary antibody bound to the label, which is specific for the primary antibody, e.g.","a goat anti-rabbit antibody.","The principal differences in immunolabeling methods and materials reside in the way that the label is attached to the antibody-antigen complex, the type of label that is used, and the means by which the antibody-antigen complex is detected.","Limitations for direct labeling primary antibodies include the need for buffers free of primary amines, or carrier proteins such as bovine serum albumin (BSA), and other compounds such as tris-(hydroxymethyl)aminomethane (TRIS), glycine, and ammonium ions.","These materials are, however, common components in antibody buffers and purification methods, and it may not be possible or feasible to remove them prior to the coupling reaction.","In particular, many monoclonal antibodies are available only as ascites fluid or in hybridoma culture supernatants, or diluted with carrier proteins, such as albumins.","Thus, direct labeling of antibodies in ascites fluid or other medias containing interfering compounds is not attainable.","The indirect immunolabeling method typically involves a multi-step process in which an unlabeled first antibody (typically a primary antibody) is directly added to the sample to form a complex with the antigen in the sample.","Subsequently, a labeled secondary antibody, specific for the primary antibody, is added to the sample, where it attaches noncovalently to the primary antibody-antigen complex.","Alternatively, a detectable label is covalently attached to an immunoglobulin-binding protein such as protein A and protein G to detect the antibody-antigen complex that has previously been formed with the target in the sample.","Using ligands, such as streptavidin, that are meant to amplify the detectable signal also expands this cascade binding.","Indirect immunolabeling often results in false positives and high background.","This is due to the fact that secondary antibodies, even when purified by adsorption against related species, nevertheless can exhibit significant residual cross-reactivity when used in the same sample.","For example, when mouse tissue is probed with a mouse monoclonal antibody, the secondary antibody must necessarily be a labeled anti-mouse antibody.","This anti-mouse antibody will detect the antibody of interest but will inevitably and additionally detect irrelevant, endogenous mouse immunoglobulins inherent in mouse tissue.","This causes a significant background problem, especially in diseased tissues, which reduces the usefulness and sensitivity of the assay.","Thus, the simultaneous detection of more than one primary antibody in a sample without this significant background interference depends on the availability of secondary antibodies that 1) do not cross-react with proteins intrinsic to the sample being examined, 2) recognize only one of the primary antibodies, and 3) do not recognize each other (Brelje, et al., METHODS IN CELL BIOLOGY 38, 97-181, especially 111-118 (1993)).","To address the background problem in indirect labeling, a number of strategies have been developed to block access of the anti-mouse secondary antibodies to the endogenous mouse immunoglobulins.","One such strategy for blocking involves complexing the primary antibody with a selected biotinylated secondary antibody to produce a complex of the primary and secondary antibodies, which is then mixed with diluted normal murine serum (Trojanowski et al., U.S. Pat No.","5,281,521 (1994)).","This method is limited by the necessity to utilize an appropriate ratio of primary-secondary complex.","Too low a ratio of primary-secondary complex will cause a decrease in specific staining and increased background levels due to the uncomplexed secondary anti-mouse antibody binding to endogenous mouse antibodies.","However, the ability of a whole IgG antibody (as was used in the referenced method) to simultaneously bind and cross-link two antigens results in too high a ratio, causing the complex to precipitate or form complexes that are too large to penetrate into the cell or tissue.","Another strategy for blocking access to endogenous immunoglobulins in the sample involves pre-incubating the sample with a monovalent antibody, such as Fab′ fragments, from an irrelevant species that recognize endogenous immunoglobulins.","This approach requires large quantities of expensive Fab′ fragments and gives mixed results and adds at least two steps (block and wash) to the overall staining procedure.","The addition of a cross-linking reagent has resulted in improved reduction of background levels (Tsao, et al., U.S. Pat.","No.","5,869,274 (1997)) but this is problematic when used with fluorophore-labeled antibodies.","The cross-linking causes an increase in the levels of autofluorescence and thus the background (J. Neurosci.","Meth.","83, 97 (1998); Mosiman et al., Methods 77, 191 (1997); Commun.","Clin.","Cytometry 30,151 (1997); Beisker et al., Cytometry 8, 235 (1987)).","In addition, pre-incubation with a cross-linking reagent often masks or prevents the antibody from binding to its antigen (J. Histochem.","Cytochem.","45, 327 (1997); J. Histochem.","Cytochem.","39, 741 (1991); J. Histochem.","Cytochem.","43,193 (1995); Appl.","Immunohistochem.","Molecul.","Morphol.","9,176 (2001)).","In a variation of this blocking strategy, a multi-step sequential-labeling procedure is used to overcome the problems of cross-reactivity.","The sample is incubated with a first antibody to form a complex with the first antigen, followed by incubation of the sample with a fluorophore-labeled goat Fab anti-mouse IgG to label the first antibody and block it from subsequently complexing when the second antibody is added.","In the third step, a second mouse antibody forms a complex with the second antigen.","Because the second antibody is blocked from cross-reacting with the first antibody, the second mouse antibody is detected with a standard indirect-labeling method using a goat anti-mouse antibody conjugated to a different fluorescent dye (J. Histochem.","Cytochem.","34, 703 (1986)).","This process requires multiple incubation steps and washing steps and it still cannot be used with mouse antibodies to probe mouse tissue.","Another blocking method is disclosed in the animal research kit (ARK) developed by DAKO.","In this kit, a primary antibody is complexed with biotin-labeled goat Fab anti-mouse IgG and excess free Fab is blocked with normal mouse serum.","However, since the Fab used in this process is generated from the intact IgG (rather than a selected region) there is a potential for the formation of anti-paratope or anti-idiotype antibodies that will block the antigen-binding site and prevent immunolabeling.","The biotinylated antibody also requires subsequent addition of a labeled avidin or streptavidin conjugate for its subsequent visualization.","The present invention is advantageous over previously described methods and compositions in that it provides the benefits of indirect labeling with the easy and flexibility of direct labeling for determination of a desired target in a biological sample.","The present invention provides labeled monovalent proteins specific for a target-binding antibody, which are complexed prior to addition with a biological sample.","Because these monovalent proteins are not bivalent antibodies, precipitation and cross-linking are not a problem.","Therefore the compositions of the present invention can be used with immunologically similar monoclonal or polyclonal antibodies of either an identical isotype or different isotypes.","The monovalent labeling reagents are specific for the Fc region of target-binding antibodies, these reagents will not interfere with the binding region of the primary antibody.","In addition, the monovalent labeling proteins are not negatively affected by the presence of primary amines like BSA, gelatin, hybridoma culture supernatants or ascites fluid, thus primary antibodies present in these media can be effectively labeled with the labeling reagents of the present invention.","Thus, the present invention provides numerous advantages over the conventional methods of immunolabeling.","SUMMARY OF THE INVENTION The present invention provides labeling reagents and methods for labeling primary antibodies and for detecting a target in a sample using an immuno-labeled complex that comprises a target-binding antibody and one or more labeling reagents.","The labeling reagents comprise monovalent antibody fragments or non-antibody monomeric proteins whereby the labeling proteins have affinity for a specific region of the target-binding antibody and are covalently attached to a label.","Typically, the labeling reagent is an anti-Fc Fab or Fab′ fragment that was generated by immunizing a goat or rabbit with the Fc fragment of an antibody.","The methods for labeling a target-binding antibody with a labeling reagent comprise a) contacting a solution of target-binding antibodies with a labeling reagent, b) incubating said target-binding antibodies and said labeling reagent wherein a region of said target binding antibody is selectively bound by labeling reagent, and c) optionally removing unbound labeling reagent by adding a capture reagent comprising immunoglobulin proteins or fragments thereof that are optionally immobilized on a matrix.","The labeling of the target-binding antibody can be performed irrespective of the solution that the antibody is present in and includes proteins that are normally present in serum or ascites.","This feature of the labeling process of the target-binding antibody eliminates the need to purify and concentrate the target-binding antibody.","The time required for the labeling reagent to selectively bind to the target-binding antibody is typically very short, often less than 10 minutes.","Often the labeling reagent binds the target-binding antibody in the amount of time it takes to add and mix the labeling reagent with the target-binding antibody.","This formation of an immuno-labeled complex—a target-binding antibody and a labeling reagent—results in the formation of an target detection solution that is used to detect a target in a sample.","The labeling steps of the target-binding antibody are optionally repeated to form a panel of subsets, these immuno-labeled complex subsets may be used individually or pooled wherein each subset is distinguished from another subset by i) the target-binding antibody, or ii) a ratio of label to labeling reagent, or iii) a ratio of labeling reagent to the target-binding antibody or iv) by a physical property of the label.","Thus, it is appreciated that a wide range of subsets can be formed wherein the subsets can be used individually to detect a target in a sample or pooled to simultaneously detect multiple targets in a sample.","The simultaneous detection of multiple targets in a sample is especially useful in methods that utilize flow cytometry or methods that immobilize a population of cells or tissue on a surface.","The methods for determining a target in a sample using immuno-labeled subsets comprises forming a subset of immuno-labeled complexes, as described above, contacting a sample with said immuno-labeled complexes, incubating the sample for a time sufficient to allow the immuno-labeled complex to selectively bind to a desired target, and illuminating the immuno-labeled complex whereby the target is detected.","The sample is any material that may contain a target and typically comprises a population of cells, cellular extract, subcellular component, proteins, peptides, tissue culture, tissue, a bodily fluid, or a portion or combination thereof.","When multiple targets are detected a pooled subset of immuno-labeled complexes are formed and incubated with the sample or individual subsets are add sequentially to a sample.","For methods using flow cytometry the population of cells is illuminated when they pass through an optical examination zone and the data collected about the label determines the identity and quantity of the targets.","BRIEF DESCRIPTION OF THE DRAWINGS FIG.","1: Shows a schematic representation of the formation of the immuno-labeled complex (target-binding antibody and labeling reagent).","FIG.","2: Shows species specificity of goat Fab anti-(mouse Fc), as observed using a microplate coated with IgG of various species.","The various species were blocked with BSA, reacted with biotinylated goat Fab anti-(mouse Fc), washed, and then treated with streptavidin-horseradish peroxidase (HRP), followed by hydrogen peroxide (H2O2) and the Amplex Red peroxidase detection reagent.","FIG.","3: Shows a preferred molar ratio of a goat Fab anti-(mouse Fc) labeling reagent.","Varying amounts of an Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse Fc) were added to a constant amount of anti-biotin monoclonal antibody (mAb).","This mixture was equilibrated for 20 minutes, and then added to biotinylated-BSA in a microplate well.","After allowing time to bind, the plates were washed and the remaining fluorescence was quantitated.","The analysis was performed in triplicate (circles).","Control experiments were performed, as described above, but without adding the primary anti-biotin antibody (solid squares).","FIG.","4: Shows a comparison of the fluorescence intensity (Example 6) for labeling reagent prepared in homogeneous solution (Example 4) and labeling reagent prepared on a column (Example 5).","FIG.","5: Shows detection of multiple targets on T cells using a labeling reagent attached to a R-phycoerythrin (R-PE) (FIG.","5A) to detect CD3-positive T cells, a labeling reagent attached to Alexa Fluor 647 dye (FIG.","5B) to detect CD4-positive T cells and a labeling reagent attached to Alexa Fluor 488 dye (FIG.","5B) to detect CD8-positive T cells (Example 18).","The CD-3 detected T cells are shown in the upper left (UL) and upper right (UR) quadrants.","The relative percentages of total lymphocytes that are CD3-positive cells are 83.3% (UL+UR).","The relative percentage of CD8-positive Alexa Fluor 488 dye-stained lymphocytes and CD3-positive R-PE dye-stained lymphocytes is 35.1% (UR quadrant).","The lower left quadrant (LL, 20.4%) shows CD3-negative lymphocytes (i.e.","non-T cells) comprised of NK cells, B cells and some monocytes.","In the lower right (LR, 2.7%) region are non-T cells, which are nonspecifically stained.","FIG.","5B further shows CD3-positive T-cells subdivided into Alexa Fluor 647 dye CD4-positive and Alexa Fluor 488 dye CD8-positive.","CD4-positive cells represent 50.9% of total lymphocytes (UL quadrant) and CD8-positive cells represent 24.5% of the total lymphocytes (LR quadrant).","The 23.1% of cells in the LL quadrant are non-T cells, while the 1.5% of cells in UR quadrant are likely nonspecifically stained lymphocytes.","FIG.","6: Shows high-performance size-exclusion chromatographic analysis of Alexa Fluor 488 dye-labeled goat Fab anti-(mouse Fc) labeling reagent binding to a mouse IgG, target-binding antibody.","The labeling reagent, alone, appears as a peak at 38 minutes; the target-binding antibody, alone, appears as a peak at 33 minutes.","When labeling reagent and target-binding antibody are mixed together at a molar ratio of -5:1 (labeling reagent:target-binding antibody), the resulting immunolabeling complex appears as a peak at 29 minutes (Example 10).","FIG.","7: Shows the production of labeling reagent wherein the label is attached to the labeling reagent when immobilized on a column.","DETAILED DESCRIPTION OF THE INVENTION I. Definitions Before describing the present invention in detail, it is to be understood that this invention is not limited to specific compositions or process steps, as such may vary.","It should be noted that, as used in this specification and the appended claims, the singular form “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise.","Thus, for example, reference to “a protein labeling complex” includes a plurality of complexes and reference to “a target-binding protein” includes a plurality of proteins and the like.","Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related.","The following terms are defined for purposes of the invention as described herein.","The term “affinity” as used herein refers to the strength of the binding interaction of two molecules, such as an antibody and an antigen or a positively charged moiety and a negatively charged moiety.","For bivalent molecules such as antibodies, affinity is typically defined as the binding strength of one binding domain for the antigen, e.g.","one Fab fragment for the antigen.","The binding strength of both binding domains together for the antigen is referred to as “avidity”.","As used herein “High affinity” refers to a ligand that binds to an antibody having an affinity constant (Ka) greater than 104 M−1, typically 105-1011 M−1; as determined by inhibition ELISA or an equivalent affinity determined by comparable techniques such as, for example, Scatchard plots or using Kd/dissociation constant, which is the reciprocal of the Ka, etc.","The term “antibody” as used herein refers to a protein of the immunoglobulin (Ig) superfamily that binds noncovalently to certain substances (e.g.","antigens and immunogens) to form an antibody-antigen complex.","Antibodies can be endogenous, or polyclonal wherein an animal is immunized to elicit a polyclonal antibody response or by recombinant methods resulting in monoclonal antibodies produced from hybridoma cells or other cell lines.","It is understood that the term “antibody” as used herein includes within its scope any of the various classes or sub-classes of immunoglobulin derived from any of the animals conventionally used.","The term “antibody fragments” as used herein refers to fragments of antibodies that retain the principal selective binding characteristics of the whole antibody.","Particular fragments are well-known in the art, for example, Fab, Fab′, and F(ab′)2, which are obtained by digestion with various proteases, pepsin or papain, and which lack the Fc fragment of an intact antibody or the so-called “half-molecule” fragments obtained by reductive cleavage of the disulfide bonds connecting the heavy chain components in the intact antibody.","Such fragments also include isolated fragments consisting of the light-chain-variable region, “Fv” fragments consisting of the variable regions of the heavy and light chains, and recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker.","Other examples of binding fragments include (i) the Fd fragment, consisting of the VH and CH1 domains; (ii) the dAb fragment (Ward, et al., Nature 341, 544 (1989)), which consists of a VH domain; (iii) isolated CDR regions; and (iv) single-chain Fv molecules (scFv) described above.","In addition, arbitrary fragments can be made using recombinant technology that retains antigen-recognition characteristics.","The term “antigen” as used herein refers to a molecule that induces, or is capable of inducing, the formation of an antibody or to which an antibody binds selectively, including but not limited to a biological material.","Antigen also refers to “immunogen”.","The target-binding antibodies selectively bind an antigen, as such the term can be used herein interchangeably with the term “target”.","The term “anti-region antibody” as used herein refers to an antibody that was produced by immunizing an animal with a select region that is a fragment of a foreign antibody wherein only the fragment is used as the immunogen.","Anti-region antibodies include monoclonal and polyclonal antibodies.","The term “anti-region fragment” as used herein refers to a monovalent fragment that was generated from an anti-region antibody of the present invention by enzymatic cleavage.","The term “biotin” as used herein refers to any biotin derivative, including without limitation, substituted and unsubstituted biotin, and analogs and derivatives thereof, as well as substituted and unsubstituted derivatives of caproylamidobiotin, biocytin, desthiobiotin, desthiobiocytin, iminobiotin, and biotin sulfone.","The term “biotin-binding protein” as used herein refers to any protein that binds selectively and with high affinity to biotin, including without limitation, substituted or unsubstituted avidin, and analogs and derivatives thereof, as well as substituted and unsubstituted derivatives of streptavidin, ferritin avidin, nitroavidin, nitrostreptavidin, and Neutravidin™ avidin (a de-glycosylated modified avidin having an isoelectric point near neutral).","The term “buffer” as used herein refers to a system that acts to minimize the change in acidity or basicity of the solution against addition or depletion of chemical substances.","The term “capture reagent” refers to a non-specific immunoglobulin that is used to remove excess labeling reagent after the formation of the immuno-labeled complex.","The capture reagent is optionally attached a matrix to facilitate removal of the excess labeling regent.","A matrix typically includes a microsphere, an agarose bead or any solid surface that the excess labeling reagent can be passed by.","The term “chromophore” as used herein refers to a label that emits light in the visible spectra that can be observed without the aid of instrumentation.","The term “complex” as used herein refers to the association of two or more molecules, usually by non-covalent bonding, e.g., the association between an antibody and an antigen or the labeling reagent and the target-binding antibody.","The term “detectable response” as used herein refers to an occurrence of, or a change in, a signal that is directly or indirectly detectable either by observation or by instrumentation.","Typically, the detectable response is an occurrence of a signal wherein the fluorophore is inherently fluorescent and does not produce a change in signal upon binding to a metal ion or biological compound.","Alternatively, the detectable response is an optical response resulting in a change in the wavelength distribution patterns or intensity of absorbance or fluorescence or a change in light scatter, fluorescence lifetime, fluorescence polarization, or a combination of the above parameters.","Other detectable responses include, for example, chemiluminescence, phosphorescence, radiation from radioisotopes, magnetic attraction, and electron density.","The term “detectably distinct” as used herein refers to a signal that is distinguishable or separable by a physical property either by observation or by instrumentation.","For example, a fluorophore is readily distinguishable either by spectral characteristics or by fluorescence intensity, lifetime, polarization or photo-bleaching rate from another fluorophore in the sample, as well as from additional materials that are optionally present.","The term “directly detectable” as used herein refers to the presence of a material or the signal generated from the material is immediately detectable by observation, instrumentation, or film without requiring chemical modifications or additional substances.","The term “examination zone” as used herein refers to an optical zone of a flow cytometer, or a similar instrument, wherein cells are passed through essentially one at a time in a thin stream whereby the bound immuno-labeled complex is illuminated and the intensity and emission spectra of the fluorophore is detected and recorded.","This includes instruments wherein the examination zone moves and the sample is held in place.","The term “fluorophore” as used herein refers to a composition that is inherently fluorescent or demonstrates a change in fluorescence upon binding to a biological compound or metal ion, i.e., fluorogenic.","Fluorophores may contain substitutents that alter the solubility, spectral properties or physical properties of the fluorophore.","Numerous fluorophores are known to those skilled in the art and include, but are not limited to coumarin, cyanine, benzofuran, a quinoline, a quinazolinone, an indole, a benzazole, a borapolyazaindacene and xanthenes including fluoroscein, rhodamine and rhodol as well as other fluorophores described in RICHARD P. HAUGLAND, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (9th edition, CD-ROM, September 2002).","The term “immuno-labeled complex” refers to the complex of target-binding antibody that is non-covalently attached to a labeling reagent.","The term “immuno-labeled complex subset” as used herein refers to a discrete set of immuno-labeled complexes that are homogenous and can be distinguished from another subset of immuno-labeled complex by the physical properties of the label, or the ratio of the label to labeling reagent, or the ratio of labeling reagent to target-binding antibody, or the target-binding antibody.","Typically an immuno-labeled complex subset is present in a buffer to provide a “target detection solution”.","The term “kit” as used herein refers to a packaged set of related components, typically one or more compounds or compositions.","The term “label” as used herein refers to a chemical moiety or protein that retains it's native properties (e.g.","spectral properties, conformation and activity) when attached to a labeling reagent and used in the present methods.","The label can be directly detectable (fluorophore) or indirectly detectable (hapten or enzyme).","Such labels include, but are not limited to, radiolabels that can be measured with radiation-counting devices; pigments, dyes or other chromogens that can be visually observed or measured with a spectrophotometer; spin labels that can be measured with a spin label analyzer; and fluorescent labels (fluorophores), where the output signal is generated by the excitation of a suitable molecular adduct and that can be visualized by excitation with light that is absorbed by the dye or can be measured with standard fluorometers or imaging systems, for example.","The label can be a chemiluminescent substance, where the output signal is generated by chemical modification of the signal compound; a metal-containing substance; or an enzyme, where there occurs an enzyme-dependent secondary generation of signal, such as the formation of a colored product from a colorless substrate.","The term label can also refer to a “tag” or hapten that can bind selectively to a conjugated molecule such that the conjugated molecule, when added subsequently along with a substrate, is used to generate a detectable signal.","For example, one can use biotin as a tag and then use an avidin or streptavidin conjugate of horseradish peroxidate (HRP) to bind to the tag, and then use a colorimetric substrate (e.g., tetramethylbenzidine (TMB)) or a fluorogenic substrate such as Amplex Red reagent (Molecular Probes, Inc.) to detect the presence of HRP.","Numerous labels are know by those of skill in the art and include, but are not limited to, particles, fluorophores, haptens, enzymes and their calorimetric, fluorogenic and chemiluminescent substrates and other labels that are described in RICHARD P. HAUGLAND, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH PRODUCTS (9th edition, CD-ROM, September 2002), supra.","The term “labeling reagent” as used herein refers to a monovalent antibody fragment or a non-antibody monomeric protein provided that the labeling reagent has affinity for a selected region of the target-binding antibody and is covalently attached to a label.","The term “labeling reagent subset” as used herein refers to a discrete set of labeling reagents that are homogenous and can be distinguished from another subset of labeling reagent either by the physical properties of the label or the ratio of the label to labeling reagent.","The term “labeling solution” as used herein refers to a solution that is used to form an immuno-labeled complex wherein the solution comprises labeling reagents and a buffer.","The term “matrix” as used herein refers to a solid or semi-solid surface that a biological molecule can be attached to, such as a sample of the present invention or a capture reagent.","Examples include, but are not limited to, agarose, polyacrylamide gel, polymers, microspheres, glass surface, plastic surface, membrane, margnetic surface, and an array.","The term “monovalent antibody fragment” as used herein refers to an antibody fragment that has only one antigen-binding site.","Examples of monovalent antibody fragments include, but are not limited to, Fab fragments (no hinge region), Fab′ fragments (monovalent fragments that contain a heavy chain hinge region), and single-chain fragment variable (ScFv) proteins.","The term “non-antibody monomeric protein” as used herein refers to a protein that binds selectively and non-covalently to a member of the Ig superfamily of proteins, including but not limited to proteins A, G, and L, hybrids thereof (A/G), recombinant versions and cloned versions thereof, fusions of these proteins with detectable protein labels, and lectins but the protein itself is not an antibody or an antibody fragment.","The terms “protein” and “polypeptide” are used herein in a generic sense to include polymers of amino acid residues of any length.","The term “peptide” is used herein to refer to polypeptides having less than 100 amino acid residues, typically less than 10 amino acid residues.","The terms apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.","The term “purified” as used herein refers to a preparation of a target-binding antibody that is essentially free from contaminating proteins that normally would be present in association with the antibody, e.g., in a cellular mixture or milieu in which the protein or complex is found endogenously such as serum proteins or hybridoma supernatant.","The term “sample” as used herein refers to any material that may contain a target, as defined below.","Typically, the sample comprises a population of cells, cellular extract, subcellular components, tissue culture, a bodily fluid, and tissue.","The sample may be in an aqueous solution, a viable cell culture or immobilized on a solid or semi solid surface such as a gel, a membrane, a glass surface, a microparticle or on a microarray.","The term “target” as used herein refers to any entity that a target-binding antibody has affinity for such as an epitope or antigen.","This target includes not only the discrete epitope that the target-binding antibody has affinity for but also includes any subsequently bound molecules or structures.","In this way an epitope serves as a marker for the intended target.","For example, a cell is a target wherein the target-binding antibody binds a cell surface protein such as CD3 on a T cell wherein the target marker is CD3 and the target is the T cell.","The term “target-binding antibody” as used herein refers to an antibody that has affinity for a discrete epitope or antigen that can be used with the methods of the present invention.","Typically the discrete epitope is the target but the epitope can be a marker for the target such as CD3 on T cells.","The term can be used interchangeably with the term “primary antibody” when describing methods that use an antibody that binds directly to the antigen as opposed to a “secondary antibody” that binds to a region of the primary antibody.","II.","Compositions and Methods of Use In accordance with the present invention, labeling reagents, methods for labeling target-binging antibodies and methods for using the labeled antibodies to detect a target in a sample are provided.","The labeling reagents comprise monovalent antibody fragments or non-antibody monomeric proteins that are covalently attached to a label of the present invention.","The label covalently attached to a labeling reagent is directly detectable such as a fluorophore or functions as an indirect label that requires an additional component such as a calorimetric enzyme substrate or an enzyme conjugate.","The labeling reagents have affinity for a specific region of the target-binding antibody.","The target-binding antibodies are defined as any antibody known to one skilled in the art that has an affinity for a target in a sample.","The target-binding antibodies are labeled with the labeling reagent in a labeling method to form immuno-labeled complexes and then added to a sample to detect a target.","The labeling reagent and the methods of the present invention provide for detection of one or multiple targets in a sample.","Multiple targets are detected when either pooled subsets of immuno-labeled complexes or a panel of subsets that are sequentially added to a sample.","The subset of immuno-labeled complexes begins with labeling reagent subsets wherein a labeling reagent subset is distinguished by the ratio of label to labeling reagent or by the physical characteristics of the label.","The discrete labeling reagents subsets are added to the target-binding antibodies wherein the affinity of the antibody and ratio of labeling reagent to target-binding antibody determines the subsets of immuno-labeled complexes.","This results in an infinite number of immuno-labeled complex subsets that are distinguished by i) the target-binding antibody, or ii) a ratio of label to labeling reagent, or iii) a ratio of labeling reagent to the target-binding antibody or iv) by a physical property of the label.","These subsets can be used individually in a method of the present invention to detect a single or multiple targets in a sample or pooled and used to simultaneously detect multiple targets in a sample.","These pooled subsets allow for not only detection but also identification and quantitation of the targets.","A. Labelinq Reagents 1.Monovalent Antibody Fragments and Monomeric Non-Antibody Proteins The labeling reagents of the present invention are monovalent antibody fragments or non-antibody monomeric proteins that have affinity for a region of a target-binding antibody.","The regions of the target-binding antibody that can be bound by a labeling reagent include the Fc region, the kappa or lambda light chain region or a heavy chain region.","When the labeling reagent is derived from an antibody the monovalent fragment can be, anti-Fc, an anti-Fc isotype, anti-kappa light chain, anti-lambda light chain, or a single-chain fragment variable protein.","Labeling reagents that are a non-antibody peptide or protein, are for example but not limited to, soluble Fc receptor, protein G, protein A, protein L, lectins, or a fragment thereof.","The labeling reagents typically have affinity for the Fc region of the target-binding antibody but any region, except the binding domain, may be used as a binding site for the labeling reagent.","The Fc region is preferable because it is the farthest from the binding domain of the target-binding antibody and is unlikely to cause steric hinderance, when bound by a labeling reagent, of the binding domain for the target.","Antibody is a term of the art denoting the soluble substance or molecule secreted or produced by an animal in response to an antigen, and which has the particular property of combining specifically with the antigen that induced its formation.","Antibodies themselves also serve are antigens or immunogens because they are glycoproteins and therefore are used to generate anti-species antibodies.","Antibodies, also known as immunoglobulins, are classified into five distinct classes—IgG, IgA, IgM, IgD, and IgE.","The basic IgG immunoglobulin structure consists of two identical light polypeptide chains and two identical heavy polypeptide chains (linked together by disulfide bonds).","When IgG is treated with the enzyme papain, a monovalent antigen-binding fragment can be isolated, referred herein to as a Fab fragment.","When IgG is treated with pepsin (another proteolytic enzyme), a larger fragment is produced, F(ab′)2.This fragment can be split in half by treating with a mild reducing buffer that results in the monovalent Fab′ fragment.","The Fab′ fragment is slightly larger than the Fab and contains one or more free sulfhydryls from the hinge region (which are not found in the smaller Fab fragment).","The term “antibody fragment” is used herein to define both the Fab′ and Fab portions of the antibody.","It is well known in the art to treat antibody molecules with pepsin and papain in order to produce antibody fragments (Gorevic et al., Methods of Enzyol., 116:3 (1985)).","The monovalent Fab fragments of the present invention are produced from either murine monoclonal antibodies or polyclonal antibodies generated in a variety of animals that have been immunized with a foreign antibody or fragment thereof, U.S. Pat.","No.","4,196,265 discloses a method of producing monoclonal antibodies.","Typically, labeling reagents are derived from a polyclonal antibody that has been produced in a rabbit or goat but any animal known to one skilled in the art to produce polyclonal antibodies can be used to generate anti-species antibodies.","However, monoclonal antibodies are equal, and in some cases, preferred over polyclonal antibodies provided that the target-binding antibody is compatible with the monoclonal antibodies that are typically produced from murine hybridoma cell lines using methods well known to one skilled in the art.","Example 1 describes production of polyclonal antibodies raised in animals immunized with the Fc region of a foreign antibody.","It is a preferred embodiment of the present invention that the labeling reagents be generated against only the Fc region of a foreign antibody.","Essentially, the animal is immunized with only the Fc region fragment of a foreign antibody, such as murine.","The polyclonal antibodies are collected from subsequent bleeds, digested with an enzyme, pepsin or papain, to produce monovalent fragments.","The fragments are then affinity purified on a column comprising whole immunoglobulin protein that the animal was immunized against or just the Fc fragments.","As described in detail below, the labeling reagents are also covalently labeled with fluorophore labels when bound to the affinity column to eliminate incorporating label into the binding domain of the monovalent fragment.","One of skill in the art will appreciate that this method can be used to generate monovalent fragments against any region of a target-binding protein and that selected peptide fragments of the target-binding antibody could also be used to generate fragments.","Alternatively, a non-antibody protein or peptide such as protein G, or other suitable proteins, can be used alone or coupled with albumin wherein albumin is attached with a label of the present invention.","Preferred albumins of the invention include human and bovine serum albumins or ovalbumin.","Protein A, G and L are defined to include those proteins know to one skilled in the art or derivatives thereof that comprise at least one binding domain for IgG, i.e.","proteins that have affinity for IgG.","These proteins can be modified but do not need to be and are labeled in the same manner as the monovalent Fab fragments of the invention.","2.Labels The labels of the present invention include any directly or indirectly detectable label known by one skilled in the art that can be covalently attached to the labeling reagent of the present invention.","Labels include, without limitation, a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme and a radioisotope.","Preferred labels include fluorophores, fluorescent proteins, haptens, and enzymes.","A fluorophore of the present invention is any chemical moiety that exhibits an absorption maximum beyond 280 nm, and when covalently attached to a labeling reagent retains its spectral properties.","Fluorophores of the present invention include, without limitation; a pyrene (including any of the corresponding derivative compounds disclosed in U.S. Pat.","No.","5,132,432), an anthracene, a naphthalene, an acridine, a stilbene, an indole or benzindole, an oxazole or benzoxazole, a thiazole or benzothiazole, a 4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), a cyanine (including any corresponding compounds in U.S. Ser.","Nos.","09/968,401 and 09/969,853), a carbocyanine (including any corresponding compounds in U.S. Ser.","Nos.","09/557,275; 09/969,853 and 09/968,401; U.S. Pat.","Nos.","4,981,977; 5,268,486; 5,569,587; 5,569,766; 5,486,616; 5,627,027; 5,808,044; 5,877,310; 6,002,003; 6,004,536; 6,008,373; 6,043,025; 6,127,134; 6,130,094; 6,133,445; and publications WO 02/26891, WO 97/40104, WO 99/51702, WO 01/21624; EP 1 065 250 A1), a carbostyryl, a porphyrin, a salicylate, an anthranilate, an azulene, a perylene, a pyridine, a quinoline, a borapolyazaindacene (including any corresponding compounds disclosed in U.S. Pat.","Nos.","4,774,339; 5,187,288; 5,248,782; 5,274,113; and 5,433,896), a xanthene (including any corresponding compounds disclosed in U.S. Pat.","No.","6,162,931; 6,130,101; 6,229,055; 6,339,392; 5,451,343 and U.S. Ser.","No.","09/922,333), an oxazine (including any corresponding compounds disclosed in U.S. Pat.","No.","4,714,763) or a benzoxazine, a carbazine (including any corresponding compounds disclosed in U.S. Pat.","No.","4,810,636), a phenalenone, a coumarin (including an corresponding compounds disclosed in U.S. Pat.","Nos.","5,696,157; 5,459,276; 5,501,980 and 5,830,912), a benzofuran (including an corresponding compounds disclosed in U.S. Pat.","Nos.","4,603,209 and 4,849,362) and benzphenalenone (including any corresponding compounds disclosed in U.S. Pat.","No.","4,812,409) and derivatives thereof.","As used herein, oxazines include resorufins (including any corresponding compounds disclosed in Pat.","No.","5,242,805), aminooxazinones, diaminooxazines, and their benzo-substituted analogs.","When the fluorophore is a xanthene, the fluorophore is optionally a fluorescein, a rhodol (including any corresponding compounds disclosed in U.S. Pat.","Nos.","5,227,487 and 5,442,045), or a rhodamine (including any corresponding compounds in U.S. Pat.","Nos.","5,798,276; 5,846,737; U.S. Ser.","No.","09/129,015).","As used herein, fluorescein includes benzo- or dibenzofluoresceins, seminaphthofluoresceins, or naphthofluoresceins.","Similarly, as used herein rhodol includes seminaphthorhodafluors (including any corresponding compounds disclosed in U.S. Pat.","No.","4,945,171).","Alternatively, the fluorophore is a xanthene that is bound via a linkage that is a single covalent bond at the 9-position of the xanthene.","Preferred xanthenes include derivatives of 3H-xanthen-6-ol-3-one attached at the 9-position, derivatives of 6-amino-3H-xanthen-3-one attached at the 9-position, or derivatives of 6-amino-3H-xanthen-3-imine attached at the 9-position.","Preferred fluorophores of the invention include xanthene (rhodol, rhodamine, fluorescein and derivatives thereof) coumarin, cyanine, pyrene, oxazine and borapolyazaindacene.","Most preferred are sulfonated xanthenes, fluorinated xanthenes, sulfonated coumarins, fluorinated coumarins and sulfonated cyanines.","The choice of the fluorophore attached to the labeling reagent will determine the absorption and fluorescence emission properties of the labeling reagent and immuno-labeled complex.","Physical properties of a fluorophore label include spectral characteristics (absorption, emission and stokes shift), fluorescence intensity, lifetime, polarization and photo-bleaching rate all of which can be used to distinguish one fluorophore from another.","Typically the fluorophore contains one or more aromatic or heteroaromatic rings, that are optionally substituted one or more times by a variety of substituents, including without limitation, halogen, nitro, cyano, alkyl, perfluoroalkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, arylalkyl, acyl, aryl or heteroaryl ring system, benzo, or other substituents typically present on fluorophores known in the art.","In one aspect of the invention, the fluorophore has an absorption maximum beyond 480 nm.","In a particularly useful embodiment, the fluorophore absorbs at or near 488 nm to 514 nm (particularly suitable for excitation by the output of the argon-ion laser excitation source) or near 546 nm (particularly suitable for excitation by a mercury arc lamp).","Many of fluorophores can also function as chromophores and thus the described fluorophores are also preferred chromophores of the present invention.","In addition to fluorophores, enzymes also find use as labels for the labeling reagents.","Enzymes are desirable labels because amplification of the detectable signal can be obtained resulting in increased assay sensitivity.","The enzyme itself does not produce a detectable response but functions to break down a substrate when it is contacted by an appropriate substrate such that the converted substrate produces a fluorescent, colorimetric or luminescent signal.","Enzymes amplify the detectable signal because one enzyme on a labeling reagent can result in multiple substrates being converted to a detectable signal.","This is advantageous where there is a low quantity of target present in the sample or a fluorophore does not exist that will give comparable or stronger signal than the enzyme.","However, fluorophores are most preferred because they do not require additional assay steps and thus reduce the overall time required to complete an assay.","The enzyme substrate is selected to yield the preferred measurable product, e.g.","calorimetric, fluorescent or chemiluminescence.","Such substrates are extensively used in the art, many of which are described in the MOLECULAR PROBES HANDBOOK, supra.","A preferred calorimetric or fluorogenic substrate and enzyme combination uses oxidoreductases such as horseradish peroxidase and a substrate such as 3,3′-diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC), which yield a distinguishing color (brown and red, respectively).","Other calorimetric oxidoreductase substrates that yield detectable products include, but are not limited to: 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD), 3,3′,5,5′-tetramethylbenzidine (TMB), o-dianisidine, 5-aminosalicylic acid, 4-chloro-1-naphthol.","Fluorogenic substrates include, but are not limited to, homovanillic acid or 4-hydroxy-3-methoxyphenylacetic acid, reduced phenoxazines and reduced benzothiazines, including Amplexe Red reagent and its variants (U.S. Pat.","No.","4,384,042) and reduced dihydroxanthenes, including dihydrofluoresceins (U.S. Pat.","No.","6,162,931) and dihydrorhodamines including dihydrorhodamine 123.Peroxidase substrates that are tyramides (U.S. Pat.","Nos.","5,196,306; 5,583,001 and 5,731,158) represent a unique class of peroxidase substrates in that they can be intrinsically detectable before action of the enzyme but are “fixed in place” by the action of a peroxidase in the process described as tyramide signal amplification (TSA).","These substrates are extensively utilized to label targets in samples that are cells, tissues or arrays for their subsequent detection by microscopy, flow cytometry, optical scanning and fluorometry.","Another preferred calorimetric (and in some cases fluorogenic) substrate and enzyme combination uses a phosphatase enzyme such as an acid phosphatase, an alkaline phosphatase or a recombinant version of such a phosphatase in combination with a calorimetric substrate such as 5-bromo-6-chloro-3-indolyl phosphate (BCIP), 6-chloro-3-indolyl phosphate, 5-bromo-6-chloro-3-indolyl phosphate, p-nitrophenyl phosphate, or o-nitrophenyl phosphate or with a fluorogenic substrate such as 4-methylumbelliferyl phosphate, 6,8-difluoro-7-hydroxy4-methylcoumarinyl phosphate (DiFMUP, U.S. Pat.","No.","5,830,912) fluorescein diphosphate, 3-O-methylfluorescein phosphate, resorufin phosphate, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate (DDAO phosphate), or ELF 97, ELF 39 or related phosphates (U.S. Pat.","Nos.","5,316,906 and 5,443,986).","Glycosidases, in particular beta-galactosidase, beta-glucuronidase and beta-glucosidase, are additional suitable enzymes.","Appropriate colorimetric substrates include, but are not limited to, 5-bromo4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) and similar indolyl galactosides, glucosides, and glucuronides, o-nitrophenyl beta-D-galactopyranoside (ONPG) and p-nitrophenyl beta-D-galactopyranoside.","Preferred fluorogenic substrates include resorufin beta-D-galactopyranoside, fluorescein digalactoside (FDG), fluorescein diglucuronide and their structural variants (U.S. Pat.","Nos.","5,208,148; 5,242,805; 5,362,628; 5,576,424 and 5,773,236), 4-methylumbelliferyl beta-D-galactopyranoside, carboxyumbelliferyl beta-D-galactopyranoside and fluorinated coumarin beta-D-galactopyranosides (U.S. Pat.","No.","5,830,912).","Additional enzymes include, but are not limited to, hydrolases such as cholinesterases and peptidases, oxidases such as glucose oxidase and cytochrome oxidases, and reductases for which suitable substrates are known.","Enzymes and their appropriate substrates that produce chemiluminescence are preferred for some assays.","These include, but are not limited to, natural and recombinant forms of luciferases and aequorins.","Chemiluminescence-producing substrates for phosphatases, glycosidases and oxidases such as those containing stable dioxetanes, luminol, isoluminol and acridinium esters are additionally useful.","In addition to enzymes, haptens such as biotin are also preferred labels.","Biotin is useful because it can function in an enzyme system to further amplify the detectable signal, and it can function as a tag to be used in affinity chromatography for isolation purposes.","For detection purposes, an enzyme conjugate that has affinity for biotin is used, such as avidin-HRP.","Subsequently a peroxidase substrate is added to produce a detectable signal.","Haptens also include hormones, naturally occurring and synthetic drugs, pollutants, allergens, affector molecules, growth factors, chemokines, cytokines, lymphokines, amino acids, peptides, chemical intermediates, nucleotides and the like.","Fluorescent proteins also find use as labels for the labeling reagents of the present invention.","Examples of fluorescent proteins include green fluorescent protein (GFP) and the phycobiliproteins and the derivatives thereof.","The fluorescent proteins, especially phycobiliprotein, are particularly useful for creating tandem dye labeled labeling reagents.","These tandem dyes comprise a fluorescent protein and a fluorophore for the purposes of obtaining a larger stokes shift wherein the emission spectra is farther shifted from the wavelength of the fluorescent protein's absorption spectra.","This is particularly advantageous for detecting a low quantity of a target in a sample wherein the emitted fluorescent light is maximally optimized, in other words little to none of the emitted light is reabsorbed by the fluorescent protein.","For this to work, the fluorescent protein and fluorophore function as an energy transfer pair wherein the fluorescent protein emits at the wavelength that the fluorophore absorbs at and the fluorphore then emits at a wavelength farther from the fluorescent proteins than could have been obtained with only the fluorescent protein.","A particularly useful combination is the phycobiliproteins disclosed in U.S. Pat.","Nos.","4,520,110; 4,859,582; 5,055,556 and the sulforhodamine fluorophores disclosed in Pat.","No.","5,798,276, or the sulfonated cyanine fluorophores disclosed in U.S. Ser.","Nos.","09/968/401 and 09/969/853; or the sulfonated xanthene derivatives disclosed in Pat.","No.","6,130,101 and those combinations disclosed in U.S. Pat.","4,542,104.Alternatively, the fluorophore functions as the energy donor and the fluorescent protein is the energy acceptor.","3.Covalent Attachment of Labels to the Labeling Reagents The labeling reagents can be independently attached to one or more labels of the present invention by a number of methods known to one skilled in the art and modification of such methods.","Methods include, labeling in a solution or on an affinity column.","For labeling in solution the labeling reagent is optionally modified to contain a reactive group and the label is modified to contain a reactive group or is synthesized to contain a reactive group, as is typically the case with fluorophore labels wherein the reactive group facilitates covalent attachment.","The modification of the labeling reagent to contain a reactive group includes (1) chemical addition of such a reactive group or (2) alternatively takes advantage of the disulfide bonds of the F(ab′)2 fragment wherein the fragment is reduced to break the bond and expose the thiol group that readily reacts with a reactive group on a label, as disclosed in U.S. Pat.","No.","5,360,895.Typically, covalent attachment of the label to the fragment is the result of a chemical reaction between an electrophilic group and a nucleophilic group.","However, when a label is used that is photoactivated the covalent attachment results when the labeling solution is illuminated.","A method for covalently attaching a label, particularly an enzyme, a fluorescent protein or a particle, comprises the following steps: a) cleaving an intact anti-region antibody with an enzyme resulting in a F(ab′)2 fragment; b) contacting said F(ab′)2 fragment with a reducing agent to produce Fab′ fragments containing a thiol group; c) contacting said Fab′ fragments with a solution comprising a label that contains a reactive group; and, d) isolating Fab′ fragments of step d) that are covalently attached to a label by size exclusion or affinity chromatography.","The whole anti-region antibody is cleaved with pepsin to generate a bivalent F(ab)′2 fragment.","This fragment is typically affinity purified on a column comprising immunoglobulin proteins such as IgG that is immobilized on agarose.","The fragment is then reduced to break the disulfide bond of the hinge region that connects the two Fab fragments resulting in a Fab′ fragment with an exposed thiol group.","This is typically accomplished by adding a mild reducing buffer to the affinity purified F(ab′)2 fragments such as a buffer comprising 0.01 M EDTA and 0.01 M cysteine in phosphate buffer saline (PBS).","The resulting thiol group readily reacts with a reactive group on a label to covalently attach the label to the fragment.","Thus, a solution containing a label that has been chemically modified to contain a reactive group, using methods well known to one skilled in the art, is added to the solution of reduced Fab′ fragments.","This method is particularly useful for covalently attaching enzyme and other protein labels due to their size and the lack of exposed amine groups on the Fab fragments.","One of skill in the art will appreciate that this method requires the use of Fab′ fragments as apposed to Fab fragments due to the disulfide bonds of the Fab′ fragment and that the use of the enzyme papain or the like results in such a fragment.","An alternative labeling of monovalent antibody fragments and the monomeric non-antibody proteins is also accomplished in a solution.","The method comprises the steps: a) contacting a Fab fragment or non-antibody monomeric protein with a solution comprising a label that contains a reactive group; and, b) isolating labeled anti-region Fab fragment or non-antibody monomeric protein by size exclusion or affinity chromatography.","When a Fab fragment is to be labeled the whole antibody is cleaved with an enzyme, such as papain, to generate Fab monovalent fragments and the fragments are typically purified on an affinity column prior to addition of the label.","The Fab fragment or non-antibody monomeric proteins are optionally chemically modified to contain a reactive group.","However, for covalently attaching reactive fluorophore labels it has been found that this modification of the fragment of non-antibody protein is not necessary.","The reactive label, typically a fluorophore or hapten, are added to a solution of Fab fragments or non-antibody proteins and the labeling reagent is separated from excess label by size exclusion or affinity chromatorgraphy.","The labeling reagents are then stored in an appropriate buffer.","Labeling in solution can have some drawbacks, especially when labeling of Fab fragments or non-antibody proteins with fluorophores.","Thus, Fab fragments and non-antibody proteins of the present invention are preferably covalently attached to a fluorophore label when immobilized on an affinity column.","The fragments and non-antibody proteins are immobilized on an affinity column that comprises a protein that the fragment has affinity for, typically IgG, and after immobilization a reactive fluorophore is added to the column wherein the fragments are labeled and unreacted fluorophores pass through the column.","The use of this affinity chromatography method avoids the incorporation of label into the binding domain of the Fab fragment or non-antibody protein.","When Fab fragments are labeled with fluorophores using this method unexpected advantages were obtained wherein the fluorescent signal form fragments labeled on a column are brighter than fragments labeled in solution when the fluorophore and ratio of fluorophore to labeling reagent are held constant.","Without wishing to be bound by a theory it is possible that the decreased brightness observed from the fragments labeled in solution is due to quenching of fluorphores that are bound in or near the binding domain by the high concentration of amine groups in the binding domain.","Thus, a preferred embodiment of the invention for covalently attaching fluorphore labels to Fab fragments comprises the following steps: a) cleaving an intact anti region antibody with an enzyme that generates Fab fragments; b) isolating the anti-region Fab fragments of step a); c) contacting a matrix comprising intact immunoglobulin proteins or fragments thereof that specifically bind anti-region Fab fragments with a solution comprising said anti-region fragments of step b) wherein said Fab fragments are immobilized; d) contacting said matrix of step c) with a solution comprising a fluorophore label that contains a reactive group; e) washing said matrix to remove unbound label, and; f) eluting said labeling reagent from said matrix whereby said labeling reagent is manufactured comprising a label and being isolated from other proteins and fragments thereof.","The matrix is typically an agarose column that comprises either the selected region, such as the Fc region, or the entire antibody provided that the antibody or fragment thereof is the same species and isotype that was used to produce the antibodies that the labeling reagent was generated from.","However any matrix known to one skilled in the art can be used that allows for immobilization of labeling reagent and removal following attachment of the fluorophore label.","Fab and Fab′ fragments can both be labeled in this manner.","However a free thiol group is not necessary and therefore Fab fragments are typically labeled using this method.","Due to the unique properties of the labeling reagent and the attached labels it is a preferred embodiment of the present invention that enzyme or other protein labels are covalently attached to Fab′ fragments in solution utilizing the free thiol group of the Fab′ fragment.","It is another preferred embodiment that fluorophore labels be covalently attached to the labeling reagent when the reagent is immobilized on a affinity column wherein the labeling reagent is typically an Fab fragment or a non-antibody monomeric protein.","The attachment of the label to the fragments or the non-antibody proteins results in multiple subsets that are distinguished by the ratio of the label to the labeling reagent and the physical properties of the label.","A labeling reagent subset as used herein refers to a discrete set of labeling reagents that are homogenous and can be distinguished from another subset of labeling reagent either by the physical properties of the label or the ratio of the label to labeling reagent.","The physical properties include differences within a group of labels, such as emission spectra of fluorphores, or across groups of labels, such as the difference between an enzyme and a fluorophore.","For fluorphore labels, the physical properties typically relates to the emission spectra, this includes modification of the same label, e.g.","a cyanine with different substitutions that shifts the emission wavelength, or different fluorophores, e.g.","a cyanine and a coumarin on the same labeling reagent.","The difference in physical properties also includes the use of tandem dyes, which is specifically defined to include an energy transfer pair wherein one is a protein and the other is a fluorophore or both are fluorophores, or the pairing of other labels that are not necessarily energy transfer pairs.","A few examples of labeling reagent subsets includes, but are not limited to, a first subset comprising a single fluorophore at a known ration attached to a anti-Fc Fab fragment; a second subset comprises the same fluorophore on the Fab fragment at a different known ration from the first subset, a third subset comprises the same fluorophore but that has a shifted wavelength due to a substitution on the fluorophore.","Thus, the attachment of labels to the labeling reagents results in an extensive selection of subsets that when complexed with a target-binding antibody results in a unique method to detect one or multiple targets in a sample whereby the target is identified and quantitated.","B. Immuno-Labeled Complex The subsets of labeling reagent are complexed with target-binding antibodies to produce subsets of immuno-labeled complex that for the target detection solution.","The methods for forming the immuno-labeled complex comprises the following steps: a) contacting a solution of target-binding antibodies with a labeling reagent subset, wherein said labeling reagent subsets are distinguished by i) ratio of label to labeling reagent or ii) a physical properties of said label; b) incubating said target-binding antibodies and said labeling reagent for a time period sufficient for one or more labeling reagents to form an immuno-labeled complex with a target-binding antibody wherein a region of said target binding antibody is selectively bound by labeling reagent; c) optionally removing unbound labeling reagent by adding a capture reagent comprising immunoglobulin proteins or fragments thereof; and, d) optionally repeating said steps a), b), and c) to form individual or pooled subsets of immuno-labeling complexes wherein each subset is distinguished from another subset by i) a ratio of label to labeling reagent, or ii) a physical property of said label, or iii) a ratio of labeling reagent to said target-binding antibody, or iv) by said target-binding antibody.","A particular advantage for the use of labeling reagent of the present invention to label target-binding antibodies is that the process is relatively insensitive to the solution the antibodies are in.","Due to the physical nature of the labeling reagents, small monovalent fragments, the reagents do not cross-link and fall out of solution in the presence of high concentration of proteins.","For this reason, target-binding antibodies can be complexed when present in ascites fluid, tissue culture supernatant, serum or other solutions where there is a high concentration of proteins.","This eliminates the need to purify target-binding proteins prior to labeling.","When preparing the immuno-labeled complex using purified target-binding antibody, stock solutions of both the labeling reagent and the target-binding antibody are typically near 1 mg/mL in an appropriate buffer, although more or less concentrated solutions are also suitable.","Generally, the labeling reagent is mixed in a molar ratio of at least one to 50 moles of labeling reagent to one mole of the target-binding antibody to be complexed.","More commonly a ratio of at least one to as many as 10 moles of labeling reagent per mole of target-binding antibody is combined.","With an anti-Fc region Fab to a target-binding antibody, a molar ratio of approximately 2 to 10 is typical, more typically 3 to 5 (particularly for complexes in which the labeling reagent has been labeled while immobilized on an affinity matrix).","The ease of formation of the complex permits rapid optimization of the complex and assessment of the effect of variation in experimental parameters.","A particularly unique advantage of the invention is that the stoichiometry of the complex is easily adjusted to provide complexes with different ratios of labeling reagent to target-binding antibody, and thus there is control over the ultimate detectability of the target in the sample.","Complexes that have been labeled with the same dye but at different molar ratios can be separately detected by the differences in their intensities.","Complex formation appears to occur almost within the mixing time of the solutions (<1 minute) but the reaction typically is allowed to proceed for at least 5 minutes and can be longer before combining the immuno-labeled complex with the sample.","Although complex formation can be reversed by addition of an unlabeled antibody that contains the same binding region, reversibility is very slow; furthermore, following binding of the immuno-labeled complex to a target in a sample, the sample can be “fixed” using aldehyde-based fixatives by methods that are commonly practiced by those skilled in the art of immunolabeling.","The labeling process optionally further comprises the addition of a capture component to remove excess labeling reagent.","For applications in which immunolabeling complexes of multiple primary antibodies from the same species (e.g.","mouse monoclonal antibodies) or cross-reacting species (e.g.","mouse and human antibodies) are to be used simultaneously or sequentially, it is necessary to quench or otherwise remove any excess labeling reagent by use of a capture component or by other means to avoid inappropriate labeling of the sample.","The most effective capturing components to capture excess labeling reagent are those that contain the binding site of the labeling reagent but are themselves not labeled, preferably an antibody or antibody fragment.","Capture components may be free in solution or immobilized on a matrix, such as agarose, cellulose, or a natural or synthetic polymer, to facilitate separation of the excess capture component from the immuno-labeled complex.","The capture component is optionally attached to a microsphere or magnetic particle.","However, separation of excess labeling reagent is not essential for successful utilization of the invention, particularly when using a single target-binding antibody.","The steps of the labeling process for the target-binding antibodies can be repeated to form discrete immuno-labeled complex subsets that can be used individually or pooled in an assay to detect individual or multiple targets.","As used herein the term immuno-labeled complex subsets refers to subsets that are distinguished from each other i) a ratio of label to labeling reagent, or ii) a physical property of the label, or iii) a ratio of labeling reagent to the target-binding antibody, or iv) by the target-binding antibody, or a combination thereof.","For example a panel of subsets may comprise a target-binding antibody that is bound by a labeling reagent comprising a subset of different ratios of the same label on the labeling reagent resulting in a discrete subset of immuno-labeled complexes.","This subset of immuno-labeled complexes can be used individually wherein a target is identified by the intensity of the detectable label or used in combination with another subset of immunocomplexes that differ in the target-binding antibody to identify multiple targets.","C. Methods of Use The labeling reagents, target-binding antibodies and resulting immuno-labeled complex that forms the target detection solution can be used in a wide range of immunoassays, essentially in any assay a traditional secondary antibody is used including some assays that secondary antibodies are not used because of their size and ability to cross-link.","Examples of such assays used to detect a target in a sample include immunoblots, direct detection in a gel, flow cytometry, immunohistochemistry, confocal microscopy, fluorometry, ELISA and other modified immunoassays.","A method of the present invention for detecting a single target in a sample comprises the following steps: a) contacting a solution of target-binding antibodies with a labeling reagent subset, wherein said labeling reagent subsets are distinguished by i) ratio of label to labeling reagent or ii) a physical properties of said label; b) incubating said target-binding antibodies and said labeling reagent subset for a time period sufficient for one or more labeling reagents to form an immuno-labeled complex with a target-binding antibody wherein a region of said target binding antibody is selectively bound by labeling reagent; c) contacting said sample with said immuno-labeled complex of step b); d) incubating said sample of step c) for a time sufficient to allow said immuno-labeled complex to selectively bind to said target; and, e) illuminating said immuno-labeled complex whereby said target is detected.","A sample is incubated with a preformed immuno-labeled complex that comprises a labeling reagent and a target-binding antibody.","While this method describes the identification of a single target, subsets of labeling reagents bound to the same target-binding antibody can be used to identify and provide additional information about such targets.","For example, subsets of labeling reagent can be prepared wherein two discrete subsets are generate each with a distinct fluorophore label that is distinguished by their emission spectra, e.g.","one that emits in the green spectra and one that emits in the red spectra.","The labeling reagent subsets are then added to a solution of target-binding antibody in a controlled ratio, e.g.","two parts one labeling reagent (green emission) and one part the other labeling reagent (red emission) per target binding antibody.","In this way the immuno-labeled complexes can be used to detect a target.","If another immuno-labeled complex were added to the sample the original target could be distinguished from the subsequently detected target.","The methods of the present invention also provide for the detection of multiple targets in a sample.","Multiple targets include the discrete epitope that the target-binding antibody has affinity for as well as molecules or structures that the epitiope is bound to.","Thus, multiple target identification includes phenotyping of cells based on the concentration of the same cell surface marker on different cells.","In this way multiple target identification is not limited to the discrete epitope that the target binding antibody binds, although this is clearly a way that multiple targets can be identified, i.e.","based on the affinity of the target-binding antibody.","Therefore, a method for detecting multiple targets in a sample comprises the following steps: a) contacting a solution of target-binding antibodies with a labeling reagent subset, wherein said labeling reagent subsets are distinguished by i) ratio of label to labeling reagent or ii) a physical properties of said label; b) incubating said target-binding antibodies and said labeling reagent subset for a time period sufficient for one or more labeling reagents to form an immuno-labeled complex with a target-binding antibody wherein a region of said target-binding antibody is selectively bound by labeling reagent, wherein steps a) and b) are repeated to form discrete immuno-labeling complex subsets; c) contacting said sample with a solution comprising A) a pooled subset of immuno-labeled complexes, wherein each subset is distinguished from another subset by i) a ratio of label to labeling reagent, or ii) a physical property of said label, or iii) a ratio of labeling reagent to said target-binding antibody, or iv) by said target-binding antibody or B) an individual subset wherein step c) with a solution comprising an individual subset is repeated; d) incubating said sample of step c) for a time sufficient to allow said immuno-labeled complex to selectively bind to said target; and, e) illuminating said immuno-labeled complex whereby said target is detected.","A selected target-binding antibody and a subset of labeling reagent are incubated to form an immuno-labeled complex subset.","This procedure is repeated to form a panel of immuno-labeled complex subsets that may be pooled and added to a sample.","Altematively each immuno-labeled complex subset is added stepwise to a sample.","The immuno-labeled complex subsets are distinguished by four characteristics resulting in an infinite number of immuno-labeled complex subsets.","First (i) the subsets can be distinguished by the target-binding antibody that is determined by the end user for the information that is desired from a sample.","This means that each subset is distinguished based on the affinity of the target-binding antibody.","The target-binding antibody typically distinguishes immuno-labeled complexes when multiple targets are identified, however this is normally combined with another characteristic to gain information form a sample or increase the number of targets that can be detected at one time.","The second (ii) distinguishing feature used is the ratio of label to labeling reagent, as discussed in detail above.","A subset based on this feature would have for example a ratio of two fluorophore per each labeling reagent.","The third (iii) distinguishing feature is the ratio of labeling reagent to target-binding antibody.","This is accomplished using a controlled concentration of target-binding antibody mixed with a controlled concentration of a labeling reagent subset and the subset would comprise a target-binding antibody that is bound by a discrete number of labeling proteins.","The fourth (iv) feature is the physical feature of the label.","Typically this refers to the physical properties of the fluorophore labels wherein a subset of this group is distinguished by the label itself such as a green emitting fluorophore compared to a red emitting fluorophore.","One of skill in the art will appreciate that while immuno-labeling complex subsets can be distinguished based on one feature the subsets are typically, and most useful, when discretely identified based on a combination of the distinguishing characteristics.","Another example of detection of multiple targets utilizes the following immuno-labeled subsets, all of which comprise a different target-binding antibody but differ in the label and ratio of label.","The first subset comprises a fluorophore label that emits red-fluorescent light, a second subset comprises a fluorophore label that emits green fluorescent light, a third subset comprises a ratio of 1:1 red to green fluorophore label; a fourth subset comprises a ratio of 2:1 red to green fluorophore label and a fifth subset comprises a ratio of 1:2 red to green fluorophore label.","These subsets allow for the simultaneous detection of five targets in a sample.","This aspect of the present invention is particularly important due to the limited range of fluorophores available wherein the labeling reagents can be utilized to increase the number of targets that can be detected at one time.","One of skill in the art can appreciate that these subsets could be expanded by altering the ratio of label to labeling reagent instead of just the ratio of labeling reagent to target-binding antibody.","This same methodology can also be applied to a single fluorophore label wherein the ratios are altered and a target is detected based on the intensity of the signal instead of the color and the ratio of the color to another color.","Following the formation of the immuno-labeled complex subsets the subsets can be pooled and added to a sample or added stepwise to a sample, either of which is determined by the end user and the particular assay format.","This method of the present invention provides for maximum flexibility and ease of determining multiple targets in a sample.","Another method of the present invention provides for the determination of multiple targets in a sample specifically using the flow cytometry assay format.","Traditionally targets identified using flow cytometry used either directly labeled primary antibody or labeled microspheres that were covalently attached to a primary antibody wherein the microsphere is the label.","Examples include the fluorescent encapsulated microsphere beads sold by Luminex.","The labeling reagents and the present invention overcome both the need for directly labeled primary antibody and the need for expensive microspheres.","Thus, a method of the present invention for determining identity and quantity of targets in a sample by detecting multiple targets comprises the following steps: a) contacting a solution of target-binding antibodies with a labeling reagent subset, wherein said labeling reagent subsets are distinguished by i) ratio of label to labeling reagent or ii) a physical properties of said label; b) incubating said target-binding antibodies and said labeling reagent for a time period sufficient for one or more labeling reagents to form an immuno-labeled complex with a target-binding antibody wherein a region of said target binding antibody is selectively bound by labeling reagent, wherein steps a) and b) are repeated to form a pooled subset of immuno-labeling complexes; c) contacting a population of cells in a sample with a solution comprising a pooled subset of immuno-labeled complexes, wherein each subset is distinguished from another subset by i) a ratio of label to labeling reagent, or ii) a physical property of said label, or iii) a ratio of labeling reagent to said target-binding antibody, or iv) by said target-binding antibody; d) incubating said cells for a time period sufficient to allow said immuno-labeled complex to bind said targets; e) passing said incubated population of cells through an examination zone; and, f) collecting data from said cells that were passed through said examination zone wherein said multiple targets are detected whereby the identity and quantity of said targets is determined.","In one aspect, a target-binding antibody is pre-complexed to the target-binding antibody to form a subset and that subset or a panel of subsets are added to a sample, that are typically distinguished by the target binding antibody.","This method then avoids the need for a directly labeled primary.","Secondly, when the panel of subsets is distinguished, for example, by the ratio of label to labeling reagent or the ratio of labeling reagent to target-binding antibody the immuno-labeled complex can function similar to the microsphere beads of Luminex.","For example, this is accomplished wherein three immuno-labeled complex subsets are distinguished by the target binding antibody and the fluorophore attached to the labeling reagent and within one of the subsets is another set of subsets that are distinguished based on the ratio of label to labeling reagent.","In this way three different epitopes are detected and one of the epitopes is further distinguished and a phenotype distinction made based on the intensity of the signal generated from the labeled-immuno complex subsets based on the ratio of fluorophore to labeling reagent.","This determination of targets is facilitated when a population of cells or cellular organelles is passed through the examination zone of a flow cytometer wherein the fluorescent signal and intensity is recorded for each cell resulting in a histogram of the cell population or cellular organelles based on the detected epitopes.","In another aspect of the invention, additional detection reagents are combined with the sample concurrently with or following the addition of immuno-labeled complex subsets.","Such additional detection reagents include, but are not limited to reagents that selectively detect cells or subcellular components, ions, or indicate the cell viability, life cycle, or proliferation state.","For example, the additional detection reagent is a labeled target-binding antibody that is directly or indirectly detectable and another additional detection reagent is a stain for nucleic acids, for F-actin, or for a cellular organelle.","1.Sample Preparation The sample is defined to include any material that may contain a target to which an antibody has affinity for.","Typically the sample is biological in origin and comprises tissue, cell or a population of cells, cell extracts, cell homogenates, purified or reconstituted proteins, recombinant proteins, bodily and other biological fluids, viruses or viral particles, prions, subcellular components, or synthesized proteins.","Possible sources of cellular material used to prepare the sample of the invention include without limitation plants, animals, fungi, bacteria, archae, or cell lines derived from such organisms.","The sample can be a biological fluid such as whole blood, plasma, serum, nasal secretions, sputum, saliva, urine, sweat, transdermal exudates, cerebrospinal fluid, or the like.","Alternatively, the sample may be whole organs, tissue or cells from an animal.","Examples of sources of such samples include muscle, eye, skin, gonads, lymph nodes, heart, brain, lung, liver, kidney, spleen, solid tumors, macrophages, mesothelium, and the like.","Prior to combination with the immuno-labeled complexes, the sample is prepared in a way that makes the target, which is determined by the end user, in the sample accessible to the immuno-labeled complexes.","Typically, the samples used in the invention are comprised of tissue, cells, cell extracts, cell homogenates, purified or reconstituted proteins, recombinant proteins, biological fluids, or synthesized proteins.","Large macromolecules such as immuno-labeled complexes tend to be impermeant to membranes of live biological cells.","Treatments that permeabilize the plasma membrane, such as electroporation, shock treatments, or high extracellular ATP, can be used to introduce the immuno-labeled complexes into cells.","Alternatively, the immuno-labeled complexes can be physically inserted into cells, e.g.","by pressure microinjection, scrape loading, patch-clamp methods, or phagocytosis.","However, the desired target may require purification or separation prior to addition of the immuno-labeled complexes, which will depend on the way the antigenic determinants are contained in the sample.","For example, when the sample is to be separated on a SDS-polyacrylamide gel the sample is first equilibrated in an appropriate buffer, such as a SDS-sample buffer containing Tris, glycerol, DTT, SDS, and bromophenol blue.","When the sample contains purified target materials, the purified target materials may still be mixtures of different materials.","For example, purified protein or nucleic acid mixtures may contain several different proteins or nucleic acids.","Alternatively, the purified target materials may be electrophoresed on gels such as agarose or polyacrylamide gels to provide individual species of target materials that may be subsequently blotted onto a polymeric membrane or detected within the gel matrix.","Preparation of a sample containing purified nucleic acids or proteins generally includes denaturation and neutralization.","DNA may be denatured by incubation with base (such as sodium hydroxide) or heat.","RNA is also denatured by heating (for dot blots) or by electrophoresing in the presence of denaturants such as urea, glyoxal, or formaldehyde, rather than through exposure to base (for Northern blots).","Proteins are denatured by heating in combination with incubation or electrophoresis in the presence of detergents such as sodium dodecyl sulfate.","The nucleic acids are then neutralized by the addition of an acid (e.g., hydrochloric acid), chilling, or addition of buffer (e.g., Tris, phosphate or citrate buffer), as appropriate.","Preferably, the preparation of a sample containing purified target materials further comprises immobilization of the target materials on a solid or semi-solid support.","Purified nucleic acids are generally spotted onto filter membranes such as nitrocellulose filters or nylon membranes in the presence of appropriate salts (such as sodium chloride or ammonium acetate) for DNA spot blots.","Alternatively, the purified nucleic acids are transferred to nitrocellulose filters by capillary blotting or electroblotting under appropriate buffer conditions (for Northern or Southern blots).","To permanently bind nucleic acids to the filter membranes, standard cross-linking techniques are used (for example, nitrocellulose filters are baked at 80° C. in vacuum; nylon membranes are subjected to illumination with 360 nm light).","The filter membranes are then incubated with solutions designed to prevent nonspecific binding of the nucleic acid probe (such as BSA, casein hydrolysate, single-stranded nucleic acids from a species not related to the probe, etc.)","and hybridized to probes in a similar solution.","Purified proteins are generally spotted onto nitrocellulose or nylon filter membranes after heat and/or detergent denaturation.","Alternatively, the purified proteins are transferred to filter membranes by capillary blotting or electroblotting under appropriate buffer conditions (for Western blots).","Nonspecifically bound probe is washed from the filters with a solution such as saline-citrate or phosphate buffer.","Filters are again blocked, to prevent nonspecific adherence of immuno-labeled complexes.","Finally, samples are mixed with immuno-labeled complexes.","Nonspecifically bound immuno-labeled complexes are typically removed by washing.","When the sample contains cellular nucleic acids (such as chromosomal or plasmid-borne genes within cells, RNA or DNA viruses or mycoplasma infecting cells, or intracellular RNA) or proteins, preparation of the sample involves lysing or permeabilizing the cell, in addition to the denaturation and neutralization already described.","Cells are lysed by exposure to agents such as detergent (for example sodium dodecyl sulfate, Tween, sarkosyl, or Triton), lysozyme, base (for example sodium, lithium, or potassium hydroxide), chloroform, or heat.","Cells are permeabilized by conventional methods, such as by formaldehyde in buffer.","As with samples containing purified target materials, preparation of the sample containing cellular target materials typically further comprises immobilization of the target materials on a surface such as a solid or semi-solid matrix.","The targets may be arrayed on the support in a regular pattern or randomly.","These supports include such materials as slides, polymeric beads including latex, optical fibers, and membranes.","The beads are preferably fluorescent or nonfluorescent polystyrene, the slides and optical fibers are preferably glass or plastic, and the membrane is preferably poly(vinylidene difluoride) or nitrocellulose.","Thus, for example, when the sample contains lysed cells, cells in suspension are spotted onto or filtered through nitrocellulose or nylon membranes, or colonies of cells are grown directly on membranes that are in contact with appropriate growth media, and the cellular components, such as proteins and nucleic acids, are permanently bound to filters as described above.","Permeabilized cells are typically fixed on microscope slides with known techniques used for in situ hybridization and hybridization to chromosome “squashes” and “spreads,” (e.g., with a reagent such as formaldehyde in a buffered solution).","Alternatively, the samples used may be in a gel or solution.","In a particular aspect of the invention, the sample comprises of cells in a fluid, such as ascites, hybridoma supernatant, or serum, wherein the presence or absence of the target in such cells is detected by using an automated instrument that sorts cells according to the detectable fluorescence response of the detectable moieties in the immunolabeling complexes bound to such cells, such as by fluorescence activated cell sorting (FACS).","For methods using flow cytometry a cell population typically comprises individually isolated cells that have been isolated from other proteins and connective tissue by means well known in the art.","For example, lymphocyte cells are isolated from blood using centrifugation and a density gradient.","The cells are washed and pelleted and the labeling solution added to the pelleted cells.","2.Illumination At any time after addition of the immuno-labeled complex to the sample, the sample is illuminated with a wavelength of light selected to give a detectable optical response, and observed with a means for detecting the optical response.","Equipment that is useful for illuminating the fluorescent compounds of the present invention includes, but is not limited to, hand-held ultraviolet lamps, mercury arc lamps, xenon lamps, lasers and laser diodes.","These illumination sources are optically integrated into laser scanners, fluorescent microplate readers or standard or microfluorometers.","The degree and/or location of signal, compared with a standard or expected response, indicates whether and to what degree the sample possesses a given characteristic, i.e.","desired target.","The optical response is optionally detected by visual inspection, or by use of any of the following devices: CCD camera, video camera, photographic film, laser-scanning devices, fluorometers, photodiodes, quantum counters, epifluorescence microscopes, scanning microscopes, flow cytometers, fluorescence microplate readers, or by means for amplifying the signal such as photomultiplier tubes.","Where the sample is examined using a flow cytometer, examination of the sample optionally includes sorting portions of the sample according to their fluorescence response.","When an indirectly detectable label is used then the step of illuminating typically includes the addition of a reagent that facilitates a detectable signal such as colorimetric enzyme substrate.","Radioisotopes are also considered indirectly detectable wherein an additional reagent is not required but instead the radioisotope must be exposed to X-ray film or some other mechanism for recording and measuring the radioisotope signal.","This can also be true for some chemiluminescent signals that are best observed after expose to film.","III.","Kits of the Invention Suitable kits for preparing an immuno-labeled complex and for detection of a target in a sample also form part of the invention.","Such kits can be prepared from readily available materials and reagents and can come in a variety of embodiments.","The contents of the kit will depend on the design of the assay protocol or reagent for detection or measurement.","Generally, the kits will contain instructions, appropriate reagents and labels, and solid supports, as needed.","Typically, instructions include a tangible expression describing the reagent concentration or at least one assay method parameter such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions and the like to allow the user to carry out any one of the methods or preparations described above.","A preferred kit of the present invention comprises: a) a labeling solution comprising a labeling reagent that is independently attached to one or more labels and b) a solution comprising a capture reagent.","A preferred emodiment of this kit provides a labeling reagent that is anti-Fc Fab fragment, protein G or protein G complexed with albumin.","In a more particular embodiment of this kit, the capture component is purified mouse IgG or non-immune mouse serum and the albumin is human albumin, bovine serum albumin, or ovalbumin.","In a more preferred embodiment the albumin is ovalbumin.","The labeling solution is either a homogenous mixture of labeling reagents or comprises a pooled subset of labeling reagents.","Alternatively the kit comprises a panel of labeling reagent subsets that can be used to make a subset of immuno-labeled complexes.","Additionally the kits may comprise one or more additional components that include (a) stains for characterization of cellular organelles, cell viability, or cell proliferation state, (b) enzyme substrates or (c) enzyme conjugates such as avidin-HRP.","A wide variety of kits and components can be prepared according to the present invention, depending upon the intended user of the kit and the particular needs of the user.","It is understood by one skilled in the art, that any of the labeling reagents contemplated by the present invention can be used to in a labeling solution to be included in a kit.","The labeling reagents are not intended to be limited to only the described preferred embodiments.","IV.","Applications The instant invention has useful applications in basic research, high-throughput screening, immunohistochemistry, fluorescence in situ hybridization (FISH), microarray technology, flow cytometry, diagnostics, and medical therapeutics.","The invention can be used in a variety of assay formats for diagnostic applications in the disciplines of microbiology, immunology, hematology and blood transfusion, tissue pathology, forensic pathology, and veterinary pathology.","The invention is particularly useful in the characterization and selection of optimized antibodies from hybridoma supernatants.","Additionally, the invention can be used to deliver therapeutics to a specific target.","In general, the current invention provides a versatile and convenient method to enhance any assay that uses an antibody as part of its detection methodology.","The instant invention can be used to study biological phenomena, such as, for example, cell proliferation, signal transduction in cells, or apoptosis.","For illustration purposes only and not limitation, one could study thymidine analog 5-bromo-2′-deoxyuridine (BrdU) incorporation.","BrdU is a marker for both cell proliferation and apoptosis, as it is readily incorporated into newly synthesized DNA that has progressed through the S-phase of the cell cycle and also into DNA break sites by deoxynucleotidyl transferase (TdT).","Anti-BrdU antibodies are used to detect cells marked by BrdU incorporation.","By being able to directly label the anti-BrdU antibodies, the current invention provides a convenient method to allow for detection of the incorporated BrdU by conventional immunohistochemistry or fluorescence, depending on detection method required.","Additionally, the current invention has the advantage of allowing staining for multiple targets in one cocktail, thereby reducing the need for more samples or processing steps per experiment.","This is particularly important when analyzing precious samples (e.g., pediatric samples, leukocytes isolated from biopsies, rare antigen-specific lymphocytes and mouse tissues that yield a small number of cells).","Although it is currently possible to simultaneously measure up to 11 distinct fluorescent colors through a convoluted series of novel developments in flow cytometry hardware, software, and dye chemistry, the use of these advances has been severely limited by the lack of commercial availability of spectrally distinct directly labeled primary and secondary antibodies.","Although labeled secondary antibodies directed at individual isotype-specific targeting antibodies (e.g., anti-lgG, isotype antibodies) exist, it is not possible to use this type of labeled antibody to detect more than one of the same isotype of an antibody (e.g., an IgG1 isotype antibody) in a single sample due to cross-reactivity.","The current invention overcomes these limitations by providing for a convenient and extremely versatile method of rapidly labeling either small or large quantities of any primary antibody including primary antibodies of the same isotype to be used in, for example, multicolor flow cytometry and on Western blots.","This advance in multicolor systems has a number of advantages over current two- and three-color flow cytometric measurements.","For example, no combination of one-color stains can accurately enumerate or be used to isolate CD3+ CD4+ CD8− T cells (excluding, for example CD3+ CD4+ CD8+ T cells and small CD4+ monocytes).","The use of cell membrane markers to study leukocyte composition in blood and tissue serves as an example of an analytical monoclonal antibody application, particularly in combination with flow cytometry.","It is also the example most relevant to studies of the immune system, because the cellular composition of blood and lymphoid tissue provides a ‘window’, allowing the analysis and monitoring of the immune system.","The methods of the invention can also be used in immunofluorescence histochemistry.","This technique involves the use of antibodies labeled with fluorophores to detect substances within a specimen.","The pathologist derives a great deal of information of diagnostic value by examining thin sections of tissue in the microscope.","Tissue pathology is particularly relevant to, for example, the early diagnosis of cancer or premalignant states, and to the assessment of immunologically mediated disorders, including inflammation and transplant rejection.","The problems associated with immunofluorescence histochemistry, however, stem from the limitations of the methods currently available for use in such application.","For example, directly labeling an antibody can result in antibody inactivation and requires a relatively large of amount of antibody and time to do the conjugation.","It is also expensive and impractical to prepare directly labeled antibodies having variable degrees of label substitution.","Similarly, indirect labeling of an antibody has problems, such as lack of secondary antibody specificity, and reliance upon primary antibody differences, including antibody isotypes and available fluorophores, to do multicolor labeling.","Secondary antibody labeling is not practical where the primary antibody is from the same species or of the same isotypes.","Combinations of fluorophores or other detectable labels on the same target-binding antibody, which can be readily prepared in multiple mixtures by the methods on this invention, greatly increase the number of distinguishable signals in multicolor protocols.","Lack of secondary antibody specificity arises when the specimen containing the targeted moiety and target-binding antibody are from homologous species.","For example, BrdU-labeled DNA in rodent tissue is detected by immunohistochemical staining.","The target-binding antibody is conventionally mouse anti-BrdU, and the detecting antibody system uses an anti-mouse immunoglobulin antibody, labeled with fluorescein.","Because there is homology between mouse immunoglobulin and immunoglobulins from a number of rodent species (for example, rats, mice, hamsters, etc.","), the detecting antibody not only binds to the target-binding antibody, but also nonspecifically binds to immunoglobulin in the tissue.","The current invention eliminates this problem by pre-forming the immunolabeling complex and allows for a simple, rapid and convenient method to proceed with labeling with two, three or more fluorescent antibodies in one experiment.","Very significantly, it can always be used with primary antibodies of either the same or different isotype, and always on tissue of the same or similar species as the primary antibody.","The instant invention also has application in the field of microarrays.","Microarray technology is a powerful platform for biological exploration (Schena (Ed.","), Microarray Biochip Technology, (2000)).","Many current applications of arrays, also known as “biochips,” can be used in functional genomics as scientists seek characteristic patterns of gene expression in different physiopathological states or tissues.","A common method used in gene and protein microarray technology involves the use of biotin, digoxigenin (DIG), or dinitrophenyl (DNP) as an epitope or a “tag” such as an oligohistidine, glutathione transferase, hemagglutinin (HA), or c-myc.","In this case a detectably labeled anti-biotin, anti-DIG, anti-DNP, anti-oligohistidine, anti-glutathione transferase, anti-HA, or anti-c-myc is used as the detection reagent.","The instant invention allows for the use of multiple fluorophore- or enzyme-labeled antibodies, thereby greatly expanding the detection modalities and also providing for enhanced multiplexing and two-dimensional analysis capabilities.","Similarly, the invention can be used with protein microarrays and on Western blots.","Protein microarrays can provide a practical means to characterize patterns of variation in hundreds of thousands of different proteins in clinical or research applications.","Antibody arrays have been successfully employed that used a set of 115 antibody/antigen pairs for detection and quantitation of multiple proteins in complex mixtures (Haab et al., Genome Biology, 2, 4.1 (2001)).","However, protein microarrays use very low sample volumes, which historically have significantly limited the use of antibody technology for this application.","The invention of the application readily overcomes this limitation and provides a means to label antibodies with the fluorescent dyes using a very low sample volume and to automate formation of the staining complex and the staining process.","The present invention also provides a means for the specific detection, monitoring, and/or treatment of disease and contemplates the use of immunolabeling complexes to detect the presence of particular targets in vitro.","In such immunoassays, the sample may be utilized in liquid phase, in a gel, or bound to a solid-phase carrier, such as an array of fluorophore-labeled microspheres (e.g., U.S. Pat.","No.","5,981,180 and 5,736,330).","For example, a sample can be attached to a polymer, such as aminodextran, in order to link the sample to an insoluble support such as a polymer-coated bead, plate, or tube.","For instance, but not as a limitation, using the methods of the present invention in an in vitro assay, antibodies that specifically recognize an antigen of a particular disease are used to determine the presence and amounts of this antigen.","Likewise, the immunolabeling complexes of the present invention can be used to detect the presence of a particular target in tissue sections prepared from a histological specimen.","Preferably, the tissue to be assayed will be obtained by surgical procedures, e.g., biopsy.","The excised tissue will be assayed by procedures generally known in the art, e.g.","immunohistochemistry, for the presence of a desired target that is recognized by an immunolabeling complex, as described above.","The tissue may be fixed or frozen to permit histological sectioning.","The immunolabeling complex may be labeled, for example with a dye or fluorescent label, chemical, heavy metal or radioactive marker to permit the detection and localization of the target-binding antibody in the assayed tissue.","In situ detection can be accomplished by applying a detectable immunolabeling complex to the tissue sections.","In situ detection can be used to determine the presence of a particular target and to determine the distribution of the target in the examined tissue.","General techniques of in situ detection are well known to those of ordinary skill.","See, for example, Ponder, “Cell Marking Techniques and Their Application,” in MAMMALIAN DEVELOPMENT: A PRACTICAL APPROACH, Monk (ed.","), 115 (1987).","For diagnosing and classifying disease types, tissues are probed with an immuno-labeled complex, as defined above, that comprises a target-binding antibody to a target antigen associated with the disease, e.g., by immunohistochemical methods.","Where the disease antigen is present in body fluids, such immuno-labeled complexes comprising a target-binding antibody to the disease antigen are preferably used in immunoassays to detect a secreted disease antigen target.","Detection can be by a variety of methods including, for example, but not limited to, flow cytometry and diagnostic imaging.","When using flow cytometry for the detection method, the use of microspheres, beads, or other particles as solid supports for antigen-antibody reactions in order to detect antigens or antibodies in serum and other body fluids is particularly attractive.","Flow cytometers have the capacity to detect particle size and light scattering differences and are highly sensitive fluorescence detectors.","Microfluidic devices provide a means to perform flow-based analyses on very small samples.","Alternatively, one can use diagnostic imaging.","The method of diagnostic imaging with radiolabeled antibodies is well known.","See, for example, Srivastava (ed.","), RADIOLABELED MONOCLONAL ANTIBODIES FOR IMAGING AND THERAPY, Plenum Press (1988); Chase, “Medical Applications of Radioisotopes,” in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, Gennaro et al.","(eds.)","Mack Publishing Co., 624 (1990); and Brown, “Clinical Use of Monoclonal Antibodies,” in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al.","(eds.","), Chapman & Hall, 227 (1993).","This technique, also known as immunoscintigraphy, uses a gamma camera to detect the location of gamma-emitting radioisotopes conjugated to antibodies.","Diagnostic imaging is used, in particular, to diagnose cardiovascular disease and infectious disease.","Thus, the present invention contemplates the use of immuno-labeled complexes to diagnose cardiovascular disease.","For example, immuno-labeled complexes comprising anti-myosin antibodies can be used for imaging myocardial necrosis associated with acute myocardial infarction.","Immuno-labeled complexes comprising antibodies that bind platelets and fibrin can be used for imaging deep-vein thrombosis.","Moreover, immuno-labeled complexes comprising antibodies that bind to activated platelets can be used for imaging atherosclerotic plaque.","Immuno-labeled complexes of the present invention also can be used in the diagnosis of infectious diseases.","For example, immuno-labeled complexes comprising antibodies that bind specific bacterial antigens can be used to localize abscesses.","In addition, immuno-labeled complexes comprising antibodies that bind granulocytes and inflammatory leukocytes can be used to localize sites of bacterial infection.","Similarly, the immuno-labeled complexes of the present invention can be used to detect signal transduction in cells, the products of signal transduction, and defects, inhibitors, and activators of signal transduction.","Numerous studies have evaluated the use of antibodies for scintigraphic detection of cancer.","Investigations have covered the major types of solid tumors such as melanoma, colorectal carcinoma, ovarian carcinoma, breast carcinoma, sarcoma, and lung carcinoma.","Thus, the present invention contemplates the detection of cancer using immuno-labeled complexes comprising antibodies that bind tumor markers (targets) to detect cancer.","Examples of such tumor markers include carcinoembryonic antigen, a-fetoprotein, oncogene products, tumor-associated cell surface antigens, and necrosis-associated intracellular antigens.","In addition to diagnosis, antibody imaging can be used to monitor therapeutic responses, detect recurrences of a disease, and guide subsequent clinical decisions and surgical procedures.","In vivo diagnostic imaging using fluorescent complexes that absorb and emit light in the near infrared (such as those of the Alexa Fluor 700 and Alexa Fluor 750 dyes) is also known.","EXAMPLES The following examples describe specific aspects of the invention to illustrate the invention and to provide a description of the methods for those of skill in the art.","The examples should not be construed as limiting the invention, as the examples merely provide specific methodology useful in understanding and practicing the invention.","Example 1 Preparation of Fc Antigen Purified mouse and rabbit IgG was fragmented with the proteolytic enzyme papain (CURRENT PROTOCOLS IN CELL BIOLOGY, 16.4.1-16.4.10 (2000)).","A 12 mL solution of mouse IgG was prepared at ˜2 mg/mL in phosphate-buffered saline (PBS).","A solution containing 0.1 mg of papain in digestion buffer (PBS, 0.02 M EDTA, 0.02 M cysteine) was added to the antibody and allowed to react at 37° C. for 16 hours.","The digestion was terminated by the addition 20 μL of 0.3 M iodoacetamide in PBS.","The fragments were dialyzed against 2 L of PBS for 16 hours at 4° C. The Fc fragment was purified on a protein G-Sepharose CL-4B column.","The bound fraction containing the Fc fragment was eluted from the column using 50-100 mM glycine/HCl buffer, pH 2.5-2.8.The eluate was collected in 1 mL fractions.","The pH of the protein fractions was immediately raised to neutral by addition of 100 μL of either 500 mM phosphate or Tris buffer, pH 7.6, to each 1 mL fraction.","The solution was then loaded onto a Sephacryl S-200 Superfine size-exclusion column and fractions corresponding to a molecular weight of -50 kDa were collected and analyzed by SDS-PAGE and HPLC.","Example 2 Production of Anti-Fc Antibodies Polyclonal antibodies specific for the Fc region of an antibody were raised in goats against the purified FC region of an antibody from a different species (Example 1).","Methods of immunizing animals are well known in the art, and suitable immunization protocols and immunogen concentrations can be readily determined by those skilled in the art (Current Protocols in Immunology 2.4.1-9 (1995); ILAR Journal 37, 93 (1995)).","Briefly, individual goats were immunized with purified mouse Fc or purified rabbit Fc fragments.","The initial immunization in 50% Freund's complete adjuvant (1000 μg conjugate (half subcutaneous, half intramuscularly)) was followed by 500 pg conjugate per goat in Freund's incomplete adjuvant two and four weeks later and at monthly intervals thereafter.","Antibodies were purified from serum using protein A-Sepharose chromatography.","Antibodies against mouse Fc isotypes can be prepared by starting with isotype-selected mouse Fc antigens.","Rabbits have a single Fc isotype.","Characterization of the selectivity and cross-reactivity of isotype-specific antibodies is by standard techniques, including HPLC.","Example 3 Preparation of Fab Fragments Fragmentation of the goat anti-(mouse Fc) antibody to the monovalent Fab fragment was carried out using the proteolytic enzyme, papain, as described in Example 1.Following dialysis against PBS, the Fab fragment was purified on a protein A-Sepharose CL-4B column.","The unbound fraction containing the Fab fragment and the papain was collected.","This solution was then loaded onto a Sephacryl S-200 Superfine size-exclusion column and fractions corresponding to a molecular weight of ˜50 kDa were collected and analyzed by SDS-PAGE.","The Fab fragments of goat anti-(rabbit Fc) can be prepared similarly.","Example 4 Preparation of the Labeled Antibody Immunoglobulin-Binding Protein or the Non-Antibody Immunoglobulin-Binding Peptide and Protein Conjugates in Homogeneous Solution Conjugates of antibody immunoglobulin-binding protein or the non-antibody immunoglobulin-binding peptides or proteins with low molecular weight dyes and haptens such as biotin or digoxigenin are typically prepared from succinimidyl esters of the dye or hapten, although reactive dyes and haptens having other protein-reactive functional groups are also suitable.","The typical method for protein conjugation with succinimidyl esters is as follows.","Variations in molar ratios of dye-to-protein, protein concentration, time, temperature, buffer composition and other variables that are well known in the art are possible that still yield useful conjugates.","A protein solution of the Fab fragment of goat anti-(rabbit Fc), goat anti-(mouse Fc), protein A, protein G, or protein L or an immunoglobulin-binding peptide (e.g., a peptide identified by screening a library of peptides) is prepared at ˜10 mg/mL in 0.1 M sodium bicarbonate (pH ˜8.3).","The labeling reagents are dissolved in a suitable solvent such as DMF at ˜10 mg/mL.","Predetermined amounts of the labeling reagents are added to the protein solution with stirring.","A molar ratio of 10 moles of dye to 1 mole of protein is typical, though the optimal amount can be varied with the particular labeling reagent, the protein being labeled and the protein's concentration.","The optimal ratio was determined empirically.","When optimizing the fluorescence yield and determining the effect of degree of substitution (DOS) on the conjugate's brightness, it is typical to vary the ratio of reactive dye to protein over a several-fold range.","The reaction mixture is incubated at room temperature for a period that is typically one hour or on ice for several hours.","The dye-protein conjugate is typically separated from unreacted reagents by size-exclusion chromatography, such as on BIO-RAD P-30 resin equilibrated with PBS.","The initial, protein-containing band is collected and the DOS is determined from the absorbance at the absorbance maximum of each fluorophore, using the extinction coefficient of the free fluorophore.","The DOS of nonchromophoric labels, such as biotin, is determined as described in Haugland (Haugland et al., Meth.","Mol.","Biol.","45, 205 (1995); Haugland, Meth.","Mol.","Biol.","45, 223 (1995); Haugland, Meth.","Mol.","Biol.","45, 235 (1995); Haugland, Current Protocols in Cell Biol.","16.5.1-16.5.22 (2000)).","Using the above procedures, conjugates of goat anti-(mouse Fc) and goat anti-(rabbit Fc) were prepared with several different Alexa Fluor dyes, with Oregon Green dyes, with biotin-X succinimidyl ester, with desthiobiotin-X succinimidyl ester, with succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and with succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate (SMCC).","Some dye conjugates of protein A and protein G, including those of some Alexa Fluor dyes, are commercially available, such as from Molecular Probes.","Inc. (Eugene, Oreg.).","The interspecies specificity and approximate affinity of some other non-antibody immunoglobulin-binding proteins bind to segments of a target antibody, such as that of protein A and protein G are known (Langone, Adv.","Immunol.","32,157 (1982); Surolia et al., Trends Biochem.","Sci.","7, 74 (1982); Notani et al., J. Histochem.","Cytochem.","27, 1438 (1979); Goding, J. Immunol.","Meth.","20, 241 (1978); J. Immunol.","Meth.","127, 215 (1990); Bjorck et al., J. Immunol.","133, 969 (1984)).","In addition, labeling proteins (goat Fab anti-(mouse Fc), goat Fab anti-(mouse lambda light chain), goat Fab anti-(mouse kappa light chain), protein A, protein G, protein L, lectins, single-chain fragment variable antibodies (ScFv) ) conjugated to the detectable labels of R-phycoerythrin (R-PE), allophycocyanin (APC), tandem conjugates of phycobiliproteins with chemical dyes including several Alexa Fluor dyes, horseradish peroxidase (HRP), Coprinus cinereus peroxidase, Arthromyces ramosus peroxidase, glucose oxidase and alkaline phosphatase (AP) were or can be prepared by standard means (Haugland et al., Meth.","Mol.","Biol.","45, 205 (1995); Haugland, Meth.","Mol.","Biol.","45, 223 (1995); Haugland, Meth.","Mol.","Biol.","45, 235 (1995); Haugland, Current Protocols in Cell Biol 16.5.1-16.5.22 (2000)).","Fusion proteins, such as of protein G or protein A with detectable labels such as luciferin, aequorin, green-fluorescent protein and alkaline phosphatase are also known that are suitable for practice of the invention (Sun et al., J. Immunol.","Meth.","152, 43 (1992); Eliasson et al., J. Biol.","Chem.","263, 4323 (1988); Eliasson et al., J. Immunol.","142, 575 (1989)).","Immunoglobulin heavy and light chains, like most secreted and membrane bound proteins, are synthesized on membrane-bound ribosomes in the rough endoplasmic endoplasmic reticulum where N-linked glycosylation occurs.","The specificity of lectins for carbohydrates, including N-linked glycoproteins, is also known (EY laboratories, Inc. Lectin Conjugates Catalog, 1998).","Example 5 Preparation of the Labeled Antibody Immunoglobulin-Binding Protein or the Non-Antibody Immunoglobulin-Binding Peptide and Protein Conjugates While Bound to an Affinity Matrix Unlabeled Fab fragment for goat anti-(mouse Fc) (prepared as in Example 3) was bound to agarose-immobilized mouse IgG for one hour.","Following a wash step with bicarbonate buffer, pH 8.3, the complex of immobilized IgG and unlabeled Fab was labeled for one hour at room temperature with the succinimidyl ester of the amine-reactive label.","Unconjugated dye was eluted with bicarbonate buffer, and then the covalently labeled Fab fragment was eluted with 50-100 mM glycine/HCl buffer, pH 2.5-2.8.The eluate was collected in 1 mL fractions.","The pH of the protein fractions was immediately raised to neutral by addition of 100 μL of either 500 mM phosphate or Tris buffer, pH 7.6, to each 1 mL fraction.","Variations of the reagent concentrations, labeling times, buffer composition, elution methods and other variables are possible that can yield equivalent results.","Conjugates of the Fab fragment of goat anti-(rabbit Fc) and of protein G and protein A are prepared similarly.","Example 6 Comparison of the Alexa Fluor 488 Dye-Labeled Fab Fragments of Goat Anti-(Mouse Fc) Prepared as in Example 4 and as in Example 5 Conjugates of the Fab fragment of goat anti-(mouse Fc) with the Alexa Fluor 488 succinimidyl ester were separately prepared, as described in Examples 4 and 5.The conjugates had estimated degrees of substitution of ˜1.9 (labeled as in Example 4) and ˜3.0 (labeled as in Example 5), respectively, and virtually identical absorption and emission spectral maxima.","When excited at 488 nm, conjugates prepared using the fragment prepared as described in Example 5 were about 3.2-times more fluorescent than using the fragments that were prepared in Example 4 (FIG.","8) as detected by flow cytometry when bound to CD3 on Jurkat T cells.","Similar results were observed with other dyes.","Example 7 Preparation of a Labeling Protein from Protein G and Albumins Native protein G has a high affinity binding (nanomolar) site for albumins, in particular ovalbumin.","Equal weights of protein G and Texas Red ovalbumin (Molecular Probes.","Inc.) were dissolved in PBS, pH 7.5.After one hour, the resulting complex was separated on a Sephacryl S-200 Superfine size-exclusion column and analyzed by SDS-PAGE and HPLC.","Alternatively, the protein G is combined with a labeled albumin while the protein G is immobilized on any of the several immunoglobulins to which it binds, and the excess labeled albumin is washed away preceding elution of the albumin-labeled protein G complex from the matrix.","Example 8 Preparation of an Immunolabeling Complex on a Very Small Scale Submicrogram quantities of a target-binding antibody were complexed with submicrograms of a labeling protein in varying molar ratios of between about 1:1 and 1:20 to prepare an immunolabeling complex that was suitable for staining a sample.","For instance, 0.1 μg of mouse monoclonal anti-tubulin in 1 μL PBS with 0.1% BSA was complexed with 0.5 μg of the Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse Fc) (prepared as in Example 4) or with 0.1 μg of the Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse Fc) (prepared as in Example 5) in 5 μL of PBS for 10 minutes at room temperature.","The immunolabeling complex can be used immediately for staining tubulin in fixed-cell preparations (Example 16) or any excess unbound Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse Fc) in the immunolabeling complex can be captured with non-immune mouse IgG (Example 9) for combination with other antibody conjugates, including those of targeting antibodies that have been directly conjugated to other labels.","Rabbit antibodies were labeled similarly using labeled goat anti-(rabbit Fc).","Labeling of targeting antibodies with a labeled protein A, protein L, protein G, protein G complexed with a labeled albumin, or other immunoglobulin-binding peptides or proteins proceeds similarly.","In the case of a mouse (or rat) monoclonal antibody, it is preferred to use a labeled protein that is selective for the specific isotype of the primary antibody (e.g.","anti-(mouse IgG,) for a mouse IgG1 isotype primary antibody).","Although some cross-reactivity for other mouse (or rat) isotypes was observed using a goat antibody that was selective for mouse IgG, isotype monoclonal antibodies, routine and optimal use for labeling unmatched mouse isotypes required greater amounts of immunolabeling complexes and was somewhat less reliable.","Example 9 Capturing Excess Immunoglobulin-Binding Protein by a Capturing Component Immunolabeling complexes were prepared as described in Example 8.To the immunolabeling complex was added to each tube 25 μL of a 14.1 mg/mL stock solution of unlabeled mouse IgG to capture excess immunolabeling complexes.","As shown in FIG.","1, not all of the immunoglobulin-binding protein was necessarily complexed with the target-binding antibody to form an immunolabeling complex.","Consequently, particularly for applications in which labeling complexes of multiple primary antibodies from the same species (e.g.","mouse monoclonal antibodies) or crossreacting species (e.g.","mouse and human antibodies, FIG.","2, Table 1) were to be used simultaneously or sequentially, it is necessary to quench or otherwise remove any excess immunoglobulin-binding protein by use of a capturing component or by other means to avoid inappropriate labeling of the sample.","The most effective capturing component to capture excess immunoglobulin-binding protein is one that contains the binding site of the targeting agent.","For instance, whole mouse IgG or mouse serum was shown to be an effective and inexpensive reagent when the immunoglobulin-binding protein was bound to a segment of a mouse monoclonal antibody.","The mouse IgG was added in excess to the amount of immunoglobulin-binding protein and incubated for a period of approximately 1-5 minutes, or longer.","It is preferred to prepare the immunolabeling complex and then add the capturing component shortly before the experiment.","The rapid quenching effect permits this to be done within minutes of performing labeling of the sample by the immunolabeling complex.","If desired, the excess capturing component can be removed following labeling of the sample by a simple wash step.","Alternatively, fixation of the stained sample by aldehyde-based fixatives or other reagents or methods subsequent to incubation with the immunolabeling complex can provide permanent immobilization of the immunolabeling complex on its target in the sample.","As an alternative to adding a soluble capturing component to the immunolabeling complex, the capturing component can be immobilized on an insoluble matrix such as agarose and the immunolabeling complex contacted with that matrix.","A preferred matrix when labeling mouse antibodies to mouse antigens is mouse IgG immobilized on agarose.","Excess labeled anti-rabbit antibodies can be captured using rabbit IgG that is free in solution or immobilized.","Alternatively, the immunolabeling complex can be separated from any capturing component by chromatographic or electrophoretic means.","Example 10 HPLC Analysis of a Labeling Complex In order to analyze the success and extent of complex formation of the labeling protein with the target-binding antibody, size exclusion HPLC of the samples was performed.","For instance, a complex of Alexa Fluor 488 dye-labeled goat Fab anti-(mouse Fc) with a monoclonal mouse anti-tubulin in molar ratios of approximately 1:1, 3:1, 5:1 and 10:1.These were separated by analytical HPLC using a BioSep S-3000 column and eluting with 0.1 M NaPi, 0.1 M NaCl, pH 6.8, at a flow rate of 0.25 mLs/min.","An example of the separation using the 5:1 molar ratio (FIG.","6) demonstrates that, using this molar ratio, formation of the labeled complex is essentially quantitative.","Example 11 Cross-Reactivity of Goat Fab Anti-(Mouse Fc) to Other Species of IgG Microplates were equilibrated overnight with IgG from a mouse or non-mouse species, and then further blocked with BSA.","Variable amounts of the biotinylated Fab fragment of goat anti-(mouse Fc) were added to each well and allowed to bind.","After washing, streptavidin-HRP and the Amplex Red peroxidase substrate were added.","HRP activity was detected by the addition of H2O2 using the Amplex Red Peroxidase Assay Kit (Molecular Probes, Inc., Eugene, Oreg.).","Reactions containing 200 μM Amplex Red reagent, 1 U/mL HRP and 1 mM H2O2 (3% solution) in 50 mM sodium phosphate buffer, pH 7.4, were incubated for 30 minutes at room temperature.","Fluorescence was measured with a fluorescence microplate reader using excitation at 560±10 nm and fluorescence detection at 590±10 nm.","Background fluorescence, determined for a no-H2O2 control reaction, was subtracted from each value (Table 1 and FIG.","2).","Table 1 shows that the goat anti-(mouse Fc) antibody because of the highly conserved structure of the Fc region of an antibody it can be used to complex other non-mouse antibodies, including rat, and human antibodies.","The goat anti-mouse IgG antibody reaction with mouse antibody was set at 100% and the crossreacting antibodies were expressed as a percentage compared the mouse on mouse data.","The data in Table 1 show that the Fab fragment of the goat anti-(mouse Fc) antibody of the current invention does not strongly bind to the goat or sheep Fc domain; however, one skilled in the art could generate antibodies that will react with the goat and sheep Fc domain or the Fc domain of any other species.","Biotinylated Fab goat anti-(mouse Fc) was used in this example because it provided a convenient method to quantitate the amount of crossreactivity in a conventional method but it could have been accomplished using a fluorophore Fab labeled goat anti-(mouse Fc).","It was demonstrated by HPLC (as in Example 10) that Alexa Fluor 488 dye-labeled goat anti-(rabbit Fc) bound to rabbit primary antibodies.","TABLE 1 Cross-reactivity of goat anti-mouse IgG antibody with other non-mouse antibodies.","Species Crossreactivity % Fluorescence Mouse ++++ 100 Rat +++ 80.7 Human ++ 66.7 Rabbit + 16.9 Goat − 6.5 Sheep − 5.7 Example 12 Determination of the Optimal Molar Ratio of Immunoglobulin-Binding Protein to Target Antibody Using a Microplate Assay To 1.6 μg of mouse monoclonal anti-biotin (MW ˜145,000) in 8.0 μL PBS was added varying amounts of the Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse Fc) (MW ˜50,000) (prepared as in Example 4) to form an immunolabeling complex.","After equilibration for 20 min, a 100 μL aliquot was added to a 96-well microplate coated with biotinylated BSA.","After 30 minutes, the plates were washed and the residual fluorescence was quantitated using a fluorescence microplate reader using excitation at 485±10 nm and detecting emission at 530±12.5 nm.","As shown in FIG.","3, a molar ratio of the Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse Fc) to the anti-biotin between 5 to 20 was sufficient to form appreciably detectable complexes (FIG.","3; fluorescence quantitated, performed in triplicate (circles); control experiments performed but without adding the primary anti-biotin antibody (solid squares)).","A molar ratio of about 5 to about 10 was preferred for this pair of immunoglobulin-binding protein and target antibody.","This ratio can be varied somewhat to increase or decrease the signal or to affect the consumption of valuable reagents.","The weight ratio of immunoglobulin-binding protein to target-binding antibody is particularly affected by the actual molecular weight of the immunoglobulin-binding protein.","For instance, equal weights of the dye-labeled goat Fab anti-(mouse Fc) (prepared as in Example 5) and an intact mouse primary antibody, which corresponds to an approximately 3 to 1 molar ratio, usually yields suitable labeling complexes.","Fluorescence intensity (or enzymatic activity) of the immunolabeling complex is readily adjusted by a corresponding adjustment of the amount of labeled Fab fragment used.","Similar analyses of the ratio for other labeling proteins (including those of labeled protein A, protein G, protein L, IgG-binding peptides and antibodies to other segments of the primary antibody), and for conjugates of labels other than Alexa Fluor 488 dye (including enzymes in combination with the appropriate enzyme substrates) are done essentially as described in this example.","Example 13 Dissociation Rate of the Immunolabeling Complex A pre-equilibrated immunolabeling complex was prepared from 50 μg of an Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse Fc) and 15 μg of an anti-biotin monoclonal antibody (mAb).","The immunolabeling complex was rapidly diluted with capturing component sufficient to give a 6.2 molar excess over the anti-biotin mAb.","At various times, an aliquot was taken and added to a microplate well containing an excess of biotinylated BSA.","After 30 minutes, the plates were washed and the remaining fluorescence was quantitated.","Displacement of the labeling protein from the target-binding antibody through exchange was measured by any time-dependent decrease in fluorescence in the microplate well.","For example the fragments prepared as described in Example 4 had 68 percent fragments bound to the target-binding antibody after 30 minutes compared to 87 percent of bound fragments that were prepared according to Example 5.One hour showed a similar decrease, 56 percent and 68 percent respectively.","The labeling protein was shown to undergo a stable interaction with the target-binding antibody, with a lifetime for half exchange under these conditions of 3.5 hours.","Dissociation rates were measured for labeling protein prepared according to Example 4 and for labeling protein prepared according to Example 5, demonstrating the greater stability of immunolabeling complexes made using the labeling proteins prepared according to Example 5.Example 14 Protocol for Staining Cultured Cells with a Single Immunolabeling Complex Culturable cells, such as bovine pulmonary artery endothelial cells (BPAEC), were grown on a 22 x 22 mm glass coverslip.","The cells were fixed for 10 minutes using 3.7% formaldehyde in DMEM with fetal calf serum (FCS) at 37° C. The fixed cells were washed 3 times with PBS.","The cells were permeabilized for 10 min with 0.02% Triton X-100 in PBS, washed 3X with PBS and blocked for 30 min with 1% BSA in PBS.","Variations of the cell type and cell preparation, fixation, and permeabilization methods, including methods for antigen retrieval, are well known to scientists familiar with the art.","An immunolabeling complex was prepared as described in Example 8.The immunolabeling complex was added directly to the fixed and permeabilized cells in an amount sufficient to give a detectable signal if there is a binding site for the primary antibody present in the sample.","After an incubation period that was typically 1060 minutes (usually about 15-30 minutes), the cells were washed with fresh medium and the labeling was evaluated by methods suitable for detection of the label.","Staining by the immunolabeling complex can be additionally preceded, followed by or combined with staining by additional reagents, such as DAPI, which yields blue-fluorescent nuclei.","Example 15 Protocol for Staining Cultured Cells with Multiple Immunolabeling Complexes Cells were fixed and permeabilized as described in Example 14.Multiple immunolabeling complexes were individually prepared from a variety of labeling proteins, according to the procedure described in Example 8.The multiple immunolabeling complexes were either used individually or sequentially to stain the cells, according to the procedure described in Example 14, or two or more immunolabeling complexes were formed then co-mixed in a single staining solution and used to simultaneously stain the sample.","The optimal method for cell fixation and permeabilization and the best ratio for combination of the immunolabeling complexes are typically determined by preliminary experimentation using single immunolabeling complexes or multiple immunolabeling complexes used in combination.","A first immunolabeling complex was prepared from an Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse Fc) and mouse monoclonal anti-α-tubulin, a second immunolabeling complex was prepared from an Alexa Fluor 568 dye-labeled Fab fragment of goat anti-(mouse Fc) and mouse monoclonal anti-vimentin (anti-vimentin was an ascites fluid preparation) and a third immunolabeling complex was prepared from an Alexa Fluor 647 dye-labeled Fab fragment of goat anti-(mouse Fc) and mouse monoclonal anti-cdc6 peptide antibody (Molecular Probes).","Aliquots of the three different immunolabeling complexes were combined and used to stain BPAE cells for 30 minutes, washed with fresh medium and observed by fluorescence microscopy using optical filters appropriate for the three dyes.","In this example, some cells showed cytoplasmic staining by the anti-vimentin antibody, nuclear staining by the anti-cdc6 peptide antibody and staining of mitotic spindles by the anti-α-tubulin antibody, indicative of a cell in mitosis.","Staining by the immunolabeling complexes was additionally preceded, followed by or combined with staining by additional reagents, such as Alexa Fluor 350 phalloidin, which yielded blue-fluorescent actin filaments in the above example.","The immunolabeling complexes that are used in combination do not have to be targeted toward antibodies from the same species.","For instance, complexes of Alexa Fluor 488 dye-labeled goat anti-(mouse IgG, Fc) with a mouse IgG, monoclonal target-binding antibody and an Alexa Fluor 594 dye-labeled goat anti-(rabbit Fc) with a rabbit primary target-binding antibody can be prepared and used in combined staining protocols.","Example 16 Protocol for Staining Tissue with a Single Immunolabeling Complex A mouse intestine cryosection (University of Oregon histology core facility), a cross-section of about 16 μm thickness, was mounted on a slide.","The intestine was perfused and fixed with 4% formaldehyde prior to dissection, embedding, and sectioning.","The tissue section was rehydrated for 20 minutes in PBS.","An immunolabeling complex was prepared as described in Example 8.Briefly, 0.1 μg of mouse monoclonal anti-cdc6 peptide (a nuclear antigen) in 1 μL PBS with 0.1 % BSA was complexed with 0.5 μg of the Alexa Fluor 350 dye-labeled Fab fragment of goat anti-(mouse IgG1 Fc) (prepared as in Example 4) in 5 μL of PBS for 10 minutes at room temperature.","Excess Fab fragment of goat anti-(mouse IgG, Fc) was captured with 25 μL of a 14.1 mg/mL stock of unlabeled mouse IgG.","The tissue was permeabilized with 0.1% Triton X-100 for 10 min.","The tissue was washed two times with PBS and was blocked in 1 % BSA for 30 min.","The immunolabeling complex was added directly to the tissue for 30 minutes and washed three times in PBS.","The sample was mounted in Molecular Probes' Prolong antifade mounting medium and observed by fluorescence microscopy using optical filters appropriate for the Alexa Fluor 350 dye.","Results showed that the mouse monoclonal anti-cdc6 peptide immunolabeling complex showed specific nuclear labeling in the mouse intestine tissue section.","Variations of the tissue type and tissue preparation, fixation and permeabilization methods, mounting methods, including methods for antigen retrieval, are well known to scientists familiar with the art.","Example 17 Staining of a Tissue Target in Combination with Tyramide Signal Amplification (TSA) Mouse brain cryosections were labeled with a pre-formed complex of horseradish peroxidase (HRP)-labeled goat anti-(mouse IgG, Fc) antibody and a mouse IgG1 monoclonal anti-(glial fibrillary acidic protein (GFAP)) prepared essentially as in Example 8 using a molar ratio of labeling protein to monoclonal antibody of 3.Staining of the mouse tissues was essentially as in Example 16.The staining localization and intensity was compared to that of (a) goat anti-mouse IgG HRP conjugate and mouse anti-GFAP, (b) the Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse IgG, Fc) antibody complex of mouse anti-GFAP, (c) Alexa Fluor 488 goat anti-mouse IgG secondary antibody and mouse anti-GFAP, and (d) a direct conjugate of the Alexa Fluor 488 dye with mouse anti-GFAP.","The HRP-conjugated probes were incubated with Alexa Fluor 488 tyramide using TSA Kit #2 (Molecular Probes, Inc.) according to standard procedures.","The tissue staining patterns in each case were similar and consistent with the expected staining pattern of mouse anti-GFAP and staining was essentially free of nonspecific background.","The relative fluorescence intensities of staining measured by digital imaging were sequentially: 541 relative intensity units for the HRP-goat anti-(mouse IgG, Fc) complex of mouse anti-GFAP and (using the combinations indicated by the letters above): (a) 539, (b) 234, (c) 294, and (d) 255 relative intensity units.","Example 18 Staining of Live Cells by Multiple Immunolabeling Complexes A first immunolabeling complex was prepared from an Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse IgG, Fc) and mouse monoclonal anti-(human CD8), a second immunolabeling complex was prepared from an R-phycoerythrin-conjugated Fab fragment of goat anti-(mouse IgG1 Fc) and mouse anti-(human CD3), and a third immunolabeling complex was prepared from an Alexa Fluor 647 dye-labeled Fab fragment of goat anti-(mouse IgG, Fc) and mouse anti-(human CD4).","The complexes were prepared as described in Example 8 and were each blocked with 20 μg (1.3 μL of 14.1 μg/mL) of mouse IgG for 10 minutes at room temperature.","The first immunolabeling complex was added to 100 μL of whole blood and incubated for 15 min.","The cells were washed with PBS and 280.5 μL of the second immunolabeling complex was added and incubated for 15 min.","The cells were again washed, and 46.2 μL of the third labeling complex was added and incubated for 15 min.","After the final incubation, the red blood cells were lysed with cell-lysis buffer.","The cells were resuspended in 1% formaldehyde/PBS and analyzed on a FACS Vantage flow cytometer using a 488 nm argon-ion laser for excitation of the first and second immunolabeling complexes and a 633 nm red He-Ne laser for excitation of the third immunolabeling complex (FIGS.","5a, 5b).","The emission band pass filters used for selective detection of the dyes are 525±10 nm for the Alexa Fluor 488 (CD8), 585±21 nm for R-PE (CD3) and 675±10 nm for the Alexa Fluor 647 dye (CD4).","FIGS.","5a and 5b show that the instant invention can be used in a 3-color immunophenotyping experiment using peripheral blood lymphocytes.","CD3-positive T cells were stained with the R-phycoerythrin-conjugated Fab fragment of goat anti-(mouse Fc) and mouse anti-(human CD3), upper left (UL) quadrant, FIG.","5a.","CD4-positive cells, a T cell subset, are identified using Alexa Fluor 647 dye-labeled Fab fragment of goat anti-(mouse IgG1 Fc) and mouse anti-(human CD4), UL quadrant, FIG.","5b and CD8-positive T cells, a T cell subset, were identified using Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse IgG1 Fc) and mouse monoclonal anti-(human CD8), lower right (LR) quadrant, FIG.","5b.","Exposed antigens of live cells, including cultured cells and cells from biological fluids such as blood and cerebrospinal fluid can be simultaneously or sequentially stained by combinations of immunolabeling complexes, including antibodies to the same target labeled with two or more separately detectable immunoglobulin-binding proteins.","Example 19 The Dye-Labeled Fab Fragment of Goat Anti-(Mouse Fc) can be Utilized for the Combinatorial Labeling of Primary Antibodies, to Generate a Multitude of Colored Targets A first immuno-labeled complex was made by combining 2.5 μg Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse IgG, Fc) with 0.5 μg mouse anti-human CD3 (Caltag at 200 μg/mL), according to the procedure described in Example 4.A second immunolabeling complex was made by combining 5.0 μg Alexa Fluor 647 dye-labeled Fab fragment of goat anti-(mouse IgG, Fc) with 0.5 μg mouse anti-human CD3, according to the procedure in Example 4.Each complex was separately incubated at room temperature for 5 minutes, and each complex was then separately combined with an excess of mouse IgG (14.1 mg/mL) for 5 min at room temperature to capture excess unbound dye-labeled Fab fragments.","The two immunolabeling complexes were then added in different percentage combinations (see Table 2) to 100 μL of washed heparinized blood.","The cells were incubated with the respective combinations of complexes for 20 min on ice.","The red blood cells were then lysed with a cell-lysis buffer.","The cells were resuspended in 1 % formaldehyde/PBS and analyzed on a FacVantage flow cytometer using a 488 nm argon 633 HeNe laser for excitation and a 530 +/-10 nm band pass emission filter (FL1), and a 640 long pass filter (FL4).","Five samples of different combined percentages (Table 2) were compared by flow cytometry, with signals being collected in FL1 and FL4.To determine the percentage of cells detected with each type of emission, the FL1 and FL4 intensities for each percentage combination were normalized by dividing the FL1 and FL4 channel intensities for such combinations by the intensities of the 100% Alexa Fluor 488 dye-and 100% Alexa Fluor 647 dye-labeled cells, respectively.","TABLE 2 Theoretical versus recovered dye-labeled Fab fragment of goat anti- (mouse IgG1 Fc) combinatorial experiment.","Recovered Experimentally Experimentally percentage of mixed Recovered mixed measured cells percentage percentage of percentage labeled with of cells measured cells of cells Alexa Fluor labeled with labeled with labeled with 647 dye- Alexa Fluor Alexa Fluor Alexa Fluor 647 labeled Fab 488 dye-labeled 488 dye-labeled dye-labeled Fab fragment of Fab fragment of Fab fragment fragment of goat goat anti- goat anti-(mouse of goat anti- anti-(mouse (mouse IgG1 Fc) (mouse IgG1 Fc) IgG1 Fc) IgG1 Fc) 100% 100% 0% 0% 75% 81% 25% 14% 50% 63% 50% 38% 25% 35% 75% 73% 0% 0% 100% 100% Example 20 The Immunolabeling Complex can be Used to Detect Antigens on a Western Blot Bovine heart mitochondria were isolated (Hanson et al., Electrophoresis 22, 950 (2001)).","The isolated mitochondria were resuspended to ˜10 mg/mL in 100 mM Tris-HCl, pH 7.8,1 mM phenylmethylsulfonyl fluoride (a protease inhibitor), 2% SDS and insoluble material was removed by centrifugation for 10 minutes at 10,000×g in a tabletop centrifuge.","The protein concentration of the lysate was checked by the BCA assay (Pierce, Rockford, Ill.).","Samples for gel electrophoresis were prepared by mixing lysate, water, and loading buffer to the appropriate concentrations (final concentration of loading buffer in samples: 58 mM Tris/HCl, 10% glycerol, 2% SDS, 0.02 mg/mL bromphenol blue, 50 mM DTT, pH 8.6).","The samples were then heated to 90° C. for 5 minutes before loading on the gel and separated on a 13% SDS-PAGE gel.","Two-fold serial dilution of the extracts ranging from 8 μg of extract down to 20 0.03 μg were loaded on the SDS-PAGE gel.","The proteins were transferred to PVDF membrane for 1.5 hours using a semi-dry transfer system according to manufacturer's directions (The W.E.P.","Company, Concord, Calif.).","The PVDF membrane was blocked for 1 hour in 5% milk.","Immunolabeling complexes were made with mouse monoclonal antibodies that recognize two different mitochondrial proteins.","Alexa Fluor 647 dye-labeled Fab fragment of goat anti-(mouse IgG1 Fc) (5 μL of a 1 mg/mL stock, prepared as in Example 4) was incubated with 21 pL (0.88 mg/mL) mouse anti-(CV-alpha) and Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse IgG, Fc) (5 μL of a 1 mg/mL stock, prepared as in Example 4) was incubated with 19 μL (0.88 mg/mL) mouse anti-(CIII-core2) (Molecular Probes, Eugene, Oreg.).","Following a 30 minute incubation, 25 μL of a 14.1 mg/mL stock of unlabeled mouse IgG was added to each tube.","The immunolabeling complexes were then mixed together and brought up to 5 mL in 5% milk.","The blot was incubated with the mixture of immunolabeling complexes for 1 hour at room temperature.","The blot was washed twice for 5 seconds each with PBST (PBS with 0.1% Tween) and once with PBST for 15 minutes.","The blot was air dried and imaged on an EG&G Wallac Imager with the appropriate filters.","The Western blot revealed two distinct bands of the appropriate molecular weight.","The Western blot also showed that no cross-labeling of the antibodies occurred and the detection limit was 125 ng.","Example 21 High-Throughput Screening of Hybridomas for Identifying High Affinity and High IgG Producers Microplate wells containing both a fluorescent labeled antigen of one fluorescent color label and fluorescently labeled Fab fragments of goat anti-(mouse Fc) of a different fluorescent color made by the method described in Example 4 and 5.Hybridoma supernatant is harvested and added to the wells.","If the hybridoma are producing the desired antibody, i.e.","antibodies that bind to the labeled antigen, polarization of the florescence corresponding to the labeled antigen will allow visualization of those wells containing antigen specific antibody.","In addition, the amount of IgG that the hybridomas produce, can be simultaneously identified by polarization of the fluorescence corresponding to the labeled Fab fragments.","This method thus allows for both quantitation of the amount of antibody present in a specific amount of hybridoma supernatant and the affinity of the monoclonal antibodies for the antigen.","The reagents employed in the preceding examples are commercially available or can be prepared using commercially available instrumentation, methods, or reagents known in the art or whose preparation is described in the examples.","It is evident from the above description and results that the subject invention is greatly superior to the presently available methods for determining the presence of a target in a biological sample.","The subject invention overcomes the shortcomings of the currently used methods by allowing small quantities of antibodies to be labeled and in unlimited media while maintaining specificity and sensitivity.","The examples are not intended to provide an exhaustive description of the many different embodiments of the invention.","Thus, although the forgoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, those of ordinary skill in the art will realize readily that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.","All publications, patents and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference."]],"string":"[\n [\n \"Antibody complexes and methods for immunolabeling\",\n \"The present invention provides labeling reagents and methods for labeling primary antibodies and for detecting a target in a sample using an immuno-labeled complex that comprises a target-binding antibody and one or more labeling reagents.\",\n \"The labeling reagents comprise monovalent antibody fragments or non-antibody monomeric proteins whereby the labeling proteins have affinity for a specific region of the target-binding antibody and are covalently attached to a label.\",\n \"Typically, the labeling reagent is an anti-Fc Fab or Fab′ fragment that was generated by immunizing a goat or rabbit with the Fc fragment of an antibody.\",\n \"The present invention provides for discrete subsets of labeling reagent and immuno-labeled complexes that facilitate the simultaneous detection of multiple targets in a sample wherein the immuno-labeled complexes are distinguished by i) a ratio of label to labeling reagent, or ii) a physical property of said label, or iii) a ratio of labeling reagent to said target-binding antibody, or iv) by said target-binding antibody.\",\n \"This is particularly useful for fluorophore labels that can be attached to labeling reagents and subsequently immuno-labeled complexes in ratios for the detection of multiple targets.\"\n ],\n [\n \"1.A method of forming an immuno-labeled complex, wherein said method comprises the steps of: b) contacting a solution of target-binding antibodies with a discreet labeling reagent subset, wherein said labeling reagent subsets are distinguished by i) ratio of label to labeling reagent or ii) a physical properties of said label; b) incubating said target-binding antibodies and said labeling reagent subset for a time period sufficient for one or more labeling reagents to form an immuno-labeled complex with a target-binding antibody wherein a region of said target binding antibody is selectively bound by labeling reagent whereby a discreet immuno-labeled complex subset is formed; c) optionally removing unbound labeling reagent by adding a capture reagent comprising immunoglobulin proteins or fragments thereof; and, c) optionally repeating said steps a), b), and c) to form a panel of immuno-labeled complex subsets wherein each subset is distinguished from another subset by i) a ratio of label to labeling reagent, or ii) a physical property of said label, or iii) a ratio of labeling reagent to said target-binding antibody, or iv) by said target-binding antibody.\",\n \"2.The method according to claim 1, wherein said target binding antibody is a murine monoclonal antibody, a rabbit polyclonal antibody or a goat polyclonal antibody.\",\n \"3.The method according to claim 2, wherein said target-binding antibodies are in a solution comprising serum proteins or ascites proteins.\",\n \"4.The method according to claim 1, wherein said labeling reagent is a Fab or Fab′fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is a non-antibody protein selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.\",\n \"5.The method according to claim 4, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme and a radioisotope.\",\n \"6.The method according to claim 5, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine, and derivatives thereof.\",\n \"7.The method according to claim 5, wherein said fluorescent protein is a phycobiliprotein.\",\n \"8.The method according to claim 5, wherein said tandem dye is selected from the group consisting of a cyanine-phycobiliprotein derivative and xanthene-phycobiliprotein derivative.\",\n \"9.The method according to claim 5, wherein said enzyme is selected from the group consisting of a peroxidase, a phosphatase, a glycosidase, and a luciferase.\",\n \"10.A method for detecting a target in a sample, wherein said method comprises the steps of: a) contacting said sample with an immuno-labeled complex comprising a target-binding antibody and a labeling reagent; b) incubating said sample of step a) for a time sufficient to permit said immuno-labeled complex to selectively bind to said target; and, c) illuminating said immuno-labeled complex whereby said target is detected.\",\n \"11.The method according to claim 11, wherein said sample comprises a population of cells, cellular extract, subcellular component, proteins, peptides, tissue culture, tissue, a bodily fluid, or a portion or combination thereof.\",\n \"12.The method according to claim 11, wherein said sample is immobilized on a solid or semi-solid matrix.\",\n \"13.The method according to claim 12, wherein said matrix is a gel, a membrane, an array, a glass surface or a microparticle.\",\n \"14.The method according to claim 10, wherein said labeling reagent is a Fab or Fab′fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is a non-antibody protein selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.\",\n \"15.The method according to claim 14, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme and a radioisotope.\",\n \"16.The method according to claim 15, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine, and derivatives thereof.\",\n \"17.The method according to claim 15, wherein said fluorescent protein is a phycobiliprotein.\",\n \"18.The method according to claim 15, wherein said tandem dye is selected from the group consisting of a cyanine-phycobiliprotein derivative and xanthene-phycobiliprotein derivative.\",\n \"19.The method according to claim 15, wherein said enzyme selected from the group consisting of a peroxidase, a phosphatase, a glycosidase, and a luciferase.\",\n \"20.The method according to claim 19, wherein said method further comprises adding a colorimetric, fluorescent or chemiluminescent enzyme substrate.\",\n \"21.The method according to claim 10, wherein said immuno-labeled complex comprises a labeling reagent that is a Fab or Fab′ anti-Fc fragment wherein said fragment is independently covalently bonded to one or more labels that are selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a phycobiliprotein, a borapolyazaindacene, a peroxidase, a phosphatase, a tandem dye and derivatives thereof.\",\n \"22.A method for detecting multiple targets in a sample, said method comprising: a) contacting said sample with a solution comprising A) a pooled subset of immuno-labeled complexes wherein each said complex comprises a target-binding antibody and a labeling reagent, wherein each said subset is distinguished from another subset by i) a ratio of label to labeling reagent, or ii) a physical property of said label, or iii) a ratio of labeling reagent to said target-binding antibody, or iv) by said target-binding antibody or B) an individual immuno-labeled complex subset wherein multiple individual subsets are added to said sample; b) incubating said sample for a time sufficient to allow said immuno-labeled complex to selectively bind to said target; and, c) illuminating said immuno-labeled complex whereby said multiple targets are detected.\",\n \"23.The method according to claim 22, wherein said sample comprises a population of cells, cellular extract, subcellular component, tissue culture, tissue, a bodily fluid, or a portion or combination thereof.\",\n \"24.The method according to claim 23, wherein said sample is immobilized on a solid or semi-solid matrix.\",\n \"25.The method according to claim 24, wherein said matrix is a gel, a membrane, an array, a glass surface or a microparticle.\",\n \"26.The method according to claim 23, wherein said labeling reagent is a Fab or Fab′fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is a non-antibody protein selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.\",\n \"27.The method according to claim 26, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, an electron transfer agent, a hapten, an enzyme and a radioisotope.\",\n \"28.The method according to claim 27, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.\",\n \"29.The method according to claim 27, wherein said fluorescent protein is a phycobiliprotein.\",\n \"30.The method according to claim 27, wherein said tandem dye is selected from the group consisting of a cyanine-phycobiliprotein derivative and xanthene-phycobiliprotein derivative.\",\n \"31.The method according to claim 27, wherein said enzyme is selected from the group consisting of a peroxidase, a phosphatase, a glycosidase, and a luciferase.\",\n \"32.The method according to claim 31, wherein said method further comprises adding a calorimetric, fluorescent or chemiluminescent enzyme substrate.\",\n \"33.A method for identifying and quantitating multiple targets in a sample, wherein said method comprises the steps of: a) contacting a population of cells or fragments thereof in a sample with A) a solution comprising a pooled subset of immuno-labeled complexes wherein said complex comprises a target-binding antibody and a labeling reagent, wherein each subset is.\",\n \"distinguished from another subset i) a ratio of label to labeling reagent, or ii) a physical property of said label, or iii) a ratio of labeling reagent to said target-binding antibody, or iv) by said target-binding antibody or B) an individual subset of immuno-labeled complexes wherein multiple individual subsets are added to said sample; b) incubating said cells or fragments thereof for a time period sufficient to allow said immuno-labeled complex to bind said targets; c) passing said incubated population of cells or fragments thereof through an examination zone; and, d) collecting data from said cells or fragments thereof passed through said examination zone whereby said identification and quantitation of multiple targets is determined.\",\n \"34.The method according to claim 33, wherein said labeling reagent is a Fab or Fab′fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is a non-antibody protein selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.\",\n \"35.The method according to claim 34, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a radioisotope, and a hapten.\",\n \"36.The method according to claim 35, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.\",\n \"37.The method according to claim 35, wherein said fluorescent protein is a phycobiliprotein.\",\n \"38.The method according to claim 35, wherein said tandem dye is selected from the group consisting of cyanine-phycobiliprotein and xanthene-phycobiliprotein.\",\n \"39.A method of manufacturing an isolated labeling reagent, wherein said method comprises the steps of: a) cleaving an intact anti-region antibody with an enzyme to generate Fab fragments; b) isolating said anti-region Fab fragments of step a); c) contacting a matrix comprising intact immunoglobulin proteins or fragments that selectively bind anti-region Fab fragments with a solution comprising said anti-region fragments of step b) wherein said anti-region Fab fragments are immobilized on said matrix; d) contacting said matrix of step c) with a solution comprising a fluorophore label that contains a reactive group; e) washing said matrix of step d) to remove unbound label, and; f) eluting said labeling reagent from said matrix whereby said isolated labeling reagent is manufactured.\",\n \"40.The method according to claim 39, wherein said anti-region Fab fragment are selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment.\",\n \"41.The method according to claim 40, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.\",\n \"42.A method of manufacturing an isolated labeling reagent, wherein said method comprises the steps of: a) contacting a matrix comprising intact immunoglobulin proteins or fragments thereof that selectively bind non-antibody proteins with a solution comprising said non-antibody proteins wherein said non-antibody proteins are immobilized on said matrix; b) contacting said matrix of step a) with a solution comprising a fluorophore label that contains a reactive group; c) washing said matrix to remove unbound label, and; d) eluting said labeling reagent from said matrix whereby said isolated labeling reagent is manufactured that comprises a fluorophore label.\",\n \"43.The method according to claim 42, wherein said non-antibody protein is selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.\",\n \"44.The method according to claim 43, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.\",\n \"45.A method of manufacturing an isolated labeling reagent, wherein said method comprises the steps of: a) cleaving an intact anti-region antibody with an enzyme resulting in Fab or Fab′fragments; a) contacting said anti-region Fab or Fab′ fragments of step a) with a solution comprising a label that contains a reactive group; and, c) isolating labeled anti-region Fab or Fab′ fragments by size exclusion or affinity chromatography whereby an isolated labeling reagent is manufactured.\",\n \"46.The method according to claim 45, wherein said anti-region Fab or Fab′ fragment is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment.\",\n \"47.The method according to claim 46, wherein said label is a fluorescent protein or a tandem dye.\",\n \"48.The method according to claim 47, wherein said fluorescent protein is a phycobiliprotein.\",\n \"49.The method according to claim 47, wherein said tandem dye is selected from the group consisting of cyanine-phycobiliprotein and xanthene-phycobiliprotein.\",\n \"50.An isolated labeling reagent made by a process comprising: a) cleaving an intact anti-region antibody with an enzyme resulting in Fab or Fab′fragments; b) contacting said Fab or Fab′ fragments of step a) with a solution comprising a label that contains a reactive group; and, c) isolating labeled anti-region Fab or Fab′ fragments by size exclusion or affinity chromatography.\",\n \"51.The labeling reagent according to claim 50, wherein said anti-region Fab or Fab′fragment is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment.\",\n \"52.The labeling reagent according to claim 51, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a radioisotope, and a hapten.\",\n \"53.The labeling reagent according to claim 52, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.\",\n \"54.An isolated labeling reagent made by a process comprising: a) cleaving an intact anti-region antibody with an enzyme to generate F(ab′)2 fragments; b) contacting said anti-region F(ab′)2 with a reducing agent to produce anti-region Fab′ fragments containing a thiol group; c) contacting said Fab′ fragments with a solution comprising a label that contains a reactive group; and, d) isolating said Fab′ fragments of step d) that are covalently attached to a label by size exclusion or affinity chromatography.\",\n \"55.The labeling reagent according to claim 54, wherein said anti-region Fab′ fragment is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment.\",\n \"56.The method according to claim 55, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a radioisotope, and a hapten.\",\n \"57.An isolated labeling reagent made by a process comprising: a) cleaving an intact anti-region antibody with an enzyme to generate Fab fragments; b) isolating said anti-region Fab fragments of step a); c) contacting a matrix comprising intact immunoglobulin proteins or fragments thereof that specifically bind anti-region Fab fragments with a solution comprising said anti-region Fab fragments of step b) wherein said anti-region Fab fragments are immobilized; d) contacting said matrix of step c) with a solution comprising a fluorophore label that contains a reactive group; e) washing said matrix to remove unbound label, and; f) eluting said labeling reagent from said matrix whereby said labeling reagent is manufactured comprising a label and being isolated from other proteins and fragments thereof.\",\n \"58.The labeling reagent according to claim 57, wherein said anti-region Fab fragment is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment.\",\n \"59.The labeling reagent according to claim 58, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.\",\n \"60.An isolated labeling reagent made by a process comprising: a) contacting a matrix comprising intact immunoglobulin proteins or fragments that selectively bind non-antibody proteins with a solution comprising said non-antibody proteins wherein said non-antibody proteins are immobilized; b) contacting said matrix of step c) with a solution comprising a fluorophore label that contains a reactive group; c) washing said matrix to remove unbound label, and; d) eluting said labeling reagent from said matrix whereby said labeling reagent is manufactured comprising a label.\",\n \"61.The labeling reagent according to claim 60, wherein said non-antibody protein is selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.\",\n \"62.The labeling reagent according to claim 61, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.\",\n \"63.An immuno-labeled complex generated by a process comprising: a) contacting a solution of target-binding antibodies with a labeling reagent subset, wherein said labeling reagent subset is distinguished from another labeling reagent subset by i) ratio of label to labeling reagent or ii) a physical properties of said label; b) incubating said target-binding antibodies and said labeling reagent subset for a time period sufficient for one or more labeling reagents to form an immuno-labeled complex with a target-binding antibody wherein a region of said target binding antibody is selectively bound by labeling reagent; and, c) optionally removing unbound labeling reagent by adding a capture reagent comprising immunoglobulin proteins or fragments thereof whereby an immuno-labeled complex is produced.\",\n \"64.The immuno-labeled complex according to claim 63, wherein said process further comprises repeating said steps a), b), and c) to form subsets of immuno-labeled complexes wherein each subset is distinguished from another subset by i) ratio of label to labeling reagent or ii) a physical properties of said label, or iii) a ratio of labeling reagent to said target-binding antibody or iv) by said target-binding antibody.\",\n \"65.The immuno-labeled complex according to claim 63, wherein said target-binding antibody is a murine monoclonal antibody, a rabbit polyclonal antibody or a goat polyclonal antibody.\",\n \"66.The immuno-labeled complex according to claim 65, wherein said labeling reagent is a Fab or Fab′ fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is a non-antibody protein selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.\",\n \"67.The immuno-labeled complex according to claim 66, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme and a radioisotope.\",\n \"68.The immuno-labeled complex according to claim 67, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine, and derivatives thereof.\",\n \"69.A labeling solution comprising a buffer and either an individual labeling reagent subset or a pooled subset of labeling reagents, wherein each subset is distinguished from another subset by i) a ratio of label to labeling reagent, or ii) by a physical property of said label.\",\n \"70.The solution according to claim 69, wherein said labeling reagent is a Fab or Fab′fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is non-antibody protein is selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.\",\n \"71.The solution according to claim 70, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, an enzyme, a hapten and a radioisotope.\",\n \"72.The solution according to claim 71, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine, and derivatives thereof.\",\n \"73.A kit for preparing an immuno-labeling complex, said kit comprising: a) a labeling solution comprising a labeling reagent that is independently covalently bonded to one or more labels; or b) a panel of labeling solutions wherein each labeling solution comprises a subset of a labeling reagent and each subset is distinguished from another subset by i) a ratio of label to labeling reagent, or ii) by a physical property of said label; and, c) a solution comprising a capture reagent.\",\n \"74.The kit according to claim 73, wherein said labeling reagent is a Fab or Fab′fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is a non-antibody protein selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.\",\n \"75.The kit according to claim 74, wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, an enzyme, a hapten and a radioisotope.\",\n \"76.The kit according to claim 75, wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.\",\n \"77.The kit according to claim 76, wherein the kit further comprises an addition component selected from the group consisting of (a) stains for characterization of cellular organelles, cell viability, or cell proliferation state, (b) enzyme substrates and (c) enzyme conjugates.\"\n ],\n [\n \"<SOH> BACKGROUND OF THE INVENTION <EOH>Immunolabeling is a method for qualitative or quantitative determination of the presence of a target in a sample, wherein antibodies are utilized for their specific binding capacity.\",\n \"The antibodies form a complex with the target (antigen), wherein a detectable label is present on the antibody or on a secondary antibody.\",\n \"The detectable label is a key feature of immunolabeling, which can be detected directly or indirectly.\",\n \"The label provides a measurable signal by which the binding reaction is monitored providing a qualitative and/or quantitative measure of the degree of binding.\",\n \"The relative quantity and location of signal generated by the labeled antibodies can serve to indicate the location and/or concentration of the target.\",\n \"The label can also be used to select and isolate labeled targets, such as by flow sorting or using magnetic separation media.\",\n \"Examples of labels include but are not limited to radioactive nucleotides ( 125 I, 3 H, 14 C, 32 P), chemiluminescent, fluorescent, or phosphorescent compounds (e.g., dioxetanes, xanthene, or carbocyanine dyes, lanthanide chelates), particles (e.g., gold clusters, colloidal gold, microspheres, quantum dots), and enzymes (e.g., peroxidases, glycosidases, phosphatases, kinases).\",\n \"Ideally, the label is attached to the antibody in a manner that does not perturb the antibody's binding characteristics but enables the label to be measured by an appropriate detection technology.\",\n \"The choice of labels is influenced by factors such as ease and sensitivity of detection, equipment availability, background in the sample (including other labels) and the degree to which such labels are readily attached to the particular antibody.\",\n \"Both direct and indirect labeling of antibodies is utilized for immunolabeling.\",\n \"Direct labeling utilizes only a primary antibody, i.e.\",\n \"the antibody specific for the target, bound to the label.\",\n \"In contrast, indirect labeling utilizes a secondary antibody bound to the label, which is specific for the primary antibody, e.g.\",\n \"a goat anti-rabbit antibody.\",\n \"The principal differences in immunolabeling methods and materials reside in the way that the label is attached to the antibody-antigen complex, the type of label that is used, and the means by which the antibody-antigen complex is detected.\",\n \"Limitations for direct labeling primary antibodies include the need for buffers free of primary amines, or carrier proteins such as bovine serum albumin (BSA), and other compounds such as tris-(hydroxymethyl)aminomethane (TRIS), glycine, and ammonium ions.\",\n \"These materials are, however, common components in antibody buffers and purification methods, and it may not be possible or feasible to remove them prior to the coupling reaction.\",\n \"In particular, many monoclonal antibodies are available only as ascites fluid or in hybridoma culture supernatants, or diluted with carrier proteins, such as albumins.\",\n \"Thus, direct labeling of antibodies in ascites fluid or other medias containing interfering compounds is not attainable.\",\n \"The indirect immunolabeling method typically involves a multi-step process in which an unlabeled first antibody (typically a primary antibody) is directly added to the sample to form a complex with the antigen in the sample.\",\n \"Subsequently, a labeled secondary antibody, specific for the primary antibody, is added to the sample, where it attaches noncovalently to the primary antibody-antigen complex.\",\n \"Alternatively, a detectable label is covalently attached to an immunoglobulin-binding protein such as protein A and protein G to detect the antibody-antigen complex that has previously been formed with the target in the sample.\",\n \"Using ligands, such as streptavidin, that are meant to amplify the detectable signal also expands this cascade binding.\",\n \"Indirect immunolabeling often results in false positives and high background.\",\n \"This is due to the fact that secondary antibodies, even when purified by adsorption against related species, nevertheless can exhibit significant residual cross-reactivity when used in the same sample.\",\n \"For example, when mouse tissue is probed with a mouse monoclonal antibody, the secondary antibody must necessarily be a labeled anti-mouse antibody.\",\n \"This anti-mouse antibody will detect the antibody of interest but will inevitably and additionally detect irrelevant, endogenous mouse immunoglobulins inherent in mouse tissue.\",\n \"This causes a significant background problem, especially in diseased tissues, which reduces the usefulness and sensitivity of the assay.\",\n \"Thus, the simultaneous detection of more than one primary antibody in a sample without this significant background interference depends on the availability of secondary antibodies that 1) do not cross-react with proteins intrinsic to the sample being examined, 2) recognize only one of the primary antibodies, and 3) do not recognize each other (Brelje, et al., METHODS IN CELL BIOLOGY 38, 97-181, especially 111-118 (1993)).\",\n \"To address the background problem in indirect labeling, a number of strategies have been developed to block access of the anti-mouse secondary antibodies to the endogenous mouse immunoglobulins.\",\n \"One such strategy for blocking involves complexing the primary antibody with a selected biotinylated secondary antibody to produce a complex of the primary and secondary antibodies, which is then mixed with diluted normal murine serum (Trojanowski et al., U.S. Pat No.\",\n \"5,281,521 (1994)).\",\n \"This method is limited by the necessity to utilize an appropriate ratio of primary-secondary complex.\",\n \"Too low a ratio of primary-secondary complex will cause a decrease in specific staining and increased background levels due to the uncomplexed secondary anti-mouse antibody binding to endogenous mouse antibodies.\",\n \"However, the ability of a whole IgG antibody (as was used in the referenced method) to simultaneously bind and cross-link two antigens results in too high a ratio, causing the complex to precipitate or form complexes that are too large to penetrate into the cell or tissue.\",\n \"Another strategy for blocking access to endogenous immunoglobulins in the sample involves pre-incubating the sample with a monovalent antibody, such as Fab′ fragments, from an irrelevant species that recognize endogenous immunoglobulins.\",\n \"This approach requires large quantities of expensive Fab′ fragments and gives mixed results and adds at least two steps (block and wash) to the overall staining procedure.\",\n \"The addition of a cross-linking reagent has resulted in improved reduction of background levels (Tsao, et al., U.S. Pat.\",\n \"No.\",\n \"5,869,274 (1997)) but this is problematic when used with fluorophore-labeled antibodies.\",\n \"The cross-linking causes an increase in the levels of autofluorescence and thus the background (J. Neurosci.\",\n \"Meth.\",\n \"83, 97 (1998); Mosiman et al., Methods 77, 191 (1997); Commun.\",\n \"Clin.\",\n \"Cytometry 30,151 (1997); Beisker et al., Cytometry 8, 235 (1987)).\",\n \"In addition, pre-incubation with a cross-linking reagent often masks or prevents the antibody from binding to its antigen (J. Histochem.\",\n \"Cytochem.\",\n \"45, 327 (1997); J. Histochem.\",\n \"Cytochem.\",\n \"39, 741 (1991); J. Histochem.\",\n \"Cytochem.\",\n \"43,193 (1995); Appl.\",\n \"Immunohistochem.\",\n \"Molecul.\",\n \"Morphol.\",\n \"9,176 (2001)).\",\n \"In a variation of this blocking strategy, a multi-step sequential-labeling procedure is used to overcome the problems of cross-reactivity.\",\n \"The sample is incubated with a first antibody to form a complex with the first antigen, followed by incubation of the sample with a fluorophore-labeled goat Fab anti-mouse IgG to label the first antibody and block it from subsequently complexing when the second antibody is added.\",\n \"In the third step, a second mouse antibody forms a complex with the second antigen.\",\n \"Because the second antibody is blocked from cross-reacting with the first antibody, the second mouse antibody is detected with a standard indirect-labeling method using a goat anti-mouse antibody conjugated to a different fluorescent dye (J. Histochem.\",\n \"Cytochem.\",\n \"34, 703 (1986)).\",\n \"This process requires multiple incubation steps and washing steps and it still cannot be used with mouse antibodies to probe mouse tissue.\",\n \"Another blocking method is disclosed in the animal research kit (ARK) developed by DAKO.\",\n \"In this kit, a primary antibody is complexed with biotin-labeled goat Fab anti-mouse IgG and excess free Fab is blocked with normal mouse serum.\",\n \"However, since the Fab used in this process is generated from the intact IgG (rather than a selected region) there is a potential for the formation of anti-paratope or anti-idiotype antibodies that will block the antigen-binding site and prevent immunolabeling.\",\n \"The biotinylated antibody also requires subsequent addition of a labeled avidin or streptavidin conjugate for its subsequent visualization.\",\n \"The present invention is advantageous over previously described methods and compositions in that it provides the benefits of indirect labeling with the easy and flexibility of direct labeling for determination of a desired target in a biological sample.\",\n \"The present invention provides labeled monovalent proteins specific for a target-binding antibody, which are complexed prior to addition with a biological sample.\",\n \"Because these monovalent proteins are not bivalent antibodies, precipitation and cross-linking are not a problem.\",\n \"Therefore the compositions of the present invention can be used with immunologically similar monoclonal or polyclonal antibodies of either an identical isotype or different isotypes.\",\n \"The monovalent labeling reagents are specific for the Fc region of target-binding antibodies, these reagents will not interfere with the binding region of the primary antibody.\",\n \"In addition, the monovalent labeling proteins are not negatively affected by the presence of primary amines like BSA, gelatin, hybridoma culture supernatants or ascites fluid, thus primary antibodies present in these media can be effectively labeled with the labeling reagents of the present invention.\",\n \"Thus, the present invention provides numerous advantages over the conventional methods of immunolabeling.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides labeling reagents and methods for labeling primary antibodies and for detecting a target in a sample using an immuno-labeled complex that comprises a target-binding antibody and one or more labeling reagents.\",\n \"The labeling reagents comprise monovalent antibody fragments or non-antibody monomeric proteins whereby the labeling proteins have affinity for a specific region of the target-binding antibody and are covalently attached to a label.\",\n \"Typically, the labeling reagent is an anti-Fc Fab or Fab′ fragment that was generated by immunizing a goat or rabbit with the Fc fragment of an antibody.\",\n \"The methods for labeling a target-binding antibody with a labeling reagent comprise a) contacting a solution of target-binding antibodies with a labeling reagent, b) incubating said target-binding antibodies and said labeling reagent wherein a region of said target binding antibody is selectively bound by labeling reagent, and c) optionally removing unbound labeling reagent by adding a capture reagent comprising immunoglobulin proteins or fragments thereof that are optionally immobilized on a matrix.\",\n \"The labeling of the target-binding antibody can be performed irrespective of the solution that the antibody is present in and includes proteins that are normally present in serum or ascites.\",\n \"This feature of the labeling process of the target-binding antibody eliminates the need to purify and concentrate the target-binding antibody.\",\n \"The time required for the labeling reagent to selectively bind to the target-binding antibody is typically very short, often less than 10 minutes.\",\n \"Often the labeling reagent binds the target-binding antibody in the amount of time it takes to add and mix the labeling reagent with the target-binding antibody.\",\n \"This formation of an immuno-labeled complex—a target-binding antibody and a labeling reagent—results in the formation of an target detection solution that is used to detect a target in a sample.\",\n \"The labeling steps of the target-binding antibody are optionally repeated to form a panel of subsets, these immuno-labeled complex subsets may be used individually or pooled wherein each subset is distinguished from another subset by i) the target-binding antibody, or ii) a ratio of label to labeling reagent, or iii) a ratio of labeling reagent to the target-binding antibody or iv) by a physical property of the label.\",\n \"Thus, it is appreciated that a wide range of subsets can be formed wherein the subsets can be used individually to detect a target in a sample or pooled to simultaneously detect multiple targets in a sample.\",\n \"The simultaneous detection of multiple targets in a sample is especially useful in methods that utilize flow cytometry or methods that immobilize a population of cells or tissue on a surface.\",\n \"The methods for determining a target in a sample using immuno-labeled subsets comprises forming a subset of immuno-labeled complexes, as described above, contacting a sample with said immuno-labeled complexes, incubating the sample for a time sufficient to allow the immuno-labeled complex to selectively bind to a desired target, and illuminating the immuno-labeled complex whereby the target is detected.\",\n \"The sample is any material that may contain a target and typically comprises a population of cells, cellular extract, subcellular component, proteins, peptides, tissue culture, tissue, a bodily fluid, or a portion or combination thereof.\",\n \"When multiple targets are detected a pooled subset of immuno-labeled complexes are formed and incubated with the sample or individual subsets are add sequentially to a sample.\",\n \"For methods using flow cytometry the population of cells is illuminated when they pass through an optical examination zone and the data collected about the label determines the identity and quantity of the targets.\"\n ],\n [\n \"CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Ser.\",\n \"No.\",\n \"60/329,068, filed Oct. 12, 2001; U.S. Ser.\",\n \"No.\",\n \"60/369,418 filed Apr.\",\n \"1, 2002, U.S. Ser.\",\n \"No.10/118,204 filed Apr.\",\n \"5, 2002, and PCT/US02/31416 filed Oct. 2, 2002, which disclosures are herein incorporated by reference.\",\n \"FIELD OF THE INVENTION The present invention relates to immuno-labeled complexes and methods for use in the detection and measurement of one or more targets in a biological sample.\",\n \"The invention has applications in the fields of molecular biology, cell biology, immunohistochemistry, diagnostics, and therapeutics.\",\n \"BACKGROUND OF THE INVENTION Immunolabeling is a method for qualitative or quantitative determination of the presence of a target in a sample, wherein antibodies are utilized for their specific binding capacity.\",\n \"The antibodies form a complex with the target (antigen), wherein a detectable label is present on the antibody or on a secondary antibody.\",\n \"The detectable label is a key feature of immunolabeling, which can be detected directly or indirectly.\",\n \"The label provides a measurable signal by which the binding reaction is monitored providing a qualitative and/or quantitative measure of the degree of binding.\",\n \"The relative quantity and location of signal generated by the labeled antibodies can serve to indicate the location and/or concentration of the target.\",\n \"The label can also be used to select and isolate labeled targets, such as by flow sorting or using magnetic separation media.\",\n \"Examples of labels include but are not limited to radioactive nucleotides (125I, 3H, 14C, 32P), chemiluminescent, fluorescent, or phosphorescent compounds (e.g., dioxetanes, xanthene, or carbocyanine dyes, lanthanide chelates), particles (e.g., gold clusters, colloidal gold, microspheres, quantum dots), and enzymes (e.g., peroxidases, glycosidases, phosphatases, kinases).\",\n \"Ideally, the label is attached to the antibody in a manner that does not perturb the antibody's binding characteristics but enables the label to be measured by an appropriate detection technology.\",\n \"The choice of labels is influenced by factors such as ease and sensitivity of detection, equipment availability, background in the sample (including other labels) and the degree to which such labels are readily attached to the particular antibody.\",\n \"Both direct and indirect labeling of antibodies is utilized for immunolabeling.\",\n \"Direct labeling utilizes only a primary antibody, i.e.\",\n \"the antibody specific for the target, bound to the label.\",\n \"In contrast, indirect labeling utilizes a secondary antibody bound to the label, which is specific for the primary antibody, e.g.\",\n \"a goat anti-rabbit antibody.\",\n \"The principal differences in immunolabeling methods and materials reside in the way that the label is attached to the antibody-antigen complex, the type of label that is used, and the means by which the antibody-antigen complex is detected.\",\n \"Limitations for direct labeling primary antibodies include the need for buffers free of primary amines, or carrier proteins such as bovine serum albumin (BSA), and other compounds such as tris-(hydroxymethyl)aminomethane (TRIS), glycine, and ammonium ions.\",\n \"These materials are, however, common components in antibody buffers and purification methods, and it may not be possible or feasible to remove them prior to the coupling reaction.\",\n \"In particular, many monoclonal antibodies are available only as ascites fluid or in hybridoma culture supernatants, or diluted with carrier proteins, such as albumins.\",\n \"Thus, direct labeling of antibodies in ascites fluid or other medias containing interfering compounds is not attainable.\",\n \"The indirect immunolabeling method typically involves a multi-step process in which an unlabeled first antibody (typically a primary antibody) is directly added to the sample to form a complex with the antigen in the sample.\",\n \"Subsequently, a labeled secondary antibody, specific for the primary antibody, is added to the sample, where it attaches noncovalently to the primary antibody-antigen complex.\",\n \"Alternatively, a detectable label is covalently attached to an immunoglobulin-binding protein such as protein A and protein G to detect the antibody-antigen complex that has previously been formed with the target in the sample.\",\n \"Using ligands, such as streptavidin, that are meant to amplify the detectable signal also expands this cascade binding.\",\n \"Indirect immunolabeling often results in false positives and high background.\",\n \"This is due to the fact that secondary antibodies, even when purified by adsorption against related species, nevertheless can exhibit significant residual cross-reactivity when used in the same sample.\",\n \"For example, when mouse tissue is probed with a mouse monoclonal antibody, the secondary antibody must necessarily be a labeled anti-mouse antibody.\",\n \"This anti-mouse antibody will detect the antibody of interest but will inevitably and additionally detect irrelevant, endogenous mouse immunoglobulins inherent in mouse tissue.\",\n \"This causes a significant background problem, especially in diseased tissues, which reduces the usefulness and sensitivity of the assay.\",\n \"Thus, the simultaneous detection of more than one primary antibody in a sample without this significant background interference depends on the availability of secondary antibodies that 1) do not cross-react with proteins intrinsic to the sample being examined, 2) recognize only one of the primary antibodies, and 3) do not recognize each other (Brelje, et al., METHODS IN CELL BIOLOGY 38, 97-181, especially 111-118 (1993)).\",\n \"To address the background problem in indirect labeling, a number of strategies have been developed to block access of the anti-mouse secondary antibodies to the endogenous mouse immunoglobulins.\",\n \"One such strategy for blocking involves complexing the primary antibody with a selected biotinylated secondary antibody to produce a complex of the primary and secondary antibodies, which is then mixed with diluted normal murine serum (Trojanowski et al., U.S. Pat No.\",\n \"5,281,521 (1994)).\",\n \"This method is limited by the necessity to utilize an appropriate ratio of primary-secondary complex.\",\n \"Too low a ratio of primary-secondary complex will cause a decrease in specific staining and increased background levels due to the uncomplexed secondary anti-mouse antibody binding to endogenous mouse antibodies.\",\n \"However, the ability of a whole IgG antibody (as was used in the referenced method) to simultaneously bind and cross-link two antigens results in too high a ratio, causing the complex to precipitate or form complexes that are too large to penetrate into the cell or tissue.\",\n \"Another strategy for blocking access to endogenous immunoglobulins in the sample involves pre-incubating the sample with a monovalent antibody, such as Fab′ fragments, from an irrelevant species that recognize endogenous immunoglobulins.\",\n \"This approach requires large quantities of expensive Fab′ fragments and gives mixed results and adds at least two steps (block and wash) to the overall staining procedure.\",\n \"The addition of a cross-linking reagent has resulted in improved reduction of background levels (Tsao, et al., U.S. Pat.\",\n \"No.\",\n \"5,869,274 (1997)) but this is problematic when used with fluorophore-labeled antibodies.\",\n \"The cross-linking causes an increase in the levels of autofluorescence and thus the background (J. Neurosci.\",\n \"Meth.\",\n \"83, 97 (1998); Mosiman et al., Methods 77, 191 (1997); Commun.\",\n \"Clin.\",\n \"Cytometry 30,151 (1997); Beisker et al., Cytometry 8, 235 (1987)).\",\n \"In addition, pre-incubation with a cross-linking reagent often masks or prevents the antibody from binding to its antigen (J. Histochem.\",\n \"Cytochem.\",\n \"45, 327 (1997); J. Histochem.\",\n \"Cytochem.\",\n \"39, 741 (1991); J. Histochem.\",\n \"Cytochem.\",\n \"43,193 (1995); Appl.\",\n \"Immunohistochem.\",\n \"Molecul.\",\n \"Morphol.\",\n \"9,176 (2001)).\",\n \"In a variation of this blocking strategy, a multi-step sequential-labeling procedure is used to overcome the problems of cross-reactivity.\",\n \"The sample is incubated with a first antibody to form a complex with the first antigen, followed by incubation of the sample with a fluorophore-labeled goat Fab anti-mouse IgG to label the first antibody and block it from subsequently complexing when the second antibody is added.\",\n \"In the third step, a second mouse antibody forms a complex with the second antigen.\",\n \"Because the second antibody is blocked from cross-reacting with the first antibody, the second mouse antibody is detected with a standard indirect-labeling method using a goat anti-mouse antibody conjugated to a different fluorescent dye (J. Histochem.\",\n \"Cytochem.\",\n \"34, 703 (1986)).\",\n \"This process requires multiple incubation steps and washing steps and it still cannot be used with mouse antibodies to probe mouse tissue.\",\n \"Another blocking method is disclosed in the animal research kit (ARK) developed by DAKO.\",\n \"In this kit, a primary antibody is complexed with biotin-labeled goat Fab anti-mouse IgG and excess free Fab is blocked with normal mouse serum.\",\n \"However, since the Fab used in this process is generated from the intact IgG (rather than a selected region) there is a potential for the formation of anti-paratope or anti-idiotype antibodies that will block the antigen-binding site and prevent immunolabeling.\",\n \"The biotinylated antibody also requires subsequent addition of a labeled avidin or streptavidin conjugate for its subsequent visualization.\",\n \"The present invention is advantageous over previously described methods and compositions in that it provides the benefits of indirect labeling with the easy and flexibility of direct labeling for determination of a desired target in a biological sample.\",\n \"The present invention provides labeled monovalent proteins specific for a target-binding antibody, which are complexed prior to addition with a biological sample.\",\n \"Because these monovalent proteins are not bivalent antibodies, precipitation and cross-linking are not a problem.\",\n \"Therefore the compositions of the present invention can be used with immunologically similar monoclonal or polyclonal antibodies of either an identical isotype or different isotypes.\",\n \"The monovalent labeling reagents are specific for the Fc region of target-binding antibodies, these reagents will not interfere with the binding region of the primary antibody.\",\n \"In addition, the monovalent labeling proteins are not negatively affected by the presence of primary amines like BSA, gelatin, hybridoma culture supernatants or ascites fluid, thus primary antibodies present in these media can be effectively labeled with the labeling reagents of the present invention.\",\n \"Thus, the present invention provides numerous advantages over the conventional methods of immunolabeling.\",\n \"SUMMARY OF THE INVENTION The present invention provides labeling reagents and methods for labeling primary antibodies and for detecting a target in a sample using an immuno-labeled complex that comprises a target-binding antibody and one or more labeling reagents.\",\n \"The labeling reagents comprise monovalent antibody fragments or non-antibody monomeric proteins whereby the labeling proteins have affinity for a specific region of the target-binding antibody and are covalently attached to a label.\",\n \"Typically, the labeling reagent is an anti-Fc Fab or Fab′ fragment that was generated by immunizing a goat or rabbit with the Fc fragment of an antibody.\",\n \"The methods for labeling a target-binding antibody with a labeling reagent comprise a) contacting a solution of target-binding antibodies with a labeling reagent, b) incubating said target-binding antibodies and said labeling reagent wherein a region of said target binding antibody is selectively bound by labeling reagent, and c) optionally removing unbound labeling reagent by adding a capture reagent comprising immunoglobulin proteins or fragments thereof that are optionally immobilized on a matrix.\",\n \"The labeling of the target-binding antibody can be performed irrespective of the solution that the antibody is present in and includes proteins that are normally present in serum or ascites.\",\n \"This feature of the labeling process of the target-binding antibody eliminates the need to purify and concentrate the target-binding antibody.\",\n \"The time required for the labeling reagent to selectively bind to the target-binding antibody is typically very short, often less than 10 minutes.\",\n \"Often the labeling reagent binds the target-binding antibody in the amount of time it takes to add and mix the labeling reagent with the target-binding antibody.\",\n \"This formation of an immuno-labeled complex—a target-binding antibody and a labeling reagent—results in the formation of an target detection solution that is used to detect a target in a sample.\",\n \"The labeling steps of the target-binding antibody are optionally repeated to form a panel of subsets, these immuno-labeled complex subsets may be used individually or pooled wherein each subset is distinguished from another subset by i) the target-binding antibody, or ii) a ratio of label to labeling reagent, or iii) a ratio of labeling reagent to the target-binding antibody or iv) by a physical property of the label.\",\n \"Thus, it is appreciated that a wide range of subsets can be formed wherein the subsets can be used individually to detect a target in a sample or pooled to simultaneously detect multiple targets in a sample.\",\n \"The simultaneous detection of multiple targets in a sample is especially useful in methods that utilize flow cytometry or methods that immobilize a population of cells or tissue on a surface.\",\n \"The methods for determining a target in a sample using immuno-labeled subsets comprises forming a subset of immuno-labeled complexes, as described above, contacting a sample with said immuno-labeled complexes, incubating the sample for a time sufficient to allow the immuno-labeled complex to selectively bind to a desired target, and illuminating the immuno-labeled complex whereby the target is detected.\",\n \"The sample is any material that may contain a target and typically comprises a population of cells, cellular extract, subcellular component, proteins, peptides, tissue culture, tissue, a bodily fluid, or a portion or combination thereof.\",\n \"When multiple targets are detected a pooled subset of immuno-labeled complexes are formed and incubated with the sample or individual subsets are add sequentially to a sample.\",\n \"For methods using flow cytometry the population of cells is illuminated when they pass through an optical examination zone and the data collected about the label determines the identity and quantity of the targets.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS FIG.\",\n \"1: Shows a schematic representation of the formation of the immuno-labeled complex (target-binding antibody and labeling reagent).\",\n \"FIG.\",\n \"2: Shows species specificity of goat Fab anti-(mouse Fc), as observed using a microplate coated with IgG of various species.\",\n \"The various species were blocked with BSA, reacted with biotinylated goat Fab anti-(mouse Fc), washed, and then treated with streptavidin-horseradish peroxidase (HRP), followed by hydrogen peroxide (H2O2) and the Amplex Red peroxidase detection reagent.\",\n \"FIG.\",\n \"3: Shows a preferred molar ratio of a goat Fab anti-(mouse Fc) labeling reagent.\",\n \"Varying amounts of an Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse Fc) were added to a constant amount of anti-biotin monoclonal antibody (mAb).\",\n \"This mixture was equilibrated for 20 minutes, and then added to biotinylated-BSA in a microplate well.\",\n \"After allowing time to bind, the plates were washed and the remaining fluorescence was quantitated.\",\n \"The analysis was performed in triplicate (circles).\",\n \"Control experiments were performed, as described above, but without adding the primary anti-biotin antibody (solid squares).\",\n \"FIG.\",\n \"4: Shows a comparison of the fluorescence intensity (Example 6) for labeling reagent prepared in homogeneous solution (Example 4) and labeling reagent prepared on a column (Example 5).\",\n \"FIG.\",\n \"5: Shows detection of multiple targets on T cells using a labeling reagent attached to a R-phycoerythrin (R-PE) (FIG.\",\n \"5A) to detect CD3-positive T cells, a labeling reagent attached to Alexa Fluor 647 dye (FIG.\",\n \"5B) to detect CD4-positive T cells and a labeling reagent attached to Alexa Fluor 488 dye (FIG.\",\n \"5B) to detect CD8-positive T cells (Example 18).\",\n \"The CD-3 detected T cells are shown in the upper left (UL) and upper right (UR) quadrants.\",\n \"The relative percentages of total lymphocytes that are CD3-positive cells are 83.3% (UL+UR).\",\n \"The relative percentage of CD8-positive Alexa Fluor 488 dye-stained lymphocytes and CD3-positive R-PE dye-stained lymphocytes is 35.1% (UR quadrant).\",\n \"The lower left quadrant (LL, 20.4%) shows CD3-negative lymphocytes (i.e.\",\n \"non-T cells) comprised of NK cells, B cells and some monocytes.\",\n \"In the lower right (LR, 2.7%) region are non-T cells, which are nonspecifically stained.\",\n \"FIG.\",\n \"5B further shows CD3-positive T-cells subdivided into Alexa Fluor 647 dye CD4-positive and Alexa Fluor 488 dye CD8-positive.\",\n \"CD4-positive cells represent 50.9% of total lymphocytes (UL quadrant) and CD8-positive cells represent 24.5% of the total lymphocytes (LR quadrant).\",\n \"The 23.1% of cells in the LL quadrant are non-T cells, while the 1.5% of cells in UR quadrant are likely nonspecifically stained lymphocytes.\",\n \"FIG.\",\n \"6: Shows high-performance size-exclusion chromatographic analysis of Alexa Fluor 488 dye-labeled goat Fab anti-(mouse Fc) labeling reagent binding to a mouse IgG, target-binding antibody.\",\n \"The labeling reagent, alone, appears as a peak at 38 minutes; the target-binding antibody, alone, appears as a peak at 33 minutes.\",\n \"When labeling reagent and target-binding antibody are mixed together at a molar ratio of -5:1 (labeling reagent:target-binding antibody), the resulting immunolabeling complex appears as a peak at 29 minutes (Example 10).\",\n \"FIG.\",\n \"7: Shows the production of labeling reagent wherein the label is attached to the labeling reagent when immobilized on a column.\",\n \"DETAILED DESCRIPTION OF THE INVENTION I. Definitions Before describing the present invention in detail, it is to be understood that this invention is not limited to specific compositions or process steps, as such may vary.\",\n \"It should be noted that, as used in this specification and the appended claims, the singular form “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise.\",\n \"Thus, for example, reference to “a protein labeling complex” includes a plurality of complexes and reference to “a target-binding protein” includes a plurality of proteins and the like.\",\n \"Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related.\",\n \"The following terms are defined for purposes of the invention as described herein.\",\n \"The term “affinity” as used herein refers to the strength of the binding interaction of two molecules, such as an antibody and an antigen or a positively charged moiety and a negatively charged moiety.\",\n \"For bivalent molecules such as antibodies, affinity is typically defined as the binding strength of one binding domain for the antigen, e.g.\",\n \"one Fab fragment for the antigen.\",\n \"The binding strength of both binding domains together for the antigen is referred to as “avidity”.\",\n \"As used herein “High affinity” refers to a ligand that binds to an antibody having an affinity constant (Ka) greater than 104 M−1, typically 105-1011 M−1; as determined by inhibition ELISA or an equivalent affinity determined by comparable techniques such as, for example, Scatchard plots or using Kd/dissociation constant, which is the reciprocal of the Ka, etc.\",\n \"The term “antibody” as used herein refers to a protein of the immunoglobulin (Ig) superfamily that binds noncovalently to certain substances (e.g.\",\n \"antigens and immunogens) to form an antibody-antigen complex.\",\n \"Antibodies can be endogenous, or polyclonal wherein an animal is immunized to elicit a polyclonal antibody response or by recombinant methods resulting in monoclonal antibodies produced from hybridoma cells or other cell lines.\",\n \"It is understood that the term “antibody” as used herein includes within its scope any of the various classes or sub-classes of immunoglobulin derived from any of the animals conventionally used.\",\n \"The term “antibody fragments” as used herein refers to fragments of antibodies that retain the principal selective binding characteristics of the whole antibody.\",\n \"Particular fragments are well-known in the art, for example, Fab, Fab′, and F(ab′)2, which are obtained by digestion with various proteases, pepsin or papain, and which lack the Fc fragment of an intact antibody or the so-called “half-molecule” fragments obtained by reductive cleavage of the disulfide bonds connecting the heavy chain components in the intact antibody.\",\n \"Such fragments also include isolated fragments consisting of the light-chain-variable region, “Fv” fragments consisting of the variable regions of the heavy and light chains, and recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker.\",\n \"Other examples of binding fragments include (i) the Fd fragment, consisting of the VH and CH1 domains; (ii) the dAb fragment (Ward, et al., Nature 341, 544 (1989)), which consists of a VH domain; (iii) isolated CDR regions; and (iv) single-chain Fv molecules (scFv) described above.\",\n \"In addition, arbitrary fragments can be made using recombinant technology that retains antigen-recognition characteristics.\",\n \"The term “antigen” as used herein refers to a molecule that induces, or is capable of inducing, the formation of an antibody or to which an antibody binds selectively, including but not limited to a biological material.\",\n \"Antigen also refers to “immunogen”.\",\n \"The target-binding antibodies selectively bind an antigen, as such the term can be used herein interchangeably with the term “target”.\",\n \"The term “anti-region antibody” as used herein refers to an antibody that was produced by immunizing an animal with a select region that is a fragment of a foreign antibody wherein only the fragment is used as the immunogen.\",\n \"Anti-region antibodies include monoclonal and polyclonal antibodies.\",\n \"The term “anti-region fragment” as used herein refers to a monovalent fragment that was generated from an anti-region antibody of the present invention by enzymatic cleavage.\",\n \"The term “biotin” as used herein refers to any biotin derivative, including without limitation, substituted and unsubstituted biotin, and analogs and derivatives thereof, as well as substituted and unsubstituted derivatives of caproylamidobiotin, biocytin, desthiobiotin, desthiobiocytin, iminobiotin, and biotin sulfone.\",\n \"The term “biotin-binding protein” as used herein refers to any protein that binds selectively and with high affinity to biotin, including without limitation, substituted or unsubstituted avidin, and analogs and derivatives thereof, as well as substituted and unsubstituted derivatives of streptavidin, ferritin avidin, nitroavidin, nitrostreptavidin, and Neutravidin™ avidin (a de-glycosylated modified avidin having an isoelectric point near neutral).\",\n \"The term “buffer” as used herein refers to a system that acts to minimize the change in acidity or basicity of the solution against addition or depletion of chemical substances.\",\n \"The term “capture reagent” refers to a non-specific immunoglobulin that is used to remove excess labeling reagent after the formation of the immuno-labeled complex.\",\n \"The capture reagent is optionally attached a matrix to facilitate removal of the excess labeling regent.\",\n \"A matrix typically includes a microsphere, an agarose bead or any solid surface that the excess labeling reagent can be passed by.\",\n \"The term “chromophore” as used herein refers to a label that emits light in the visible spectra that can be observed without the aid of instrumentation.\",\n \"The term “complex” as used herein refers to the association of two or more molecules, usually by non-covalent bonding, e.g., the association between an antibody and an antigen or the labeling reagent and the target-binding antibody.\",\n \"The term “detectable response” as used herein refers to an occurrence of, or a change in, a signal that is directly or indirectly detectable either by observation or by instrumentation.\",\n \"Typically, the detectable response is an occurrence of a signal wherein the fluorophore is inherently fluorescent and does not produce a change in signal upon binding to a metal ion or biological compound.\",\n \"Alternatively, the detectable response is an optical response resulting in a change in the wavelength distribution patterns or intensity of absorbance or fluorescence or a change in light scatter, fluorescence lifetime, fluorescence polarization, or a combination of the above parameters.\",\n \"Other detectable responses include, for example, chemiluminescence, phosphorescence, radiation from radioisotopes, magnetic attraction, and electron density.\",\n \"The term “detectably distinct” as used herein refers to a signal that is distinguishable or separable by a physical property either by observation or by instrumentation.\",\n \"For example, a fluorophore is readily distinguishable either by spectral characteristics or by fluorescence intensity, lifetime, polarization or photo-bleaching rate from another fluorophore in the sample, as well as from additional materials that are optionally present.\",\n \"The term “directly detectable” as used herein refers to the presence of a material or the signal generated from the material is immediately detectable by observation, instrumentation, or film without requiring chemical modifications or additional substances.\",\n \"The term “examination zone” as used herein refers to an optical zone of a flow cytometer, or a similar instrument, wherein cells are passed through essentially one at a time in a thin stream whereby the bound immuno-labeled complex is illuminated and the intensity and emission spectra of the fluorophore is detected and recorded.\",\n \"This includes instruments wherein the examination zone moves and the sample is held in place.\",\n \"The term “fluorophore” as used herein refers to a composition that is inherently fluorescent or demonstrates a change in fluorescence upon binding to a biological compound or metal ion, i.e., fluorogenic.\",\n \"Fluorophores may contain substitutents that alter the solubility, spectral properties or physical properties of the fluorophore.\",\n \"Numerous fluorophores are known to those skilled in the art and include, but are not limited to coumarin, cyanine, benzofuran, a quinoline, a quinazolinone, an indole, a benzazole, a borapolyazaindacene and xanthenes including fluoroscein, rhodamine and rhodol as well as other fluorophores described in RICHARD P. HAUGLAND, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (9th edition, CD-ROM, September 2002).\",\n \"The term “immuno-labeled complex” refers to the complex of target-binding antibody that is non-covalently attached to a labeling reagent.\",\n \"The term “immuno-labeled complex subset” as used herein refers to a discrete set of immuno-labeled complexes that are homogenous and can be distinguished from another subset of immuno-labeled complex by the physical properties of the label, or the ratio of the label to labeling reagent, or the ratio of labeling reagent to target-binding antibody, or the target-binding antibody.\",\n \"Typically an immuno-labeled complex subset is present in a buffer to provide a “target detection solution”.\",\n \"The term “kit” as used herein refers to a packaged set of related components, typically one or more compounds or compositions.\",\n \"The term “label” as used herein refers to a chemical moiety or protein that retains it's native properties (e.g.\",\n \"spectral properties, conformation and activity) when attached to a labeling reagent and used in the present methods.\",\n \"The label can be directly detectable (fluorophore) or indirectly detectable (hapten or enzyme).\",\n \"Such labels include, but are not limited to, radiolabels that can be measured with radiation-counting devices; pigments, dyes or other chromogens that can be visually observed or measured with a spectrophotometer; spin labels that can be measured with a spin label analyzer; and fluorescent labels (fluorophores), where the output signal is generated by the excitation of a suitable molecular adduct and that can be visualized by excitation with light that is absorbed by the dye or can be measured with standard fluorometers or imaging systems, for example.\",\n \"The label can be a chemiluminescent substance, where the output signal is generated by chemical modification of the signal compound; a metal-containing substance; or an enzyme, where there occurs an enzyme-dependent secondary generation of signal, such as the formation of a colored product from a colorless substrate.\",\n \"The term label can also refer to a “tag” or hapten that can bind selectively to a conjugated molecule such that the conjugated molecule, when added subsequently along with a substrate, is used to generate a detectable signal.\",\n \"For example, one can use biotin as a tag and then use an avidin or streptavidin conjugate of horseradish peroxidate (HRP) to bind to the tag, and then use a colorimetric substrate (e.g., tetramethylbenzidine (TMB)) or a fluorogenic substrate such as Amplex Red reagent (Molecular Probes, Inc.) to detect the presence of HRP.\",\n \"Numerous labels are know by those of skill in the art and include, but are not limited to, particles, fluorophores, haptens, enzymes and their calorimetric, fluorogenic and chemiluminescent substrates and other labels that are described in RICHARD P. HAUGLAND, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH PRODUCTS (9th edition, CD-ROM, September 2002), supra.\",\n \"The term “labeling reagent” as used herein refers to a monovalent antibody fragment or a non-antibody monomeric protein provided that the labeling reagent has affinity for a selected region of the target-binding antibody and is covalently attached to a label.\",\n \"The term “labeling reagent subset” as used herein refers to a discrete set of labeling reagents that are homogenous and can be distinguished from another subset of labeling reagent either by the physical properties of the label or the ratio of the label to labeling reagent.\",\n \"The term “labeling solution” as used herein refers to a solution that is used to form an immuno-labeled complex wherein the solution comprises labeling reagents and a buffer.\",\n \"The term “matrix” as used herein refers to a solid or semi-solid surface that a biological molecule can be attached to, such as a sample of the present invention or a capture reagent.\",\n \"Examples include, but are not limited to, agarose, polyacrylamide gel, polymers, microspheres, glass surface, plastic surface, membrane, margnetic surface, and an array.\",\n \"The term “monovalent antibody fragment” as used herein refers to an antibody fragment that has only one antigen-binding site.\",\n \"Examples of monovalent antibody fragments include, but are not limited to, Fab fragments (no hinge region), Fab′ fragments (monovalent fragments that contain a heavy chain hinge region), and single-chain fragment variable (ScFv) proteins.\",\n \"The term “non-antibody monomeric protein” as used herein refers to a protein that binds selectively and non-covalently to a member of the Ig superfamily of proteins, including but not limited to proteins A, G, and L, hybrids thereof (A/G), recombinant versions and cloned versions thereof, fusions of these proteins with detectable protein labels, and lectins but the protein itself is not an antibody or an antibody fragment.\",\n \"The terms “protein” and “polypeptide” are used herein in a generic sense to include polymers of amino acid residues of any length.\",\n \"The term “peptide” is used herein to refer to polypeptides having less than 100 amino acid residues, typically less than 10 amino acid residues.\",\n \"The terms apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.\",\n \"The term “purified” as used herein refers to a preparation of a target-binding antibody that is essentially free from contaminating proteins that normally would be present in association with the antibody, e.g., in a cellular mixture or milieu in which the protein or complex is found endogenously such as serum proteins or hybridoma supernatant.\",\n \"The term “sample” as used herein refers to any material that may contain a target, as defined below.\",\n \"Typically, the sample comprises a population of cells, cellular extract, subcellular components, tissue culture, a bodily fluid, and tissue.\",\n \"The sample may be in an aqueous solution, a viable cell culture or immobilized on a solid or semi solid surface such as a gel, a membrane, a glass surface, a microparticle or on a microarray.\",\n \"The term “target” as used herein refers to any entity that a target-binding antibody has affinity for such as an epitope or antigen.\",\n \"This target includes not only the discrete epitope that the target-binding antibody has affinity for but also includes any subsequently bound molecules or structures.\",\n \"In this way an epitope serves as a marker for the intended target.\",\n \"For example, a cell is a target wherein the target-binding antibody binds a cell surface protein such as CD3 on a T cell wherein the target marker is CD3 and the target is the T cell.\",\n \"The term “target-binding antibody” as used herein refers to an antibody that has affinity for a discrete epitope or antigen that can be used with the methods of the present invention.\",\n \"Typically the discrete epitope is the target but the epitope can be a marker for the target such as CD3 on T cells.\",\n \"The term can be used interchangeably with the term “primary antibody” when describing methods that use an antibody that binds directly to the antigen as opposed to a “secondary antibody” that binds to a region of the primary antibody.\",\n \"II.\",\n \"Compositions and Methods of Use In accordance with the present invention, labeling reagents, methods for labeling target-binging antibodies and methods for using the labeled antibodies to detect a target in a sample are provided.\",\n \"The labeling reagents comprise monovalent antibody fragments or non-antibody monomeric proteins that are covalently attached to a label of the present invention.\",\n \"The label covalently attached to a labeling reagent is directly detectable such as a fluorophore or functions as an indirect label that requires an additional component such as a calorimetric enzyme substrate or an enzyme conjugate.\",\n \"The labeling reagents have affinity for a specific region of the target-binding antibody.\",\n \"The target-binding antibodies are defined as any antibody known to one skilled in the art that has an affinity for a target in a sample.\",\n \"The target-binding antibodies are labeled with the labeling reagent in a labeling method to form immuno-labeled complexes and then added to a sample to detect a target.\",\n \"The labeling reagent and the methods of the present invention provide for detection of one or multiple targets in a sample.\",\n \"Multiple targets are detected when either pooled subsets of immuno-labeled complexes or a panel of subsets that are sequentially added to a sample.\",\n \"The subset of immuno-labeled complexes begins with labeling reagent subsets wherein a labeling reagent subset is distinguished by the ratio of label to labeling reagent or by the physical characteristics of the label.\",\n \"The discrete labeling reagents subsets are added to the target-binding antibodies wherein the affinity of the antibody and ratio of labeling reagent to target-binding antibody determines the subsets of immuno-labeled complexes.\",\n \"This results in an infinite number of immuno-labeled complex subsets that are distinguished by i) the target-binding antibody, or ii) a ratio of label to labeling reagent, or iii) a ratio of labeling reagent to the target-binding antibody or iv) by a physical property of the label.\",\n \"These subsets can be used individually in a method of the present invention to detect a single or multiple targets in a sample or pooled and used to simultaneously detect multiple targets in a sample.\",\n \"These pooled subsets allow for not only detection but also identification and quantitation of the targets.\",\n \"A. Labelinq Reagents 1.Monovalent Antibody Fragments and Monomeric Non-Antibody Proteins The labeling reagents of the present invention are monovalent antibody fragments or non-antibody monomeric proteins that have affinity for a region of a target-binding antibody.\",\n \"The regions of the target-binding antibody that can be bound by a labeling reagent include the Fc region, the kappa or lambda light chain region or a heavy chain region.\",\n \"When the labeling reagent is derived from an antibody the monovalent fragment can be, anti-Fc, an anti-Fc isotype, anti-kappa light chain, anti-lambda light chain, or a single-chain fragment variable protein.\",\n \"Labeling reagents that are a non-antibody peptide or protein, are for example but not limited to, soluble Fc receptor, protein G, protein A, protein L, lectins, or a fragment thereof.\",\n \"The labeling reagents typically have affinity for the Fc region of the target-binding antibody but any region, except the binding domain, may be used as a binding site for the labeling reagent.\",\n \"The Fc region is preferable because it is the farthest from the binding domain of the target-binding antibody and is unlikely to cause steric hinderance, when bound by a labeling reagent, of the binding domain for the target.\",\n \"Antibody is a term of the art denoting the soluble substance or molecule secreted or produced by an animal in response to an antigen, and which has the particular property of combining specifically with the antigen that induced its formation.\",\n \"Antibodies themselves also serve are antigens or immunogens because they are glycoproteins and therefore are used to generate anti-species antibodies.\",\n \"Antibodies, also known as immunoglobulins, are classified into five distinct classes—IgG, IgA, IgM, IgD, and IgE.\",\n \"The basic IgG immunoglobulin structure consists of two identical light polypeptide chains and two identical heavy polypeptide chains (linked together by disulfide bonds).\",\n \"When IgG is treated with the enzyme papain, a monovalent antigen-binding fragment can be isolated, referred herein to as a Fab fragment.\",\n \"When IgG is treated with pepsin (another proteolytic enzyme), a larger fragment is produced, F(ab′)2.This fragment can be split in half by treating with a mild reducing buffer that results in the monovalent Fab′ fragment.\",\n \"The Fab′ fragment is slightly larger than the Fab and contains one or more free sulfhydryls from the hinge region (which are not found in the smaller Fab fragment).\",\n \"The term “antibody fragment” is used herein to define both the Fab′ and Fab portions of the antibody.\",\n \"It is well known in the art to treat antibody molecules with pepsin and papain in order to produce antibody fragments (Gorevic et al., Methods of Enzyol., 116:3 (1985)).\",\n \"The monovalent Fab fragments of the present invention are produced from either murine monoclonal antibodies or polyclonal antibodies generated in a variety of animals that have been immunized with a foreign antibody or fragment thereof, U.S. Pat.\",\n \"No.\",\n \"4,196,265 discloses a method of producing monoclonal antibodies.\",\n \"Typically, labeling reagents are derived from a polyclonal antibody that has been produced in a rabbit or goat but any animal known to one skilled in the art to produce polyclonal antibodies can be used to generate anti-species antibodies.\",\n \"However, monoclonal antibodies are equal, and in some cases, preferred over polyclonal antibodies provided that the target-binding antibody is compatible with the monoclonal antibodies that are typically produced from murine hybridoma cell lines using methods well known to one skilled in the art.\",\n \"Example 1 describes production of polyclonal antibodies raised in animals immunized with the Fc region of a foreign antibody.\",\n \"It is a preferred embodiment of the present invention that the labeling reagents be generated against only the Fc region of a foreign antibody.\",\n \"Essentially, the animal is immunized with only the Fc region fragment of a foreign antibody, such as murine.\",\n \"The polyclonal antibodies are collected from subsequent bleeds, digested with an enzyme, pepsin or papain, to produce monovalent fragments.\",\n \"The fragments are then affinity purified on a column comprising whole immunoglobulin protein that the animal was immunized against or just the Fc fragments.\",\n \"As described in detail below, the labeling reagents are also covalently labeled with fluorophore labels when bound to the affinity column to eliminate incorporating label into the binding domain of the monovalent fragment.\",\n \"One of skill in the art will appreciate that this method can be used to generate monovalent fragments against any region of a target-binding protein and that selected peptide fragments of the target-binding antibody could also be used to generate fragments.\",\n \"Alternatively, a non-antibody protein or peptide such as protein G, or other suitable proteins, can be used alone or coupled with albumin wherein albumin is attached with a label of the present invention.\",\n \"Preferred albumins of the invention include human and bovine serum albumins or ovalbumin.\",\n \"Protein A, G and L are defined to include those proteins know to one skilled in the art or derivatives thereof that comprise at least one binding domain for IgG, i.e.\",\n \"proteins that have affinity for IgG.\",\n \"These proteins can be modified but do not need to be and are labeled in the same manner as the monovalent Fab fragments of the invention.\",\n \"2.Labels The labels of the present invention include any directly or indirectly detectable label known by one skilled in the art that can be covalently attached to the labeling reagent of the present invention.\",\n \"Labels include, without limitation, a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme and a radioisotope.\",\n \"Preferred labels include fluorophores, fluorescent proteins, haptens, and enzymes.\",\n \"A fluorophore of the present invention is any chemical moiety that exhibits an absorption maximum beyond 280 nm, and when covalently attached to a labeling reagent retains its spectral properties.\",\n \"Fluorophores of the present invention include, without limitation; a pyrene (including any of the corresponding derivative compounds disclosed in U.S. Pat.\",\n \"No.\",\n \"5,132,432), an anthracene, a naphthalene, an acridine, a stilbene, an indole or benzindole, an oxazole or benzoxazole, a thiazole or benzothiazole, a 4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), a cyanine (including any corresponding compounds in U.S. Ser.\",\n \"Nos.\",\n \"09/968,401 and 09/969,853), a carbocyanine (including any corresponding compounds in U.S. Ser.\",\n \"Nos.\",\n \"09/557,275; 09/969,853 and 09/968,401; U.S. Pat.\",\n \"Nos.\",\n \"4,981,977; 5,268,486; 5,569,587; 5,569,766; 5,486,616; 5,627,027; 5,808,044; 5,877,310; 6,002,003; 6,004,536; 6,008,373; 6,043,025; 6,127,134; 6,130,094; 6,133,445; and publications WO 02/26891, WO 97/40104, WO 99/51702, WO 01/21624; EP 1 065 250 A1), a carbostyryl, a porphyrin, a salicylate, an anthranilate, an azulene, a perylene, a pyridine, a quinoline, a borapolyazaindacene (including any corresponding compounds disclosed in U.S. Pat.\",\n \"Nos.\",\n \"4,774,339; 5,187,288; 5,248,782; 5,274,113; and 5,433,896), a xanthene (including any corresponding compounds disclosed in U.S. Pat.\",\n \"No.\",\n \"6,162,931; 6,130,101; 6,229,055; 6,339,392; 5,451,343 and U.S. Ser.\",\n \"No.\",\n \"09/922,333), an oxazine (including any corresponding compounds disclosed in U.S. Pat.\",\n \"No.\",\n \"4,714,763) or a benzoxazine, a carbazine (including any corresponding compounds disclosed in U.S. Pat.\",\n \"No.\",\n \"4,810,636), a phenalenone, a coumarin (including an corresponding compounds disclosed in U.S. Pat.\",\n \"Nos.\",\n \"5,696,157; 5,459,276; 5,501,980 and 5,830,912), a benzofuran (including an corresponding compounds disclosed in U.S. Pat.\",\n \"Nos.\",\n \"4,603,209 and 4,849,362) and benzphenalenone (including any corresponding compounds disclosed in U.S. Pat.\",\n \"No.\",\n \"4,812,409) and derivatives thereof.\",\n \"As used herein, oxazines include resorufins (including any corresponding compounds disclosed in Pat.\",\n \"No.\",\n \"5,242,805), aminooxazinones, diaminooxazines, and their benzo-substituted analogs.\",\n \"When the fluorophore is a xanthene, the fluorophore is optionally a fluorescein, a rhodol (including any corresponding compounds disclosed in U.S. Pat.\",\n \"Nos.\",\n \"5,227,487 and 5,442,045), or a rhodamine (including any corresponding compounds in U.S. Pat.\",\n \"Nos.\",\n \"5,798,276; 5,846,737; U.S. Ser.\",\n \"No.\",\n \"09/129,015).\",\n \"As used herein, fluorescein includes benzo- or dibenzofluoresceins, seminaphthofluoresceins, or naphthofluoresceins.\",\n \"Similarly, as used herein rhodol includes seminaphthorhodafluors (including any corresponding compounds disclosed in U.S. Pat.\",\n \"No.\",\n \"4,945,171).\",\n \"Alternatively, the fluorophore is a xanthene that is bound via a linkage that is a single covalent bond at the 9-position of the xanthene.\",\n \"Preferred xanthenes include derivatives of 3H-xanthen-6-ol-3-one attached at the 9-position, derivatives of 6-amino-3H-xanthen-3-one attached at the 9-position, or derivatives of 6-amino-3H-xanthen-3-imine attached at the 9-position.\",\n \"Preferred fluorophores of the invention include xanthene (rhodol, rhodamine, fluorescein and derivatives thereof) coumarin, cyanine, pyrene, oxazine and borapolyazaindacene.\",\n \"Most preferred are sulfonated xanthenes, fluorinated xanthenes, sulfonated coumarins, fluorinated coumarins and sulfonated cyanines.\",\n \"The choice of the fluorophore attached to the labeling reagent will determine the absorption and fluorescence emission properties of the labeling reagent and immuno-labeled complex.\",\n \"Physical properties of a fluorophore label include spectral characteristics (absorption, emission and stokes shift), fluorescence intensity, lifetime, polarization and photo-bleaching rate all of which can be used to distinguish one fluorophore from another.\",\n \"Typically the fluorophore contains one or more aromatic or heteroaromatic rings, that are optionally substituted one or more times by a variety of substituents, including without limitation, halogen, nitro, cyano, alkyl, perfluoroalkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, arylalkyl, acyl, aryl or heteroaryl ring system, benzo, or other substituents typically present on fluorophores known in the art.\",\n \"In one aspect of the invention, the fluorophore has an absorption maximum beyond 480 nm.\",\n \"In a particularly useful embodiment, the fluorophore absorbs at or near 488 nm to 514 nm (particularly suitable for excitation by the output of the argon-ion laser excitation source) or near 546 nm (particularly suitable for excitation by a mercury arc lamp).\",\n \"Many of fluorophores can also function as chromophores and thus the described fluorophores are also preferred chromophores of the present invention.\",\n \"In addition to fluorophores, enzymes also find use as labels for the labeling reagents.\",\n \"Enzymes are desirable labels because amplification of the detectable signal can be obtained resulting in increased assay sensitivity.\",\n \"The enzyme itself does not produce a detectable response but functions to break down a substrate when it is contacted by an appropriate substrate such that the converted substrate produces a fluorescent, colorimetric or luminescent signal.\",\n \"Enzymes amplify the detectable signal because one enzyme on a labeling reagent can result in multiple substrates being converted to a detectable signal.\",\n \"This is advantageous where there is a low quantity of target present in the sample or a fluorophore does not exist that will give comparable or stronger signal than the enzyme.\",\n \"However, fluorophores are most preferred because they do not require additional assay steps and thus reduce the overall time required to complete an assay.\",\n \"The enzyme substrate is selected to yield the preferred measurable product, e.g.\",\n \"calorimetric, fluorescent or chemiluminescence.\",\n \"Such substrates are extensively used in the art, many of which are described in the MOLECULAR PROBES HANDBOOK, supra.\",\n \"A preferred calorimetric or fluorogenic substrate and enzyme combination uses oxidoreductases such as horseradish peroxidase and a substrate such as 3,3′-diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC), which yield a distinguishing color (brown and red, respectively).\",\n \"Other calorimetric oxidoreductase substrates that yield detectable products include, but are not limited to: 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD), 3,3′,5,5′-tetramethylbenzidine (TMB), o-dianisidine, 5-aminosalicylic acid, 4-chloro-1-naphthol.\",\n \"Fluorogenic substrates include, but are not limited to, homovanillic acid or 4-hydroxy-3-methoxyphenylacetic acid, reduced phenoxazines and reduced benzothiazines, including Amplexe Red reagent and its variants (U.S. Pat.\",\n \"No.\",\n \"4,384,042) and reduced dihydroxanthenes, including dihydrofluoresceins (U.S. Pat.\",\n \"No.\",\n \"6,162,931) and dihydrorhodamines including dihydrorhodamine 123.Peroxidase substrates that are tyramides (U.S. Pat.\",\n \"Nos.\",\n \"5,196,306; 5,583,001 and 5,731,158) represent a unique class of peroxidase substrates in that they can be intrinsically detectable before action of the enzyme but are “fixed in place” by the action of a peroxidase in the process described as tyramide signal amplification (TSA).\",\n \"These substrates are extensively utilized to label targets in samples that are cells, tissues or arrays for their subsequent detection by microscopy, flow cytometry, optical scanning and fluorometry.\",\n \"Another preferred calorimetric (and in some cases fluorogenic) substrate and enzyme combination uses a phosphatase enzyme such as an acid phosphatase, an alkaline phosphatase or a recombinant version of such a phosphatase in combination with a calorimetric substrate such as 5-bromo-6-chloro-3-indolyl phosphate (BCIP), 6-chloro-3-indolyl phosphate, 5-bromo-6-chloro-3-indolyl phosphate, p-nitrophenyl phosphate, or o-nitrophenyl phosphate or with a fluorogenic substrate such as 4-methylumbelliferyl phosphate, 6,8-difluoro-7-hydroxy4-methylcoumarinyl phosphate (DiFMUP, U.S. Pat.\",\n \"No.\",\n \"5,830,912) fluorescein diphosphate, 3-O-methylfluorescein phosphate, resorufin phosphate, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate (DDAO phosphate), or ELF 97, ELF 39 or related phosphates (U.S. Pat.\",\n \"Nos.\",\n \"5,316,906 and 5,443,986).\",\n \"Glycosidases, in particular beta-galactosidase, beta-glucuronidase and beta-glucosidase, are additional suitable enzymes.\",\n \"Appropriate colorimetric substrates include, but are not limited to, 5-bromo4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) and similar indolyl galactosides, glucosides, and glucuronides, o-nitrophenyl beta-D-galactopyranoside (ONPG) and p-nitrophenyl beta-D-galactopyranoside.\",\n \"Preferred fluorogenic substrates include resorufin beta-D-galactopyranoside, fluorescein digalactoside (FDG), fluorescein diglucuronide and their structural variants (U.S. Pat.\",\n \"Nos.\",\n \"5,208,148; 5,242,805; 5,362,628; 5,576,424 and 5,773,236), 4-methylumbelliferyl beta-D-galactopyranoside, carboxyumbelliferyl beta-D-galactopyranoside and fluorinated coumarin beta-D-galactopyranosides (U.S. Pat.\",\n \"No.\",\n \"5,830,912).\",\n \"Additional enzymes include, but are not limited to, hydrolases such as cholinesterases and peptidases, oxidases such as glucose oxidase and cytochrome oxidases, and reductases for which suitable substrates are known.\",\n \"Enzymes and their appropriate substrates that produce chemiluminescence are preferred for some assays.\",\n \"These include, but are not limited to, natural and recombinant forms of luciferases and aequorins.\",\n \"Chemiluminescence-producing substrates for phosphatases, glycosidases and oxidases such as those containing stable dioxetanes, luminol, isoluminol and acridinium esters are additionally useful.\",\n \"In addition to enzymes, haptens such as biotin are also preferred labels.\",\n \"Biotin is useful because it can function in an enzyme system to further amplify the detectable signal, and it can function as a tag to be used in affinity chromatography for isolation purposes.\",\n \"For detection purposes, an enzyme conjugate that has affinity for biotin is used, such as avidin-HRP.\",\n \"Subsequently a peroxidase substrate is added to produce a detectable signal.\",\n \"Haptens also include hormones, naturally occurring and synthetic drugs, pollutants, allergens, affector molecules, growth factors, chemokines, cytokines, lymphokines, amino acids, peptides, chemical intermediates, nucleotides and the like.\",\n \"Fluorescent proteins also find use as labels for the labeling reagents of the present invention.\",\n \"Examples of fluorescent proteins include green fluorescent protein (GFP) and the phycobiliproteins and the derivatives thereof.\",\n \"The fluorescent proteins, especially phycobiliprotein, are particularly useful for creating tandem dye labeled labeling reagents.\",\n \"These tandem dyes comprise a fluorescent protein and a fluorophore for the purposes of obtaining a larger stokes shift wherein the emission spectra is farther shifted from the wavelength of the fluorescent protein's absorption spectra.\",\n \"This is particularly advantageous for detecting a low quantity of a target in a sample wherein the emitted fluorescent light is maximally optimized, in other words little to none of the emitted light is reabsorbed by the fluorescent protein.\",\n \"For this to work, the fluorescent protein and fluorophore function as an energy transfer pair wherein the fluorescent protein emits at the wavelength that the fluorophore absorbs at and the fluorphore then emits at a wavelength farther from the fluorescent proteins than could have been obtained with only the fluorescent protein.\",\n \"A particularly useful combination is the phycobiliproteins disclosed in U.S. Pat.\",\n \"Nos.\",\n \"4,520,110; 4,859,582; 5,055,556 and the sulforhodamine fluorophores disclosed in Pat.\",\n \"No.\",\n \"5,798,276, or the sulfonated cyanine fluorophores disclosed in U.S. Ser.\",\n \"Nos.\",\n \"09/968/401 and 09/969/853; or the sulfonated xanthene derivatives disclosed in Pat.\",\n \"No.\",\n \"6,130,101 and those combinations disclosed in U.S. Pat.\",\n \"4,542,104.Alternatively, the fluorophore functions as the energy donor and the fluorescent protein is the energy acceptor.\",\n \"3.Covalent Attachment of Labels to the Labeling Reagents The labeling reagents can be independently attached to one or more labels of the present invention by a number of methods known to one skilled in the art and modification of such methods.\",\n \"Methods include, labeling in a solution or on an affinity column.\",\n \"For labeling in solution the labeling reagent is optionally modified to contain a reactive group and the label is modified to contain a reactive group or is synthesized to contain a reactive group, as is typically the case with fluorophore labels wherein the reactive group facilitates covalent attachment.\",\n \"The modification of the labeling reagent to contain a reactive group includes (1) chemical addition of such a reactive group or (2) alternatively takes advantage of the disulfide bonds of the F(ab′)2 fragment wherein the fragment is reduced to break the bond and expose the thiol group that readily reacts with a reactive group on a label, as disclosed in U.S. Pat.\",\n \"No.\",\n \"5,360,895.Typically, covalent attachment of the label to the fragment is the result of a chemical reaction between an electrophilic group and a nucleophilic group.\",\n \"However, when a label is used that is photoactivated the covalent attachment results when the labeling solution is illuminated.\",\n \"A method for covalently attaching a label, particularly an enzyme, a fluorescent protein or a particle, comprises the following steps: a) cleaving an intact anti-region antibody with an enzyme resulting in a F(ab′)2 fragment; b) contacting said F(ab′)2 fragment with a reducing agent to produce Fab′ fragments containing a thiol group; c) contacting said Fab′ fragments with a solution comprising a label that contains a reactive group; and, d) isolating Fab′ fragments of step d) that are covalently attached to a label by size exclusion or affinity chromatography.\",\n \"The whole anti-region antibody is cleaved with pepsin to generate a bivalent F(ab)′2 fragment.\",\n \"This fragment is typically affinity purified on a column comprising immunoglobulin proteins such as IgG that is immobilized on agarose.\",\n \"The fragment is then reduced to break the disulfide bond of the hinge region that connects the two Fab fragments resulting in a Fab′ fragment with an exposed thiol group.\",\n \"This is typically accomplished by adding a mild reducing buffer to the affinity purified F(ab′)2 fragments such as a buffer comprising 0.01 M EDTA and 0.01 M cysteine in phosphate buffer saline (PBS).\",\n \"The resulting thiol group readily reacts with a reactive group on a label to covalently attach the label to the fragment.\",\n \"Thus, a solution containing a label that has been chemically modified to contain a reactive group, using methods well known to one skilled in the art, is added to the solution of reduced Fab′ fragments.\",\n \"This method is particularly useful for covalently attaching enzyme and other protein labels due to their size and the lack of exposed amine groups on the Fab fragments.\",\n \"One of skill in the art will appreciate that this method requires the use of Fab′ fragments as apposed to Fab fragments due to the disulfide bonds of the Fab′ fragment and that the use of the enzyme papain or the like results in such a fragment.\",\n \"An alternative labeling of monovalent antibody fragments and the monomeric non-antibody proteins is also accomplished in a solution.\",\n \"The method comprises the steps: a) contacting a Fab fragment or non-antibody monomeric protein with a solution comprising a label that contains a reactive group; and, b) isolating labeled anti-region Fab fragment or non-antibody monomeric protein by size exclusion or affinity chromatography.\",\n \"When a Fab fragment is to be labeled the whole antibody is cleaved with an enzyme, such as papain, to generate Fab monovalent fragments and the fragments are typically purified on an affinity column prior to addition of the label.\",\n \"The Fab fragment or non-antibody monomeric proteins are optionally chemically modified to contain a reactive group.\",\n \"However, for covalently attaching reactive fluorophore labels it has been found that this modification of the fragment of non-antibody protein is not necessary.\",\n \"The reactive label, typically a fluorophore or hapten, are added to a solution of Fab fragments or non-antibody proteins and the labeling reagent is separated from excess label by size exclusion or affinity chromatorgraphy.\",\n \"The labeling reagents are then stored in an appropriate buffer.\",\n \"Labeling in solution can have some drawbacks, especially when labeling of Fab fragments or non-antibody proteins with fluorophores.\",\n \"Thus, Fab fragments and non-antibody proteins of the present invention are preferably covalently attached to a fluorophore label when immobilized on an affinity column.\",\n \"The fragments and non-antibody proteins are immobilized on an affinity column that comprises a protein that the fragment has affinity for, typically IgG, and after immobilization a reactive fluorophore is added to the column wherein the fragments are labeled and unreacted fluorophores pass through the column.\",\n \"The use of this affinity chromatography method avoids the incorporation of label into the binding domain of the Fab fragment or non-antibody protein.\",\n \"When Fab fragments are labeled with fluorophores using this method unexpected advantages were obtained wherein the fluorescent signal form fragments labeled on a column are brighter than fragments labeled in solution when the fluorophore and ratio of fluorophore to labeling reagent are held constant.\",\n \"Without wishing to be bound by a theory it is possible that the decreased brightness observed from the fragments labeled in solution is due to quenching of fluorphores that are bound in or near the binding domain by the high concentration of amine groups in the binding domain.\",\n \"Thus, a preferred embodiment of the invention for covalently attaching fluorphore labels to Fab fragments comprises the following steps: a) cleaving an intact anti region antibody with an enzyme that generates Fab fragments; b) isolating the anti-region Fab fragments of step a); c) contacting a matrix comprising intact immunoglobulin proteins or fragments thereof that specifically bind anti-region Fab fragments with a solution comprising said anti-region fragments of step b) wherein said Fab fragments are immobilized; d) contacting said matrix of step c) with a solution comprising a fluorophore label that contains a reactive group; e) washing said matrix to remove unbound label, and; f) eluting said labeling reagent from said matrix whereby said labeling reagent is manufactured comprising a label and being isolated from other proteins and fragments thereof.\",\n \"The matrix is typically an agarose column that comprises either the selected region, such as the Fc region, or the entire antibody provided that the antibody or fragment thereof is the same species and isotype that was used to produce the antibodies that the labeling reagent was generated from.\",\n \"However any matrix known to one skilled in the art can be used that allows for immobilization of labeling reagent and removal following attachment of the fluorophore label.\",\n \"Fab and Fab′ fragments can both be labeled in this manner.\",\n \"However a free thiol group is not necessary and therefore Fab fragments are typically labeled using this method.\",\n \"Due to the unique properties of the labeling reagent and the attached labels it is a preferred embodiment of the present invention that enzyme or other protein labels are covalently attached to Fab′ fragments in solution utilizing the free thiol group of the Fab′ fragment.\",\n \"It is another preferred embodiment that fluorophore labels be covalently attached to the labeling reagent when the reagent is immobilized on a affinity column wherein the labeling reagent is typically an Fab fragment or a non-antibody monomeric protein.\",\n \"The attachment of the label to the fragments or the non-antibody proteins results in multiple subsets that are distinguished by the ratio of the label to the labeling reagent and the physical properties of the label.\",\n \"A labeling reagent subset as used herein refers to a discrete set of labeling reagents that are homogenous and can be distinguished from another subset of labeling reagent either by the physical properties of the label or the ratio of the label to labeling reagent.\",\n \"The physical properties include differences within a group of labels, such as emission spectra of fluorphores, or across groups of labels, such as the difference between an enzyme and a fluorophore.\",\n \"For fluorphore labels, the physical properties typically relates to the emission spectra, this includes modification of the same label, e.g.\",\n \"a cyanine with different substitutions that shifts the emission wavelength, or different fluorophores, e.g.\",\n \"a cyanine and a coumarin on the same labeling reagent.\",\n \"The difference in physical properties also includes the use of tandem dyes, which is specifically defined to include an energy transfer pair wherein one is a protein and the other is a fluorophore or both are fluorophores, or the pairing of other labels that are not necessarily energy transfer pairs.\",\n \"A few examples of labeling reagent subsets includes, but are not limited to, a first subset comprising a single fluorophore at a known ration attached to a anti-Fc Fab fragment; a second subset comprises the same fluorophore on the Fab fragment at a different known ration from the first subset, a third subset comprises the same fluorophore but that has a shifted wavelength due to a substitution on the fluorophore.\",\n \"Thus, the attachment of labels to the labeling reagents results in an extensive selection of subsets that when complexed with a target-binding antibody results in a unique method to detect one or multiple targets in a sample whereby the target is identified and quantitated.\",\n \"B. Immuno-Labeled Complex The subsets of labeling reagent are complexed with target-binding antibodies to produce subsets of immuno-labeled complex that for the target detection solution.\",\n \"The methods for forming the immuno-labeled complex comprises the following steps: a) contacting a solution of target-binding antibodies with a labeling reagent subset, wherein said labeling reagent subsets are distinguished by i) ratio of label to labeling reagent or ii) a physical properties of said label; b) incubating said target-binding antibodies and said labeling reagent for a time period sufficient for one or more labeling reagents to form an immuno-labeled complex with a target-binding antibody wherein a region of said target binding antibody is selectively bound by labeling reagent; c) optionally removing unbound labeling reagent by adding a capture reagent comprising immunoglobulin proteins or fragments thereof; and, d) optionally repeating said steps a), b), and c) to form individual or pooled subsets of immuno-labeling complexes wherein each subset is distinguished from another subset by i) a ratio of label to labeling reagent, or ii) a physical property of said label, or iii) a ratio of labeling reagent to said target-binding antibody, or iv) by said target-binding antibody.\",\n \"A particular advantage for the use of labeling reagent of the present invention to label target-binding antibodies is that the process is relatively insensitive to the solution the antibodies are in.\",\n \"Due to the physical nature of the labeling reagents, small monovalent fragments, the reagents do not cross-link and fall out of solution in the presence of high concentration of proteins.\",\n \"For this reason, target-binding antibodies can be complexed when present in ascites fluid, tissue culture supernatant, serum or other solutions where there is a high concentration of proteins.\",\n \"This eliminates the need to purify target-binding proteins prior to labeling.\",\n \"When preparing the immuno-labeled complex using purified target-binding antibody, stock solutions of both the labeling reagent and the target-binding antibody are typically near 1 mg/mL in an appropriate buffer, although more or less concentrated solutions are also suitable.\",\n \"Generally, the labeling reagent is mixed in a molar ratio of at least one to 50 moles of labeling reagent to one mole of the target-binding antibody to be complexed.\",\n \"More commonly a ratio of at least one to as many as 10 moles of labeling reagent per mole of target-binding antibody is combined.\",\n \"With an anti-Fc region Fab to a target-binding antibody, a molar ratio of approximately 2 to 10 is typical, more typically 3 to 5 (particularly for complexes in which the labeling reagent has been labeled while immobilized on an affinity matrix).\",\n \"The ease of formation of the complex permits rapid optimization of the complex and assessment of the effect of variation in experimental parameters.\",\n \"A particularly unique advantage of the invention is that the stoichiometry of the complex is easily adjusted to provide complexes with different ratios of labeling reagent to target-binding antibody, and thus there is control over the ultimate detectability of the target in the sample.\",\n \"Complexes that have been labeled with the same dye but at different molar ratios can be separately detected by the differences in their intensities.\",\n \"Complex formation appears to occur almost within the mixing time of the solutions (<1 minute) but the reaction typically is allowed to proceed for at least 5 minutes and can be longer before combining the immuno-labeled complex with the sample.\",\n \"Although complex formation can be reversed by addition of an unlabeled antibody that contains the same binding region, reversibility is very slow; furthermore, following binding of the immuno-labeled complex to a target in a sample, the sample can be “fixed” using aldehyde-based fixatives by methods that are commonly practiced by those skilled in the art of immunolabeling.\",\n \"The labeling process optionally further comprises the addition of a capture component to remove excess labeling reagent.\",\n \"For applications in which immunolabeling complexes of multiple primary antibodies from the same species (e.g.\",\n \"mouse monoclonal antibodies) or cross-reacting species (e.g.\",\n \"mouse and human antibodies) are to be used simultaneously or sequentially, it is necessary to quench or otherwise remove any excess labeling reagent by use of a capture component or by other means to avoid inappropriate labeling of the sample.\",\n \"The most effective capturing components to capture excess labeling reagent are those that contain the binding site of the labeling reagent but are themselves not labeled, preferably an antibody or antibody fragment.\",\n \"Capture components may be free in solution or immobilized on a matrix, such as agarose, cellulose, or a natural or synthetic polymer, to facilitate separation of the excess capture component from the immuno-labeled complex.\",\n \"The capture component is optionally attached to a microsphere or magnetic particle.\",\n \"However, separation of excess labeling reagent is not essential for successful utilization of the invention, particularly when using a single target-binding antibody.\",\n \"The steps of the labeling process for the target-binding antibodies can be repeated to form discrete immuno-labeled complex subsets that can be used individually or pooled in an assay to detect individual or multiple targets.\",\n \"As used herein the term immuno-labeled complex subsets refers to subsets that are distinguished from each other i) a ratio of label to labeling reagent, or ii) a physical property of the label, or iii) a ratio of labeling reagent to the target-binding antibody, or iv) by the target-binding antibody, or a combination thereof.\",\n \"For example a panel of subsets may comprise a target-binding antibody that is bound by a labeling reagent comprising a subset of different ratios of the same label on the labeling reagent resulting in a discrete subset of immuno-labeled complexes.\",\n \"This subset of immuno-labeled complexes can be used individually wherein a target is identified by the intensity of the detectable label or used in combination with another subset of immunocomplexes that differ in the target-binding antibody to identify multiple targets.\",\n \"C. Methods of Use The labeling reagents, target-binding antibodies and resulting immuno-labeled complex that forms the target detection solution can be used in a wide range of immunoassays, essentially in any assay a traditional secondary antibody is used including some assays that secondary antibodies are not used because of their size and ability to cross-link.\",\n \"Examples of such assays used to detect a target in a sample include immunoblots, direct detection in a gel, flow cytometry, immunohistochemistry, confocal microscopy, fluorometry, ELISA and other modified immunoassays.\",\n \"A method of the present invention for detecting a single target in a sample comprises the following steps: a) contacting a solution of target-binding antibodies with a labeling reagent subset, wherein said labeling reagent subsets are distinguished by i) ratio of label to labeling reagent or ii) a physical properties of said label; b) incubating said target-binding antibodies and said labeling reagent subset for a time period sufficient for one or more labeling reagents to form an immuno-labeled complex with a target-binding antibody wherein a region of said target binding antibody is selectively bound by labeling reagent; c) contacting said sample with said immuno-labeled complex of step b); d) incubating said sample of step c) for a time sufficient to allow said immuno-labeled complex to selectively bind to said target; and, e) illuminating said immuno-labeled complex whereby said target is detected.\",\n \"A sample is incubated with a preformed immuno-labeled complex that comprises a labeling reagent and a target-binding antibody.\",\n \"While this method describes the identification of a single target, subsets of labeling reagents bound to the same target-binding antibody can be used to identify and provide additional information about such targets.\",\n \"For example, subsets of labeling reagent can be prepared wherein two discrete subsets are generate each with a distinct fluorophore label that is distinguished by their emission spectra, e.g.\",\n \"one that emits in the green spectra and one that emits in the red spectra.\",\n \"The labeling reagent subsets are then added to a solution of target-binding antibody in a controlled ratio, e.g.\",\n \"two parts one labeling reagent (green emission) and one part the other labeling reagent (red emission) per target binding antibody.\",\n \"In this way the immuno-labeled complexes can be used to detect a target.\",\n \"If another immuno-labeled complex were added to the sample the original target could be distinguished from the subsequently detected target.\",\n \"The methods of the present invention also provide for the detection of multiple targets in a sample.\",\n \"Multiple targets include the discrete epitope that the target-binding antibody has affinity for as well as molecules or structures that the epitiope is bound to.\",\n \"Thus, multiple target identification includes phenotyping of cells based on the concentration of the same cell surface marker on different cells.\",\n \"In this way multiple target identification is not limited to the discrete epitope that the target binding antibody binds, although this is clearly a way that multiple targets can be identified, i.e.\",\n \"based on the affinity of the target-binding antibody.\",\n \"Therefore, a method for detecting multiple targets in a sample comprises the following steps: a) contacting a solution of target-binding antibodies with a labeling reagent subset, wherein said labeling reagent subsets are distinguished by i) ratio of label to labeling reagent or ii) a physical properties of said label; b) incubating said target-binding antibodies and said labeling reagent subset for a time period sufficient for one or more labeling reagents to form an immuno-labeled complex with a target-binding antibody wherein a region of said target-binding antibody is selectively bound by labeling reagent, wherein steps a) and b) are repeated to form discrete immuno-labeling complex subsets; c) contacting said sample with a solution comprising A) a pooled subset of immuno-labeled complexes, wherein each subset is distinguished from another subset by i) a ratio of label to labeling reagent, or ii) a physical property of said label, or iii) a ratio of labeling reagent to said target-binding antibody, or iv) by said target-binding antibody or B) an individual subset wherein step c) with a solution comprising an individual subset is repeated; d) incubating said sample of step c) for a time sufficient to allow said immuno-labeled complex to selectively bind to said target; and, e) illuminating said immuno-labeled complex whereby said target is detected.\",\n \"A selected target-binding antibody and a subset of labeling reagent are incubated to form an immuno-labeled complex subset.\",\n \"This procedure is repeated to form a panel of immuno-labeled complex subsets that may be pooled and added to a sample.\",\n \"Altematively each immuno-labeled complex subset is added stepwise to a sample.\",\n \"The immuno-labeled complex subsets are distinguished by four characteristics resulting in an infinite number of immuno-labeled complex subsets.\",\n \"First (i) the subsets can be distinguished by the target-binding antibody that is determined by the end user for the information that is desired from a sample.\",\n \"This means that each subset is distinguished based on the affinity of the target-binding antibody.\",\n \"The target-binding antibody typically distinguishes immuno-labeled complexes when multiple targets are identified, however this is normally combined with another characteristic to gain information form a sample or increase the number of targets that can be detected at one time.\",\n \"The second (ii) distinguishing feature used is the ratio of label to labeling reagent, as discussed in detail above.\",\n \"A subset based on this feature would have for example a ratio of two fluorophore per each labeling reagent.\",\n \"The third (iii) distinguishing feature is the ratio of labeling reagent to target-binding antibody.\",\n \"This is accomplished using a controlled concentration of target-binding antibody mixed with a controlled concentration of a labeling reagent subset and the subset would comprise a target-binding antibody that is bound by a discrete number of labeling proteins.\",\n \"The fourth (iv) feature is the physical feature of the label.\",\n \"Typically this refers to the physical properties of the fluorophore labels wherein a subset of this group is distinguished by the label itself such as a green emitting fluorophore compared to a red emitting fluorophore.\",\n \"One of skill in the art will appreciate that while immuno-labeling complex subsets can be distinguished based on one feature the subsets are typically, and most useful, when discretely identified based on a combination of the distinguishing characteristics.\",\n \"Another example of detection of multiple targets utilizes the following immuno-labeled subsets, all of which comprise a different target-binding antibody but differ in the label and ratio of label.\",\n \"The first subset comprises a fluorophore label that emits red-fluorescent light, a second subset comprises a fluorophore label that emits green fluorescent light, a third subset comprises a ratio of 1:1 red to green fluorophore label; a fourth subset comprises a ratio of 2:1 red to green fluorophore label and a fifth subset comprises a ratio of 1:2 red to green fluorophore label.\",\n \"These subsets allow for the simultaneous detection of five targets in a sample.\",\n \"This aspect of the present invention is particularly important due to the limited range of fluorophores available wherein the labeling reagents can be utilized to increase the number of targets that can be detected at one time.\",\n \"One of skill in the art can appreciate that these subsets could be expanded by altering the ratio of label to labeling reagent instead of just the ratio of labeling reagent to target-binding antibody.\",\n \"This same methodology can also be applied to a single fluorophore label wherein the ratios are altered and a target is detected based on the intensity of the signal instead of the color and the ratio of the color to another color.\",\n \"Following the formation of the immuno-labeled complex subsets the subsets can be pooled and added to a sample or added stepwise to a sample, either of which is determined by the end user and the particular assay format.\",\n \"This method of the present invention provides for maximum flexibility and ease of determining multiple targets in a sample.\",\n \"Another method of the present invention provides for the determination of multiple targets in a sample specifically using the flow cytometry assay format.\",\n \"Traditionally targets identified using flow cytometry used either directly labeled primary antibody or labeled microspheres that were covalently attached to a primary antibody wherein the microsphere is the label.\",\n \"Examples include the fluorescent encapsulated microsphere beads sold by Luminex.\",\n \"The labeling reagents and the present invention overcome both the need for directly labeled primary antibody and the need for expensive microspheres.\",\n \"Thus, a method of the present invention for determining identity and quantity of targets in a sample by detecting multiple targets comprises the following steps: a) contacting a solution of target-binding antibodies with a labeling reagent subset, wherein said labeling reagent subsets are distinguished by i) ratio of label to labeling reagent or ii) a physical properties of said label; b) incubating said target-binding antibodies and said labeling reagent for a time period sufficient for one or more labeling reagents to form an immuno-labeled complex with a target-binding antibody wherein a region of said target binding antibody is selectively bound by labeling reagent, wherein steps a) and b) are repeated to form a pooled subset of immuno-labeling complexes; c) contacting a population of cells in a sample with a solution comprising a pooled subset of immuno-labeled complexes, wherein each subset is distinguished from another subset by i) a ratio of label to labeling reagent, or ii) a physical property of said label, or iii) a ratio of labeling reagent to said target-binding antibody, or iv) by said target-binding antibody; d) incubating said cells for a time period sufficient to allow said immuno-labeled complex to bind said targets; e) passing said incubated population of cells through an examination zone; and, f) collecting data from said cells that were passed through said examination zone wherein said multiple targets are detected whereby the identity and quantity of said targets is determined.\",\n \"In one aspect, a target-binding antibody is pre-complexed to the target-binding antibody to form a subset and that subset or a panel of subsets are added to a sample, that are typically distinguished by the target binding antibody.\",\n \"This method then avoids the need for a directly labeled primary.\",\n \"Secondly, when the panel of subsets is distinguished, for example, by the ratio of label to labeling reagent or the ratio of labeling reagent to target-binding antibody the immuno-labeled complex can function similar to the microsphere beads of Luminex.\",\n \"For example, this is accomplished wherein three immuno-labeled complex subsets are distinguished by the target binding antibody and the fluorophore attached to the labeling reagent and within one of the subsets is another set of subsets that are distinguished based on the ratio of label to labeling reagent.\",\n \"In this way three different epitopes are detected and one of the epitopes is further distinguished and a phenotype distinction made based on the intensity of the signal generated from the labeled-immuno complex subsets based on the ratio of fluorophore to labeling reagent.\",\n \"This determination of targets is facilitated when a population of cells or cellular organelles is passed through the examination zone of a flow cytometer wherein the fluorescent signal and intensity is recorded for each cell resulting in a histogram of the cell population or cellular organelles based on the detected epitopes.\",\n \"In another aspect of the invention, additional detection reagents are combined with the sample concurrently with or following the addition of immuno-labeled complex subsets.\",\n \"Such additional detection reagents include, but are not limited to reagents that selectively detect cells or subcellular components, ions, or indicate the cell viability, life cycle, or proliferation state.\",\n \"For example, the additional detection reagent is a labeled target-binding antibody that is directly or indirectly detectable and another additional detection reagent is a stain for nucleic acids, for F-actin, or for a cellular organelle.\",\n \"1.Sample Preparation The sample is defined to include any material that may contain a target to which an antibody has affinity for.\",\n \"Typically the sample is biological in origin and comprises tissue, cell or a population of cells, cell extracts, cell homogenates, purified or reconstituted proteins, recombinant proteins, bodily and other biological fluids, viruses or viral particles, prions, subcellular components, or synthesized proteins.\",\n \"Possible sources of cellular material used to prepare the sample of the invention include without limitation plants, animals, fungi, bacteria, archae, or cell lines derived from such organisms.\",\n \"The sample can be a biological fluid such as whole blood, plasma, serum, nasal secretions, sputum, saliva, urine, sweat, transdermal exudates, cerebrospinal fluid, or the like.\",\n \"Alternatively, the sample may be whole organs, tissue or cells from an animal.\",\n \"Examples of sources of such samples include muscle, eye, skin, gonads, lymph nodes, heart, brain, lung, liver, kidney, spleen, solid tumors, macrophages, mesothelium, and the like.\",\n \"Prior to combination with the immuno-labeled complexes, the sample is prepared in a way that makes the target, which is determined by the end user, in the sample accessible to the immuno-labeled complexes.\",\n \"Typically, the samples used in the invention are comprised of tissue, cells, cell extracts, cell homogenates, purified or reconstituted proteins, recombinant proteins, biological fluids, or synthesized proteins.\",\n \"Large macromolecules such as immuno-labeled complexes tend to be impermeant to membranes of live biological cells.\",\n \"Treatments that permeabilize the plasma membrane, such as electroporation, shock treatments, or high extracellular ATP, can be used to introduce the immuno-labeled complexes into cells.\",\n \"Alternatively, the immuno-labeled complexes can be physically inserted into cells, e.g.\",\n \"by pressure microinjection, scrape loading, patch-clamp methods, or phagocytosis.\",\n \"However, the desired target may require purification or separation prior to addition of the immuno-labeled complexes, which will depend on the way the antigenic determinants are contained in the sample.\",\n \"For example, when the sample is to be separated on a SDS-polyacrylamide gel the sample is first equilibrated in an appropriate buffer, such as a SDS-sample buffer containing Tris, glycerol, DTT, SDS, and bromophenol blue.\",\n \"When the sample contains purified target materials, the purified target materials may still be mixtures of different materials.\",\n \"For example, purified protein or nucleic acid mixtures may contain several different proteins or nucleic acids.\",\n \"Alternatively, the purified target materials may be electrophoresed on gels such as agarose or polyacrylamide gels to provide individual species of target materials that may be subsequently blotted onto a polymeric membrane or detected within the gel matrix.\",\n \"Preparation of a sample containing purified nucleic acids or proteins generally includes denaturation and neutralization.\",\n \"DNA may be denatured by incubation with base (such as sodium hydroxide) or heat.\",\n \"RNA is also denatured by heating (for dot blots) or by electrophoresing in the presence of denaturants such as urea, glyoxal, or formaldehyde, rather than through exposure to base (for Northern blots).\",\n \"Proteins are denatured by heating in combination with incubation or electrophoresis in the presence of detergents such as sodium dodecyl sulfate.\",\n \"The nucleic acids are then neutralized by the addition of an acid (e.g., hydrochloric acid), chilling, or addition of buffer (e.g., Tris, phosphate or citrate buffer), as appropriate.\",\n \"Preferably, the preparation of a sample containing purified target materials further comprises immobilization of the target materials on a solid or semi-solid support.\",\n \"Purified nucleic acids are generally spotted onto filter membranes such as nitrocellulose filters or nylon membranes in the presence of appropriate salts (such as sodium chloride or ammonium acetate) for DNA spot blots.\",\n \"Alternatively, the purified nucleic acids are transferred to nitrocellulose filters by capillary blotting or electroblotting under appropriate buffer conditions (for Northern or Southern blots).\",\n \"To permanently bind nucleic acids to the filter membranes, standard cross-linking techniques are used (for example, nitrocellulose filters are baked at 80° C. in vacuum; nylon membranes are subjected to illumination with 360 nm light).\",\n \"The filter membranes are then incubated with solutions designed to prevent nonspecific binding of the nucleic acid probe (such as BSA, casein hydrolysate, single-stranded nucleic acids from a species not related to the probe, etc.)\",\n \"and hybridized to probes in a similar solution.\",\n \"Purified proteins are generally spotted onto nitrocellulose or nylon filter membranes after heat and/or detergent denaturation.\",\n \"Alternatively, the purified proteins are transferred to filter membranes by capillary blotting or electroblotting under appropriate buffer conditions (for Western blots).\",\n \"Nonspecifically bound probe is washed from the filters with a solution such as saline-citrate or phosphate buffer.\",\n \"Filters are again blocked, to prevent nonspecific adherence of immuno-labeled complexes.\",\n \"Finally, samples are mixed with immuno-labeled complexes.\",\n \"Nonspecifically bound immuno-labeled complexes are typically removed by washing.\",\n \"When the sample contains cellular nucleic acids (such as chromosomal or plasmid-borne genes within cells, RNA or DNA viruses or mycoplasma infecting cells, or intracellular RNA) or proteins, preparation of the sample involves lysing or permeabilizing the cell, in addition to the denaturation and neutralization already described.\",\n \"Cells are lysed by exposure to agents such as detergent (for example sodium dodecyl sulfate, Tween, sarkosyl, or Triton), lysozyme, base (for example sodium, lithium, or potassium hydroxide), chloroform, or heat.\",\n \"Cells are permeabilized by conventional methods, such as by formaldehyde in buffer.\",\n \"As with samples containing purified target materials, preparation of the sample containing cellular target materials typically further comprises immobilization of the target materials on a surface such as a solid or semi-solid matrix.\",\n \"The targets may be arrayed on the support in a regular pattern or randomly.\",\n \"These supports include such materials as slides, polymeric beads including latex, optical fibers, and membranes.\",\n \"The beads are preferably fluorescent or nonfluorescent polystyrene, the slides and optical fibers are preferably glass or plastic, and the membrane is preferably poly(vinylidene difluoride) or nitrocellulose.\",\n \"Thus, for example, when the sample contains lysed cells, cells in suspension are spotted onto or filtered through nitrocellulose or nylon membranes, or colonies of cells are grown directly on membranes that are in contact with appropriate growth media, and the cellular components, such as proteins and nucleic acids, are permanently bound to filters as described above.\",\n \"Permeabilized cells are typically fixed on microscope slides with known techniques used for in situ hybridization and hybridization to chromosome “squashes” and “spreads,” (e.g., with a reagent such as formaldehyde in a buffered solution).\",\n \"Alternatively, the samples used may be in a gel or solution.\",\n \"In a particular aspect of the invention, the sample comprises of cells in a fluid, such as ascites, hybridoma supernatant, or serum, wherein the presence or absence of the target in such cells is detected by using an automated instrument that sorts cells according to the detectable fluorescence response of the detectable moieties in the immunolabeling complexes bound to such cells, such as by fluorescence activated cell sorting (FACS).\",\n \"For methods using flow cytometry a cell population typically comprises individually isolated cells that have been isolated from other proteins and connective tissue by means well known in the art.\",\n \"For example, lymphocyte cells are isolated from blood using centrifugation and a density gradient.\",\n \"The cells are washed and pelleted and the labeling solution added to the pelleted cells.\",\n \"2.Illumination At any time after addition of the immuno-labeled complex to the sample, the sample is illuminated with a wavelength of light selected to give a detectable optical response, and observed with a means for detecting the optical response.\",\n \"Equipment that is useful for illuminating the fluorescent compounds of the present invention includes, but is not limited to, hand-held ultraviolet lamps, mercury arc lamps, xenon lamps, lasers and laser diodes.\",\n \"These illumination sources are optically integrated into laser scanners, fluorescent microplate readers or standard or microfluorometers.\",\n \"The degree and/or location of signal, compared with a standard or expected response, indicates whether and to what degree the sample possesses a given characteristic, i.e.\",\n \"desired target.\",\n \"The optical response is optionally detected by visual inspection, or by use of any of the following devices: CCD camera, video camera, photographic film, laser-scanning devices, fluorometers, photodiodes, quantum counters, epifluorescence microscopes, scanning microscopes, flow cytometers, fluorescence microplate readers, or by means for amplifying the signal such as photomultiplier tubes.\",\n \"Where the sample is examined using a flow cytometer, examination of the sample optionally includes sorting portions of the sample according to their fluorescence response.\",\n \"When an indirectly detectable label is used then the step of illuminating typically includes the addition of a reagent that facilitates a detectable signal such as colorimetric enzyme substrate.\",\n \"Radioisotopes are also considered indirectly detectable wherein an additional reagent is not required but instead the radioisotope must be exposed to X-ray film or some other mechanism for recording and measuring the radioisotope signal.\",\n \"This can also be true for some chemiluminescent signals that are best observed after expose to film.\",\n \"III.\",\n \"Kits of the Invention Suitable kits for preparing an immuno-labeled complex and for detection of a target in a sample also form part of the invention.\",\n \"Such kits can be prepared from readily available materials and reagents and can come in a variety of embodiments.\",\n \"The contents of the kit will depend on the design of the assay protocol or reagent for detection or measurement.\",\n \"Generally, the kits will contain instructions, appropriate reagents and labels, and solid supports, as needed.\",\n \"Typically, instructions include a tangible expression describing the reagent concentration or at least one assay method parameter such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions and the like to allow the user to carry out any one of the methods or preparations described above.\",\n \"A preferred kit of the present invention comprises: a) a labeling solution comprising a labeling reagent that is independently attached to one or more labels and b) a solution comprising a capture reagent.\",\n \"A preferred emodiment of this kit provides a labeling reagent that is anti-Fc Fab fragment, protein G or protein G complexed with albumin.\",\n \"In a more particular embodiment of this kit, the capture component is purified mouse IgG or non-immune mouse serum and the albumin is human albumin, bovine serum albumin, or ovalbumin.\",\n \"In a more preferred embodiment the albumin is ovalbumin.\",\n \"The labeling solution is either a homogenous mixture of labeling reagents or comprises a pooled subset of labeling reagents.\",\n \"Alternatively the kit comprises a panel of labeling reagent subsets that can be used to make a subset of immuno-labeled complexes.\",\n \"Additionally the kits may comprise one or more additional components that include (a) stains for characterization of cellular organelles, cell viability, or cell proliferation state, (b) enzyme substrates or (c) enzyme conjugates such as avidin-HRP.\",\n \"A wide variety of kits and components can be prepared according to the present invention, depending upon the intended user of the kit and the particular needs of the user.\",\n \"It is understood by one skilled in the art, that any of the labeling reagents contemplated by the present invention can be used to in a labeling solution to be included in a kit.\",\n \"The labeling reagents are not intended to be limited to only the described preferred embodiments.\",\n \"IV.\",\n \"Applications The instant invention has useful applications in basic research, high-throughput screening, immunohistochemistry, fluorescence in situ hybridization (FISH), microarray technology, flow cytometry, diagnostics, and medical therapeutics.\",\n \"The invention can be used in a variety of assay formats for diagnostic applications in the disciplines of microbiology, immunology, hematology and blood transfusion, tissue pathology, forensic pathology, and veterinary pathology.\",\n \"The invention is particularly useful in the characterization and selection of optimized antibodies from hybridoma supernatants.\",\n \"Additionally, the invention can be used to deliver therapeutics to a specific target.\",\n \"In general, the current invention provides a versatile and convenient method to enhance any assay that uses an antibody as part of its detection methodology.\",\n \"The instant invention can be used to study biological phenomena, such as, for example, cell proliferation, signal transduction in cells, or apoptosis.\",\n \"For illustration purposes only and not limitation, one could study thymidine analog 5-bromo-2′-deoxyuridine (BrdU) incorporation.\",\n \"BrdU is a marker for both cell proliferation and apoptosis, as it is readily incorporated into newly synthesized DNA that has progressed through the S-phase of the cell cycle and also into DNA break sites by deoxynucleotidyl transferase (TdT).\",\n \"Anti-BrdU antibodies are used to detect cells marked by BrdU incorporation.\",\n \"By being able to directly label the anti-BrdU antibodies, the current invention provides a convenient method to allow for detection of the incorporated BrdU by conventional immunohistochemistry or fluorescence, depending on detection method required.\",\n \"Additionally, the current invention has the advantage of allowing staining for multiple targets in one cocktail, thereby reducing the need for more samples or processing steps per experiment.\",\n \"This is particularly important when analyzing precious samples (e.g., pediatric samples, leukocytes isolated from biopsies, rare antigen-specific lymphocytes and mouse tissues that yield a small number of cells).\",\n \"Although it is currently possible to simultaneously measure up to 11 distinct fluorescent colors through a convoluted series of novel developments in flow cytometry hardware, software, and dye chemistry, the use of these advances has been severely limited by the lack of commercial availability of spectrally distinct directly labeled primary and secondary antibodies.\",\n \"Although labeled secondary antibodies directed at individual isotype-specific targeting antibodies (e.g., anti-lgG, isotype antibodies) exist, it is not possible to use this type of labeled antibody to detect more than one of the same isotype of an antibody (e.g., an IgG1 isotype antibody) in a single sample due to cross-reactivity.\",\n \"The current invention overcomes these limitations by providing for a convenient and extremely versatile method of rapidly labeling either small or large quantities of any primary antibody including primary antibodies of the same isotype to be used in, for example, multicolor flow cytometry and on Western blots.\",\n \"This advance in multicolor systems has a number of advantages over current two- and three-color flow cytometric measurements.\",\n \"For example, no combination of one-color stains can accurately enumerate or be used to isolate CD3+ CD4+ CD8− T cells (excluding, for example CD3+ CD4+ CD8+ T cells and small CD4+ monocytes).\",\n \"The use of cell membrane markers to study leukocyte composition in blood and tissue serves as an example of an analytical monoclonal antibody application, particularly in combination with flow cytometry.\",\n \"It is also the example most relevant to studies of the immune system, because the cellular composition of blood and lymphoid tissue provides a ‘window’, allowing the analysis and monitoring of the immune system.\",\n \"The methods of the invention can also be used in immunofluorescence histochemistry.\",\n \"This technique involves the use of antibodies labeled with fluorophores to detect substances within a specimen.\",\n \"The pathologist derives a great deal of information of diagnostic value by examining thin sections of tissue in the microscope.\",\n \"Tissue pathology is particularly relevant to, for example, the early diagnosis of cancer or premalignant states, and to the assessment of immunologically mediated disorders, including inflammation and transplant rejection.\",\n \"The problems associated with immunofluorescence histochemistry, however, stem from the limitations of the methods currently available for use in such application.\",\n \"For example, directly labeling an antibody can result in antibody inactivation and requires a relatively large of amount of antibody and time to do the conjugation.\",\n \"It is also expensive and impractical to prepare directly labeled antibodies having variable degrees of label substitution.\",\n \"Similarly, indirect labeling of an antibody has problems, such as lack of secondary antibody specificity, and reliance upon primary antibody differences, including antibody isotypes and available fluorophores, to do multicolor labeling.\",\n \"Secondary antibody labeling is not practical where the primary antibody is from the same species or of the same isotypes.\",\n \"Combinations of fluorophores or other detectable labels on the same target-binding antibody, which can be readily prepared in multiple mixtures by the methods on this invention, greatly increase the number of distinguishable signals in multicolor protocols.\",\n \"Lack of secondary antibody specificity arises when the specimen containing the targeted moiety and target-binding antibody are from homologous species.\",\n \"For example, BrdU-labeled DNA in rodent tissue is detected by immunohistochemical staining.\",\n \"The target-binding antibody is conventionally mouse anti-BrdU, and the detecting antibody system uses an anti-mouse immunoglobulin antibody, labeled with fluorescein.\",\n \"Because there is homology between mouse immunoglobulin and immunoglobulins from a number of rodent species (for example, rats, mice, hamsters, etc.\",\n \"), the detecting antibody not only binds to the target-binding antibody, but also nonspecifically binds to immunoglobulin in the tissue.\",\n \"The current invention eliminates this problem by pre-forming the immunolabeling complex and allows for a simple, rapid and convenient method to proceed with labeling with two, three or more fluorescent antibodies in one experiment.\",\n \"Very significantly, it can always be used with primary antibodies of either the same or different isotype, and always on tissue of the same or similar species as the primary antibody.\",\n \"The instant invention also has application in the field of microarrays.\",\n \"Microarray technology is a powerful platform for biological exploration (Schena (Ed.\",\n \"), Microarray Biochip Technology, (2000)).\",\n \"Many current applications of arrays, also known as “biochips,” can be used in functional genomics as scientists seek characteristic patterns of gene expression in different physiopathological states or tissues.\",\n \"A common method used in gene and protein microarray technology involves the use of biotin, digoxigenin (DIG), or dinitrophenyl (DNP) as an epitope or a “tag” such as an oligohistidine, glutathione transferase, hemagglutinin (HA), or c-myc.\",\n \"In this case a detectably labeled anti-biotin, anti-DIG, anti-DNP, anti-oligohistidine, anti-glutathione transferase, anti-HA, or anti-c-myc is used as the detection reagent.\",\n \"The instant invention allows for the use of multiple fluorophore- or enzyme-labeled antibodies, thereby greatly expanding the detection modalities and also providing for enhanced multiplexing and two-dimensional analysis capabilities.\",\n \"Similarly, the invention can be used with protein microarrays and on Western blots.\",\n \"Protein microarrays can provide a practical means to characterize patterns of variation in hundreds of thousands of different proteins in clinical or research applications.\",\n \"Antibody arrays have been successfully employed that used a set of 115 antibody/antigen pairs for detection and quantitation of multiple proteins in complex mixtures (Haab et al., Genome Biology, 2, 4.1 (2001)).\",\n \"However, protein microarrays use very low sample volumes, which historically have significantly limited the use of antibody technology for this application.\",\n \"The invention of the application readily overcomes this limitation and provides a means to label antibodies with the fluorescent dyes using a very low sample volume and to automate formation of the staining complex and the staining process.\",\n \"The present invention also provides a means for the specific detection, monitoring, and/or treatment of disease and contemplates the use of immunolabeling complexes to detect the presence of particular targets in vitro.\",\n \"In such immunoassays, the sample may be utilized in liquid phase, in a gel, or bound to a solid-phase carrier, such as an array of fluorophore-labeled microspheres (e.g., U.S. Pat.\",\n \"No.\",\n \"5,981,180 and 5,736,330).\",\n \"For example, a sample can be attached to a polymer, such as aminodextran, in order to link the sample to an insoluble support such as a polymer-coated bead, plate, or tube.\",\n \"For instance, but not as a limitation, using the methods of the present invention in an in vitro assay, antibodies that specifically recognize an antigen of a particular disease are used to determine the presence and amounts of this antigen.\",\n \"Likewise, the immunolabeling complexes of the present invention can be used to detect the presence of a particular target in tissue sections prepared from a histological specimen.\",\n \"Preferably, the tissue to be assayed will be obtained by surgical procedures, e.g., biopsy.\",\n \"The excised tissue will be assayed by procedures generally known in the art, e.g.\",\n \"immunohistochemistry, for the presence of a desired target that is recognized by an immunolabeling complex, as described above.\",\n \"The tissue may be fixed or frozen to permit histological sectioning.\",\n \"The immunolabeling complex may be labeled, for example with a dye or fluorescent label, chemical, heavy metal or radioactive marker to permit the detection and localization of the target-binding antibody in the assayed tissue.\",\n \"In situ detection can be accomplished by applying a detectable immunolabeling complex to the tissue sections.\",\n \"In situ detection can be used to determine the presence of a particular target and to determine the distribution of the target in the examined tissue.\",\n \"General techniques of in situ detection are well known to those of ordinary skill.\",\n \"See, for example, Ponder, “Cell Marking Techniques and Their Application,” in MAMMALIAN DEVELOPMENT: A PRACTICAL APPROACH, Monk (ed.\",\n \"), 115 (1987).\",\n \"For diagnosing and classifying disease types, tissues are probed with an immuno-labeled complex, as defined above, that comprises a target-binding antibody to a target antigen associated with the disease, e.g., by immunohistochemical methods.\",\n \"Where the disease antigen is present in body fluids, such immuno-labeled complexes comprising a target-binding antibody to the disease antigen are preferably used in immunoassays to detect a secreted disease antigen target.\",\n \"Detection can be by a variety of methods including, for example, but not limited to, flow cytometry and diagnostic imaging.\",\n \"When using flow cytometry for the detection method, the use of microspheres, beads, or other particles as solid supports for antigen-antibody reactions in order to detect antigens or antibodies in serum and other body fluids is particularly attractive.\",\n \"Flow cytometers have the capacity to detect particle size and light scattering differences and are highly sensitive fluorescence detectors.\",\n \"Microfluidic devices provide a means to perform flow-based analyses on very small samples.\",\n \"Alternatively, one can use diagnostic imaging.\",\n \"The method of diagnostic imaging with radiolabeled antibodies is well known.\",\n \"See, for example, Srivastava (ed.\",\n \"), RADIOLABELED MONOCLONAL ANTIBODIES FOR IMAGING AND THERAPY, Plenum Press (1988); Chase, “Medical Applications of Radioisotopes,” in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, Gennaro et al.\",\n \"(eds.)\",\n \"Mack Publishing Co., 624 (1990); and Brown, “Clinical Use of Monoclonal Antibodies,” in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al.\",\n \"(eds.\",\n \"), Chapman & Hall, 227 (1993).\",\n \"This technique, also known as immunoscintigraphy, uses a gamma camera to detect the location of gamma-emitting radioisotopes conjugated to antibodies.\",\n \"Diagnostic imaging is used, in particular, to diagnose cardiovascular disease and infectious disease.\",\n \"Thus, the present invention contemplates the use of immuno-labeled complexes to diagnose cardiovascular disease.\",\n \"For example, immuno-labeled complexes comprising anti-myosin antibodies can be used for imaging myocardial necrosis associated with acute myocardial infarction.\",\n \"Immuno-labeled complexes comprising antibodies that bind platelets and fibrin can be used for imaging deep-vein thrombosis.\",\n \"Moreover, immuno-labeled complexes comprising antibodies that bind to activated platelets can be used for imaging atherosclerotic plaque.\",\n \"Immuno-labeled complexes of the present invention also can be used in the diagnosis of infectious diseases.\",\n \"For example, immuno-labeled complexes comprising antibodies that bind specific bacterial antigens can be used to localize abscesses.\",\n \"In addition, immuno-labeled complexes comprising antibodies that bind granulocytes and inflammatory leukocytes can be used to localize sites of bacterial infection.\",\n \"Similarly, the immuno-labeled complexes of the present invention can be used to detect signal transduction in cells, the products of signal transduction, and defects, inhibitors, and activators of signal transduction.\",\n \"Numerous studies have evaluated the use of antibodies for scintigraphic detection of cancer.\",\n \"Investigations have covered the major types of solid tumors such as melanoma, colorectal carcinoma, ovarian carcinoma, breast carcinoma, sarcoma, and lung carcinoma.\",\n \"Thus, the present invention contemplates the detection of cancer using immuno-labeled complexes comprising antibodies that bind tumor markers (targets) to detect cancer.\",\n \"Examples of such tumor markers include carcinoembryonic antigen, a-fetoprotein, oncogene products, tumor-associated cell surface antigens, and necrosis-associated intracellular antigens.\",\n \"In addition to diagnosis, antibody imaging can be used to monitor therapeutic responses, detect recurrences of a disease, and guide subsequent clinical decisions and surgical procedures.\",\n \"In vivo diagnostic imaging using fluorescent complexes that absorb and emit light in the near infrared (such as those of the Alexa Fluor 700 and Alexa Fluor 750 dyes) is also known.\",\n \"EXAMPLES The following examples describe specific aspects of the invention to illustrate the invention and to provide a description of the methods for those of skill in the art.\",\n \"The examples should not be construed as limiting the invention, as the examples merely provide specific methodology useful in understanding and practicing the invention.\",\n \"Example 1 Preparation of Fc Antigen Purified mouse and rabbit IgG was fragmented with the proteolytic enzyme papain (CURRENT PROTOCOLS IN CELL BIOLOGY, 16.4.1-16.4.10 (2000)).\",\n \"A 12 mL solution of mouse IgG was prepared at ˜2 mg/mL in phosphate-buffered saline (PBS).\",\n \"A solution containing 0.1 mg of papain in digestion buffer (PBS, 0.02 M EDTA, 0.02 M cysteine) was added to the antibody and allowed to react at 37° C. for 16 hours.\",\n \"The digestion was terminated by the addition 20 μL of 0.3 M iodoacetamide in PBS.\",\n \"The fragments were dialyzed against 2 L of PBS for 16 hours at 4° C. The Fc fragment was purified on a protein G-Sepharose CL-4B column.\",\n \"The bound fraction containing the Fc fragment was eluted from the column using 50-100 mM glycine/HCl buffer, pH 2.5-2.8.The eluate was collected in 1 mL fractions.\",\n \"The pH of the protein fractions was immediately raised to neutral by addition of 100 μL of either 500 mM phosphate or Tris buffer, pH 7.6, to each 1 mL fraction.\",\n \"The solution was then loaded onto a Sephacryl S-200 Superfine size-exclusion column and fractions corresponding to a molecular weight of -50 kDa were collected and analyzed by SDS-PAGE and HPLC.\",\n \"Example 2 Production of Anti-Fc Antibodies Polyclonal antibodies specific for the Fc region of an antibody were raised in goats against the purified FC region of an antibody from a different species (Example 1).\",\n \"Methods of immunizing animals are well known in the art, and suitable immunization protocols and immunogen concentrations can be readily determined by those skilled in the art (Current Protocols in Immunology 2.4.1-9 (1995); ILAR Journal 37, 93 (1995)).\",\n \"Briefly, individual goats were immunized with purified mouse Fc or purified rabbit Fc fragments.\",\n \"The initial immunization in 50% Freund's complete adjuvant (1000 μg conjugate (half subcutaneous, half intramuscularly)) was followed by 500 pg conjugate per goat in Freund's incomplete adjuvant two and four weeks later and at monthly intervals thereafter.\",\n \"Antibodies were purified from serum using protein A-Sepharose chromatography.\",\n \"Antibodies against mouse Fc isotypes can be prepared by starting with isotype-selected mouse Fc antigens.\",\n \"Rabbits have a single Fc isotype.\",\n \"Characterization of the selectivity and cross-reactivity of isotype-specific antibodies is by standard techniques, including HPLC.\",\n \"Example 3 Preparation of Fab Fragments Fragmentation of the goat anti-(mouse Fc) antibody to the monovalent Fab fragment was carried out using the proteolytic enzyme, papain, as described in Example 1.Following dialysis against PBS, the Fab fragment was purified on a protein A-Sepharose CL-4B column.\",\n \"The unbound fraction containing the Fab fragment and the papain was collected.\",\n \"This solution was then loaded onto a Sephacryl S-200 Superfine size-exclusion column and fractions corresponding to a molecular weight of ˜50 kDa were collected and analyzed by SDS-PAGE.\",\n \"The Fab fragments of goat anti-(rabbit Fc) can be prepared similarly.\",\n \"Example 4 Preparation of the Labeled Antibody Immunoglobulin-Binding Protein or the Non-Antibody Immunoglobulin-Binding Peptide and Protein Conjugates in Homogeneous Solution Conjugates of antibody immunoglobulin-binding protein or the non-antibody immunoglobulin-binding peptides or proteins with low molecular weight dyes and haptens such as biotin or digoxigenin are typically prepared from succinimidyl esters of the dye or hapten, although reactive dyes and haptens having other protein-reactive functional groups are also suitable.\",\n \"The typical method for protein conjugation with succinimidyl esters is as follows.\",\n \"Variations in molar ratios of dye-to-protein, protein concentration, time, temperature, buffer composition and other variables that are well known in the art are possible that still yield useful conjugates.\",\n \"A protein solution of the Fab fragment of goat anti-(rabbit Fc), goat anti-(mouse Fc), protein A, protein G, or protein L or an immunoglobulin-binding peptide (e.g., a peptide identified by screening a library of peptides) is prepared at ˜10 mg/mL in 0.1 M sodium bicarbonate (pH ˜8.3).\",\n \"The labeling reagents are dissolved in a suitable solvent such as DMF at ˜10 mg/mL.\",\n \"Predetermined amounts of the labeling reagents are added to the protein solution with stirring.\",\n \"A molar ratio of 10 moles of dye to 1 mole of protein is typical, though the optimal amount can be varied with the particular labeling reagent, the protein being labeled and the protein's concentration.\",\n \"The optimal ratio was determined empirically.\",\n \"When optimizing the fluorescence yield and determining the effect of degree of substitution (DOS) on the conjugate's brightness, it is typical to vary the ratio of reactive dye to protein over a several-fold range.\",\n \"The reaction mixture is incubated at room temperature for a period that is typically one hour or on ice for several hours.\",\n \"The dye-protein conjugate is typically separated from unreacted reagents by size-exclusion chromatography, such as on BIO-RAD P-30 resin equilibrated with PBS.\",\n \"The initial, protein-containing band is collected and the DOS is determined from the absorbance at the absorbance maximum of each fluorophore, using the extinction coefficient of the free fluorophore.\",\n \"The DOS of nonchromophoric labels, such as biotin, is determined as described in Haugland (Haugland et al., Meth.\",\n \"Mol.\",\n \"Biol.\",\n \"45, 205 (1995); Haugland, Meth.\",\n \"Mol.\",\n \"Biol.\",\n \"45, 223 (1995); Haugland, Meth.\",\n \"Mol.\",\n \"Biol.\",\n \"45, 235 (1995); Haugland, Current Protocols in Cell Biol.\",\n \"16.5.1-16.5.22 (2000)).\",\n \"Using the above procedures, conjugates of goat anti-(mouse Fc) and goat anti-(rabbit Fc) were prepared with several different Alexa Fluor dyes, with Oregon Green dyes, with biotin-X succinimidyl ester, with desthiobiotin-X succinimidyl ester, with succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and with succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate (SMCC).\",\n \"Some dye conjugates of protein A and protein G, including those of some Alexa Fluor dyes, are commercially available, such as from Molecular Probes.\",\n \"Inc. (Eugene, Oreg.).\",\n \"The interspecies specificity and approximate affinity of some other non-antibody immunoglobulin-binding proteins bind to segments of a target antibody, such as that of protein A and protein G are known (Langone, Adv.\",\n \"Immunol.\",\n \"32,157 (1982); Surolia et al., Trends Biochem.\",\n \"Sci.\",\n \"7, 74 (1982); Notani et al., J. Histochem.\",\n \"Cytochem.\",\n \"27, 1438 (1979); Goding, J. Immunol.\",\n \"Meth.\",\n \"20, 241 (1978); J. Immunol.\",\n \"Meth.\",\n \"127, 215 (1990); Bjorck et al., J. Immunol.\",\n \"133, 969 (1984)).\",\n \"In addition, labeling proteins (goat Fab anti-(mouse Fc), goat Fab anti-(mouse lambda light chain), goat Fab anti-(mouse kappa light chain), protein A, protein G, protein L, lectins, single-chain fragment variable antibodies (ScFv) ) conjugated to the detectable labels of R-phycoerythrin (R-PE), allophycocyanin (APC), tandem conjugates of phycobiliproteins with chemical dyes including several Alexa Fluor dyes, horseradish peroxidase (HRP), Coprinus cinereus peroxidase, Arthromyces ramosus peroxidase, glucose oxidase and alkaline phosphatase (AP) were or can be prepared by standard means (Haugland et al., Meth.\",\n \"Mol.\",\n \"Biol.\",\n \"45, 205 (1995); Haugland, Meth.\",\n \"Mol.\",\n \"Biol.\",\n \"45, 223 (1995); Haugland, Meth.\",\n \"Mol.\",\n \"Biol.\",\n \"45, 235 (1995); Haugland, Current Protocols in Cell Biol 16.5.1-16.5.22 (2000)).\",\n \"Fusion proteins, such as of protein G or protein A with detectable labels such as luciferin, aequorin, green-fluorescent protein and alkaline phosphatase are also known that are suitable for practice of the invention (Sun et al., J. Immunol.\",\n \"Meth.\",\n \"152, 43 (1992); Eliasson et al., J. Biol.\",\n \"Chem.\",\n \"263, 4323 (1988); Eliasson et al., J. Immunol.\",\n \"142, 575 (1989)).\",\n \"Immunoglobulin heavy and light chains, like most secreted and membrane bound proteins, are synthesized on membrane-bound ribosomes in the rough endoplasmic endoplasmic reticulum where N-linked glycosylation occurs.\",\n \"The specificity of lectins for carbohydrates, including N-linked glycoproteins, is also known (EY laboratories, Inc. Lectin Conjugates Catalog, 1998).\",\n \"Example 5 Preparation of the Labeled Antibody Immunoglobulin-Binding Protein or the Non-Antibody Immunoglobulin-Binding Peptide and Protein Conjugates While Bound to an Affinity Matrix Unlabeled Fab fragment for goat anti-(mouse Fc) (prepared as in Example 3) was bound to agarose-immobilized mouse IgG for one hour.\",\n \"Following a wash step with bicarbonate buffer, pH 8.3, the complex of immobilized IgG and unlabeled Fab was labeled for one hour at room temperature with the succinimidyl ester of the amine-reactive label.\",\n \"Unconjugated dye was eluted with bicarbonate buffer, and then the covalently labeled Fab fragment was eluted with 50-100 mM glycine/HCl buffer, pH 2.5-2.8.The eluate was collected in 1 mL fractions.\",\n \"The pH of the protein fractions was immediately raised to neutral by addition of 100 μL of either 500 mM phosphate or Tris buffer, pH 7.6, to each 1 mL fraction.\",\n \"Variations of the reagent concentrations, labeling times, buffer composition, elution methods and other variables are possible that can yield equivalent results.\",\n \"Conjugates of the Fab fragment of goat anti-(rabbit Fc) and of protein G and protein A are prepared similarly.\",\n \"Example 6 Comparison of the Alexa Fluor 488 Dye-Labeled Fab Fragments of Goat Anti-(Mouse Fc) Prepared as in Example 4 and as in Example 5 Conjugates of the Fab fragment of goat anti-(mouse Fc) with the Alexa Fluor 488 succinimidyl ester were separately prepared, as described in Examples 4 and 5.The conjugates had estimated degrees of substitution of ˜1.9 (labeled as in Example 4) and ˜3.0 (labeled as in Example 5), respectively, and virtually identical absorption and emission spectral maxima.\",\n \"When excited at 488 nm, conjugates prepared using the fragment prepared as described in Example 5 were about 3.2-times more fluorescent than using the fragments that were prepared in Example 4 (FIG.\",\n \"8) as detected by flow cytometry when bound to CD3 on Jurkat T cells.\",\n \"Similar results were observed with other dyes.\",\n \"Example 7 Preparation of a Labeling Protein from Protein G and Albumins Native protein G has a high affinity binding (nanomolar) site for albumins, in particular ovalbumin.\",\n \"Equal weights of protein G and Texas Red ovalbumin (Molecular Probes.\",\n \"Inc.) were dissolved in PBS, pH 7.5.After one hour, the resulting complex was separated on a Sephacryl S-200 Superfine size-exclusion column and analyzed by SDS-PAGE and HPLC.\",\n \"Alternatively, the protein G is combined with a labeled albumin while the protein G is immobilized on any of the several immunoglobulins to which it binds, and the excess labeled albumin is washed away preceding elution of the albumin-labeled protein G complex from the matrix.\",\n \"Example 8 Preparation of an Immunolabeling Complex on a Very Small Scale Submicrogram quantities of a target-binding antibody were complexed with submicrograms of a labeling protein in varying molar ratios of between about 1:1 and 1:20 to prepare an immunolabeling complex that was suitable for staining a sample.\",\n \"For instance, 0.1 μg of mouse monoclonal anti-tubulin in 1 μL PBS with 0.1% BSA was complexed with 0.5 μg of the Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse Fc) (prepared as in Example 4) or with 0.1 μg of the Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse Fc) (prepared as in Example 5) in 5 μL of PBS for 10 minutes at room temperature.\",\n \"The immunolabeling complex can be used immediately for staining tubulin in fixed-cell preparations (Example 16) or any excess unbound Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse Fc) in the immunolabeling complex can be captured with non-immune mouse IgG (Example 9) for combination with other antibody conjugates, including those of targeting antibodies that have been directly conjugated to other labels.\",\n \"Rabbit antibodies were labeled similarly using labeled goat anti-(rabbit Fc).\",\n \"Labeling of targeting antibodies with a labeled protein A, protein L, protein G, protein G complexed with a labeled albumin, or other immunoglobulin-binding peptides or proteins proceeds similarly.\",\n \"In the case of a mouse (or rat) monoclonal antibody, it is preferred to use a labeled protein that is selective for the specific isotype of the primary antibody (e.g.\",\n \"anti-(mouse IgG,) for a mouse IgG1 isotype primary antibody).\",\n \"Although some cross-reactivity for other mouse (or rat) isotypes was observed using a goat antibody that was selective for mouse IgG, isotype monoclonal antibodies, routine and optimal use for labeling unmatched mouse isotypes required greater amounts of immunolabeling complexes and was somewhat less reliable.\",\n \"Example 9 Capturing Excess Immunoglobulin-Binding Protein by a Capturing Component Immunolabeling complexes were prepared as described in Example 8.To the immunolabeling complex was added to each tube 25 μL of a 14.1 mg/mL stock solution of unlabeled mouse IgG to capture excess immunolabeling complexes.\",\n \"As shown in FIG.\",\n \"1, not all of the immunoglobulin-binding protein was necessarily complexed with the target-binding antibody to form an immunolabeling complex.\",\n \"Consequently, particularly for applications in which labeling complexes of multiple primary antibodies from the same species (e.g.\",\n \"mouse monoclonal antibodies) or crossreacting species (e.g.\",\n \"mouse and human antibodies, FIG.\",\n \"2, Table 1) were to be used simultaneously or sequentially, it is necessary to quench or otherwise remove any excess immunoglobulin-binding protein by use of a capturing component or by other means to avoid inappropriate labeling of the sample.\",\n \"The most effective capturing component to capture excess immunoglobulin-binding protein is one that contains the binding site of the targeting agent.\",\n \"For instance, whole mouse IgG or mouse serum was shown to be an effective and inexpensive reagent when the immunoglobulin-binding protein was bound to a segment of a mouse monoclonal antibody.\",\n \"The mouse IgG was added in excess to the amount of immunoglobulin-binding protein and incubated for a period of approximately 1-5 minutes, or longer.\",\n \"It is preferred to prepare the immunolabeling complex and then add the capturing component shortly before the experiment.\",\n \"The rapid quenching effect permits this to be done within minutes of performing labeling of the sample by the immunolabeling complex.\",\n \"If desired, the excess capturing component can be removed following labeling of the sample by a simple wash step.\",\n \"Alternatively, fixation of the stained sample by aldehyde-based fixatives or other reagents or methods subsequent to incubation with the immunolabeling complex can provide permanent immobilization of the immunolabeling complex on its target in the sample.\",\n \"As an alternative to adding a soluble capturing component to the immunolabeling complex, the capturing component can be immobilized on an insoluble matrix such as agarose and the immunolabeling complex contacted with that matrix.\",\n \"A preferred matrix when labeling mouse antibodies to mouse antigens is mouse IgG immobilized on agarose.\",\n \"Excess labeled anti-rabbit antibodies can be captured using rabbit IgG that is free in solution or immobilized.\",\n \"Alternatively, the immunolabeling complex can be separated from any capturing component by chromatographic or electrophoretic means.\",\n \"Example 10 HPLC Analysis of a Labeling Complex In order to analyze the success and extent of complex formation of the labeling protein with the target-binding antibody, size exclusion HPLC of the samples was performed.\",\n \"For instance, a complex of Alexa Fluor 488 dye-labeled goat Fab anti-(mouse Fc) with a monoclonal mouse anti-tubulin in molar ratios of approximately 1:1, 3:1, 5:1 and 10:1.These were separated by analytical HPLC using a BioSep S-3000 column and eluting with 0.1 M NaPi, 0.1 M NaCl, pH 6.8, at a flow rate of 0.25 mLs/min.\",\n \"An example of the separation using the 5:1 molar ratio (FIG.\",\n \"6) demonstrates that, using this molar ratio, formation of the labeled complex is essentially quantitative.\",\n \"Example 11 Cross-Reactivity of Goat Fab Anti-(Mouse Fc) to Other Species of IgG Microplates were equilibrated overnight with IgG from a mouse or non-mouse species, and then further blocked with BSA.\",\n \"Variable amounts of the biotinylated Fab fragment of goat anti-(mouse Fc) were added to each well and allowed to bind.\",\n \"After washing, streptavidin-HRP and the Amplex Red peroxidase substrate were added.\",\n \"HRP activity was detected by the addition of H2O2 using the Amplex Red Peroxidase Assay Kit (Molecular Probes, Inc., Eugene, Oreg.).\",\n \"Reactions containing 200 μM Amplex Red reagent, 1 U/mL HRP and 1 mM H2O2 (3% solution) in 50 mM sodium phosphate buffer, pH 7.4, were incubated for 30 minutes at room temperature.\",\n \"Fluorescence was measured with a fluorescence microplate reader using excitation at 560±10 nm and fluorescence detection at 590±10 nm.\",\n \"Background fluorescence, determined for a no-H2O2 control reaction, was subtracted from each value (Table 1 and FIG.\",\n \"2).\",\n \"Table 1 shows that the goat anti-(mouse Fc) antibody because of the highly conserved structure of the Fc region of an antibody it can be used to complex other non-mouse antibodies, including rat, and human antibodies.\",\n \"The goat anti-mouse IgG antibody reaction with mouse antibody was set at 100% and the crossreacting antibodies were expressed as a percentage compared the mouse on mouse data.\",\n \"The data in Table 1 show that the Fab fragment of the goat anti-(mouse Fc) antibody of the current invention does not strongly bind to the goat or sheep Fc domain; however, one skilled in the art could generate antibodies that will react with the goat and sheep Fc domain or the Fc domain of any other species.\",\n \"Biotinylated Fab goat anti-(mouse Fc) was used in this example because it provided a convenient method to quantitate the amount of crossreactivity in a conventional method but it could have been accomplished using a fluorophore Fab labeled goat anti-(mouse Fc).\",\n \"It was demonstrated by HPLC (as in Example 10) that Alexa Fluor 488 dye-labeled goat anti-(rabbit Fc) bound to rabbit primary antibodies.\",\n \"TABLE 1 Cross-reactivity of goat anti-mouse IgG antibody with other non-mouse antibodies.\",\n \"Species Crossreactivity % Fluorescence Mouse ++++ 100 Rat +++ 80.7 Human ++ 66.7 Rabbit + 16.9 Goat − 6.5 Sheep − 5.7 Example 12 Determination of the Optimal Molar Ratio of Immunoglobulin-Binding Protein to Target Antibody Using a Microplate Assay To 1.6 μg of mouse monoclonal anti-biotin (MW ˜145,000) in 8.0 μL PBS was added varying amounts of the Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse Fc) (MW ˜50,000) (prepared as in Example 4) to form an immunolabeling complex.\",\n \"After equilibration for 20 min, a 100 μL aliquot was added to a 96-well microplate coated with biotinylated BSA.\",\n \"After 30 minutes, the plates were washed and the residual fluorescence was quantitated using a fluorescence microplate reader using excitation at 485±10 nm and detecting emission at 530±12.5 nm.\",\n \"As shown in FIG.\",\n \"3, a molar ratio of the Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse Fc) to the anti-biotin between 5 to 20 was sufficient to form appreciably detectable complexes (FIG.\",\n \"3; fluorescence quantitated, performed in triplicate (circles); control experiments performed but without adding the primary anti-biotin antibody (solid squares)).\",\n \"A molar ratio of about 5 to about 10 was preferred for this pair of immunoglobulin-binding protein and target antibody.\",\n \"This ratio can be varied somewhat to increase or decrease the signal or to affect the consumption of valuable reagents.\",\n \"The weight ratio of immunoglobulin-binding protein to target-binding antibody is particularly affected by the actual molecular weight of the immunoglobulin-binding protein.\",\n \"For instance, equal weights of the dye-labeled goat Fab anti-(mouse Fc) (prepared as in Example 5) and an intact mouse primary antibody, which corresponds to an approximately 3 to 1 molar ratio, usually yields suitable labeling complexes.\",\n \"Fluorescence intensity (or enzymatic activity) of the immunolabeling complex is readily adjusted by a corresponding adjustment of the amount of labeled Fab fragment used.\",\n \"Similar analyses of the ratio for other labeling proteins (including those of labeled protein A, protein G, protein L, IgG-binding peptides and antibodies to other segments of the primary antibody), and for conjugates of labels other than Alexa Fluor 488 dye (including enzymes in combination with the appropriate enzyme substrates) are done essentially as described in this example.\",\n \"Example 13 Dissociation Rate of the Immunolabeling Complex A pre-equilibrated immunolabeling complex was prepared from 50 μg of an Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse Fc) and 15 μg of an anti-biotin monoclonal antibody (mAb).\",\n \"The immunolabeling complex was rapidly diluted with capturing component sufficient to give a 6.2 molar excess over the anti-biotin mAb.\",\n \"At various times, an aliquot was taken and added to a microplate well containing an excess of biotinylated BSA.\",\n \"After 30 minutes, the plates were washed and the remaining fluorescence was quantitated.\",\n \"Displacement of the labeling protein from the target-binding antibody through exchange was measured by any time-dependent decrease in fluorescence in the microplate well.\",\n \"For example the fragments prepared as described in Example 4 had 68 percent fragments bound to the target-binding antibody after 30 minutes compared to 87 percent of bound fragments that were prepared according to Example 5.One hour showed a similar decrease, 56 percent and 68 percent respectively.\",\n \"The labeling protein was shown to undergo a stable interaction with the target-binding antibody, with a lifetime for half exchange under these conditions of 3.5 hours.\",\n \"Dissociation rates were measured for labeling protein prepared according to Example 4 and for labeling protein prepared according to Example 5, demonstrating the greater stability of immunolabeling complexes made using the labeling proteins prepared according to Example 5.Example 14 Protocol for Staining Cultured Cells with a Single Immunolabeling Complex Culturable cells, such as bovine pulmonary artery endothelial cells (BPAEC), were grown on a 22 x 22 mm glass coverslip.\",\n \"The cells were fixed for 10 minutes using 3.7% formaldehyde in DMEM with fetal calf serum (FCS) at 37° C. The fixed cells were washed 3 times with PBS.\",\n \"The cells were permeabilized for 10 min with 0.02% Triton X-100 in PBS, washed 3X with PBS and blocked for 30 min with 1% BSA in PBS.\",\n \"Variations of the cell type and cell preparation, fixation, and permeabilization methods, including methods for antigen retrieval, are well known to scientists familiar with the art.\",\n \"An immunolabeling complex was prepared as described in Example 8.The immunolabeling complex was added directly to the fixed and permeabilized cells in an amount sufficient to give a detectable signal if there is a binding site for the primary antibody present in the sample.\",\n \"After an incubation period that was typically 1060 minutes (usually about 15-30 minutes), the cells were washed with fresh medium and the labeling was evaluated by methods suitable for detection of the label.\",\n \"Staining by the immunolabeling complex can be additionally preceded, followed by or combined with staining by additional reagents, such as DAPI, which yields blue-fluorescent nuclei.\",\n \"Example 15 Protocol for Staining Cultured Cells with Multiple Immunolabeling Complexes Cells were fixed and permeabilized as described in Example 14.Multiple immunolabeling complexes were individually prepared from a variety of labeling proteins, according to the procedure described in Example 8.The multiple immunolabeling complexes were either used individually or sequentially to stain the cells, according to the procedure described in Example 14, or two or more immunolabeling complexes were formed then co-mixed in a single staining solution and used to simultaneously stain the sample.\",\n \"The optimal method for cell fixation and permeabilization and the best ratio for combination of the immunolabeling complexes are typically determined by preliminary experimentation using single immunolabeling complexes or multiple immunolabeling complexes used in combination.\",\n \"A first immunolabeling complex was prepared from an Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse Fc) and mouse monoclonal anti-α-tubulin, a second immunolabeling complex was prepared from an Alexa Fluor 568 dye-labeled Fab fragment of goat anti-(mouse Fc) and mouse monoclonal anti-vimentin (anti-vimentin was an ascites fluid preparation) and a third immunolabeling complex was prepared from an Alexa Fluor 647 dye-labeled Fab fragment of goat anti-(mouse Fc) and mouse monoclonal anti-cdc6 peptide antibody (Molecular Probes).\",\n \"Aliquots of the three different immunolabeling complexes were combined and used to stain BPAE cells for 30 minutes, washed with fresh medium and observed by fluorescence microscopy using optical filters appropriate for the three dyes.\",\n \"In this example, some cells showed cytoplasmic staining by the anti-vimentin antibody, nuclear staining by the anti-cdc6 peptide antibody and staining of mitotic spindles by the anti-α-tubulin antibody, indicative of a cell in mitosis.\",\n \"Staining by the immunolabeling complexes was additionally preceded, followed by or combined with staining by additional reagents, such as Alexa Fluor 350 phalloidin, which yielded blue-fluorescent actin filaments in the above example.\",\n \"The immunolabeling complexes that are used in combination do not have to be targeted toward antibodies from the same species.\",\n \"For instance, complexes of Alexa Fluor 488 dye-labeled goat anti-(mouse IgG, Fc) with a mouse IgG, monoclonal target-binding antibody and an Alexa Fluor 594 dye-labeled goat anti-(rabbit Fc) with a rabbit primary target-binding antibody can be prepared and used in combined staining protocols.\",\n \"Example 16 Protocol for Staining Tissue with a Single Immunolabeling Complex A mouse intestine cryosection (University of Oregon histology core facility), a cross-section of about 16 μm thickness, was mounted on a slide.\",\n \"The intestine was perfused and fixed with 4% formaldehyde prior to dissection, embedding, and sectioning.\",\n \"The tissue section was rehydrated for 20 minutes in PBS.\",\n \"An immunolabeling complex was prepared as described in Example 8.Briefly, 0.1 μg of mouse monoclonal anti-cdc6 peptide (a nuclear antigen) in 1 μL PBS with 0.1 % BSA was complexed with 0.5 μg of the Alexa Fluor 350 dye-labeled Fab fragment of goat anti-(mouse IgG1 Fc) (prepared as in Example 4) in 5 μL of PBS for 10 minutes at room temperature.\",\n \"Excess Fab fragment of goat anti-(mouse IgG, Fc) was captured with 25 μL of a 14.1 mg/mL stock of unlabeled mouse IgG.\",\n \"The tissue was permeabilized with 0.1% Triton X-100 for 10 min.\",\n \"The tissue was washed two times with PBS and was blocked in 1 % BSA for 30 min.\",\n \"The immunolabeling complex was added directly to the tissue for 30 minutes and washed three times in PBS.\",\n \"The sample was mounted in Molecular Probes' Prolong antifade mounting medium and observed by fluorescence microscopy using optical filters appropriate for the Alexa Fluor 350 dye.\",\n \"Results showed that the mouse monoclonal anti-cdc6 peptide immunolabeling complex showed specific nuclear labeling in the mouse intestine tissue section.\",\n \"Variations of the tissue type and tissue preparation, fixation and permeabilization methods, mounting methods, including methods for antigen retrieval, are well known to scientists familiar with the art.\",\n \"Example 17 Staining of a Tissue Target in Combination with Tyramide Signal Amplification (TSA) Mouse brain cryosections were labeled with a pre-formed complex of horseradish peroxidase (HRP)-labeled goat anti-(mouse IgG, Fc) antibody and a mouse IgG1 monoclonal anti-(glial fibrillary acidic protein (GFAP)) prepared essentially as in Example 8 using a molar ratio of labeling protein to monoclonal antibody of 3.Staining of the mouse tissues was essentially as in Example 16.The staining localization and intensity was compared to that of (a) goat anti-mouse IgG HRP conjugate and mouse anti-GFAP, (b) the Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse IgG, Fc) antibody complex of mouse anti-GFAP, (c) Alexa Fluor 488 goat anti-mouse IgG secondary antibody and mouse anti-GFAP, and (d) a direct conjugate of the Alexa Fluor 488 dye with mouse anti-GFAP.\",\n \"The HRP-conjugated probes were incubated with Alexa Fluor 488 tyramide using TSA Kit #2 (Molecular Probes, Inc.) according to standard procedures.\",\n \"The tissue staining patterns in each case were similar and consistent with the expected staining pattern of mouse anti-GFAP and staining was essentially free of nonspecific background.\",\n \"The relative fluorescence intensities of staining measured by digital imaging were sequentially: 541 relative intensity units for the HRP-goat anti-(mouse IgG, Fc) complex of mouse anti-GFAP and (using the combinations indicated by the letters above): (a) 539, (b) 234, (c) 294, and (d) 255 relative intensity units.\",\n \"Example 18 Staining of Live Cells by Multiple Immunolabeling Complexes A first immunolabeling complex was prepared from an Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse IgG, Fc) and mouse monoclonal anti-(human CD8), a second immunolabeling complex was prepared from an R-phycoerythrin-conjugated Fab fragment of goat anti-(mouse IgG1 Fc) and mouse anti-(human CD3), and a third immunolabeling complex was prepared from an Alexa Fluor 647 dye-labeled Fab fragment of goat anti-(mouse IgG, Fc) and mouse anti-(human CD4).\",\n \"The complexes were prepared as described in Example 8 and were each blocked with 20 μg (1.3 μL of 14.1 μg/mL) of mouse IgG for 10 minutes at room temperature.\",\n \"The first immunolabeling complex was added to 100 μL of whole blood and incubated for 15 min.\",\n \"The cells were washed with PBS and 280.5 μL of the second immunolabeling complex was added and incubated for 15 min.\",\n \"The cells were again washed, and 46.2 μL of the third labeling complex was added and incubated for 15 min.\",\n \"After the final incubation, the red blood cells were lysed with cell-lysis buffer.\",\n \"The cells were resuspended in 1% formaldehyde/PBS and analyzed on a FACS Vantage flow cytometer using a 488 nm argon-ion laser for excitation of the first and second immunolabeling complexes and a 633 nm red He-Ne laser for excitation of the third immunolabeling complex (FIGS.\",\n \"5a, 5b).\",\n \"The emission band pass filters used for selective detection of the dyes are 525±10 nm for the Alexa Fluor 488 (CD8), 585±21 nm for R-PE (CD3) and 675±10 nm for the Alexa Fluor 647 dye (CD4).\",\n \"FIGS.\",\n \"5a and 5b show that the instant invention can be used in a 3-color immunophenotyping experiment using peripheral blood lymphocytes.\",\n \"CD3-positive T cells were stained with the R-phycoerythrin-conjugated Fab fragment of goat anti-(mouse Fc) and mouse anti-(human CD3), upper left (UL) quadrant, FIG.\",\n \"5a.\",\n \"CD4-positive cells, a T cell subset, are identified using Alexa Fluor 647 dye-labeled Fab fragment of goat anti-(mouse IgG1 Fc) and mouse anti-(human CD4), UL quadrant, FIG.\",\n \"5b and CD8-positive T cells, a T cell subset, were identified using Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse IgG1 Fc) and mouse monoclonal anti-(human CD8), lower right (LR) quadrant, FIG.\",\n \"5b.\",\n \"Exposed antigens of live cells, including cultured cells and cells from biological fluids such as blood and cerebrospinal fluid can be simultaneously or sequentially stained by combinations of immunolabeling complexes, including antibodies to the same target labeled with two or more separately detectable immunoglobulin-binding proteins.\",\n \"Example 19 The Dye-Labeled Fab Fragment of Goat Anti-(Mouse Fc) can be Utilized for the Combinatorial Labeling of Primary Antibodies, to Generate a Multitude of Colored Targets A first immuno-labeled complex was made by combining 2.5 μg Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse IgG, Fc) with 0.5 μg mouse anti-human CD3 (Caltag at 200 μg/mL), according to the procedure described in Example 4.A second immunolabeling complex was made by combining 5.0 μg Alexa Fluor 647 dye-labeled Fab fragment of goat anti-(mouse IgG, Fc) with 0.5 μg mouse anti-human CD3, according to the procedure in Example 4.Each complex was separately incubated at room temperature for 5 minutes, and each complex was then separately combined with an excess of mouse IgG (14.1 mg/mL) for 5 min at room temperature to capture excess unbound dye-labeled Fab fragments.\",\n \"The two immunolabeling complexes were then added in different percentage combinations (see Table 2) to 100 μL of washed heparinized blood.\",\n \"The cells were incubated with the respective combinations of complexes for 20 min on ice.\",\n \"The red blood cells were then lysed with a cell-lysis buffer.\",\n \"The cells were resuspended in 1 % formaldehyde/PBS and analyzed on a FacVantage flow cytometer using a 488 nm argon 633 HeNe laser for excitation and a 530 +/-10 nm band pass emission filter (FL1), and a 640 long pass filter (FL4).\",\n \"Five samples of different combined percentages (Table 2) were compared by flow cytometry, with signals being collected in FL1 and FL4.To determine the percentage of cells detected with each type of emission, the FL1 and FL4 intensities for each percentage combination were normalized by dividing the FL1 and FL4 channel intensities for such combinations by the intensities of the 100% Alexa Fluor 488 dye-and 100% Alexa Fluor 647 dye-labeled cells, respectively.\",\n \"TABLE 2 Theoretical versus recovered dye-labeled Fab fragment of goat anti- (mouse IgG1 Fc) combinatorial experiment.\",\n \"Recovered Experimentally Experimentally percentage of mixed Recovered mixed measured cells percentage percentage of percentage labeled with of cells measured cells of cells Alexa Fluor labeled with labeled with labeled with 647 dye- Alexa Fluor Alexa Fluor Alexa Fluor 647 labeled Fab 488 dye-labeled 488 dye-labeled dye-labeled Fab fragment of Fab fragment of Fab fragment fragment of goat goat anti- goat anti-(mouse of goat anti- anti-(mouse (mouse IgG1 Fc) (mouse IgG1 Fc) IgG1 Fc) IgG1 Fc) 100% 100% 0% 0% 75% 81% 25% 14% 50% 63% 50% 38% 25% 35% 75% 73% 0% 0% 100% 100% Example 20 The Immunolabeling Complex can be Used to Detect Antigens on a Western Blot Bovine heart mitochondria were isolated (Hanson et al., Electrophoresis 22, 950 (2001)).\",\n \"The isolated mitochondria were resuspended to ˜10 mg/mL in 100 mM Tris-HCl, pH 7.8,1 mM phenylmethylsulfonyl fluoride (a protease inhibitor), 2% SDS and insoluble material was removed by centrifugation for 10 minutes at 10,000×g in a tabletop centrifuge.\",\n \"The protein concentration of the lysate was checked by the BCA assay (Pierce, Rockford, Ill.).\",\n \"Samples for gel electrophoresis were prepared by mixing lysate, water, and loading buffer to the appropriate concentrations (final concentration of loading buffer in samples: 58 mM Tris/HCl, 10% glycerol, 2% SDS, 0.02 mg/mL bromphenol blue, 50 mM DTT, pH 8.6).\",\n \"The samples were then heated to 90° C. for 5 minutes before loading on the gel and separated on a 13% SDS-PAGE gel.\",\n \"Two-fold serial dilution of the extracts ranging from 8 μg of extract down to 20 0.03 μg were loaded on the SDS-PAGE gel.\",\n \"The proteins were transferred to PVDF membrane for 1.5 hours using a semi-dry transfer system according to manufacturer's directions (The W.E.P.\",\n \"Company, Concord, Calif.).\",\n \"The PVDF membrane was blocked for 1 hour in 5% milk.\",\n \"Immunolabeling complexes were made with mouse monoclonal antibodies that recognize two different mitochondrial proteins.\",\n \"Alexa Fluor 647 dye-labeled Fab fragment of goat anti-(mouse IgG1 Fc) (5 μL of a 1 mg/mL stock, prepared as in Example 4) was incubated with 21 pL (0.88 mg/mL) mouse anti-(CV-alpha) and Alexa Fluor 488 dye-labeled Fab fragment of goat anti-(mouse IgG, Fc) (5 μL of a 1 mg/mL stock, prepared as in Example 4) was incubated with 19 μL (0.88 mg/mL) mouse anti-(CIII-core2) (Molecular Probes, Eugene, Oreg.).\",\n \"Following a 30 minute incubation, 25 μL of a 14.1 mg/mL stock of unlabeled mouse IgG was added to each tube.\",\n \"The immunolabeling complexes were then mixed together and brought up to 5 mL in 5% milk.\",\n \"The blot was incubated with the mixture of immunolabeling complexes for 1 hour at room temperature.\",\n \"The blot was washed twice for 5 seconds each with PBST (PBS with 0.1% Tween) and once with PBST for 15 minutes.\",\n \"The blot was air dried and imaged on an EG&G Wallac Imager with the appropriate filters.\",\n \"The Western blot revealed two distinct bands of the appropriate molecular weight.\",\n \"The Western blot also showed that no cross-labeling of the antibodies occurred and the detection limit was 125 ng.\",\n \"Example 21 High-Throughput Screening of Hybridomas for Identifying High Affinity and High IgG Producers Microplate wells containing both a fluorescent labeled antigen of one fluorescent color label and fluorescently labeled Fab fragments of goat anti-(mouse Fc) of a different fluorescent color made by the method described in Example 4 and 5.Hybridoma supernatant is harvested and added to the wells.\",\n \"If the hybridoma are producing the desired antibody, i.e.\",\n \"antibodies that bind to the labeled antigen, polarization of the florescence corresponding to the labeled antigen will allow visualization of those wells containing antigen specific antibody.\",\n \"In addition, the amount of IgG that the hybridomas produce, can be simultaneously identified by polarization of the fluorescence corresponding to the labeled Fab fragments.\",\n \"This method thus allows for both quantitation of the amount of antibody present in a specific amount of hybridoma supernatant and the affinity of the monoclonal antibodies for the antigen.\",\n \"The reagents employed in the preceding examples are commercially available or can be prepared using commercially available instrumentation, methods, or reagents known in the art or whose preparation is described in the examples.\",\n \"It is evident from the above description and results that the subject invention is greatly superior to the presently available methods for determining the presence of a target in a biological sample.\",\n \"The subject invention overcomes the shortcomings of the currently used methods by allowing small quantities of antibodies to be labeled and in unlimited media while maintaining specificity and sensitivity.\",\n \"The examples are not intended to provide an exhaustive description of the many different embodiments of the invention.\",\n \"Thus, although the forgoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, those of ordinary skill in the art will realize readily that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.\",\n \"All publications, patents and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10467550"}}},{"rowIdx":374,"cells":{"text":{"kind":"list like","value":[["Gssp4 polynucleotides and polypeptides and uses thereof","The present invention relates to the field of metabolic research.","Metabolic disorders, such as obesity, are a public health problem that is serious and widespread.","GSSP4 polypeptides have been identified that are believed to be beneficial in the treatment of metabolic disorders.","These compounds should be effective for reducing cholesterol levels, body mass, body fat, and for treating metabolic-related diseases and disorders.","The metabolic-related diseases or disorders envisioned to be treated by the methods of the invention include, but are not limited to: obesity, hyperlipidemia, hypercholesterolemia, atherosclerosis, diabetes, glucose intolerance, insulin resistance and hypertension."],["1.A method of reducing circulating free fatty acid levels in an individual comprising administering to said individual a physiologically acceptable composition comprising a carrier and a polypeptide sequence comprising at least 6 consecutive amino acids of SEQ ID NO:3 with metabolic-related activity, wherein said method optionally reduces body mass.","2.A method of reducing circulating glucose levels in an individual comprising administering to said individual a physiologically acceptable composition comprising a carrier and a polypeptide sequence comprising at least 6 consecutive amino acids of SEQ ID NO:3 with metabolic-related activity, wherein said method optionally reduces body mass.","3.A method of reducing circulating triglyceride levels in an individual comprising administering to said individual a physiologically acceptable composition comprising a carrier and a polypeptide sequence comprising at least 6 consecutive amino acids of SEQ ID NO:3 with metabolic-related activity, wherein said method optionally reduces body mass.","4.A method of reducing circulating cholesterol levels in an individual comprising administering to said individual a physiologically acceptable composition comprising a carrier and a polypeptide sequence comprising at least 6 consecutive amino acids of SEQ ID NO:3 with metabolic-related activity, wherein said method optionally reduces body mass.","5.An isolated polypeptide comprising: a) an amino acid sequence at least 50% identical to the full length polypeptide of SEQ ID NO: 3; b) a polypeptide fragment of at least six consecutive amino acids of SEQ ID NO: 3; c) the polypeptide of SEQ ID NO: 3; d) homomultimers or heteromultimers of the polypeptide of SEQ ID NO: 3; e) a heterologous polypeptide fused to the polypeptide of a), b), c), or d); or f) a polypeptide according to a), b), c), d), or e) that has been differentially modified.","6.A composition comprising an isolated polypeptide according to claim 3 and a pharmaceutically or physiologically acceptable carrier.","7.An isolated or purified polynucleotide: a) encoding a polypeptide comprising an amino acid sequence at least 50% identical to the full length polypeptide of SEQ ID NO: 3; b) encoding a polypeptide comprising a fragment of at least six consecutive amino acids of SEQ ID NO: 3; c) encoding a polypeptide comprising SEQ ID NO: 3; d) encoding homomultimers or heteromultimers of the polypeptide of SEQ ID NO: 3; e) encoding a polypeptide comprising a heterologous polypeptide fused to the polypeptide of a), b), c), or d); f) encoding a polypeptide according to a), b), c), d), or e) that has been differentially modified; g) comprising a polynucleotide according to a), b), c), d), e), or f) operably linked to a promoter, other regulatory element, or selectable marker; or h) comprising a vector comprising a polynucleotide according to a), b), c), d), e), f), or g).","8.A recombinant cell comprising a polynucleotide according to claim 7.9.An antibody that specifically binds to a polypeptide according to claim 5.10.A method of producing a polypeptide comprising culturing a recombinant cell according to claim 8 under conditions that allow for the expression of said polypeptide.","11.The method according to claim 10, further comprising the isolation, recovery, or purification of said polypeptide.","12.A method of treating a obesity, a metabolic disease or a metabolic disorder comprising the administration of a therapeutically effective amount of a composition comprising: a) an isolated polypeptide comprising: 1) an amino acid sequence at least 50% identical to the full length polypeptide of SEQ ID NO: 3; 2) a polypeptide fragment of at least six consecutive amino acids of SEQ ID NO: 3; 3) the polypeptide of SEQ ID NO: 3; 4) homomultimers or heteromultimers of the polypeptide of SEQ ID NO: 3; 5) a heterologous polypeptide fused to the polypeptide of a(1), a(2), a(3), or a(4); or 6) a polypeptide according to a(1), a(2), a(3), a(4), or a(5) that has been differentially modified; b) an isolated or purified polynucleotide: 1) encoding a polypeptide comprising an amino acid sequence at least 50% identical to the full length polypeptide of SEQ ID NO: 3; 2) encoding a polypeptide comprising a fragment of at least six consecutive amino acids of SEQ ID NO: 3; 3) encoding a polypeptide comprising SEQ ID NO: 3; 4) encoding homomultimers or heteromultimers of the polypeptide of SEQ ID NO: 3; 5) encoding a polypeptide comprising a heterologous polypeptide fused to the polypeptide of b(1), b(2), b(3), or b(4); 6) encoding a polypeptide according to b(1), b(2), b(3), b(4), or b(5) that has been differentially modified; 7) comprising a polynucleotide according to b(1), b(2), b(3), b(4), b(5), or b(6) operably linked to a promoter, other regulatory element, or selectable marker; or 8) comprising a vector comprising a polynucleotide according to b(1), b(2), b(3), b(4), b(5), b(6), or b(7); or c) a recombinant cell comprising: 1) a polynucleotide encoding a polypeptide comprising an amino acid sequence at least 50% identical to the full length polypeptide of SEQ ID NO: 3; 2) a polynucleotide encoding a polypeptide comprising a fragment of at least six consecutive amino acids of SEQ ID NO: 3; 3) a polynucleotide encoding a polypeptide comprising SEQ ID NO: 3; 4) a polynucleotide encoding homomultimers or heteromultimers of the polypeptide of SEQ ID NO: 3; 5) a polynucleotide encoding a polypeptide comprising a heterologous polypeptide fused to the polypeptide of c(1), c(2), c(3), or c(4); 6) a polynucleotide encoding a polypeptide according to c(1), c(2), c(3), c(4), or c(5) that has been differentially modified; 7) a polynucleotide according to c(1), c(2), c(3), c(4), c(5), or c(6) operably linked to a promoter, other regulatory element, or selectable marker; or 8) comprising a vector comprising a polynucleotide according to c(1), c(2), c(3), c(4), c(5), c(6), or c(7).","13.A transgenic animal comprising a polynucleotide according to claim 7."],["<SOH> BACKGROUND OF TH INVENTION <EOH>The following discussion is intended to facilitate the understanding of the invention, but is not intended nor admitted to be prior art to the invention.","Obesity is a public health problem that is serious, widespread, and increasing.","In the United States, 20 percent of the population is obese; in Europe, a slightly lower percentage is obese (Friedman (2000) Nature 404:632-634).","Obesity is associated with increased risk of hypertension, cardiovascular disease, diabetes, and cancer as well as respiratory complications and osteoarthritis (Kopelman (2000) Nature 404:635-643).","Even modest weight loss ameliorates these associated conditions.","While still acknowledging that lifestyle factors including environment, diet, age and exercise play a role in obesity, twin studies, analyses of familial aggregation, and adoption studies all indicate that obesity is largely the result of genetic factors (Barsh et al (2000) Nature 404:644-651).","In agreement with these studies, is the fact that an increasing number of obesity-related genes are being identified.","Some of the more extensively studied genes include those encoding leptin (ob) and its receptor (db), pro-opiomelanocortin (Pomc), melanocortin-4-receptor (Mc4r), agouti protein (A y ), carboxypeptidase E (fat), 5-hydroxytryptamine receptor 2C (Htr2c), nescient basic helix-loop-helix 2 (Nhlh2), prohormone convertase 1 IPCSK1), and tubby protein (tubby) (rev'd in Barsh et al (2000) Nature 404:644-651)."],["<SOH> SUMMARY OF THE INVENTION <EOH>The instant invention is based on the discovery that GSSP4 polypeptides have unexpected effects in vitro and in vivo, including utility for weight reduction, prevention of weight gain, reduction of cholesterol levels, and control of blood glucose levels in humans and other mammals.","These unexpected effects of administration of GSSP4 polypeptides in mammals also include reduction of elevated free fatty acid levels caused by administration of epinephrine, i.v.","injection of “intralipid”, or administration of a high fat test meal, as well as increased fatty acid oxidation in muscle cells, reduction of circulating cholesterol levels, modulation of blood glucose and weight reduction in mammals, particularly those consuming a high fat/high carbohydrate diet.","These effects are unexpected and surprising given that proteins of similar structure or homology (such as colipase and mamba intestinal toxin 1) have not been shown to have utility for weight reduction, prevention of weight gain, reduction of cholesterol levels, and control of blood glucose levels.","However, the GSSP4 polypeptides of the invention are effective and can be provided at levels that are feasible for treatments in humans.","Thus, the invention is drawn to GSSP4 polypeptides, polynucleotides encoding said polypeptides, vectors comprising said GSSP4 polynucleotides, and cells recombinant for said GSSP4 polynucleotides, as well as to pharmaceutical and physiologically acceptable compositions comprising said GSSP4 polypeptide and methods of administering said GSSP4 polypeptides or polynucleotides in a pharmaceutical and physiologically acceptable compositions in order to reduce body weight, cholesterol levels or glucose levels, or to treat metabolic-related diseases and disorders.","Assays for identifying agonists and antagonists of metabolic-related activity are also part of the invention.","In a first aspect, the invention features a purified, isolated, or recombinant GSSP4 polypeptides.","In preferred embodiments, said polypeptides comprise, consist essentially of, or consist of, those having significant activity wherein the said activity is selected from the group consisiting of cholesterol reduction, cholesterol regulation, lipid partitioning, lipid metabolism, glucose control, and insulin-like activity.","In preferred embodiments, said polypeptides comprise, consist essentially of, or consist of, the full length polypeptide of SEQ ID NO:3 or a fragment of consecutive amino acids of the full length polypeptide sequence of SEQ ID NO:3.In other preferred embodiments, said polypeptides comprise an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding consecutive amino acids of the polypeptide sequences identified in SEQ ID NO:3.In a further preferred embodiment, the GSSP4 polypeptide is able to lower circulating (either blood, serum or plasma) levels (concentration) of: (i) free fatty acids, (ii) glucose, and/or (iii) triglycerides.","Further preferred GSSP4 polypeptides are those that significantly stimulate muscle lipid or free fatty acid oxidation.","Further preferred GSSP4 polypeptides are those that cause C2C12 cells differentiated in the presence of said polypeptides to undergo at least 10%, 20%, 30%, 35%, or 40% more oleate oxidation as compared to untreated cells.","Further preferred GSSP4 polypeptides are those that are at least 30% more efficient than untreated cells at increasing leptin uptake in a liver cell line (preferably BPRCL mouse liver cells (ATCC CRL-2217)).","Further preferred GSSP4 polypeptides are those that significantly reduce the postprandial increase in plasma free fatty acids, particularly following a high fat meal.","Further preferred GSSP4 polypeptides are those that significantly reduce or eliminate ketone body production, particularly following a high fat meal.","Further preferred GSSP4 polypeptides are those that increase glucose uptake in skeletal muscle cells.","Further preferred GSSP4 polypeptides are those that increase glucose uptake in adipose cells.","Further preferred GSSP4 polypeptides are those that increase glucose uptake in neuronal cells.","Further preferred GSSP4 polypeptides are those that increase glucose uptake in red blood cells.","Further preferred GSSP4 polypeptides are those that increase glucose uptake in the brain.","Further preferred GSSP4 polypeptides are those that significantly reduce the postprandial increase in plasma glucose following a meal, particularly a high carbohydrate meal.","Further preferred GSSP4 polypeptides are those that significantly prevent the postprandial increase in plasma glucose following a meal, particularly a high fat or a high carbohydrate meal.","Further preferred GSSP4 polypeptides are those that improve insulin sensitivity.","Further preferred GSSP4 polypeptides are those that modulate food intake or food selection.","Further preferred GSSP4 polypeptides are those that modulate satiety.","Further preferred GSSP4 polypeptides are those that modulate fatty acid metabolism.","Further preferred GSSP4 polypeptides are those that modulate cholesterol metabolism, particularly in steroidogenic tissues.","Therefore, said polypeptides have a potential role in effecting, either directly or indirectly or both, levels of reproductive hormones (eg.","estradiol, progesterone, testosterone).","Further preferred GSSP4 polypeptides are those that modulate cortisol levels.","Further preferred GSSP4 polypeptides are those that modulate aldosterone levels.","Therefore, said polypeptides have a potential role in effecting, either directly or indirectly or both, levels of sodium and potassium.","Further preferred GSSP4 polypeptides are those that modulate blood pressure preferably to normalize blood pressure within a normal range.","Further preferred GSSP4 polypeptides are those that form multimers (e.g., heteromultimers or homomultimers) in vitro and/or in vivo.","Preferred multimers are homodimers or homotrimers.","Other preferred multimers are homomultimers comprising at least 4, 6, 8, 9, 10 or 12 GSSP4 polypeptides.","Other preferred mulimers are hetero multimers comprising GSSP4 polypeptides of the invention.","Further preferred embodiments include heterologous polypeptides comprising a GSSP4 polypeptide of the invention.","In a second aspect, the invention features purified, isolated, or recombinant polynucleotides encoding said GSSP4 polypeptides described in the first aspect, or the complement thereof.","A further preferred embodiment of the invention is a recombinant, purified or isolated polynucleotide comprising, or consisting of a mammalian genomic sequence, gene, cDNA, or fragments thereof.","In one aspect the sequence is derived from a human, mouse or other mammal.","In a preferred aspect, the genomic sequence includes isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 22, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, 1000, 2000, 5000, 6000 or 7500 nucleotides of any one of the polynucleotide sequences described in SEQ ID NO:1, 2, or the complements thereof, wherein said contiguous span comprises a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding nucleotide sequence in SEQ ID NO: 1, 2, or 3.In further embodiments the polynucleotides are DNA, RNA, DNA/RNA hybrids, single-stranded, and double-stranded.","Further preferred are GSSP4 polynucleotides and polypeptides that have cholesterol regulating activies.","Further preferred are GSSP4 polynucleotides and polypeptides that have body weight regulating activies.","Further preferred are GSSP4 polynucleotides and polypeptides that have body fat regulating activies.","Further preferred are GSSP4 polynucleotides and polypeptides that have glucose regulating activies.","Further preferred are GSSP4 polynucleotides and polypeptides that have lipid regulating activies.","In a third aspect, the invention features a recombinant vector comprising, consisting essentially of, or consisting of, said polynucleotide described in the second aspect.","In a fourth aspect, the invention features a recombinant cell comprising, consisting essentially of, or consisting of, said recombinant vector described in the third aspect.","A further embodiment includes a host cell recombinant for a polynucleotide of the invention.","In a fifth aspect, the invention features a pharmaceutical or physiologically acceptable composition comprising, consisting essentially of, or consisting of, said GSSP4 polypeptides described in the first aspect and, a pharmaceutical or physiologically acceptable diluent.","In a sixth aspect, the invention features a method of controlling cholesterol levels comprising, providing, or administering to individuals with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.","In further preferred embodiments, the invention features a method of lowering body weight comprising, providing, or administering to individuals with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.","In further preferred embodiments, the invention features a method of lowering body fat comprising, providing, or administering to individuals with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.","In further preferred embodiments, the invention features a method of lowering controlling blood glucose comprising, providing, or administering to individuals with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.","In a seventh aspect, the invention features a method of preventing or treating a metabolic-related disease or disorder comprising, providing or administering to an individual in need of such treatment said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.","Preferably, said obesity-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM or Type II diabetes), Insulin dependent diabetes mellitus (IDDM or Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.","Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.","In preferred embodiments, said individual is a mammal, preferably a human.","In a further preferred embodiment, a pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect suggest that a compound may have utility in alleviating insulin resistance in individuals, particularly those that are obese or overweight.","In a further preferred embodiment, the present invention may be used in complementary therapy in individuals to improve their cholesterol, weight or glucose level, comprising a pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect in combination with known agents.","The present invention further provides a method of improving the cholesterol levels, body weight or glucose control in individuals comprising the administration of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect alone, without known agents.","In a further preferred embodiment, the present invention may be administered either concomitantly or concurrently, with known agents for example in the form of separate dosage units to be used simultaneously, separately or sequentially (either before or after the known agent).","Accordingly, the present invention further provides a product containing a composition a pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect and a known agent as a combined preparation for simultaneous, separate or sequential use for the improvement of cholesterol levels, body weight or glucose control in individuals, particularly those who are obese or overweight.","The ratio of the present composition to known agent is such that the quantity of each active ingredient employed will be such as to provide a therapeutically effective level, but will not be larger than the quantity recommended as safe for administration.","In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect can be used as a method to improve insulin sensitivity in some persons, particularly those with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) or noninsulin dependent diabetics (Type II) in combination with insulin therapy.","In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect can be used as a method to improve insulin sensitivity in some persons, particularly those with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) or noninsulin dependent diabetes mellitus (NIDDM, Type II) in combination with alternate therapies.","In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used as a method in prophylaxis of long-term detrimental effects caused by prolonged high dosage of insulin in humans having IDDM or NIDDM.","In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing hypersecretion of insulin and disorders or conditions resulting therefrom.","In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing obesity and consequences or complications thereof.","In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing hypercholesterolemia and consequences or complications thereof.","In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing NIDDM or IDDM and consequences or complications thereof.","In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing impaired glucose tolerance (IGT).","Further preferred embodiment thus provides therapeutics and methods for normalizing insulin resistance.","Further preferred embodiment thus provides therapeutics and methods for reducing, slowing or preventing the progression to NIDDM.","In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing the appearance of insulin-resistance syndrome.","In further preferred embodiments, other conditions, particularly obesity, associated with insulin resistance are treated or prevented according to the methods of the invention.","Thus, by preventing or treating obesity, the methods of the invention will allow an individual to have a more comfortable life and avoid the onset of various diseases triggered by obesity.","In further preferred embodiments, the target of the methods according to the present invention includes individuals with normal glucose tolerance (NGT) who are obese or who have fasting hyperinsulinemia, or who have both.","In an eighth aspect, the invention features a method of controlling blood free fatty acid (FFA) levels and lipid metabolism comprising, providing, or administering to individuals in need of increasing mobilization and utilization of fat stores and decreasing total fat stores with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.","In a further preferred embodiment, the identification of said individuals in need of increasing mobilization and utilization of fat stores and decreasing total fat stores to be treated with said pharmaceutical or physiologically acceptable composition comprises a person who is involved in physical activity which increases metabolic demand.","Furthermore, increasing mobilization and utilization of fat stores and decreasing total fat stores would provide a means to decrease body weight, preventing weight gain, decrease body fat in overweight and obese individuals.","Reduction in weight and obesity will thus decrease the risk of chronic disease associated with obesity such as but not limited to the onset of various lipid metabolism disorders, hypertension, Type II diabetes, atherosclerosis, cardiovascular disease and stroke.","In related aspects, embodiments of the present invention includes methods of causing or inducing a desired biological response in an individual comprising the steps of: providing or administering to an individual a composition comprising a GSSP4 polypeptide, wherein said biological response is selected from the group consisting of: (a) lowering circulating (either blood, serum, or plasma) levels (concentration) of free fatty acids; (b) lowering circulating (either blood, serum or plasma) levels (concentration) of glucose; (c) lowering circulating (either blood, serum or plasma) levels (concentration) of triglycerides; (d) stimulating muscle lipid or free fatty acid oxidation; (e) increasing leptin uptake in the liver or liver cells; (f) reducing the postprandial increase in plasma free fatty acids, particularly following a high fat meal; and, (g) reducing or eliminating ketone body production, particularly following a high fat meal; (h) increasing tissue sensitivity to insulin, particularly muscle, adipose, liver or brain, (i) reducing cholesterol levels, particularly in those with elevated cholesterol (ie.","greater than 200 mg/dl); (j) modulating circulating (either blood, serum or plasma) levels (concentration) of glucose within physiological range, preferably maintaining glucose between 60-190 mg/dl; (k) modulating circulating (either blood, serum or plasma) levels (concentration) of FFA within physiological range preferably maintaining FFA between 190-420 mg/dl; (l) modulating ketone body production as the result of a high fat meal, wherein said modulating is preferably reducing or eliminating; (m) reducing body weight particularly in individuals with a BMI of greater than 27.In a ninth aspect, the invention features a method of making the GSSP4 polypeptide described in the first aspect, wherein said method is selected from the group consisting of: proteolytic cleavage, recombinant methodology and artificial synthesis.","In a tenth aspect, the present invention provides a method of making a recombinant GSSP4 polypeptides, the method comprising providing a transgenic, non-human mammal whose milk contains said recombinant GSSP4 polypeptides, and purifying said recombinant GSSP4 polypeptides from the milk of said non-human mammal.","In one embodiment, said non-human mammal is a cow, goat, sheep, rabbit, or mouse.","In another embodiment, the method comprises purifying a recombinant GSSP4 polypeptides from said milk, and further comprises cleaving said protein in vitro to obtain a desired GSSP4 polypeptides.","In an eleventh aspect, the invention features a purified or isolated antibody capable of specifically binding to a protein comprising the sequence of one of the polypeptides of the present invention.","In one aspect of this embodiment, the antibody is capable of binding to a polypeptide comprising at least 6 consecutive amino acids, at least 8 consecutive amino acids, or at least 10 consecutive amino acids of the sequence of one of the polypeptides of the present invention.","In a twelfth aspect, the invention features a use of polypeptides described in the first aspect or polynucleotides described in the second aspect for treatment of metabolic-related diseases and disorders or reducing or increasing body mass.","Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM or Type II diabetes), Insulin dependent diabetes mellitus (IDDM or Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.","Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.","In preferred embodiments, said individual is a mammal, preferably a human.","In a thirteenth aspect, the invention features a use of polypeptides described in the first aspect or polynucleotides described in the second aspect for the preparation of a medicament for the treatment of metabolic-related diseases and disorders or for reducing body mass.","Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM or Type II diabetes), Insulin dependent diabetes mellitus (IDDM or Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.","Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.","In preferred embodiments, said individual is a mammal, preferably a human.","In a fourteenth aspect, the invention provides polypeptides of the first aspect of the invention or a composition of the fifth aspect for use in a method of treatment of the human or animal body.","In a fifteenth aspect, the invention provides polynucleotides described in the second aspect or an acceptable composition thereof, for use in a method of treatment of the human or animal body.","In a sixteenth aspect, the invention features methods of reducing body weight for cosmetic purposes comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect, or polypeptides described in the first aspect.","Preferably, for said reducing body weight said individual has a BMI of at least 20, 25, 30, 35, or 40.In a seventeenth aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect or a polypeptide described in the first aspect for reducing body mass in said individuals with a BMI of at least 30, 35, 40, or 45 or for treatment or prevention of metabolic-related diseases or disorders.","Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM or Type II diabetes), Insulin dependent diabetes mellitus (IDDM or Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.","Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.","In preferred embodiments, said individual is a mammal, preferably a human.","In preferred embodiments, the identification of said individuals to be treated with said pharmaceutical or physiologically acceptable composition comprises genotyping GSSP4 single nucleotide polymorphisms (SNPs) or measuring GSSP4 polypeptide or mRNA levels in clinical samples from said individuals.","Preferably, said clinical samples are selected from the group consisting of blood, serum, plasma, urine, and saliva.","In an eighteenth aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect for reducing body weight for cosmetic reasons.","In further preferred embodiments, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect for reducing glucose levels.","In a nineteenth aspect, the invention features methods of treating insulin resistance comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect, or a polypeptide described in the first aspect."],["FIELD OF THE INVENTION The present invention relates to the field of metabolic research, in particular the discovery of compounds effective for reducing cholesterol levels, body fat, and body mass, and useful for treating metabolic-related diseases an disorders.","The metabolic-related diseases or disorders envisioned to be treated by the methods of the invention include, but are not limited to, obesity, hyperlipidemia, hypercholesterolemia, atherosclerosis, diabetes, glucose intolerance, insulin resistance and hypertension.","BACKGROUND OF TH INVENTION The following discussion is intended to facilitate the understanding of the invention, but is not intended nor admitted to be prior art to the invention.","Obesity is a public health problem that is serious, widespread, and increasing.","In the United States, 20 percent of the population is obese; in Europe, a slightly lower percentage is obese (Friedman (2000) Nature 404:632-634).","Obesity is associated with increased risk of hypertension, cardiovascular disease, diabetes, and cancer as well as respiratory complications and osteoarthritis (Kopelman (2000) Nature 404:635-643).","Even modest weight loss ameliorates these associated conditions.","While still acknowledging that lifestyle factors including environment, diet, age and exercise play a role in obesity, twin studies, analyses of familial aggregation, and adoption studies all indicate that obesity is largely the result of genetic factors (Barsh et al (2000) Nature 404:644-651).","In agreement with these studies, is the fact that an increasing number of obesity-related genes are being identified.","Some of the more extensively studied genes include those encoding leptin (ob) and its receptor (db), pro-opiomelanocortin (Pomc), melanocortin-4-receptor (Mc4r), agouti protein (Ay), carboxypeptidase E (fat), 5-hydroxytryptamine receptor 2C (Htr2c), nescient basic helix-loop-helix 2 (Nhlh2), prohormone convertase 1 IPCSK1), and tubby protein (tubby) (rev'd in Barsh et al (2000) Nature 404:644-651).","SUMMARY OF THE INVENTION The instant invention is based on the discovery that GSSP4 polypeptides have unexpected effects in vitro and in vivo, including utility for weight reduction, prevention of weight gain, reduction of cholesterol levels, and control of blood glucose levels in humans and other mammals.","These unexpected effects of administration of GSSP4 polypeptides in mammals also include reduction of elevated free fatty acid levels caused by administration of epinephrine, i.v.","injection of “intralipid”, or administration of a high fat test meal, as well as increased fatty acid oxidation in muscle cells, reduction of circulating cholesterol levels, modulation of blood glucose and weight reduction in mammals, particularly those consuming a high fat/high carbohydrate diet.","These effects are unexpected and surprising given that proteins of similar structure or homology (such as colipase and mamba intestinal toxin 1) have not been shown to have utility for weight reduction, prevention of weight gain, reduction of cholesterol levels, and control of blood glucose levels.","However, the GSSP4 polypeptides of the invention are effective and can be provided at levels that are feasible for treatments in humans.","Thus, the invention is drawn to GSSP4 polypeptides, polynucleotides encoding said polypeptides, vectors comprising said GSSP4 polynucleotides, and cells recombinant for said GSSP4 polynucleotides, as well as to pharmaceutical and physiologically acceptable compositions comprising said GSSP4 polypeptide and methods of administering said GSSP4 polypeptides or polynucleotides in a pharmaceutical and physiologically acceptable compositions in order to reduce body weight, cholesterol levels or glucose levels, or to treat metabolic-related diseases and disorders.","Assays for identifying agonists and antagonists of metabolic-related activity are also part of the invention.","In a first aspect, the invention features a purified, isolated, or recombinant GSSP4 polypeptides.","In preferred embodiments, said polypeptides comprise, consist essentially of, or consist of, those having significant activity wherein the said activity is selected from the group consisiting of cholesterol reduction, cholesterol regulation, lipid partitioning, lipid metabolism, glucose control, and insulin-like activity.","In preferred embodiments, said polypeptides comprise, consist essentially of, or consist of, the full length polypeptide of SEQ ID NO:3 or a fragment of consecutive amino acids of the full length polypeptide sequence of SEQ ID NO:3.In other preferred embodiments, said polypeptides comprise an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding consecutive amino acids of the polypeptide sequences identified in SEQ ID NO:3.In a further preferred embodiment, the GSSP4 polypeptide is able to lower circulating (either blood, serum or plasma) levels (concentration) of: (i) free fatty acids, (ii) glucose, and/or (iii) triglycerides.","Further preferred GSSP4 polypeptides are those that significantly stimulate muscle lipid or free fatty acid oxidation.","Further preferred GSSP4 polypeptides are those that cause C2C12 cells differentiated in the presence of said polypeptides to undergo at least 10%, 20%, 30%, 35%, or 40% more oleate oxidation as compared to untreated cells.","Further preferred GSSP4 polypeptides are those that are at least 30% more efficient than untreated cells at increasing leptin uptake in a liver cell line (preferably BPRCL mouse liver cells (ATCC CRL-2217)).","Further preferred GSSP4 polypeptides are those that significantly reduce the postprandial increase in plasma free fatty acids, particularly following a high fat meal.","Further preferred GSSP4 polypeptides are those that significantly reduce or eliminate ketone body production, particularly following a high fat meal.","Further preferred GSSP4 polypeptides are those that increase glucose uptake in skeletal muscle cells.","Further preferred GSSP4 polypeptides are those that increase glucose uptake in adipose cells.","Further preferred GSSP4 polypeptides are those that increase glucose uptake in neuronal cells.","Further preferred GSSP4 polypeptides are those that increase glucose uptake in red blood cells.","Further preferred GSSP4 polypeptides are those that increase glucose uptake in the brain.","Further preferred GSSP4 polypeptides are those that significantly reduce the postprandial increase in plasma glucose following a meal, particularly a high carbohydrate meal.","Further preferred GSSP4 polypeptides are those that significantly prevent the postprandial increase in plasma glucose following a meal, particularly a high fat or a high carbohydrate meal.","Further preferred GSSP4 polypeptides are those that improve insulin sensitivity.","Further preferred GSSP4 polypeptides are those that modulate food intake or food selection.","Further preferred GSSP4 polypeptides are those that modulate satiety.","Further preferred GSSP4 polypeptides are those that modulate fatty acid metabolism.","Further preferred GSSP4 polypeptides are those that modulate cholesterol metabolism, particularly in steroidogenic tissues.","Therefore, said polypeptides have a potential role in effecting, either directly or indirectly or both, levels of reproductive hormones (eg.","estradiol, progesterone, testosterone).","Further preferred GSSP4 polypeptides are those that modulate cortisol levels.","Further preferred GSSP4 polypeptides are those that modulate aldosterone levels.","Therefore, said polypeptides have a potential role in effecting, either directly or indirectly or both, levels of sodium and potassium.","Further preferred GSSP4 polypeptides are those that modulate blood pressure preferably to normalize blood pressure within a normal range.","Further preferred GSSP4 polypeptides are those that form multimers (e.g., heteromultimers or homomultimers) in vitro and/or in vivo.","Preferred multimers are homodimers or homotrimers.","Other preferred multimers are homomultimers comprising at least 4, 6, 8, 9, 10 or 12 GSSP4 polypeptides.","Other preferred mulimers are hetero multimers comprising GSSP4 polypeptides of the invention.","Further preferred embodiments include heterologous polypeptides comprising a GSSP4 polypeptide of the invention.","In a second aspect, the invention features purified, isolated, or recombinant polynucleotides encoding said GSSP4 polypeptides described in the first aspect, or the complement thereof.","A further preferred embodiment of the invention is a recombinant, purified or isolated polynucleotide comprising, or consisting of a mammalian genomic sequence, gene, cDNA, or fragments thereof.","In one aspect the sequence is derived from a human, mouse or other mammal.","In a preferred aspect, the genomic sequence includes isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 22, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, 1000, 2000, 5000, 6000 or 7500 nucleotides of any one of the polynucleotide sequences described in SEQ ID NO:1, 2, or the complements thereof, wherein said contiguous span comprises a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding nucleotide sequence in SEQ ID NO: 1, 2, or 3.In further embodiments the polynucleotides are DNA, RNA, DNA/RNA hybrids, single-stranded, and double-stranded.","Further preferred are GSSP4 polynucleotides and polypeptides that have cholesterol regulating activies.","Further preferred are GSSP4 polynucleotides and polypeptides that have body weight regulating activies.","Further preferred are GSSP4 polynucleotides and polypeptides that have body fat regulating activies.","Further preferred are GSSP4 polynucleotides and polypeptides that have glucose regulating activies.","Further preferred are GSSP4 polynucleotides and polypeptides that have lipid regulating activies.","In a third aspect, the invention features a recombinant vector comprising, consisting essentially of, or consisting of, said polynucleotide described in the second aspect.","In a fourth aspect, the invention features a recombinant cell comprising, consisting essentially of, or consisting of, said recombinant vector described in the third aspect.","A further embodiment includes a host cell recombinant for a polynucleotide of the invention.","In a fifth aspect, the invention features a pharmaceutical or physiologically acceptable composition comprising, consisting essentially of, or consisting of, said GSSP4 polypeptides described in the first aspect and, a pharmaceutical or physiologically acceptable diluent.","In a sixth aspect, the invention features a method of controlling cholesterol levels comprising, providing, or administering to individuals with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.","In further preferred embodiments, the invention features a method of lowering body weight comprising, providing, or administering to individuals with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.","In further preferred embodiments, the invention features a method of lowering body fat comprising, providing, or administering to individuals with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.","In further preferred embodiments, the invention features a method of lowering controlling blood glucose comprising, providing, or administering to individuals with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.","In a seventh aspect, the invention features a method of preventing or treating a metabolic-related disease or disorder comprising, providing or administering to an individual in need of such treatment said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.","Preferably, said obesity-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM or Type II diabetes), Insulin dependent diabetes mellitus (IDDM or Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.","Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.","In preferred embodiments, said individual is a mammal, preferably a human.","In a further preferred embodiment, a pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect suggest that a compound may have utility in alleviating insulin resistance in individuals, particularly those that are obese or overweight.","In a further preferred embodiment, the present invention may be used in complementary therapy in individuals to improve their cholesterol, weight or glucose level, comprising a pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect in combination with known agents.","The present invention further provides a method of improving the cholesterol levels, body weight or glucose control in individuals comprising the administration of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect alone, without known agents.","In a further preferred embodiment, the present invention may be administered either concomitantly or concurrently, with known agents for example in the form of separate dosage units to be used simultaneously, separately or sequentially (either before or after the known agent).","Accordingly, the present invention further provides a product containing a composition a pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect and a known agent as a combined preparation for simultaneous, separate or sequential use for the improvement of cholesterol levels, body weight or glucose control in individuals, particularly those who are obese or overweight.","The ratio of the present composition to known agent is such that the quantity of each active ingredient employed will be such as to provide a therapeutically effective level, but will not be larger than the quantity recommended as safe for administration.","In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect can be used as a method to improve insulin sensitivity in some persons, particularly those with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) or noninsulin dependent diabetics (Type II) in combination with insulin therapy.","In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect can be used as a method to improve insulin sensitivity in some persons, particularly those with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) or noninsulin dependent diabetes mellitus (NIDDM, Type II) in combination with alternate therapies.","In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used as a method in prophylaxis of long-term detrimental effects caused by prolonged high dosage of insulin in humans having IDDM or NIDDM.","In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing hypersecretion of insulin and disorders or conditions resulting therefrom.","In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing obesity and consequences or complications thereof.","In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing hypercholesterolemia and consequences or complications thereof.","In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing NIDDM or IDDM and consequences or complications thereof.","In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing impaired glucose tolerance (IGT).","Further preferred embodiment thus provides therapeutics and methods for normalizing insulin resistance.","Further preferred embodiment thus provides therapeutics and methods for reducing, slowing or preventing the progression to NIDDM.","In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing the appearance of insulin-resistance syndrome.","In further preferred embodiments, other conditions, particularly obesity, associated with insulin resistance are treated or prevented according to the methods of the invention.","Thus, by preventing or treating obesity, the methods of the invention will allow an individual to have a more comfortable life and avoid the onset of various diseases triggered by obesity.","In further preferred embodiments, the target of the methods according to the present invention includes individuals with normal glucose tolerance (NGT) who are obese or who have fasting hyperinsulinemia, or who have both.","In an eighth aspect, the invention features a method of controlling blood free fatty acid (FFA) levels and lipid metabolism comprising, providing, or administering to individuals in need of increasing mobilization and utilization of fat stores and decreasing total fat stores with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.","In a further preferred embodiment, the identification of said individuals in need of increasing mobilization and utilization of fat stores and decreasing total fat stores to be treated with said pharmaceutical or physiologically acceptable composition comprises a person who is involved in physical activity which increases metabolic demand.","Furthermore, increasing mobilization and utilization of fat stores and decreasing total fat stores would provide a means to decrease body weight, preventing weight gain, decrease body fat in overweight and obese individuals.","Reduction in weight and obesity will thus decrease the risk of chronic disease associated with obesity such as but not limited to the onset of various lipid metabolism disorders, hypertension, Type II diabetes, atherosclerosis, cardiovascular disease and stroke.","In related aspects, embodiments of the present invention includes methods of causing or inducing a desired biological response in an individual comprising the steps of: providing or administering to an individual a composition comprising a GSSP4 polypeptide, wherein said biological response is selected from the group consisting of: (a) lowering circulating (either blood, serum, or plasma) levels (concentration) of free fatty acids; (b) lowering circulating (either blood, serum or plasma) levels (concentration) of glucose; (c) lowering circulating (either blood, serum or plasma) levels (concentration) of triglycerides; (d) stimulating muscle lipid or free fatty acid oxidation; (e) increasing leptin uptake in the liver or liver cells; (f) reducing the postprandial increase in plasma free fatty acids, particularly following a high fat meal; and, (g) reducing or eliminating ketone body production, particularly following a high fat meal; (h) increasing tissue sensitivity to insulin, particularly muscle, adipose, liver or brain, (i) reducing cholesterol levels, particularly in those with elevated cholesterol (ie.","greater than 200 mg/dl); (j) modulating circulating (either blood, serum or plasma) levels (concentration) of glucose within physiological range, preferably maintaining glucose between 60-190 mg/dl; (k) modulating circulating (either blood, serum or plasma) levels (concentration) of FFA within physiological range preferably maintaining FFA between 190-420 mg/dl; (l) modulating ketone body production as the result of a high fat meal, wherein said modulating is preferably reducing or eliminating; (m) reducing body weight particularly in individuals with a BMI of greater than 27.In a ninth aspect, the invention features a method of making the GSSP4 polypeptide described in the first aspect, wherein said method is selected from the group consisting of: proteolytic cleavage, recombinant methodology and artificial synthesis.","In a tenth aspect, the present invention provides a method of making a recombinant GSSP4 polypeptides, the method comprising providing a transgenic, non-human mammal whose milk contains said recombinant GSSP4 polypeptides, and purifying said recombinant GSSP4 polypeptides from the milk of said non-human mammal.","In one embodiment, said non-human mammal is a cow, goat, sheep, rabbit, or mouse.","In another embodiment, the method comprises purifying a recombinant GSSP4 polypeptides from said milk, and further comprises cleaving said protein in vitro to obtain a desired GSSP4 polypeptides.","In an eleventh aspect, the invention features a purified or isolated antibody capable of specifically binding to a protein comprising the sequence of one of the polypeptides of the present invention.","In one aspect of this embodiment, the antibody is capable of binding to a polypeptide comprising at least 6 consecutive amino acids, at least 8 consecutive amino acids, or at least 10 consecutive amino acids of the sequence of one of the polypeptides of the present invention.","In a twelfth aspect, the invention features a use of polypeptides described in the first aspect or polynucleotides described in the second aspect for treatment of metabolic-related diseases and disorders or reducing or increasing body mass.","Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM or Type II diabetes), Insulin dependent diabetes mellitus (IDDM or Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.","Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.","In preferred embodiments, said individual is a mammal, preferably a human.","In a thirteenth aspect, the invention features a use of polypeptides described in the first aspect or polynucleotides described in the second aspect for the preparation of a medicament for the treatment of metabolic-related diseases and disorders or for reducing body mass.","Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM or Type II diabetes), Insulin dependent diabetes mellitus (IDDM or Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.","Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.","In preferred embodiments, said individual is a mammal, preferably a human.","In a fourteenth aspect, the invention provides polypeptides of the first aspect of the invention or a composition of the fifth aspect for use in a method of treatment of the human or animal body.","In a fifteenth aspect, the invention provides polynucleotides described in the second aspect or an acceptable composition thereof, for use in a method of treatment of the human or animal body.","In a sixteenth aspect, the invention features methods of reducing body weight for cosmetic purposes comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect, or polypeptides described in the first aspect.","Preferably, for said reducing body weight said individual has a BMI of at least 20, 25, 30, 35, or 40.In a seventeenth aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect or a polypeptide described in the first aspect for reducing body mass in said individuals with a BMI of at least 30, 35, 40, or 45 or for treatment or prevention of metabolic-related diseases or disorders.","Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM or Type II diabetes), Insulin dependent diabetes mellitus (IDDM or Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.","Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.","In preferred embodiments, said individual is a mammal, preferably a human.","In preferred embodiments, the identification of said individuals to be treated with said pharmaceutical or physiologically acceptable composition comprises genotyping GSSP4 single nucleotide polymorphisms (SNPs) or measuring GSSP4 polypeptide or mRNA levels in clinical samples from said individuals.","Preferably, said clinical samples are selected from the group consisting of blood, serum, plasma, urine, and saliva.","In an eighteenth aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect for reducing body weight for cosmetic reasons.","In further preferred embodiments, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect for reducing glucose levels.","In a nineteenth aspect, the invention features methods of treating insulin resistance comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect, or a polypeptide described in the first aspect.","BRIEF DESCRIPTION OF THE SEQENCES SEQ ID NO:1 shows GSSP4 genomic sequence indicating regulatory regions, exons, polymorphic alleles and positions, and reverse and forward microsequencing primers for haplotyping.","SEQ ID NO:2 shows GSSP4 polynucleotide sequence of cDNA SEQ ID NO:3 shows GSSP4 polypeptide sequence SEQ ID NO:4-18 shows GSSP4 47-mer nucleotide sequence comprising polymorphic allele at position 24.The corresponding alleles and primers indicated in SEQ ID NO:1 are as follows: VLP_1206_C_A, m=C or A SEQ ID NO:4 VLP_148_A_G, r=A or G SEQ ID NO:5 VLP_1851_T_C, y=T or C SEQ ID NO:6 VLP_2551_G_A, r=G or A SEQ ID NO:7 VLP_3124_C_T, y=C or T SEQ ID NO:8 VLP_3563_G_A, r=G or A SEQ ID NO:9 VLP_3792_G_A, r=G or A SEQ ID NO:10 VLP_4417_A_C, m=A or G SEQ ID NO:11 VLP_5757_T_C, y=T or C SEQ ID NO:12 VLP_6322_A_G, r=G or A SEQ ID NO:13 VLP_816_G_A, r=G or A SEQ ID NO:14 VLP_924_G_A, r=G or A SEQ ID NO:15 VLP_99-1_174_T_C, y=T or C SEQ ID NO:16 VLP_99-1_325_C_G, s=C or G SEQ ID NO:17 VLP_99-2_389_T_C, y=T or G SEQ ID NO:18 DETAILED DESCRIPTION OF THE INVENTION Before describing the invention in greater detail, the following definitions are set forth to illustrate and define the meaning and scope of the terms used to describe the invention herein.","As used interchangeably herein, the terms “oligonucleotides”, and “polynucleotides” and nucleic acid include RNA, DNA, or RNA/DNA hybrid sequences of more than one nucleotide in either single chain or duplex form.","The terms encompass “modified nucleotides” which comprise at least one modification, including by way of example and not limitation: (a) an alternative linking group, (b) an analogous form of purine, (c) an analogous form of pyrimidine, or (d) an analogous sugar.","For examples of analogous linking groups, purines, pyrimidines, and sugars see for example PCT publication No.","WO 95/04064.The polynucleotide sequences of the invention may be prepared by any known method, including synthetic, recombinant, ex vivo generation, or a combination thereof, as well as utilizing any purification methods known in the art.","The terms polynucleotide construct, recombinant polynucleotide and recombinant polypeptide are used herein consistently with their use in the art.","The terms “upstream” and “downstream” are also used herein consistently with their use in the art.","The terms “base paired” and “Watson & Crick base paired” are used interchangeably herein and consistently with their use in the art.","Similarly, the terms “complementary”, “complement thereof”, “complement”, “complementary polynucleotide”, “complementary nucleic acid” and “complementary nucleotide sequence” are used interchangeably herein and consistently with their use in the art.","The term “purified” is used herein to describe a polynucleotide or polynucleotide vector of the invention that has been separated from other compounds including, but not limited to, other nucleic acids, carbohydrates, lipids and proteins (such as the enzymes used in the synthesis of the polynucleotide).","Purified can also refer to the separation of covalently closed polynucleotides from linear polynucleotides, or vice versa, for example.","A polynucleotide is substantially pure when at least about 50%, 60%, 75%, or 90% of a sample contains a single polynucleotide sequence.","In some cases this involves a determination between conformations (linear versus covalently closed).","A substantially pure polynucleotide typically comprises about 50, 60, 70, 80, 90, 95, 99% weight/weight of a nucleic acid sample.","Polynucleotide purity or homogeneity may be indicated by a number of means well known in the art, such as agarose or polyacrylamide gel electrophoresis of a sample, followed by visualizing a single polynucleotide band upon staining the gel.","For certain purposes higher resolution can be provided by using HPLC or other means well known in the art.","Similarly, the term “purified” is used herein to describe a polypeptide of the invention that has been separated from other compounds including, but not limited to, nucleic acids, lipids, carbohydrates and other proteins.","In some preferred embodiments, a polypeptide is substantially pure when at least about 50%, 60%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% of the polypeptide molecules of a sample have a single amino acid sequence.","In some preferred embodiments, a substantially pure polypeptide typically comprises about 50%, 60%, 70%, 80%, 90% 95%, 96%, 97%, 98%, 99% or 99.5% weight/weight of a protein sample.","Polypeptide purity or homogeneity is indicated by a number of methods well known in the art, such as agarose or polyacrylamide gel electrophoresis of a sample, followed by visualizing a single polypeptide band upon staining the gel.","For certain purposes higher resolution can be provided by using HPLC or other methods well known in the art.","Further, as used herein, the term “purified” does not require absolute purity; rather, it is intended as a relative definition.","Purification of starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.","Alternatively, purification may be expressed as “at least” a percent purity relative to heterologous polynucleotides (DNA, RNA or both) or polypeptides.","As a preferred embodiment, the polynucleotides or polypeptides of the present invention are at least; 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 96%, 98%, 99%, 99.5% or 100% pure relative to heterologous polynucleotides or polypeptides.","As a further preferred embodiment the polynucleotides or polypeptides have an “at least” purity ranging from any number, to the thousandth position, between 90% and 100% (e.g., at least 99.995% pure) relative to heterologous polynucleotides or polypeptides.","Additionally, purity of the polynucleotides or polypeptides may be expressed as a percentage (as descried above) relative to all materials and compounds other than the carrier solution.","Each number, to the thousandth position, may be claimed as individual species of purity.","The term “isolated” requires that the material be removed from its original environment (e.g., the natural environment if it is naturally occurring).","For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.","Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition, and still be isolated in that the vector or composition is not part of its natural environment.","Specifically excluded from the definition of “isolated” are: naturally occurring chromosomes (e.g., chromosome spreads), artificial chromosome libraries, genomic libraries, and cDNA libraries that exist either as an in vitro nucleic acid preparation or as a transfected/transformed host cell preparation, wherein the host cells are either an in vitro heterogeneous preparation or plated as a heterogeneous population of single colonies.","Also specifically excluded are the above libraries wherein a 5′ EST makes up less than 5% (or alternatively 1%, 2%, 3%, 4%, 10%, 25%, 50%, 75%, or 90%, 95%, or 99%) of the number of nucleic acid inserts in the vector molecules.","Further specifically excluded are whole cell genomic DNA or whole cell RNA preparations (including said whole cell preparations which are mechanically sheared or enzymatically digested).","Further specifically excluded are the above whole cell preparations as either an in vitro preparation or as a heterogeneous mixture separated by electrophoresis (including blot transfers of the same) wherein the polynucleotide of the invention have not been further separated from the heterologous polynucleotides in the electrophoresis medium (e.g., further separating by excising a single band from a heterogeneous band population in an agarose gel or nylon blot).","The term “primer” denotes a specific oligonucleotide sequence which is complementary to a target nucleotide sequence and used to hybridize to the target nucleotide sequence.","A primer serves as an initiation point for nucleotide polymerization catalyzed by DNA polymerase, RNA polymerase, or reverse transcriptase.","The term “probe” denotes a defined nucleic acid segment (or nucleotide analog segment, e.g., PNA as defined hereinbelow) which can be used to identify a specific polynucleotide sequence present in a sample, said nucleic acid segment comprising a nucleotide sequence complementary to the specific polynucleotide sequence to be identified.","The term “polypeptide” refers to a polymer of amino acids without regard to the length of the polymer.","Thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide.","This term also does not specify or exclude post-expression modifications of polypeptides.","For example, polypeptides that include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide.","Also included within the definition are polypeptides which contain one or more analogs of an amino acid (including, for example, non-naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.","), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.","The phrases “control of blood glucose”, “glucose regulation” “regulation of blood glucose”, “modulating blood glucose”, and “glucose control” refer to maintaining or regulating blood, serum, and plasma levels of glucose between 70-190 mg/dl.","Without being limited by theory, the compounds/polypeptides of the invention feature a method of chronic control of blood glucose within a narrow and more physiological range compared to the control of blood glucose attained with current therapies which treat disorders of glucose metabolism or insulin action, including but not limited to hyperglycemia, insulin resistance, insulin dependent diabetes mellitus and noninsulin dependent diabetes mellitus.","Without being limited by theory, the compounds/polypeptides of the invention are capable of modulating the control of blood glucose (as defined above), directing or partitioning glucose between the liver and the peripheral tissues, and are thus believed to treat “diseases involving the control of blood glucose between the liver and peripheral tissues”.","The term “peripheral tissues” is meant to include the blood, brain, muscle and adipose tissue.","In preferred embodiments, the compounds/polypeptides of the invention direct or partition glucose towards the liver, muscle and brain.","In alternative preferred embodiments, glucose is directed or partitioned towards the adipose tissue.","In other preferred embodiments, glucose is directed or partitioned towards the liver.","In other preferred embodiments, glucose is directed or partitioned towards the brain.","In other preferred embodiments, glucose is directed towards the blood.","In yet other preferred embodiments, the compounds/polypeptides of the invention increase or decrease the oxidation of glucose, preferably by the muscle.","Without being limited by theory, the compounds/polypeptides of the invention are capable of modulating the partitioning of dietary or endogenous lipids between the liver and peripheral tissues, and are thus believed to treat “diseases involving the partitioning of lipids between the liver and peripheral tissues.” The term “peripheral tissues” is meant to include the blood, muscle and adipose tissue.","In preferred embodiments, the compounds/polypeptides of the invention partition the lipids toward the muscle.","In alternative preferred embodiments, the lipids are partitioned toward the blood.","In alternative preferred embodiments, the lipids are partitioned toward the adipose tissue.","In other preferred embodiments, the lipids are partitioned toward the liver.","In yet other preferred embodiments, the compounds/polypeptides of the invention increase or decrease the oxidation of dietary or endogenous lipids, preferably free fatty acids (FFA) by the muscle.","Dietary and endogenous lipids include, but are not limited to triglycerides (TG) and FFA.","“Preferred diseases” believed to involve the the control of cholesterol, body fat, lipid metabolism, blood glucose, partitioning of blood glucose, and partitioning of lipids include obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, reproductive cancers, hypercortisolism, aldosteronism, hyperandrogenism, hyperkalemia, hypernatremia, hyperlipoproteinemia, hyperinsulinemia, hyperglycemia, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM, Type II diabetes), Insulin dependent diabetes mellitus (IDDM, Type I diabetes), diabetes-related complications (eg.","ketosis), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be prevented or treated by the methods of the invention.","Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.","The term “heterologous”, when used herein, is intended to designate any polypeptide or polynucleotide other than a GSSP4 polypeptide or a polynucleotide encoding a GSSP4 polypeptide of the present invention.","The terms “comprising”, “consisting of” and “consisting essentially of” are defined according to their standard meaning.","A defined meaning set forth in the M.P.E.P.","controls over a defined meaning in the art and a defined meaning set forth in controlling Federal Circuit case law controls over a meaning set forth in the M.P.E.P.","With this in mind, the terms may be substituted for one another throughout the instant application in order to attach the specific meaning associated with each term.","The term “host cell recombinant for” a particular polynucleotide of the present invention, means a host cell that has been altered by the hands of man to contain said polynucleotide in a way not naturally found in said cell.","For example, said host cell may be transiently or stably transfected or transduced with said polynucleotide of the present invention.","The term “obesity” as used herein is defined by the National Heart, Lung and Blood Institute Expert Panel (J Amer Diet Assoc 98:1178-1191, 1998) based on Body Mass Index (BMI).","BMI is calculated as body weight in kilograms divided by the square of height in meters, ie kg/m2.A BMI of less than 18.5 kg/m2 is considered “Underweight”; a BMI of 18.5-24.9 kg/m2 is considered “Normal” (healthy); a BMI of 25.0-29.9 kg/m2 is considered “Overweight”; a BMI of 30.0-34.9 kg/m2 is considered “Class I Obesity”; a BMI of 35.0-39.9 kg/m2 is considered “Class II Obesity”; a BMI of greater than 39.9 kg/m2 is considered “Class III Obesity”.","Waist circumference can also be used to indicate a risk of metabolic complications where in men a circumference of greater than or equal to 94 cm indicates an increased risk, and greater than or equal to 102 cm indicates a substantially increased risk Similarly for women, greater than or equal to 88 cm indicates an increased risk, and greater than or equal to 88 cm indicates a substantially increased risk.","The waist circumference is measured in cm at midpoint between lower border of ribs and upper border of the pelvis.","Other measures of obesity include, but are not limited to, skinfold thickness which is a measurement in cm of skinfold thickness using calipers and bioimpedance which is based on the principle that lean mass and water will conduct electrical current and measurement of resistance to a weak current (impedance) applied across extremities provides an estimate of body fat using an empirically derived equation.","The term “Insulin Dependent Diabetes Mellitus”, “IDDM”, “Type I Diabetes” and “Type I diabetics” are synomous, inclusive, or interchangable.","The term “diabetes-related complications” refer to pathologic or physiologic states experienced by Type I diabetics (as defined previously) or Type II diabetics (as defined previously) or both.","The term “Noninsulin Dependent Diabetes Mellitus”, “NIDDM”, “Type II Diabetes” and “Type II diabetics” are synomous, inclusive, or interchangable.","The term “agent acting on the control of blood glucose, directing glucose between the liver and peripheral tissues” refers to a compound or polypeptide of the invention that modulates the control of blood glucose as previously described.","Preferably, the agent increases or decreases the oxidation of glucose, preferably by the muscle.","Preferably the agent decreases or increases the body weight of individuals or is used to treat or prevent an metabolic-related disease or disorder such as obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM, Type II diabetes), Insulin dependent diabetes mellitus (IDDM, Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.","Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.","The terms “response to an agent acting on the control of blood glucose, directing glucose between the liver and peripheral tissues” refer to drug efficacy, including but not limited to, ability to metabolize a compound, ability to convert a pro-drug to an active drug, and the pharmacokinetics (absorption, distribution, elimination) and the pharmacodynamics (receptor-related) of a drug in an individual.","The terms “side effects to an agent acting on the control of blood glucose, directing glucose between the liver and peripheral tissues” refer to adverse effects of therapy resulting from extensions of the principal pharmacological action of the drug or to idiosyncratic adverse reactions resulting from an interaction of the drug with unique host factors.","“Side effects to an agent acting on the partitioning of dietary lipids between the liver and peripheral tissues” can include, but are not limited to, adverse reactions such as dermatologic, hematologic or hepatologic toxicities and further includes gastric and intestinal ulceration, disturbance in platelet function, renal injury, nephritis, vasomotor rhinitis with profuse watery secretions, angioneurotic edema, generalized urticaria, and bronchial asthma to laryngeal edema and bronchoconstriction, hypotension, and shock.","The term “agent acting on the partitioning of glucose between the liver and peripheral tissues” refers to a compound or polypeptide of the invention that modulates the partitioning of glucose between the liver and the peripheral tissues as previously described.","Preferably, the agent increases or decreases blood glucose levels.","Preferably, the agent increases or decreases oxidation of glucose, preferably by the muscle.","Preferably the agent decreases or increases the body weight of individuals or is used to treat or prevent an metabolic-related disease or disorder such as obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM, Type II diabetes), Insulin dependent diabetes mellitus (IDDM, Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.","Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.","The terms “response to an agent acting on the partitioning of glucose between the liver and peripheral tissues” refer to drug efficacy, including but not limited to, ability to metabolize a compound, ability to convert a pro-drug to an active drug, and the pharmacokinetics (absorption, distribution, elimination) and the pharmacodynamics (receptor-related) of a drug in an individual.","The terms “side effects to an agent acting on the partitioning of glucose between the liver and peripheral tissues” refer to adverse effects of therapy resulting from extensions of the principal pharmacological action of the drug or to idiosyncratic adverse reactions resulting from an interaction of the drug with unique host factors.","“Side effects to an agent acting on the partitioning of dietary lipids between the liver and peripheral tissues” can include, but are not limited to, adverse reactions such as dermatologic, hematologic or hepatologic toxicities and further includes gastric and intestinal ulceration, disturbance in platelet function, renal injury, nephritis, vasomotor rhinitis with profuse watery secretions, angioneurotic edema, generalized urticaria, and bronchial asthma to laryngeal edema and bronchoconstriction, hypotension, and shock.","The term “agent acting on the partitioning of dietary lipids between the liver and peripheral tissues” refers to a compound or polypeptide of the invention that modulates the partitioning of dietary lipids between the liver and the peripheral tissues as previously described.","Preferably, the agent increases or decreases the oxidation of dietary lipids, preferably free fatty acids (FFA) by the muscle.","Preferably the agent decreases or increases the body weight of individuals or is used to treat or prevent an metabolic-related disease or disorder such as obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM, Type II diabetes), Insulin dependent diabetes mellitus (IDDM, Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.","Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.","The terms “response to an agent acting on the partitioning of dietary lipids between the liver and peripheral tissues” refer to drug efficacy, including but not limited to, ability to metabolize a compound, ability to convert a pro-drug to an active drug, and the pharmacokinetics (absorption, distribution, elimination) and the pharmacodynamics (receptor-related) of a drug in an individual.","The terms “side effects to an agent acting on the partitioning of dietary lipids between the liver and peripheral tissues” refer to adverse effects of therapy resulting from extensions of the principal pharmacological action of the drug or to idiosyncratic adverse reactions resulting from an interaction of the drug with unique host factors.","“Side effects to an agent acting on the partitioning of dietary lipids between the liver and peripheral tissues” can include, but are not limited to, adverse reactions such as dermatologic, hematologic or hepatologic toxicities and further includes gastric and intestinal ulceration, disturbance in platelet function, renal injury, nephritis, vasomotor rhinitis with profuse watery secretions, angioneurotic edema, generalized urticaria, and bronchial asthma to laryngeal edema and bronchoconstriction, hypotension, and shock.","The term “agent acting on the partitioning of endogenous lipids between the liver and peripheral tissues” refers to a compound or polypeptide of the invention that modulates the partitioning of endogenous lipids between the liver and the peripheral tissues as previously described.","Preferably, the agent increases or decreases the oxidation of endogenous lipids, preferably free fatty acids (FFA) by the muscle.","Preferably the agent decreases or increases the body weight of individuals or is used to treat or prevent an metabolic-related disease or disorder such as obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM, Type II diabetes), Insulin dependent diabetes mellitus (IDDM, Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.","Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.","The terms “response to an agent acting on the partitioning of endogenous lipids between the liver and peripheral tissues” refer to drug efficacy, including but not limited to, ability to metabolize a compound, ability to convert a pro-drug to an active drug, and the pharmacokinetics (absorption, distribution, elimination) and the pharmacodynamics (receptor-related) of a drug in an individual.","The terms “side effects to an agent acting on the partitioning of endogenous lipids between the liver and peripheral tissues” refer to adverse effects of therapy resulting from extensions of the principal pharmacological action of the drug or to idiosyncratic adverse reactions resulting from an interaction of the drug with unique host factors.","“Side effects to an agent acting on the partitioning of endogenous lipids between the liver and peripheral tissues” can include, but are not limited to, adverse reactions such as dermatologic, hematologic or hepatologic toxicities and further includes gastric and intestinal ulceration, disturbance in platelet function, renal injury, nephritis, vasomotor rhinitis with profuse watery secretions, angioneurotic edema, generalized urticaria, and bronchial asthma to laryngeal edema and bronchoconstriction, hypotension, and shock.","The term “GSSP4-related diseases and disorders” as used herein refers to any disease or disorder comprising an aberrant functioning of a GSSP4, or which could be treated or prevented by modulating GSSP4 levels or activity.","“Aberrant functioning of a GSSP4” includes, but is not limited to, aberrant levels of expression of a GSSP4 polypeptide (either increased or decreased, but preferably decreased), aberrant activity of a GSSP4 polypeptide (either increased or decreased), and aberrant interactions with ligands or binding partners (either increased or decreased).","By “aberrant” is meant a change from the type, or level of activity seen in normal cells, tissues, or patients, or seen previously in the cell, tissue, or patient prior to the onset of the illness.","In preferred embodiments, these GSSP4-related diseases and disorders include obesity and the metabolic-related diseases and disorders described previously.","The term “cosmetic treatments” is meant to include treatments with compounds or polypeptides of the invention that increase or decrease the body mass of an individual where the individual is not clinically obese or clinically underweight as defined previously.","Thus, these individuals have a body mass index (BMI) below the cut-off for clinical obesity (e.g.","below 30 kg/m2) and above the cut-off for clinical thinness (e.g.","above 18.5 kg/m2).","In addition, these individuals are preferably healthy (e.g.","do not have an metabolic-related disease or disorder of the invention).","“Cosmetic treatments” are also meant to encompass, in some circumstances, more localized increases in adipose tissue, for example, gains or losses specifically around the waist or hips, or around the hips and thighs, for example.","These localized gains or losses of adipose tissue can be identified by increases or decreases in waist or hip size, for example.","The term “prevent” or “preventing” as used herein refers to administering a compound prior to the onset of clinical symptoms of a disease or condition so as to prevent a physical manifestation of aberrations associated with obesity or insulin resistance to some extent.","The term “prevent” or “preventing” does not mean the result is necessarily absolute, but rather effective for providing some degree of prevention or amelioration of the progression of the metabolic or GSSP4-related disorder (i.e., provide protective effects), amelioration of the symptoms of the disorder, and amelioration of the reoccurrence of the metabolic or GSSP4-related disorder.","The term “treat” or “treating” as used herein refers to administering a compound after the onset of clinical symptoms.","The term “treat” or “treating” means to ameliorate, alleviate symptoms, eliminate the causation of the symptoms either on a temporary or permanent basis, or to prevent or slow the appearance of symptoms of the named disorder or condition.","The term “in need of treatment” as used herein refers to a judgment made by a caregiver (e.g.","physician, nurse, nurse practitioner, etc in the case of humans; veterinarian in the case of animals, including non-human mammals) that an individual or animal requires or will benefit from treatment.","This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, but that include the knowledge that the individual or animal is ill, or will be ill, as the result of a condition that is treatable by the compounds of the invention.","The term “perceives a need for treatment” refers to a sub-clinical determination that an individual desires to reduce weight for cosmetic reasons as discussed under “cosmetic treatment” above.","The term “perceives a need for treatment” in other embodiments can refer to the decision that an owner of an animal makes for cosmetic treatment of the animal.","The term “patient” or “individual” as used herein refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans.","The term may specify male or female or both, or exclude male or female.","The term “non-human animal” refers to any non-human vertebrate, including birds and more usually mammals, preferably primates, animals such as swine, goats, sheep, donkeys, horses, cats, dogs, rabbits or rodents, more preferably rats or mice.","Both the terms “animal” and “mammal” expressly embrace human subjects unless preceded with the term “non-human”.","The inventors believe GSSP4 polypeptides are able to significantly reduce the postprandial response of glucose in an individual, particularly following a high fat/high carbohydrate meal, while not effecting the levels of insulin.","Further, GSSP4 polypeptides of the invention are believed to modulate weight gain, particularly in individuals are fed a high fat/high carbohydrate diet.","The instant invention encompasses the use of GSSP4 polypeptides in the modulation of glucose as an important new tool to control energy homeostasis and glucose regulation.","The instant invention encompasses the use of GSSP4 polypeptides in the partitioning of glucose as an important new tool to control energy homeostasis and glucose regulation.","The instant invention encompasses the use of GSSP4 polypeptides in the partitioning of lipids as an important new tool to control energy homeostasis and lipid regulation.","The instant invention encompasses the use of GSSP4 polypeptides in the partitioning of dietary lipids as an important new tool to control energy homeostasis and lipid regulation.","The instant invention encompasses the use of GSSP4 polypeptides in the partitioning of endogenous lipids as an important new tool to control energy homeostasis and lipid regulation.","PREFERRED EMBODIMENTS OF THE INVENTION I. GSSP4 Polypeptides of the Invention GSSP4 polypeptides that have measurable activity in vitro and in vivo have been identified.","These activities include, but are not limited to, reduction of the postprandial response of plasma free fatty acids, glucose, and triglycerides, particularly in mice fed a high fat/carbohydrate meal, increase in muscle free fatty acid oxidation in vitro and ex vivo, and sustained weight loss in mice on a high fat/carbohydrate diet.","Other assays for GSSP4 polypeptide activity in vitro and in vivo are also provided (Examples 2-13, for example), and equivalent assays can be designed by those with skill in the art.","The term “obesity-related” or “metabolic-related” activity as used herein refers to at least one, and preferably all, of the activities described herein for GSSP4 polypeptides.","Assays for the determination of these activities are provided herein, and equivalent assays can be designed by those with ordinary skill in the art.","The term “metabolic-related activity” as used herein refers to at least one, and preferably all, of the activities described herein for GSSP4 polypeptides.","Assays for the determination of these activities are provided herein, known in the art, or can be designed by those with ordinary skill in the art.","Optionally, “metabolic-related activity” can be selected from the group consisting of control of blood glucose, partitioning of glucose, glucose metabolism, lipid partitioning, lipid metabolism, and insulin-like activity, or an activity within one of these categories.","Optionally, “obesity-related” activity can be selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity, or an activity within one of these categories.","By “lipid partitioning” activity is meant the ability to effect the location of dietary lipids among the major tissue groups including, adipose tissue, liver, and muscle.","The inventors have shown that GSSP4 polypeptides of the invention play a role in the partitioning of lipids to the muscle, liver or adipose tissue.","By “lipid metabolism” activity is meant the ability to influence the metabolism of lipids.","The inventors have shown that GSSP4 polypeptides of the invention have the ability to affect the level of free fatty acids in the plasma as well as to increase the metabolism of lipids in the muscle through free fatty acid oxidation experiments (Examples 4, 8, 10, 11, and 12) and to transiently affect the levels of triglycerides in the plasma and the muscle (Examples 7, 10, and 13).","By “insulin-like” activity is meant the ability of GSSP4 polypeptides to modulate the levels of glucose in the plasma.","The inventors have found that GSSP4 polypeptides do not significantly impact insulin levels but do impact glucose levels similarly to the effects of insulin (Examples 9 & 10).","These effects are not seen in the presence of the intact (full-length) GSSP4 polypeptide or are significantly greater in the presence of the GSSP4 polypeptides compared with the full-length GSSP4 polypeptide.","By “intact” or “full-length” GSSP4 polypeptide as used herein is meant the full length polypeptide sequence of the GSSP4 polypeptide of SEQ ID NO:3, from the N-terminal methionine to the C-terminal stop codon.","Preferred GSSP4 polypeptides have a biological activity described herein, and as they would be useful in making antibodies, diagnostic assays, etc.","As a further preferred embodiment, GSSP4 polypeptides which allow a rise in plasma glucose to not more than 190 mg/dl, particularly following consumption of food, or which prevent a drop in serum glucose to not less than 70 mg/dl, particularly following consumption of food.","As a further preferred embodiment, GSSP4 polypeptides which allow a rise in plasma fatty acids to not more than 420 mg/dl, particularly following consumption of food, or which prevent a drop in serum fatty acids to not less than 190 mg/dl, particularly following consumption of food.","By “significantly” as used herein is meant statistically significant as it is typically determined by those with ordinary skill in the art.","For example, data are typically calculated as a mean±SEM, and a p-value≦0.05 is considered statistically significant.","Statistical analysis is typically done using either the unpaired Student's t test or the paired Student's t test, as appropriate in each study.","Representative “metabolic-related assays” are provided in Examples below.","These assays include, but are not limited to, in vivo and in vitro methods of measuring the postprandial response, methods of measuring glucose uptake, glucose oxidation, glucose concentration, lipid concentration, free fatty acid levels, fatty acid oxidation and methods of measuring weight modulation.","In preferred embodiments, the post-prandial response is measured in non-human animals, preferably rodents.","In preferred embodiments physiologic parameters are measured including, but not limited to, levels of glucose, fatty acids, insulin, and leptin.","In other preferred embodiments, free fatty acid oxidation is measured in cells in vitro or ex vivo, preferably in muscle cells or tissue of non-human animals, preferably rodents.","In yet other preferred embodiments weight modulation is measured in human or non-human animals, preferably rodents (rats or mice), primates, canines, felines or procines on a high fat/carbohydrate diet.","Optionally, “metabolic-related activity” includes other activities not specifically identified herein.","In general, “measurable parameters” relating to obesity and the field of metabolic research can be selected from the group consisting of free fatty acid levels, free fatty acid oxidation, triglyceride levels, glucose levels, insulin levels, leptin levels, food intake, and body weight.","In these metabolic-related assays, preferred GSSP4 polypeptides or polynucleotides or both would cause a significant change in at least one of the measurable parameters selected from the group consisting of post-prandial lipemia, free fatty acid levels, triglyceride levels, glucose levels, glucose oxidation, glucose uptake, free fatty acid oxidation, and weight.","Alternatively, preferred GSSP4 polypeptides or polynucleotides or both would have a significant change in at least one of the measurable parameters selected from the group consisting of an increase in blood fatty acid levels (FFA), an decrease in blood glucose levels, a decrease in insulin levels, an increase in glucose oxidation and an increase in FFA oxidation.","The invention is drawn, inter alia, to isolated, purified or recombinant GSSP4 polypeptides.","GSSP4 polypeptides of the invention are useful for treating or preventing insulin resistance, and reducing body weight or increasing body weight (using antagonists of GSSP4 polypeptides) either as a cosmetic treatment or for treatment or prevention of metabolic-related diseases and disorders.","GSSP4 polypeptides are also useful inter alia in screening assays for agonists or antagonists of GSSP4 polypeptide activity, for raising GSSP4 polypeptide-specific antibodies, and in diagnostic assays.","The GSSP4 polypeptides of the present invention are preferably provided in an isolated form, and may be partially or substantially purified.","A recombinantly produced version of any one of the GSSP4 polypeptides can be substantially purified by the one-step method described by Smith et al.","((1988) Gene 67(1):31-40) or by the methods described herein or known in the art.","Polypeptides of the invention also can be purified from natural or recombinant sources using antibodies directed against the polypeptides of the invention by methods known in the art of protein purification.","Preparations of GSSP4 polypeptides of the invention involving a partial purification of or selection for the GSSP4 polypeptides are also specifically contemplated.","These crude preparations are envisioned to be the result of the concentration of cells expressing GSSP4 polypeptides with perhaps a few additional purification steps, but prior to complete purification of the polypeptides.","The cells expressing GSSP4 polypeptides are present in a pellet, they are lysed, or the crude polypeptide is lyophilized, for example.","GSSP4 polypeptides can be any integer in length from at least 6 consecutive amino acids to 1 amino acids less than a full length GSSP4 polypeptide of SEQ ID NO:4.Thus, for SEQ ID NO:4, a GSSP4 polypeptide can be: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105 consecutive amino acids.","Each GSSP4 polypeptide as described above can be further specified in terms of its N-terminal and C-terminal positions.","For example, every combination of a N-terminal and C-terminal position that polypeptides of from 6 contiguous amino acids to 1 amino acids less than the full length GSSP4 polypeptide could occupy, on any given intact and contiguous full length GSSP4 polypeptide sequence are included in the present invention.","Thus, a 6 consecutive amino acid fragment could occupy positions selected from the group consisting of 1-6, 2-7, 3-8, 4-9, 5-10, 6-11, 7-12, 8-13, 9-14, 10-15, 11-16, 12-17, 13-18, 14-19, 15-20, 16-21, 17-22, 18-23, 19-24, 20-25, 21-26, 22-27, 23-28, 24-29, 25-30, 26-31, 27-32, 28-33, 29-34, 30-35, 31-36, 32-37, 33-38, 34-39, 35-40, 36-41, 37-42, 38-43, 39-44, 40-45, 41-46, 42-47, 43-48, 44-49, 45-50, 46-51, 47-52, 48-53, 49-54, 50-55, 51-56, 52-57, 53-58, 54-59, 55-60, 56-61, 57-62, 58-63, 59-64, 60-65, 61-66, 62-67, 63-68, 64-69, 65-70, 66-71, 67-72, 68-73, 69-74, 70-75, 71-76, 72-77, 73-78, 74-79, 75-80, 76-81, 77-82, 78-83, 79-84, 80-85, 81-86, 82-87, 83-88, 84-89, 85-90, 86-91, 87-92, 88-93, 89-94, 90-95, 91-96, 92-97, 93-98, 94-99, 95-100, 96-101, 97-102, 98-103, 99-104, and 100-105, of a 105 consecutive amino acid fragment.","Similarly, the positions occupied by all the other fragments of sizes between 6 amino acids and 105 amino acids of SEQ ID NO:3 are included in the present invention and can also be immediately envisaged based on these two examples and therefore, are not individually listed solely for the purpose of not unnecessarily lengthening the specification.","Furthermore, the positions occupied by fragments of 6 to 105 consecutive amino acids of SEQ ID NO:3 are included in the present invention and can also be immediately envisaged based on these two examples and therefore are not individually listed solely for the purpose of not unnecessarily lengthening the specification.","In addition, the positions occupied by fragments of 6 consecutive amino acids to 1 amino acid less than any other full length GSSP4 polypeptide can also be envisaged based on these two examples and therefore are not individually listed solely for the purpose of not unnecessarily lengthening the specification.","The GSSP4 polypeptides of the present invention may alternatively be described by the formula “n to c” (inclusive); where “n” equals the N-terminal most amino acid position (as defined by the sequence listing) and “c” equals the C-terminal most amino acid position (as defined by the sequence listing) of the polypeptide; and further where “n” equals an integer between 1 and 99; and where “c” equals an integer between 7 and 105, the number of amino acids of the full length polypeptide sequence; and where “n” is an integer smaller then “c” by at least 6.Therefore, for the sequences provided in SEQ ID NO:3, “n” is any integer selected from the list consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, and 99; and “c” is any integer selected from the group consisting of: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, and 105.Every combination of “n” and “c” positions are included as specific embodiments of the invention.","Moreover, the formula “n” to “c” may be modified as “n1-n2” to “c1-c2”, wherein “n1-n2” and “c1-c2” represent positional ranges selected from any two integers above which represent amino acid positions of the sequence listing.","Alternative formulas include “n1-n2” to “c” and “n” to “c1-c2”.","These specific embodiments, and other polypeptide and polynucleotide fragment embodiments described herein may be modified as being “at least”, “equal to”, “equal to or less than”, “less than”, “at least ______ but not greater than ______” or “from ______ to ______”.","a specified size or specified N-terminal and/or C-terminal positions.","It is noted that all ranges used to describe any embodiment of the present invention are inclusive unless specifically set forth otherwise.","The present invention also provides for the exclusion of any individual fragment specified by N-terminal and C-terminal positions or of any fragment specified by size in amino acid residues as described above.","In addition, any number of fragments specified by N-terminal and C-terminal positions or by size in amino acid residues as described above may be excluded as individual species.","Further, any number of fragments specified by N-terminal and C-terminal positions or by size in amino acid residues as described above may make up a polypeptide fragment in any combination and may optionally include non-GSSP4 polypeptide sequence as well.","The term “GSSP4 fragment” as used herein refers to fragments of a full-length GSSP4 polypeptides that comprise at least 6 and any other integer number of amino acids up to 104 of the full-length GSSP4 polypeptide (defined above).","GSSP4 polypeptides of the invention include variants, fragments, analogs and derivatives of the GSSP4 polypeptides described above, including modified GSSP4 polypeptides.","Variants It will be recognized by one of ordinary skill in the art that some amino acids of the GSSP4 polypeptide sequences of the present invention can be varied without significant effect on the structure or function of the proteins; there will be critical amino acids in the sequence that determine activity.","Thus, the invention further includes variants of GSSP4 polypeptides that have metabolic-related activity as described above.","Such variants include GSSP4 polypeptide sequences with one or more amino acid deletions, insertions, inversions, repeats, and substitutions either from natural mutations or human manipulation selected according to general rules known in the art so as to have little effect on activity.","Guidance concerning how to make phenotypically silent amino acid substitutions is provided below.","There are two main approaches for studying the tolerance of an amino acid sequence to change (see, Bowie, et al.","(1990) Science, 247, 1306-10).","The first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection.","The second approach uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene and selections or screens to identify sequences that maintain functionality.","These studies have revealed that proteins are surprisingly tolerant of amino acid substitutions and indicate which amino acid changes are likely to be permissive at a certain position of the protein.","For example, most buried amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved.","Other such phenotypically silent substitutions are described by Bowie et al.","(supra) and the references cited therein.","In the case of an amino acid substitution in the amino acid sequence of a polypeptide according to the invention, one or several amino acids can be replaced by “equivalent” amino acids.","The expression “equivalent” amino acid is used herein to designate any amino acid that may be substituted for one of the amino acids having similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged.","In particular embodiments, conservative substitutions of interest are shown in Table 1 under the heading of preferred substitutions.","If such substitutions result in a change in biological activity, then more substantial changes, or as further described below in reference to amino acid classes, are introduced and the products screened.","TABLE 1 Original Residue Exemplary Substitutions Preferred Substitutions Ala (A) val; leu; ile val Arg (R) lys; gin; asn lys Asn (N) gin; his; lys; arg gin Asp (D) glu glu Cys (C) ser ser Gin (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His (H) asn; gin; lys; arg arg Ile (I) leu; val; met; ala; phe; norleucine leu Leu (L) norleucine; ile; val; met; ala; phe ile Lys (K) arg; gin; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; ala; norleucine leu Substantial modifications in function or immunological identity of the GSSP4 polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.","Naturally occurring residues are divided into groups based on common side-chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn, gln, his, lys, arg; (5) residues that influence chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.","Non-conservative substitutions will entail exchanging a member of one of these classes for another class.","Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.","The variations can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis.","Site-directed mutagenesis [Carter et al., Nucl.","Acids Res., 13:4331 (1986); Zoller et al., Nucl.","Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)], restriction selection mutagenesis [Wells et al., Philos.","Trans.","R. Soc.","London SerA, 317:415 (1986)] or other known techniques can be performed on the cloned DNA to produce the GSSP4 variant DNA.","Amino acids in the GSSP4 polypeptide sequences of the invention that are essential for function can also be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham, et al.","(1989) Science 244(4908):1081-5).","The latter procedure introduces single alanine mutations at every residue in the molecule.","The resulting mutant molecules are then tested for metabolic-related activity using assays as described above.","Of special interest are substitutions of charged amino acids with other charged or neutral amino acids that may produce proteins with highly desirable improved characteristics, such as less aggregation.","Aggregation may not only reduce activity but also be problematic when preparing pharmaceutical or physiologically acceptable formulations, because aggregates can be immunogenic (see, e.g., Pinckard, et al., (1967) Clin.","Exp.","Immunol 2:331-340; Robbins, et al., (1987) Diabetes July;36(7):838-41; and Cleland, et al., (1993) Crit Rev Ther Drug Carrier Syst.","10(4):307-77).","Thus, the fragment, derivative, analog, or homolog of the GSSP4 polypeptides of the present invention may be, for example: (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code (i.e.","may be a non-naturally occurring amino acid); or (ii) one in which one or more of the amino acid residues includes a substituent group; or (iii) one in which the GSSP4 polypeptide is fused with another compound, such as a compound to increase the half-life of the fragment (for example, polyethylene glycol); or (iv) one in which the additional amino acids are fused to the above form of the fragment, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the above form of the fragment or a pro-protein sequence.","Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.","A further embodiment of the invention relates to a polypeptide which comprises the amino acid sequence of GSSP4 polypeptide having an amino acid sequence which contains at least one conservative amino acid substitution, but not more than 50 conservative amino acid substitutions, not more than 40 conservative amino acid substitutions, not more than 30 conservative amino acid substitutions, and not more than 20 conservative amino acid substitutions.","Also provided are polypeptides which comprise the amino acid sequence of a GSSP4 fragment, having at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid substitutions.","Another specific embodiment of a modified GSSP4 polypeptide of the invention is a polypeptide that is resistant to proteolysis, for example a GSSP4 polypeptide in which a —CONH— peptide bond is modified and replaced by one or more of the following: a (CH2NH) reduced bond; a (NHCO) retro inverso bond; a (CH2—O) methylene-oxy bond; a (CH2—S) thiomethylene bond; a (CH2CH2) carba bond; a (CO—CH2) cetomethylene bond; a (CHOH—CH2) hydroxyethylene bond); a (N—N) bound; a E-alcene bond; or a —CH═CH— bond.","Thus, the invention also encompasses a GSSP4 polypeptide or a variant thereof in which at least one peptide bond has been modified as described above.","In addition, amino acids have chirality within the body of either L or D. In some embodiments it is preferable to alter the chirality of the amino acids in the GSSP4 polypeptides of the invention in order to extend half-life within the body.","Thus, in some embodiments, one or more of the amino acids are preferably in the L configuration.","In other embodiments, one or more of the amino acids are preferably in the D configuration.","Percent Identity The polypeptides of the present invention also include polypeptides having an amino acid sequence at least 50% identical, at least 60% identical, or 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a GSSP4 polypeptide as described above.","By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a GSSP4 polypeptide amino acid sequence is meant that the amino acid sequence is identical to the GSSP4 polypeptide sequence except that it may include up to five amino acid alterations per each 100 amino acids of the GSSP4 polypeptide amino acid sequence.","The reference sequence is the GSSP4 polypeptide with a sequence corresponding to the sequences provided in SEQ ID NO:3.Thus, to obtain a polypeptide having an amino acid sequence at least 95% identical to a GSSP4 polypeptide amino acid sequence, up to 5% (5 of 100) of the amino acid residues in the sequence may be inserted, deleted, or substituted with another amino acid compared with the GSSP4 polypeptide sequence.","These alterations may occur at the amino or carboxy termini or anywhere between those terminal positions, interspersed either individually among residues in the sequence or in one or more contiguous groups within the sequence.","As a practical matter, whether any particular polypeptide is a percentage identical to a GSSP4 polypeptide can be determined conventionally using known computer programs.","Such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, (1988) Proc Natl Acad Sci USA April;85(8):2444-8; Altschul et al., (1990) J. Mol.","Biol.","215(3):403-410; Thompson et al., (1994) Nucleic Acids Res.","22(2):4673-4680; Higgins et al., (1996) Meth.","Enzymol.","266:383-402; Altschul et al., (1997) Nuc.","Acids Res.","25:3389-3402; Altschul et al., (1993) Nature Genetics 3:266-272).","In a particularly preferred embodiment, protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool (“BLAST”), which is well known in the art (See, e.g., Karlin and Altschul (1990) Proc Natl Acad Sci USA March;87(6):2264-8; Altschul et al., 1990, 1993, 1997, all supra).","In particular, five specific BLAST programs are used to perform the following tasks: (1) BLASTP and BLAST3 compare an amino acid query sequence against a protein sequence database; (2) BLASTN compares a nucleotide query sequence against a nucleotide sequence database; (3) BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database; (4) TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands); and (5) TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.","The BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as “high-scoring segment pairs,” between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database.","High-scoring segment pairs are preferably identified (i.e., aligned) by means of a scoring matrix, many of which are known in the art.","Preferably, the scoring matrix used is the BLOSUM62 matrix (see, Gonnet et al., (1992) Science June 5;256(5062): 1443-5; Henikoff and Henikoff (1993) Proteins September;17(1):49-61).","Less preferably, the PAM or PAM250 matrices may also be used (See, e.g., Schwartz and Dayhoff, eds, (1978) Matrices for Detecting Distance Relationships: Atlas of Protein Sequence and Structure, Washington: National Biomedical Research Foundation).","The BLAST programs evaluate the statistical significance of all high-scoring segment pairs identified, and preferably selects those segments which satisfy a user-specified threshold of significance, such as a user-specified percent homology.","Preferably, the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula of Karlin (See, e.g., Karlin and Altschul, (1990) Proc Natl Acad Sci USA March;87(6):2264-8).","The BLAST programs may be used with the default parameters or with modified parameters provided by the user.","Preferably, the parameters are default parameters.","A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al.","(1990) Comp.","App.","Biosci.","6:237-245.In a sequence alignment the query and subject sequences are both amino acid sequences.","The result of said global sequence alignment is in percent identity.","Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group=25 Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=247 or the length of the subject amino acid sequence, whichever is shorter.","If the subject sequence is shorter than the query sequence due to N-or C-terminal deletions, not because of internal deletions, the results, in percent identity, must be manually corrected because the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity.","For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, that are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence.","Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment.","This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score.","This final percent identity score is what is used for the purposes of the present invention.","Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score.","That is, only query amino acid residues outside the farthest N- and C-terminal residues of the subject sequence.","For example, a 90 amino acid residue subject sequence is aligned with a 100-residue query sequence to determine percent identity.","The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not match/align with the first residues at the N-terminus.","The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program.","If the remaining 90 residues are perfectly matched the final percent identity would be 90%.","In another example, a 90-residue subject sequence is compared with a 100-residue query sequence.","This time the deletions are internal so there are no residues at the N- or C-termini of the subject sequence, which are not matched/aligned with the query.","In this case, the percent identity calculated by FASTDB is not manually corrected.","Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected.","No other manual corrections are made for the purposes of the present invention.","Production Note, throughout the disclosure, wherever GSSP4 polypeptides are discussed, GSSP4 fragments are specifically intended to be included as a preferred subset of GSSP4 polypeptides.","Specific production methods are addressed in detail in sections III, IV, V, and VI of the present specification.","In brief, GSSP4 polypeptides are preferably isolated from mammalian tissue samples, preferably human samples, or expressed from mammalian genes, preferably human genes, in mammalian cells, preferably human cells.","The GSSP4 polypeptides of the invention can be made using expression methods known in the art.","The polynucleotides encoding the desired polypeptides of the invention, including fragments thereof, are ligated into an expression vector suitable for the particular host used.","Both eukaryotic and prokaryotic host systems can be used in forming recombinant polypeptides.","The polypeptides are isolated from lysed cells or from the culture medium and purified to the extent needed for its intended use.","In addition, shorter protein fragments may be produced by chemical synthesis.","Purification is by techniques known in the art, for example, differential extraction, salt fractionation, chromatography, centrifugation, and the like.","Nucleotides comprising the coding sequence of the polynucleotides of the invention, or fragments thereof, are cloned into expression vectors by anyone skilled in the art.","Preferred expression vectors include eukaryotic and prokaryotic expression vectors.","Further preferred is the pGEX-3X expression vector.","Purification of GSSP4 polypeptides may be facilitated by recombinant fusion of a heterologous peptide to the C-or N-terminus of the GSSP4 polypeptides.","Preferred fusion polypeptides expressed include heterologous polypeptides containing a His-tag added to the C-terminus, a glutathione-S-transferase (GST) tag and a factor Xa protease digestion site.","A preferred method of making the polypeptides of the invention includes a method comprising the following steps: Escherichia coli cells, preferably BL21, and preferably comprising the pGEX-3X vector containing polynucleotides of the invention containing a His tag added to the C-terminus, GST tag and Xa protease site, are grown to subconfluence, preferably absorbance of 0.8, and induced with isopropyl beta-D-thiogalactoside.","Cells are pelleted, washed and lysed in buffer, preferably in buffer containing guanidine hydrochloride.","Polypeptides are allowed to bind to nickel, preferably Ni-NTA containing beads, and are washed with buffers, preferably buffers containing urea.","Polypeptide bound to nickel are equilibrated with buffer, preferably buffer containing sodium chloride and calcium chloride, preferably at pH 7.5.Polypeptides bound to Nickel are digested, ie.","treated with buffer comprising protease, preferably protease factor Xa.","Digestion is preferably carried out at room temperature for 12 to 20 hours.","The cleaved GST tag, if any, is washed away with buffer, preferably buffer with urea, preferably at pH 5.9.Polypeptides of the invention are eluted with buffer, preferably buffer with urea, preferably at pH 4.5.Polypeptides of the invention are refolded, preferably by methods comprising the following steps: Polypeptides are diluted in buffer common in the art, preferably to a concentration of 100 microgram/ml, preferably in buffer containing urea at pH 4.5.Polypeptides are dialyzed against buffer, preferably dialysis buffer comprising 4M urea, 5 mM cysteine, 0.02% Tween-20, 10% glycerol, 10 mM Tris, 150 mM sodium chloride, and 100 mM NaH2PO4, preferably at pH 8.3.Dialysis buffer comprising 2 M urea is used to replace the initial dialysis buffer, and dialysis buffer is replaced at least 1, 2, or 3 times over at least 1, 2, 3, 4, 5, or 6 days.","The refolded polypeptide is desalted by any method known in the art, preferably using a spin column.","Polypeptides of the invention are further purified by methods known in the art.","Preferably polpeptides are purified with reverse phase HPLC.","Polypeptides are preferably eluted with 0.08% trifluoroacetic acid and a 10-50% acetonitrile gradient, and elution is monitored preferably at 206 nm.","Acetonitrile and trifluoroacetic acid are removed for the compositions comprising the purified polypeptide, preferably by evaporation by lyophilization.","Further examples of methods useful for the expression or purification of polypeptides of the invention are found described in Methods in Enzymology.","The nucleic acid encoding a GSSP4 fragment can be obtained by PCR from a vector containing the GSSP4 nucleotide sequence using oligonucleotide primers complementary to the desired GSSP4 cDNA and containing restriction endonuclease sequences.","Transfection of a GSSP4 fragment-expressing vector into mouse NIH 3T3 cells is one embodiment of introducing polynucleotides into host cells.","Introduction of a polynucleotide encoding a polypeptide into a host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods.","Such methods are described in many standard laboratory manuals, such as Davis et al.","((1986) Methods in Molecular Biology, Elsevier Science Publishing Co., Inc., Amsterdam).","It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.","A polypeptide of this invention (i.e.","a GSSP4 fragment) can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography.","Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.","Polypeptides of the present invention, and preferably the secreted form, can also be recovered from: products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells.","Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated.","Preferably the polypeptides of the invention are non-glycosylated.","In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.","In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with the polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polynucleotides.","For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequences via homologous recombination, see, e.g., U.S. Pat.","No.","5,641,670, issued Jun.","24, 1997; International Publication No.","WO 96/29411, published Sep. 26, 1996; International Publication No.","WO 94/12650, published Aug. 4, 1994; Koller et al., (1989) Proc Natl Acad Sci USA November;86(22):8932-5; Koller et al., (1989) Proc Natl Acad Sci USA November;86(22):8927-31; and Zijlstra et al.","(1989) Nature November 23;342(6248):435-8; the disclosures of each of which are incorporated by reference in their entireties).","Modifications In addition, polypeptides of the invention can be chemically synthesized using techniques known in the art (See, e.g., Creighton, 1983 Proteins.","New York, N.Y.: W. H. Freeman and Company; and Hunkapiller et al., (1984) Nature July 12-18;310(5973): 105-11).","For example, a relative short fragment of the invention can be synthesized by use of a peptide synthesizer.","Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the fragment sequence.","Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoroamino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general.","Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).","The invention encompasses polypeptide fragments which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc.","Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.","Additional post-translational modifications encompassed by the invention include, for example, N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression.","The polypeptide fragments may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the polypeptide.","Also provided by the invention are chemically modified derivatives of the polypeptides of the invention that may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity.","See U.S. Pat.","No.","4,179,337.The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like.","The polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.","The polymer may be of any molecular weight, and may be branched or unbranched.","For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term “about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing.","Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).","The polyethylene glycol molecules (or other chemical moieties) should be attached to the polypeptide with consideration of effects on functional or antigenic domains of the polypeptide.","There are a number of attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al.","(1992) Exp Hematol.","September;20(8):1028-35, reporting pegylation of GM-CSF using tresyl chloride).","Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.","Multimers The polypeptide fragments of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers).","Accordingly, the present invention relates to monomers and multimers of the polypeptide fragments of the invention, their preparation, and compositions (preferably, pharmaceutical or physiologically acceptable compositions) containing them.","In specific embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers.","In additional embodiments, the multimers of the invention are at least dimers, at least trimers, or at least tetramers.","Multimers encompassed by the invention may be homomers or heteromers.","As used herein, the term homomer, refers to a multimer containing only polypeptides corresponding to the GSSP4 polypeptides of the invention (including polypeptide fragments, variants, splice variants, and fusion proteins corresponding to these polypeptide fragments as described herein).","These homomers may contain polypeptide fragments having identical or different amino acid sequences.","In a specific embodiment, a homomer of the invention is a multimer containing only polypeptide fragments having an identical amino acid sequence.","In another specific embodiment, a homomer of the invention is a multimer containing polypeptide fragments having different amino acid sequences.","In specific embodiments, the multimer of the invention is a homodimer (e.g., containing polypeptide fragments having identical or different amino acid sequences) or a homotrimer (e.g., containing polypeptide fragments having identical and/or different amino acid sequences).","In additional embodiments, the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.","As used herein, the term heteromer refers to a multimer containing one or more heterologous polypeptides (i.e., corresponding to different proteins or polypeptide fragments thereof) in addition to the polypeptides of the invention.","In a specific embodiment, the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer.","In additional embodiments, the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.","Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation.","Thus, in one embodiment, multimers of the invention, such as, for example, homodimers or homotrimers, are formed when polypeptides of the invention contact one another in solution.","In another embodiment, heteromultimers of the invention, such as, for example, heterotrimers or heterotetramers, are formed when polypeptides of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution.","In other embodiments, multimers of the invention are formed by covalent associations with and/or between the polypeptides of the invention.","Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (e.g., that recited in the sequence listing, or contained in the polypeptide encoded by a deposited clone).","In one instance, the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences, which interact in the native (i.e., naturally occurring) polypeptide.","In another instance, the covalent associations are the consequence of chemical or recombinant manipulation.","Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a fusion protein of the invention.","In one example, covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., U.S. Pat.","No.","5,478,925).","In a specific example, the covalent associations are between the heterologous sequence contained in an Fc fusion protein of the invention (as described herein).","In another specific example, covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from another protein that is capable of forming covalently associated multimers, such as for example, oseteoprotegerin (see, e.g., International Publication NO: WO 98/49305, the contents of which are herein incorporated by reference in its entirety).","In another embodiment, two or more polypeptides of the invention are joined through peptide linkers.","Examples include those peptide linkers described in U.S. Pat.","No.","5,073,627 (hereby incorporated by reference).","Proteins comprising multiple polypeptides of the invention separated by peptide linkers may be produced using conventional recombinant DNA technology.","Another method for preparing multimer polypeptides of the invention involves use of polypeptides of the invention fused to a leucine zipper or isoleucine zipper polypeptide sequence.","Examples of leucine zipper domains suitable for producing soluble multimeric proteins of the invention are those described in PCT application WO 94/10308, hereby incorporated by reference.","Recombinant fusion proteins comprising a polypeptide of the invention fused to a polypeptide sequence that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric fusion protein is recovered from the culture supernatant using techniques known in the art.","Trimeric polypeptides of the invention may offer the advantage of enhanced biological activity.","Preferred leucine zipper moieties and isoleucine moieties are those that preferentially form trimers.","One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al.","FEBS Letters (1994) May 16;344(2-3):191-5.and in U.S. patent application Ser.","No.","08/446,922, hereby incorporated by reference.","Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric polypeptides of the invention.","In another example, proteins of the invention are associated by interactions between Flag® & polypeptide sequence contained in fusion proteins of the invention containing Flag® polypeptide sequence.","In a further embodiment, proteins of the invention are associated by interactions between heterologous polypeptide sequence contained in Flag® fusion proteins of the invention and anti Flag® antibody.","The multimers of the invention may be generated using chemical techniques known in the art.","For example, polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., U.S. Pat.","No.","5,478,925, which is herein incorporated by reference in its entirety).","Additionally, multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., U.S. Pat.","No.","5,478,925, which is herein incorporated by reference in its entirety).","Further, polypeptides of the invention may be routinely modified by the addition of cysteine or biotin to the C-terminus or N-terminus of the polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., U.S. Pat.","No.","5,478,925, which is herein incorporated by reference in its entirety).","Additionally, at least 30 techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, e.g., U.S. Pat.","No.","5,478,925, which is herein incorporated by reference in its entirety).","Alternatively, multimers of the invention may be generated using genetic engineering techniques known in the art.","In one embodiment, polypeptides contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., U.S. Pat.","No.","5,478,925, which is herein incorporated by reference in its entirety).","In a specific embodiment, polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., U.S. Pat.","No.","5,478,925, which is herein incorporated by reference in its entirety).","In another embodiment, recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain (or hyrophobic or signal peptide) and which can be incorporated by membrane reconstitution techniques into liposomes (See, e.g., U.S. Pat.","No.","5,478,925, which is herein incorporated by reference in its entirety).","II.","GSSP4 Polynucleotides of the Invention Preferred polynucleotides are those that encode GSSP4 polypeptides of the invention.","The recombinant polynucleotides encoding GSSP4 polypeptides can be used in a variety of ways, including, but not limited to, expressing the polypeptides in recombinant cells for use in screening assays for antagonists and agonists of its activity as well as to facilitate its purification for use in a variety of ways including, but not limited to screening assays for agonists and antagonists of its activity, diagnostic screens, and raising antibodies, as well as treatment and/or prevention of metabolic-related diseases and disorders and/or to reduce body mass.","The invention relates to the polynucleotides encoding GSSP4 polypeptides and variant polypeptide fragments thereof as described herein.","These polynucleotides may be purified, isolated, and/or recombinant.","In all cases, the desired GSSP4 polynucleotides of the invention are those that encode GSSP4 polypeptides of the invention having metabolic-related activity as described and discussed herein.","Fragments A polynucleotide fragment is a polynucleotide having a sequence that entirely is the same as part, but not all, of the full length GSSP4 polypeptide or a specified GSSP4 polypeptide nucleotide sequence.","Such fragments may be “free-standing”, i.e.","not part of or fused to other polynucleotides, or they may be comprised within another non-GSSP4 (heterologous) polynucleotide of which they form a part or region.","However, several GSSP4 polynucleotide fragments may be comprised within a single polynucleotide.","The GSSP4 polynucleotides of the invention comprise from 18 consecutive bases to 18 consecutive bases less than the full length polynucleotide sequences encoding the intact GSSP4 polypeptides, for example the GSSP4 polynucleotide sequences in SEQ ID NO:1 or 2.In one aspect of this embodiment, the polynucleotide comprises at least 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, or 648 consecutive nucleotides of a polynucleotide of the present invention.","In addition to the above preferred nucleic acid sizes, further preferred nucleic acids comprise at least 18 nucleotides, wherein “at least 18” is defined as any integer between 18 and the integer representing 18 nucleotides less than the 3′ most nucleotide position of the intact GSSP4 polypeptides cDNA as set forth in SEQ ID NO: 2, or elsewhere herein.","Further included as preferred polynucleotides of the present invention are nucleic acid fragments at least 18 nucleotides in length, as described above, that are further specified in terms of their 5′ and 3′ position set forth in the sequence listing below.","For allelic and degenerate and other variants, position 1 is defined as the 5′ most nucleotide of the ORF, i.e., the nucleotide “A” of the start codon (ATG) with the remaining nucleotides numbered consecutively.","Therefore, every combination of a 5′ and 3′ nucleotide position that a polynucleotide fragment invention, at least 18 contiguous nucleotides in length, could occupy on an intact GSSP4 polypeptide polynucleotide of the present invention is included in the invention as an individual species.","The polynucleotide fragments specified by 5′ and 3′ positions can be immediately envisaged and are therefore not individually listed solely for the purpose of not unnecessarily lengthening the specification.","It is noted that the above species of polynucleotide fragments of the present invention may alternatively be described by the formula “x to y”; where “x” equals the 5′ most nucleotide position and “y” equals the 3′ most nucleotide position of the polynucleotide; and further where “x” equals an integer between 1 and the number of nucleotides of the polynucleotide sequence of the present invention minus 18, and where “y” equals an integer between 19 and the number of nucleotides of the polynucleotide sequence of the present invention minus 18 nucleotides; and where “x” is an integer smaller then “y” by at least 18.The GSSP4 polynucleotide fragments of the invention comprise from 18 consecutive bases to the full length polynucleotide sequence encoding the GSSP4 fragments described in Section II of the Preferred Embodiments of the Invention.","In one aspect of this embodiment, the polynucleotide comprises at least 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 740, 770, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100 or 2200 consecutive nucleotides of a polynucleotide of the present invention.","In addition to the above preferred nucleic acid sizes, further preferred nucleic acids comprise at least 18 nucleotides, wherein “at least 18” is defined as any integer between 18 and the integer corresponding to the 3′ most nucleotide position of a GSSP4 fragment cDNA herein.","Further included as preferred polynucleotides of the present invention are nucleic acid fragments at least 18 nucleotides in length, as described above, that are further specified in terms of their 5′ and 3′ position.","The 5′ and 3′ positions are represented by the position numbers set forth in the sequence listing below.","For allelic and degenerate and other variants, position 1 is defined as the 5′ most nucleotide of the open reading frame (ORF), i.e., the nucleotide “A” of the start codon (ATG) with the remaining nucleotides numbered consecutively.","Therefore, every combination of a 5′ and 3′ nucleotide position that a polynucleotide fragment invention, at least 18 contiguous nucleotides in length, could occupy on a GSSP4 fragment polynucleotide of the present invention is included in the invention as an individual species.","The polynucleotide fragments specified by 5′ and 3′ positions can be immediately envisaged and are therefore not individually listed solely for the purpose of not unnecessarily lengthening the specification.","It is noted that the above species of polynucleotide fragments of the present invention may alternatively be described by the formula “x to y”; where “x” equals the 5′ most nucleotide position and “y” equals the 3′ most nucleotide position of the polynucleotide; and further where “x” equals an integer between 1 and the number of nucleotides of the GSSP4 polynucleotide sequences of the present invention minus 18, and where “y” equals an integer between 9 and the number of nucleotides of the GSSP4 polynucleotide sequences of the present invention; and where “x” is an integer smaller than “y” by at least 18.Every combination of “x” and ‘y’ positions are included as specific embodiments of the invention.","Moreover, the formula “x” to “y” may be modified as “‘x1-x2” to “y1-y2’”, wherein “x1-x2” and “y1-y2” represent positional ranges selected from any two nucleotide positions of the sequence listing.","Alternative formulas include “‘x1-x2” to “y’” and “‘x” to “y1-y2’”.","These specific embodiments, and other polynucleotide fragment embodiments described herein may be modified as being “at least”, “equal to”, “equal to or less than”, “less than”, “at least ______ but not greater than ______”or “from ______ to ______”.","a specified size or specified 5′ and/or 3′ positions.","The present invention also provides for the exclusion of any species of polynucleotide fragments of the present invention specified by 5′ and 3′ positions or polynucleotides specified by size in nucleotides as described above.","Any number of fragments specified by 5′ and 3′ positions or by size in nucleotides, as described above, may be excluded.","Variants In other preferred embodiments, variants of GSSP4 polynucleotides encoding GSSP4 polypeptides are envisioned.","Variants of polynucleotides, as the term is used herein, are polynucleotides whose sequence differs from a reference polynucleotide.","A variant of a polynucleotide may be a naturally occurring variant such as a naturally occurring allelic variant, or it may be a variant that is not known to occur naturally.","Such non-naturally occurring variants of the polynucleotide may be made by mutagenesis techniques, including those applied to polynucleotides, cells or organisms.","Generally, differences are limited so that the nucleotide sequences of the reference and the variant are closely similar overall and, in many regions, identical.","Polynucleotide variants that comprise a sequence substantially different from those described above but that, due to the degeneracy of the genetic code, still encode GSSP4 polypeptides of the present invention are also specifically envisioned.","It would also be routine for one skilled in the art to generate the degenerate variants described above, for instance, to optimize codon expression for a particular host (e.g., change codons in the human mRNA to those preferred by other mammalian or bacterial host cells).","As stated above, variant polynucleotides may occur naturally, such as a natural allelic variant, or by recombinant methods.","By an “allelic variant” is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism (See, e.g., B. Lewin, (1990) Genes IV, Oxford University Press, New York).","Non-naturally occurring variants may be produced using art-known mutagenesis techniques.","Such nucleic acid variants include those produced by nucleotide substitutions, deletions, or additions.","The substitutions, deletions, or additions may involve one or more nucleotides.","Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions.","Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of GSSP4 polypeptides of the invention.","Also preferred in this regard are conservative substitutions.","Nucleotide changes present in a variant polynucleotide are preferably silent, which means that they do not alter the amino acids encoded by the polynucleotide.","However, nucleotide changes may also result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence.","In cases where the nucleotide substitutions result in one or more amino acid changes, preferred GSSP4 polypeptides include those that retain one or more metabolic-related activity as described in Section I of the Preferred Embodiments of the Invention.","By “retain the same activities” is meant that the activity measured using the polypeptide encoded by the variant GSSP4 polynucleotide in assays is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, and not more than 101%, 102%, 103%, 104%, 105%, 110%, 115%, 120% or 125% of the activity measured using a GSSP4 polypeptide described in the Examples Section herein.","By the activity being “increased” is meant that the activity measured using the polypeptide encoded by the variant GSSP4 polynucleotide in assays is at least 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 170%, 180%, 190%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 450%, or 500% of the activity measured using a GSSP4 polypeptide described in the Examples Section herein.","By the activity being “decrease” is meant that the activity measured using the polypeptide encoded by the variant GSSP4 polynucleotide in assays is decreased by at least 25%, 30%, 35%, 40%, 45%, or 50% of the activity measured using a GSSP4 polypeptide described in the Examples Section herein.","Percent Identity The present invention is further directed to nucleic acid molecules having sequences at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequences of SEQ ID NO:1, or 2 or fragments thereof that encode a polypeptide having metabolic-related activity as described in Section I of the Preferred Embodiments of the Invention.","Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequences shown in SEQ ID NO:1, or 2 or fragments thereof will encode a polypeptide having biological activity.","In fact, since degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assay.","It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having biological activity.","This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly affect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid), as further described previously in Section I of the Preferred Embodiments of the Invention.","By a polynucleotide having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the GSSP4 polypeptide.","In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted, inserted, or substituted with another nucleotide.","The query sequence may be an entire sequence or any fragment specified as described herein.","The methods of determining and defining whether any particular nucleic acid molecule or polypeptide is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be done by using known computer programs.","A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al., ((1990) Comput Appl Biosci.","July;6(3):237-45).","In a sequence alignment the query and subject sequences are both DNA sequences.","An RNA sequence can be compared by first converting U's to T's.","The result of said global sequence alignment is in percent identity.","Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter.","If the subject sequence is shorter than the query sequence because of 5′ or 3′ deletions, not because of internal deletions, a manual correction must be made to the results.","This is because the FASTDB program does not account for 5′ and 3′ truncations of the subject sequence when calculating percent identity.","For subject sequences truncated at the 5′ or 3′ ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5′ and 3′ of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence.","Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment.","This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score.","This corrected score is what is used for the purposes of the present invention.","Only nucleotides outside the 5′ and 3′ nucleotides of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.","No other manual corrections are made for the purposes of the present invention.","Fusions Further included in the present invention are polynucleotides encoding the polypeptides of the present invention that are fused in frame to the coding sequences for additional heterologous amino acid sequences.","Also included in the present invention are nucleic acids encoding polypeptides of the present invention together with additional, non-coding sequences, including for example, but not limited to non-coding 5′ and 3′ sequences, vector sequence, sequences used for purification, probing, or priming.","For example, heterologous sequences include transcribed, non-translated sequences that may play a role in transcription, and mRNA processing, for example, ribosome binding and stability of mRNA.","The heterologous sequences may alternatively comprise additional coding sequences that provide additional functionalities.","Thus, a nucleotide sequence encoding a polypeptide may be fused to a tag sequence, such as a sequence encoding a peptide that facilitates purification of the fused polypeptide.","In certain preferred embodiments of this aspect of the invention, the tag amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available.","For instance, hexa-histidine provides for convenient purification of the fusion protein (See, Gentz et al., (1989) Proc Natl Acad Sci USA February;86(3):821-4).","The “HA” tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein (See, Wilson et al., (1984) Cell 37(3):767-78).","As discussed above, other such fusion proteins include GSSP4 fragment cDNA fused to Fc at the N- or C-terminus.","III.","Recombinant Vectors of the Invention The term “vector” is used herein to designate either a circular or a linear DNA or RNA molecule, that is either double-stranded or single-stranded, and that comprises at least one polynucleotide of interest that is sought to be transferred in a cell host or in a unicellular or multicellular host organism.","The present invention relates to recombinant vectors comprising any one of the polynucleotides described herein.","The present invention encompasses a family of recombinant vectors that comprise polynucleotides encoding GSSP4 polypeptides of the invention.","In a first preferred embodiment, a recombinant vector of the invention is used to amplify the inserted polynucleotide in a suitable cell host, this polynucleotide being amplified every time that the recombinant vector replicates.","The inserted polynucleotide can be one that encodes GSSP4 polypeptides of the invention.","A second preferred embodiment of the recombinant vectors according to the invention consists of expression vectors comprising polynucleotides encoding GSSP4 polypeptides of the invention.","Within certain embodiments, expression vectors are employed to express a GSSP4 polypeptide of the invention, preferably a modified GSSP4 fragment described in the present invention, which can be then purified and, for example, be used as a treatment for metabolic-related diseases, or simply to reduce body mass of individuals.","Expression requires that appropriate signals are provided in the vectors, said signals including various regulatory elements, such as enhancers/promoters from both viral and mammalian sources, that drive expression of the genes of interest in host cells.","Dominant drug selection markers for establishing permanent, stable, cell clones expressing the products are generally included in the expression vectors of the invention, as they are elements that link expression of the drug selection markers to expression of the polypeptide.","More particularly, the present invention relates to expression vectors which include nucleic acids encoding a GSSP4 polypeptide of the invention, or a modified GSSP4 fragment as described herein, or variants or fragments thereof, under the control of a regulatory sequence selected among GSSP4 polypeptides, or alternatively under the control of an exogenous regulatory sequence.","Consequently, preferred expression vectors of the invention are selected from the group consisting of: (a) a GSSP4 fragmentregulatory sequence and driving the expression of a coding polynucleotide operably linked thereto; and (b) a GSSP4 fragment coding sequence of the invention, operably linked to regulatory sequences allowing its expression in a suitable cell host and/or host organism.","Some of the elements which can be found in the vectors of the present invention are described in further detail in the following sections.","1) General Features of the Expression Vectors of the Invention: A recombinant vector according to the invention comprises, but is not limited to, a YAC (Yeast Artificial Chromosome), a BAC (Bacterial Artificial Chromosome), a phage, a phagemid, a cosmid, a plasmid, or even a linear DNA molecule which may consist of a chromosomal, non-chromosomal, semi-synthetic or synthetic DNA.","Such a recombinant vector can comprise a transcriptional unit comprising an assembly of: (1) a genetic element or elements having a regulatory role in gene expression, for example promoters or enhancers.","Enhancers are cis-acting elements of DNA, usually from about 10 to 300 bp in length that act on the promoter to increase the transcription; (2) a structural or coding sequence which is transcribed into mRNA and eventually translated into a polypeptide, said structural or coding sequence being operably linked to the regulatory elements described in (1); and (3) appropriate transcription initiation and termination sequences.","Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell.","Alternatively, when a recombinant protein is expressed without a leader or transport sequence, it may include a N-terminal residue.","This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.","Generally, recombinant expression vectors will include origins of replication, selectable markers permitting transformation of the host cell, and a promoter derived from a highly expressed gene to direct transcription of a downstream structural sequence.","The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably a leader sequence capable of directing secretion of the translated protein into the periplasmic space or the extracellular medium.","In a specific embodiment wherein the vector is adapted for transfecting and expressing desired sequences in mammalian host cells, preferred vectors will comprise an origin of replication in the desired host, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcriptional termination sequences, and 5′-flanking non-transcribed sequences.","DNA sequences derived from the SV40 viral genome, for example SV40 origin, early promoter, enhancer, splice and polyadenylation sites may be used to provide the required non-transcribed genetic elements.","2) Regulatory Elements Promoters The suitable promoter regions used in the expression vectors of the present invention are chosen taking into account the cell host in which the heterologous gene is expressed.","The particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of directing the expression of the nucleic acid in the targeted cell.","Thus, where a human cell is targeted, it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell, such as, for example, a human or a viral promoter.","A suitable promoter may be heterologous with respect to the nucleic acid for which it controls the expression or alternatively can be endogenous to the native polynucleotide containing the coding sequence to be expressed.","Additionally, the promoter is generally heterologous with respect to the recombinant vector sequences within which the construct promoter/coding sequence has been inserted.","Promoter regions can be selected from any desired gene using, for example, CAT (chloramphenicol transferase) vectors and more preferably pKK232-8 and pCM7 vectors.","Preferred bacterial promoters are the LacI, LacZ, the T3 or T7 bacteriophage RNA polymerase promoters, the gpt, lambda PR, PL and trp promoters (EP 0036776), the polyhedrin promoter, or the p10 protein promoter from baculovirus (Kit Novagen) (Smith et al., (1983) Mol Cell Biol December;3(12):2156-65; O'Reilly et al., 1992), the lambda PR promoter or also the trc promoter.","Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-L.","In addition, promoters specific for a particular cell type may be chosen, such as those facilitating expression in adipose tissue, muscle tissue, or liver.","Selection of a convenient vector and promoter is well within the level of ordinary skill in the art.","The choice of a promoter is well within the ability of a person skilled in the field of genetic engineering.","For example, one may refer to Sambrook et al.","(1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY, Vol.","1, 2, 3 (1989), or also to the procedures described by Fuller et al.","(1996) Immunology in Current Protocols in Molecular Biology.","Other Regulatory Elements Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript.","The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed such as human growth hormone and SV40 polyadenylation signals.","Also contemplated as an element of the expression cassette is a terminator.","These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.","3) Selectable Markers Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct.","The selectable marker genes for selection of transformed host cells are preferably dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, TRP 1 for S. cerevisiae or tetracycline, rifampicin or ampicillin resistance in E. coli, or levan saccharase for mycobacteria, this latter marker being a negative selection marker.","4) Preferred Vectors Bacterial Vectors As a representative but non-limiting example, useful expression vectors for bacterial use can comprise a selectable marker and a bacterial origin of replication derived from commercially available plasmids comprising genetic elements of pBR322 (ATCC 37017).","Such commercial vectors include, for example, pKK223-3 (Pharmacia, Uppsala, Sweden), and pGEM1 (Promega Biotec, Madison, Wis., USA).","Large numbers of other suitable vectors are known to those of skill in the art, and are commercially available, such as the following bacterial vectors: pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia); pQE-30 (QIAexpress).","Baculovirus Vectors A suitable vector for the expression of polypeptides of the invention is a baculovirus vector that can be propagated in insect cells and in insect cell lines.","A specific suitable host vector system is the pVL1392/1393 baculovirus transfer vector (Pharmingen) that is used to transfect the SF9 cell line (ATCC NoCRL 1711) which is derived from Spodoptera frugiperda.","Other suitable vectors for the expression of GSSP4 polypeptides in a baculovirus expression system include those described by Chai et al.","(1993; Biotechnol Appl Biochem.","December; 18 (Pt 3):259-73); Vlasak et al.","(1983; Eur J Biochem September 1;135(1):123-6); and Lenhard et al.","(1996; Gene March 9;169(2):187-90).","Viral Vectors In one specific embodiment, the vector is derived from an adenovirus.","Preferred adenovirus vectors according to the invention are those described by Feldman and Steg (1996; Semin Interv Cardiol September;1(3):203-8) or Ohno et al.","(1994; Science August 5;265(5173):781-4).","Another preferred recombinant adenovirus according to this specific embodiment of the present invention is the human adenovirus type 2 or 5 (Ad 2 or Ad 5) or an adenovirus of animal origin (French patent application No.","FR-93.05954).","Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery systems of choice for the transfer of exogenous polynucleotides in vivo, particularly to mammals, including humans.","These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host.","Particularly preferred retroviruses for the preparation or construction of retroviral in vitro or in vivo gene delivery vehicles of the present invention include retroviruses selected from the group consisting of Mink-Cell Focus Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis virus and Rous Sarcoma virus.","Particularly preferred Murine Leukemia Viruses include the 4070A and the 1504A viruses, Abelson (ATCC No VR-999), Friend (ATCC No VR-245), Gross (ATCC No VR-590), Rauscher (ATCC No VR-998) and Moloney Murine Leukemia Virus (ATCC No VR-190; PCT Application No WO 94/24298).","Particularly preferred Rous Sarcoma Viruses include Bryan high titer (ATCC Nos VR-334, VR-657, VR-726, VR-659 and VR-728).","Other preferred retroviral vectors are those described in Roth et al.","(1996), PCT Application No WO 93/25234, PCT Application No WO 94/06920, Roux et al., ((1989) Proc Natl Acad Sci USA December;86(23):9079-83), Julan et al., (1992) J. Gen. Virol.","3:3251-3255 and Neda et al., ((1991) J Biol Chem August 5;266(22):14143-6).","Yet another viral vector system that is contemplated by the invention consists of the adeno-associated virus (AAV).","The adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle (Muzyczka et al., (1992) Curr Top Microbiol Immunol;158:97-129).","It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (Flotte et al., (1992) Am J Respir Cell Mol Biol September;7(3):349-56; Samulski et al., (1989) J Virol September;63(9):3822-8; McLaughlin et al., (1989) Am.","J. Hum.","Genet.","59:561-569).","One advantageous feature of AAV derives from its reduced efficacy for transducing primary cells relative to transformed cells.","5) Delivery of the Recombinant Vectors In order to effect expression of the polynucleotides of the invention, these constructs must be delivered into a cell.","This delivery may be accomplished in vitro, as in laboratory procedures for transforming cell lines, or in vivo or ex vivo, as in the treatment of certain disease states.","One mechanism is viral infection where the expression construct is encapsulated in an infectious viral particle.","Several non-viral methods for the transfer of polynucleotides into cultured mammalian cells are also contemplated by the present invention, and include, without being limited to, calcium phosphate precipitation (Graham et al., (1973) Virology August;54(2):536-9; Chen et al., (1987) Mol Cell Biol August;7(8):2745-52), DEAE-dextran (Gopal, (1985) Mol Cell Biol May;5(5): 1188-90), electroporation (Tur-Kaspa et al., (1986) Mol Cell Biol February;6(2):716-8; Potter et al., (1984) Proc Natl Acad Sci USA November;81(22):7161-5.","), direct microinjection (Harland et al., (1985) J Cell Biol September;101(3):1094-9), DNA-loaded liposomes (Nicolau et al., (1982) Biochim Biophys Acta October 11;721(2):185-90; Fraley et al., (1979) Proc Natl Acad Sci USA July;76(7):3348-52), and receptor-mediated transfection (Wu and Wu, (1987) J Biol Chem April 5;262(10):4429-32; Wu and Wu (1988) Biochemistry February 9;27(3):887-92).","Some of these techniques may be successfully adapted for in vivo or ex vivo use.","Once the expression polynucleotide has been delivered into the cell, it may be stably integrated into the genome of the recipient cell.","This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non specific location (gene augmentation).","In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA.","Such nucleic acid segments or “episomes” encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle.","One specific embodiment for a method for delivering a protein or peptide to the interior of a cell of a vertebrate in vivo comprises the step of introducing a preparation comprising a physiologically acceptable carrier and a naked polynucleotide operatively coding for the polypeptide of interest into the interstitial space of a tissue comprising the cell, whereby the naked polynucleotide is taken up into the interior of the cell and has a physiological effect.","This is particularly applicable for transfer in vitro but it may be applied to in vivo as well.","Compositions for use in vitro and in vivo comprising a “naked” polynucleotide are described in PCT application No.","WO 90/11092 (Vical Inc.) and also in PCT application No.","WO 95/11307 (Institut Pasteur, INSERM, Universit{acute over (e )} d'Ottawa) as well as in the articles of Tascon et al.","(1996) Nature Medicine.","2(8):888-892 and of Huygen et al.","((1996) Nat Med August;2(8):893-8).","In still another embodiment of the invention, the transfer of a naked polynucleotide of the invention, including a polynucleotide construct of the invention, into cells may be proceeded with a particle bombardment (biolistic), said particles being DNA-coated microprojectiles accelerated to a high velocity allowing them to pierce cell membranes and enter cells without killing them, such as described by Klein et al.","((1990) Curr Genet February;17(2):97-103).","In a further embodiment, the polynucleotide of the invention may be entrapped in a liposome (Ghosh and Bacchawat, (1991) Targeted Diagn Ther;4:87-103; Wong et al., (1980) Gene 10:87-94; Nicolau et al., (1987) Methods Enzymol.;149:157-76).","These liposomes may further be targeted to cells expressing LSR by incorporating leptin, triglycerides, ACRP30, or other known LSR ligands into the liposome membrane.","In a specific embodiment, the invention provides a composition for the in vivo production of an GSSP4 polypeptides described herein.","It comprises a naked polynucleotide operatively coding for this polypeptide, in solution in a physiologically acceptable carrier, and suitable for introduction into a tissue to cause cells of the tissue to express the said polypeptide.","The amount of vector to be injected to the desired host organism varies according to the site of injection.","As an indicative dose, it will be injected between 0.1 and 100 μg of the vector in an animal body, preferably a mammal body, for example a mouse body.","In another embodiment of the vector according to the invention, it may be introduced in vitro in a host cell, preferably in a host cell previously harvested from the animal to be treated and more preferably a somatic cell such as a muscle cell.","In a subsequent step, the cell that has been transformed with the vector coding for the desired GSSP4 polypeptides or the desired fragment thereof is reintroduced into the animal body in order to deliver the recombinant protein within the body either locally or systemically.","IV.","Recombinant Cells of the Invention Another object of the invention consists of host cells recombinant for, i.e., that have been transformed or transfected with one of the polynucleotides described herein, and more precisely a polynucleotide comprising a polynucleotide encoding a GSSP4 polypeptide of the invention such as any one of those described in “Polynucleotides of the Invention”.","These polynucleotides can be present in cells as a result of transient or stable transfection.","The invention includes host cells that are transformed (prokaryotic cells) or that are transfected (eukaryotic cells) with a recombinant vector such as any one of those described in “Recombinant Vectors of the Invention”.","Generally, a recombinant host cell of the invention comprises at least one of the polynucleotides or the recombinant vectors of the invention that are described herein.","Preferred host cells used as recipients for the recombinant vectors of the invention are the following: a) Prokaryotic host cells: Escherichia coli strains (I.E.","DH5-α strain), Bacillus subtilis, Salmonella typhimurium, and strains from species like Pseudomonas, Streptomyces and Staphylococcus, and b) Eukaryotic host cells: HeLa cells (ATCC NoCCL2; NoCCL2.1; NoCCL2.2), Cv 1 cells (ATCC NoCCL70), COS cells (ATCC NoCRL1650; NoCRL1651), Sf-9 cells (ATCC NoCRL1711), C127 cells (ATCC No CRL-1804), 3T3 (ATCC No CRL-6361), CHO (ATCC No CCL-61), human kidney 293 (ATCC No 45504; No CRL-1573), BHK (ECACC No 84100501; No 84111301), PLC cells, HepG2, and Hep3B.","The constructs in the host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.","Following transformation of a suitable host and growth of the host to an appropriate cell density, the selected promoter is induced by appropriate means, such as temperature shift or chemical induction, and cells are cultivated for an additional period.","Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.","Microbial cells employed in the expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.","Such methods are well known by the skilled artisan.","Further, according to the invention, these recombinant cells can be created in vitro or in vivo in an animal, preferably a mammal, most preferably selected from the group consisting of mice, rats, dogs, pigs, sheep, cattle, and primates, not to include humans.","Recombinant cells created in vitro can also be later surgically implanted in an animal, for example.","Methods to create recombinant cells in vivo in animals are well-known in the art.","The present invention also encompasses primary, secondary, and immortalized homologously recombinant host cells of vertebrate origin, preferably mammalian origin and particularly human origin, that have been engineered to: a) insert exogenous (heterologous) polynucleotides into the endogenous chromosomal DNA of a targeted gene, b) delete endogenous chromosomal DNA, and/or c) replace endogenous chromosomal DNA with exogenous polynucleotides.","Insertions, deletions, and/or replacements of polynucleotide sequences may be to the coding sequences of the targeted gene and/or to regulatory regions, such as promoter and enhancer sequences, operably associated with the targeted gene.","The present invention further relates to a method of making a homologously recombinant host cell in vitro or in vivo, wherein the expression of a targeted gene not normally expressed in the cell is altered.","Preferably the alteration causes expression of the targeted gene under normal growth conditions or under conditions suitable for producing the polypeptide encoded by the targeted gene.","The method comprises the steps of: (a) transfecting the cell in vitro or in vivo with a polynucleotide construct, the polynucleotide construct comprising; (i) a targeting sequence; (ii) a regulatory sequence and/or a coding sequence; and (iii) an unpaired splice donor site, if necessary, thereby producing a transfected cell; and (b) maintaining the transfected cell in vitro or in vivo under conditions appropriate for homologous recombination.","The present invention further relates to a method of altering the expression of a targeted gene in a cell in vitro or in vivo wherein the gene is not normally expressed in the cell, comprising the steps of: (a) transfecting the cell in vitro or in vivo with a polynucleotide construct, the polynucleotide construct comprising: (i) a targeting sequence; (ii) a regulatory sequence and/or a coding sequence; and (iii) an unpaired splice donor site, if necessary, thereby producing a transfected cell; and (b) maintaining the transfected cell in vitro or in vivo under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell; and (c) maintaining the homologously recombinant cell in vitro or in vivo under conditions appropriate for expression of the gene.","The present invention further relates to a method of making a polypeptide of the present invention by altering the expression of a targeted endogenous gene in a cell in vitro or in vivo wherein the gene is not normally expressed in the cell, comprising the steps of: a) transfecting the cell in vitro with a polynucleotide construct, the polynucleotide construct comprising: (i) a targeting sequence; (ii) a regulatory sequence and/or a coding sequence; and (iii) an unpaired splice donor site, if necessary, thereby producing a transfected cell; (b) maintaining the transfected cell in vitro or in vivo under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell; and c) maintaining the homologously recombinant cell in vitro or in vivo under conditions appropriate for expression of the gene thereby making the polypeptide.","The present invention further relates to a polynucleotide construct that alters the expression of a targeted gene in a cell type in which the gene is not normally expressed.","This occurs when a polynucleotide construct is inserted into the chromosomal DNA of the target cell, wherein the polynucleotide construct comprises: a) a targeting sequence; b) a regulatory sequence and/or coding sequence; and c) an unpaired splice-donor site, if necessary.","Further included are polynucleotide constructs, as described above, wherein the construct further comprises a polynucleotide which encodes a polypeptide and is in-frame with the targeted endogenous gene after homologous recombination with chromosomal DNA.","The compositions may be produced, and methods performed, by techniques known in the art, such as those described in U.S. Pat.","Nos.","6,054,288; 6,048,729; 6,048,724; 6,048,524; 5,994,127; 5,968,502; 5,965,125; 5,869,239; 5,817,789; 5,783,385; 5,733,761; 5,641,670; 5,580,734; International Publication Nos:WO96/29411, WO 94/12650; and scientific articles described by Koller et al., (1994) Annu.","Rev.","Immunol.","10:705-730; the disclosures of each of which are incorporated by reference in their entireties).","The expression of GSSP4s in mammalian, and typically human, cells may be rendered defective, or alternatively it may be enhanced, with the insertion of a GSSP4 genomic or cDNA sequence with the replacement of the GSSP4 gene counterpart in the genome of an animal cell by a GSSP4 polynucleotide according to the invention.","These genetic alterations may be generated by homologous recombination events using specific DNA constructs that have been previously described.","One kind of host cell that may be used are mammalian zygotes, such as murine zygotes.","For example, murine zygotes may undergo microinjection with a purified DNA molecule of interest, for example a purified DNA molecule that has previously been adjusted to a concentration range from 1 ng/ml—for BAC inserts—3 ng/μl —for P1 bacteriophage inserts—in 10 mM Tris-HCl, pH 7.4, 250 μM EDTA containing 100 mM NaCl, 30 μM spermine, and 70 μM spermidine.","When the DNA to be microinjected has a large size, polyamines and high salt concentrations can be used in order to avoid mechanical breakage of this DNA, as described by Schedl et al ((1993) Nature March 18;362(6417):258-61).","Any one of the polynucleotides of the invention, including the DNA constructs described herein, may be introduced in an embryonic stem (ES) cell line, preferably a mouse ES cell line.","ES cell lines are derived from pluripotent, uncommitted cells of the inner cell mass of pre-implantation blastocysts.","Preferred ES cell lines are the following: ES-E14TG2a (ATCC No.CRL-1821), ES-D3 (ATCC No.CRL1934 and No.","CRL-11632), YS001 (ATCC No.","CRL-11776), 36.5 (ATCC No.","CRL-11116).","To maintain ES cells in an uncommitted state, they are cultured in the presence of growth inhibited feeder cells which provide the appropriate signals to preserve this embryonic phenotype and serve as a matrix for ES cell adherence.","Preferred feeder cells are primary embryonic fibroblasts that are established from tissue of day 13-day 14 embryos of virtually any mouse strain, that are maintained in culture, such as described by Abbondanzo et al.","(1993; Methods Enzymol;225:803-23) and are inhibited in growth by irradiation, such as described by Robertson ((1987) Embryo-derived stem cell lines.","In: E. J. Robertson Ed.","Teratocarcinomas and embrionic stem cells: a practical approach.","IRL Press, Oxford), or by the presence of an inhibitory concentration of LIF, such as described by Pease and Williams (1990; Exp Cell Res.","October;190(2):209-11).","The constructs in the host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.","Following transformation of a suitable host and growth of the host to an appropriate cell density, the selected promoter is induced by appropriate means, such as temperature shift or chemical induction, and cells are cultivated for an additional period.","Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.","Microbial cells employed in the expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.","Such methods are well known by the skilled artisan.","V. Transgenic Animals The present invention also provides methods and compositions for the generation of non-human animals and plants that express recombinant GSSP4 polypeptides, i.e.","recombinant GSSP4 fragments or full-length GSSP4 polypeptides.","The animals or plants can be transgenic, i.e.","each of their cells contains a gene encoding a GSSP4 polypeptide, or, alternatively, a polynucleotide encoding a GSSP4 polypeptide can be introduced into somatic cells of the animal or plant, e.g.","into mammary secretory epithelial cells of a mammal.","In preferred embodiments, the non-human animal is a mammal such as a cow, sheep, goat, pig, or rabbit.","Methods of making transgenic animals such as mammals are well known to those of skill in the art, and any such method can be used in the present invention.","Briefly, transgenic mammals can be produced, e.g., by transfecting a pluripotential stem cell such as an ES cell with a polynucleotide encoding a polypeptide of interest.","Successfully transformed ES cells can then be introduced into an early stage embryo which is then implanted into the uterus of a mammal of the same species.","In certain cases, the transformed (“transgenic”) cells will comprise part of the germ line of the resulting animal, and adult animals comprising the transgenic cells in the germ line can then be mated to other animals, thereby eventually producing a population of transgenic animals that have the transgene in each of their cells, and which can stably transmit the transgene to each of their offspring.","Other methods of introducing the polynucleotide can be used, for example introducing the polynucleotide encoding the polypeptide of interest into a fertilized egg or early stage embryo via microinjection.","Alternatively, the transgene may be introduced into an animal by infection of zygotes with a retrovirus containing the transgene (Jaenisch, R. (1976) Proc.","Natl.","Acad.","Sci.","USA 73, 1260-1264).","Methods of making transgenic mammals are described, e.g., in Wall et al.","(1992) J Cell Biochem 1992 June;49(2): 113-20; Hogan, et al.","(1986) in Manipulating the mouse embryo.","A Laboratory Manual.","Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; in WO 91/08216, or in U.S. Pat.","No.","4,736,866.In a preferred method, the polynucleotides are microinjected into the fertilized oocyte.","Typically, fertilized oocytes are microinjected using standard techniques, and then cultured in vitrountil a “pre-implantation embryo” is obtained.","Such pre-implantation embryos preferably contain approximately 16 to 150 cells.","Methods for culturing fertilized oocytes to the pre-implantation stage are described, e.g., by Gordon et al.","((1984) Methods in Enzymology, 101, 414); Hogan et al.","((1986) in Manipulating the mouse embryo.","A Laboratory Manual.","Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y) (for the mouse embryo); Hammer et al.","((1985) Nature, 315, 680) (for rabbit and porcine embryos); Gandolfi et al.","((1987) J. Reprod.","Fert.","81, 23-28); Rexroad et al.","((1988) J. Anim.","Sci.","66, 947-953) (for ovine embryos); and Eyestone et al.","((1989) J. Reprod.","Fert.","85, 715-720); Camous et al.","((1984) J. Reprod.","Fert.","72, 779-785); and Heyman et al.","((1987) Theriogenology 27, 5968) (for bovine embryos); the disclosures of each of which are incorporated herein in their entireties.","Pre-implantation embryos are then transferred to an appropriate female by standard methods to permit the birth of a transgenic or chimeric animal, depending upon the stage of development when the transgene is introduced.","As the frequency of transgene incorporation is often low, the detection of transgene integration in pre-implantation embryos is often desirable using any of the herein-described methods.","Any of a number of methods can be used to detect the presence of a transgene in a pre-implantation embryo.","For example, one or more cells may be removed from the pre-implantation embryo, and the presence or absence of the transgene in the removed cell or cells can be detected using any standard method e.g.","PCR.","Alternatively, the presence of a transgene can be detected in utero or post partum using standard methods.","In a particularly preferred embodiment of the present invention, transgenic mammals are generated that secrete recombinant GSSP4 polypeptides in their milk.","As the mammary gland is a highly efficient protein-producing organ, such methods can be used to produce protein concentrations in the gram per liter range, and often significantly more.","Preferably, expression in the mammary gland is accomplished by operably linking the polynucleotide encoding the GSSP4 polypeptide to a mammary gland specific promoter and, optionally, other regulatory elements.","Suitable promoters and other elements include, but are not limited to, those derived from mammalian short and long WAP, alpha, beta, and kappa, casein, alpha and beta lactoglobulin, beta-CN 5′ genes, as well as the the mouse mammary tumor virus (MMTV) promoter.","Such promoters and other elements may be derived from any mammal, including, but not limited to, cows, goats, sheep, pigs, mice, rabbits, and guinea pigs.","Promoter and other regulatory sequences, vectors, and other relevant teachings are provided, e.g., by Clark (1998) J Mammary Gland Biol Neoplasia 3:337-50; Jost et al.","(1999) Nat.","Biotechnol 17:160-4; U.S. Pat.","Nos.","5,994,616; 6,140,552; 6,013,857; Sohn et al.","(1999) DNA Cell Biol.","18:845-52; Kim et al.","(1999) J. Biochem.","(Japan) 126:320-5; Soulier et al.","(1999) Euro.","J. Biochem.","260:533-9; Zhang et al.","(1997) Chin.","J. Biotech.","13:271-6; Rijnkels et al.","(1998) Transgen.","Res.","7:5-14; Korhonen et al.","(1997) Euro.","J. Biochem.","245:482-9; Uusi-Oukari et al.","(1997) Transgen.","Res.","6:75-84; Hitchin et al.","(1996) Prot.","Expr.","Purif.","7:247-52; Platenburg et al.","(1994) Transgen.","Res.","3:99-108; Heng-Cherl et al.","(1993) Animal Biotech 4:89-107; and Christa et al.","(2000) Euro.","J. Biochem.","267:1665-71; the entire disclosures of each of which is herein incorporated by reference.","In another embodiment, the polypeptides of the invention can be produced in milk by introducing polynucleotides encoding the polypeptides into somatic cells of the mammary gland in vivo, e.g.","mammary secreting epithelial cells.","For example, plasmid DNA can be infused through the nipple canal, e.g.","in association with DEAE-dextran (see, e.g., Hens et al.","(2000) Biochim.","Biophys.","Acta 1523:161-171), in association with a ligand that can lead to receptor-mediated endocytosis of the construct (see, e.g., Sobolev et al.","(1998) 273:7928-33), or in a viral vector such as a retroviral vector, e.g.","the Gibbon ape leukemia virus (see, e.g., Archer et al.","(1994) PNAS 91:6840-6844).","In any of these embodiments, the polynucleotide may be operably linked to a mammary gland specific promoter, as described above, or, alternatively, any strongly expressing promoter such as CMV or MoMLV LTR.","The suitability of any vector, promoter, regulatory element, etc.","for use in the present invention can be assessed beforehand by transfecting cells such as mammary epithelial cells, e.g.","MacT cells (bovine mammary epithelial cells) or GME cells (goat mammary epithelial cells), in vitro and assessing the efficiency of transfection and expression of the transgene in the cells.","For in vivo administration, the polynucleotides can be administered in any suitable formulation, at any of a range of concentrations (e.g.","1-500 μg/ml, preferably 50-100 μg/ml), at any volume (e.g.","1-100 ml, preferably 1 to 20 ml), and can be administered any number of times (e.g.","1, 2, 3, 5, or 10 times), at any frequency (e.g.","every 1, 2, 3, 5, 10, or any number of days).","Suitable concentrations, frequencies, modes of administration, etc.","will depend upon the particular polynucleotide, vector, animal, etc., and can readily be determined by one of skill in the art.","In a preferred embodiment, a retroviral vector such as as Gibbon ape leukemia viral vector is used, as described in Archer et al.","((1994) PNAS 91:6840-6844).","As retroviral infection typically requires cell division, cell division in the mammary glands can be stimulated in conjunction with the administration of the vector, e.g.","using a factor such as estrodiol benzoate, progesterone, reserpine, or dexamethasone.","Further, retroviral and other methods of infection can be facilitated using accessory compounds such as polybrene.","In any of the herein-described methods for obtaining GSSP4 polypeptides from milk, the quantity of milk obtained, and thus the quantity of GSSP4 polypeptides produced, can be enhanced using any standard method of lactation induction, e.g.","using hexestrol, estrogen, and/or progesterone.","The polynucleotides used in such embodiments can either encode a full-length GSSP4 polypeptide or a GSSP4 fragment.","Typically, the encoded polypeptide will include a signal sequence to ensure the secretion of the protein into the milk.","Where a full length GSSP4 sequence is used, the full length protein can, e.g., be isolated from milk and cleaved in vitro using a suitable protease.","Alternatively, a second, protease-encoding polynucleotide can be introduced into the animal or into the mammary gland cells, whereby expression of the protease results in the cleavage of the GSSP4 polypeptide in vivo, thereby allowing the direct isolation of GSSP4 fragments from milk.","VI.","Pharmaceutical or Physiologically Acceptable Compositions of the Invention The GSSP4 polypeptides of the invention can be administered to non-human animals and/or humans, alone or in pharmaceutical or physiologically acceptable compositions where they are mixed with suitable carriers or excipient(s).","The pharmaceutical or physiologically acceptable composition is then provided at a therapeutically effective dose.","A therapeutically effective dose refers to that amount of a GSSP4 polypeptide sufficient to result in prevention or amelioration of symptoms or physiological status of metabolic-related diseases or disorders as determined by the methods described herein.","A therapeutically effective dose can also refer to the amount of a GSSP4 polypeptide necessary for a reduction in weight or a prevention of an increase in weight or prevention of an increase in the rate of weight gain in persons desiring this affect for cosmetic reasons.","A therapeutically effective dosage of a GSSP4 polypeptide of the invention is that dosage that is adequate to promote weight loss or weight gain with continued periodic use or administration.","Techniques for formulation and administration of GSSP4 polypeptides may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition.","Other diseases or disorders that GSSP4 polypeptides of the invention could be used to treat or prevent include, but are not limited to, obesity and metabolic-related diseases and disorders such as obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, non-insulin-dependent diabetes and Type II diabetes.","Type II diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, and renal lesions.","Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.","Other metabolic-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.","The GSSP4 polypeptides may also be used to enhance physical performance during work or exercise or enhance a feeling of general well-being.","Physical performance activities include walking, running, jumping, lifting and/or climbing.","The GSSP4 polypeptides or antagonists thereof may also be used to treat dyslexia, attention-deficit disorder (ADD), attention-deficit/hyperactivity disorder (ADHD), and psychiatric disorders such as schizophrenia by modulating fatty acid metabolism, more specifically, the production of certain long-chain polyunsaturated fatty acids.","It is expressly considered that the GSSP4 polypeptides of the invention may be provided alone or in combination with other pharmaceutically or physiologically acceptable compounds.","Other compounds useful for the treatment of obesity and other diseases and disorders are currently well-known in the art.","In a preferred embodiment, the GSSP4 polypeptides are useful for, and used in, the treatment of insulin resistance and diabetes using methods described herein and known in the art.","More particularly, a preferred embodiments relates to process for the therapeutic modification and regulation of glucose metabolism in an animal or human subject, which comprises administering to a subject in need of treatment (alternatively on a timed daily basis) GSSP4 polypeptide (or polynucleotide encoding said polypeptide) in dosage amount and for a period sufficient to reduce plasma glucose levels in said animal or human subject.","Further preferred embodiments relate to methods for the prophylaxis or treatment of diabetes comprising administering to a subject in need of treatment (alternatively on a timed daily basis) a GSSP4 polypeptide (or polynucleotide encoding said polypeptide) in dosage amount and for a period sufficient to reduce plasma glucose levels in said animal or human subject.","Routes of Administration.","Suitable routes of administration include oral, nasal, rectal, transmucosal, or intestinal administration, parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intrapulmonary (inhaled) or intraocular injections using methods known in the art.","A particularly useful method of administering compounds for promoting weight loss involves surgical implantation, for example into the abdominal cavity of the recipient, of a device for delivering GSSP4 polypeptidesover an extended period of time.","Other particularly preferred routes of administration are aerosol and depot formulation.","Sustained release formulations, particularly depot of the invented medicaments are expressly contemplated.","Composition/Formulation Pharmaceutical or physiologically acceptable compositions and medicaments for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries.","Proper formulation is dependent upon the route of administration chosen.","Certain of the medicaments described herein will include a pharmaceutically or physiologically acceptable acceptable carrier and at least one polypeptide that is a GSSP4 polypeptide of the invention.","For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer such as a phosphate or bicarbonate buffer.","For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation.","Such penetrants are generally known in the art.","Pharmaceutical or physiologically acceptable preparations that can be taken orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.","The push-fit capsules can contain the active ingredients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.","In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.","In addition, stabilizers may be added.","All formulations for oral administration should be in dosages suitable for such administration.","For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.","For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable gaseous propellant, e.g.","carbon dioxide.","In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount.","Capsules and cartridges of, e.g.","gelatin, for use in an inhaler or insufflator, may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.","The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.","Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.","The compositions may take such forms as suspensions, solutions or emulsions in aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.","Pharmaceutical or physiologically acceptable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form.","Aqueous suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.","Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.","Alternatively, the active ingredient may be in powder or lyophilized form for constitution with a suitable vehicle, such as sterile pyrogen-free water, before use.","In addition to the formulations described previously, the compounds may also be formulated as a depot preparation.","Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.","Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.","Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.","Various sustained release materials have been established and are well known by those skilled in the art.","Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.","Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.","The pharmaceutical or physiologically acceptable compositions also may comprise suitable solid or gel phase carriers or excipients.","Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.","Effective Dosage.","Pharmaceutical or physiologically acceptable compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve their intended purpose.","More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated.","Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.","For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays.","For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes or encompasses a concentration point or range shown to increase leptin or lipoprotein uptake or binding in an in vitro system.","Such information can be used to more accurately determine useful doses in humans.","A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms in a patient.","Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50, (the dose lethal to 50% of the test population) and the ED50 (the dose therapeutically effective in 50% of the population).","The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD5O and ED5O.","Compounds that exhibit high therapeutic indices are preferred.","The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.","The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50, with little or no toxicity.","The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.","The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.","(See, e.g., Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.","1).","Dosage amount and interval may be adjusted individually to provide plasma levels of the active compound which are sufficient to maintain or prevent weight loss or gain, depending on the particular situation.","Dosages necessary to achieve these effects will depend on individual characteristics and route of administration.","Dosage intervals can also be determined using the value for the minimum effective concentration.","Compounds should be administered using a regimen that maintains plasma levels above the minimum effective concentration for 10-90% of the time, preferably between 30-90%; and most preferably between 50-90%.","In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.","The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.","A preferred dosage range for the amount of a GSSP4 polypeptide of the invention, which can be administered on a daily or regular basis to achieve desired results, including a reduction in levels of circulating plasma triglyceride-rich lipoproteins, range from 0.01-0.5 mg/kg body mass.","A more preferred dosage range is from 0.05-0.1 mg/kg.","Of course, these daily dosages can be delivered or administered in small amounts periodically during the course of a day.","It is noted that these dosage ranges are only preferred ranges and are not meant to be limiting to the invention.","VII.","Methods of Treatment The invention is drawn inter alia to methods of preventing or treating metabolic-related diseases and disorders comprising providing an individual in need of such treatment with a GSSP4 polypeptide of the invention.","Preferably, the GSSP4 polypeptide has metabolic-related activity either in vitro or in vivo.","Preferably the GSSP4 polypeptide is provided to the individual in a pharmaceutical composition that is preferably taken orally.","Preferably the individual is a mammal, and most preferably a human.","In preferred embodiments, the metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM, Type II diabetes), Insulin dependent diabetes mellitus (IDDM, Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.","Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.","In highly preferred embodiments, GSSP4 polypeptides in pharmaceutical compositions are used to modulate body weight in healthy individuals for cosmetic reasons.","The invention also features a method of preventing or treating metabolic-related diseases and disorders comprising providing an individual in need of such treatment with a compound identified by assays of the invention (described in Section VI of the Preferred Embodiments of the Invention and in the Examples).","Preferably these compounds antagonize or agonize effects of GSSP4 polypeptides in cells in vitro, muscles ex vivo, or in animal models.","Alternatively, these compounds agonize or antagonize the effects of GSSP4 polypeptides on glucose metabolism, fatty acid metabolism, or lipid metabolism.","Preferably, the compound is provided to the individual in a pharmaceutical composition that is preferably taken orally.","Preferably the individual is a mammal, and most preferably a human.","In preferred embodiments, the metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM, Type II diabetes), Insulin dependent diabetes mellitus (IDDM, Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.","Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.","In highly preferred embodiments, the pharmaceutical compositions are used to modulate glucose levels.","In highly preferred embodiments, the pharmaceutical compositions are used to modulate body weight for cosmetic reasons.","In a further preferred embodiment, NIDDM patients are often treated with oral insulin secretagogues, such as 1,1-dimethyl-2-(2-morpholinophenyl)guanidine fumarate (BTS67582) or sulfonylureas including tolbutamide, tolazamide, chlorpropamide, glibendamide, glimepiride, glipizide and glidazide, or with insulin sensitising agents including metformin, ciglitazone, trogitazone and pioglitazone.","A further use of the present invention is in therapy of NIDDM patients to improve their weight and glucose control, comprising a pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect in combination with an oral insulin secretagogue or an insulin sensitising agent.","Preferably, the oral insulin secretagogue is 1,1-dimethyl-2-(2-morpholino phenyl)guanidine fumarate (BTS67582) or a sulphonylurea selected from tolbutamide, tolazamide, chlorpropamide, glibenclamide, glimepiride, glipizide and glidazide.","Preferably, the insulin sensitising agent is selected from metformin, ciglitazone, troglitazone and pioglitazone.","The present invention further provides a method of improving the weight and glucose control of NIDDM patients comprising the administration of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect alone, without an oral insulin secretagogue or an insulin sensitising agent.","In a further preferred embodiment, the present invention may be administered either concomitantly or concurrently, with the oral insulin secretagogue or insulin sensitising agent for example in the form of separate dosage units to be used simultaneously, separately or sequentially (either before or after the secretagogue or either before or after the sensitising agent).","Accordingly, the present invention further provides a product containing a composition of said a pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect and an oral insulin secretagogue or insulin sensitising agent as a combined preparation for simultaneous, separate or sequential use for the improvement of weight and glucose control in NIDDM patients.","The ratio of the present composition to the oral insulin secretagogue or insulin sensitising agent is such that the quantity of each active ingredient employed will be such as to provide a therapeutically effective level, but will not be larger than the quantity recommended as safe for administration.","The action of reducing insulin resistance by the present invention indicates that compounds of such invention may be useful in the manufacture of a medicament which can be used as an insulin sensitiser.","Accordingly, the present invention further provides for the use in the manufacture of a medicament which is an insulin sensitiser.","In further embodiments, some patients who are diagnosed with Insulin Dependent Diabetes Mellitus (IDDM, Type I) can also show a certain amount of insulin resistance.","Therefore, there may be benefits in treating these patients with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect in order to reduce their insulin resistance.","This would mean that these patients would require a lower dosage of insulin in order to maintain similar or better control of their diabetes since the insulin dose would be associated with a greater blood glucose lowering efficacy.","Such therapy would provide long-term benefits in terms of reducing the detrimental effects which can be caused by prolonged high-dosage of insulin treatment.","Additionally, some Noninsulin Dependent Diabetes Mellitus (NIDDM, Type II) patients are also treated with insulin and have insulin resistance.","Accordingly the present invention further provides a method for, and the use thereof in the manufacture of the medicament for, reducing the amount of insulin required daily by a human having NIDDM.","The present invention also provides a method for, and the use of the composition in the manufacture of a medicament for, the prophylaxis of long-term detrimental effects caused by prolonged high dosage of insulin in humans having IDDM.","A consequence of the resistance is that glucose concentrations rise.","This leads, in turn, to an increased release of insulin.","Hyperinsulinemia, both in the fasting and postprandial states, is a hallmark of insulin resistance.","Hyperinsulinemia is also caused by stimulation of gluconeogenesis.","Epidemilogical studies have shown that hyperinsulinemia is a risk factor for morbidity and mortality in cardiovascular disease (Smith U.","(1994) Am.","J. Clin.","Nutr.","59, suppl.","686S).","Accordingly, the invention also provides therapeutics and methods for reducing or preventing hypersecretion of insulin and disorders or conditions resulting therefrom.","NIDDM is associated with various complications.","As defined herein, “complications of NIDDM” is referred to as cardiovascular complications or several of the metabolic and circulatory disturbances that are associated with hyperglycemia, e.g., insulin resistance, hyperinsulinemia and/or hyperproinsulinemia, delayed insulin release, dyslipidemia, retinopathy, peripheral neuropathy, hypertension, and other coronary artery diseases (CADs).","CAD is a major cause of morbidity and mortality in patients with NIDDM.","Thus, by providing therapeutics and methods for reducing glucose levels, the invention provides therapeutics and methods for treating and preventing NIDDM and consequences thereof.","The invention also provides therapeutics and methods for treating and preventing having impaired glucose tolerance (IGT).","The usual meaning of impaired glucose tolerance is that it is a condition associated with insulin-resistance which is intermediate between frank, NIDDM and normal glucose tolerance (NGT).","A high percentage of the IGT population is known to progress to NIDDM relative to persons with normal glucose tolerance (Sad, et al., New Engl.","J. Med.","1988; 319:1500-6).","Thus, by providing therapeutics and methods for reducing or preventing IGT, i.e., for normalizing insulin resistance, the progression to NIDDM can be delayed or prevented.","IGT is diagnosed by a procedure wherein an affected person's postprandial glucose response is determined to be abnormal as assessed by 2-hour postprandial plasma glucose levels.","In this test, a measured amount of glucose is given to the patient and blood glucose levels measured regular intervals, usually every half hour for the first two hours and every hour thereafter.","In a “normal” or non-IGT individual glucose levels rise during the first two hours to level less than 140 mg/dl and then drop rapidly.","In an IGT individual, the blood glucose levels are higher and the drop-off level is at a slower rate.","Resistance to insulin-stimulated glucose uptake in individuals who do not become frankly hyperglycemic nevertheless increases the likelihood of these individuals to develop numerous other diseases.","In particular, an attempt to compensate for insulin resistance sets in motion a series of events that play an important role in the development of both hypertension and coronary artery disease (CAD), such as premature atherosclerotic vascular disease.","This cluster of abnormalities is commonly called the “Metabolic Syndrome”, or the “Insulin-Resistance Syndrome” or “Syndrome X”.","Increased plasma triglyceride and decreased HDL-cholesterol concentrations, conditions which are known to be associated with CAD, have also been reported to be associated with insulin resistance.","Thus, by providing therapeutics and methods for reducing or preventing insulin resistance, the invention provides methods for reducing and/or preventing the appearance of insulin-resistance syndrome.","Yet other diseases are associated with insulin resistance and, thus, be treated or prevented according to the methods of the invention.","For example, obesity, which is the result of an imbalance between caloric intake and energy expenditure is highly correlated with insulin resistance and diabetes (Hotamisligil, Spiegelman et al., Science, 1993, 259:87-91).","In humans obesity can be defined as a body weight exceeding 20% of the desirable body weight for individuals of the same sex, height and frame (Slans, L. B., in Endocrinology & Metabolism, 2d Ed., McGraw-Hill, New York 1987, pp.","1203-1244; see also, R H. Williams, Textbook of Endocrinology, 1974, pp.","904-916).","In other animals (or also in humans) obesity can be determined by body weight patterns correlated with prolactin profiles given that members of a species that are young, lean and “healthy” (i.e., free of any disorders, not just metabolic disorders) have daily plasma prolactin level profiles that follow a regular pattern that is highly reproducible with a small standard deviation.","Obesity, or excess fat deposits, correlate with and may trigger the onset of various lipid metabolism disorders, e.g.","hypertension, Type II diabetes (NIDDM), atherosclerosis, cardiovascular disease, etc.","Even in the absence of clinical obesity (according to the above definition) the reduction of body fat stores (notably visceral fat stores) in man especially on a long-term or permanent basis would be of significant benefit, both cosmetically and physiologically.","Thus, by preventing or treating obesity, the methods of the invention will allow an individual to have a more comfortable life and avoid the onset of various diseases triggered by obesity.","In yet another embodiment, the invention provides a method for treating a subject having polycystic ovary syndrome (PCOS).","PCOS is among the most common disorders of premenopausal women, affecting 5-10% of this population.","It is a syndrome of unknown Etiology characterized by hyperandrogenism, chronic anovulation, defects in insulin action, insulin secretion, ovarian steroidogenesis and fibrinolysis.","Women with PCOS frequently are insulin resistant and at increased risk to develop glucose intolerance or NIDDM in the third and fourth decades of life (Dunaif et al.","(1996) J. Clin.","Endocrinol.","Metab.","81:3299).","Hyperandrogenism also is a feature of a variety of diverse insulin-resistant states, from the type A syndrome, through leprechaunism and lipoatrophic diabetes, to the type B syndrome, when these conditions occur in premenopausal women.","It has been suggested that hyperinsulinemia per se causes hyperandrogenism.","Insulin-sensitizing agents, e.g., troglitazone, have been shown to be effective in PCOS and that, in particular, the defects in insulin action, insulin secretion, ovarian steroidogenosis and fibrinolysis are improved (Ehrman et al.","(1997) J. Clin.","Invest.","100:1230), such as in insulin-resistant humans.","Accordingly, the invention provides methods for reducing insulin resistance, normalizing blood glucose thus treating and/or preventing PCOS.","Insulin resistance is also often associated with infections and cancer.","Thus, prevention or reducing insulin resistance according to the methods of the invention may prevent or reduce infections and cancer.","Insulin resistance can be diagnosed by various methods, such as by the intravenous glucose tolerance test or by measuring the fasting insulin level.","It is well known that there is an excellent correlation between the height of the fasting insulin level and the degree of insulin resistance.","Therefore, one could use elevated fasting insulin levels as a surrogate marker for insulin resistance for the purpose of identifying which normal glucose tolerance (NGT) individuals have insulin resistance.","Another way to do this is to follow the approach as disclosed in The New England Journal of Medicine, No.","3, pp.","1188 (1995), i.e.","to select obese subjects as an initial criteria for entry into the treatment group.","Some obese subjects have impaired glucose tolerance (IGT) while others have normal glucose tolerance (NGT).","Since essentially all obese subjects are insulin resistant, i.e.","even the NGT obese subjects are insulin resistant, they have fasting hyperinsulinemia.","Therefore, the target of the treatment according to the present invention can be defined as NGT individuals who are obese or who have fasting hyperinsulinemia, or who have both.","Insulin resistance can also be diagnosed by the euglycemic glucose clamp test.","This test involves the simultaneous administration of a constant insulin infusion and a variable rate glucose infusion.","During the test, which lasts 3-4 hours, the plasma glucose concentration is kept constant at euglycemic levels by measuring the glucose level every 5-10 minutes and then adjusting the variable rate glucose infusion to keep the plasma glucose level unchanged.","Under these circumstances, the rate of glucose entry into the bloodstream is equal to the overall rate of glucose disposal in the body.","The difference between the rate of glucose disposal in the basal state (no insulin infusion) and the insulin infused state, represents insulin mediated glucose uptake.","In normal individuals, insulin causes brisk and large increase in overall body glucose disposal, whereas in NIDDM subjects, this effect of insulin is greatly blunted, and is only 20-30% of normal.","In insulin resistant subjects with either IGT or NGT, the rate of insulin stimulated glucose disposal is about half way between normal and NIDDM.","For example, at a steady state plasma insulin concentration of about 100 uU/ml (a physiologic level) the glucose disposal rate in normal subjects is about 7 mg/kg/min.","In NIDDM subjects, it is about 2.5 mg/.kg/min., and in patients with IGT (or insulin resistant subjects with NGT) it is about 4-5 mg/kg/min.","This is a highly reproducible and precise test, and can distinguish patients within these categories.","It is also known, that as subjects become more insulin resistant, the fasting insulin level rises.","There is an excellent positive correlation between the height of the fasting insulin level and the magnitude of the insulin resistance as measured by euglycemic glucose clamp tests and, therefore, this provides the rationale for using fasting insulin levels as a surrogate measure of insulin resistance.","Thus, any of the above-described tests or other tests known in the art can be used to determine that a subject is insulin-resistant, which patient can then be treated according to the methods of the invention to reduce or cure the insulin-resistance.","Alternatively, the methods of the invention can also be used to prevent the development of insulin resistance in a subject, e.g., those known to have an increased risk of developing insulin-resistance.","More generally, the instant invention is drawn to treatment with GSSP4 polypeptides where an individual is shown to have a particular genotype for GSSP4 marker.","Treatment comprises providing pharmaceutically acceptable GSSP4 polypeptides to the individual.","The exact amount of GSSP4 polypeptide provided would be determined through clinical trials under the guidance of qualified physicians, but would be expected to be in the range of 5-7 mg per individual per day.","In general, a preferred range would be from 0.5 to 14 mg per individual per day, with a highly preferred range being between 1 and 10 mg per individual per day.","Individuals who could benefit from treatment with GSSP4 polypeptides could be identified through genotyping.","GSSP4 Genotyping The methods treatment using genotyping to identify individuals that would benefit from treatments of the invention are based on the finding that single nucleotide polymorphisms (SNPs) in GSSP4 have been identified that show an association in obese adolescents with free fatty acid (FFA) and respiratory quotient levels, others that show an association with the relationship between BMI and leptin, and still others that show an association with glucose levels.","Further, a combination of GSSP4 SNPs associated with FFA and leptin metabolism may also predict people who will be seriously overweight.","Briefly, the term “genotype” as used herein refers to the identity of the alleles present in an individual or a sample.","The term “genotyping” a sample or an individual for a biallelic marker consists of determining the specific allele or the specific nucleotide carried by an individual at a biallelic marker.","Biallelic markers generally consist of a polymorphism at one single base position.","Each biallelic marker therefore corresponds to two forms of a polynucleotide sequence which, when compared with one another, present a nucleotide modification at one position.","Usually, the nucleotide modification involves the substitution of one nucleotide for another, optionally either the original or the alternative allele of the biallelic markers disclosed in SEQ ID NO:1.Optionally either the original or the alternative allele of these biallelic markers may be specified as being present.","Preferred polynucleotides may consist of, consist essentially of, or comprise a contiguous span of nucleotides upstream and down-stream of the alternate allele position noted in SEQ ID No:1 as well as sequences which are complementary thereto.","The “contiguous span” may be at least 8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 70, 80, 100, 250, 500 or 1000 nucleotides in length, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID.","Methods of genotyping comprise determining the identity of a nucleotide at GSSP4 biallelic marker site by any method known in the art.","Preferably, microsequencing is used.","The genotype is used to determine whether an individual should be treated with GSSP4 polypeptides.","Thus, these genotyping methods are performed on nucleic acid samples derived from a single individual.","These methods are well-known in the art, and discussed fully in the applications referenced briefly below.","Any method known in the art can be used to identify the nucleotide present at a biallelic marker site.","Since the biallelic marker allele to be detected has been identified and specified in the present invention, detection will prove simple for one of ordinary skill in the art by employing any of a number of techniques.","Many genotyping methods require the previous amplification of the DNA region carrying the biallelic marker of interest.","While the amplification of target or signal is often preferred at present, ultrasensitive detection methods that do not require amplification are also encompassed by the present genotyping methods.","Methods well-known to those skilled in the art that can be used to detect biallelic polymorphisms include methods such as conventional dot blot analysis, single strand conformational polymorphism analysis (SSCP; Orita et al.","(1989) Proc Natl Acad Sci USA April;86(8):2766-70), denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis, mismatch cleavage detection, and other conventional techniques as described in Sheffield et al.","(1991; Am J Hum Genet October;49(4):699-706); White et al.","(1992), Grompe et al.","((1989) Proc Natl Acad Sci USA August;86(15):5888-92; (1993) Nat Genet.","October;5(2):111-7).","Another method for determining the identity of the nucleotide present at a particular polymorphic site employs a specialized exonuclease-resistant nucleotide derivative as described in U.S. Pat.","No.","4,656,127.Preferred methods involve directly determining the identity of the nucleotide present at a biallelic marker site by sequencing assay, allele-specific amplification assay, or hybridization assay.","The following is a description of some preferred methods.","A highly preferred method is the microsequencing technique.","The term “sequencing” is used herein to refer to polymerase extension of duplex primer/template complexes and includes both traditional sequencing and microsequencing.","Preferred biallelic markers, as shown in SEQ ID NO:1 and corresponding 47 mers as shown in SEQ ID NOs:4-18, include but are not limited to: 1.Biallelic marker 1: position base 148, alternate alleles A/G 2.Biallelic marker 2: position base 2551, alternate alleles G/A 3.Biallelic marker 3: position base 4417, alternate alleles A/C 4.Biallelic marker 4: position base 6322, alternate alleles A/G (amino acid change isoleucine to valine) 5.Biallelic marker 7: position base 816, alternate alleles G/A 6.Biallelic marker 8: position base 924, alternate alleles G/A 7.Biallelic marker 9: position base 1206, alternate alleles C/A 8.Biallelic marker 10: position base 1851, alternate alleles T/C 9.Biallelic marker 11: position base 3124, alternate alleles C/T 10.Biallelic marker 12: position base 3563, alternate alleles G/A 11.Biallelic marker 13: position base 3792, alternate alleles G/A 12.Biallelic marker 14: position base 5757, alternate alleles T/C 1) Sequencing Assays The nucleotide present at a polymorphic site can be determined by sequencing methods.","In a preferred embodiment, DNA samples are subjected to PCR amplification before sequencing using any method known in the art.","Preferably, the amplified DNA is subjected to automated dideoxy terminator sequencing reactions using a dye-primer cycle sequencing protocol.","Sequence analysis allows the identification of the base present at the biallelic marker site.","2) Microsequencing Assays In microsequencing methods, the nucleotide at a polymorphic site in a target DNA is detected by a single nucleotide primer extension reaction.","This method involves appropriate microsequencing primers that hybridize just upstream of the polymorphic base of interest in the target nucleic acid.","A polymerase is used to specifically extend the 3′ end of the primer with one single ddNTP (chain terminator) complementary to the nucleotide at the polymorphic site.","The identity of the incorporated nucleotide is then determined in any suitable way.","Preferred microsequencing primers are described in SEQ ID NO:1.Typically, microsequencing reactions are carried out using fluorescent ddNTPs and the extended microsequencing primers are analyzed by electrophoresis on ABI 377 sequencing machines to determine the identity of the incorporated nucleotide as described in EP 412 883.Alternatively capillary electrophoresis can be used in order to process a higher number of assays simultaneously.","Different approaches can be used for the labeling and detection of ddNTPs.","A homogeneous phase detection method based on fluorescence resonance energy transfer has been described by Chen and Kwok ((1997) Nucleic Acids Res.","January 15;25(2):347-53) and Chen et al.","((1997) Proc Natl Acad Sci USA September 30;94(20):10756-61).","In this method, amplified genomic DNA fragments containing polymorphic sites are incubated with a 5′-fluorescein-labeled primer in the presence of allelic dye-labeled dideoxyribonucleoside triphosphates and a modified Taq polymerase.","The dye-labeled primer is extended one base by the dye-terminator specific for the allele present on the template.","At the end of the genotyping reaction, the fluorescence intensities of the two dyes in the reaction mixture are analyzed directly without separation or purification.","All these steps can be performed in the same tube and the fluorescence changes can be monitored in real time.","Alternatively, the extended primer may be analyzed by MALDI-TOF Mass Spectrometry.","The base at the polymorphic site is identified by the mass added onto the microsequencing primer (see Haff and Smirnov, (1997) Nucleic Acids Res.","September 15;25(18):3749-50; (1997) Genome Res.","April;7(4):378-88).","Microsequencing may be achieved by the established microsequencing method or by developments or derivatives thereof.","Alternative methods include several solid-phase microsequencing techniques.","The basic microsequencing protocol is the same as described previously, except that the method is conducted as a heterogeneous phase assay, in which the primer or the target molecule is immobilized or captured onto a solid support.","To simplify the primer separation and the terminal nucleotide addition analysis, oligonucleotides are attached to solid supports or are modified in such ways that permit affinity separation as well as polymerase extension.","The 5′ ends and internal nucleotides of synthetic oligonucleotides can be modified in a number of different ways to permit different affinity separation approaches, e.g., biotinylation.","If a single affinity group is used on the oligonucleotides, the oligonucleotides can be separated from the incorporated terminator regent.","This eliminates the need of physical or size separation.","More than one oligonucleotide can be separated from the terminator reagent and analyzed simultaneously if more than one affinity group is used.","This permits the analysis of several nucleic acid species or more nucleic acid sequence information per extension reaction.","The affinity group need not be on the priming oligonucleotide but could alternatively be present on the template.","For example, immobilization can be carried out via an interaction between biotinylated DNA and streptavidin-coated microtitration wells or avidin-coated polystyrene particles.","In the same manner, oligonucleotides or templates may be attached to a solid support in a high-density format.","In such solid phase microsequencing reactions, incorporated ddNTPs can be radiolabeled (Syvänen, (1994) Clin Chim Acta.","May;226(2):225-36) or linked to fluorescein (Livak and Hainer, (1994) Hum Mutat.;3(4):379-85).","The detection of radiolabeled ddNTPs can be achieved through scintillation-based techniques.","The detection of fluorescein-linked ddNTPs can be based on the binding of antifluorescein antibody conjugated with alkaline phosphatase, followed by incubation with a chromogenic substrate (such as p-nitrophenyl phosphate).","Other possible reporter-detection pairs include: ddNTP linked to dinitrophenyl (DNP) and anti-DNP alkaline phosphatase conjugate (Harju et al., (1993) Clin Chem.","November;39(11 Pt 1):2282-7) or biotinylated ddNTP and horseradish peroxidase-conjugated streptavidin with o-phenylenediamine as a substrate (WO 92/15712).","As yet another alternative solid-phase microsequencing procedure, Nyren et al.","((1993) Anal Biochem.","January;208(1):171-5).","described a method relying on the detection of DNA polymerase activity by an enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA).","Pastinen et al.","((1997) Genome Res.","June;7(6):606-14) describe a method for multiplex detection of single nucleotide polymorphism in which the solid phase minisequencing principle is applied to an oligonucleotide array format.","High-density arrays of DNA probes attached to a solid support (DNA chips) are further described below.","It will be appreciated that any primer having a 3′ end immediately adjacent to the polymorphic nucleotide may be used.","Similarly, it will be appreciated that microsequencing analysis may be performed for any biallelic marker or any combination of biallelic markers of the present invention.","3) Allele-Specific Amplification Assay Methods Discrimination between the two alleles of a biallelic marker can also be achieved by allele specific amplification, a selective strategy, whereby one of the alleles is amplified without, or at a much higher rate than, amplification of the other allele.","This is accomplished by placing the polymorphic base at the 3′ end of one of the amplification primers.","Because the extension forms from the 3′ end of the primer, a mismatch at or near this position has an inhibitory effect on amplification.","Therefore, under appropriate amplification conditions, these primers only direct amplification on their complementary allele.","Determining the precise location of the mismatch and the corresponding assay conditions are well with the ordinary skill in the art.","The “Oligonucleotide Ligation Assay” (OLA) uses two oligonucleotides which are designed to be capable of hybridizing to abutting sequences of a single strand of a target molecules.","One of the oligonucleotides is biotinylated, and the other is detectably labeled.","If the precise complementary sequence is found in a target molecule, the oligonucleotides will hybridize such that their termini abut, and create a ligation substrate that can be captured and detected.","OLA is capable of detecting single nucleotide polymorphisms and may be advantageously combined with PCR as described by Nickerson et al.","((1990) Proc Natl Acad Sci USA November;87(22):8923-7).","In this method, PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA.","Other amplification methods which are particularly suited for the detection of single nucleotide polymorphism include LCR (ligase chain reaction) and Gap LCR (GLCR).","LCR uses two pairs of probes to exponentially amplify a specific target.","The sequences of each pair of oligonucleotides are selected to permit the pair to hybridize to abutting sequences of the same strand of the target.","Such hybridization forms a substrate for a template-dependant ligase.","In accordance with the present invention, LCR can be performed with oligonucleotides having the proximal and distal sequences of the same strand of a biallelic marker site.","In one embodiment, either oligonucleotide will be designed to include the biallelic marker site.","In such an embodiment, the reaction conditions are selected such that the oligonucleotides can be ligated together only if the target molecule either contains or lacks the specific nucleotide that is complementary to the biallelic marker on the oligonucleotide.","In an alternative embodiment, the oligonucleotides will not include the biallelic marker, such that when they hybridize to the target molecule, a “gap” is created as described in WO 90/01069.This gap is then “filled” with complementary dNTPs (as mediated by DNA polymerase), or by an additional pair of oligonucleotides.","Thus at the end of each cycle, each single strand has a complement capable of serving as a target during the next cycle and exponential allele-specific amplification of the desired sequence is obtained.","Ligase/Polymerase-mediated Genetic Bit Analysis™ is another method for determining the identity of a nucleotide at a preselected site in a nucleic acid molecule (WO 95/21271).","This method involves the incorporation of a nucleoside triphosphate that is complementary to the nucleotide present at the preselected site onto the terminus of a primer molecule, and their subsequent ligation to a second oligonucleotide.","The reaction is monitored by detecting a specific label attached to the reaction's solid phase or by detection in solution.","4) Hybridization Assay Methods A preferred method of determining the identity of the nucleotide present at a biallelic marker site involves nucleic acid hybridization.","The hybridization probes, which can be conveniently used in such reactions, preferably include probes specific for GSSP4 cDNA surrounding GSSP4 biallelic markers.","Any hybridization assay may be used including Southern hybridization, Northern hybridization, dot blot hybridization and solid-phase hybridization (see Sambrook et al., supra).","Hybridization refers to the formation of a duplex structure by two single stranded nucleic acids due to complementary base pairing.","Hybridization can occur between exactly complementary nucleic acid strands or between nucleic acid strands that contain minor regions of mismatch.","Specific probes can be designed that hybridize to one form of a biallelic marker and not to the other and therefore are able to discriminate between different allelic forms.","Allele-specific probes are often used in pairs, one member of a pair showing perfect match to a target sequence containing the original allele and the other showing a perfect match to the target sequence containing the alternative allele.","Hybridization conditions should be sufficiently stringent that there is a significant difference in hybridization intensity between alleles, and preferably an essentially binary response, whereby a probe hybridizes to only one of the alleles.","Stringent, sequence specific hybridization conditions, under which a probe will hybridize only to the exactly complementary target sequence are well known in the art (Sambrook et al., supra).","Stringent conditions are sequence dependent and will be different in different circumstances.","Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.","Although such hybridizations can be performed in solution, it is preferred to employ a solid-phase hybridization assay.","The target DNA comprising a biallelic marker of the present invention may be amplified prior to the hybridization reaction.","The presence of a specific allele in the sample is determined by detecting the presence or the absence of stable hybrid duplexes formed between the probe and the target DNA.","The detection of hybrid duplexes can be carried out by a number of methods.","Various detection assay formats are well known which utilize detectable labels bound to either the target or the probe to enable detection of the hybrid duplexes.","Typically, hybridization duplexes are separated from unhybridized nucleic acids and the labels bound to the duplexes are then detected.","Those skilled in the art will recognize that wash steps may be employed to wash away excess target DNA or probe as well as unbound conjugate.","Further, standard heterogeneous assay formats are suitable for detecting the hybrids using the labels present on the primers and probes.","Two recently developed assays allow hybridization-based allele discrimination with no need for separations or washes (see Landegren U. et al., (1998) Genome Res.","August;8(8):769-76).","The TaqMan assay takes advantage of the 5′ nuclease activity of Taq DNA polymerase to digest a DNA probe annealed specifically to the accumulating amplification product.","TaqMan probes are labeled with a donor-acceptor dye pair that interacts via fluorescence resonance energy transfer (FRET).","Cleavage of the TaqMan probe by the advancing polymerase during amplification dissociates the donor dye from the quenching acceptor dye, greatly increasing the donor fluorescence.","All reagents necessary to detect two allelic variants can be assembled at the beginning of the reaction and the results are monitored in real time (see Livak et al., 1995).","In an alternative homogeneous hybridization based procedure, molecular beacons are used for allele discriminations.","Molecular beacons are hairpin-shaped oligonucleotide probes that report the presence of specific nucleic acids in homogeneous solutions.","When they bind to their targets they undergo a conformational reorganization that restores the fluorescence of an internally quenched fluorophore (Tyagi et al., (1998) Nat Biotechnol.","January;16(1):49-53).","The polynucleotides provided herein can be used to produce probes which can be used in hybridization assays for the detection of biallelic marker alleles in biological samples.","These probes are characterized in that they preferably comprise between 8 and 50 nucleotides, and in that they are sufficiently complementary to a sequence comprising a biallelic marker of the present invention to hybridize thereto and preferably sufficiently specific to be able to discriminate the targeted sequence for only one nucleotide variation.","A particularly preferred probe is 25 nucleotides in length.","Preferably the biallelic marker is within 4 nucleotides of the center of the polynucleotide probe.","In particularly preferred probes, the biallelic marker is at the center of said polynucleotide.","In preferred embodiments the polymorphic base is within 5, 4, 3, 2, 1, nucleotides of the center of the said polynucleotide, more preferably at the center of said polynucleotide.","Preferably the probes of the present invention are labeled or immobilized on a solid support.","By assaying the hybridization to an allele specific probe, one can detect the presence or absence of a biallelic marker allele in a given sample.","High-Throughput parallel hybridizations in array format are specifically encompassed within “hybridization assays” and are described below.","5) Hybridization To Addressable Arrays Of Oligonucleotides Hybridization assays based on oligonucleotide arrays rely on the differences in hybridization stability of short oligonucleotides to perfectly matched and mismatched target sequence variants.","Efficient access to polymorphism information is obtained through a basic structure comprising high-density arrays of oligonucleotide probes attached to a solid support (e.g., the chip) at selected positions.","Each DNA chip can contain thousands to millions of individual synthetic DNA probes arranged in a grid-like pattern and miniaturized to the size of a dime.","The chip technology has already been applied with success in numerous cases.","For example, the screening of mutations has been undertaken in the BRCA1 gene, in S. cerevisiae mutant strains, and in the protease gene of HIV-1 virus (Hacia et al., (1996) Nat Genet.","December; 14(4):441-7; Shoemaker et al., (1996) Nat Genet December; 14(4):450-6; Kozal et al., (1996) Nat Med.","July;2(7):753-9).","Chips of various formats for use in detecting biallelic polymorphisms can be produced on a customized basis by Affymetrix (GeneChip™), Hyseq (HyChip and HyGnostics), and Protogene Laboratories.","In general, these methods employ arrays of oligonucleotide probes that are complementary to target nucleic acid sequence segments from an individual, which target sequences include a polymorphic marker.","EP 785280 describes a tiling strategy for the detection of single nucleotide polymorphisms.","Briefly, arrays may generally be “tiled” for a large number of specific polymorphisms.","By “tiling” is generally meant the synthesis of a defined set of oligonucleotide probes which is made up of a sequence complementary to the target sequence of interest, as well as preselected variations of that sequence, e.g., substitution of one or more given positions with one or more members of the basis set of monomers, i.e.","nucleotides.","Tiling strategies are further described in PCT application No.","WO 95/11995.In a particular aspect, arrays are tiled for a number of specific, identified biallelic marker sequences.","In particular, the array is tiled to include a number of detection blocks, each detection block being specific for a specific biallelic marker or a set of biallelic markers.","For example, a detection block may be tiled to include a number of probes, which span the sequence segment that includes a specific polymorphism.","To ensure probes that are complementary to each allele, the probes are synthesized in pairs differing at the biallelic marker.","In addition to the probes differing at the polymorphic base, monosubstituted probes are also generally tiled within the detection block.","These monosubstituted probes have bases at and up to a certain number of bases in either direction from the polymorphism, substituted with the remaining nucleotides (selected from A, T, G, C and U).","Typically the probes in a tiled detection block will include substitutions of the sequence positions up to and including those that are 5 bases away from the biallelic marker.","The monosubstituted probes provide internal controls for the tiled array, to distinguish actual hybridization from artefactual cross-hybridization.","Upon completion of hybridization with the target sequence and washing of the array, the array is scanned to determine the position on the array to which the target sequence hybridizes.","The hybridization data from the scanned array is then analyzed to identify which allele or alleles of the biallelic marker are present in the sample.","Hybridization and scanning may be carried out as described in PCT application No.","WO 92/10092 and WO 95/11995 and U.S. Pat.","No.","5,424,186.Thus, in some embodiments, the chips may comprise an array of nucleic acid sequences of fragments of about 15 nucleotides in length.","In preferred embodiments the polymorphic base is within 5, 4, 3, 2, 1, nucleotides of the center of the said polynucleotide, more preferably at the center of said polynucleotide.","In some embodiments, the chip may comprise an array of at least 2, 3, 4, 5, 6, 7, 8 or more of these polynucleotides.","6) Integrated Systems Another technique, which may be used to analyze polymorphisms, includes multicomponent integrated systems, which miniaturize and compartmentalize processes such as PCR and capillary electrophoresis reactions in a single functional device.","An example of such technique is disclosed in U.S. Pat.","No.","5,589,136, which describes the integration of PCR amplification and capillary electrophoresis in chips.","Integrated systems can be envisaged mainly when microfluidic systems are used.","These systems comprise a pattern of microchannels designed onto a glass, silicon, quartz, or plastic wafer included on a microchip.","The movements of the samples are controlled by electric, electroosmotic or hydrostatic forces applied across different areas of the microchip to create functional microscopic valves and pumps with no moving parts.","Varying the voltage controls the liquid flow at intersections between the micro-machined channels and changes the liquid flow rate for pumping across different sections of the microchip.","For genotyping biallelic markers, the microfluidic system may integrate nucleic acid amplification, microsequencing, capillary electrophoresis and a detection method such as laser-induced fluorescence detection.","In a first step, the DNA samples are amplified, preferably by PCR.","Then, the amplification products are subjected to automated microsequencing reactions using ddNTPs (specific fluorescence for each ddNTP) and the appropriate oligonucleotide microsequencing primers which hybridize just upstream of the targeted polymorphic base.","Once the extension at the 3′ end is completed, the primers are separated from the unincorporated fluorescent ddNTPs by capillary electrophoresis.","The separation medium used in capillary electrophoresis can for example be polyacrylamide, polyethyleneglycol or dextran.","The incorporated ddNTPs in the single-nucleotide primer extension products are identified by fluorescence detection.","This microchip can be used to process at least 96 to 384 samples in parallel.","It can use the usual four color laser induced fluorescence detection of the ddNTPs.","GSSP4 Association Studies Association studies focus on population frequencies and rely on the phenomenon of linkage disequilibrium.","Linkage disequilibrium is the deviation from random of the occurrence of pairs of specific alleles at different loci on the same chromosome.","If a specific allele in a given gene is directly associated with a particular trait, its frequency will be statistically increased in an affected (trait positive) population, when compared to the frequency in a trait negative population or in a random control population.","As a consequence of the existence of linkage disequilibrium, the frequency of all other alleles present in the haplotype carrying the trait-causing allele will also be increased in trait positive individuals compared to trait negative individuals or random controls.","Therefore, association between the trait and any allele (specifically a biallelic marker allele) in linkage disequilibrium with the trait-causing allele will suffice to suggest the presence of a trait-related gene in that particular region.","Case-control populations can be genotyped for biallelic markers to identify associations that narrowly locate a trait causing allele, as any marker in linkage disequilibrium with one given marker associated with a trait will be associated with the trait.","Linkage disequilibrium allows the relative frequencies in case-control populations of a limited number of genetic polymorphisms (specifically biallelic markers) to be analyzed as an alternative to screening all possible functional polymorphisms in order to find trait-causing alleles.","Association studies compare the frequency of marker alleles in unrelated case-control populations, and represent powerful tools for the dissection of complex traits.","Case-Control Populations (Inclusion Criteria) Population-based association studies do not concern familial inheritance, but compare the prevalence of a particular genetic marker, or a set of markers, in case-control populations.","They are case-control studies based on comparison of unrelated case (affected or trait positive) individuals and unrelated control (unaffected, trait negative or random) individuals.","Preferably, the control group is composed of unaffected or trait negative individuals.","Further, the control group is ethnically matched to the case population.","Moreover, the control group is preferably matched to the case-population for the main known confusion factor for the trait under study (for example age-matched for an age-dependent trait).","Ideally, individuals in the two samples are paired in such a way that they are expected to differ only in their disease status.","The terms “trait positive population”, “case population” and “affected population” are used interchangeably herein.","An important step in the dissection of complex traits using association studies is the choice of case-control populations (see, Lander and Schork, (1994) Science, September 30;265(5181):2037-48).","A major step in the choice of case-control populations is the clinical definition of a given trait or phenotype.","Any genetic trait may be analyzed by the association method proposed here by carefully selecting the individuals to be included in the trait positive and trait negative phenotypic groups.","Four criteria are often useful: clinical phenotype, age at onset, family history and severity.","The selection procedure for continuous or quantitative traits (such as blood pressure for example) involves selecting individuals at opposite ends of the phenotype distribution of the trait under study, so as to include in these trait positive and trait negative populations individuals with non-overlapping phenotypes.","Preferably, case-control populations consist of phenotypically homogeneous populations.","Trait positive and trait negative populations consist of phenotypically uniform populations of individuals representing each between 1 and 98%, preferably between 1 and 80%, more preferably between 1 and 50%, and more preferably between 1 and 30%, most preferably between 1 and 20% of the total population under study, and preferably selected among individuals exhibiting non-overlapping phenotypes.","The clearer the difference between the two trait phenotypes, the greater the probability of detecting an association with biallelic markers.","The selection of those drastically different but relatively uniform phenotypes enables efficient comparisons in association studies and the possible detection of marked differences at the genetic level, provided that the sample sizes of the populations under study are significant enough.","In preferred embodiments, a first group of between 50 and 300 trait positive individuals, preferably about 100 individuals, are recruited according to their phenotypes.","A similar number of trait negative individuals are included in such studies.","In the present invention, typical examples of inclusion criteria include obesity and disorders related to obesity as well as physiologic parameters associated with obesity, such as free fatty acid levels, glucose levels, insulin levels, leptin levels, triglyceride levels, free fatty acid oxidation levels, and weight loss.","Association Analysis The general strategy to perform association studies using biallelic markers derived from a region carrying a candidate gene is to scan two groups of individuals (case-control populations) in order to measure and statistically compare the allele frequencies of the biallelic markers of the present invention in both groups.","If a statistically significant association with a trait is identified for at least one or more of the analyzed biallelic markers, one can assume that: either the associated allele is directly responsible for causing the trait (i.e.","the associated allele is the trait causing allele), or more likely the associated allele is in linkage disequilibrium with the trait causing allele.","The specific characteristics of the associated allele with respect to the candidate gene function usually give further insight into the relationship between the associated allele and the trait (causal or in linkage disequilibrium).","If the evidence indicates that the associated allele within the candidate gene is most probably not the trait-causing allele but is in linkage disequilibrium with the real trait-causing allele, then the trait-causing allele can be found by sequencing the vicinity of the associated marker, and performing further association studies with the polymorphisms that are revealed in an iterative manner.","Association studies are usually run in two successive steps.","In a first phase, the frequencies of a reduced number of biallelic markers from the candidate gene are determined in the trait positive and trait negative populations.","In a second phase of the analysis, the position of the genetic loci responsible for the given trait is further refined using a higher density of markers from the relevant region.","However, if the candidate gene under study is relatively small in length a single phase may be sufficient to establish significant associations.","Haplotype Analysis As described above, when a chromosome carrying a disease allele first appears in a population as a result of either mutation or migration, the mutant allele necessarily resides on a chromosome having a set of linked markers: the ancestral haplotype.","This haplotype can be tracked through populations and its statistical association with a given trait can be analyzed.","Complementing single point (allelic) association studies with multi-point association studies also called haplotype studies increases the statistical power of association studies.","Thus, a haplotype association study allows one to define the frequency and the type of the ancestral carrier haplotype.","A haplotype analysis is important in that it increases the statistical power of an analysis involving individual markers.","In a first stage of a haplotype frequency analysis, the frequency of the possible haplotypes based on various combinations of the identified biallelic markers of the invention is determined.","The haplotype frequency is then compared for distinct populations of trait positive and control individuals.","The number of trait positive individuals, which should be, subjected to this analysis to obtain statistically significant results usually ranges between 30 and 300, with a preferred number of individuals ranging between 50 and 150.The same considerations apply to the number of unaffected individuals (or random control) used in the study.","The results of this first analysis provide haplotype frequencies in case-control populations, for each evaluated haplotype frequency a p-value and an odds ratio are calculated.","If a statistically significant association is found the relative risk for an individual carrying the given haplotype of being affected with the trait under study can be approximated.","Interaction Analysis The biallelic markers of the present invention may also be used to identify patterns of biallelic markers associated with detectable traits resulting from polygenic interactions.","The analysis of genetic interaction between alleles at unlinked loci requires individual genotyping using the techniques described herein.","The analysis of allelic interaction among a selected set of biallelic markers with appropriate level of statistical significance can be considered as a haplotype analysis.","Interaction analysis consists in stratifying the case-control populations with respect to a given haplotype for the first loci and performing a haplotype analysis with the second loci with each subpopulation.","VIII.","Assays for Identifying Modulators of GSSP4 Polypeptide Activity The invention features methods of screening for one or more compounds that modulate the activity of GSSP4s in cells, which includes providing potential compounds to be tested to the cells.","Exemplary assays that may be used are described in the Examples 2, 5-7,9-11.To these assays would be added compounds to be tested for their inhibitory or stimulatory activity as compared to the effects of GSSP4 polypeptides alone.","Other assays in which an effect is observed based on the addition of GSSP4 polypeptides can also be used to screen for modulators of GSSP4 polypeptide activity or effects of the presence of GSSP4 polypeptides on cells.","The essential step is to apply an unknown compound and then to monitor an assay for a change from what is seen when only GSSP4 polypeptides are applied to the cell.","A change is defined as something that is significantly different in the presence of the compound plus GSSP4 polypeptide compared to GSSP4 polypeptide alone.","In this case, significantly different would be an “increase” or a “decrease” in a measurable effect of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%.","The term “modulation” as used herein refers to a measurable change in an activity.","Examples include, but are not limited to, lipolysis stimulated receptor (LSR) modulation, leptin modulation, lipoprotein modulation, plasma FFA levels, FFA oxidation, TG levels, glucose levels, and weight.","These effects can be in vitro or preferably in vivo.","Modulation of an activity can be either an increase or a decrease in the activity.","Thus, LSR activity can be increased or decreased, leptin activity can be increased or decreased, and lipoprotein activity can be increased or decreased.","Similarly, FFA, TG, and glucose levels (and weight) can be increased or decreased in vivo Free Fatty Acid oxidation can be increased or decreased in vivo or ex vivo.","By “LSR” activity is meant expression of LSR on the surface of the cell, or in a particular conformation, as well as its ability to bind, uptake, and degrade leptin and lipoprotein.","By “leptin” activity is meant its binding, uptake and degradation by LSR, as well as its transport across a blood brain barrier, and potentially these occurrences where LSR is not necessarily the mediating factor or the only mediating factor.","Similarly, by “lipoprotein” activity is meant its binding, uptake and degradation by LSR, as well as these occurrences where LSR is not necessarily the mediating factor or the only mediating factor.","Exemplary assays are provided in Examples 2, 5-7,9-11.These assay and other comparable assays can be used to determine/identify compounds that modulate GSSP4 polypeptide activity.","In some cases it may be important to identify compounds that modulate some but not all of the GSSP4 polypeptide activities, although preferably all activities are modified.","The term “increasing” as used herein refers to the ability of a compound to increase the activity of GSSP4 polypeptides in some measurable way compared to the effect of GSSP4 polypeptides in its absence.","As a result of the presence of the compound leptin binding and/or uptake might increase, for example, as compared to controls in the presence of the GSSP4 polypeptide alone.","Preferably, an increase in activity is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% compared to the level of activity in the presence of the GSSP4 polypeptide.","Similarly, the term “decreasing” as used herein refers to the ability of a compound to decrease an activity in some measurable way compared to the effect of a GSSP4 polypeptide in its absence.","For example, the presence of the compound decreases the plasma concentrations of FFA, TG, and glucose in mice.","Also as a result of the presence of a compound leptin binding and/or uptake might decrease, for example, as compared to controls in the presence of the GSSP4 polypeptide alone.","Preferably, an decrease in activity is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% as compared to the level of activity in the presence of the GSSP4 polypeptide alone.","The invention features a method for identifying a potential compound to modulate body mass in individuals in need of modulating body mass comprising: a) contacting a cell with a GSSP4 polypeptide and a candidate compound; b) detecting a result selected from the group consisting of LSR modulation, leptin modulation, lipoprotein modulation; FFA oxidation modulation; and c) wherein said result identifies said potential compound if said result differs from said result when said cell is contacted with the GSSP4 polypeptide alone.","In preferred embodiments, said contacting further comprises a ligand of said LSR.","Preferably said ligand is selected from the group consisting of cytokine, lipoprotein, free fatty acids, and C1q, and more preferably said cytokine is leptin, and most preferably said leptin is a leptin polypeptide fragment as described in U.S.","Provisional application No.","60/155,506 hereby incorporated by reference herein in its entirety including any figures, drawings, or tables.","In other preferred embodiments, said GSSP4 polypeptide is mouse or is human.","In other preferred embodiments, said cell is selected from the group consisting of PLC, CHO-K1, Hep3B, and HepG2.In yet other preferred embodiments, said lipoprotein modulation is selected from the group consisting of binding, uptake, and degradation.","Preferably, said modulation is an increase in said binding, uptake, or degradation.","Alternatively, said modulation is a decrease in said binding, uptake, or degradation.","In other preferred embodiments, leptin modulation is selected from the group consisting of binding, uptake, degradation, and transport.","Preferably, said modulation is an increase in said binding, uptake, degradation, or transport.","Alternatively, said modulation is a decrease in said binding, uptake, degradation, or transport.","Preferably, said transport is across a blood-brain barrier.","In yet other preferred embodiments, said LSR modulation is expression on the surface of said cell.","Preferably, said detecting comprises FACS, more preferably said detecting further comprises antibodies that bind specifically to said LSR, and most preferably said antibodies bind specifically to the carboxy terminus of said LSR.","In still other preferred embodiments, said potential compound is selected from the group consisting of peptides, peptide libraries, non-peptide libraries, peptoids, fatty acids, lipoproteins, medicaments, antibodies, small molecules, and proteases.","IX.","Epitopes and Antibody Fusions A preferred embodiment of the present invention is directed to eiptope-bearing polypeptides and epitope-bearing polypeptide fragments.","These epitopes may be “antigenic epitopes” or both an “antigenic epitope” and an “immunogenic epitope”.","An “immunogenic epitope” is defined as a part of a protein that elicits an antibody response in vivo when the polypeptide is the immunogen.","On the other hand, a region of polypeptide to which an antibody binds is defined as an “antigenic determinant” or “antigenic epitope.” The number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes.","See, e.g., Geysen, et al.","(1983) Proc.","Natl.","Acad.","Sci.","USA 81:39984002.It is particularly noted that although a particular epitope may not be immunogenic, it is nonetheless useful since antibodies can be made in vitro to any epitope.","An epitope can comprise as few as 3 amino acids in a spatial conformation which is unique to the epitope.","Generally an epitope consists of at least 6 such amino acids, and more often at least 8-10 such amino acids.","In preferred embodiment, antigenic epitopes comprise a number of amino acids that is any integer between 3 and 50.Fragments which function as epitopes may be produced by any conventional means.","See, e.g., Houghten, R. A., Proc.","Natl.","Acad.","Sci.","USA 82:5131-5135 (1985), further described in U.S. Pat.","No.","4,631,211.Methods for determining the amino acids which make up an immunogenic epitope include x-ray crystallography, 2-dimensional nuclear magnetic resonance, and epitope mapping, e.g., the Pepscan method described by H. Mario Geysen et al.","(1984); Proc.","Natl.","Acad.","Sci.","U.S.A. 81:3998-4002; PCT Publication No.","WO 84/03564; and PCT Publication No.","WO 84/03506.Another example is the algorithm of Jameson and Wolf, Comp.","Appl.","Biosci.","4:181-186 (1988) (said references incorporated by reference in their entireties).","The Jameson-Wolf antigenic analysis, for example, may be performed using the computer program PROTEAN, using default parameters (Version 4.0 Windows, DNASTAR, Inc., 1228 South Park Street Madison, Wis.).","The epitope-bearing fragments of the present invention preferably comprises 6 to 50 amino acids (i.e.","any integer between 6 and 50, inclusive) of a polypeptide of the present invention.","Also, included in the present invention are antigenic fragments between the integers of 6 and the full length sequence of the sequence listing.","All combinations of sequences between the integers of 6 and the full-length sequence of a polypeptide of the present invention are included.","The epitope-bearing fragments may be specified by either the number of contiguous amino acid residues (as a sub-genus) or by specific N-terminal and C-terminal positions (as species) as described above for the polypeptide fragments of the present invention.","Any number of epitope-bearing fragments of the present invention may also be excluded in the same manner.","Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies that specifically bind the epitope (See, Wilson et al., 1984; and Sutcliffe, J. G. et al., 1983).","The antibodies are then used in various techniques such as diagnostic and tissue/cell identification techniques, as described herein, and in purification methods.","Similarly, immunogenic epitopes can be used to induce antibodies according to methods well known in the art (See, Sutcliffe et al., supra; Wilson et al., supra; Chow, M. et al.",";(1985) and Bittle, F. J. et al., (1985).","A preferred immunogenic epitope includes the polypeptides of the sequence listing.","The immunogenic epitopes may be presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse) if nessary.","Immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting.).","Epitope-bearing polypeptides of the present invention are used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods (See, e.g., Sutcliffe, et al., supra; Wilson, et al., supra, and Bittle, et al., 1985).","If in vivo immunization is used, animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling of the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid.","For instance, peptides containing cysteine residues may be coupled to a carrier using a linker such as—maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde.","Animals such as rabbits, rats and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 μgs of peptide or carrier protein and Freund's adjuvant.","Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody, which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface.","The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.","As one of skill in the art will appreciate, and discussed above, the polypeptides of the present invention including, but not limited to, polypeptides comprising an immunogenic or antigenic epitope can be fused to heterologous polypeptide sequences.","For example, the polypeptides of the present invention may be fused with the constant region comprising portions of immunoglobulins (IgA, IgE, IgG, IgM), or portions of the constant region (CH1, CH2, CH3, any combination thereof including both entire domains and portions thereof) resulting in chimeric polypeptides.","These fusion proteins facilitate purification, and show an increased half-life in vivo.","This has been shown, e.g., for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (See, e.g., EPA 0,394,827; and Traunecker et al., 1988).","Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion can also be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone (See, e.g., Fountoulakis et al., 1995).","Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag to aid in detection and purification of the expressed polypeptide.","Additonal fusion proteins of the invention may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, or codon-shuffling (collectively referred to as “DNA shuffling”).","DNA shuffling may be employed to modulate the activities of polypeptides of the present invention thereby effectively generating agonists and antagonists of the polypeptides.","See, for example, U.S. Pat.","Nos.","5,605,793; 5,811,238; 5,834,252; 5,837,458; and Patten, P. A., et al., (1997); Harayama, S., (1998); Hansson, L. O., et al (1999); and Lorenzo, M. M. and Blasco, R., (1998).","(Each of these documents are hereby incorporated by reference).","In one embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of coding polynucleotides of the invention, or the polypeptides encoded thereby may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc.","of one or more heterologous molecules.","Antibodies: The present invention further relates to antibodies and T-cell antigen receptors (TCR), which specifically bind the polypeptides, and more specifically, the epitopes of the polyepeptides of the present invention.","The antibodies of the present invention include IgG (including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM, and IgY.","As used herein, the term “antibody” (Ab) is meant to include whole antibodies, including single-chain whole antibodies, and antigen binding fragments thereof.","In a preferred embodiment the antibodies are human antigen binding antibody fragments of the present invention include, but are not limited to, Fab, Fab′ F(ab)2 and F(ab)2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain.","The antibodies may be from any animal origin including birds and mammals.","Preferably, the antibodies are human, murine, rabbit, goat, guinea pig, camel, horse, or chicken.","Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entire or partial of the following: hinge region, CH1, CH2, and CH3 domains.","Also included in the invention are any combinations of variable region(s) and hinge region, CH1, CH2, and CH3 domains.","The present invention further includes chimeric, humanized, and human monoclonal and polyclonal antibodies, which specifically bind the polypeptides of the present invention.","The present invention further includes antibodies that are anti-idiotypic to the antibodies of the present invention.","The antibodies of the present invention may be monospecific, bispecific, and trispecific or have greater multispecificity.","Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for heterologous compositions, such as a heterologous polypeptide or solid support material.","See, e.g., WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, A. et al.","(1991); U.S. Pat.","Nos.","5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648; Kostelny, S. A. et al.","(1992).","Antibodies of the present invention may be described or specified in terms of the epitope(s) or epitope-bearing portion(s) of a polypeptide of the present invention, which are recognized or specifically bound by the antibody.","In the case of proteins of the present invention secreted proteins, the antibodies may specifically bind a full-length protein encoded by a nucleic acid of the present invention, a mature protein (i.e., the protein generated by cleavage of the signal peptide) encoded by a nucleic acid of the present invention, a signal peptide encoded by a nucleic acid of the present invention, or any other polypeptide of the present invention.","Therefore, the epitope(s) or epitope bearing polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous amino acid residues, or otherwise described herein (including the sequence listing).","Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded as individual species.","Therefore, the present invention includes antibodies that specifically bind specified polypeptides of the present invention, and allows for the exclusion of the same.","Antibodies of the present invention may also be described or specified in terms of their cross-reactivity.","Antibodies that do not specifically bind any other analog, ortholog, or homolog of the polypeptides of the present invention are included.","Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein, eg., using FASTDB and the parameters set forth herein) to a polypeptide of the present invention are also included in the present invention.","Further included in the present invention are antibodies, which only bind polypeptides encoded by polynucleotides, which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described herein).","Antibodies of the present invention may also be described or specified in terms of their binding affinity.","Preferred binding affinities include those with a dissociation constant or Kd value less than 5×10−6M, 10−6M, 5×10−7M, 10−7M, 5×10−8M, 10−8M, 5×10−9M, 10−9M, 5×10−10M, 10−10M, 5×10−11M, 10−11M, 5×10−12M, 10−12M, 5×10−13M, 10−13M, 5×10−14M, 10−14M, 5×10−15M, and 10−15M.","Antibodies of the present invention have uses that include, but are not limited to, methods known in the art to purify, detect, and target the polypeptides of the present invention including both in vitro and in vivo diagnostic and therapeutic methods.","For example, the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples (See, e.g., Harlow et al., 1988).","The antibodies of the present invention may be used either alone or in combination with other compositions.","The antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions.","For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, or toxins.","See, e.g., WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat.","No.","5,314,995; and EP 0 396 387.The antibodies of the present invention may be prepared by any suitable method known in the art.","For example, a polypeptide of the present invention or an antigenic fragment thereof can be administered to an animal in order to induce the production of sera containing polyclonal antibodies.","The term “monoclonal antibody” is not limited to antibodies produced through hybridoma technology.","The term “antibody” refers to a polypeptide or group of polypeptides which are comprised of at least one binding domain, where a binding domain is formed from the folding of variable domains of an antibody molecule to form three-dimensional binding spaces with an internal surface shape and charge distribution complementary to the features of an antigenic determinant of an antigen, which allows an immunological reaction with the antigen.","The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.","Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technology.","Hybridoma techniques include those known in the art (See, e.g., Harlow et al.","1988); Hammerling, et al, 1981).","(Said references incorporated by reference in their entireties).","Fab and F(ab′)2 fragments may be produced, for example, from hybridoma-produced antibodies by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments).","Alternatively, antibodies of the present invention can be produced through the application of recombinant DNA technology or through synthetic chemistry using methods known in the art.","For example, the antibodies of the present invention can be prepared using various phage display methods known in the art.","In phage display methods, functional antibody domains are displayed on the surface of a phage particle, which carries polynucleotide sequences encoding them.","Phage with a desired binding property are selected from a repertoire or combinatorial antibody library (e.g.","human or murine) by selecting directly with antigen, typically antigen bound or captured to a solid surface or bead.","Phage used in these methods are typically filamentous phage including fd and M13 with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein.","Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman U. et al.","(1995); Ames, R. S. et al.","(1995); Kettleborough, C. A. et al.","(1994); Persic, L. et al.","(1997); Burton, D. R. et al.","(1994); PCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat.","Nos.","5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727 and 5,733,743.As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host including mammalian cells, insect cells, plant cells, yeast, and bacteria.","For example, techniques to recombinantly produce Fab, Fab′ F(ab)2 and F(ab′)2 fragments can also be employed using methods known in the art such as those disclosed in WO 92/22324; Mullinax, R. L. et al.","(1992); and Sawai, H. et al.","(1995); and Better, M. et al.","(1988).","Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat.","Nos.","4,946,778 and 5,258,498; Huston et al.","(1991); Shu, L. et al.","(1993); and Skerra, A. et al.","(1988).","For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies.","Methods for producing chimeric antibodies are known in the art.","See e.g., Morrison, (1985); Oi et al., (1986); Gillies, S. D. et al.","(1989); and U.S. Pat.","No.","5,807,715.Antibodies can be humanized using a variety of techniques including CDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat.","Nos.","5,530,101; and 5,585,089), veneering or resurfacing, (EP 0 592 106; EP 0 519 596; Padlan E. A., 1991; Studnicka G. M. et al., 1994; Roguska M. A. et al., 1994), and chain shuffling (U.S. Pat.","No.","5,565,332).","Human antibodies can be made by a variety of methods known in the art including phage display methods described above.","See also, U.S. Pat.","Nos.","4,444,887,4,716,111, 5,545,806, and 5,814,318; WO 98/46645; WO 98/50433; WO 98/24893; WO 96/34096; WO 96/33735; and WO 91/10741.Further included in the present invention are antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide of the present invention.","The antibodies may be specific for antigens other than polypeptides of the present invention.","For example, antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors.","Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vitro immunoassays and purification methods using methods known in the art (See e.g., Harbor et al.","supra; WO 93/21232; EP 0 439 095; Naramura, M. et al.","1994; U.S. Pat.","No.","5,474,981; Gillies, S. O. et al., 1992; Fell, H. P. et al., 1991).","The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions.","For example, the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof.","The antibody portion fused to a polypeptide of the present invention may comprise the hinge region, CH1 domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof.","The polypeptides of the present invention may be fused or conjugated to the above antibody portions to increase the in vivo half-life of the polypeptides or for use in immunoassays using methods known in the art.","The polypeptides may also be fused or conjugated to the above antibody portions to form multimers.","For example, Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions.","Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM.","Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art.","See e.g., U.S. Pat.","Nos.","5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,112,946; EP 0 307 434, EP 0 367 166; WO 96/04388, WO 91/06570; Ashkenazi, A. et al.","(1991); Zheng, X. X. et al.","(1995); and Vil, H. et al.","(1992).","The invention further relates to antibodies that act as agonists or antagonists of the polypeptides of the present invention.","For example, the present invention includes antibodies that disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully.","Included are both receptor-specific antibodies and ligand-specific antibodies.","Included are receptor-specific antibodies, which do not prevent ligand binding but prevent receptor activation.","Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art.","Also include are receptor-specific antibodies which both prevent ligand binding and receptor activation.","Likewise, included are neutralizing antibodies that bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies that bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor.","Further included are antibodies that activate the receptor.","These antibodies may act as agonists for either all or less than all of the biological activities affected by ligand-mediated receptor activation.","The antibodies may be specified as agonists or antagonists for biological activities comprising specific activities disclosed herein.","The above antibody agonists can be made using methods known in the art.","See e.g., WO 96/40281; U.S. Pat.","No.","5,811,097; Deng, B. et al.","(1998); Chen, Z. et al.","(1998); Harrop, J.","A. et al.","(1998); Zhu, Z. et al.","(1998); Yoon, D. Y. et al.","(1998); Prat, M. et al.","(1998) J.; Pitard, V. et al.","(1997); Liautard, J. et al.","(1997); Carlson, N. G. et al.","(1997) J.; Taryman, R. E. et al.","(1995); Muller, Y.","A. et al.","(1998); Bartunek, P. et al.","(1996).","As discussed above, antibodies of the polypeptides of the invention can, in turn, be utilized to generate anti-idiotypic antibodies that “mimic” polypeptides of the invention using techniques well known to those skilled in the art (See, e.g.","Greenspan and Bona (1989); and Nissinoff (1991).","For example, antibodies which bind to and competitively inhibit polypeptide multimerization or binding of a polypeptide of the invention to ligand can be used to generate anti-idiotypes that “mimic” the polypeptide multimerization or binding domain and, as a consequence, bind to and neutralize polypeptide or its ligand.","Such neutralization anti-idiotypic antibodies can be used to bind a polypeptide of the invention or to bind its ligands/receptors, and therby block its biological activity, The invention also concerns a purified or isolated antibody capable of specifically binding to a mutated full length or mature polypeptide of the present invention or to a fragment or variant thereof comprising an epitope of the mutated polypeptide.","In another preferred embodiment, the present invention concerns an antibody capable of binding to a polypeptide comprising at least 10 consecutive amino acids of a polypeptide of the present invention and including at least one of the amino acids which can be encoded by the trait causing mutations.","Non-human animals or mammals, whether wild-type or transgenic, which express a different species of a polypeptide of the present invention than the one to which antibody binding is desired, and animals which do not express a polypeptide of the present invention (i.e.","a knock out animal) are particularly useful for preparing antibodies.","Gene knock out animals will recognize all or most of the exposed regions of a polypeptide of the present invention as foreign antigens, and therefore produce antibodies with a wider array of epitopes.","Moreover, smaller polypeptides with only 10 to 30 amino acids may be useful in obtaining specific binding to any one of the polypeptides of the present invention.","In addition, the humoral immune system of animals which produce a species of a polypeptide of the present invention that resembles the antigenic sequence will preferentially recognize the differences between the animal's native polypeptide species and the antigen sequence, and produce antibodies to these unique sites in the antigen sequence.","Such a technique will be particularly useful in obtaining antibodies that specifically bind to any one of the polypeptides of the present invention.","Antibody preparations prepared according to either protocol are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample.","The antibodies may also be used in therapeutic compositions for killing cells expressing the protein or reducing the levels of the protein in the body.","The antibodies of the invention may be labeled by any one of the radioactive, fluorescent or enzymatic labels known in the art.","Consequently, the invention is also directed to a method for detecting specifically the presence of a polypeptide of the present invention according to the invention in a biological sample, said method comprising the following steps: a) bringing into contact the biological sample with a polyclonal or monoclonal antibody that specifically binds a polypeptide of the present invention; and b) detecting the antigen-antibody complex formed.","The invention also concerns a diagnostic kit for detecting in vitro the presence of a polypeptide of the present invention in a biological sample, wherein said kit comprises: a) a polyclonal or monoclonal antibody that specifically binds a polypeptide of the present invention, optionally labeled; b) a reagent allowing the detection of the antigen-antibody complexes formed, said reagent carrying optionally a label, or being able to be recognized itself by a labeled reagent, more particularly in the case when the above-mentioned monoclonal or polyclonal antibody is not labeled by itself.","The antibodies of the invention may be labeled by any one of the radioactive, fluorescent or enzymatic labels known in the art.","EXAMPLES The following Examples are provided for illustrative purposes and not as a means of limitation to support the finding that GSSP4 polypeptides have efficacy in reducing insulin resistance and improving glucose and lipid metabolism and may have an insulin sensitising action.","One of ordinary skill in the art would be able to design equivalent assays and methods based on the disclosure herein all of which form part of the instant invention.","Example 1 Northern Analysis of GSSP4 RNA Analysis of GSSP4 expression in different human tissues (adult and fetal) and cell lines, as well as mouse embryos in different stages of development, is accomplished by using poly A+ RNA blots purchased from Clontech (e.g.","#7780-1, 7757-1, 7756-1, 7768-1and 7763-1).","Labeling of RNA probes is performed using the RNA Strip-EZ kit from Ambion as per manufacture's instructions.","Hybridization of RNA probes to RNA blots is performed Ultrahyb hybridization solution (Ambion).","Briefly, blots are prehybridized for 30 min at 58° C. (low-strigency) or 65° C. (high stringency).","After adding the labeled probe (2×106 cpm/ml), blots are hybridized overnight (14-24 hrs), and is washed 2×20 min at 50° C. with 2× SSC/0.1% SDS (low stringency), 2×20 min at 58° C. with 1× SSC/0.1% SDS (medium stringency) and 2×20 min at 65° C. with 1× SSC/0.1% SDS (high stringency).","After washings are completed blots are exposed on the phosphoimager (Molecular Dynamics) for 1-3 days.","Example 2 In Vitro Tests of Metabolic-Related Activity The activity of various preparations of GSSP4 polypeptides are assessed using various in vitro assays including those provided below.","These assays are also exemplary of those that can be used to develop GSSP4 polypeptide antagonists and agonists.","To do that, the effect of GSSP4 polypeptides in the above assays, e.g.","on glucose uptake and fatty acid oxidation and partitioning in the presence of the candidate molecules would be compared with the effect of GSSP4 polypeptides in the assays in the absence of the candidate molecules.","In Vitro Muscle Cells Glucose Uptake L6 Muscle cells are obtained from the European Culture Collection (Porton Down) and are used at passages 7-11.Cells are maintained in standard tissue culture medium DMEM, and glucose uptake is assessed using [.sup.3H]-2-deoxyglucose (2DG) with or without GSSP4 polypeptides in the presence or absence of insulin (10.sup.-8 M) as has been previously described (Walker P S et al, Glucose transport activity in L6 muscle cells is regulated by the coordinate control of subcellular glucose transporter distribution, biosynthesis, and mRNA transcription, JBC, 1990;265(3), 1516-1523, and Kilp A et al, Stimulation of hexose transport by metformin in L6 muscle cells in culture, Endocrinology,1992;130(5), 2535-2544).","Uptake of 2DG is expressed as the percentage change compared with control (no added insulin or GSSP4).","Values are presented as mean .+−.SEM of sets of 4 wells per experiment.","Differences between sets of wells are evaluated by Student's t test, probability values p<0.05 are considered to be significant.","Effect on Muscle Cell Fatty Acid Oxidation C2C12 cells are differentiated in the presence or absence of 2 μg/mL GSSP4 protein for 4 days.","On day 4, oleate oxidation rates are determined by measuring conversion of 1-14C-oleate (0.2 mM) to 14CO2 for 90 min This experiment can be used to screen for active fragments and peptides as well as agonists and antagonists or activators and inhibitors of GSSP4 polypeptides.","The effect of polypeptides on the rate of oleate oxidation can be compared in differentiated C2C12 cells (murine skeletal muscle cells; ATCC, Manassas, Va. CRL-1772) and in a hepatocyte cell line (Hepa1-6; ATCC, Manassas, Va. CRL-1830).","Cultured cells are maintained according to manufacturer's instructions.","The oleate oxidation assay is performed as previously described (Muoio et al (1999) Biochem J 338;783-791).","Briefly, nearly confluent myocytes are kept in low serum differentiation media (DMEM, 2.5% Horse serum) for 4 days, at which time formation of myotubes became maximal.","Hepatocytes are kept in the same DMEM medium supplemented with 10% FCS for 2 days.","One hour prior to the experiment the media is removed and 1 mL of preincubation media (MEM, 2.5% Horse serum, 3 mM glucose, 4 mM Glutamine, 25 mM Hepes, 1% FFA free BSA, 0.25 mM Oleate, 5 μg/mL gentamycin) is added.","At the start of the oxidation experiment 14C-Oleic acid (1 μCi/mL, American Radiolabeled Chemical Inc., St. Louis, Mo.)","is added and cells are incubated for 90 min at 37° C. in the absence/presence of 2.5 μg/mL GSSP4 polypeptides.","After the incubation period 0.75 mL of the media is removed and assayed for 14C-oxidation products as described below for the muscle FFA oxidation experiment.","Triglyceride and Protein Partitioning following Oleate Oxidaiton in Cultured Cells Following transfer of media for oleate oxidation assay, cells are placed on ice.","To determine triglyceride and protein content, cells are washed with 1 mL of 1×PBS to remove residual media.","To each well 300 μL of cell dissociation solution (Sigma) is added and incubated at 37° C. for 10 min.","Plates are tapped to loosen cells, and 0.5 mL of 1×PBS is added.","The cell suspension is transferred to an eppendorf tube, each well is rinsed with an additional 0.5 mL of 1×PBS, and is transferred to appropriate eppendorf tube.","Samples are centrifuged at 1000 rpm for 10 minutes at room temperature.","Supernatant is discarded and 750 μL of 1×PBS/2% chaps is added to cell pellet.","Cell suspension is vortexed and placed on ice for 1 hour.","Samples are then centrifuged at 13000 rpm for 20 min at 4° C. Supernatants are transferred to new tube and frozen at −20° C. until analyzed.","Quantitative measure of triglyceride level in each sample is determined using Sigma Diagnostics GPO-TRINDER enzymatic kit.","The procedure outlined in the manual is adhered to, with the following exceptions: assay is performed in 48 well plate, 350 μL of sample volume is assayed, control blank consisted of 350 μL PBS/2% chaps, and standard contained 10 μL standard provide in kit plus 690 μL PBS/2% chaps.","Analysis of samples is carried out on a Packard Spectra Count at a wavelength of 550 nm.","Protein analysis is carried out on 25 μL of each supernatant sample using the BCA protein assay (Pierce) following manufacturer's instructions.","Analysis of samples is carried out on a Packard Spectra Count at a wavelength of 550 nm.","Cellular Binding and Uptake of GSSP4 Polypeptides as Detected by Fluorescence Microscopy Fluorecein isothiocyanate (FITC) conjugation of GSSP4 polypeptides: Purified GSSP4 proteins at 1 mg/mL concentration are labeled with FITC using Sigma's FluoroTag FITC conjugation kit (Stock No.","FITC-1).","Protocol outlined in the Sigma Handbook for small scale conjugation is followed for GSSP4 protein labeling.","Cell Culture: C2C12 mouse skeletal muscle cells (ATCC, Manassas, Va. CRL-1772) and Hepa-1-6 mouse hepatocytes (ATCC, Manassas, Va. CRL-1830) are seeded into 6 well plates at a cell density of 2×105 cells per well.","C2C12 and Hepa-1-6 cells are cultured according to repository's instructions for 24-48 hours prior to analysis.","Assay is performed when cells are 80% confluent.","FITC labeled GSSP4 proteincellular binding and uptake using microscopy: C2C12 and Hepa 1-6 cells are incubated in the presence/absence of antibody directed against human LSR (81B: N-terminal sequence of human LSR; does not cross react with mouse LSR and 93A: c-terminal sequence, cross reacts with mouse LSR) or an antiserum directed against gC1qr (953) for 1 hour at 37° C., 5% CO2.LSR antibodies are added to the media at a concentration of 2 μg/mL.","The anti-gC1qr antiserum is added to the media at a volume of 2.5 μL undiluted serum (high concentration) or 1:100 dilution (low concentration).","Following incubation with specified antibody, FITC-GSSP4 polypeptide (50 nM/mL) is added to each cell culture well.","Cells are again incubated for 1 hour at 37° C., 5% CO2.Cells are washed 2× with PBS, cells are scraped from well into 1 mL of PBS.","Cell suspension is transferred to an eppendorf tube and centrifuged at 1000 rpm for 2 minutes.","Supernatant is removed and cells resuspended in 200 μL of PBS.","Binding and uptake of FITC-GSSP4 polypeptide is analyzed by fluorescence microscopy under 40× magnification.","This assay may be useful for identifying agents that facilitate or prevent the uptake and/or binding of GSSP4 polypeptides to cells.","Example 3 In Vivo Tests for Metabolic-Related Activity in Rodent Diabetes Models As metabolic profiles differ among various animal models of obesity and diabetes, analysis of multiple models is undertaken to separate the effects GSSP4 polypeptides on hyperglycemia, hyperinsulinemia, hyperlipidemia and obesity.","Mutation in colonies of laboratory animals and different sensitivities to dietary regimens have made the development of animal models with non-insulin dependent diabetes associated with obesity and insulin resistance possible.","Genetic models such as db/db and ob/ob (See Diabetes, (1982) 31(1): 1-6) in mice and fa/fa in zucker rats have been developed by the various laboratories for understanding the pathophysiology of disease and testing the efficacy of new antidiabetic compounds (Diabetes, (1983) 32: 830-838; Annu.","Rep. Sankyo Res.","Lab.","(1994).","46: 1-57).","The homozygous animals, C57 BL/KsJ-db/db mice developed by Jackson Laboratory, US, are obese, hyperglycemic, hyperinsulinemic and insulin resistant (J. Clin.","Invest., (1990) 85: 962-967), whereas heterozygous are lean and normoglycemic.","In db/db model, mouse progressively develops insulinopenia with age, a feature commonly observed in late stages of human type II diabetes when blood sugar levels are insufficiently controlled.","The state of pancreas and its course vary according to the models.","Since this model resembles that of type II diabetes mellitus, the compounds of the present invention are tested for blood sugar and triglycerides lowering activities.","Zucker (fa/fa) rats are severely obese, hyperinsulinemic, and insulin resistant (Coleman, Diabetes 31:1, 1982; E. Shafrir, in Diabetes Mellitus; H. Rifkin and D. Porte, Jr. Eds.","(Elsevier Science Publishing Co., Inc., New York, ed.","4, 1990), pp.","299-340), and the fa/fa mutation may be the rat equivalent of the murine db mutation (Friedman et al., Cell 69:217-220, 1992; Truett et al., Proc.","Natl.","Acad.","Sci.","USA 88:7806, 1991).","Tubby (tub/tub) mice are characterized by obesity, moderate insulin resistance and hyperinsulinemia without significant hyperglycemia (Coleman et al., J. Heredity 81:424, 1990).","Previously, leptin was reported to reverse insulin resistance and diabetes mellitus in mice with congenital lipodystrophy (Shimomura et al.","Nature 401: 73-76 (1999).","Leptin is found to be less effective in a different lipodystrophic mouse model of lipoatrophic diabetes (Gavrilova et al Nature 403: 850 (2000); hereby incorporated herein in its entirety including any drawings, figures, or tables).","The streptozotocin (STZ) model for chemically-induced diabetes is tested to examine the effects of hyperglycemia in the absence of obesity.","STZ-treated animals are deficient in insulin and severely hyperglycemic (Coleman, Diabetes 31:1, 1982; E. Shafrir, in Diabetes Mellitus; H. Rifkin and D. Porte, Jr. Eds.","(Elsevier Science Publishing Co., Inc., New York, ed.","4, 1990), pp.","299-340).","The monosodium glutamate (MSG) model for chemically-induced obesity (Olney, Science 164:719, 1969; Cameron et al., Cli.","Exp.","Pharmacol.","Physiol.","5:41, 1978), in which obesity is less severe than in the genetic models and develops without hyperphagia, hyperinsulinemia and insulin resistance, is also examined.","Finally, a non-chemical, non-genetic model for induction of obesity includes feeding rodents a high fat/high carbohydrate (cafeteria diet) diet ad libitum.","The instant invention encompasses the use of GSSP4 polypeptides for reducing the insulin resistance and hyperglycemia in any or all of the above rodent diabetes models or in humans with Type I or Type II diabetes or other prefered metabolic diseases described previously or models based on other mammals.","In the compositions of the present invention the GSSP4 polypeptides may, if desired, be associated with other compatible pharmacologically-active antidiabetic agents such as insulin, leptin (U.S. provisional application No.","60/155,506), or troglitazone, either alone or in combination.","Assays include that described previously in Gavrilova et al.","((2000) Diabetes November;49(11): 1910-6; (2000) Nature February 24;403(6772):850) using A-ZIP/F-1 mice, except that GSSP4 polypeptides are administered intraperotineally, subcutaneously, intramuscularly or intravenously.","The glucose and insulin levels of the mice would be tested, and the food intake and liver weight monitored, as well as other factors, such as leptin, FFA, and TG levels, typically measured in our experiments.","In Vivo Assay for Anti-Hyperglycemic Activity of GSSP4 Polypeptides Genetically altered obese diabetic mice (db/db) (male, 7-9 weeks old) are housed (7-9 mice/cage) under standard laboratory conditions at 22.degree.","C. and 50% relative humidity, and maintained on a diet of Purina rodent chow and water ad libitum.","Prior to treatment, blood is collected from the tail vein of each animal and blood glucose concentrations are determined using One Touch BasicGlucose Monitor System (Lifescan).","Mice that have plasma glucose levels between 250 to 500 mg/dl are used.","Each treatment group consists of seven mice that are distributed so that the mean glucose levels are equivalent in each group at the start of the study.","db/db mice are dosed by micro-osmotic pumps, inserted using isoflurane anesthesia, to provide GSSP4 polypeptides, saline, and an irrelevant peptide to the mice subcutaneously (s.c.).","Blood is sampled from the tail vein hourly for 4 hours and at 24, 30 h post-dosing and analyzed for blood glucose concentrations.","Food is withdrawn from 0-4 h post dosing and reintroduced thereafter.","Individual body weights and mean food consumption (each cage) are also measured after 24 h. Significant differences between groups (comparing GSSP4 treated to saline-treated) are evaluated using Student t-test.","In Vivo Insulin Sensitivity Assay In vivo insulin sensitivity is examined by utilizing two-step hyperinsulinemic-euglycemic clamps according to the following protocol.","Rodents from any or all of the various models described in Example 2 are housed for at least a week prior to experimental procedures.","Surgeries for the placement of jugular vein and carotid artery catheters are performed under sterile conditions using ketamine and xylazine (i.m.)","anesthesia.","After surgery, all rodents are allowed to regain consciousness and placed in individual cages.","GSSP4 polypeptides or vehicle is administered through the jugular vein after complete recovery and for the following two days.","Sixteen hours after the last treatment, hyperinsulinemic-euglycemic clamps are performed.","Rodents are placed in restrainers and a bolus of 4 .mu Ci [3-.sup.3 H] glucose (NEN) is administered, followed by a continuous infusion of the tracer at a dose of 0.2 mu.Ci/min (20 mu.1/min).","Two hours after the start of the tracer infusion, 3 blood samples (0.3 ml each) are collected at 10 minute intervals (−20-0 min) for basal measurements.","An insulin infusion is then started (5 mU/kg/min), and 100 .mu.1 blood samples are taken every 10 min.","to monitor plasma glucose.","A 30% glucose solution is infused using a second pump based on the plasma glucose levels in order to reach and maintain euglycemia.","Once a steady state is established at 5 mU/kg/min insulin (stable glucose infusion rate and plasma glucose), 3 additional blood samples (0.3 ml each) are obtained for measurements of glucose, [3-.sup.3H] glucose and insulin (100-120 min.).","A higher dose of insulin (25 mU/kg/min.)","is then administered and glucose infusion rates are adjusted for the second euglycemic clamp and blood samples are taken at min.","220-240.Glucose specific activity is determined in deproteinized plasma and the calculations of Rd and hepatic glucose output (HGO) are made, as described (Lang et al., Endocrinology 130:43, 1992).","Plasma insulin levels at basal period and after 5 and 25 mU/kg/min.","infusions are then determined and compared between GSSP4 treated and vehicle treated rodents.","Insulin regulation of glucose homeostasis has two major components; stimulation of peripheral glucose uptake and suppression of hepatic glucose output.","Using tracer studies in the glucose clamps, it is possible to determine which portion of the insulin response is affected by the GSSP4 polypeptides.","Example 4 Effect of GSSP4 Polypeptides on Mice Fed a High-Fat Diet Experiments are performed using approximately 6 week old C57B1/6 mice (8 per group).","All mice are housed individually.","The mice are maintained on a high fat diet throughout each experiment.","The high fat diet (cafeteria diet; D12331 from Research Diets, Inc.) has the following composition: protein kcal % 16, carbohydrate kcal % 26, and fat kcal % 58.The fat is primarily composed of coconut oil, hydrogenated.","After the mice are fed a high fat diet for 6 days, micro-osmotic pumps are inserted using isoflurane anesthesia, and are used to provide full-length GSSP4 polypeptides, GSSP4 polypeptide fragments, saline, and an irrelevant peptide to the mice subcutaneously (s.c.) for 18 days.","GSSP4 polypeptides are provided at doses of 100, 50, 25, and 2.5 μg/day and the irrelevant peptide is provided at 10 μg/day.","Body weight is measured on the first, third and fifth day of the high fat diet, and then daily after the start of treatment.","Final body weight and final blood samples are taken by cardiac puncture and are used to determine triglyceride (TG), total cholesterol (TC), glucose, leptin, and insulin levels.","The amount of food consumed per day is also determined for each group.","Plasma glucose is determined by a glucose oxidase procedure (Analox GM7) and plasma insulin determined by radioimmunoassay (Amerlex, Amersham).","Example 5 Effect of GSSP4 Polypeptides on Plasma Free Fatty Acid in C57 BL/6 Mice The effect of GSSP4 polypeptides on postprandial lipemia (PPL) in normal C57BL6/J mice is tested.","The mice used in this experiment are fasted for 2 hours prior to the experiment after which a baseline blood sample is taken.","All blood samples are taken from the tail using EDTA coated capillary tubes (50 μL each time point).","At time 0 (8:30 AM), a standard high fat meal (6 g butter, 6 g sunflower oil, 10 g nonfat dry milk, 10 g sucrose, 12 mL distilled water prepared fresh following Nb#6, JF, pg.1) is given by gavage (vol.=1% of body weight) to all animals.","Immediately following the high fat meal, 25 μg a GSSP4 polypeptide is injected i.p.","in 100 μL saline.","The same dose (25 μg/mL in 100 μL) is again injected at 45 min and at 1 hr 45 min.","Control animals are injected with saline (3×100 μL).","Untreated and treated animals are handled in an alternating mode.","Blood samples are taken in hourly intervals, and are immediately put on ice.","Plasma is prepared by centrifugation following each time point.","Plasma is kept at −20° C. and free fatty acids (FFA), triglycerides (TG) and glucose are determined within 24 hours using standard test kits (Sigma and Wako).","Due to the limited amount of plasma available, glucose is determined in duplicate using pooled samples.","For each time point, equal volumes of plasma from all 8 animals per treatment group are pooled.","Example 6 Effect of GSSP4 Polypeptides on Plasma Leptin and Insulin in C57 BL/6 Mice The effect of GSSP4 polypeptides on plasma leptin and insulin levels during postprandial lipemia (PPL) in normal C57BL6/J mice is tested.","The experimental procedure is the same as previously described, except that blood is drawn only at 0, 2 and 4 hours to allow for greater blood samples needed for the determination of leptin and insulin by RIA.","Briefly, 16 mice are fasted for 2 hours prior to the experiment after which a baseline blood sample is taken.","All blood samples are taken from the tail using EDTA coated capillary tubes (100 μL each time point).","At time 0 (9:00 AM), a standard high fat meal is given by gavage (vol.=1% of body weight) to all animals.","Immediately following the high fat meal, 25 μg of a GSSP4 polypeptide is injected i.p.","in 100 μL saline.","The same dose (25 μg in 100 μL) is again injected at 45 min and at 1 hr 45 min (treated group).","Control animals are injected with saline (3×100 μL).","Untreated and treated animals are handled in an alternating mode.","Blood samples are immediately put on ice and plasma is prepared by centrifugation following each time point.","Plasma is kept at −20° C. and free fatty acids (FFA) are determined within 24 hours using a standard test kit (Wako).","Leptin and Insulin are determined by RIA (ML-82K and SRI-13K, LINCO Research, Inc., St. Charles, Mo.)","following the manufacturer's protocol.","However, only 20 μL plasma is used.","Each determination is done in duplicate.","Due to the limited amount of plasma available, leptin and insulin are determined in 4 pools of 2 animals each in both treatment groups.","Example 7 Effect of GSSP4 Polypeptides on Plasma FFA, TG and Glucose in C57 BL/6 Mice The effect of GSSP4 polypeptides on plasma FFA, TG, glucose, leptin and insulin levels during postprandial lipemia (PPL) in normal C57BL6/J mice has been described.","Weight loss resulting from GSSP4 polypeptides (2.5 μg/day) given to normal C57BL6/J mice on a high fat diet is shown.","The experimental procedure is similar to described previously.","Briefly, 14 mice are fasted for 2 hours prior to the experiment after which a baseline blood sample is taken.","All blood samples are taken from the tail using EDTA coated capillary tubes (50 μL each time point).","At time 0 (9:00 AM), a standard high fat meal is given by gavage (vol.=1% of body weight) to all animals.","Immediately following the high fat meal, 4 mice are injected 25 μg of a GSSP4 polypeptide i.p.","in 100 μL saline.","The same dose (25 μg in 100 μL) is again injected at 45 min and at 1 hr 45 min.","A second treatment group receives 3 times 50 μg GSSP4 polypeptide at the same intervals.","Control animals are injected with saline (3×100 μL).","Untreated and treated animals are handled in an alternating mode.","Blood samples are immediately put on ice.","Plasma is prepared by centrifugation following each time point.","Plasma is kept at −20° C. and free fatty acids (FFA), triglycerides (TG) and glucose are determined within 24 hours using standard test kits (Sigma and Wako).","Example 8 Effect of GSSP4 Polypeptides on FFA Following Epinephrine Injection In mice, plasma free fatty acids increase after intragastric administration of a high fat/carbohydrate test meal.","These free fatty acids are mostly produced by the activity of lipolytic enzymes i.e.","lipoprotein lipase (LPL) and hepatic lipase (HL).","In this species, these enzymes are found in significant amounts both bound to endothelium and freely circulating in plasma.","Another source of plasma free fatty acids is hormone sensitive lipase (HSL) that releases free fatty acids from adipose tissue after β-adrenergic stimulation.","To test whether GSSP4 polypeptides also regulate the metabolism of free fatty acid released by HSL, mice are injected with epinephrine.","Two groups of mice are given epinephrine (5 μg) by intraperitoneal injection.","A treated group is injected with a GSSP4 polypeptide (25 μg) one hour before and again together with epinephrine, while control animals receive saline.","Plasma is isolated and free fatty acids and glucose are measured as described above.","Example 9 Effect of GSSP4 Polypeptides on Muscle FFA Oxidation To investigate the effect of GSSP4 polypeptides on muscle free fatty acid oxidation, intact hind limb muscles from C57BL/6J mice are isolated and FFA oxidation is measured using oleate as substrate (Clee et al (2000) J Lipid Res 41:521-531; Muoio et al (1999) Am J Physiol 276:E913-921).","Oleate oxidation in isolated muscle is measured as previously described (Cuendet et al (1976) J Clin Invest 58:1078-1088; Le Marchand-Brustel (1978) Am J Physiol 234:E348-E358).","Briefly, mice are sacrificed by cervical dislocation and soleus and EDL muscles are rapidly isolated from the hind limbs.","The distal tendon of each muscle is tied to a piece of suture to facilitate transfer among different media.","All incubations are carried out at 30° C. in 1.5 mL of Krebs-Henseleit bicarbonate buffer (118.6 mM NaCl, 4.76 mM KCl, 1.19 mM KH2PO4, 1.19 mM MgSO4, 2.54 mM CaCl2, 25 mM NaHCO3, 10 mM Hepes, pH 7.4) supplemented with 4% FFA free bovine serum albumin (fraction V, RIA grade, Sigma) and 5 mM glucose (Sigma).","The total concentration of oleate (Sigma) throughout the experiment is 0.25 mM.","All media are oxygenated (95% O2; 5% CO2) prior to incubation.","The gas mixture is hydrated throughout the experiment by bubbling through a gas washer (Kontes Inc., Vineland, N.J.).","Muscles are rinsed for 30 min in incubation media with oxygenation.","The muscles are then transferred to fresh media (1.5 mL) and incubated at 30° C. in the presence of 1 μCi/mL [1-14C] oleic acid (American Radiolabeled Chemicals).","The incubation vials containing this media are sealed with a rubber septum from which a center well carrying a piece of Whatman paper (1.5 cm×11.5 cm) is suspended.","After an initial incubation period of 10 min with constant oxygenation, gas circulation is removed to close the system to the outside environment and the muscles are incubated for 90 min at 30° C. At the end of this period, 0.45 mL of Solvable (Packard Instruments, Meriden, Conn.) is injected onto the Whatman paper in the center well and oleate oxidation by the muscle is stopped by transferring the vial onto ice.","After 5 min, the muscle is removed from the medium, and an aliquot of 0.5 mL medium is also removed.","The vials are closed again and 1 mL of 35% perchloric acid is injected with a syringe into the media by piercing through the rubber septum.","The CO2 released from the acidified media is collected by the Solvable in the center well.","After a 90 min collection period at 30° C., the Whatman paper is removed from the center well and placed in scintillation vials containing 15 mL of scintillation fluid (HionicFlour, Packard Instruments, Meriden, Conn.).","The amount of 14C radioactivity is quantitated by liquid scintillation counting.","The rate of oleate oxidation is expressed as nmol oleate produced in 90 min/g muscle.","To test the effect of gACRP30 or ACRP30 on oleate oxidation, these proteins are added to the media at a final concentration of 2.5 μg/mL and maintained in the media throughout the procedure.","Example 10 Effect of GSSP4 Polypeptides on Triglyceride in Muscle & Liver Isolated from Mice To determine whether the increased FFA oxidation induced by GSSP4 polypeptides is also accompanied by increased FFA delivery into muscle or liver, the hindlimb muscle and liver triglyceride content is measured after the GSSP4 polypeptide treatment of mice.","Hind limb muscles as well as liver samples are removed from treated and untreated animals and the triglyceride and free fatty acid concentration is determined following a standard lipid extraction method (Shimabukuro et al (1997) Proc Natl Acad Sci USA 94:4637-4641) followed by TG and FFA analysis using standard test kits.","Example 11 Effect of GSSP4 Polypeptides on FFA following Intralipid Injection Two groups of mice are intravenously (tail vein) injected with 30 μL bolus of Intralipid-20% (Clintec) to generate a sudden rise in plasma FFAs, thus by-passing intestinal absorption.","(Intralipid is an intravenous fat emulsion used in nutritional therapy).","A treated group (GSSP4 polypeptide-treated) is injected with a GSSP4 polypeptide (25 μg) at 30 and 60 minutes before Intralipid is given, while control animals (▴ control) received saline.","Plasma is isolated and FFAs are measured as described previously.","The effect of GSSP4 polypeptides on the decay in plasma FFAs following the peak induced by Intralipid injection is then monitored.","Example 12 Tests of Obesity-Related Activity in Humans Tests of the efficacy of in humans are performed in accordance with a physician's recommendations and with established guidelines.","The parameters tested in mice are also tested in humans (e.g.","food intake, weight, TG, TC, glucose, insulin, leptin, FFA).","It is expected that the physiological factors would show changes over the short term.","Changes in weight gain might require a longer period of time.","In addition, the diet is carefully monitored.","GSSP4 is given in daily doses of about 6 mg protein per 70 kg person or about 10 mg per day.","Other doses are tested, for instance 1 mg or 5 mg per day up to 20 mg, 50 mg, or 100 mg per day.","Example 13 Tests of Obesity-Related Activity in a Murine Lipoatrophic Diabetes Model Leptin was reported to reverse insulin resistance and diabetes mellitus in mice with congenital lipodystrophy (Shimomura et al.","Nature 401: 73-76 (1999).","Leptin is found to be less effective in a different lipodystrophic mouse model of lipoatrophic diabetes (Gavrilova et al Nature 403: 850 (2000); hereby incorporated herein in its entirety including any drawings, figures, or tables).","The instant invention encompasses the use of GSSP4 or polypeptide fragments for reducing the insulin resistance and hyperglycaemia in this model either alone or in combination with leptin, the leptin peptide (U.S. provisional application No.","60/155,506), or other compounds.","Assays include that described previously in Gavrilova et al.","((2000) Diabetes November;49(11): 1910-6; (2000) Nature February 24;403(6772):850) using A-ZIP/F-1 mice, except that would be administered using the methods previously described in Example 5 (or Examples 8-10).","The glucose and insulin levels of the mice would be tested, and the food intake and liver weight monitored, as well as other factors, such as leptin, FFA, and TG levels, typically measured in our experiments (see Example 5, above, or Examples 8-10)."]],"string":"[\n [\n \"Gssp4 polynucleotides and polypeptides and uses thereof\",\n \"The present invention relates to the field of metabolic research.\",\n \"Metabolic disorders, such as obesity, are a public health problem that is serious and widespread.\",\n \"GSSP4 polypeptides have been identified that are believed to be beneficial in the treatment of metabolic disorders.\",\n \"These compounds should be effective for reducing cholesterol levels, body mass, body fat, and for treating metabolic-related diseases and disorders.\",\n \"The metabolic-related diseases or disorders envisioned to be treated by the methods of the invention include, but are not limited to: obesity, hyperlipidemia, hypercholesterolemia, atherosclerosis, diabetes, glucose intolerance, insulin resistance and hypertension.\"\n ],\n [\n \"1.A method of reducing circulating free fatty acid levels in an individual comprising administering to said individual a physiologically acceptable composition comprising a carrier and a polypeptide sequence comprising at least 6 consecutive amino acids of SEQ ID NO:3 with metabolic-related activity, wherein said method optionally reduces body mass.\",\n \"2.A method of reducing circulating glucose levels in an individual comprising administering to said individual a physiologically acceptable composition comprising a carrier and a polypeptide sequence comprising at least 6 consecutive amino acids of SEQ ID NO:3 with metabolic-related activity, wherein said method optionally reduces body mass.\",\n \"3.A method of reducing circulating triglyceride levels in an individual comprising administering to said individual a physiologically acceptable composition comprising a carrier and a polypeptide sequence comprising at least 6 consecutive amino acids of SEQ ID NO:3 with metabolic-related activity, wherein said method optionally reduces body mass.\",\n \"4.A method of reducing circulating cholesterol levels in an individual comprising administering to said individual a physiologically acceptable composition comprising a carrier and a polypeptide sequence comprising at least 6 consecutive amino acids of SEQ ID NO:3 with metabolic-related activity, wherein said method optionally reduces body mass.\",\n \"5.An isolated polypeptide comprising: a) an amino acid sequence at least 50% identical to the full length polypeptide of SEQ ID NO: 3; b) a polypeptide fragment of at least six consecutive amino acids of SEQ ID NO: 3; c) the polypeptide of SEQ ID NO: 3; d) homomultimers or heteromultimers of the polypeptide of SEQ ID NO: 3; e) a heterologous polypeptide fused to the polypeptide of a), b), c), or d); or f) a polypeptide according to a), b), c), d), or e) that has been differentially modified.\",\n \"6.A composition comprising an isolated polypeptide according to claim 3 and a pharmaceutically or physiologically acceptable carrier.\",\n \"7.An isolated or purified polynucleotide: a) encoding a polypeptide comprising an amino acid sequence at least 50% identical to the full length polypeptide of SEQ ID NO: 3; b) encoding a polypeptide comprising a fragment of at least six consecutive amino acids of SEQ ID NO: 3; c) encoding a polypeptide comprising SEQ ID NO: 3; d) encoding homomultimers or heteromultimers of the polypeptide of SEQ ID NO: 3; e) encoding a polypeptide comprising a heterologous polypeptide fused to the polypeptide of a), b), c), or d); f) encoding a polypeptide according to a), b), c), d), or e) that has been differentially modified; g) comprising a polynucleotide according to a), b), c), d), e), or f) operably linked to a promoter, other regulatory element, or selectable marker; or h) comprising a vector comprising a polynucleotide according to a), b), c), d), e), f), or g).\",\n \"8.A recombinant cell comprising a polynucleotide according to claim 7.9.An antibody that specifically binds to a polypeptide according to claim 5.10.A method of producing a polypeptide comprising culturing a recombinant cell according to claim 8 under conditions that allow for the expression of said polypeptide.\",\n \"11.The method according to claim 10, further comprising the isolation, recovery, or purification of said polypeptide.\",\n \"12.A method of treating a obesity, a metabolic disease or a metabolic disorder comprising the administration of a therapeutically effective amount of a composition comprising: a) an isolated polypeptide comprising: 1) an amino acid sequence at least 50% identical to the full length polypeptide of SEQ ID NO: 3; 2) a polypeptide fragment of at least six consecutive amino acids of SEQ ID NO: 3; 3) the polypeptide of SEQ ID NO: 3; 4) homomultimers or heteromultimers of the polypeptide of SEQ ID NO: 3; 5) a heterologous polypeptide fused to the polypeptide of a(1), a(2), a(3), or a(4); or 6) a polypeptide according to a(1), a(2), a(3), a(4), or a(5) that has been differentially modified; b) an isolated or purified polynucleotide: 1) encoding a polypeptide comprising an amino acid sequence at least 50% identical to the full length polypeptide of SEQ ID NO: 3; 2) encoding a polypeptide comprising a fragment of at least six consecutive amino acids of SEQ ID NO: 3; 3) encoding a polypeptide comprising SEQ ID NO: 3; 4) encoding homomultimers or heteromultimers of the polypeptide of SEQ ID NO: 3; 5) encoding a polypeptide comprising a heterologous polypeptide fused to the polypeptide of b(1), b(2), b(3), or b(4); 6) encoding a polypeptide according to b(1), b(2), b(3), b(4), or b(5) that has been differentially modified; 7) comprising a polynucleotide according to b(1), b(2), b(3), b(4), b(5), or b(6) operably linked to a promoter, other regulatory element, or selectable marker; or 8) comprising a vector comprising a polynucleotide according to b(1), b(2), b(3), b(4), b(5), b(6), or b(7); or c) a recombinant cell comprising: 1) a polynucleotide encoding a polypeptide comprising an amino acid sequence at least 50% identical to the full length polypeptide of SEQ ID NO: 3; 2) a polynucleotide encoding a polypeptide comprising a fragment of at least six consecutive amino acids of SEQ ID NO: 3; 3) a polynucleotide encoding a polypeptide comprising SEQ ID NO: 3; 4) a polynucleotide encoding homomultimers or heteromultimers of the polypeptide of SEQ ID NO: 3; 5) a polynucleotide encoding a polypeptide comprising a heterologous polypeptide fused to the polypeptide of c(1), c(2), c(3), or c(4); 6) a polynucleotide encoding a polypeptide according to c(1), c(2), c(3), c(4), or c(5) that has been differentially modified; 7) a polynucleotide according to c(1), c(2), c(3), c(4), c(5), or c(6) operably linked to a promoter, other regulatory element, or selectable marker; or 8) comprising a vector comprising a polynucleotide according to c(1), c(2), c(3), c(4), c(5), c(6), or c(7).\",\n \"13.A transgenic animal comprising a polynucleotide according to claim 7.\"\n ],\n [\n \"<SOH> BACKGROUND OF TH INVENTION <EOH>The following discussion is intended to facilitate the understanding of the invention, but is not intended nor admitted to be prior art to the invention.\",\n \"Obesity is a public health problem that is serious, widespread, and increasing.\",\n \"In the United States, 20 percent of the population is obese; in Europe, a slightly lower percentage is obese (Friedman (2000) Nature 404:632-634).\",\n \"Obesity is associated with increased risk of hypertension, cardiovascular disease, diabetes, and cancer as well as respiratory complications and osteoarthritis (Kopelman (2000) Nature 404:635-643).\",\n \"Even modest weight loss ameliorates these associated conditions.\",\n \"While still acknowledging that lifestyle factors including environment, diet, age and exercise play a role in obesity, twin studies, analyses of familial aggregation, and adoption studies all indicate that obesity is largely the result of genetic factors (Barsh et al (2000) Nature 404:644-651).\",\n \"In agreement with these studies, is the fact that an increasing number of obesity-related genes are being identified.\",\n \"Some of the more extensively studied genes include those encoding leptin (ob) and its receptor (db), pro-opiomelanocortin (Pomc), melanocortin-4-receptor (Mc4r), agouti protein (A y ), carboxypeptidase E (fat), 5-hydroxytryptamine receptor 2C (Htr2c), nescient basic helix-loop-helix 2 (Nhlh2), prohormone convertase 1 IPCSK1), and tubby protein (tubby) (rev'd in Barsh et al (2000) Nature 404:644-651).\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>The instant invention is based on the discovery that GSSP4 polypeptides have unexpected effects in vitro and in vivo, including utility for weight reduction, prevention of weight gain, reduction of cholesterol levels, and control of blood glucose levels in humans and other mammals.\",\n \"These unexpected effects of administration of GSSP4 polypeptides in mammals also include reduction of elevated free fatty acid levels caused by administration of epinephrine, i.v.\",\n \"injection of “intralipid”, or administration of a high fat test meal, as well as increased fatty acid oxidation in muscle cells, reduction of circulating cholesterol levels, modulation of blood glucose and weight reduction in mammals, particularly those consuming a high fat/high carbohydrate diet.\",\n \"These effects are unexpected and surprising given that proteins of similar structure or homology (such as colipase and mamba intestinal toxin 1) have not been shown to have utility for weight reduction, prevention of weight gain, reduction of cholesterol levels, and control of blood glucose levels.\",\n \"However, the GSSP4 polypeptides of the invention are effective and can be provided at levels that are feasible for treatments in humans.\",\n \"Thus, the invention is drawn to GSSP4 polypeptides, polynucleotides encoding said polypeptides, vectors comprising said GSSP4 polynucleotides, and cells recombinant for said GSSP4 polynucleotides, as well as to pharmaceutical and physiologically acceptable compositions comprising said GSSP4 polypeptide and methods of administering said GSSP4 polypeptides or polynucleotides in a pharmaceutical and physiologically acceptable compositions in order to reduce body weight, cholesterol levels or glucose levels, or to treat metabolic-related diseases and disorders.\",\n \"Assays for identifying agonists and antagonists of metabolic-related activity are also part of the invention.\",\n \"In a first aspect, the invention features a purified, isolated, or recombinant GSSP4 polypeptides.\",\n \"In preferred embodiments, said polypeptides comprise, consist essentially of, or consist of, those having significant activity wherein the said activity is selected from the group consisiting of cholesterol reduction, cholesterol regulation, lipid partitioning, lipid metabolism, glucose control, and insulin-like activity.\",\n \"In preferred embodiments, said polypeptides comprise, consist essentially of, or consist of, the full length polypeptide of SEQ ID NO:3 or a fragment of consecutive amino acids of the full length polypeptide sequence of SEQ ID NO:3.In other preferred embodiments, said polypeptides comprise an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding consecutive amino acids of the polypeptide sequences identified in SEQ ID NO:3.In a further preferred embodiment, the GSSP4 polypeptide is able to lower circulating (either blood, serum or plasma) levels (concentration) of: (i) free fatty acids, (ii) glucose, and/or (iii) triglycerides.\",\n \"Further preferred GSSP4 polypeptides are those that significantly stimulate muscle lipid or free fatty acid oxidation.\",\n \"Further preferred GSSP4 polypeptides are those that cause C2C12 cells differentiated in the presence of said polypeptides to undergo at least 10%, 20%, 30%, 35%, or 40% more oleate oxidation as compared to untreated cells.\",\n \"Further preferred GSSP4 polypeptides are those that are at least 30% more efficient than untreated cells at increasing leptin uptake in a liver cell line (preferably BPRCL mouse liver cells (ATCC CRL-2217)).\",\n \"Further preferred GSSP4 polypeptides are those that significantly reduce the postprandial increase in plasma free fatty acids, particularly following a high fat meal.\",\n \"Further preferred GSSP4 polypeptides are those that significantly reduce or eliminate ketone body production, particularly following a high fat meal.\",\n \"Further preferred GSSP4 polypeptides are those that increase glucose uptake in skeletal muscle cells.\",\n \"Further preferred GSSP4 polypeptides are those that increase glucose uptake in adipose cells.\",\n \"Further preferred GSSP4 polypeptides are those that increase glucose uptake in neuronal cells.\",\n \"Further preferred GSSP4 polypeptides are those that increase glucose uptake in red blood cells.\",\n \"Further preferred GSSP4 polypeptides are those that increase glucose uptake in the brain.\",\n \"Further preferred GSSP4 polypeptides are those that significantly reduce the postprandial increase in plasma glucose following a meal, particularly a high carbohydrate meal.\",\n \"Further preferred GSSP4 polypeptides are those that significantly prevent the postprandial increase in plasma glucose following a meal, particularly a high fat or a high carbohydrate meal.\",\n \"Further preferred GSSP4 polypeptides are those that improve insulin sensitivity.\",\n \"Further preferred GSSP4 polypeptides are those that modulate food intake or food selection.\",\n \"Further preferred GSSP4 polypeptides are those that modulate satiety.\",\n \"Further preferred GSSP4 polypeptides are those that modulate fatty acid metabolism.\",\n \"Further preferred GSSP4 polypeptides are those that modulate cholesterol metabolism, particularly in steroidogenic tissues.\",\n \"Therefore, said polypeptides have a potential role in effecting, either directly or indirectly or both, levels of reproductive hormones (eg.\",\n \"estradiol, progesterone, testosterone).\",\n \"Further preferred GSSP4 polypeptides are those that modulate cortisol levels.\",\n \"Further preferred GSSP4 polypeptides are those that modulate aldosterone levels.\",\n \"Therefore, said polypeptides have a potential role in effecting, either directly or indirectly or both, levels of sodium and potassium.\",\n \"Further preferred GSSP4 polypeptides are those that modulate blood pressure preferably to normalize blood pressure within a normal range.\",\n \"Further preferred GSSP4 polypeptides are those that form multimers (e.g., heteromultimers or homomultimers) in vitro and/or in vivo.\",\n \"Preferred multimers are homodimers or homotrimers.\",\n \"Other preferred multimers are homomultimers comprising at least 4, 6, 8, 9, 10 or 12 GSSP4 polypeptides.\",\n \"Other preferred mulimers are hetero multimers comprising GSSP4 polypeptides of the invention.\",\n \"Further preferred embodiments include heterologous polypeptides comprising a GSSP4 polypeptide of the invention.\",\n \"In a second aspect, the invention features purified, isolated, or recombinant polynucleotides encoding said GSSP4 polypeptides described in the first aspect, or the complement thereof.\",\n \"A further preferred embodiment of the invention is a recombinant, purified or isolated polynucleotide comprising, or consisting of a mammalian genomic sequence, gene, cDNA, or fragments thereof.\",\n \"In one aspect the sequence is derived from a human, mouse or other mammal.\",\n \"In a preferred aspect, the genomic sequence includes isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 22, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, 1000, 2000, 5000, 6000 or 7500 nucleotides of any one of the polynucleotide sequences described in SEQ ID NO:1, 2, or the complements thereof, wherein said contiguous span comprises a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding nucleotide sequence in SEQ ID NO: 1, 2, or 3.In further embodiments the polynucleotides are DNA, RNA, DNA/RNA hybrids, single-stranded, and double-stranded.\",\n \"Further preferred are GSSP4 polynucleotides and polypeptides that have cholesterol regulating activies.\",\n \"Further preferred are GSSP4 polynucleotides and polypeptides that have body weight regulating activies.\",\n \"Further preferred are GSSP4 polynucleotides and polypeptides that have body fat regulating activies.\",\n \"Further preferred are GSSP4 polynucleotides and polypeptides that have glucose regulating activies.\",\n \"Further preferred are GSSP4 polynucleotides and polypeptides that have lipid regulating activies.\",\n \"In a third aspect, the invention features a recombinant vector comprising, consisting essentially of, or consisting of, said polynucleotide described in the second aspect.\",\n \"In a fourth aspect, the invention features a recombinant cell comprising, consisting essentially of, or consisting of, said recombinant vector described in the third aspect.\",\n \"A further embodiment includes a host cell recombinant for a polynucleotide of the invention.\",\n \"In a fifth aspect, the invention features a pharmaceutical or physiologically acceptable composition comprising, consisting essentially of, or consisting of, said GSSP4 polypeptides described in the first aspect and, a pharmaceutical or physiologically acceptable diluent.\",\n \"In a sixth aspect, the invention features a method of controlling cholesterol levels comprising, providing, or administering to individuals with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.\",\n \"In further preferred embodiments, the invention features a method of lowering body weight comprising, providing, or administering to individuals with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.\",\n \"In further preferred embodiments, the invention features a method of lowering body fat comprising, providing, or administering to individuals with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.\",\n \"In further preferred embodiments, the invention features a method of lowering controlling blood glucose comprising, providing, or administering to individuals with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.\",\n \"In a seventh aspect, the invention features a method of preventing or treating a metabolic-related disease or disorder comprising, providing or administering to an individual in need of such treatment said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.\",\n \"Preferably, said obesity-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM or Type II diabetes), Insulin dependent diabetes mellitus (IDDM or Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.\",\n \"Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.\",\n \"In preferred embodiments, said individual is a mammal, preferably a human.\",\n \"In a further preferred embodiment, a pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect suggest that a compound may have utility in alleviating insulin resistance in individuals, particularly those that are obese or overweight.\",\n \"In a further preferred embodiment, the present invention may be used in complementary therapy in individuals to improve their cholesterol, weight or glucose level, comprising a pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect in combination with known agents.\",\n \"The present invention further provides a method of improving the cholesterol levels, body weight or glucose control in individuals comprising the administration of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect alone, without known agents.\",\n \"In a further preferred embodiment, the present invention may be administered either concomitantly or concurrently, with known agents for example in the form of separate dosage units to be used simultaneously, separately or sequentially (either before or after the known agent).\",\n \"Accordingly, the present invention further provides a product containing a composition a pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect and a known agent as a combined preparation for simultaneous, separate or sequential use for the improvement of cholesterol levels, body weight or glucose control in individuals, particularly those who are obese or overweight.\",\n \"The ratio of the present composition to known agent is such that the quantity of each active ingredient employed will be such as to provide a therapeutically effective level, but will not be larger than the quantity recommended as safe for administration.\",\n \"In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect can be used as a method to improve insulin sensitivity in some persons, particularly those with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) or noninsulin dependent diabetics (Type II) in combination with insulin therapy.\",\n \"In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect can be used as a method to improve insulin sensitivity in some persons, particularly those with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) or noninsulin dependent diabetes mellitus (NIDDM, Type II) in combination with alternate therapies.\",\n \"In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used as a method in prophylaxis of long-term detrimental effects caused by prolonged high dosage of insulin in humans having IDDM or NIDDM.\",\n \"In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing hypersecretion of insulin and disorders or conditions resulting therefrom.\",\n \"In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing obesity and consequences or complications thereof.\",\n \"In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing hypercholesterolemia and consequences or complications thereof.\",\n \"In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing NIDDM or IDDM and consequences or complications thereof.\",\n \"In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing impaired glucose tolerance (IGT).\",\n \"Further preferred embodiment thus provides therapeutics and methods for normalizing insulin resistance.\",\n \"Further preferred embodiment thus provides therapeutics and methods for reducing, slowing or preventing the progression to NIDDM.\",\n \"In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing the appearance of insulin-resistance syndrome.\",\n \"In further preferred embodiments, other conditions, particularly obesity, associated with insulin resistance are treated or prevented according to the methods of the invention.\",\n \"Thus, by preventing or treating obesity, the methods of the invention will allow an individual to have a more comfortable life and avoid the onset of various diseases triggered by obesity.\",\n \"In further preferred embodiments, the target of the methods according to the present invention includes individuals with normal glucose tolerance (NGT) who are obese or who have fasting hyperinsulinemia, or who have both.\",\n \"In an eighth aspect, the invention features a method of controlling blood free fatty acid (FFA) levels and lipid metabolism comprising, providing, or administering to individuals in need of increasing mobilization and utilization of fat stores and decreasing total fat stores with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.\",\n \"In a further preferred embodiment, the identification of said individuals in need of increasing mobilization and utilization of fat stores and decreasing total fat stores to be treated with said pharmaceutical or physiologically acceptable composition comprises a person who is involved in physical activity which increases metabolic demand.\",\n \"Furthermore, increasing mobilization and utilization of fat stores and decreasing total fat stores would provide a means to decrease body weight, preventing weight gain, decrease body fat in overweight and obese individuals.\",\n \"Reduction in weight and obesity will thus decrease the risk of chronic disease associated with obesity such as but not limited to the onset of various lipid metabolism disorders, hypertension, Type II diabetes, atherosclerosis, cardiovascular disease and stroke.\",\n \"In related aspects, embodiments of the present invention includes methods of causing or inducing a desired biological response in an individual comprising the steps of: providing or administering to an individual a composition comprising a GSSP4 polypeptide, wherein said biological response is selected from the group consisting of: (a) lowering circulating (either blood, serum, or plasma) levels (concentration) of free fatty acids; (b) lowering circulating (either blood, serum or plasma) levels (concentration) of glucose; (c) lowering circulating (either blood, serum or plasma) levels (concentration) of triglycerides; (d) stimulating muscle lipid or free fatty acid oxidation; (e) increasing leptin uptake in the liver or liver cells; (f) reducing the postprandial increase in plasma free fatty acids, particularly following a high fat meal; and, (g) reducing or eliminating ketone body production, particularly following a high fat meal; (h) increasing tissue sensitivity to insulin, particularly muscle, adipose, liver or brain, (i) reducing cholesterol levels, particularly in those with elevated cholesterol (ie.\",\n \"greater than 200 mg/dl); (j) modulating circulating (either blood, serum or plasma) levels (concentration) of glucose within physiological range, preferably maintaining glucose between 60-190 mg/dl; (k) modulating circulating (either blood, serum or plasma) levels (concentration) of FFA within physiological range preferably maintaining FFA between 190-420 mg/dl; (l) modulating ketone body production as the result of a high fat meal, wherein said modulating is preferably reducing or eliminating; (m) reducing body weight particularly in individuals with a BMI of greater than 27.In a ninth aspect, the invention features a method of making the GSSP4 polypeptide described in the first aspect, wherein said method is selected from the group consisting of: proteolytic cleavage, recombinant methodology and artificial synthesis.\",\n \"In a tenth aspect, the present invention provides a method of making a recombinant GSSP4 polypeptides, the method comprising providing a transgenic, non-human mammal whose milk contains said recombinant GSSP4 polypeptides, and purifying said recombinant GSSP4 polypeptides from the milk of said non-human mammal.\",\n \"In one embodiment, said non-human mammal is a cow, goat, sheep, rabbit, or mouse.\",\n \"In another embodiment, the method comprises purifying a recombinant GSSP4 polypeptides from said milk, and further comprises cleaving said protein in vitro to obtain a desired GSSP4 polypeptides.\",\n \"In an eleventh aspect, the invention features a purified or isolated antibody capable of specifically binding to a protein comprising the sequence of one of the polypeptides of the present invention.\",\n \"In one aspect of this embodiment, the antibody is capable of binding to a polypeptide comprising at least 6 consecutive amino acids, at least 8 consecutive amino acids, or at least 10 consecutive amino acids of the sequence of one of the polypeptides of the present invention.\",\n \"In a twelfth aspect, the invention features a use of polypeptides described in the first aspect or polynucleotides described in the second aspect for treatment of metabolic-related diseases and disorders or reducing or increasing body mass.\",\n \"Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM or Type II diabetes), Insulin dependent diabetes mellitus (IDDM or Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.\",\n \"Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.\",\n \"In preferred embodiments, said individual is a mammal, preferably a human.\",\n \"In a thirteenth aspect, the invention features a use of polypeptides described in the first aspect or polynucleotides described in the second aspect for the preparation of a medicament for the treatment of metabolic-related diseases and disorders or for reducing body mass.\",\n \"Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM or Type II diabetes), Insulin dependent diabetes mellitus (IDDM or Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.\",\n \"Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.\",\n \"In preferred embodiments, said individual is a mammal, preferably a human.\",\n \"In a fourteenth aspect, the invention provides polypeptides of the first aspect of the invention or a composition of the fifth aspect for use in a method of treatment of the human or animal body.\",\n \"In a fifteenth aspect, the invention provides polynucleotides described in the second aspect or an acceptable composition thereof, for use in a method of treatment of the human or animal body.\",\n \"In a sixteenth aspect, the invention features methods of reducing body weight for cosmetic purposes comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect, or polypeptides described in the first aspect.\",\n \"Preferably, for said reducing body weight said individual has a BMI of at least 20, 25, 30, 35, or 40.In a seventeenth aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect or a polypeptide described in the first aspect for reducing body mass in said individuals with a BMI of at least 30, 35, 40, or 45 or for treatment or prevention of metabolic-related diseases or disorders.\",\n \"Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM or Type II diabetes), Insulin dependent diabetes mellitus (IDDM or Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.\",\n \"Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.\",\n \"In preferred embodiments, said individual is a mammal, preferably a human.\",\n \"In preferred embodiments, the identification of said individuals to be treated with said pharmaceutical or physiologically acceptable composition comprises genotyping GSSP4 single nucleotide polymorphisms (SNPs) or measuring GSSP4 polypeptide or mRNA levels in clinical samples from said individuals.\",\n \"Preferably, said clinical samples are selected from the group consisting of blood, serum, plasma, urine, and saliva.\",\n \"In an eighteenth aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect for reducing body weight for cosmetic reasons.\",\n \"In further preferred embodiments, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect for reducing glucose levels.\",\n \"In a nineteenth aspect, the invention features methods of treating insulin resistance comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect, or a polypeptide described in the first aspect.\"\n ],\n [\n \"FIELD OF THE INVENTION The present invention relates to the field of metabolic research, in particular the discovery of compounds effective for reducing cholesterol levels, body fat, and body mass, and useful for treating metabolic-related diseases an disorders.\",\n \"The metabolic-related diseases or disorders envisioned to be treated by the methods of the invention include, but are not limited to, obesity, hyperlipidemia, hypercholesterolemia, atherosclerosis, diabetes, glucose intolerance, insulin resistance and hypertension.\",\n \"BACKGROUND OF TH INVENTION The following discussion is intended to facilitate the understanding of the invention, but is not intended nor admitted to be prior art to the invention.\",\n \"Obesity is a public health problem that is serious, widespread, and increasing.\",\n \"In the United States, 20 percent of the population is obese; in Europe, a slightly lower percentage is obese (Friedman (2000) Nature 404:632-634).\",\n \"Obesity is associated with increased risk of hypertension, cardiovascular disease, diabetes, and cancer as well as respiratory complications and osteoarthritis (Kopelman (2000) Nature 404:635-643).\",\n \"Even modest weight loss ameliorates these associated conditions.\",\n \"While still acknowledging that lifestyle factors including environment, diet, age and exercise play a role in obesity, twin studies, analyses of familial aggregation, and adoption studies all indicate that obesity is largely the result of genetic factors (Barsh et al (2000) Nature 404:644-651).\",\n \"In agreement with these studies, is the fact that an increasing number of obesity-related genes are being identified.\",\n \"Some of the more extensively studied genes include those encoding leptin (ob) and its receptor (db), pro-opiomelanocortin (Pomc), melanocortin-4-receptor (Mc4r), agouti protein (Ay), carboxypeptidase E (fat), 5-hydroxytryptamine receptor 2C (Htr2c), nescient basic helix-loop-helix 2 (Nhlh2), prohormone convertase 1 IPCSK1), and tubby protein (tubby) (rev'd in Barsh et al (2000) Nature 404:644-651).\",\n \"SUMMARY OF THE INVENTION The instant invention is based on the discovery that GSSP4 polypeptides have unexpected effects in vitro and in vivo, including utility for weight reduction, prevention of weight gain, reduction of cholesterol levels, and control of blood glucose levels in humans and other mammals.\",\n \"These unexpected effects of administration of GSSP4 polypeptides in mammals also include reduction of elevated free fatty acid levels caused by administration of epinephrine, i.v.\",\n \"injection of “intralipid”, or administration of a high fat test meal, as well as increased fatty acid oxidation in muscle cells, reduction of circulating cholesterol levels, modulation of blood glucose and weight reduction in mammals, particularly those consuming a high fat/high carbohydrate diet.\",\n \"These effects are unexpected and surprising given that proteins of similar structure or homology (such as colipase and mamba intestinal toxin 1) have not been shown to have utility for weight reduction, prevention of weight gain, reduction of cholesterol levels, and control of blood glucose levels.\",\n \"However, the GSSP4 polypeptides of the invention are effective and can be provided at levels that are feasible for treatments in humans.\",\n \"Thus, the invention is drawn to GSSP4 polypeptides, polynucleotides encoding said polypeptides, vectors comprising said GSSP4 polynucleotides, and cells recombinant for said GSSP4 polynucleotides, as well as to pharmaceutical and physiologically acceptable compositions comprising said GSSP4 polypeptide and methods of administering said GSSP4 polypeptides or polynucleotides in a pharmaceutical and physiologically acceptable compositions in order to reduce body weight, cholesterol levels or glucose levels, or to treat metabolic-related diseases and disorders.\",\n \"Assays for identifying agonists and antagonists of metabolic-related activity are also part of the invention.\",\n \"In a first aspect, the invention features a purified, isolated, or recombinant GSSP4 polypeptides.\",\n \"In preferred embodiments, said polypeptides comprise, consist essentially of, or consist of, those having significant activity wherein the said activity is selected from the group consisiting of cholesterol reduction, cholesterol regulation, lipid partitioning, lipid metabolism, glucose control, and insulin-like activity.\",\n \"In preferred embodiments, said polypeptides comprise, consist essentially of, or consist of, the full length polypeptide of SEQ ID NO:3 or a fragment of consecutive amino acids of the full length polypeptide sequence of SEQ ID NO:3.In other preferred embodiments, said polypeptides comprise an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding consecutive amino acids of the polypeptide sequences identified in SEQ ID NO:3.In a further preferred embodiment, the GSSP4 polypeptide is able to lower circulating (either blood, serum or plasma) levels (concentration) of: (i) free fatty acids, (ii) glucose, and/or (iii) triglycerides.\",\n \"Further preferred GSSP4 polypeptides are those that significantly stimulate muscle lipid or free fatty acid oxidation.\",\n \"Further preferred GSSP4 polypeptides are those that cause C2C12 cells differentiated in the presence of said polypeptides to undergo at least 10%, 20%, 30%, 35%, or 40% more oleate oxidation as compared to untreated cells.\",\n \"Further preferred GSSP4 polypeptides are those that are at least 30% more efficient than untreated cells at increasing leptin uptake in a liver cell line (preferably BPRCL mouse liver cells (ATCC CRL-2217)).\",\n \"Further preferred GSSP4 polypeptides are those that significantly reduce the postprandial increase in plasma free fatty acids, particularly following a high fat meal.\",\n \"Further preferred GSSP4 polypeptides are those that significantly reduce or eliminate ketone body production, particularly following a high fat meal.\",\n \"Further preferred GSSP4 polypeptides are those that increase glucose uptake in skeletal muscle cells.\",\n \"Further preferred GSSP4 polypeptides are those that increase glucose uptake in adipose cells.\",\n \"Further preferred GSSP4 polypeptides are those that increase glucose uptake in neuronal cells.\",\n \"Further preferred GSSP4 polypeptides are those that increase glucose uptake in red blood cells.\",\n \"Further preferred GSSP4 polypeptides are those that increase glucose uptake in the brain.\",\n \"Further preferred GSSP4 polypeptides are those that significantly reduce the postprandial increase in plasma glucose following a meal, particularly a high carbohydrate meal.\",\n \"Further preferred GSSP4 polypeptides are those that significantly prevent the postprandial increase in plasma glucose following a meal, particularly a high fat or a high carbohydrate meal.\",\n \"Further preferred GSSP4 polypeptides are those that improve insulin sensitivity.\",\n \"Further preferred GSSP4 polypeptides are those that modulate food intake or food selection.\",\n \"Further preferred GSSP4 polypeptides are those that modulate satiety.\",\n \"Further preferred GSSP4 polypeptides are those that modulate fatty acid metabolism.\",\n \"Further preferred GSSP4 polypeptides are those that modulate cholesterol metabolism, particularly in steroidogenic tissues.\",\n \"Therefore, said polypeptides have a potential role in effecting, either directly or indirectly or both, levels of reproductive hormones (eg.\",\n \"estradiol, progesterone, testosterone).\",\n \"Further preferred GSSP4 polypeptides are those that modulate cortisol levels.\",\n \"Further preferred GSSP4 polypeptides are those that modulate aldosterone levels.\",\n \"Therefore, said polypeptides have a potential role in effecting, either directly or indirectly or both, levels of sodium and potassium.\",\n \"Further preferred GSSP4 polypeptides are those that modulate blood pressure preferably to normalize blood pressure within a normal range.\",\n \"Further preferred GSSP4 polypeptides are those that form multimers (e.g., heteromultimers or homomultimers) in vitro and/or in vivo.\",\n \"Preferred multimers are homodimers or homotrimers.\",\n \"Other preferred multimers are homomultimers comprising at least 4, 6, 8, 9, 10 or 12 GSSP4 polypeptides.\",\n \"Other preferred mulimers are hetero multimers comprising GSSP4 polypeptides of the invention.\",\n \"Further preferred embodiments include heterologous polypeptides comprising a GSSP4 polypeptide of the invention.\",\n \"In a second aspect, the invention features purified, isolated, or recombinant polynucleotides encoding said GSSP4 polypeptides described in the first aspect, or the complement thereof.\",\n \"A further preferred embodiment of the invention is a recombinant, purified or isolated polynucleotide comprising, or consisting of a mammalian genomic sequence, gene, cDNA, or fragments thereof.\",\n \"In one aspect the sequence is derived from a human, mouse or other mammal.\",\n \"In a preferred aspect, the genomic sequence includes isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 22, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, 1000, 2000, 5000, 6000 or 7500 nucleotides of any one of the polynucleotide sequences described in SEQ ID NO:1, 2, or the complements thereof, wherein said contiguous span comprises a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding nucleotide sequence in SEQ ID NO: 1, 2, or 3.In further embodiments the polynucleotides are DNA, RNA, DNA/RNA hybrids, single-stranded, and double-stranded.\",\n \"Further preferred are GSSP4 polynucleotides and polypeptides that have cholesterol regulating activies.\",\n \"Further preferred are GSSP4 polynucleotides and polypeptides that have body weight regulating activies.\",\n \"Further preferred are GSSP4 polynucleotides and polypeptides that have body fat regulating activies.\",\n \"Further preferred are GSSP4 polynucleotides and polypeptides that have glucose regulating activies.\",\n \"Further preferred are GSSP4 polynucleotides and polypeptides that have lipid regulating activies.\",\n \"In a third aspect, the invention features a recombinant vector comprising, consisting essentially of, or consisting of, said polynucleotide described in the second aspect.\",\n \"In a fourth aspect, the invention features a recombinant cell comprising, consisting essentially of, or consisting of, said recombinant vector described in the third aspect.\",\n \"A further embodiment includes a host cell recombinant for a polynucleotide of the invention.\",\n \"In a fifth aspect, the invention features a pharmaceutical or physiologically acceptable composition comprising, consisting essentially of, or consisting of, said GSSP4 polypeptides described in the first aspect and, a pharmaceutical or physiologically acceptable diluent.\",\n \"In a sixth aspect, the invention features a method of controlling cholesterol levels comprising, providing, or administering to individuals with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.\",\n \"In further preferred embodiments, the invention features a method of lowering body weight comprising, providing, or administering to individuals with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.\",\n \"In further preferred embodiments, the invention features a method of lowering body fat comprising, providing, or administering to individuals with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.\",\n \"In further preferred embodiments, the invention features a method of lowering controlling blood glucose comprising, providing, or administering to individuals with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.\",\n \"In a seventh aspect, the invention features a method of preventing or treating a metabolic-related disease or disorder comprising, providing or administering to an individual in need of such treatment said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.\",\n \"Preferably, said obesity-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM or Type II diabetes), Insulin dependent diabetes mellitus (IDDM or Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.\",\n \"Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.\",\n \"In preferred embodiments, said individual is a mammal, preferably a human.\",\n \"In a further preferred embodiment, a pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect suggest that a compound may have utility in alleviating insulin resistance in individuals, particularly those that are obese or overweight.\",\n \"In a further preferred embodiment, the present invention may be used in complementary therapy in individuals to improve their cholesterol, weight or glucose level, comprising a pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect in combination with known agents.\",\n \"The present invention further provides a method of improving the cholesterol levels, body weight or glucose control in individuals comprising the administration of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect alone, without known agents.\",\n \"In a further preferred embodiment, the present invention may be administered either concomitantly or concurrently, with known agents for example in the form of separate dosage units to be used simultaneously, separately or sequentially (either before or after the known agent).\",\n \"Accordingly, the present invention further provides a product containing a composition a pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect and a known agent as a combined preparation for simultaneous, separate or sequential use for the improvement of cholesterol levels, body weight or glucose control in individuals, particularly those who are obese or overweight.\",\n \"The ratio of the present composition to known agent is such that the quantity of each active ingredient employed will be such as to provide a therapeutically effective level, but will not be larger than the quantity recommended as safe for administration.\",\n \"In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect can be used as a method to improve insulin sensitivity in some persons, particularly those with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) or noninsulin dependent diabetics (Type II) in combination with insulin therapy.\",\n \"In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect can be used as a method to improve insulin sensitivity in some persons, particularly those with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) or noninsulin dependent diabetes mellitus (NIDDM, Type II) in combination with alternate therapies.\",\n \"In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used as a method in prophylaxis of long-term detrimental effects caused by prolonged high dosage of insulin in humans having IDDM or NIDDM.\",\n \"In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing hypersecretion of insulin and disorders or conditions resulting therefrom.\",\n \"In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing obesity and consequences or complications thereof.\",\n \"In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing hypercholesterolemia and consequences or complications thereof.\",\n \"In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing NIDDM or IDDM and consequences or complications thereof.\",\n \"In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing impaired glucose tolerance (IGT).\",\n \"Further preferred embodiment thus provides therapeutics and methods for normalizing insulin resistance.\",\n \"Further preferred embodiment thus provides therapeutics and methods for reducing, slowing or preventing the progression to NIDDM.\",\n \"In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect is used in therapeutics or methods for reducing or preventing the appearance of insulin-resistance syndrome.\",\n \"In further preferred embodiments, other conditions, particularly obesity, associated with insulin resistance are treated or prevented according to the methods of the invention.\",\n \"Thus, by preventing or treating obesity, the methods of the invention will allow an individual to have a more comfortable life and avoid the onset of various diseases triggered by obesity.\",\n \"In further preferred embodiments, the target of the methods according to the present invention includes individuals with normal glucose tolerance (NGT) who are obese or who have fasting hyperinsulinemia, or who have both.\",\n \"In an eighth aspect, the invention features a method of controlling blood free fatty acid (FFA) levels and lipid metabolism comprising, providing, or administering to individuals in need of increasing mobilization and utilization of fat stores and decreasing total fat stores with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect.\",\n \"In a further preferred embodiment, the identification of said individuals in need of increasing mobilization and utilization of fat stores and decreasing total fat stores to be treated with said pharmaceutical or physiologically acceptable composition comprises a person who is involved in physical activity which increases metabolic demand.\",\n \"Furthermore, increasing mobilization and utilization of fat stores and decreasing total fat stores would provide a means to decrease body weight, preventing weight gain, decrease body fat in overweight and obese individuals.\",\n \"Reduction in weight and obesity will thus decrease the risk of chronic disease associated with obesity such as but not limited to the onset of various lipid metabolism disorders, hypertension, Type II diabetes, atherosclerosis, cardiovascular disease and stroke.\",\n \"In related aspects, embodiments of the present invention includes methods of causing or inducing a desired biological response in an individual comprising the steps of: providing or administering to an individual a composition comprising a GSSP4 polypeptide, wherein said biological response is selected from the group consisting of: (a) lowering circulating (either blood, serum, or plasma) levels (concentration) of free fatty acids; (b) lowering circulating (either blood, serum or plasma) levels (concentration) of glucose; (c) lowering circulating (either blood, serum or plasma) levels (concentration) of triglycerides; (d) stimulating muscle lipid or free fatty acid oxidation; (e) increasing leptin uptake in the liver or liver cells; (f) reducing the postprandial increase in plasma free fatty acids, particularly following a high fat meal; and, (g) reducing or eliminating ketone body production, particularly following a high fat meal; (h) increasing tissue sensitivity to insulin, particularly muscle, adipose, liver or brain, (i) reducing cholesterol levels, particularly in those with elevated cholesterol (ie.\",\n \"greater than 200 mg/dl); (j) modulating circulating (either blood, serum or plasma) levels (concentration) of glucose within physiological range, preferably maintaining glucose between 60-190 mg/dl; (k) modulating circulating (either blood, serum or plasma) levels (concentration) of FFA within physiological range preferably maintaining FFA between 190-420 mg/dl; (l) modulating ketone body production as the result of a high fat meal, wherein said modulating is preferably reducing or eliminating; (m) reducing body weight particularly in individuals with a BMI of greater than 27.In a ninth aspect, the invention features a method of making the GSSP4 polypeptide described in the first aspect, wherein said method is selected from the group consisting of: proteolytic cleavage, recombinant methodology and artificial synthesis.\",\n \"In a tenth aspect, the present invention provides a method of making a recombinant GSSP4 polypeptides, the method comprising providing a transgenic, non-human mammal whose milk contains said recombinant GSSP4 polypeptides, and purifying said recombinant GSSP4 polypeptides from the milk of said non-human mammal.\",\n \"In one embodiment, said non-human mammal is a cow, goat, sheep, rabbit, or mouse.\",\n \"In another embodiment, the method comprises purifying a recombinant GSSP4 polypeptides from said milk, and further comprises cleaving said protein in vitro to obtain a desired GSSP4 polypeptides.\",\n \"In an eleventh aspect, the invention features a purified or isolated antibody capable of specifically binding to a protein comprising the sequence of one of the polypeptides of the present invention.\",\n \"In one aspect of this embodiment, the antibody is capable of binding to a polypeptide comprising at least 6 consecutive amino acids, at least 8 consecutive amino acids, or at least 10 consecutive amino acids of the sequence of one of the polypeptides of the present invention.\",\n \"In a twelfth aspect, the invention features a use of polypeptides described in the first aspect or polynucleotides described in the second aspect for treatment of metabolic-related diseases and disorders or reducing or increasing body mass.\",\n \"Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM or Type II diabetes), Insulin dependent diabetes mellitus (IDDM or Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.\",\n \"Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.\",\n \"In preferred embodiments, said individual is a mammal, preferably a human.\",\n \"In a thirteenth aspect, the invention features a use of polypeptides described in the first aspect or polynucleotides described in the second aspect for the preparation of a medicament for the treatment of metabolic-related diseases and disorders or for reducing body mass.\",\n \"Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM or Type II diabetes), Insulin dependent diabetes mellitus (IDDM or Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.\",\n \"Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.\",\n \"In preferred embodiments, said individual is a mammal, preferably a human.\",\n \"In a fourteenth aspect, the invention provides polypeptides of the first aspect of the invention or a composition of the fifth aspect for use in a method of treatment of the human or animal body.\",\n \"In a fifteenth aspect, the invention provides polynucleotides described in the second aspect or an acceptable composition thereof, for use in a method of treatment of the human or animal body.\",\n \"In a sixteenth aspect, the invention features methods of reducing body weight for cosmetic purposes comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect, or polypeptides described in the first aspect.\",\n \"Preferably, for said reducing body weight said individual has a BMI of at least 20, 25, 30, 35, or 40.In a seventeenth aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect or a polypeptide described in the first aspect for reducing body mass in said individuals with a BMI of at least 30, 35, 40, or 45 or for treatment or prevention of metabolic-related diseases or disorders.\",\n \"Preferably, said metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM or Type II diabetes), Insulin dependent diabetes mellitus (IDDM or Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.\",\n \"Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.\",\n \"In preferred embodiments, said individual is a mammal, preferably a human.\",\n \"In preferred embodiments, the identification of said individuals to be treated with said pharmaceutical or physiologically acceptable composition comprises genotyping GSSP4 single nucleotide polymorphisms (SNPs) or measuring GSSP4 polypeptide or mRNA levels in clinical samples from said individuals.\",\n \"Preferably, said clinical samples are selected from the group consisting of blood, serum, plasma, urine, and saliva.\",\n \"In an eighteenth aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect for reducing body weight for cosmetic reasons.\",\n \"In further preferred embodiments, the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect for reducing glucose levels.\",\n \"In a nineteenth aspect, the invention features methods of treating insulin resistance comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect, or a polypeptide described in the first aspect.\",\n \"BRIEF DESCRIPTION OF THE SEQENCES SEQ ID NO:1 shows GSSP4 genomic sequence indicating regulatory regions, exons, polymorphic alleles and positions, and reverse and forward microsequencing primers for haplotyping.\",\n \"SEQ ID NO:2 shows GSSP4 polynucleotide sequence of cDNA SEQ ID NO:3 shows GSSP4 polypeptide sequence SEQ ID NO:4-18 shows GSSP4 47-mer nucleotide sequence comprising polymorphic allele at position 24.The corresponding alleles and primers indicated in SEQ ID NO:1 are as follows: VLP_1206_C_A, m=C or A SEQ ID NO:4 VLP_148_A_G, r=A or G SEQ ID NO:5 VLP_1851_T_C, y=T or C SEQ ID NO:6 VLP_2551_G_A, r=G or A SEQ ID NO:7 VLP_3124_C_T, y=C or T SEQ ID NO:8 VLP_3563_G_A, r=G or A SEQ ID NO:9 VLP_3792_G_A, r=G or A SEQ ID NO:10 VLP_4417_A_C, m=A or G SEQ ID NO:11 VLP_5757_T_C, y=T or C SEQ ID NO:12 VLP_6322_A_G, r=G or A SEQ ID NO:13 VLP_816_G_A, r=G or A SEQ ID NO:14 VLP_924_G_A, r=G or A SEQ ID NO:15 VLP_99-1_174_T_C, y=T or C SEQ ID NO:16 VLP_99-1_325_C_G, s=C or G SEQ ID NO:17 VLP_99-2_389_T_C, y=T or G SEQ ID NO:18 DETAILED DESCRIPTION OF THE INVENTION Before describing the invention in greater detail, the following definitions are set forth to illustrate and define the meaning and scope of the terms used to describe the invention herein.\",\n \"As used interchangeably herein, the terms “oligonucleotides”, and “polynucleotides” and nucleic acid include RNA, DNA, or RNA/DNA hybrid sequences of more than one nucleotide in either single chain or duplex form.\",\n \"The terms encompass “modified nucleotides” which comprise at least one modification, including by way of example and not limitation: (a) an alternative linking group, (b) an analogous form of purine, (c) an analogous form of pyrimidine, or (d) an analogous sugar.\",\n \"For examples of analogous linking groups, purines, pyrimidines, and sugars see for example PCT publication No.\",\n \"WO 95/04064.The polynucleotide sequences of the invention may be prepared by any known method, including synthetic, recombinant, ex vivo generation, or a combination thereof, as well as utilizing any purification methods known in the art.\",\n \"The terms polynucleotide construct, recombinant polynucleotide and recombinant polypeptide are used herein consistently with their use in the art.\",\n \"The terms “upstream” and “downstream” are also used herein consistently with their use in the art.\",\n \"The terms “base paired” and “Watson & Crick base paired” are used interchangeably herein and consistently with their use in the art.\",\n \"Similarly, the terms “complementary”, “complement thereof”, “complement”, “complementary polynucleotide”, “complementary nucleic acid” and “complementary nucleotide sequence” are used interchangeably herein and consistently with their use in the art.\",\n \"The term “purified” is used herein to describe a polynucleotide or polynucleotide vector of the invention that has been separated from other compounds including, but not limited to, other nucleic acids, carbohydrates, lipids and proteins (such as the enzymes used in the synthesis of the polynucleotide).\",\n \"Purified can also refer to the separation of covalently closed polynucleotides from linear polynucleotides, or vice versa, for example.\",\n \"A polynucleotide is substantially pure when at least about 50%, 60%, 75%, or 90% of a sample contains a single polynucleotide sequence.\",\n \"In some cases this involves a determination between conformations (linear versus covalently closed).\",\n \"A substantially pure polynucleotide typically comprises about 50, 60, 70, 80, 90, 95, 99% weight/weight of a nucleic acid sample.\",\n \"Polynucleotide purity or homogeneity may be indicated by a number of means well known in the art, such as agarose or polyacrylamide gel electrophoresis of a sample, followed by visualizing a single polynucleotide band upon staining the gel.\",\n \"For certain purposes higher resolution can be provided by using HPLC or other means well known in the art.\",\n \"Similarly, the term “purified” is used herein to describe a polypeptide of the invention that has been separated from other compounds including, but not limited to, nucleic acids, lipids, carbohydrates and other proteins.\",\n \"In some preferred embodiments, a polypeptide is substantially pure when at least about 50%, 60%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% of the polypeptide molecules of a sample have a single amino acid sequence.\",\n \"In some preferred embodiments, a substantially pure polypeptide typically comprises about 50%, 60%, 70%, 80%, 90% 95%, 96%, 97%, 98%, 99% or 99.5% weight/weight of a protein sample.\",\n \"Polypeptide purity or homogeneity is indicated by a number of methods well known in the art, such as agarose or polyacrylamide gel electrophoresis of a sample, followed by visualizing a single polypeptide band upon staining the gel.\",\n \"For certain purposes higher resolution can be provided by using HPLC or other methods well known in the art.\",\n \"Further, as used herein, the term “purified” does not require absolute purity; rather, it is intended as a relative definition.\",\n \"Purification of starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.\",\n \"Alternatively, purification may be expressed as “at least” a percent purity relative to heterologous polynucleotides (DNA, RNA or both) or polypeptides.\",\n \"As a preferred embodiment, the polynucleotides or polypeptides of the present invention are at least; 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 96%, 98%, 99%, 99.5% or 100% pure relative to heterologous polynucleotides or polypeptides.\",\n \"As a further preferred embodiment the polynucleotides or polypeptides have an “at least” purity ranging from any number, to the thousandth position, between 90% and 100% (e.g., at least 99.995% pure) relative to heterologous polynucleotides or polypeptides.\",\n \"Additionally, purity of the polynucleotides or polypeptides may be expressed as a percentage (as descried above) relative to all materials and compounds other than the carrier solution.\",\n \"Each number, to the thousandth position, may be claimed as individual species of purity.\",\n \"The term “isolated” requires that the material be removed from its original environment (e.g., the natural environment if it is naturally occurring).\",\n \"For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.\",\n \"Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition, and still be isolated in that the vector or composition is not part of its natural environment.\",\n \"Specifically excluded from the definition of “isolated” are: naturally occurring chromosomes (e.g., chromosome spreads), artificial chromosome libraries, genomic libraries, and cDNA libraries that exist either as an in vitro nucleic acid preparation or as a transfected/transformed host cell preparation, wherein the host cells are either an in vitro heterogeneous preparation or plated as a heterogeneous population of single colonies.\",\n \"Also specifically excluded are the above libraries wherein a 5′ EST makes up less than 5% (or alternatively 1%, 2%, 3%, 4%, 10%, 25%, 50%, 75%, or 90%, 95%, or 99%) of the number of nucleic acid inserts in the vector molecules.\",\n \"Further specifically excluded are whole cell genomic DNA or whole cell RNA preparations (including said whole cell preparations which are mechanically sheared or enzymatically digested).\",\n \"Further specifically excluded are the above whole cell preparations as either an in vitro preparation or as a heterogeneous mixture separated by electrophoresis (including blot transfers of the same) wherein the polynucleotide of the invention have not been further separated from the heterologous polynucleotides in the electrophoresis medium (e.g., further separating by excising a single band from a heterogeneous band population in an agarose gel or nylon blot).\",\n \"The term “primer” denotes a specific oligonucleotide sequence which is complementary to a target nucleotide sequence and used to hybridize to the target nucleotide sequence.\",\n \"A primer serves as an initiation point for nucleotide polymerization catalyzed by DNA polymerase, RNA polymerase, or reverse transcriptase.\",\n \"The term “probe” denotes a defined nucleic acid segment (or nucleotide analog segment, e.g., PNA as defined hereinbelow) which can be used to identify a specific polynucleotide sequence present in a sample, said nucleic acid segment comprising a nucleotide sequence complementary to the specific polynucleotide sequence to be identified.\",\n \"The term “polypeptide” refers to a polymer of amino acids without regard to the length of the polymer.\",\n \"Thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide.\",\n \"This term also does not specify or exclude post-expression modifications of polypeptides.\",\n \"For example, polypeptides that include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide.\",\n \"Also included within the definition are polypeptides which contain one or more analogs of an amino acid (including, for example, non-naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.\",\n \"), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.\",\n \"The phrases “control of blood glucose”, “glucose regulation” “regulation of blood glucose”, “modulating blood glucose”, and “glucose control” refer to maintaining or regulating blood, serum, and plasma levels of glucose between 70-190 mg/dl.\",\n \"Without being limited by theory, the compounds/polypeptides of the invention feature a method of chronic control of blood glucose within a narrow and more physiological range compared to the control of blood glucose attained with current therapies which treat disorders of glucose metabolism or insulin action, including but not limited to hyperglycemia, insulin resistance, insulin dependent diabetes mellitus and noninsulin dependent diabetes mellitus.\",\n \"Without being limited by theory, the compounds/polypeptides of the invention are capable of modulating the control of blood glucose (as defined above), directing or partitioning glucose between the liver and the peripheral tissues, and are thus believed to treat “diseases involving the control of blood glucose between the liver and peripheral tissues”.\",\n \"The term “peripheral tissues” is meant to include the blood, brain, muscle and adipose tissue.\",\n \"In preferred embodiments, the compounds/polypeptides of the invention direct or partition glucose towards the liver, muscle and brain.\",\n \"In alternative preferred embodiments, glucose is directed or partitioned towards the adipose tissue.\",\n \"In other preferred embodiments, glucose is directed or partitioned towards the liver.\",\n \"In other preferred embodiments, glucose is directed or partitioned towards the brain.\",\n \"In other preferred embodiments, glucose is directed towards the blood.\",\n \"In yet other preferred embodiments, the compounds/polypeptides of the invention increase or decrease the oxidation of glucose, preferably by the muscle.\",\n \"Without being limited by theory, the compounds/polypeptides of the invention are capable of modulating the partitioning of dietary or endogenous lipids between the liver and peripheral tissues, and are thus believed to treat “diseases involving the partitioning of lipids between the liver and peripheral tissues.” The term “peripheral tissues” is meant to include the blood, muscle and adipose tissue.\",\n \"In preferred embodiments, the compounds/polypeptides of the invention partition the lipids toward the muscle.\",\n \"In alternative preferred embodiments, the lipids are partitioned toward the blood.\",\n \"In alternative preferred embodiments, the lipids are partitioned toward the adipose tissue.\",\n \"In other preferred embodiments, the lipids are partitioned toward the liver.\",\n \"In yet other preferred embodiments, the compounds/polypeptides of the invention increase or decrease the oxidation of dietary or endogenous lipids, preferably free fatty acids (FFA) by the muscle.\",\n \"Dietary and endogenous lipids include, but are not limited to triglycerides (TG) and FFA.\",\n \"“Preferred diseases” believed to involve the the control of cholesterol, body fat, lipid metabolism, blood glucose, partitioning of blood glucose, and partitioning of lipids include obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, reproductive cancers, hypercortisolism, aldosteronism, hyperandrogenism, hyperkalemia, hypernatremia, hyperlipoproteinemia, hyperinsulinemia, hyperglycemia, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM, Type II diabetes), Insulin dependent diabetes mellitus (IDDM, Type I diabetes), diabetes-related complications (eg.\",\n \"ketosis), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be prevented or treated by the methods of the invention.\",\n \"Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.\",\n \"The term “heterologous”, when used herein, is intended to designate any polypeptide or polynucleotide other than a GSSP4 polypeptide or a polynucleotide encoding a GSSP4 polypeptide of the present invention.\",\n \"The terms “comprising”, “consisting of” and “consisting essentially of” are defined according to their standard meaning.\",\n \"A defined meaning set forth in the M.P.E.P.\",\n \"controls over a defined meaning in the art and a defined meaning set forth in controlling Federal Circuit case law controls over a meaning set forth in the M.P.E.P.\",\n \"With this in mind, the terms may be substituted for one another throughout the instant application in order to attach the specific meaning associated with each term.\",\n \"The term “host cell recombinant for” a particular polynucleotide of the present invention, means a host cell that has been altered by the hands of man to contain said polynucleotide in a way not naturally found in said cell.\",\n \"For example, said host cell may be transiently or stably transfected or transduced with said polynucleotide of the present invention.\",\n \"The term “obesity” as used herein is defined by the National Heart, Lung and Blood Institute Expert Panel (J Amer Diet Assoc 98:1178-1191, 1998) based on Body Mass Index (BMI).\",\n \"BMI is calculated as body weight in kilograms divided by the square of height in meters, ie kg/m2.A BMI of less than 18.5 kg/m2 is considered “Underweight”; a BMI of 18.5-24.9 kg/m2 is considered “Normal” (healthy); a BMI of 25.0-29.9 kg/m2 is considered “Overweight”; a BMI of 30.0-34.9 kg/m2 is considered “Class I Obesity”; a BMI of 35.0-39.9 kg/m2 is considered “Class II Obesity”; a BMI of greater than 39.9 kg/m2 is considered “Class III Obesity”.\",\n \"Waist circumference can also be used to indicate a risk of metabolic complications where in men a circumference of greater than or equal to 94 cm indicates an increased risk, and greater than or equal to 102 cm indicates a substantially increased risk Similarly for women, greater than or equal to 88 cm indicates an increased risk, and greater than or equal to 88 cm indicates a substantially increased risk.\",\n \"The waist circumference is measured in cm at midpoint between lower border of ribs and upper border of the pelvis.\",\n \"Other measures of obesity include, but are not limited to, skinfold thickness which is a measurement in cm of skinfold thickness using calipers and bioimpedance which is based on the principle that lean mass and water will conduct electrical current and measurement of resistance to a weak current (impedance) applied across extremities provides an estimate of body fat using an empirically derived equation.\",\n \"The term “Insulin Dependent Diabetes Mellitus”, “IDDM”, “Type I Diabetes” and “Type I diabetics” are synomous, inclusive, or interchangable.\",\n \"The term “diabetes-related complications” refer to pathologic or physiologic states experienced by Type I diabetics (as defined previously) or Type II diabetics (as defined previously) or both.\",\n \"The term “Noninsulin Dependent Diabetes Mellitus”, “NIDDM”, “Type II Diabetes” and “Type II diabetics” are synomous, inclusive, or interchangable.\",\n \"The term “agent acting on the control of blood glucose, directing glucose between the liver and peripheral tissues” refers to a compound or polypeptide of the invention that modulates the control of blood glucose as previously described.\",\n \"Preferably, the agent increases or decreases the oxidation of glucose, preferably by the muscle.\",\n \"Preferably the agent decreases or increases the body weight of individuals or is used to treat or prevent an metabolic-related disease or disorder such as obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM, Type II diabetes), Insulin dependent diabetes mellitus (IDDM, Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.\",\n \"Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.\",\n \"The terms “response to an agent acting on the control of blood glucose, directing glucose between the liver and peripheral tissues” refer to drug efficacy, including but not limited to, ability to metabolize a compound, ability to convert a pro-drug to an active drug, and the pharmacokinetics (absorption, distribution, elimination) and the pharmacodynamics (receptor-related) of a drug in an individual.\",\n \"The terms “side effects to an agent acting on the control of blood glucose, directing glucose between the liver and peripheral tissues” refer to adverse effects of therapy resulting from extensions of the principal pharmacological action of the drug or to idiosyncratic adverse reactions resulting from an interaction of the drug with unique host factors.\",\n \"“Side effects to an agent acting on the partitioning of dietary lipids between the liver and peripheral tissues” can include, but are not limited to, adverse reactions such as dermatologic, hematologic or hepatologic toxicities and further includes gastric and intestinal ulceration, disturbance in platelet function, renal injury, nephritis, vasomotor rhinitis with profuse watery secretions, angioneurotic edema, generalized urticaria, and bronchial asthma to laryngeal edema and bronchoconstriction, hypotension, and shock.\",\n \"The term “agent acting on the partitioning of glucose between the liver and peripheral tissues” refers to a compound or polypeptide of the invention that modulates the partitioning of glucose between the liver and the peripheral tissues as previously described.\",\n \"Preferably, the agent increases or decreases blood glucose levels.\",\n \"Preferably, the agent increases or decreases oxidation of glucose, preferably by the muscle.\",\n \"Preferably the agent decreases or increases the body weight of individuals or is used to treat or prevent an metabolic-related disease or disorder such as obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM, Type II diabetes), Insulin dependent diabetes mellitus (IDDM, Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.\",\n \"Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.\",\n \"The terms “response to an agent acting on the partitioning of glucose between the liver and peripheral tissues” refer to drug efficacy, including but not limited to, ability to metabolize a compound, ability to convert a pro-drug to an active drug, and the pharmacokinetics (absorption, distribution, elimination) and the pharmacodynamics (receptor-related) of a drug in an individual.\",\n \"The terms “side effects to an agent acting on the partitioning of glucose between the liver and peripheral tissues” refer to adverse effects of therapy resulting from extensions of the principal pharmacological action of the drug or to idiosyncratic adverse reactions resulting from an interaction of the drug with unique host factors.\",\n \"“Side effects to an agent acting on the partitioning of dietary lipids between the liver and peripheral tissues” can include, but are not limited to, adverse reactions such as dermatologic, hematologic or hepatologic toxicities and further includes gastric and intestinal ulceration, disturbance in platelet function, renal injury, nephritis, vasomotor rhinitis with profuse watery secretions, angioneurotic edema, generalized urticaria, and bronchial asthma to laryngeal edema and bronchoconstriction, hypotension, and shock.\",\n \"The term “agent acting on the partitioning of dietary lipids between the liver and peripheral tissues” refers to a compound or polypeptide of the invention that modulates the partitioning of dietary lipids between the liver and the peripheral tissues as previously described.\",\n \"Preferably, the agent increases or decreases the oxidation of dietary lipids, preferably free fatty acids (FFA) by the muscle.\",\n \"Preferably the agent decreases or increases the body weight of individuals or is used to treat or prevent an metabolic-related disease or disorder such as obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM, Type II diabetes), Insulin dependent diabetes mellitus (IDDM, Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.\",\n \"Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.\",\n \"The terms “response to an agent acting on the partitioning of dietary lipids between the liver and peripheral tissues” refer to drug efficacy, including but not limited to, ability to metabolize a compound, ability to convert a pro-drug to an active drug, and the pharmacokinetics (absorption, distribution, elimination) and the pharmacodynamics (receptor-related) of a drug in an individual.\",\n \"The terms “side effects to an agent acting on the partitioning of dietary lipids between the liver and peripheral tissues” refer to adverse effects of therapy resulting from extensions of the principal pharmacological action of the drug or to idiosyncratic adverse reactions resulting from an interaction of the drug with unique host factors.\",\n \"“Side effects to an agent acting on the partitioning of dietary lipids between the liver and peripheral tissues” can include, but are not limited to, adverse reactions such as dermatologic, hematologic or hepatologic toxicities and further includes gastric and intestinal ulceration, disturbance in platelet function, renal injury, nephritis, vasomotor rhinitis with profuse watery secretions, angioneurotic edema, generalized urticaria, and bronchial asthma to laryngeal edema and bronchoconstriction, hypotension, and shock.\",\n \"The term “agent acting on the partitioning of endogenous lipids between the liver and peripheral tissues” refers to a compound or polypeptide of the invention that modulates the partitioning of endogenous lipids between the liver and the peripheral tissues as previously described.\",\n \"Preferably, the agent increases or decreases the oxidation of endogenous lipids, preferably free fatty acids (FFA) by the muscle.\",\n \"Preferably the agent decreases or increases the body weight of individuals or is used to treat or prevent an metabolic-related disease or disorder such as obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM, Type II diabetes), Insulin dependent diabetes mellitus (IDDM, Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.\",\n \"Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.\",\n \"The terms “response to an agent acting on the partitioning of endogenous lipids between the liver and peripheral tissues” refer to drug efficacy, including but not limited to, ability to metabolize a compound, ability to convert a pro-drug to an active drug, and the pharmacokinetics (absorption, distribution, elimination) and the pharmacodynamics (receptor-related) of a drug in an individual.\",\n \"The terms “side effects to an agent acting on the partitioning of endogenous lipids between the liver and peripheral tissues” refer to adverse effects of therapy resulting from extensions of the principal pharmacological action of the drug or to idiosyncratic adverse reactions resulting from an interaction of the drug with unique host factors.\",\n \"“Side effects to an agent acting on the partitioning of endogenous lipids between the liver and peripheral tissues” can include, but are not limited to, adverse reactions such as dermatologic, hematologic or hepatologic toxicities and further includes gastric and intestinal ulceration, disturbance in platelet function, renal injury, nephritis, vasomotor rhinitis with profuse watery secretions, angioneurotic edema, generalized urticaria, and bronchial asthma to laryngeal edema and bronchoconstriction, hypotension, and shock.\",\n \"The term “GSSP4-related diseases and disorders” as used herein refers to any disease or disorder comprising an aberrant functioning of a GSSP4, or which could be treated or prevented by modulating GSSP4 levels or activity.\",\n \"“Aberrant functioning of a GSSP4” includes, but is not limited to, aberrant levels of expression of a GSSP4 polypeptide (either increased or decreased, but preferably decreased), aberrant activity of a GSSP4 polypeptide (either increased or decreased), and aberrant interactions with ligands or binding partners (either increased or decreased).\",\n \"By “aberrant” is meant a change from the type, or level of activity seen in normal cells, tissues, or patients, or seen previously in the cell, tissue, or patient prior to the onset of the illness.\",\n \"In preferred embodiments, these GSSP4-related diseases and disorders include obesity and the metabolic-related diseases and disorders described previously.\",\n \"The term “cosmetic treatments” is meant to include treatments with compounds or polypeptides of the invention that increase or decrease the body mass of an individual where the individual is not clinically obese or clinically underweight as defined previously.\",\n \"Thus, these individuals have a body mass index (BMI) below the cut-off for clinical obesity (e.g.\",\n \"below 30 kg/m2) and above the cut-off for clinical thinness (e.g.\",\n \"above 18.5 kg/m2).\",\n \"In addition, these individuals are preferably healthy (e.g.\",\n \"do not have an metabolic-related disease or disorder of the invention).\",\n \"“Cosmetic treatments” are also meant to encompass, in some circumstances, more localized increases in adipose tissue, for example, gains or losses specifically around the waist or hips, or around the hips and thighs, for example.\",\n \"These localized gains or losses of adipose tissue can be identified by increases or decreases in waist or hip size, for example.\",\n \"The term “prevent” or “preventing” as used herein refers to administering a compound prior to the onset of clinical symptoms of a disease or condition so as to prevent a physical manifestation of aberrations associated with obesity or insulin resistance to some extent.\",\n \"The term “prevent” or “preventing” does not mean the result is necessarily absolute, but rather effective for providing some degree of prevention or amelioration of the progression of the metabolic or GSSP4-related disorder (i.e., provide protective effects), amelioration of the symptoms of the disorder, and amelioration of the reoccurrence of the metabolic or GSSP4-related disorder.\",\n \"The term “treat” or “treating” as used herein refers to administering a compound after the onset of clinical symptoms.\",\n \"The term “treat” or “treating” means to ameliorate, alleviate symptoms, eliminate the causation of the symptoms either on a temporary or permanent basis, or to prevent or slow the appearance of symptoms of the named disorder or condition.\",\n \"The term “in need of treatment” as used herein refers to a judgment made by a caregiver (e.g.\",\n \"physician, nurse, nurse practitioner, etc in the case of humans; veterinarian in the case of animals, including non-human mammals) that an individual or animal requires or will benefit from treatment.\",\n \"This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, but that include the knowledge that the individual or animal is ill, or will be ill, as the result of a condition that is treatable by the compounds of the invention.\",\n \"The term “perceives a need for treatment” refers to a sub-clinical determination that an individual desires to reduce weight for cosmetic reasons as discussed under “cosmetic treatment” above.\",\n \"The term “perceives a need for treatment” in other embodiments can refer to the decision that an owner of an animal makes for cosmetic treatment of the animal.\",\n \"The term “patient” or “individual” as used herein refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans.\",\n \"The term may specify male or female or both, or exclude male or female.\",\n \"The term “non-human animal” refers to any non-human vertebrate, including birds and more usually mammals, preferably primates, animals such as swine, goats, sheep, donkeys, horses, cats, dogs, rabbits or rodents, more preferably rats or mice.\",\n \"Both the terms “animal” and “mammal” expressly embrace human subjects unless preceded with the term “non-human”.\",\n \"The inventors believe GSSP4 polypeptides are able to significantly reduce the postprandial response of glucose in an individual, particularly following a high fat/high carbohydrate meal, while not effecting the levels of insulin.\",\n \"Further, GSSP4 polypeptides of the invention are believed to modulate weight gain, particularly in individuals are fed a high fat/high carbohydrate diet.\",\n \"The instant invention encompasses the use of GSSP4 polypeptides in the modulation of glucose as an important new tool to control energy homeostasis and glucose regulation.\",\n \"The instant invention encompasses the use of GSSP4 polypeptides in the partitioning of glucose as an important new tool to control energy homeostasis and glucose regulation.\",\n \"The instant invention encompasses the use of GSSP4 polypeptides in the partitioning of lipids as an important new tool to control energy homeostasis and lipid regulation.\",\n \"The instant invention encompasses the use of GSSP4 polypeptides in the partitioning of dietary lipids as an important new tool to control energy homeostasis and lipid regulation.\",\n \"The instant invention encompasses the use of GSSP4 polypeptides in the partitioning of endogenous lipids as an important new tool to control energy homeostasis and lipid regulation.\",\n \"PREFERRED EMBODIMENTS OF THE INVENTION I. GSSP4 Polypeptides of the Invention GSSP4 polypeptides that have measurable activity in vitro and in vivo have been identified.\",\n \"These activities include, but are not limited to, reduction of the postprandial response of plasma free fatty acids, glucose, and triglycerides, particularly in mice fed a high fat/carbohydrate meal, increase in muscle free fatty acid oxidation in vitro and ex vivo, and sustained weight loss in mice on a high fat/carbohydrate diet.\",\n \"Other assays for GSSP4 polypeptide activity in vitro and in vivo are also provided (Examples 2-13, for example), and equivalent assays can be designed by those with skill in the art.\",\n \"The term “obesity-related” or “metabolic-related” activity as used herein refers to at least one, and preferably all, of the activities described herein for GSSP4 polypeptides.\",\n \"Assays for the determination of these activities are provided herein, and equivalent assays can be designed by those with ordinary skill in the art.\",\n \"The term “metabolic-related activity” as used herein refers to at least one, and preferably all, of the activities described herein for GSSP4 polypeptides.\",\n \"Assays for the determination of these activities are provided herein, known in the art, or can be designed by those with ordinary skill in the art.\",\n \"Optionally, “metabolic-related activity” can be selected from the group consisting of control of blood glucose, partitioning of glucose, glucose metabolism, lipid partitioning, lipid metabolism, and insulin-like activity, or an activity within one of these categories.\",\n \"Optionally, “obesity-related” activity can be selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity, or an activity within one of these categories.\",\n \"By “lipid partitioning” activity is meant the ability to effect the location of dietary lipids among the major tissue groups including, adipose tissue, liver, and muscle.\",\n \"The inventors have shown that GSSP4 polypeptides of the invention play a role in the partitioning of lipids to the muscle, liver or adipose tissue.\",\n \"By “lipid metabolism” activity is meant the ability to influence the metabolism of lipids.\",\n \"The inventors have shown that GSSP4 polypeptides of the invention have the ability to affect the level of free fatty acids in the plasma as well as to increase the metabolism of lipids in the muscle through free fatty acid oxidation experiments (Examples 4, 8, 10, 11, and 12) and to transiently affect the levels of triglycerides in the plasma and the muscle (Examples 7, 10, and 13).\",\n \"By “insulin-like” activity is meant the ability of GSSP4 polypeptides to modulate the levels of glucose in the plasma.\",\n \"The inventors have found that GSSP4 polypeptides do not significantly impact insulin levels but do impact glucose levels similarly to the effects of insulin (Examples 9 & 10).\",\n \"These effects are not seen in the presence of the intact (full-length) GSSP4 polypeptide or are significantly greater in the presence of the GSSP4 polypeptides compared with the full-length GSSP4 polypeptide.\",\n \"By “intact” or “full-length” GSSP4 polypeptide as used herein is meant the full length polypeptide sequence of the GSSP4 polypeptide of SEQ ID NO:3, from the N-terminal methionine to the C-terminal stop codon.\",\n \"Preferred GSSP4 polypeptides have a biological activity described herein, and as they would be useful in making antibodies, diagnostic assays, etc.\",\n \"As a further preferred embodiment, GSSP4 polypeptides which allow a rise in plasma glucose to not more than 190 mg/dl, particularly following consumption of food, or which prevent a drop in serum glucose to not less than 70 mg/dl, particularly following consumption of food.\",\n \"As a further preferred embodiment, GSSP4 polypeptides which allow a rise in plasma fatty acids to not more than 420 mg/dl, particularly following consumption of food, or which prevent a drop in serum fatty acids to not less than 190 mg/dl, particularly following consumption of food.\",\n \"By “significantly” as used herein is meant statistically significant as it is typically determined by those with ordinary skill in the art.\",\n \"For example, data are typically calculated as a mean±SEM, and a p-value≦0.05 is considered statistically significant.\",\n \"Statistical analysis is typically done using either the unpaired Student's t test or the paired Student's t test, as appropriate in each study.\",\n \"Representative “metabolic-related assays” are provided in Examples below.\",\n \"These assays include, but are not limited to, in vivo and in vitro methods of measuring the postprandial response, methods of measuring glucose uptake, glucose oxidation, glucose concentration, lipid concentration, free fatty acid levels, fatty acid oxidation and methods of measuring weight modulation.\",\n \"In preferred embodiments, the post-prandial response is measured in non-human animals, preferably rodents.\",\n \"In preferred embodiments physiologic parameters are measured including, but not limited to, levels of glucose, fatty acids, insulin, and leptin.\",\n \"In other preferred embodiments, free fatty acid oxidation is measured in cells in vitro or ex vivo, preferably in muscle cells or tissue of non-human animals, preferably rodents.\",\n \"In yet other preferred embodiments weight modulation is measured in human or non-human animals, preferably rodents (rats or mice), primates, canines, felines or procines on a high fat/carbohydrate diet.\",\n \"Optionally, “metabolic-related activity” includes other activities not specifically identified herein.\",\n \"In general, “measurable parameters” relating to obesity and the field of metabolic research can be selected from the group consisting of free fatty acid levels, free fatty acid oxidation, triglyceride levels, glucose levels, insulin levels, leptin levels, food intake, and body weight.\",\n \"In these metabolic-related assays, preferred GSSP4 polypeptides or polynucleotides or both would cause a significant change in at least one of the measurable parameters selected from the group consisting of post-prandial lipemia, free fatty acid levels, triglyceride levels, glucose levels, glucose oxidation, glucose uptake, free fatty acid oxidation, and weight.\",\n \"Alternatively, preferred GSSP4 polypeptides or polynucleotides or both would have a significant change in at least one of the measurable parameters selected from the group consisting of an increase in blood fatty acid levels (FFA), an decrease in blood glucose levels, a decrease in insulin levels, an increase in glucose oxidation and an increase in FFA oxidation.\",\n \"The invention is drawn, inter alia, to isolated, purified or recombinant GSSP4 polypeptides.\",\n \"GSSP4 polypeptides of the invention are useful for treating or preventing insulin resistance, and reducing body weight or increasing body weight (using antagonists of GSSP4 polypeptides) either as a cosmetic treatment or for treatment or prevention of metabolic-related diseases and disorders.\",\n \"GSSP4 polypeptides are also useful inter alia in screening assays for agonists or antagonists of GSSP4 polypeptide activity, for raising GSSP4 polypeptide-specific antibodies, and in diagnostic assays.\",\n \"The GSSP4 polypeptides of the present invention are preferably provided in an isolated form, and may be partially or substantially purified.\",\n \"A recombinantly produced version of any one of the GSSP4 polypeptides can be substantially purified by the one-step method described by Smith et al.\",\n \"((1988) Gene 67(1):31-40) or by the methods described herein or known in the art.\",\n \"Polypeptides of the invention also can be purified from natural or recombinant sources using antibodies directed against the polypeptides of the invention by methods known in the art of protein purification.\",\n \"Preparations of GSSP4 polypeptides of the invention involving a partial purification of or selection for the GSSP4 polypeptides are also specifically contemplated.\",\n \"These crude preparations are envisioned to be the result of the concentration of cells expressing GSSP4 polypeptides with perhaps a few additional purification steps, but prior to complete purification of the polypeptides.\",\n \"The cells expressing GSSP4 polypeptides are present in a pellet, they are lysed, or the crude polypeptide is lyophilized, for example.\",\n \"GSSP4 polypeptides can be any integer in length from at least 6 consecutive amino acids to 1 amino acids less than a full length GSSP4 polypeptide of SEQ ID NO:4.Thus, for SEQ ID NO:4, a GSSP4 polypeptide can be: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105 consecutive amino acids.\",\n \"Each GSSP4 polypeptide as described above can be further specified in terms of its N-terminal and C-terminal positions.\",\n \"For example, every combination of a N-terminal and C-terminal position that polypeptides of from 6 contiguous amino acids to 1 amino acids less than the full length GSSP4 polypeptide could occupy, on any given intact and contiguous full length GSSP4 polypeptide sequence are included in the present invention.\",\n \"Thus, a 6 consecutive amino acid fragment could occupy positions selected from the group consisting of 1-6, 2-7, 3-8, 4-9, 5-10, 6-11, 7-12, 8-13, 9-14, 10-15, 11-16, 12-17, 13-18, 14-19, 15-20, 16-21, 17-22, 18-23, 19-24, 20-25, 21-26, 22-27, 23-28, 24-29, 25-30, 26-31, 27-32, 28-33, 29-34, 30-35, 31-36, 32-37, 33-38, 34-39, 35-40, 36-41, 37-42, 38-43, 39-44, 40-45, 41-46, 42-47, 43-48, 44-49, 45-50, 46-51, 47-52, 48-53, 49-54, 50-55, 51-56, 52-57, 53-58, 54-59, 55-60, 56-61, 57-62, 58-63, 59-64, 60-65, 61-66, 62-67, 63-68, 64-69, 65-70, 66-71, 67-72, 68-73, 69-74, 70-75, 71-76, 72-77, 73-78, 74-79, 75-80, 76-81, 77-82, 78-83, 79-84, 80-85, 81-86, 82-87, 83-88, 84-89, 85-90, 86-91, 87-92, 88-93, 89-94, 90-95, 91-96, 92-97, 93-98, 94-99, 95-100, 96-101, 97-102, 98-103, 99-104, and 100-105, of a 105 consecutive amino acid fragment.\",\n \"Similarly, the positions occupied by all the other fragments of sizes between 6 amino acids and 105 amino acids of SEQ ID NO:3 are included in the present invention and can also be immediately envisaged based on these two examples and therefore, are not individually listed solely for the purpose of not unnecessarily lengthening the specification.\",\n \"Furthermore, the positions occupied by fragments of 6 to 105 consecutive amino acids of SEQ ID NO:3 are included in the present invention and can also be immediately envisaged based on these two examples and therefore are not individually listed solely for the purpose of not unnecessarily lengthening the specification.\",\n \"In addition, the positions occupied by fragments of 6 consecutive amino acids to 1 amino acid less than any other full length GSSP4 polypeptide can also be envisaged based on these two examples and therefore are not individually listed solely for the purpose of not unnecessarily lengthening the specification.\",\n \"The GSSP4 polypeptides of the present invention may alternatively be described by the formula “n to c” (inclusive); where “n” equals the N-terminal most amino acid position (as defined by the sequence listing) and “c” equals the C-terminal most amino acid position (as defined by the sequence listing) of the polypeptide; and further where “n” equals an integer between 1 and 99; and where “c” equals an integer between 7 and 105, the number of amino acids of the full length polypeptide sequence; and where “n” is an integer smaller then “c” by at least 6.Therefore, for the sequences provided in SEQ ID NO:3, “n” is any integer selected from the list consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, and 99; and “c” is any integer selected from the group consisting of: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, and 105.Every combination of “n” and “c” positions are included as specific embodiments of the invention.\",\n \"Moreover, the formula “n” to “c” may be modified as “n1-n2” to “c1-c2”, wherein “n1-n2” and “c1-c2” represent positional ranges selected from any two integers above which represent amino acid positions of the sequence listing.\",\n \"Alternative formulas include “n1-n2” to “c” and “n” to “c1-c2”.\",\n \"These specific embodiments, and other polypeptide and polynucleotide fragment embodiments described herein may be modified as being “at least”, “equal to”, “equal to or less than”, “less than”, “at least ______ but not greater than ______” or “from ______ to ______”.\",\n \"a specified size or specified N-terminal and/or C-terminal positions.\",\n \"It is noted that all ranges used to describe any embodiment of the present invention are inclusive unless specifically set forth otherwise.\",\n \"The present invention also provides for the exclusion of any individual fragment specified by N-terminal and C-terminal positions or of any fragment specified by size in amino acid residues as described above.\",\n \"In addition, any number of fragments specified by N-terminal and C-terminal positions or by size in amino acid residues as described above may be excluded as individual species.\",\n \"Further, any number of fragments specified by N-terminal and C-terminal positions or by size in amino acid residues as described above may make up a polypeptide fragment in any combination and may optionally include non-GSSP4 polypeptide sequence as well.\",\n \"The term “GSSP4 fragment” as used herein refers to fragments of a full-length GSSP4 polypeptides that comprise at least 6 and any other integer number of amino acids up to 104 of the full-length GSSP4 polypeptide (defined above).\",\n \"GSSP4 polypeptides of the invention include variants, fragments, analogs and derivatives of the GSSP4 polypeptides described above, including modified GSSP4 polypeptides.\",\n \"Variants It will be recognized by one of ordinary skill in the art that some amino acids of the GSSP4 polypeptide sequences of the present invention can be varied without significant effect on the structure or function of the proteins; there will be critical amino acids in the sequence that determine activity.\",\n \"Thus, the invention further includes variants of GSSP4 polypeptides that have metabolic-related activity as described above.\",\n \"Such variants include GSSP4 polypeptide sequences with one or more amino acid deletions, insertions, inversions, repeats, and substitutions either from natural mutations or human manipulation selected according to general rules known in the art so as to have little effect on activity.\",\n \"Guidance concerning how to make phenotypically silent amino acid substitutions is provided below.\",\n \"There are two main approaches for studying the tolerance of an amino acid sequence to change (see, Bowie, et al.\",\n \"(1990) Science, 247, 1306-10).\",\n \"The first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection.\",\n \"The second approach uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene and selections or screens to identify sequences that maintain functionality.\",\n \"These studies have revealed that proteins are surprisingly tolerant of amino acid substitutions and indicate which amino acid changes are likely to be permissive at a certain position of the protein.\",\n \"For example, most buried amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved.\",\n \"Other such phenotypically silent substitutions are described by Bowie et al.\",\n \"(supra) and the references cited therein.\",\n \"In the case of an amino acid substitution in the amino acid sequence of a polypeptide according to the invention, one or several amino acids can be replaced by “equivalent” amino acids.\",\n \"The expression “equivalent” amino acid is used herein to designate any amino acid that may be substituted for one of the amino acids having similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged.\",\n \"In particular embodiments, conservative substitutions of interest are shown in Table 1 under the heading of preferred substitutions.\",\n \"If such substitutions result in a change in biological activity, then more substantial changes, or as further described below in reference to amino acid classes, are introduced and the products screened.\",\n \"TABLE 1 Original Residue Exemplary Substitutions Preferred Substitutions Ala (A) val; leu; ile val Arg (R) lys; gin; asn lys Asn (N) gin; his; lys; arg gin Asp (D) glu glu Cys (C) ser ser Gin (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His (H) asn; gin; lys; arg arg Ile (I) leu; val; met; ala; phe; norleucine leu Leu (L) norleucine; ile; val; met; ala; phe ile Lys (K) arg; gin; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; ala; norleucine leu Substantial modifications in function or immunological identity of the GSSP4 polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.\",\n \"Naturally occurring residues are divided into groups based on common side-chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn, gln, his, lys, arg; (5) residues that influence chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.\",\n \"Non-conservative substitutions will entail exchanging a member of one of these classes for another class.\",\n \"Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.\",\n \"The variations can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis.\",\n \"Site-directed mutagenesis [Carter et al., Nucl.\",\n \"Acids Res., 13:4331 (1986); Zoller et al., Nucl.\",\n \"Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)], restriction selection mutagenesis [Wells et al., Philos.\",\n \"Trans.\",\n \"R. Soc.\",\n \"London SerA, 317:415 (1986)] or other known techniques can be performed on the cloned DNA to produce the GSSP4 variant DNA.\",\n \"Amino acids in the GSSP4 polypeptide sequences of the invention that are essential for function can also be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham, et al.\",\n \"(1989) Science 244(4908):1081-5).\",\n \"The latter procedure introduces single alanine mutations at every residue in the molecule.\",\n \"The resulting mutant molecules are then tested for metabolic-related activity using assays as described above.\",\n \"Of special interest are substitutions of charged amino acids with other charged or neutral amino acids that may produce proteins with highly desirable improved characteristics, such as less aggregation.\",\n \"Aggregation may not only reduce activity but also be problematic when preparing pharmaceutical or physiologically acceptable formulations, because aggregates can be immunogenic (see, e.g., Pinckard, et al., (1967) Clin.\",\n \"Exp.\",\n \"Immunol 2:331-340; Robbins, et al., (1987) Diabetes July;36(7):838-41; and Cleland, et al., (1993) Crit Rev Ther Drug Carrier Syst.\",\n \"10(4):307-77).\",\n \"Thus, the fragment, derivative, analog, or homolog of the GSSP4 polypeptides of the present invention may be, for example: (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code (i.e.\",\n \"may be a non-naturally occurring amino acid); or (ii) one in which one or more of the amino acid residues includes a substituent group; or (iii) one in which the GSSP4 polypeptide is fused with another compound, such as a compound to increase the half-life of the fragment (for example, polyethylene glycol); or (iv) one in which the additional amino acids are fused to the above form of the fragment, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the above form of the fragment or a pro-protein sequence.\",\n \"Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.\",\n \"A further embodiment of the invention relates to a polypeptide which comprises the amino acid sequence of GSSP4 polypeptide having an amino acid sequence which contains at least one conservative amino acid substitution, but not more than 50 conservative amino acid substitutions, not more than 40 conservative amino acid substitutions, not more than 30 conservative amino acid substitutions, and not more than 20 conservative amino acid substitutions.\",\n \"Also provided are polypeptides which comprise the amino acid sequence of a GSSP4 fragment, having at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid substitutions.\",\n \"Another specific embodiment of a modified GSSP4 polypeptide of the invention is a polypeptide that is resistant to proteolysis, for example a GSSP4 polypeptide in which a —CONH— peptide bond is modified and replaced by one or more of the following: a (CH2NH) reduced bond; a (NHCO) retro inverso bond; a (CH2—O) methylene-oxy bond; a (CH2—S) thiomethylene bond; a (CH2CH2) carba bond; a (CO—CH2) cetomethylene bond; a (CHOH—CH2) hydroxyethylene bond); a (N—N) bound; a E-alcene bond; or a —CH═CH— bond.\",\n \"Thus, the invention also encompasses a GSSP4 polypeptide or a variant thereof in which at least one peptide bond has been modified as described above.\",\n \"In addition, amino acids have chirality within the body of either L or D. In some embodiments it is preferable to alter the chirality of the amino acids in the GSSP4 polypeptides of the invention in order to extend half-life within the body.\",\n \"Thus, in some embodiments, one or more of the amino acids are preferably in the L configuration.\",\n \"In other embodiments, one or more of the amino acids are preferably in the D configuration.\",\n \"Percent Identity The polypeptides of the present invention also include polypeptides having an amino acid sequence at least 50% identical, at least 60% identical, or 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a GSSP4 polypeptide as described above.\",\n \"By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a GSSP4 polypeptide amino acid sequence is meant that the amino acid sequence is identical to the GSSP4 polypeptide sequence except that it may include up to five amino acid alterations per each 100 amino acids of the GSSP4 polypeptide amino acid sequence.\",\n \"The reference sequence is the GSSP4 polypeptide with a sequence corresponding to the sequences provided in SEQ ID NO:3.Thus, to obtain a polypeptide having an amino acid sequence at least 95% identical to a GSSP4 polypeptide amino acid sequence, up to 5% (5 of 100) of the amino acid residues in the sequence may be inserted, deleted, or substituted with another amino acid compared with the GSSP4 polypeptide sequence.\",\n \"These alterations may occur at the amino or carboxy termini or anywhere between those terminal positions, interspersed either individually among residues in the sequence or in one or more contiguous groups within the sequence.\",\n \"As a practical matter, whether any particular polypeptide is a percentage identical to a GSSP4 polypeptide can be determined conventionally using known computer programs.\",\n \"Such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, (1988) Proc Natl Acad Sci USA April;85(8):2444-8; Altschul et al., (1990) J. Mol.\",\n \"Biol.\",\n \"215(3):403-410; Thompson et al., (1994) Nucleic Acids Res.\",\n \"22(2):4673-4680; Higgins et al., (1996) Meth.\",\n \"Enzymol.\",\n \"266:383-402; Altschul et al., (1997) Nuc.\",\n \"Acids Res.\",\n \"25:3389-3402; Altschul et al., (1993) Nature Genetics 3:266-272).\",\n \"In a particularly preferred embodiment, protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool (“BLAST”), which is well known in the art (See, e.g., Karlin and Altschul (1990) Proc Natl Acad Sci USA March;87(6):2264-8; Altschul et al., 1990, 1993, 1997, all supra).\",\n \"In particular, five specific BLAST programs are used to perform the following tasks: (1) BLASTP and BLAST3 compare an amino acid query sequence against a protein sequence database; (2) BLASTN compares a nucleotide query sequence against a nucleotide sequence database; (3) BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database; (4) TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands); and (5) TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.\",\n \"The BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as “high-scoring segment pairs,” between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database.\",\n \"High-scoring segment pairs are preferably identified (i.e., aligned) by means of a scoring matrix, many of which are known in the art.\",\n \"Preferably, the scoring matrix used is the BLOSUM62 matrix (see, Gonnet et al., (1992) Science June 5;256(5062): 1443-5; Henikoff and Henikoff (1993) Proteins September;17(1):49-61).\",\n \"Less preferably, the PAM or PAM250 matrices may also be used (See, e.g., Schwartz and Dayhoff, eds, (1978) Matrices for Detecting Distance Relationships: Atlas of Protein Sequence and Structure, Washington: National Biomedical Research Foundation).\",\n \"The BLAST programs evaluate the statistical significance of all high-scoring segment pairs identified, and preferably selects those segments which satisfy a user-specified threshold of significance, such as a user-specified percent homology.\",\n \"Preferably, the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula of Karlin (See, e.g., Karlin and Altschul, (1990) Proc Natl Acad Sci USA March;87(6):2264-8).\",\n \"The BLAST programs may be used with the default parameters or with modified parameters provided by the user.\",\n \"Preferably, the parameters are default parameters.\",\n \"A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al.\",\n \"(1990) Comp.\",\n \"App.\",\n \"Biosci.\",\n \"6:237-245.In a sequence alignment the query and subject sequences are both amino acid sequences.\",\n \"The result of said global sequence alignment is in percent identity.\",\n \"Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group=25 Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=247 or the length of the subject amino acid sequence, whichever is shorter.\",\n \"If the subject sequence is shorter than the query sequence due to N-or C-terminal deletions, not because of internal deletions, the results, in percent identity, must be manually corrected because the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity.\",\n \"For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, that are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence.\",\n \"Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment.\",\n \"This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score.\",\n \"This final percent identity score is what is used for the purposes of the present invention.\",\n \"Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score.\",\n \"That is, only query amino acid residues outside the farthest N- and C-terminal residues of the subject sequence.\",\n \"For example, a 90 amino acid residue subject sequence is aligned with a 100-residue query sequence to determine percent identity.\",\n \"The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not match/align with the first residues at the N-terminus.\",\n \"The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program.\",\n \"If the remaining 90 residues are perfectly matched the final percent identity would be 90%.\",\n \"In another example, a 90-residue subject sequence is compared with a 100-residue query sequence.\",\n \"This time the deletions are internal so there are no residues at the N- or C-termini of the subject sequence, which are not matched/aligned with the query.\",\n \"In this case, the percent identity calculated by FASTDB is not manually corrected.\",\n \"Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected.\",\n \"No other manual corrections are made for the purposes of the present invention.\",\n \"Production Note, throughout the disclosure, wherever GSSP4 polypeptides are discussed, GSSP4 fragments are specifically intended to be included as a preferred subset of GSSP4 polypeptides.\",\n \"Specific production methods are addressed in detail in sections III, IV, V, and VI of the present specification.\",\n \"In brief, GSSP4 polypeptides are preferably isolated from mammalian tissue samples, preferably human samples, or expressed from mammalian genes, preferably human genes, in mammalian cells, preferably human cells.\",\n \"The GSSP4 polypeptides of the invention can be made using expression methods known in the art.\",\n \"The polynucleotides encoding the desired polypeptides of the invention, including fragments thereof, are ligated into an expression vector suitable for the particular host used.\",\n \"Both eukaryotic and prokaryotic host systems can be used in forming recombinant polypeptides.\",\n \"The polypeptides are isolated from lysed cells or from the culture medium and purified to the extent needed for its intended use.\",\n \"In addition, shorter protein fragments may be produced by chemical synthesis.\",\n \"Purification is by techniques known in the art, for example, differential extraction, salt fractionation, chromatography, centrifugation, and the like.\",\n \"Nucleotides comprising the coding sequence of the polynucleotides of the invention, or fragments thereof, are cloned into expression vectors by anyone skilled in the art.\",\n \"Preferred expression vectors include eukaryotic and prokaryotic expression vectors.\",\n \"Further preferred is the pGEX-3X expression vector.\",\n \"Purification of GSSP4 polypeptides may be facilitated by recombinant fusion of a heterologous peptide to the C-or N-terminus of the GSSP4 polypeptides.\",\n \"Preferred fusion polypeptides expressed include heterologous polypeptides containing a His-tag added to the C-terminus, a glutathione-S-transferase (GST) tag and a factor Xa protease digestion site.\",\n \"A preferred method of making the polypeptides of the invention includes a method comprising the following steps: Escherichia coli cells, preferably BL21, and preferably comprising the pGEX-3X vector containing polynucleotides of the invention containing a His tag added to the C-terminus, GST tag and Xa protease site, are grown to subconfluence, preferably absorbance of 0.8, and induced with isopropyl beta-D-thiogalactoside.\",\n \"Cells are pelleted, washed and lysed in buffer, preferably in buffer containing guanidine hydrochloride.\",\n \"Polypeptides are allowed to bind to nickel, preferably Ni-NTA containing beads, and are washed with buffers, preferably buffers containing urea.\",\n \"Polypeptide bound to nickel are equilibrated with buffer, preferably buffer containing sodium chloride and calcium chloride, preferably at pH 7.5.Polypeptides bound to Nickel are digested, ie.\",\n \"treated with buffer comprising protease, preferably protease factor Xa.\",\n \"Digestion is preferably carried out at room temperature for 12 to 20 hours.\",\n \"The cleaved GST tag, if any, is washed away with buffer, preferably buffer with urea, preferably at pH 5.9.Polypeptides of the invention are eluted with buffer, preferably buffer with urea, preferably at pH 4.5.Polypeptides of the invention are refolded, preferably by methods comprising the following steps: Polypeptides are diluted in buffer common in the art, preferably to a concentration of 100 microgram/ml, preferably in buffer containing urea at pH 4.5.Polypeptides are dialyzed against buffer, preferably dialysis buffer comprising 4M urea, 5 mM cysteine, 0.02% Tween-20, 10% glycerol, 10 mM Tris, 150 mM sodium chloride, and 100 mM NaH2PO4, preferably at pH 8.3.Dialysis buffer comprising 2 M urea is used to replace the initial dialysis buffer, and dialysis buffer is replaced at least 1, 2, or 3 times over at least 1, 2, 3, 4, 5, or 6 days.\",\n \"The refolded polypeptide is desalted by any method known in the art, preferably using a spin column.\",\n \"Polypeptides of the invention are further purified by methods known in the art.\",\n \"Preferably polpeptides are purified with reverse phase HPLC.\",\n \"Polypeptides are preferably eluted with 0.08% trifluoroacetic acid and a 10-50% acetonitrile gradient, and elution is monitored preferably at 206 nm.\",\n \"Acetonitrile and trifluoroacetic acid are removed for the compositions comprising the purified polypeptide, preferably by evaporation by lyophilization.\",\n \"Further examples of methods useful for the expression or purification of polypeptides of the invention are found described in Methods in Enzymology.\",\n \"The nucleic acid encoding a GSSP4 fragment can be obtained by PCR from a vector containing the GSSP4 nucleotide sequence using oligonucleotide primers complementary to the desired GSSP4 cDNA and containing restriction endonuclease sequences.\",\n \"Transfection of a GSSP4 fragment-expressing vector into mouse NIH 3T3 cells is one embodiment of introducing polynucleotides into host cells.\",\n \"Introduction of a polynucleotide encoding a polypeptide into a host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods.\",\n \"Such methods are described in many standard laboratory manuals, such as Davis et al.\",\n \"((1986) Methods in Molecular Biology, Elsevier Science Publishing Co., Inc., Amsterdam).\",\n \"It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.\",\n \"A polypeptide of this invention (i.e.\",\n \"a GSSP4 fragment) can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography.\",\n \"Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.\",\n \"Polypeptides of the present invention, and preferably the secreted form, can also be recovered from: products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells.\",\n \"Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated.\",\n \"Preferably the polypeptides of the invention are non-glycosylated.\",\n \"In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.\",\n \"In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with the polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polynucleotides.\",\n \"For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequences via homologous recombination, see, e.g., U.S. Pat.\",\n \"No.\",\n \"5,641,670, issued Jun.\",\n \"24, 1997; International Publication No.\",\n \"WO 96/29411, published Sep. 26, 1996; International Publication No.\",\n \"WO 94/12650, published Aug. 4, 1994; Koller et al., (1989) Proc Natl Acad Sci USA November;86(22):8932-5; Koller et al., (1989) Proc Natl Acad Sci USA November;86(22):8927-31; and Zijlstra et al.\",\n \"(1989) Nature November 23;342(6248):435-8; the disclosures of each of which are incorporated by reference in their entireties).\",\n \"Modifications In addition, polypeptides of the invention can be chemically synthesized using techniques known in the art (See, e.g., Creighton, 1983 Proteins.\",\n \"New York, N.Y.: W. H. Freeman and Company; and Hunkapiller et al., (1984) Nature July 12-18;310(5973): 105-11).\",\n \"For example, a relative short fragment of the invention can be synthesized by use of a peptide synthesizer.\",\n \"Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the fragment sequence.\",\n \"Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoroamino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general.\",\n \"Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).\",\n \"The invention encompasses polypeptide fragments which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc.\",\n \"Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.\",\n \"Additional post-translational modifications encompassed by the invention include, for example, N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression.\",\n \"The polypeptide fragments may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the polypeptide.\",\n \"Also provided by the invention are chemically modified derivatives of the polypeptides of the invention that may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity.\",\n \"See U.S. Pat.\",\n \"No.\",\n \"4,179,337.The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like.\",\n \"The polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.\",\n \"The polymer may be of any molecular weight, and may be branched or unbranched.\",\n \"For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term “about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing.\",\n \"Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).\",\n \"The polyethylene glycol molecules (or other chemical moieties) should be attached to the polypeptide with consideration of effects on functional or antigenic domains of the polypeptide.\",\n \"There are a number of attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al.\",\n \"(1992) Exp Hematol.\",\n \"September;20(8):1028-35, reporting pegylation of GM-CSF using tresyl chloride).\",\n \"Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.\",\n \"Multimers The polypeptide fragments of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers).\",\n \"Accordingly, the present invention relates to monomers and multimers of the polypeptide fragments of the invention, their preparation, and compositions (preferably, pharmaceutical or physiologically acceptable compositions) containing them.\",\n \"In specific embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers.\",\n \"In additional embodiments, the multimers of the invention are at least dimers, at least trimers, or at least tetramers.\",\n \"Multimers encompassed by the invention may be homomers or heteromers.\",\n \"As used herein, the term homomer, refers to a multimer containing only polypeptides corresponding to the GSSP4 polypeptides of the invention (including polypeptide fragments, variants, splice variants, and fusion proteins corresponding to these polypeptide fragments as described herein).\",\n \"These homomers may contain polypeptide fragments having identical or different amino acid sequences.\",\n \"In a specific embodiment, a homomer of the invention is a multimer containing only polypeptide fragments having an identical amino acid sequence.\",\n \"In another specific embodiment, a homomer of the invention is a multimer containing polypeptide fragments having different amino acid sequences.\",\n \"In specific embodiments, the multimer of the invention is a homodimer (e.g., containing polypeptide fragments having identical or different amino acid sequences) or a homotrimer (e.g., containing polypeptide fragments having identical and/or different amino acid sequences).\",\n \"In additional embodiments, the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.\",\n \"As used herein, the term heteromer refers to a multimer containing one or more heterologous polypeptides (i.e., corresponding to different proteins or polypeptide fragments thereof) in addition to the polypeptides of the invention.\",\n \"In a specific embodiment, the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer.\",\n \"In additional embodiments, the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.\",\n \"Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation.\",\n \"Thus, in one embodiment, multimers of the invention, such as, for example, homodimers or homotrimers, are formed when polypeptides of the invention contact one another in solution.\",\n \"In another embodiment, heteromultimers of the invention, such as, for example, heterotrimers or heterotetramers, are formed when polypeptides of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution.\",\n \"In other embodiments, multimers of the invention are formed by covalent associations with and/or between the polypeptides of the invention.\",\n \"Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (e.g., that recited in the sequence listing, or contained in the polypeptide encoded by a deposited clone).\",\n \"In one instance, the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences, which interact in the native (i.e., naturally occurring) polypeptide.\",\n \"In another instance, the covalent associations are the consequence of chemical or recombinant manipulation.\",\n \"Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a fusion protein of the invention.\",\n \"In one example, covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., U.S. Pat.\",\n \"No.\",\n \"5,478,925).\",\n \"In a specific example, the covalent associations are between the heterologous sequence contained in an Fc fusion protein of the invention (as described herein).\",\n \"In another specific example, covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from another protein that is capable of forming covalently associated multimers, such as for example, oseteoprotegerin (see, e.g., International Publication NO: WO 98/49305, the contents of which are herein incorporated by reference in its entirety).\",\n \"In another embodiment, two or more polypeptides of the invention are joined through peptide linkers.\",\n \"Examples include those peptide linkers described in U.S. Pat.\",\n \"No.\",\n \"5,073,627 (hereby incorporated by reference).\",\n \"Proteins comprising multiple polypeptides of the invention separated by peptide linkers may be produced using conventional recombinant DNA technology.\",\n \"Another method for preparing multimer polypeptides of the invention involves use of polypeptides of the invention fused to a leucine zipper or isoleucine zipper polypeptide sequence.\",\n \"Examples of leucine zipper domains suitable for producing soluble multimeric proteins of the invention are those described in PCT application WO 94/10308, hereby incorporated by reference.\",\n \"Recombinant fusion proteins comprising a polypeptide of the invention fused to a polypeptide sequence that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric fusion protein is recovered from the culture supernatant using techniques known in the art.\",\n \"Trimeric polypeptides of the invention may offer the advantage of enhanced biological activity.\",\n \"Preferred leucine zipper moieties and isoleucine moieties are those that preferentially form trimers.\",\n \"One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al.\",\n \"FEBS Letters (1994) May 16;344(2-3):191-5.and in U.S. patent application Ser.\",\n \"No.\",\n \"08/446,922, hereby incorporated by reference.\",\n \"Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric polypeptides of the invention.\",\n \"In another example, proteins of the invention are associated by interactions between Flag® & polypeptide sequence contained in fusion proteins of the invention containing Flag® polypeptide sequence.\",\n \"In a further embodiment, proteins of the invention are associated by interactions between heterologous polypeptide sequence contained in Flag® fusion proteins of the invention and anti Flag® antibody.\",\n \"The multimers of the invention may be generated using chemical techniques known in the art.\",\n \"For example, polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., U.S. Pat.\",\n \"No.\",\n \"5,478,925, which is herein incorporated by reference in its entirety).\",\n \"Additionally, multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., U.S. Pat.\",\n \"No.\",\n \"5,478,925, which is herein incorporated by reference in its entirety).\",\n \"Further, polypeptides of the invention may be routinely modified by the addition of cysteine or biotin to the C-terminus or N-terminus of the polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., U.S. Pat.\",\n \"No.\",\n \"5,478,925, which is herein incorporated by reference in its entirety).\",\n \"Additionally, at least 30 techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, e.g., U.S. Pat.\",\n \"No.\",\n \"5,478,925, which is herein incorporated by reference in its entirety).\",\n \"Alternatively, multimers of the invention may be generated using genetic engineering techniques known in the art.\",\n \"In one embodiment, polypeptides contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., U.S. Pat.\",\n \"No.\",\n \"5,478,925, which is herein incorporated by reference in its entirety).\",\n \"In a specific embodiment, polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., U.S. Pat.\",\n \"No.\",\n \"5,478,925, which is herein incorporated by reference in its entirety).\",\n \"In another embodiment, recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain (or hyrophobic or signal peptide) and which can be incorporated by membrane reconstitution techniques into liposomes (See, e.g., U.S. Pat.\",\n \"No.\",\n \"5,478,925, which is herein incorporated by reference in its entirety).\",\n \"II.\",\n \"GSSP4 Polynucleotides of the Invention Preferred polynucleotides are those that encode GSSP4 polypeptides of the invention.\",\n \"The recombinant polynucleotides encoding GSSP4 polypeptides can be used in a variety of ways, including, but not limited to, expressing the polypeptides in recombinant cells for use in screening assays for antagonists and agonists of its activity as well as to facilitate its purification for use in a variety of ways including, but not limited to screening assays for agonists and antagonists of its activity, diagnostic screens, and raising antibodies, as well as treatment and/or prevention of metabolic-related diseases and disorders and/or to reduce body mass.\",\n \"The invention relates to the polynucleotides encoding GSSP4 polypeptides and variant polypeptide fragments thereof as described herein.\",\n \"These polynucleotides may be purified, isolated, and/or recombinant.\",\n \"In all cases, the desired GSSP4 polynucleotides of the invention are those that encode GSSP4 polypeptides of the invention having metabolic-related activity as described and discussed herein.\",\n \"Fragments A polynucleotide fragment is a polynucleotide having a sequence that entirely is the same as part, but not all, of the full length GSSP4 polypeptide or a specified GSSP4 polypeptide nucleotide sequence.\",\n \"Such fragments may be “free-standing”, i.e.\",\n \"not part of or fused to other polynucleotides, or they may be comprised within another non-GSSP4 (heterologous) polynucleotide of which they form a part or region.\",\n \"However, several GSSP4 polynucleotide fragments may be comprised within a single polynucleotide.\",\n \"The GSSP4 polynucleotides of the invention comprise from 18 consecutive bases to 18 consecutive bases less than the full length polynucleotide sequences encoding the intact GSSP4 polypeptides, for example the GSSP4 polynucleotide sequences in SEQ ID NO:1 or 2.In one aspect of this embodiment, the polynucleotide comprises at least 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, or 648 consecutive nucleotides of a polynucleotide of the present invention.\",\n \"In addition to the above preferred nucleic acid sizes, further preferred nucleic acids comprise at least 18 nucleotides, wherein “at least 18” is defined as any integer between 18 and the integer representing 18 nucleotides less than the 3′ most nucleotide position of the intact GSSP4 polypeptides cDNA as set forth in SEQ ID NO: 2, or elsewhere herein.\",\n \"Further included as preferred polynucleotides of the present invention are nucleic acid fragments at least 18 nucleotides in length, as described above, that are further specified in terms of their 5′ and 3′ position set forth in the sequence listing below.\",\n \"For allelic and degenerate and other variants, position 1 is defined as the 5′ most nucleotide of the ORF, i.e., the nucleotide “A” of the start codon (ATG) with the remaining nucleotides numbered consecutively.\",\n \"Therefore, every combination of a 5′ and 3′ nucleotide position that a polynucleotide fragment invention, at least 18 contiguous nucleotides in length, could occupy on an intact GSSP4 polypeptide polynucleotide of the present invention is included in the invention as an individual species.\",\n \"The polynucleotide fragments specified by 5′ and 3′ positions can be immediately envisaged and are therefore not individually listed solely for the purpose of not unnecessarily lengthening the specification.\",\n \"It is noted that the above species of polynucleotide fragments of the present invention may alternatively be described by the formula “x to y”; where “x” equals the 5′ most nucleotide position and “y” equals the 3′ most nucleotide position of the polynucleotide; and further where “x” equals an integer between 1 and the number of nucleotides of the polynucleotide sequence of the present invention minus 18, and where “y” equals an integer between 19 and the number of nucleotides of the polynucleotide sequence of the present invention minus 18 nucleotides; and where “x” is an integer smaller then “y” by at least 18.The GSSP4 polynucleotide fragments of the invention comprise from 18 consecutive bases to the full length polynucleotide sequence encoding the GSSP4 fragments described in Section II of the Preferred Embodiments of the Invention.\",\n \"In one aspect of this embodiment, the polynucleotide comprises at least 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 740, 770, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100 or 2200 consecutive nucleotides of a polynucleotide of the present invention.\",\n \"In addition to the above preferred nucleic acid sizes, further preferred nucleic acids comprise at least 18 nucleotides, wherein “at least 18” is defined as any integer between 18 and the integer corresponding to the 3′ most nucleotide position of a GSSP4 fragment cDNA herein.\",\n \"Further included as preferred polynucleotides of the present invention are nucleic acid fragments at least 18 nucleotides in length, as described above, that are further specified in terms of their 5′ and 3′ position.\",\n \"The 5′ and 3′ positions are represented by the position numbers set forth in the sequence listing below.\",\n \"For allelic and degenerate and other variants, position 1 is defined as the 5′ most nucleotide of the open reading frame (ORF), i.e., the nucleotide “A” of the start codon (ATG) with the remaining nucleotides numbered consecutively.\",\n \"Therefore, every combination of a 5′ and 3′ nucleotide position that a polynucleotide fragment invention, at least 18 contiguous nucleotides in length, could occupy on a GSSP4 fragment polynucleotide of the present invention is included in the invention as an individual species.\",\n \"The polynucleotide fragments specified by 5′ and 3′ positions can be immediately envisaged and are therefore not individually listed solely for the purpose of not unnecessarily lengthening the specification.\",\n \"It is noted that the above species of polynucleotide fragments of the present invention may alternatively be described by the formula “x to y”; where “x” equals the 5′ most nucleotide position and “y” equals the 3′ most nucleotide position of the polynucleotide; and further where “x” equals an integer between 1 and the number of nucleotides of the GSSP4 polynucleotide sequences of the present invention minus 18, and where “y” equals an integer between 9 and the number of nucleotides of the GSSP4 polynucleotide sequences of the present invention; and where “x” is an integer smaller than “y” by at least 18.Every combination of “x” and ‘y’ positions are included as specific embodiments of the invention.\",\n \"Moreover, the formula “x” to “y” may be modified as “‘x1-x2” to “y1-y2’”, wherein “x1-x2” and “y1-y2” represent positional ranges selected from any two nucleotide positions of the sequence listing.\",\n \"Alternative formulas include “‘x1-x2” to “y’” and “‘x” to “y1-y2’”.\",\n \"These specific embodiments, and other polynucleotide fragment embodiments described herein may be modified as being “at least”, “equal to”, “equal to or less than”, “less than”, “at least ______ but not greater than ______”or “from ______ to ______”.\",\n \"a specified size or specified 5′ and/or 3′ positions.\",\n \"The present invention also provides for the exclusion of any species of polynucleotide fragments of the present invention specified by 5′ and 3′ positions or polynucleotides specified by size in nucleotides as described above.\",\n \"Any number of fragments specified by 5′ and 3′ positions or by size in nucleotides, as described above, may be excluded.\",\n \"Variants In other preferred embodiments, variants of GSSP4 polynucleotides encoding GSSP4 polypeptides are envisioned.\",\n \"Variants of polynucleotides, as the term is used herein, are polynucleotides whose sequence differs from a reference polynucleotide.\",\n \"A variant of a polynucleotide may be a naturally occurring variant such as a naturally occurring allelic variant, or it may be a variant that is not known to occur naturally.\",\n \"Such non-naturally occurring variants of the polynucleotide may be made by mutagenesis techniques, including those applied to polynucleotides, cells or organisms.\",\n \"Generally, differences are limited so that the nucleotide sequences of the reference and the variant are closely similar overall and, in many regions, identical.\",\n \"Polynucleotide variants that comprise a sequence substantially different from those described above but that, due to the degeneracy of the genetic code, still encode GSSP4 polypeptides of the present invention are also specifically envisioned.\",\n \"It would also be routine for one skilled in the art to generate the degenerate variants described above, for instance, to optimize codon expression for a particular host (e.g., change codons in the human mRNA to those preferred by other mammalian or bacterial host cells).\",\n \"As stated above, variant polynucleotides may occur naturally, such as a natural allelic variant, or by recombinant methods.\",\n \"By an “allelic variant” is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism (See, e.g., B. Lewin, (1990) Genes IV, Oxford University Press, New York).\",\n \"Non-naturally occurring variants may be produced using art-known mutagenesis techniques.\",\n \"Such nucleic acid variants include those produced by nucleotide substitutions, deletions, or additions.\",\n \"The substitutions, deletions, or additions may involve one or more nucleotides.\",\n \"Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions.\",\n \"Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of GSSP4 polypeptides of the invention.\",\n \"Also preferred in this regard are conservative substitutions.\",\n \"Nucleotide changes present in a variant polynucleotide are preferably silent, which means that they do not alter the amino acids encoded by the polynucleotide.\",\n \"However, nucleotide changes may also result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence.\",\n \"In cases where the nucleotide substitutions result in one or more amino acid changes, preferred GSSP4 polypeptides include those that retain one or more metabolic-related activity as described in Section I of the Preferred Embodiments of the Invention.\",\n \"By “retain the same activities” is meant that the activity measured using the polypeptide encoded by the variant GSSP4 polynucleotide in assays is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, and not more than 101%, 102%, 103%, 104%, 105%, 110%, 115%, 120% or 125% of the activity measured using a GSSP4 polypeptide described in the Examples Section herein.\",\n \"By the activity being “increased” is meant that the activity measured using the polypeptide encoded by the variant GSSP4 polynucleotide in assays is at least 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 170%, 180%, 190%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 450%, or 500% of the activity measured using a GSSP4 polypeptide described in the Examples Section herein.\",\n \"By the activity being “decrease” is meant that the activity measured using the polypeptide encoded by the variant GSSP4 polynucleotide in assays is decreased by at least 25%, 30%, 35%, 40%, 45%, or 50% of the activity measured using a GSSP4 polypeptide described in the Examples Section herein.\",\n \"Percent Identity The present invention is further directed to nucleic acid molecules having sequences at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequences of SEQ ID NO:1, or 2 or fragments thereof that encode a polypeptide having metabolic-related activity as described in Section I of the Preferred Embodiments of the Invention.\",\n \"Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequences shown in SEQ ID NO:1, or 2 or fragments thereof will encode a polypeptide having biological activity.\",\n \"In fact, since degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assay.\",\n \"It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having biological activity.\",\n \"This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly affect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid), as further described previously in Section I of the Preferred Embodiments of the Invention.\",\n \"By a polynucleotide having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the GSSP4 polypeptide.\",\n \"In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted, inserted, or substituted with another nucleotide.\",\n \"The query sequence may be an entire sequence or any fragment specified as described herein.\",\n \"The methods of determining and defining whether any particular nucleic acid molecule or polypeptide is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be done by using known computer programs.\",\n \"A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al., ((1990) Comput Appl Biosci.\",\n \"July;6(3):237-45).\",\n \"In a sequence alignment the query and subject sequences are both DNA sequences.\",\n \"An RNA sequence can be compared by first converting U's to T's.\",\n \"The result of said global sequence alignment is in percent identity.\",\n \"Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter.\",\n \"If the subject sequence is shorter than the query sequence because of 5′ or 3′ deletions, not because of internal deletions, a manual correction must be made to the results.\",\n \"This is because the FASTDB program does not account for 5′ and 3′ truncations of the subject sequence when calculating percent identity.\",\n \"For subject sequences truncated at the 5′ or 3′ ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5′ and 3′ of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence.\",\n \"Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment.\",\n \"This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score.\",\n \"This corrected score is what is used for the purposes of the present invention.\",\n \"Only nucleotides outside the 5′ and 3′ nucleotides of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.\",\n \"No other manual corrections are made for the purposes of the present invention.\",\n \"Fusions Further included in the present invention are polynucleotides encoding the polypeptides of the present invention that are fused in frame to the coding sequences for additional heterologous amino acid sequences.\",\n \"Also included in the present invention are nucleic acids encoding polypeptides of the present invention together with additional, non-coding sequences, including for example, but not limited to non-coding 5′ and 3′ sequences, vector sequence, sequences used for purification, probing, or priming.\",\n \"For example, heterologous sequences include transcribed, non-translated sequences that may play a role in transcription, and mRNA processing, for example, ribosome binding and stability of mRNA.\",\n \"The heterologous sequences may alternatively comprise additional coding sequences that provide additional functionalities.\",\n \"Thus, a nucleotide sequence encoding a polypeptide may be fused to a tag sequence, such as a sequence encoding a peptide that facilitates purification of the fused polypeptide.\",\n \"In certain preferred embodiments of this aspect of the invention, the tag amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available.\",\n \"For instance, hexa-histidine provides for convenient purification of the fusion protein (See, Gentz et al., (1989) Proc Natl Acad Sci USA February;86(3):821-4).\",\n \"The “HA” tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein (See, Wilson et al., (1984) Cell 37(3):767-78).\",\n \"As discussed above, other such fusion proteins include GSSP4 fragment cDNA fused to Fc at the N- or C-terminus.\",\n \"III.\",\n \"Recombinant Vectors of the Invention The term “vector” is used herein to designate either a circular or a linear DNA or RNA molecule, that is either double-stranded or single-stranded, and that comprises at least one polynucleotide of interest that is sought to be transferred in a cell host or in a unicellular or multicellular host organism.\",\n \"The present invention relates to recombinant vectors comprising any one of the polynucleotides described herein.\",\n \"The present invention encompasses a family of recombinant vectors that comprise polynucleotides encoding GSSP4 polypeptides of the invention.\",\n \"In a first preferred embodiment, a recombinant vector of the invention is used to amplify the inserted polynucleotide in a suitable cell host, this polynucleotide being amplified every time that the recombinant vector replicates.\",\n \"The inserted polynucleotide can be one that encodes GSSP4 polypeptides of the invention.\",\n \"A second preferred embodiment of the recombinant vectors according to the invention consists of expression vectors comprising polynucleotides encoding GSSP4 polypeptides of the invention.\",\n \"Within certain embodiments, expression vectors are employed to express a GSSP4 polypeptide of the invention, preferably a modified GSSP4 fragment described in the present invention, which can be then purified and, for example, be used as a treatment for metabolic-related diseases, or simply to reduce body mass of individuals.\",\n \"Expression requires that appropriate signals are provided in the vectors, said signals including various regulatory elements, such as enhancers/promoters from both viral and mammalian sources, that drive expression of the genes of interest in host cells.\",\n \"Dominant drug selection markers for establishing permanent, stable, cell clones expressing the products are generally included in the expression vectors of the invention, as they are elements that link expression of the drug selection markers to expression of the polypeptide.\",\n \"More particularly, the present invention relates to expression vectors which include nucleic acids encoding a GSSP4 polypeptide of the invention, or a modified GSSP4 fragment as described herein, or variants or fragments thereof, under the control of a regulatory sequence selected among GSSP4 polypeptides, or alternatively under the control of an exogenous regulatory sequence.\",\n \"Consequently, preferred expression vectors of the invention are selected from the group consisting of: (a) a GSSP4 fragmentregulatory sequence and driving the expression of a coding polynucleotide operably linked thereto; and (b) a GSSP4 fragment coding sequence of the invention, operably linked to regulatory sequences allowing its expression in a suitable cell host and/or host organism.\",\n \"Some of the elements which can be found in the vectors of the present invention are described in further detail in the following sections.\",\n \"1) General Features of the Expression Vectors of the Invention: A recombinant vector according to the invention comprises, but is not limited to, a YAC (Yeast Artificial Chromosome), a BAC (Bacterial Artificial Chromosome), a phage, a phagemid, a cosmid, a plasmid, or even a linear DNA molecule which may consist of a chromosomal, non-chromosomal, semi-synthetic or synthetic DNA.\",\n \"Such a recombinant vector can comprise a transcriptional unit comprising an assembly of: (1) a genetic element or elements having a regulatory role in gene expression, for example promoters or enhancers.\",\n \"Enhancers are cis-acting elements of DNA, usually from about 10 to 300 bp in length that act on the promoter to increase the transcription; (2) a structural or coding sequence which is transcribed into mRNA and eventually translated into a polypeptide, said structural or coding sequence being operably linked to the regulatory elements described in (1); and (3) appropriate transcription initiation and termination sequences.\",\n \"Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell.\",\n \"Alternatively, when a recombinant protein is expressed without a leader or transport sequence, it may include a N-terminal residue.\",\n \"This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.\",\n \"Generally, recombinant expression vectors will include origins of replication, selectable markers permitting transformation of the host cell, and a promoter derived from a highly expressed gene to direct transcription of a downstream structural sequence.\",\n \"The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably a leader sequence capable of directing secretion of the translated protein into the periplasmic space or the extracellular medium.\",\n \"In a specific embodiment wherein the vector is adapted for transfecting and expressing desired sequences in mammalian host cells, preferred vectors will comprise an origin of replication in the desired host, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcriptional termination sequences, and 5′-flanking non-transcribed sequences.\",\n \"DNA sequences derived from the SV40 viral genome, for example SV40 origin, early promoter, enhancer, splice and polyadenylation sites may be used to provide the required non-transcribed genetic elements.\",\n \"2) Regulatory Elements Promoters The suitable promoter regions used in the expression vectors of the present invention are chosen taking into account the cell host in which the heterologous gene is expressed.\",\n \"The particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of directing the expression of the nucleic acid in the targeted cell.\",\n \"Thus, where a human cell is targeted, it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell, such as, for example, a human or a viral promoter.\",\n \"A suitable promoter may be heterologous with respect to the nucleic acid for which it controls the expression or alternatively can be endogenous to the native polynucleotide containing the coding sequence to be expressed.\",\n \"Additionally, the promoter is generally heterologous with respect to the recombinant vector sequences within which the construct promoter/coding sequence has been inserted.\",\n \"Promoter regions can be selected from any desired gene using, for example, CAT (chloramphenicol transferase) vectors and more preferably pKK232-8 and pCM7 vectors.\",\n \"Preferred bacterial promoters are the LacI, LacZ, the T3 or T7 bacteriophage RNA polymerase promoters, the gpt, lambda PR, PL and trp promoters (EP 0036776), the polyhedrin promoter, or the p10 protein promoter from baculovirus (Kit Novagen) (Smith et al., (1983) Mol Cell Biol December;3(12):2156-65; O'Reilly et al., 1992), the lambda PR promoter or also the trc promoter.\",\n \"Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-L.\",\n \"In addition, promoters specific for a particular cell type may be chosen, such as those facilitating expression in adipose tissue, muscle tissue, or liver.\",\n \"Selection of a convenient vector and promoter is well within the level of ordinary skill in the art.\",\n \"The choice of a promoter is well within the ability of a person skilled in the field of genetic engineering.\",\n \"For example, one may refer to Sambrook et al.\",\n \"(1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY, Vol.\",\n \"1, 2, 3 (1989), or also to the procedures described by Fuller et al.\",\n \"(1996) Immunology in Current Protocols in Molecular Biology.\",\n \"Other Regulatory Elements Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript.\",\n \"The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed such as human growth hormone and SV40 polyadenylation signals.\",\n \"Also contemplated as an element of the expression cassette is a terminator.\",\n \"These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.\",\n \"3) Selectable Markers Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct.\",\n \"The selectable marker genes for selection of transformed host cells are preferably dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, TRP 1 for S. cerevisiae or tetracycline, rifampicin or ampicillin resistance in E. coli, or levan saccharase for mycobacteria, this latter marker being a negative selection marker.\",\n \"4) Preferred Vectors Bacterial Vectors As a representative but non-limiting example, useful expression vectors for bacterial use can comprise a selectable marker and a bacterial origin of replication derived from commercially available plasmids comprising genetic elements of pBR322 (ATCC 37017).\",\n \"Such commercial vectors include, for example, pKK223-3 (Pharmacia, Uppsala, Sweden), and pGEM1 (Promega Biotec, Madison, Wis., USA).\",\n \"Large numbers of other suitable vectors are known to those of skill in the art, and are commercially available, such as the following bacterial vectors: pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia); pQE-30 (QIAexpress).\",\n \"Baculovirus Vectors A suitable vector for the expression of polypeptides of the invention is a baculovirus vector that can be propagated in insect cells and in insect cell lines.\",\n \"A specific suitable host vector system is the pVL1392/1393 baculovirus transfer vector (Pharmingen) that is used to transfect the SF9 cell line (ATCC NoCRL 1711) which is derived from Spodoptera frugiperda.\",\n \"Other suitable vectors for the expression of GSSP4 polypeptides in a baculovirus expression system include those described by Chai et al.\",\n \"(1993; Biotechnol Appl Biochem.\",\n \"December; 18 (Pt 3):259-73); Vlasak et al.\",\n \"(1983; Eur J Biochem September 1;135(1):123-6); and Lenhard et al.\",\n \"(1996; Gene March 9;169(2):187-90).\",\n \"Viral Vectors In one specific embodiment, the vector is derived from an adenovirus.\",\n \"Preferred adenovirus vectors according to the invention are those described by Feldman and Steg (1996; Semin Interv Cardiol September;1(3):203-8) or Ohno et al.\",\n \"(1994; Science August 5;265(5173):781-4).\",\n \"Another preferred recombinant adenovirus according to this specific embodiment of the present invention is the human adenovirus type 2 or 5 (Ad 2 or Ad 5) or an adenovirus of animal origin (French patent application No.\",\n \"FR-93.05954).\",\n \"Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery systems of choice for the transfer of exogenous polynucleotides in vivo, particularly to mammals, including humans.\",\n \"These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host.\",\n \"Particularly preferred retroviruses for the preparation or construction of retroviral in vitro or in vivo gene delivery vehicles of the present invention include retroviruses selected from the group consisting of Mink-Cell Focus Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis virus and Rous Sarcoma virus.\",\n \"Particularly preferred Murine Leukemia Viruses include the 4070A and the 1504A viruses, Abelson (ATCC No VR-999), Friend (ATCC No VR-245), Gross (ATCC No VR-590), Rauscher (ATCC No VR-998) and Moloney Murine Leukemia Virus (ATCC No VR-190; PCT Application No WO 94/24298).\",\n \"Particularly preferred Rous Sarcoma Viruses include Bryan high titer (ATCC Nos VR-334, VR-657, VR-726, VR-659 and VR-728).\",\n \"Other preferred retroviral vectors are those described in Roth et al.\",\n \"(1996), PCT Application No WO 93/25234, PCT Application No WO 94/06920, Roux et al., ((1989) Proc Natl Acad Sci USA December;86(23):9079-83), Julan et al., (1992) J. Gen. Virol.\",\n \"3:3251-3255 and Neda et al., ((1991) J Biol Chem August 5;266(22):14143-6).\",\n \"Yet another viral vector system that is contemplated by the invention consists of the adeno-associated virus (AAV).\",\n \"The adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle (Muzyczka et al., (1992) Curr Top Microbiol Immunol;158:97-129).\",\n \"It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (Flotte et al., (1992) Am J Respir Cell Mol Biol September;7(3):349-56; Samulski et al., (1989) J Virol September;63(9):3822-8; McLaughlin et al., (1989) Am.\",\n \"J. Hum.\",\n \"Genet.\",\n \"59:561-569).\",\n \"One advantageous feature of AAV derives from its reduced efficacy for transducing primary cells relative to transformed cells.\",\n \"5) Delivery of the Recombinant Vectors In order to effect expression of the polynucleotides of the invention, these constructs must be delivered into a cell.\",\n \"This delivery may be accomplished in vitro, as in laboratory procedures for transforming cell lines, or in vivo or ex vivo, as in the treatment of certain disease states.\",\n \"One mechanism is viral infection where the expression construct is encapsulated in an infectious viral particle.\",\n \"Several non-viral methods for the transfer of polynucleotides into cultured mammalian cells are also contemplated by the present invention, and include, without being limited to, calcium phosphate precipitation (Graham et al., (1973) Virology August;54(2):536-9; Chen et al., (1987) Mol Cell Biol August;7(8):2745-52), DEAE-dextran (Gopal, (1985) Mol Cell Biol May;5(5): 1188-90), electroporation (Tur-Kaspa et al., (1986) Mol Cell Biol February;6(2):716-8; Potter et al., (1984) Proc Natl Acad Sci USA November;81(22):7161-5.\",\n \"), direct microinjection (Harland et al., (1985) J Cell Biol September;101(3):1094-9), DNA-loaded liposomes (Nicolau et al., (1982) Biochim Biophys Acta October 11;721(2):185-90; Fraley et al., (1979) Proc Natl Acad Sci USA July;76(7):3348-52), and receptor-mediated transfection (Wu and Wu, (1987) J Biol Chem April 5;262(10):4429-32; Wu and Wu (1988) Biochemistry February 9;27(3):887-92).\",\n \"Some of these techniques may be successfully adapted for in vivo or ex vivo use.\",\n \"Once the expression polynucleotide has been delivered into the cell, it may be stably integrated into the genome of the recipient cell.\",\n \"This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non specific location (gene augmentation).\",\n \"In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA.\",\n \"Such nucleic acid segments or “episomes” encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle.\",\n \"One specific embodiment for a method for delivering a protein or peptide to the interior of a cell of a vertebrate in vivo comprises the step of introducing a preparation comprising a physiologically acceptable carrier and a naked polynucleotide operatively coding for the polypeptide of interest into the interstitial space of a tissue comprising the cell, whereby the naked polynucleotide is taken up into the interior of the cell and has a physiological effect.\",\n \"This is particularly applicable for transfer in vitro but it may be applied to in vivo as well.\",\n \"Compositions for use in vitro and in vivo comprising a “naked” polynucleotide are described in PCT application No.\",\n \"WO 90/11092 (Vical Inc.) and also in PCT application No.\",\n \"WO 95/11307 (Institut Pasteur, INSERM, Universit{acute over (e )} d'Ottawa) as well as in the articles of Tascon et al.\",\n \"(1996) Nature Medicine.\",\n \"2(8):888-892 and of Huygen et al.\",\n \"((1996) Nat Med August;2(8):893-8).\",\n \"In still another embodiment of the invention, the transfer of a naked polynucleotide of the invention, including a polynucleotide construct of the invention, into cells may be proceeded with a particle bombardment (biolistic), said particles being DNA-coated microprojectiles accelerated to a high velocity allowing them to pierce cell membranes and enter cells without killing them, such as described by Klein et al.\",\n \"((1990) Curr Genet February;17(2):97-103).\",\n \"In a further embodiment, the polynucleotide of the invention may be entrapped in a liposome (Ghosh and Bacchawat, (1991) Targeted Diagn Ther;4:87-103; Wong et al., (1980) Gene 10:87-94; Nicolau et al., (1987) Methods Enzymol.;149:157-76).\",\n \"These liposomes may further be targeted to cells expressing LSR by incorporating leptin, triglycerides, ACRP30, or other known LSR ligands into the liposome membrane.\",\n \"In a specific embodiment, the invention provides a composition for the in vivo production of an GSSP4 polypeptides described herein.\",\n \"It comprises a naked polynucleotide operatively coding for this polypeptide, in solution in a physiologically acceptable carrier, and suitable for introduction into a tissue to cause cells of the tissue to express the said polypeptide.\",\n \"The amount of vector to be injected to the desired host organism varies according to the site of injection.\",\n \"As an indicative dose, it will be injected between 0.1 and 100 μg of the vector in an animal body, preferably a mammal body, for example a mouse body.\",\n \"In another embodiment of the vector according to the invention, it may be introduced in vitro in a host cell, preferably in a host cell previously harvested from the animal to be treated and more preferably a somatic cell such as a muscle cell.\",\n \"In a subsequent step, the cell that has been transformed with the vector coding for the desired GSSP4 polypeptides or the desired fragment thereof is reintroduced into the animal body in order to deliver the recombinant protein within the body either locally or systemically.\",\n \"IV.\",\n \"Recombinant Cells of the Invention Another object of the invention consists of host cells recombinant for, i.e., that have been transformed or transfected with one of the polynucleotides described herein, and more precisely a polynucleotide comprising a polynucleotide encoding a GSSP4 polypeptide of the invention such as any one of those described in “Polynucleotides of the Invention”.\",\n \"These polynucleotides can be present in cells as a result of transient or stable transfection.\",\n \"The invention includes host cells that are transformed (prokaryotic cells) or that are transfected (eukaryotic cells) with a recombinant vector such as any one of those described in “Recombinant Vectors of the Invention”.\",\n \"Generally, a recombinant host cell of the invention comprises at least one of the polynucleotides or the recombinant vectors of the invention that are described herein.\",\n \"Preferred host cells used as recipients for the recombinant vectors of the invention are the following: a) Prokaryotic host cells: Escherichia coli strains (I.E.\",\n \"DH5-α strain), Bacillus subtilis, Salmonella typhimurium, and strains from species like Pseudomonas, Streptomyces and Staphylococcus, and b) Eukaryotic host cells: HeLa cells (ATCC NoCCL2; NoCCL2.1; NoCCL2.2), Cv 1 cells (ATCC NoCCL70), COS cells (ATCC NoCRL1650; NoCRL1651), Sf-9 cells (ATCC NoCRL1711), C127 cells (ATCC No CRL-1804), 3T3 (ATCC No CRL-6361), CHO (ATCC No CCL-61), human kidney 293 (ATCC No 45504; No CRL-1573), BHK (ECACC No 84100501; No 84111301), PLC cells, HepG2, and Hep3B.\",\n \"The constructs in the host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.\",\n \"Following transformation of a suitable host and growth of the host to an appropriate cell density, the selected promoter is induced by appropriate means, such as temperature shift or chemical induction, and cells are cultivated for an additional period.\",\n \"Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.\",\n \"Microbial cells employed in the expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.\",\n \"Such methods are well known by the skilled artisan.\",\n \"Further, according to the invention, these recombinant cells can be created in vitro or in vivo in an animal, preferably a mammal, most preferably selected from the group consisting of mice, rats, dogs, pigs, sheep, cattle, and primates, not to include humans.\",\n \"Recombinant cells created in vitro can also be later surgically implanted in an animal, for example.\",\n \"Methods to create recombinant cells in vivo in animals are well-known in the art.\",\n \"The present invention also encompasses primary, secondary, and immortalized homologously recombinant host cells of vertebrate origin, preferably mammalian origin and particularly human origin, that have been engineered to: a) insert exogenous (heterologous) polynucleotides into the endogenous chromosomal DNA of a targeted gene, b) delete endogenous chromosomal DNA, and/or c) replace endogenous chromosomal DNA with exogenous polynucleotides.\",\n \"Insertions, deletions, and/or replacements of polynucleotide sequences may be to the coding sequences of the targeted gene and/or to regulatory regions, such as promoter and enhancer sequences, operably associated with the targeted gene.\",\n \"The present invention further relates to a method of making a homologously recombinant host cell in vitro or in vivo, wherein the expression of a targeted gene not normally expressed in the cell is altered.\",\n \"Preferably the alteration causes expression of the targeted gene under normal growth conditions or under conditions suitable for producing the polypeptide encoded by the targeted gene.\",\n \"The method comprises the steps of: (a) transfecting the cell in vitro or in vivo with a polynucleotide construct, the polynucleotide construct comprising; (i) a targeting sequence; (ii) a regulatory sequence and/or a coding sequence; and (iii) an unpaired splice donor site, if necessary, thereby producing a transfected cell; and (b) maintaining the transfected cell in vitro or in vivo under conditions appropriate for homologous recombination.\",\n \"The present invention further relates to a method of altering the expression of a targeted gene in a cell in vitro or in vivo wherein the gene is not normally expressed in the cell, comprising the steps of: (a) transfecting the cell in vitro or in vivo with a polynucleotide construct, the polynucleotide construct comprising: (i) a targeting sequence; (ii) a regulatory sequence and/or a coding sequence; and (iii) an unpaired splice donor site, if necessary, thereby producing a transfected cell; and (b) maintaining the transfected cell in vitro or in vivo under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell; and (c) maintaining the homologously recombinant cell in vitro or in vivo under conditions appropriate for expression of the gene.\",\n \"The present invention further relates to a method of making a polypeptide of the present invention by altering the expression of a targeted endogenous gene in a cell in vitro or in vivo wherein the gene is not normally expressed in the cell, comprising the steps of: a) transfecting the cell in vitro with a polynucleotide construct, the polynucleotide construct comprising: (i) a targeting sequence; (ii) a regulatory sequence and/or a coding sequence; and (iii) an unpaired splice donor site, if necessary, thereby producing a transfected cell; (b) maintaining the transfected cell in vitro or in vivo under conditions appropriate for homologous recombination, thereby producing a homologously recombinant cell; and c) maintaining the homologously recombinant cell in vitro or in vivo under conditions appropriate for expression of the gene thereby making the polypeptide.\",\n \"The present invention further relates to a polynucleotide construct that alters the expression of a targeted gene in a cell type in which the gene is not normally expressed.\",\n \"This occurs when a polynucleotide construct is inserted into the chromosomal DNA of the target cell, wherein the polynucleotide construct comprises: a) a targeting sequence; b) a regulatory sequence and/or coding sequence; and c) an unpaired splice-donor site, if necessary.\",\n \"Further included are polynucleotide constructs, as described above, wherein the construct further comprises a polynucleotide which encodes a polypeptide and is in-frame with the targeted endogenous gene after homologous recombination with chromosomal DNA.\",\n \"The compositions may be produced, and methods performed, by techniques known in the art, such as those described in U.S. Pat.\",\n \"Nos.\",\n \"6,054,288; 6,048,729; 6,048,724; 6,048,524; 5,994,127; 5,968,502; 5,965,125; 5,869,239; 5,817,789; 5,783,385; 5,733,761; 5,641,670; 5,580,734; International Publication Nos:WO96/29411, WO 94/12650; and scientific articles described by Koller et al., (1994) Annu.\",\n \"Rev.\",\n \"Immunol.\",\n \"10:705-730; the disclosures of each of which are incorporated by reference in their entireties).\",\n \"The expression of GSSP4s in mammalian, and typically human, cells may be rendered defective, or alternatively it may be enhanced, with the insertion of a GSSP4 genomic or cDNA sequence with the replacement of the GSSP4 gene counterpart in the genome of an animal cell by a GSSP4 polynucleotide according to the invention.\",\n \"These genetic alterations may be generated by homologous recombination events using specific DNA constructs that have been previously described.\",\n \"One kind of host cell that may be used are mammalian zygotes, such as murine zygotes.\",\n \"For example, murine zygotes may undergo microinjection with a purified DNA molecule of interest, for example a purified DNA molecule that has previously been adjusted to a concentration range from 1 ng/ml—for BAC inserts—3 ng/μl —for P1 bacteriophage inserts—in 10 mM Tris-HCl, pH 7.4, 250 μM EDTA containing 100 mM NaCl, 30 μM spermine, and 70 μM spermidine.\",\n \"When the DNA to be microinjected has a large size, polyamines and high salt concentrations can be used in order to avoid mechanical breakage of this DNA, as described by Schedl et al ((1993) Nature March 18;362(6417):258-61).\",\n \"Any one of the polynucleotides of the invention, including the DNA constructs described herein, may be introduced in an embryonic stem (ES) cell line, preferably a mouse ES cell line.\",\n \"ES cell lines are derived from pluripotent, uncommitted cells of the inner cell mass of pre-implantation blastocysts.\",\n \"Preferred ES cell lines are the following: ES-E14TG2a (ATCC No.CRL-1821), ES-D3 (ATCC No.CRL1934 and No.\",\n \"CRL-11632), YS001 (ATCC No.\",\n \"CRL-11776), 36.5 (ATCC No.\",\n \"CRL-11116).\",\n \"To maintain ES cells in an uncommitted state, they are cultured in the presence of growth inhibited feeder cells which provide the appropriate signals to preserve this embryonic phenotype and serve as a matrix for ES cell adherence.\",\n \"Preferred feeder cells are primary embryonic fibroblasts that are established from tissue of day 13-day 14 embryos of virtually any mouse strain, that are maintained in culture, such as described by Abbondanzo et al.\",\n \"(1993; Methods Enzymol;225:803-23) and are inhibited in growth by irradiation, such as described by Robertson ((1987) Embryo-derived stem cell lines.\",\n \"In: E. J. Robertson Ed.\",\n \"Teratocarcinomas and embrionic stem cells: a practical approach.\",\n \"IRL Press, Oxford), or by the presence of an inhibitory concentration of LIF, such as described by Pease and Williams (1990; Exp Cell Res.\",\n \"October;190(2):209-11).\",\n \"The constructs in the host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.\",\n \"Following transformation of a suitable host and growth of the host to an appropriate cell density, the selected promoter is induced by appropriate means, such as temperature shift or chemical induction, and cells are cultivated for an additional period.\",\n \"Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.\",\n \"Microbial cells employed in the expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.\",\n \"Such methods are well known by the skilled artisan.\",\n \"V. Transgenic Animals The present invention also provides methods and compositions for the generation of non-human animals and plants that express recombinant GSSP4 polypeptides, i.e.\",\n \"recombinant GSSP4 fragments or full-length GSSP4 polypeptides.\",\n \"The animals or plants can be transgenic, i.e.\",\n \"each of their cells contains a gene encoding a GSSP4 polypeptide, or, alternatively, a polynucleotide encoding a GSSP4 polypeptide can be introduced into somatic cells of the animal or plant, e.g.\",\n \"into mammary secretory epithelial cells of a mammal.\",\n \"In preferred embodiments, the non-human animal is a mammal such as a cow, sheep, goat, pig, or rabbit.\",\n \"Methods of making transgenic animals such as mammals are well known to those of skill in the art, and any such method can be used in the present invention.\",\n \"Briefly, transgenic mammals can be produced, e.g., by transfecting a pluripotential stem cell such as an ES cell with a polynucleotide encoding a polypeptide of interest.\",\n \"Successfully transformed ES cells can then be introduced into an early stage embryo which is then implanted into the uterus of a mammal of the same species.\",\n \"In certain cases, the transformed (“transgenic”) cells will comprise part of the germ line of the resulting animal, and adult animals comprising the transgenic cells in the germ line can then be mated to other animals, thereby eventually producing a population of transgenic animals that have the transgene in each of their cells, and which can stably transmit the transgene to each of their offspring.\",\n \"Other methods of introducing the polynucleotide can be used, for example introducing the polynucleotide encoding the polypeptide of interest into a fertilized egg or early stage embryo via microinjection.\",\n \"Alternatively, the transgene may be introduced into an animal by infection of zygotes with a retrovirus containing the transgene (Jaenisch, R. (1976) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 73, 1260-1264).\",\n \"Methods of making transgenic mammals are described, e.g., in Wall et al.\",\n \"(1992) J Cell Biochem 1992 June;49(2): 113-20; Hogan, et al.\",\n \"(1986) in Manipulating the mouse embryo.\",\n \"A Laboratory Manual.\",\n \"Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; in WO 91/08216, or in U.S. Pat.\",\n \"No.\",\n \"4,736,866.In a preferred method, the polynucleotides are microinjected into the fertilized oocyte.\",\n \"Typically, fertilized oocytes are microinjected using standard techniques, and then cultured in vitrountil a “pre-implantation embryo” is obtained.\",\n \"Such pre-implantation embryos preferably contain approximately 16 to 150 cells.\",\n \"Methods for culturing fertilized oocytes to the pre-implantation stage are described, e.g., by Gordon et al.\",\n \"((1984) Methods in Enzymology, 101, 414); Hogan et al.\",\n \"((1986) in Manipulating the mouse embryo.\",\n \"A Laboratory Manual.\",\n \"Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y) (for the mouse embryo); Hammer et al.\",\n \"((1985) Nature, 315, 680) (for rabbit and porcine embryos); Gandolfi et al.\",\n \"((1987) J. Reprod.\",\n \"Fert.\",\n \"81, 23-28); Rexroad et al.\",\n \"((1988) J. Anim.\",\n \"Sci.\",\n \"66, 947-953) (for ovine embryos); and Eyestone et al.\",\n \"((1989) J. Reprod.\",\n \"Fert.\",\n \"85, 715-720); Camous et al.\",\n \"((1984) J. Reprod.\",\n \"Fert.\",\n \"72, 779-785); and Heyman et al.\",\n \"((1987) Theriogenology 27, 5968) (for bovine embryos); the disclosures of each of which are incorporated herein in their entireties.\",\n \"Pre-implantation embryos are then transferred to an appropriate female by standard methods to permit the birth of a transgenic or chimeric animal, depending upon the stage of development when the transgene is introduced.\",\n \"As the frequency of transgene incorporation is often low, the detection of transgene integration in pre-implantation embryos is often desirable using any of the herein-described methods.\",\n \"Any of a number of methods can be used to detect the presence of a transgene in a pre-implantation embryo.\",\n \"For example, one or more cells may be removed from the pre-implantation embryo, and the presence or absence of the transgene in the removed cell or cells can be detected using any standard method e.g.\",\n \"PCR.\",\n \"Alternatively, the presence of a transgene can be detected in utero or post partum using standard methods.\",\n \"In a particularly preferred embodiment of the present invention, transgenic mammals are generated that secrete recombinant GSSP4 polypeptides in their milk.\",\n \"As the mammary gland is a highly efficient protein-producing organ, such methods can be used to produce protein concentrations in the gram per liter range, and often significantly more.\",\n \"Preferably, expression in the mammary gland is accomplished by operably linking the polynucleotide encoding the GSSP4 polypeptide to a mammary gland specific promoter and, optionally, other regulatory elements.\",\n \"Suitable promoters and other elements include, but are not limited to, those derived from mammalian short and long WAP, alpha, beta, and kappa, casein, alpha and beta lactoglobulin, beta-CN 5′ genes, as well as the the mouse mammary tumor virus (MMTV) promoter.\",\n \"Such promoters and other elements may be derived from any mammal, including, but not limited to, cows, goats, sheep, pigs, mice, rabbits, and guinea pigs.\",\n \"Promoter and other regulatory sequences, vectors, and other relevant teachings are provided, e.g., by Clark (1998) J Mammary Gland Biol Neoplasia 3:337-50; Jost et al.\",\n \"(1999) Nat.\",\n \"Biotechnol 17:160-4; U.S. Pat.\",\n \"Nos.\",\n \"5,994,616; 6,140,552; 6,013,857; Sohn et al.\",\n \"(1999) DNA Cell Biol.\",\n \"18:845-52; Kim et al.\",\n \"(1999) J. Biochem.\",\n \"(Japan) 126:320-5; Soulier et al.\",\n \"(1999) Euro.\",\n \"J. Biochem.\",\n \"260:533-9; Zhang et al.\",\n \"(1997) Chin.\",\n \"J. Biotech.\",\n \"13:271-6; Rijnkels et al.\",\n \"(1998) Transgen.\",\n \"Res.\",\n \"7:5-14; Korhonen et al.\",\n \"(1997) Euro.\",\n \"J. Biochem.\",\n \"245:482-9; Uusi-Oukari et al.\",\n \"(1997) Transgen.\",\n \"Res.\",\n \"6:75-84; Hitchin et al.\",\n \"(1996) Prot.\",\n \"Expr.\",\n \"Purif.\",\n \"7:247-52; Platenburg et al.\",\n \"(1994) Transgen.\",\n \"Res.\",\n \"3:99-108; Heng-Cherl et al.\",\n \"(1993) Animal Biotech 4:89-107; and Christa et al.\",\n \"(2000) Euro.\",\n \"J. Biochem.\",\n \"267:1665-71; the entire disclosures of each of which is herein incorporated by reference.\",\n \"In another embodiment, the polypeptides of the invention can be produced in milk by introducing polynucleotides encoding the polypeptides into somatic cells of the mammary gland in vivo, e.g.\",\n \"mammary secreting epithelial cells.\",\n \"For example, plasmid DNA can be infused through the nipple canal, e.g.\",\n \"in association with DEAE-dextran (see, e.g., Hens et al.\",\n \"(2000) Biochim.\",\n \"Biophys.\",\n \"Acta 1523:161-171), in association with a ligand that can lead to receptor-mediated endocytosis of the construct (see, e.g., Sobolev et al.\",\n \"(1998) 273:7928-33), or in a viral vector such as a retroviral vector, e.g.\",\n \"the Gibbon ape leukemia virus (see, e.g., Archer et al.\",\n \"(1994) PNAS 91:6840-6844).\",\n \"In any of these embodiments, the polynucleotide may be operably linked to a mammary gland specific promoter, as described above, or, alternatively, any strongly expressing promoter such as CMV or MoMLV LTR.\",\n \"The suitability of any vector, promoter, regulatory element, etc.\",\n \"for use in the present invention can be assessed beforehand by transfecting cells such as mammary epithelial cells, e.g.\",\n \"MacT cells (bovine mammary epithelial cells) or GME cells (goat mammary epithelial cells), in vitro and assessing the efficiency of transfection and expression of the transgene in the cells.\",\n \"For in vivo administration, the polynucleotides can be administered in any suitable formulation, at any of a range of concentrations (e.g.\",\n \"1-500 μg/ml, preferably 50-100 μg/ml), at any volume (e.g.\",\n \"1-100 ml, preferably 1 to 20 ml), and can be administered any number of times (e.g.\",\n \"1, 2, 3, 5, or 10 times), at any frequency (e.g.\",\n \"every 1, 2, 3, 5, 10, or any number of days).\",\n \"Suitable concentrations, frequencies, modes of administration, etc.\",\n \"will depend upon the particular polynucleotide, vector, animal, etc., and can readily be determined by one of skill in the art.\",\n \"In a preferred embodiment, a retroviral vector such as as Gibbon ape leukemia viral vector is used, as described in Archer et al.\",\n \"((1994) PNAS 91:6840-6844).\",\n \"As retroviral infection typically requires cell division, cell division in the mammary glands can be stimulated in conjunction with the administration of the vector, e.g.\",\n \"using a factor such as estrodiol benzoate, progesterone, reserpine, or dexamethasone.\",\n \"Further, retroviral and other methods of infection can be facilitated using accessory compounds such as polybrene.\",\n \"In any of the herein-described methods for obtaining GSSP4 polypeptides from milk, the quantity of milk obtained, and thus the quantity of GSSP4 polypeptides produced, can be enhanced using any standard method of lactation induction, e.g.\",\n \"using hexestrol, estrogen, and/or progesterone.\",\n \"The polynucleotides used in such embodiments can either encode a full-length GSSP4 polypeptide or a GSSP4 fragment.\",\n \"Typically, the encoded polypeptide will include a signal sequence to ensure the secretion of the protein into the milk.\",\n \"Where a full length GSSP4 sequence is used, the full length protein can, e.g., be isolated from milk and cleaved in vitro using a suitable protease.\",\n \"Alternatively, a second, protease-encoding polynucleotide can be introduced into the animal or into the mammary gland cells, whereby expression of the protease results in the cleavage of the GSSP4 polypeptide in vivo, thereby allowing the direct isolation of GSSP4 fragments from milk.\",\n \"VI.\",\n \"Pharmaceutical or Physiologically Acceptable Compositions of the Invention The GSSP4 polypeptides of the invention can be administered to non-human animals and/or humans, alone or in pharmaceutical or physiologically acceptable compositions where they are mixed with suitable carriers or excipient(s).\",\n \"The pharmaceutical or physiologically acceptable composition is then provided at a therapeutically effective dose.\",\n \"A therapeutically effective dose refers to that amount of a GSSP4 polypeptide sufficient to result in prevention or amelioration of symptoms or physiological status of metabolic-related diseases or disorders as determined by the methods described herein.\",\n \"A therapeutically effective dose can also refer to the amount of a GSSP4 polypeptide necessary for a reduction in weight or a prevention of an increase in weight or prevention of an increase in the rate of weight gain in persons desiring this affect for cosmetic reasons.\",\n \"A therapeutically effective dosage of a GSSP4 polypeptide of the invention is that dosage that is adequate to promote weight loss or weight gain with continued periodic use or administration.\",\n \"Techniques for formulation and administration of GSSP4 polypeptides may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition.\",\n \"Other diseases or disorders that GSSP4 polypeptides of the invention could be used to treat or prevent include, but are not limited to, obesity and metabolic-related diseases and disorders such as obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, non-insulin-dependent diabetes and Type II diabetes.\",\n \"Type II diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, and renal lesions.\",\n \"Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.\",\n \"Other metabolic-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.\",\n \"The GSSP4 polypeptides may also be used to enhance physical performance during work or exercise or enhance a feeling of general well-being.\",\n \"Physical performance activities include walking, running, jumping, lifting and/or climbing.\",\n \"The GSSP4 polypeptides or antagonists thereof may also be used to treat dyslexia, attention-deficit disorder (ADD), attention-deficit/hyperactivity disorder (ADHD), and psychiatric disorders such as schizophrenia by modulating fatty acid metabolism, more specifically, the production of certain long-chain polyunsaturated fatty acids.\",\n \"It is expressly considered that the GSSP4 polypeptides of the invention may be provided alone or in combination with other pharmaceutically or physiologically acceptable compounds.\",\n \"Other compounds useful for the treatment of obesity and other diseases and disorders are currently well-known in the art.\",\n \"In a preferred embodiment, the GSSP4 polypeptides are useful for, and used in, the treatment of insulin resistance and diabetes using methods described herein and known in the art.\",\n \"More particularly, a preferred embodiments relates to process for the therapeutic modification and regulation of glucose metabolism in an animal or human subject, which comprises administering to a subject in need of treatment (alternatively on a timed daily basis) GSSP4 polypeptide (or polynucleotide encoding said polypeptide) in dosage amount and for a period sufficient to reduce plasma glucose levels in said animal or human subject.\",\n \"Further preferred embodiments relate to methods for the prophylaxis or treatment of diabetes comprising administering to a subject in need of treatment (alternatively on a timed daily basis) a GSSP4 polypeptide (or polynucleotide encoding said polypeptide) in dosage amount and for a period sufficient to reduce plasma glucose levels in said animal or human subject.\",\n \"Routes of Administration.\",\n \"Suitable routes of administration include oral, nasal, rectal, transmucosal, or intestinal administration, parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intrapulmonary (inhaled) or intraocular injections using methods known in the art.\",\n \"A particularly useful method of administering compounds for promoting weight loss involves surgical implantation, for example into the abdominal cavity of the recipient, of a device for delivering GSSP4 polypeptidesover an extended period of time.\",\n \"Other particularly preferred routes of administration are aerosol and depot formulation.\",\n \"Sustained release formulations, particularly depot of the invented medicaments are expressly contemplated.\",\n \"Composition/Formulation Pharmaceutical or physiologically acceptable compositions and medicaments for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries.\",\n \"Proper formulation is dependent upon the route of administration chosen.\",\n \"Certain of the medicaments described herein will include a pharmaceutically or physiologically acceptable acceptable carrier and at least one polypeptide that is a GSSP4 polypeptide of the invention.\",\n \"For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer such as a phosphate or bicarbonate buffer.\",\n \"For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation.\",\n \"Such penetrants are generally known in the art.\",\n \"Pharmaceutical or physiologically acceptable preparations that can be taken orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.\",\n \"The push-fit capsules can contain the active ingredients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.\",\n \"In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.\",\n \"In addition, stabilizers may be added.\",\n \"All formulations for oral administration should be in dosages suitable for such administration.\",\n \"For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.\",\n \"For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable gaseous propellant, e.g.\",\n \"carbon dioxide.\",\n \"In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount.\",\n \"Capsules and cartridges of, e.g.\",\n \"gelatin, for use in an inhaler or insufflator, may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.\",\n \"The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.\",\n \"Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.\",\n \"The compositions may take such forms as suspensions, solutions or emulsions in aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.\",\n \"Pharmaceutical or physiologically acceptable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form.\",\n \"Aqueous suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.\",\n \"Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.\",\n \"Alternatively, the active ingredient may be in powder or lyophilized form for constitution with a suitable vehicle, such as sterile pyrogen-free water, before use.\",\n \"In addition to the formulations described previously, the compounds may also be formulated as a depot preparation.\",\n \"Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.\",\n \"Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.\",\n \"Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.\",\n \"Various sustained release materials have been established and are well known by those skilled in the art.\",\n \"Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.\",\n \"Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.\",\n \"The pharmaceutical or physiologically acceptable compositions also may comprise suitable solid or gel phase carriers or excipients.\",\n \"Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.\",\n \"Effective Dosage.\",\n \"Pharmaceutical or physiologically acceptable compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve their intended purpose.\",\n \"More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated.\",\n \"Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.\",\n \"For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays.\",\n \"For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes or encompasses a concentration point or range shown to increase leptin or lipoprotein uptake or binding in an in vitro system.\",\n \"Such information can be used to more accurately determine useful doses in humans.\",\n \"A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms in a patient.\",\n \"Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50, (the dose lethal to 50% of the test population) and the ED50 (the dose therapeutically effective in 50% of the population).\",\n \"The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD5O and ED5O.\",\n \"Compounds that exhibit high therapeutic indices are preferred.\",\n \"The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.\",\n \"The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50, with little or no toxicity.\",\n \"The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.\",\n \"The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.\",\n \"(See, e.g., Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.\",\n \"1).\",\n \"Dosage amount and interval may be adjusted individually to provide plasma levels of the active compound which are sufficient to maintain or prevent weight loss or gain, depending on the particular situation.\",\n \"Dosages necessary to achieve these effects will depend on individual characteristics and route of administration.\",\n \"Dosage intervals can also be determined using the value for the minimum effective concentration.\",\n \"Compounds should be administered using a regimen that maintains plasma levels above the minimum effective concentration for 10-90% of the time, preferably between 30-90%; and most preferably between 50-90%.\",\n \"In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.\",\n \"The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.\",\n \"A preferred dosage range for the amount of a GSSP4 polypeptide of the invention, which can be administered on a daily or regular basis to achieve desired results, including a reduction in levels of circulating plasma triglyceride-rich lipoproteins, range from 0.01-0.5 mg/kg body mass.\",\n \"A more preferred dosage range is from 0.05-0.1 mg/kg.\",\n \"Of course, these daily dosages can be delivered or administered in small amounts periodically during the course of a day.\",\n \"It is noted that these dosage ranges are only preferred ranges and are not meant to be limiting to the invention.\",\n \"VII.\",\n \"Methods of Treatment The invention is drawn inter alia to methods of preventing or treating metabolic-related diseases and disorders comprising providing an individual in need of such treatment with a GSSP4 polypeptide of the invention.\",\n \"Preferably, the GSSP4 polypeptide has metabolic-related activity either in vitro or in vivo.\",\n \"Preferably the GSSP4 polypeptide is provided to the individual in a pharmaceutical composition that is preferably taken orally.\",\n \"Preferably the individual is a mammal, and most preferably a human.\",\n \"In preferred embodiments, the metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM, Type II diabetes), Insulin dependent diabetes mellitus (IDDM, Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.\",\n \"Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.\",\n \"In highly preferred embodiments, GSSP4 polypeptides in pharmaceutical compositions are used to modulate body weight in healthy individuals for cosmetic reasons.\",\n \"The invention also features a method of preventing or treating metabolic-related diseases and disorders comprising providing an individual in need of such treatment with a compound identified by assays of the invention (described in Section VI of the Preferred Embodiments of the Invention and in the Examples).\",\n \"Preferably these compounds antagonize or agonize effects of GSSP4 polypeptides in cells in vitro, muscles ex vivo, or in animal models.\",\n \"Alternatively, these compounds agonize or antagonize the effects of GSSP4 polypeptides on glucose metabolism, fatty acid metabolism, or lipid metabolism.\",\n \"Preferably, the compound is provided to the individual in a pharmaceutical composition that is preferably taken orally.\",\n \"Preferably the individual is a mammal, and most preferably a human.\",\n \"In preferred embodiments, the metabolic-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance (IGT), insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin dependent diabetes mellitus (NIDDM, Type II diabetes), Insulin dependent diabetes mellitus (IDDM, Type I diabetes), diabetes-related complications (such as elevated ketone bodies), microangiopathy, retinopathy, ocular lesions, neuropathy, nephropathy, polycystic ovarian syndrome (PCOS), and microangiopathic lesions, as well as syndromes such as acanthosis nigricans, leprechaunism, and lipoatrophy to be treated by the methods of the invention.\",\n \"Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.\",\n \"In highly preferred embodiments, the pharmaceutical compositions are used to modulate glucose levels.\",\n \"In highly preferred embodiments, the pharmaceutical compositions are used to modulate body weight for cosmetic reasons.\",\n \"In a further preferred embodiment, NIDDM patients are often treated with oral insulin secretagogues, such as 1,1-dimethyl-2-(2-morpholinophenyl)guanidine fumarate (BTS67582) or sulfonylureas including tolbutamide, tolazamide, chlorpropamide, glibendamide, glimepiride, glipizide and glidazide, or with insulin sensitising agents including metformin, ciglitazone, trogitazone and pioglitazone.\",\n \"A further use of the present invention is in therapy of NIDDM patients to improve their weight and glucose control, comprising a pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect in combination with an oral insulin secretagogue or an insulin sensitising agent.\",\n \"Preferably, the oral insulin secretagogue is 1,1-dimethyl-2-(2-morpholino phenyl)guanidine fumarate (BTS67582) or a sulphonylurea selected from tolbutamide, tolazamide, chlorpropamide, glibenclamide, glimepiride, glipizide and glidazide.\",\n \"Preferably, the insulin sensitising agent is selected from metformin, ciglitazone, troglitazone and pioglitazone.\",\n \"The present invention further provides a method of improving the weight and glucose control of NIDDM patients comprising the administration of said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect alone, without an oral insulin secretagogue or an insulin sensitising agent.\",\n \"In a further preferred embodiment, the present invention may be administered either concomitantly or concurrently, with the oral insulin secretagogue or insulin sensitising agent for example in the form of separate dosage units to be used simultaneously, separately or sequentially (either before or after the secretagogue or either before or after the sensitising agent).\",\n \"Accordingly, the present invention further provides a product containing a composition of said a pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect and an oral insulin secretagogue or insulin sensitising agent as a combined preparation for simultaneous, separate or sequential use for the improvement of weight and glucose control in NIDDM patients.\",\n \"The ratio of the present composition to the oral insulin secretagogue or insulin sensitising agent is such that the quantity of each active ingredient employed will be such as to provide a therapeutically effective level, but will not be larger than the quantity recommended as safe for administration.\",\n \"The action of reducing insulin resistance by the present invention indicates that compounds of such invention may be useful in the manufacture of a medicament which can be used as an insulin sensitiser.\",\n \"Accordingly, the present invention further provides for the use in the manufacture of a medicament which is an insulin sensitiser.\",\n \"In further embodiments, some patients who are diagnosed with Insulin Dependent Diabetes Mellitus (IDDM, Type I) can also show a certain amount of insulin resistance.\",\n \"Therefore, there may be benefits in treating these patients with said pharmaceutical or physiologically acceptable composition described in the fifth aspect or polynucleotides described in the second aspect in order to reduce their insulin resistance.\",\n \"This would mean that these patients would require a lower dosage of insulin in order to maintain similar or better control of their diabetes since the insulin dose would be associated with a greater blood glucose lowering efficacy.\",\n \"Such therapy would provide long-term benefits in terms of reducing the detrimental effects which can be caused by prolonged high-dosage of insulin treatment.\",\n \"Additionally, some Noninsulin Dependent Diabetes Mellitus (NIDDM, Type II) patients are also treated with insulin and have insulin resistance.\",\n \"Accordingly the present invention further provides a method for, and the use thereof in the manufacture of the medicament for, reducing the amount of insulin required daily by a human having NIDDM.\",\n \"The present invention also provides a method for, and the use of the composition in the manufacture of a medicament for, the prophylaxis of long-term detrimental effects caused by prolonged high dosage of insulin in humans having IDDM.\",\n \"A consequence of the resistance is that glucose concentrations rise.\",\n \"This leads, in turn, to an increased release of insulin.\",\n \"Hyperinsulinemia, both in the fasting and postprandial states, is a hallmark of insulin resistance.\",\n \"Hyperinsulinemia is also caused by stimulation of gluconeogenesis.\",\n \"Epidemilogical studies have shown that hyperinsulinemia is a risk factor for morbidity and mortality in cardiovascular disease (Smith U.\",\n \"(1994) Am.\",\n \"J. Clin.\",\n \"Nutr.\",\n \"59, suppl.\",\n \"686S).\",\n \"Accordingly, the invention also provides therapeutics and methods for reducing or preventing hypersecretion of insulin and disorders or conditions resulting therefrom.\",\n \"NIDDM is associated with various complications.\",\n \"As defined herein, “complications of NIDDM” is referred to as cardiovascular complications or several of the metabolic and circulatory disturbances that are associated with hyperglycemia, e.g., insulin resistance, hyperinsulinemia and/or hyperproinsulinemia, delayed insulin release, dyslipidemia, retinopathy, peripheral neuropathy, hypertension, and other coronary artery diseases (CADs).\",\n \"CAD is a major cause of morbidity and mortality in patients with NIDDM.\",\n \"Thus, by providing therapeutics and methods for reducing glucose levels, the invention provides therapeutics and methods for treating and preventing NIDDM and consequences thereof.\",\n \"The invention also provides therapeutics and methods for treating and preventing having impaired glucose tolerance (IGT).\",\n \"The usual meaning of impaired glucose tolerance is that it is a condition associated with insulin-resistance which is intermediate between frank, NIDDM and normal glucose tolerance (NGT).\",\n \"A high percentage of the IGT population is known to progress to NIDDM relative to persons with normal glucose tolerance (Sad, et al., New Engl.\",\n \"J. Med.\",\n \"1988; 319:1500-6).\",\n \"Thus, by providing therapeutics and methods for reducing or preventing IGT, i.e., for normalizing insulin resistance, the progression to NIDDM can be delayed or prevented.\",\n \"IGT is diagnosed by a procedure wherein an affected person's postprandial glucose response is determined to be abnormal as assessed by 2-hour postprandial plasma glucose levels.\",\n \"In this test, a measured amount of glucose is given to the patient and blood glucose levels measured regular intervals, usually every half hour for the first two hours and every hour thereafter.\",\n \"In a “normal” or non-IGT individual glucose levels rise during the first two hours to level less than 140 mg/dl and then drop rapidly.\",\n \"In an IGT individual, the blood glucose levels are higher and the drop-off level is at a slower rate.\",\n \"Resistance to insulin-stimulated glucose uptake in individuals who do not become frankly hyperglycemic nevertheless increases the likelihood of these individuals to develop numerous other diseases.\",\n \"In particular, an attempt to compensate for insulin resistance sets in motion a series of events that play an important role in the development of both hypertension and coronary artery disease (CAD), such as premature atherosclerotic vascular disease.\",\n \"This cluster of abnormalities is commonly called the “Metabolic Syndrome”, or the “Insulin-Resistance Syndrome” or “Syndrome X”.\",\n \"Increased plasma triglyceride and decreased HDL-cholesterol concentrations, conditions which are known to be associated with CAD, have also been reported to be associated with insulin resistance.\",\n \"Thus, by providing therapeutics and methods for reducing or preventing insulin resistance, the invention provides methods for reducing and/or preventing the appearance of insulin-resistance syndrome.\",\n \"Yet other diseases are associated with insulin resistance and, thus, be treated or prevented according to the methods of the invention.\",\n \"For example, obesity, which is the result of an imbalance between caloric intake and energy expenditure is highly correlated with insulin resistance and diabetes (Hotamisligil, Spiegelman et al., Science, 1993, 259:87-91).\",\n \"In humans obesity can be defined as a body weight exceeding 20% of the desirable body weight for individuals of the same sex, height and frame (Slans, L. B., in Endocrinology & Metabolism, 2d Ed., McGraw-Hill, New York 1987, pp.\",\n \"1203-1244; see also, R H. Williams, Textbook of Endocrinology, 1974, pp.\",\n \"904-916).\",\n \"In other animals (or also in humans) obesity can be determined by body weight patterns correlated with prolactin profiles given that members of a species that are young, lean and “healthy” (i.e., free of any disorders, not just metabolic disorders) have daily plasma prolactin level profiles that follow a regular pattern that is highly reproducible with a small standard deviation.\",\n \"Obesity, or excess fat deposits, correlate with and may trigger the onset of various lipid metabolism disorders, e.g.\",\n \"hypertension, Type II diabetes (NIDDM), atherosclerosis, cardiovascular disease, etc.\",\n \"Even in the absence of clinical obesity (according to the above definition) the reduction of body fat stores (notably visceral fat stores) in man especially on a long-term or permanent basis would be of significant benefit, both cosmetically and physiologically.\",\n \"Thus, by preventing or treating obesity, the methods of the invention will allow an individual to have a more comfortable life and avoid the onset of various diseases triggered by obesity.\",\n \"In yet another embodiment, the invention provides a method for treating a subject having polycystic ovary syndrome (PCOS).\",\n \"PCOS is among the most common disorders of premenopausal women, affecting 5-10% of this population.\",\n \"It is a syndrome of unknown Etiology characterized by hyperandrogenism, chronic anovulation, defects in insulin action, insulin secretion, ovarian steroidogenesis and fibrinolysis.\",\n \"Women with PCOS frequently are insulin resistant and at increased risk to develop glucose intolerance or NIDDM in the third and fourth decades of life (Dunaif et al.\",\n \"(1996) J. Clin.\",\n \"Endocrinol.\",\n \"Metab.\",\n \"81:3299).\",\n \"Hyperandrogenism also is a feature of a variety of diverse insulin-resistant states, from the type A syndrome, through leprechaunism and lipoatrophic diabetes, to the type B syndrome, when these conditions occur in premenopausal women.\",\n \"It has been suggested that hyperinsulinemia per se causes hyperandrogenism.\",\n \"Insulin-sensitizing agents, e.g., troglitazone, have been shown to be effective in PCOS and that, in particular, the defects in insulin action, insulin secretion, ovarian steroidogenosis and fibrinolysis are improved (Ehrman et al.\",\n \"(1997) J. Clin.\",\n \"Invest.\",\n \"100:1230), such as in insulin-resistant humans.\",\n \"Accordingly, the invention provides methods for reducing insulin resistance, normalizing blood glucose thus treating and/or preventing PCOS.\",\n \"Insulin resistance is also often associated with infections and cancer.\",\n \"Thus, prevention or reducing insulin resistance according to the methods of the invention may prevent or reduce infections and cancer.\",\n \"Insulin resistance can be diagnosed by various methods, such as by the intravenous glucose tolerance test or by measuring the fasting insulin level.\",\n \"It is well known that there is an excellent correlation between the height of the fasting insulin level and the degree of insulin resistance.\",\n \"Therefore, one could use elevated fasting insulin levels as a surrogate marker for insulin resistance for the purpose of identifying which normal glucose tolerance (NGT) individuals have insulin resistance.\",\n \"Another way to do this is to follow the approach as disclosed in The New England Journal of Medicine, No.\",\n \"3, pp.\",\n \"1188 (1995), i.e.\",\n \"to select obese subjects as an initial criteria for entry into the treatment group.\",\n \"Some obese subjects have impaired glucose tolerance (IGT) while others have normal glucose tolerance (NGT).\",\n \"Since essentially all obese subjects are insulin resistant, i.e.\",\n \"even the NGT obese subjects are insulin resistant, they have fasting hyperinsulinemia.\",\n \"Therefore, the target of the treatment according to the present invention can be defined as NGT individuals who are obese or who have fasting hyperinsulinemia, or who have both.\",\n \"Insulin resistance can also be diagnosed by the euglycemic glucose clamp test.\",\n \"This test involves the simultaneous administration of a constant insulin infusion and a variable rate glucose infusion.\",\n \"During the test, which lasts 3-4 hours, the plasma glucose concentration is kept constant at euglycemic levels by measuring the glucose level every 5-10 minutes and then adjusting the variable rate glucose infusion to keep the plasma glucose level unchanged.\",\n \"Under these circumstances, the rate of glucose entry into the bloodstream is equal to the overall rate of glucose disposal in the body.\",\n \"The difference between the rate of glucose disposal in the basal state (no insulin infusion) and the insulin infused state, represents insulin mediated glucose uptake.\",\n \"In normal individuals, insulin causes brisk and large increase in overall body glucose disposal, whereas in NIDDM subjects, this effect of insulin is greatly blunted, and is only 20-30% of normal.\",\n \"In insulin resistant subjects with either IGT or NGT, the rate of insulin stimulated glucose disposal is about half way between normal and NIDDM.\",\n \"For example, at a steady state plasma insulin concentration of about 100 uU/ml (a physiologic level) the glucose disposal rate in normal subjects is about 7 mg/kg/min.\",\n \"In NIDDM subjects, it is about 2.5 mg/.kg/min., and in patients with IGT (or insulin resistant subjects with NGT) it is about 4-5 mg/kg/min.\",\n \"This is a highly reproducible and precise test, and can distinguish patients within these categories.\",\n \"It is also known, that as subjects become more insulin resistant, the fasting insulin level rises.\",\n \"There is an excellent positive correlation between the height of the fasting insulin level and the magnitude of the insulin resistance as measured by euglycemic glucose clamp tests and, therefore, this provides the rationale for using fasting insulin levels as a surrogate measure of insulin resistance.\",\n \"Thus, any of the above-described tests or other tests known in the art can be used to determine that a subject is insulin-resistant, which patient can then be treated according to the methods of the invention to reduce or cure the insulin-resistance.\",\n \"Alternatively, the methods of the invention can also be used to prevent the development of insulin resistance in a subject, e.g., those known to have an increased risk of developing insulin-resistance.\",\n \"More generally, the instant invention is drawn to treatment with GSSP4 polypeptides where an individual is shown to have a particular genotype for GSSP4 marker.\",\n \"Treatment comprises providing pharmaceutically acceptable GSSP4 polypeptides to the individual.\",\n \"The exact amount of GSSP4 polypeptide provided would be determined through clinical trials under the guidance of qualified physicians, but would be expected to be in the range of 5-7 mg per individual per day.\",\n \"In general, a preferred range would be from 0.5 to 14 mg per individual per day, with a highly preferred range being between 1 and 10 mg per individual per day.\",\n \"Individuals who could benefit from treatment with GSSP4 polypeptides could be identified through genotyping.\",\n \"GSSP4 Genotyping The methods treatment using genotyping to identify individuals that would benefit from treatments of the invention are based on the finding that single nucleotide polymorphisms (SNPs) in GSSP4 have been identified that show an association in obese adolescents with free fatty acid (FFA) and respiratory quotient levels, others that show an association with the relationship between BMI and leptin, and still others that show an association with glucose levels.\",\n \"Further, a combination of GSSP4 SNPs associated with FFA and leptin metabolism may also predict people who will be seriously overweight.\",\n \"Briefly, the term “genotype” as used herein refers to the identity of the alleles present in an individual or a sample.\",\n \"The term “genotyping” a sample or an individual for a biallelic marker consists of determining the specific allele or the specific nucleotide carried by an individual at a biallelic marker.\",\n \"Biallelic markers generally consist of a polymorphism at one single base position.\",\n \"Each biallelic marker therefore corresponds to two forms of a polynucleotide sequence which, when compared with one another, present a nucleotide modification at one position.\",\n \"Usually, the nucleotide modification involves the substitution of one nucleotide for another, optionally either the original or the alternative allele of the biallelic markers disclosed in SEQ ID NO:1.Optionally either the original or the alternative allele of these biallelic markers may be specified as being present.\",\n \"Preferred polynucleotides may consist of, consist essentially of, or comprise a contiguous span of nucleotides upstream and down-stream of the alternate allele position noted in SEQ ID No:1 as well as sequences which are complementary thereto.\",\n \"The “contiguous span” may be at least 8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 70, 80, 100, 250, 500 or 1000 nucleotides in length, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID.\",\n \"Methods of genotyping comprise determining the identity of a nucleotide at GSSP4 biallelic marker site by any method known in the art.\",\n \"Preferably, microsequencing is used.\",\n \"The genotype is used to determine whether an individual should be treated with GSSP4 polypeptides.\",\n \"Thus, these genotyping methods are performed on nucleic acid samples derived from a single individual.\",\n \"These methods are well-known in the art, and discussed fully in the applications referenced briefly below.\",\n \"Any method known in the art can be used to identify the nucleotide present at a biallelic marker site.\",\n \"Since the biallelic marker allele to be detected has been identified and specified in the present invention, detection will prove simple for one of ordinary skill in the art by employing any of a number of techniques.\",\n \"Many genotyping methods require the previous amplification of the DNA region carrying the biallelic marker of interest.\",\n \"While the amplification of target or signal is often preferred at present, ultrasensitive detection methods that do not require amplification are also encompassed by the present genotyping methods.\",\n \"Methods well-known to those skilled in the art that can be used to detect biallelic polymorphisms include methods such as conventional dot blot analysis, single strand conformational polymorphism analysis (SSCP; Orita et al.\",\n \"(1989) Proc Natl Acad Sci USA April;86(8):2766-70), denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis, mismatch cleavage detection, and other conventional techniques as described in Sheffield et al.\",\n \"(1991; Am J Hum Genet October;49(4):699-706); White et al.\",\n \"(1992), Grompe et al.\",\n \"((1989) Proc Natl Acad Sci USA August;86(15):5888-92; (1993) Nat Genet.\",\n \"October;5(2):111-7).\",\n \"Another method for determining the identity of the nucleotide present at a particular polymorphic site employs a specialized exonuclease-resistant nucleotide derivative as described in U.S. Pat.\",\n \"No.\",\n \"4,656,127.Preferred methods involve directly determining the identity of the nucleotide present at a biallelic marker site by sequencing assay, allele-specific amplification assay, or hybridization assay.\",\n \"The following is a description of some preferred methods.\",\n \"A highly preferred method is the microsequencing technique.\",\n \"The term “sequencing” is used herein to refer to polymerase extension of duplex primer/template complexes and includes both traditional sequencing and microsequencing.\",\n \"Preferred biallelic markers, as shown in SEQ ID NO:1 and corresponding 47 mers as shown in SEQ ID NOs:4-18, include but are not limited to: 1.Biallelic marker 1: position base 148, alternate alleles A/G 2.Biallelic marker 2: position base 2551, alternate alleles G/A 3.Biallelic marker 3: position base 4417, alternate alleles A/C 4.Biallelic marker 4: position base 6322, alternate alleles A/G (amino acid change isoleucine to valine) 5.Biallelic marker 7: position base 816, alternate alleles G/A 6.Biallelic marker 8: position base 924, alternate alleles G/A 7.Biallelic marker 9: position base 1206, alternate alleles C/A 8.Biallelic marker 10: position base 1851, alternate alleles T/C 9.Biallelic marker 11: position base 3124, alternate alleles C/T 10.Biallelic marker 12: position base 3563, alternate alleles G/A 11.Biallelic marker 13: position base 3792, alternate alleles G/A 12.Biallelic marker 14: position base 5757, alternate alleles T/C 1) Sequencing Assays The nucleotide present at a polymorphic site can be determined by sequencing methods.\",\n \"In a preferred embodiment, DNA samples are subjected to PCR amplification before sequencing using any method known in the art.\",\n \"Preferably, the amplified DNA is subjected to automated dideoxy terminator sequencing reactions using a dye-primer cycle sequencing protocol.\",\n \"Sequence analysis allows the identification of the base present at the biallelic marker site.\",\n \"2) Microsequencing Assays In microsequencing methods, the nucleotide at a polymorphic site in a target DNA is detected by a single nucleotide primer extension reaction.\",\n \"This method involves appropriate microsequencing primers that hybridize just upstream of the polymorphic base of interest in the target nucleic acid.\",\n \"A polymerase is used to specifically extend the 3′ end of the primer with one single ddNTP (chain terminator) complementary to the nucleotide at the polymorphic site.\",\n \"The identity of the incorporated nucleotide is then determined in any suitable way.\",\n \"Preferred microsequencing primers are described in SEQ ID NO:1.Typically, microsequencing reactions are carried out using fluorescent ddNTPs and the extended microsequencing primers are analyzed by electrophoresis on ABI 377 sequencing machines to determine the identity of the incorporated nucleotide as described in EP 412 883.Alternatively capillary electrophoresis can be used in order to process a higher number of assays simultaneously.\",\n \"Different approaches can be used for the labeling and detection of ddNTPs.\",\n \"A homogeneous phase detection method based on fluorescence resonance energy transfer has been described by Chen and Kwok ((1997) Nucleic Acids Res.\",\n \"January 15;25(2):347-53) and Chen et al.\",\n \"((1997) Proc Natl Acad Sci USA September 30;94(20):10756-61).\",\n \"In this method, amplified genomic DNA fragments containing polymorphic sites are incubated with a 5′-fluorescein-labeled primer in the presence of allelic dye-labeled dideoxyribonucleoside triphosphates and a modified Taq polymerase.\",\n \"The dye-labeled primer is extended one base by the dye-terminator specific for the allele present on the template.\",\n \"At the end of the genotyping reaction, the fluorescence intensities of the two dyes in the reaction mixture are analyzed directly without separation or purification.\",\n \"All these steps can be performed in the same tube and the fluorescence changes can be monitored in real time.\",\n \"Alternatively, the extended primer may be analyzed by MALDI-TOF Mass Spectrometry.\",\n \"The base at the polymorphic site is identified by the mass added onto the microsequencing primer (see Haff and Smirnov, (1997) Nucleic Acids Res.\",\n \"September 15;25(18):3749-50; (1997) Genome Res.\",\n \"April;7(4):378-88).\",\n \"Microsequencing may be achieved by the established microsequencing method or by developments or derivatives thereof.\",\n \"Alternative methods include several solid-phase microsequencing techniques.\",\n \"The basic microsequencing protocol is the same as described previously, except that the method is conducted as a heterogeneous phase assay, in which the primer or the target molecule is immobilized or captured onto a solid support.\",\n \"To simplify the primer separation and the terminal nucleotide addition analysis, oligonucleotides are attached to solid supports or are modified in such ways that permit affinity separation as well as polymerase extension.\",\n \"The 5′ ends and internal nucleotides of synthetic oligonucleotides can be modified in a number of different ways to permit different affinity separation approaches, e.g., biotinylation.\",\n \"If a single affinity group is used on the oligonucleotides, the oligonucleotides can be separated from the incorporated terminator regent.\",\n \"This eliminates the need of physical or size separation.\",\n \"More than one oligonucleotide can be separated from the terminator reagent and analyzed simultaneously if more than one affinity group is used.\",\n \"This permits the analysis of several nucleic acid species or more nucleic acid sequence information per extension reaction.\",\n \"The affinity group need not be on the priming oligonucleotide but could alternatively be present on the template.\",\n \"For example, immobilization can be carried out via an interaction between biotinylated DNA and streptavidin-coated microtitration wells or avidin-coated polystyrene particles.\",\n \"In the same manner, oligonucleotides or templates may be attached to a solid support in a high-density format.\",\n \"In such solid phase microsequencing reactions, incorporated ddNTPs can be radiolabeled (Syvänen, (1994) Clin Chim Acta.\",\n \"May;226(2):225-36) or linked to fluorescein (Livak and Hainer, (1994) Hum Mutat.;3(4):379-85).\",\n \"The detection of radiolabeled ddNTPs can be achieved through scintillation-based techniques.\",\n \"The detection of fluorescein-linked ddNTPs can be based on the binding of antifluorescein antibody conjugated with alkaline phosphatase, followed by incubation with a chromogenic substrate (such as p-nitrophenyl phosphate).\",\n \"Other possible reporter-detection pairs include: ddNTP linked to dinitrophenyl (DNP) and anti-DNP alkaline phosphatase conjugate (Harju et al., (1993) Clin Chem.\",\n \"November;39(11 Pt 1):2282-7) or biotinylated ddNTP and horseradish peroxidase-conjugated streptavidin with o-phenylenediamine as a substrate (WO 92/15712).\",\n \"As yet another alternative solid-phase microsequencing procedure, Nyren et al.\",\n \"((1993) Anal Biochem.\",\n \"January;208(1):171-5).\",\n \"described a method relying on the detection of DNA polymerase activity by an enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA).\",\n \"Pastinen et al.\",\n \"((1997) Genome Res.\",\n \"June;7(6):606-14) describe a method for multiplex detection of single nucleotide polymorphism in which the solid phase minisequencing principle is applied to an oligonucleotide array format.\",\n \"High-density arrays of DNA probes attached to a solid support (DNA chips) are further described below.\",\n \"It will be appreciated that any primer having a 3′ end immediately adjacent to the polymorphic nucleotide may be used.\",\n \"Similarly, it will be appreciated that microsequencing analysis may be performed for any biallelic marker or any combination of biallelic markers of the present invention.\",\n \"3) Allele-Specific Amplification Assay Methods Discrimination between the two alleles of a biallelic marker can also be achieved by allele specific amplification, a selective strategy, whereby one of the alleles is amplified without, or at a much higher rate than, amplification of the other allele.\",\n \"This is accomplished by placing the polymorphic base at the 3′ end of one of the amplification primers.\",\n \"Because the extension forms from the 3′ end of the primer, a mismatch at or near this position has an inhibitory effect on amplification.\",\n \"Therefore, under appropriate amplification conditions, these primers only direct amplification on their complementary allele.\",\n \"Determining the precise location of the mismatch and the corresponding assay conditions are well with the ordinary skill in the art.\",\n \"The “Oligonucleotide Ligation Assay” (OLA) uses two oligonucleotides which are designed to be capable of hybridizing to abutting sequences of a single strand of a target molecules.\",\n \"One of the oligonucleotides is biotinylated, and the other is detectably labeled.\",\n \"If the precise complementary sequence is found in a target molecule, the oligonucleotides will hybridize such that their termini abut, and create a ligation substrate that can be captured and detected.\",\n \"OLA is capable of detecting single nucleotide polymorphisms and may be advantageously combined with PCR as described by Nickerson et al.\",\n \"((1990) Proc Natl Acad Sci USA November;87(22):8923-7).\",\n \"In this method, PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA.\",\n \"Other amplification methods which are particularly suited for the detection of single nucleotide polymorphism include LCR (ligase chain reaction) and Gap LCR (GLCR).\",\n \"LCR uses two pairs of probes to exponentially amplify a specific target.\",\n \"The sequences of each pair of oligonucleotides are selected to permit the pair to hybridize to abutting sequences of the same strand of the target.\",\n \"Such hybridization forms a substrate for a template-dependant ligase.\",\n \"In accordance with the present invention, LCR can be performed with oligonucleotides having the proximal and distal sequences of the same strand of a biallelic marker site.\",\n \"In one embodiment, either oligonucleotide will be designed to include the biallelic marker site.\",\n \"In such an embodiment, the reaction conditions are selected such that the oligonucleotides can be ligated together only if the target molecule either contains or lacks the specific nucleotide that is complementary to the biallelic marker on the oligonucleotide.\",\n \"In an alternative embodiment, the oligonucleotides will not include the biallelic marker, such that when they hybridize to the target molecule, a “gap” is created as described in WO 90/01069.This gap is then “filled” with complementary dNTPs (as mediated by DNA polymerase), or by an additional pair of oligonucleotides.\",\n \"Thus at the end of each cycle, each single strand has a complement capable of serving as a target during the next cycle and exponential allele-specific amplification of the desired sequence is obtained.\",\n \"Ligase/Polymerase-mediated Genetic Bit Analysis™ is another method for determining the identity of a nucleotide at a preselected site in a nucleic acid molecule (WO 95/21271).\",\n \"This method involves the incorporation of a nucleoside triphosphate that is complementary to the nucleotide present at the preselected site onto the terminus of a primer molecule, and their subsequent ligation to a second oligonucleotide.\",\n \"The reaction is monitored by detecting a specific label attached to the reaction's solid phase or by detection in solution.\",\n \"4) Hybridization Assay Methods A preferred method of determining the identity of the nucleotide present at a biallelic marker site involves nucleic acid hybridization.\",\n \"The hybridization probes, which can be conveniently used in such reactions, preferably include probes specific for GSSP4 cDNA surrounding GSSP4 biallelic markers.\",\n \"Any hybridization assay may be used including Southern hybridization, Northern hybridization, dot blot hybridization and solid-phase hybridization (see Sambrook et al., supra).\",\n \"Hybridization refers to the formation of a duplex structure by two single stranded nucleic acids due to complementary base pairing.\",\n \"Hybridization can occur between exactly complementary nucleic acid strands or between nucleic acid strands that contain minor regions of mismatch.\",\n \"Specific probes can be designed that hybridize to one form of a biallelic marker and not to the other and therefore are able to discriminate between different allelic forms.\",\n \"Allele-specific probes are often used in pairs, one member of a pair showing perfect match to a target sequence containing the original allele and the other showing a perfect match to the target sequence containing the alternative allele.\",\n \"Hybridization conditions should be sufficiently stringent that there is a significant difference in hybridization intensity between alleles, and preferably an essentially binary response, whereby a probe hybridizes to only one of the alleles.\",\n \"Stringent, sequence specific hybridization conditions, under which a probe will hybridize only to the exactly complementary target sequence are well known in the art (Sambrook et al., supra).\",\n \"Stringent conditions are sequence dependent and will be different in different circumstances.\",\n \"Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.\",\n \"Although such hybridizations can be performed in solution, it is preferred to employ a solid-phase hybridization assay.\",\n \"The target DNA comprising a biallelic marker of the present invention may be amplified prior to the hybridization reaction.\",\n \"The presence of a specific allele in the sample is determined by detecting the presence or the absence of stable hybrid duplexes formed between the probe and the target DNA.\",\n \"The detection of hybrid duplexes can be carried out by a number of methods.\",\n \"Various detection assay formats are well known which utilize detectable labels bound to either the target or the probe to enable detection of the hybrid duplexes.\",\n \"Typically, hybridization duplexes are separated from unhybridized nucleic acids and the labels bound to the duplexes are then detected.\",\n \"Those skilled in the art will recognize that wash steps may be employed to wash away excess target DNA or probe as well as unbound conjugate.\",\n \"Further, standard heterogeneous assay formats are suitable for detecting the hybrids using the labels present on the primers and probes.\",\n \"Two recently developed assays allow hybridization-based allele discrimination with no need for separations or washes (see Landegren U. et al., (1998) Genome Res.\",\n \"August;8(8):769-76).\",\n \"The TaqMan assay takes advantage of the 5′ nuclease activity of Taq DNA polymerase to digest a DNA probe annealed specifically to the accumulating amplification product.\",\n \"TaqMan probes are labeled with a donor-acceptor dye pair that interacts via fluorescence resonance energy transfer (FRET).\",\n \"Cleavage of the TaqMan probe by the advancing polymerase during amplification dissociates the donor dye from the quenching acceptor dye, greatly increasing the donor fluorescence.\",\n \"All reagents necessary to detect two allelic variants can be assembled at the beginning of the reaction and the results are monitored in real time (see Livak et al., 1995).\",\n \"In an alternative homogeneous hybridization based procedure, molecular beacons are used for allele discriminations.\",\n \"Molecular beacons are hairpin-shaped oligonucleotide probes that report the presence of specific nucleic acids in homogeneous solutions.\",\n \"When they bind to their targets they undergo a conformational reorganization that restores the fluorescence of an internally quenched fluorophore (Tyagi et al., (1998) Nat Biotechnol.\",\n \"January;16(1):49-53).\",\n \"The polynucleotides provided herein can be used to produce probes which can be used in hybridization assays for the detection of biallelic marker alleles in biological samples.\",\n \"These probes are characterized in that they preferably comprise between 8 and 50 nucleotides, and in that they are sufficiently complementary to a sequence comprising a biallelic marker of the present invention to hybridize thereto and preferably sufficiently specific to be able to discriminate the targeted sequence for only one nucleotide variation.\",\n \"A particularly preferred probe is 25 nucleotides in length.\",\n \"Preferably the biallelic marker is within 4 nucleotides of the center of the polynucleotide probe.\",\n \"In particularly preferred probes, the biallelic marker is at the center of said polynucleotide.\",\n \"In preferred embodiments the polymorphic base is within 5, 4, 3, 2, 1, nucleotides of the center of the said polynucleotide, more preferably at the center of said polynucleotide.\",\n \"Preferably the probes of the present invention are labeled or immobilized on a solid support.\",\n \"By assaying the hybridization to an allele specific probe, one can detect the presence or absence of a biallelic marker allele in a given sample.\",\n \"High-Throughput parallel hybridizations in array format are specifically encompassed within “hybridization assays” and are described below.\",\n \"5) Hybridization To Addressable Arrays Of Oligonucleotides Hybridization assays based on oligonucleotide arrays rely on the differences in hybridization stability of short oligonucleotides to perfectly matched and mismatched target sequence variants.\",\n \"Efficient access to polymorphism information is obtained through a basic structure comprising high-density arrays of oligonucleotide probes attached to a solid support (e.g., the chip) at selected positions.\",\n \"Each DNA chip can contain thousands to millions of individual synthetic DNA probes arranged in a grid-like pattern and miniaturized to the size of a dime.\",\n \"The chip technology has already been applied with success in numerous cases.\",\n \"For example, the screening of mutations has been undertaken in the BRCA1 gene, in S. cerevisiae mutant strains, and in the protease gene of HIV-1 virus (Hacia et al., (1996) Nat Genet.\",\n \"December; 14(4):441-7; Shoemaker et al., (1996) Nat Genet December; 14(4):450-6; Kozal et al., (1996) Nat Med.\",\n \"July;2(7):753-9).\",\n \"Chips of various formats for use in detecting biallelic polymorphisms can be produced on a customized basis by Affymetrix (GeneChip™), Hyseq (HyChip and HyGnostics), and Protogene Laboratories.\",\n \"In general, these methods employ arrays of oligonucleotide probes that are complementary to target nucleic acid sequence segments from an individual, which target sequences include a polymorphic marker.\",\n \"EP 785280 describes a tiling strategy for the detection of single nucleotide polymorphisms.\",\n \"Briefly, arrays may generally be “tiled” for a large number of specific polymorphisms.\",\n \"By “tiling” is generally meant the synthesis of a defined set of oligonucleotide probes which is made up of a sequence complementary to the target sequence of interest, as well as preselected variations of that sequence, e.g., substitution of one or more given positions with one or more members of the basis set of monomers, i.e.\",\n \"nucleotides.\",\n \"Tiling strategies are further described in PCT application No.\",\n \"WO 95/11995.In a particular aspect, arrays are tiled for a number of specific, identified biallelic marker sequences.\",\n \"In particular, the array is tiled to include a number of detection blocks, each detection block being specific for a specific biallelic marker or a set of biallelic markers.\",\n \"For example, a detection block may be tiled to include a number of probes, which span the sequence segment that includes a specific polymorphism.\",\n \"To ensure probes that are complementary to each allele, the probes are synthesized in pairs differing at the biallelic marker.\",\n \"In addition to the probes differing at the polymorphic base, monosubstituted probes are also generally tiled within the detection block.\",\n \"These monosubstituted probes have bases at and up to a certain number of bases in either direction from the polymorphism, substituted with the remaining nucleotides (selected from A, T, G, C and U).\",\n \"Typically the probes in a tiled detection block will include substitutions of the sequence positions up to and including those that are 5 bases away from the biallelic marker.\",\n \"The monosubstituted probes provide internal controls for the tiled array, to distinguish actual hybridization from artefactual cross-hybridization.\",\n \"Upon completion of hybridization with the target sequence and washing of the array, the array is scanned to determine the position on the array to which the target sequence hybridizes.\",\n \"The hybridization data from the scanned array is then analyzed to identify which allele or alleles of the biallelic marker are present in the sample.\",\n \"Hybridization and scanning may be carried out as described in PCT application No.\",\n \"WO 92/10092 and WO 95/11995 and U.S. Pat.\",\n \"No.\",\n \"5,424,186.Thus, in some embodiments, the chips may comprise an array of nucleic acid sequences of fragments of about 15 nucleotides in length.\",\n \"In preferred embodiments the polymorphic base is within 5, 4, 3, 2, 1, nucleotides of the center of the said polynucleotide, more preferably at the center of said polynucleotide.\",\n \"In some embodiments, the chip may comprise an array of at least 2, 3, 4, 5, 6, 7, 8 or more of these polynucleotides.\",\n \"6) Integrated Systems Another technique, which may be used to analyze polymorphisms, includes multicomponent integrated systems, which miniaturize and compartmentalize processes such as PCR and capillary electrophoresis reactions in a single functional device.\",\n \"An example of such technique is disclosed in U.S. Pat.\",\n \"No.\",\n \"5,589,136, which describes the integration of PCR amplification and capillary electrophoresis in chips.\",\n \"Integrated systems can be envisaged mainly when microfluidic systems are used.\",\n \"These systems comprise a pattern of microchannels designed onto a glass, silicon, quartz, or plastic wafer included on a microchip.\",\n \"The movements of the samples are controlled by electric, electroosmotic or hydrostatic forces applied across different areas of the microchip to create functional microscopic valves and pumps with no moving parts.\",\n \"Varying the voltage controls the liquid flow at intersections between the micro-machined channels and changes the liquid flow rate for pumping across different sections of the microchip.\",\n \"For genotyping biallelic markers, the microfluidic system may integrate nucleic acid amplification, microsequencing, capillary electrophoresis and a detection method such as laser-induced fluorescence detection.\",\n \"In a first step, the DNA samples are amplified, preferably by PCR.\",\n \"Then, the amplification products are subjected to automated microsequencing reactions using ddNTPs (specific fluorescence for each ddNTP) and the appropriate oligonucleotide microsequencing primers which hybridize just upstream of the targeted polymorphic base.\",\n \"Once the extension at the 3′ end is completed, the primers are separated from the unincorporated fluorescent ddNTPs by capillary electrophoresis.\",\n \"The separation medium used in capillary electrophoresis can for example be polyacrylamide, polyethyleneglycol or dextran.\",\n \"The incorporated ddNTPs in the single-nucleotide primer extension products are identified by fluorescence detection.\",\n \"This microchip can be used to process at least 96 to 384 samples in parallel.\",\n \"It can use the usual four color laser induced fluorescence detection of the ddNTPs.\",\n \"GSSP4 Association Studies Association studies focus on population frequencies and rely on the phenomenon of linkage disequilibrium.\",\n \"Linkage disequilibrium is the deviation from random of the occurrence of pairs of specific alleles at different loci on the same chromosome.\",\n \"If a specific allele in a given gene is directly associated with a particular trait, its frequency will be statistically increased in an affected (trait positive) population, when compared to the frequency in a trait negative population or in a random control population.\",\n \"As a consequence of the existence of linkage disequilibrium, the frequency of all other alleles present in the haplotype carrying the trait-causing allele will also be increased in trait positive individuals compared to trait negative individuals or random controls.\",\n \"Therefore, association between the trait and any allele (specifically a biallelic marker allele) in linkage disequilibrium with the trait-causing allele will suffice to suggest the presence of a trait-related gene in that particular region.\",\n \"Case-control populations can be genotyped for biallelic markers to identify associations that narrowly locate a trait causing allele, as any marker in linkage disequilibrium with one given marker associated with a trait will be associated with the trait.\",\n \"Linkage disequilibrium allows the relative frequencies in case-control populations of a limited number of genetic polymorphisms (specifically biallelic markers) to be analyzed as an alternative to screening all possible functional polymorphisms in order to find trait-causing alleles.\",\n \"Association studies compare the frequency of marker alleles in unrelated case-control populations, and represent powerful tools for the dissection of complex traits.\",\n \"Case-Control Populations (Inclusion Criteria) Population-based association studies do not concern familial inheritance, but compare the prevalence of a particular genetic marker, or a set of markers, in case-control populations.\",\n \"They are case-control studies based on comparison of unrelated case (affected or trait positive) individuals and unrelated control (unaffected, trait negative or random) individuals.\",\n \"Preferably, the control group is composed of unaffected or trait negative individuals.\",\n \"Further, the control group is ethnically matched to the case population.\",\n \"Moreover, the control group is preferably matched to the case-population for the main known confusion factor for the trait under study (for example age-matched for an age-dependent trait).\",\n \"Ideally, individuals in the two samples are paired in such a way that they are expected to differ only in their disease status.\",\n \"The terms “trait positive population”, “case population” and “affected population” are used interchangeably herein.\",\n \"An important step in the dissection of complex traits using association studies is the choice of case-control populations (see, Lander and Schork, (1994) Science, September 30;265(5181):2037-48).\",\n \"A major step in the choice of case-control populations is the clinical definition of a given trait or phenotype.\",\n \"Any genetic trait may be analyzed by the association method proposed here by carefully selecting the individuals to be included in the trait positive and trait negative phenotypic groups.\",\n \"Four criteria are often useful: clinical phenotype, age at onset, family history and severity.\",\n \"The selection procedure for continuous or quantitative traits (such as blood pressure for example) involves selecting individuals at opposite ends of the phenotype distribution of the trait under study, so as to include in these trait positive and trait negative populations individuals with non-overlapping phenotypes.\",\n \"Preferably, case-control populations consist of phenotypically homogeneous populations.\",\n \"Trait positive and trait negative populations consist of phenotypically uniform populations of individuals representing each between 1 and 98%, preferably between 1 and 80%, more preferably between 1 and 50%, and more preferably between 1 and 30%, most preferably between 1 and 20% of the total population under study, and preferably selected among individuals exhibiting non-overlapping phenotypes.\",\n \"The clearer the difference between the two trait phenotypes, the greater the probability of detecting an association with biallelic markers.\",\n \"The selection of those drastically different but relatively uniform phenotypes enables efficient comparisons in association studies and the possible detection of marked differences at the genetic level, provided that the sample sizes of the populations under study are significant enough.\",\n \"In preferred embodiments, a first group of between 50 and 300 trait positive individuals, preferably about 100 individuals, are recruited according to their phenotypes.\",\n \"A similar number of trait negative individuals are included in such studies.\",\n \"In the present invention, typical examples of inclusion criteria include obesity and disorders related to obesity as well as physiologic parameters associated with obesity, such as free fatty acid levels, glucose levels, insulin levels, leptin levels, triglyceride levels, free fatty acid oxidation levels, and weight loss.\",\n \"Association Analysis The general strategy to perform association studies using biallelic markers derived from a region carrying a candidate gene is to scan two groups of individuals (case-control populations) in order to measure and statistically compare the allele frequencies of the biallelic markers of the present invention in both groups.\",\n \"If a statistically significant association with a trait is identified for at least one or more of the analyzed biallelic markers, one can assume that: either the associated allele is directly responsible for causing the trait (i.e.\",\n \"the associated allele is the trait causing allele), or more likely the associated allele is in linkage disequilibrium with the trait causing allele.\",\n \"The specific characteristics of the associated allele with respect to the candidate gene function usually give further insight into the relationship between the associated allele and the trait (causal or in linkage disequilibrium).\",\n \"If the evidence indicates that the associated allele within the candidate gene is most probably not the trait-causing allele but is in linkage disequilibrium with the real trait-causing allele, then the trait-causing allele can be found by sequencing the vicinity of the associated marker, and performing further association studies with the polymorphisms that are revealed in an iterative manner.\",\n \"Association studies are usually run in two successive steps.\",\n \"In a first phase, the frequencies of a reduced number of biallelic markers from the candidate gene are determined in the trait positive and trait negative populations.\",\n \"In a second phase of the analysis, the position of the genetic loci responsible for the given trait is further refined using a higher density of markers from the relevant region.\",\n \"However, if the candidate gene under study is relatively small in length a single phase may be sufficient to establish significant associations.\",\n \"Haplotype Analysis As described above, when a chromosome carrying a disease allele first appears in a population as a result of either mutation or migration, the mutant allele necessarily resides on a chromosome having a set of linked markers: the ancestral haplotype.\",\n \"This haplotype can be tracked through populations and its statistical association with a given trait can be analyzed.\",\n \"Complementing single point (allelic) association studies with multi-point association studies also called haplotype studies increases the statistical power of association studies.\",\n \"Thus, a haplotype association study allows one to define the frequency and the type of the ancestral carrier haplotype.\",\n \"A haplotype analysis is important in that it increases the statistical power of an analysis involving individual markers.\",\n \"In a first stage of a haplotype frequency analysis, the frequency of the possible haplotypes based on various combinations of the identified biallelic markers of the invention is determined.\",\n \"The haplotype frequency is then compared for distinct populations of trait positive and control individuals.\",\n \"The number of trait positive individuals, which should be, subjected to this analysis to obtain statistically significant results usually ranges between 30 and 300, with a preferred number of individuals ranging between 50 and 150.The same considerations apply to the number of unaffected individuals (or random control) used in the study.\",\n \"The results of this first analysis provide haplotype frequencies in case-control populations, for each evaluated haplotype frequency a p-value and an odds ratio are calculated.\",\n \"If a statistically significant association is found the relative risk for an individual carrying the given haplotype of being affected with the trait under study can be approximated.\",\n \"Interaction Analysis The biallelic markers of the present invention may also be used to identify patterns of biallelic markers associated with detectable traits resulting from polygenic interactions.\",\n \"The analysis of genetic interaction between alleles at unlinked loci requires individual genotyping using the techniques described herein.\",\n \"The analysis of allelic interaction among a selected set of biallelic markers with appropriate level of statistical significance can be considered as a haplotype analysis.\",\n \"Interaction analysis consists in stratifying the case-control populations with respect to a given haplotype for the first loci and performing a haplotype analysis with the second loci with each subpopulation.\",\n \"VIII.\",\n \"Assays for Identifying Modulators of GSSP4 Polypeptide Activity The invention features methods of screening for one or more compounds that modulate the activity of GSSP4s in cells, which includes providing potential compounds to be tested to the cells.\",\n \"Exemplary assays that may be used are described in the Examples 2, 5-7,9-11.To these assays would be added compounds to be tested for their inhibitory or stimulatory activity as compared to the effects of GSSP4 polypeptides alone.\",\n \"Other assays in which an effect is observed based on the addition of GSSP4 polypeptides can also be used to screen for modulators of GSSP4 polypeptide activity or effects of the presence of GSSP4 polypeptides on cells.\",\n \"The essential step is to apply an unknown compound and then to monitor an assay for a change from what is seen when only GSSP4 polypeptides are applied to the cell.\",\n \"A change is defined as something that is significantly different in the presence of the compound plus GSSP4 polypeptide compared to GSSP4 polypeptide alone.\",\n \"In this case, significantly different would be an “increase” or a “decrease” in a measurable effect of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%.\",\n \"The term “modulation” as used herein refers to a measurable change in an activity.\",\n \"Examples include, but are not limited to, lipolysis stimulated receptor (LSR) modulation, leptin modulation, lipoprotein modulation, plasma FFA levels, FFA oxidation, TG levels, glucose levels, and weight.\",\n \"These effects can be in vitro or preferably in vivo.\",\n \"Modulation of an activity can be either an increase or a decrease in the activity.\",\n \"Thus, LSR activity can be increased or decreased, leptin activity can be increased or decreased, and lipoprotein activity can be increased or decreased.\",\n \"Similarly, FFA, TG, and glucose levels (and weight) can be increased or decreased in vivo Free Fatty Acid oxidation can be increased or decreased in vivo or ex vivo.\",\n \"By “LSR” activity is meant expression of LSR on the surface of the cell, or in a particular conformation, as well as its ability to bind, uptake, and degrade leptin and lipoprotein.\",\n \"By “leptin” activity is meant its binding, uptake and degradation by LSR, as well as its transport across a blood brain barrier, and potentially these occurrences where LSR is not necessarily the mediating factor or the only mediating factor.\",\n \"Similarly, by “lipoprotein” activity is meant its binding, uptake and degradation by LSR, as well as these occurrences where LSR is not necessarily the mediating factor or the only mediating factor.\",\n \"Exemplary assays are provided in Examples 2, 5-7,9-11.These assay and other comparable assays can be used to determine/identify compounds that modulate GSSP4 polypeptide activity.\",\n \"In some cases it may be important to identify compounds that modulate some but not all of the GSSP4 polypeptide activities, although preferably all activities are modified.\",\n \"The term “increasing” as used herein refers to the ability of a compound to increase the activity of GSSP4 polypeptides in some measurable way compared to the effect of GSSP4 polypeptides in its absence.\",\n \"As a result of the presence of the compound leptin binding and/or uptake might increase, for example, as compared to controls in the presence of the GSSP4 polypeptide alone.\",\n \"Preferably, an increase in activity is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% compared to the level of activity in the presence of the GSSP4 polypeptide.\",\n \"Similarly, the term “decreasing” as used herein refers to the ability of a compound to decrease an activity in some measurable way compared to the effect of a GSSP4 polypeptide in its absence.\",\n \"For example, the presence of the compound decreases the plasma concentrations of FFA, TG, and glucose in mice.\",\n \"Also as a result of the presence of a compound leptin binding and/or uptake might decrease, for example, as compared to controls in the presence of the GSSP4 polypeptide alone.\",\n \"Preferably, an decrease in activity is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% as compared to the level of activity in the presence of the GSSP4 polypeptide alone.\",\n \"The invention features a method for identifying a potential compound to modulate body mass in individuals in need of modulating body mass comprising: a) contacting a cell with a GSSP4 polypeptide and a candidate compound; b) detecting a result selected from the group consisting of LSR modulation, leptin modulation, lipoprotein modulation; FFA oxidation modulation; and c) wherein said result identifies said potential compound if said result differs from said result when said cell is contacted with the GSSP4 polypeptide alone.\",\n \"In preferred embodiments, said contacting further comprises a ligand of said LSR.\",\n \"Preferably said ligand is selected from the group consisting of cytokine, lipoprotein, free fatty acids, and C1q, and more preferably said cytokine is leptin, and most preferably said leptin is a leptin polypeptide fragment as described in U.S.\",\n \"Provisional application No.\",\n \"60/155,506 hereby incorporated by reference herein in its entirety including any figures, drawings, or tables.\",\n \"In other preferred embodiments, said GSSP4 polypeptide is mouse or is human.\",\n \"In other preferred embodiments, said cell is selected from the group consisting of PLC, CHO-K1, Hep3B, and HepG2.In yet other preferred embodiments, said lipoprotein modulation is selected from the group consisting of binding, uptake, and degradation.\",\n \"Preferably, said modulation is an increase in said binding, uptake, or degradation.\",\n \"Alternatively, said modulation is a decrease in said binding, uptake, or degradation.\",\n \"In other preferred embodiments, leptin modulation is selected from the group consisting of binding, uptake, degradation, and transport.\",\n \"Preferably, said modulation is an increase in said binding, uptake, degradation, or transport.\",\n \"Alternatively, said modulation is a decrease in said binding, uptake, degradation, or transport.\",\n \"Preferably, said transport is across a blood-brain barrier.\",\n \"In yet other preferred embodiments, said LSR modulation is expression on the surface of said cell.\",\n \"Preferably, said detecting comprises FACS, more preferably said detecting further comprises antibodies that bind specifically to said LSR, and most preferably said antibodies bind specifically to the carboxy terminus of said LSR.\",\n \"In still other preferred embodiments, said potential compound is selected from the group consisting of peptides, peptide libraries, non-peptide libraries, peptoids, fatty acids, lipoproteins, medicaments, antibodies, small molecules, and proteases.\",\n \"IX.\",\n \"Epitopes and Antibody Fusions A preferred embodiment of the present invention is directed to eiptope-bearing polypeptides and epitope-bearing polypeptide fragments.\",\n \"These epitopes may be “antigenic epitopes” or both an “antigenic epitope” and an “immunogenic epitope”.\",\n \"An “immunogenic epitope” is defined as a part of a protein that elicits an antibody response in vivo when the polypeptide is the immunogen.\",\n \"On the other hand, a region of polypeptide to which an antibody binds is defined as an “antigenic determinant” or “antigenic epitope.” The number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes.\",\n \"See, e.g., Geysen, et al.\",\n \"(1983) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 81:39984002.It is particularly noted that although a particular epitope may not be immunogenic, it is nonetheless useful since antibodies can be made in vitro to any epitope.\",\n \"An epitope can comprise as few as 3 amino acids in a spatial conformation which is unique to the epitope.\",\n \"Generally an epitope consists of at least 6 such amino acids, and more often at least 8-10 such amino acids.\",\n \"In preferred embodiment, antigenic epitopes comprise a number of amino acids that is any integer between 3 and 50.Fragments which function as epitopes may be produced by any conventional means.\",\n \"See, e.g., Houghten, R. A., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 82:5131-5135 (1985), further described in U.S. Pat.\",\n \"No.\",\n \"4,631,211.Methods for determining the amino acids which make up an immunogenic epitope include x-ray crystallography, 2-dimensional nuclear magnetic resonance, and epitope mapping, e.g., the Pepscan method described by H. Mario Geysen et al.\",\n \"(1984); Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"U.S.A. 81:3998-4002; PCT Publication No.\",\n \"WO 84/03564; and PCT Publication No.\",\n \"WO 84/03506.Another example is the algorithm of Jameson and Wolf, Comp.\",\n \"Appl.\",\n \"Biosci.\",\n \"4:181-186 (1988) (said references incorporated by reference in their entireties).\",\n \"The Jameson-Wolf antigenic analysis, for example, may be performed using the computer program PROTEAN, using default parameters (Version 4.0 Windows, DNASTAR, Inc., 1228 South Park Street Madison, Wis.).\",\n \"The epitope-bearing fragments of the present invention preferably comprises 6 to 50 amino acids (i.e.\",\n \"any integer between 6 and 50, inclusive) of a polypeptide of the present invention.\",\n \"Also, included in the present invention are antigenic fragments between the integers of 6 and the full length sequence of the sequence listing.\",\n \"All combinations of sequences between the integers of 6 and the full-length sequence of a polypeptide of the present invention are included.\",\n \"The epitope-bearing fragments may be specified by either the number of contiguous amino acid residues (as a sub-genus) or by specific N-terminal and C-terminal positions (as species) as described above for the polypeptide fragments of the present invention.\",\n \"Any number of epitope-bearing fragments of the present invention may also be excluded in the same manner.\",\n \"Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies that specifically bind the epitope (See, Wilson et al., 1984; and Sutcliffe, J. G. et al., 1983).\",\n \"The antibodies are then used in various techniques such as diagnostic and tissue/cell identification techniques, as described herein, and in purification methods.\",\n \"Similarly, immunogenic epitopes can be used to induce antibodies according to methods well known in the art (See, Sutcliffe et al., supra; Wilson et al., supra; Chow, M. et al.\",\n \";(1985) and Bittle, F. J. et al., (1985).\",\n \"A preferred immunogenic epitope includes the polypeptides of the sequence listing.\",\n \"The immunogenic epitopes may be presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse) if nessary.\",\n \"Immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting.).\",\n \"Epitope-bearing polypeptides of the present invention are used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods (See, e.g., Sutcliffe, et al., supra; Wilson, et al., supra, and Bittle, et al., 1985).\",\n \"If in vivo immunization is used, animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling of the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid.\",\n \"For instance, peptides containing cysteine residues may be coupled to a carrier using a linker such as—maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde.\",\n \"Animals such as rabbits, rats and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 μgs of peptide or carrier protein and Freund's adjuvant.\",\n \"Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody, which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface.\",\n \"The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.\",\n \"As one of skill in the art will appreciate, and discussed above, the polypeptides of the present invention including, but not limited to, polypeptides comprising an immunogenic or antigenic epitope can be fused to heterologous polypeptide sequences.\",\n \"For example, the polypeptides of the present invention may be fused with the constant region comprising portions of immunoglobulins (IgA, IgE, IgG, IgM), or portions of the constant region (CH1, CH2, CH3, any combination thereof including both entire domains and portions thereof) resulting in chimeric polypeptides.\",\n \"These fusion proteins facilitate purification, and show an increased half-life in vivo.\",\n \"This has been shown, e.g., for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (See, e.g., EPA 0,394,827; and Traunecker et al., 1988).\",\n \"Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion can also be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone (See, e.g., Fountoulakis et al., 1995).\",\n \"Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag to aid in detection and purification of the expressed polypeptide.\",\n \"Additonal fusion proteins of the invention may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, or codon-shuffling (collectively referred to as “DNA shuffling”).\",\n \"DNA shuffling may be employed to modulate the activities of polypeptides of the present invention thereby effectively generating agonists and antagonists of the polypeptides.\",\n \"See, for example, U.S. Pat.\",\n \"Nos.\",\n \"5,605,793; 5,811,238; 5,834,252; 5,837,458; and Patten, P. A., et al., (1997); Harayama, S., (1998); Hansson, L. O., et al (1999); and Lorenzo, M. M. and Blasco, R., (1998).\",\n \"(Each of these documents are hereby incorporated by reference).\",\n \"In one embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of coding polynucleotides of the invention, or the polypeptides encoded thereby may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc.\",\n \"of one or more heterologous molecules.\",\n \"Antibodies: The present invention further relates to antibodies and T-cell antigen receptors (TCR), which specifically bind the polypeptides, and more specifically, the epitopes of the polyepeptides of the present invention.\",\n \"The antibodies of the present invention include IgG (including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM, and IgY.\",\n \"As used herein, the term “antibody” (Ab) is meant to include whole antibodies, including single-chain whole antibodies, and antigen binding fragments thereof.\",\n \"In a preferred embodiment the antibodies are human antigen binding antibody fragments of the present invention include, but are not limited to, Fab, Fab′ F(ab)2 and F(ab)2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain.\",\n \"The antibodies may be from any animal origin including birds and mammals.\",\n \"Preferably, the antibodies are human, murine, rabbit, goat, guinea pig, camel, horse, or chicken.\",\n \"Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entire or partial of the following: hinge region, CH1, CH2, and CH3 domains.\",\n \"Also included in the invention are any combinations of variable region(s) and hinge region, CH1, CH2, and CH3 domains.\",\n \"The present invention further includes chimeric, humanized, and human monoclonal and polyclonal antibodies, which specifically bind the polypeptides of the present invention.\",\n \"The present invention further includes antibodies that are anti-idiotypic to the antibodies of the present invention.\",\n \"The antibodies of the present invention may be monospecific, bispecific, and trispecific or have greater multispecificity.\",\n \"Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for heterologous compositions, such as a heterologous polypeptide or solid support material.\",\n \"See, e.g., WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, A. et al.\",\n \"(1991); U.S. Pat.\",\n \"Nos.\",\n \"5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648; Kostelny, S. A. et al.\",\n \"(1992).\",\n \"Antibodies of the present invention may be described or specified in terms of the epitope(s) or epitope-bearing portion(s) of a polypeptide of the present invention, which are recognized or specifically bound by the antibody.\",\n \"In the case of proteins of the present invention secreted proteins, the antibodies may specifically bind a full-length protein encoded by a nucleic acid of the present invention, a mature protein (i.e., the protein generated by cleavage of the signal peptide) encoded by a nucleic acid of the present invention, a signal peptide encoded by a nucleic acid of the present invention, or any other polypeptide of the present invention.\",\n \"Therefore, the epitope(s) or epitope bearing polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous amino acid residues, or otherwise described herein (including the sequence listing).\",\n \"Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded as individual species.\",\n \"Therefore, the present invention includes antibodies that specifically bind specified polypeptides of the present invention, and allows for the exclusion of the same.\",\n \"Antibodies of the present invention may also be described or specified in terms of their cross-reactivity.\",\n \"Antibodies that do not specifically bind any other analog, ortholog, or homolog of the polypeptides of the present invention are included.\",\n \"Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein, eg., using FASTDB and the parameters set forth herein) to a polypeptide of the present invention are also included in the present invention.\",\n \"Further included in the present invention are antibodies, which only bind polypeptides encoded by polynucleotides, which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described herein).\",\n \"Antibodies of the present invention may also be described or specified in terms of their binding affinity.\",\n \"Preferred binding affinities include those with a dissociation constant or Kd value less than 5×10−6M, 10−6M, 5×10−7M, 10−7M, 5×10−8M, 10−8M, 5×10−9M, 10−9M, 5×10−10M, 10−10M, 5×10−11M, 10−11M, 5×10−12M, 10−12M, 5×10−13M, 10−13M, 5×10−14M, 10−14M, 5×10−15M, and 10−15M.\",\n \"Antibodies of the present invention have uses that include, but are not limited to, methods known in the art to purify, detect, and target the polypeptides of the present invention including both in vitro and in vivo diagnostic and therapeutic methods.\",\n \"For example, the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples (See, e.g., Harlow et al., 1988).\",\n \"The antibodies of the present invention may be used either alone or in combination with other compositions.\",\n \"The antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions.\",\n \"For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, or toxins.\",\n \"See, e.g., WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat.\",\n \"No.\",\n \"5,314,995; and EP 0 396 387.The antibodies of the present invention may be prepared by any suitable method known in the art.\",\n \"For example, a polypeptide of the present invention or an antigenic fragment thereof can be administered to an animal in order to induce the production of sera containing polyclonal antibodies.\",\n \"The term “monoclonal antibody” is not limited to antibodies produced through hybridoma technology.\",\n \"The term “antibody” refers to a polypeptide or group of polypeptides which are comprised of at least one binding domain, where a binding domain is formed from the folding of variable domains of an antibody molecule to form three-dimensional binding spaces with an internal surface shape and charge distribution complementary to the features of an antigenic determinant of an antigen, which allows an immunological reaction with the antigen.\",\n \"The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.\",\n \"Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technology.\",\n \"Hybridoma techniques include those known in the art (See, e.g., Harlow et al.\",\n \"1988); Hammerling, et al, 1981).\",\n \"(Said references incorporated by reference in their entireties).\",\n \"Fab and F(ab′)2 fragments may be produced, for example, from hybridoma-produced antibodies by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments).\",\n \"Alternatively, antibodies of the present invention can be produced through the application of recombinant DNA technology or through synthetic chemistry using methods known in the art.\",\n \"For example, the antibodies of the present invention can be prepared using various phage display methods known in the art.\",\n \"In phage display methods, functional antibody domains are displayed on the surface of a phage particle, which carries polynucleotide sequences encoding them.\",\n \"Phage with a desired binding property are selected from a repertoire or combinatorial antibody library (e.g.\",\n \"human or murine) by selecting directly with antigen, typically antigen bound or captured to a solid surface or bead.\",\n \"Phage used in these methods are typically filamentous phage including fd and M13 with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein.\",\n \"Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman U. et al.\",\n \"(1995); Ames, R. S. et al.\",\n \"(1995); Kettleborough, C. A. et al.\",\n \"(1994); Persic, L. et al.\",\n \"(1997); Burton, D. R. et al.\",\n \"(1994); PCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat.\",\n \"Nos.\",\n \"5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727 and 5,733,743.As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host including mammalian cells, insect cells, plant cells, yeast, and bacteria.\",\n \"For example, techniques to recombinantly produce Fab, Fab′ F(ab)2 and F(ab′)2 fragments can also be employed using methods known in the art such as those disclosed in WO 92/22324; Mullinax, R. L. et al.\",\n \"(1992); and Sawai, H. et al.\",\n \"(1995); and Better, M. et al.\",\n \"(1988).\",\n \"Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat.\",\n \"Nos.\",\n \"4,946,778 and 5,258,498; Huston et al.\",\n \"(1991); Shu, L. et al.\",\n \"(1993); and Skerra, A. et al.\",\n \"(1988).\",\n \"For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies.\",\n \"Methods for producing chimeric antibodies are known in the art.\",\n \"See e.g., Morrison, (1985); Oi et al., (1986); Gillies, S. D. et al.\",\n \"(1989); and U.S. Pat.\",\n \"No.\",\n \"5,807,715.Antibodies can be humanized using a variety of techniques including CDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat.\",\n \"Nos.\",\n \"5,530,101; and 5,585,089), veneering or resurfacing, (EP 0 592 106; EP 0 519 596; Padlan E. A., 1991; Studnicka G. M. et al., 1994; Roguska M. A. et al., 1994), and chain shuffling (U.S. Pat.\",\n \"No.\",\n \"5,565,332).\",\n \"Human antibodies can be made by a variety of methods known in the art including phage display methods described above.\",\n \"See also, U.S. Pat.\",\n \"Nos.\",\n \"4,444,887,4,716,111, 5,545,806, and 5,814,318; WO 98/46645; WO 98/50433; WO 98/24893; WO 96/34096; WO 96/33735; and WO 91/10741.Further included in the present invention are antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide of the present invention.\",\n \"The antibodies may be specific for antigens other than polypeptides of the present invention.\",\n \"For example, antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors.\",\n \"Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vitro immunoassays and purification methods using methods known in the art (See e.g., Harbor et al.\",\n \"supra; WO 93/21232; EP 0 439 095; Naramura, M. et al.\",\n \"1994; U.S. Pat.\",\n \"No.\",\n \"5,474,981; Gillies, S. O. et al., 1992; Fell, H. P. et al., 1991).\",\n \"The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions.\",\n \"For example, the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof.\",\n \"The antibody portion fused to a polypeptide of the present invention may comprise the hinge region, CH1 domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof.\",\n \"The polypeptides of the present invention may be fused or conjugated to the above antibody portions to increase the in vivo half-life of the polypeptides or for use in immunoassays using methods known in the art.\",\n \"The polypeptides may also be fused or conjugated to the above antibody portions to form multimers.\",\n \"For example, Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions.\",\n \"Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM.\",\n \"Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art.\",\n \"See e.g., U.S. Pat.\",\n \"Nos.\",\n \"5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,112,946; EP 0 307 434, EP 0 367 166; WO 96/04388, WO 91/06570; Ashkenazi, A. et al.\",\n \"(1991); Zheng, X. X. et al.\",\n \"(1995); and Vil, H. et al.\",\n \"(1992).\",\n \"The invention further relates to antibodies that act as agonists or antagonists of the polypeptides of the present invention.\",\n \"For example, the present invention includes antibodies that disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully.\",\n \"Included are both receptor-specific antibodies and ligand-specific antibodies.\",\n \"Included are receptor-specific antibodies, which do not prevent ligand binding but prevent receptor activation.\",\n \"Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art.\",\n \"Also include are receptor-specific antibodies which both prevent ligand binding and receptor activation.\",\n \"Likewise, included are neutralizing antibodies that bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies that bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor.\",\n \"Further included are antibodies that activate the receptor.\",\n \"These antibodies may act as agonists for either all or less than all of the biological activities affected by ligand-mediated receptor activation.\",\n \"The antibodies may be specified as agonists or antagonists for biological activities comprising specific activities disclosed herein.\",\n \"The above antibody agonists can be made using methods known in the art.\",\n \"See e.g., WO 96/40281; U.S. Pat.\",\n \"No.\",\n \"5,811,097; Deng, B. et al.\",\n \"(1998); Chen, Z. et al.\",\n \"(1998); Harrop, J.\",\n \"A. et al.\",\n \"(1998); Zhu, Z. et al.\",\n \"(1998); Yoon, D. Y. et al.\",\n \"(1998); Prat, M. et al.\",\n \"(1998) J.; Pitard, V. et al.\",\n \"(1997); Liautard, J. et al.\",\n \"(1997); Carlson, N. G. et al.\",\n \"(1997) J.; Taryman, R. E. et al.\",\n \"(1995); Muller, Y.\",\n \"A. et al.\",\n \"(1998); Bartunek, P. et al.\",\n \"(1996).\",\n \"As discussed above, antibodies of the polypeptides of the invention can, in turn, be utilized to generate anti-idiotypic antibodies that “mimic” polypeptides of the invention using techniques well known to those skilled in the art (See, e.g.\",\n \"Greenspan and Bona (1989); and Nissinoff (1991).\",\n \"For example, antibodies which bind to and competitively inhibit polypeptide multimerization or binding of a polypeptide of the invention to ligand can be used to generate anti-idiotypes that “mimic” the polypeptide multimerization or binding domain and, as a consequence, bind to and neutralize polypeptide or its ligand.\",\n \"Such neutralization anti-idiotypic antibodies can be used to bind a polypeptide of the invention or to bind its ligands/receptors, and therby block its biological activity, The invention also concerns a purified or isolated antibody capable of specifically binding to a mutated full length or mature polypeptide of the present invention or to a fragment or variant thereof comprising an epitope of the mutated polypeptide.\",\n \"In another preferred embodiment, the present invention concerns an antibody capable of binding to a polypeptide comprising at least 10 consecutive amino acids of a polypeptide of the present invention and including at least one of the amino acids which can be encoded by the trait causing mutations.\",\n \"Non-human animals or mammals, whether wild-type or transgenic, which express a different species of a polypeptide of the present invention than the one to which antibody binding is desired, and animals which do not express a polypeptide of the present invention (i.e.\",\n \"a knock out animal) are particularly useful for preparing antibodies.\",\n \"Gene knock out animals will recognize all or most of the exposed regions of a polypeptide of the present invention as foreign antigens, and therefore produce antibodies with a wider array of epitopes.\",\n \"Moreover, smaller polypeptides with only 10 to 30 amino acids may be useful in obtaining specific binding to any one of the polypeptides of the present invention.\",\n \"In addition, the humoral immune system of animals which produce a species of a polypeptide of the present invention that resembles the antigenic sequence will preferentially recognize the differences between the animal's native polypeptide species and the antigen sequence, and produce antibodies to these unique sites in the antigen sequence.\",\n \"Such a technique will be particularly useful in obtaining antibodies that specifically bind to any one of the polypeptides of the present invention.\",\n \"Antibody preparations prepared according to either protocol are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample.\",\n \"The antibodies may also be used in therapeutic compositions for killing cells expressing the protein or reducing the levels of the protein in the body.\",\n \"The antibodies of the invention may be labeled by any one of the radioactive, fluorescent or enzymatic labels known in the art.\",\n \"Consequently, the invention is also directed to a method for detecting specifically the presence of a polypeptide of the present invention according to the invention in a biological sample, said method comprising the following steps: a) bringing into contact the biological sample with a polyclonal or monoclonal antibody that specifically binds a polypeptide of the present invention; and b) detecting the antigen-antibody complex formed.\",\n \"The invention also concerns a diagnostic kit for detecting in vitro the presence of a polypeptide of the present invention in a biological sample, wherein said kit comprises: a) a polyclonal or monoclonal antibody that specifically binds a polypeptide of the present invention, optionally labeled; b) a reagent allowing the detection of the antigen-antibody complexes formed, said reagent carrying optionally a label, or being able to be recognized itself by a labeled reagent, more particularly in the case when the above-mentioned monoclonal or polyclonal antibody is not labeled by itself.\",\n \"The antibodies of the invention may be labeled by any one of the radioactive, fluorescent or enzymatic labels known in the art.\",\n \"EXAMPLES The following Examples are provided for illustrative purposes and not as a means of limitation to support the finding that GSSP4 polypeptides have efficacy in reducing insulin resistance and improving glucose and lipid metabolism and may have an insulin sensitising action.\",\n \"One of ordinary skill in the art would be able to design equivalent assays and methods based on the disclosure herein all of which form part of the instant invention.\",\n \"Example 1 Northern Analysis of GSSP4 RNA Analysis of GSSP4 expression in different human tissues (adult and fetal) and cell lines, as well as mouse embryos in different stages of development, is accomplished by using poly A+ RNA blots purchased from Clontech (e.g.\",\n \"#7780-1, 7757-1, 7756-1, 7768-1and 7763-1).\",\n \"Labeling of RNA probes is performed using the RNA Strip-EZ kit from Ambion as per manufacture's instructions.\",\n \"Hybridization of RNA probes to RNA blots is performed Ultrahyb hybridization solution (Ambion).\",\n \"Briefly, blots are prehybridized for 30 min at 58° C. (low-strigency) or 65° C. (high stringency).\",\n \"After adding the labeled probe (2×106 cpm/ml), blots are hybridized overnight (14-24 hrs), and is washed 2×20 min at 50° C. with 2× SSC/0.1% SDS (low stringency), 2×20 min at 58° C. with 1× SSC/0.1% SDS (medium stringency) and 2×20 min at 65° C. with 1× SSC/0.1% SDS (high stringency).\",\n \"After washings are completed blots are exposed on the phosphoimager (Molecular Dynamics) for 1-3 days.\",\n \"Example 2 In Vitro Tests of Metabolic-Related Activity The activity of various preparations of GSSP4 polypeptides are assessed using various in vitro assays including those provided below.\",\n \"These assays are also exemplary of those that can be used to develop GSSP4 polypeptide antagonists and agonists.\",\n \"To do that, the effect of GSSP4 polypeptides in the above assays, e.g.\",\n \"on glucose uptake and fatty acid oxidation and partitioning in the presence of the candidate molecules would be compared with the effect of GSSP4 polypeptides in the assays in the absence of the candidate molecules.\",\n \"In Vitro Muscle Cells Glucose Uptake L6 Muscle cells are obtained from the European Culture Collection (Porton Down) and are used at passages 7-11.Cells are maintained in standard tissue culture medium DMEM, and glucose uptake is assessed using [.sup.3H]-2-deoxyglucose (2DG) with or without GSSP4 polypeptides in the presence or absence of insulin (10.sup.-8 M) as has been previously described (Walker P S et al, Glucose transport activity in L6 muscle cells is regulated by the coordinate control of subcellular glucose transporter distribution, biosynthesis, and mRNA transcription, JBC, 1990;265(3), 1516-1523, and Kilp A et al, Stimulation of hexose transport by metformin in L6 muscle cells in culture, Endocrinology,1992;130(5), 2535-2544).\",\n \"Uptake of 2DG is expressed as the percentage change compared with control (no added insulin or GSSP4).\",\n \"Values are presented as mean .+−.SEM of sets of 4 wells per experiment.\",\n \"Differences between sets of wells are evaluated by Student's t test, probability values p<0.05 are considered to be significant.\",\n \"Effect on Muscle Cell Fatty Acid Oxidation C2C12 cells are differentiated in the presence or absence of 2 μg/mL GSSP4 protein for 4 days.\",\n \"On day 4, oleate oxidation rates are determined by measuring conversion of 1-14C-oleate (0.2 mM) to 14CO2 for 90 min This experiment can be used to screen for active fragments and peptides as well as agonists and antagonists or activators and inhibitors of GSSP4 polypeptides.\",\n \"The effect of polypeptides on the rate of oleate oxidation can be compared in differentiated C2C12 cells (murine skeletal muscle cells; ATCC, Manassas, Va. CRL-1772) and in a hepatocyte cell line (Hepa1-6; ATCC, Manassas, Va. CRL-1830).\",\n \"Cultured cells are maintained according to manufacturer's instructions.\",\n \"The oleate oxidation assay is performed as previously described (Muoio et al (1999) Biochem J 338;783-791).\",\n \"Briefly, nearly confluent myocytes are kept in low serum differentiation media (DMEM, 2.5% Horse serum) for 4 days, at which time formation of myotubes became maximal.\",\n \"Hepatocytes are kept in the same DMEM medium supplemented with 10% FCS for 2 days.\",\n \"One hour prior to the experiment the media is removed and 1 mL of preincubation media (MEM, 2.5% Horse serum, 3 mM glucose, 4 mM Glutamine, 25 mM Hepes, 1% FFA free BSA, 0.25 mM Oleate, 5 μg/mL gentamycin) is added.\",\n \"At the start of the oxidation experiment 14C-Oleic acid (1 μCi/mL, American Radiolabeled Chemical Inc., St. Louis, Mo.)\",\n \"is added and cells are incubated for 90 min at 37° C. in the absence/presence of 2.5 μg/mL GSSP4 polypeptides.\",\n \"After the incubation period 0.75 mL of the media is removed and assayed for 14C-oxidation products as described below for the muscle FFA oxidation experiment.\",\n \"Triglyceride and Protein Partitioning following Oleate Oxidaiton in Cultured Cells Following transfer of media for oleate oxidation assay, cells are placed on ice.\",\n \"To determine triglyceride and protein content, cells are washed with 1 mL of 1×PBS to remove residual media.\",\n \"To each well 300 μL of cell dissociation solution (Sigma) is added and incubated at 37° C. for 10 min.\",\n \"Plates are tapped to loosen cells, and 0.5 mL of 1×PBS is added.\",\n \"The cell suspension is transferred to an eppendorf tube, each well is rinsed with an additional 0.5 mL of 1×PBS, and is transferred to appropriate eppendorf tube.\",\n \"Samples are centrifuged at 1000 rpm for 10 minutes at room temperature.\",\n \"Supernatant is discarded and 750 μL of 1×PBS/2% chaps is added to cell pellet.\",\n \"Cell suspension is vortexed and placed on ice for 1 hour.\",\n \"Samples are then centrifuged at 13000 rpm for 20 min at 4° C. Supernatants are transferred to new tube and frozen at −20° C. until analyzed.\",\n \"Quantitative measure of triglyceride level in each sample is determined using Sigma Diagnostics GPO-TRINDER enzymatic kit.\",\n \"The procedure outlined in the manual is adhered to, with the following exceptions: assay is performed in 48 well plate, 350 μL of sample volume is assayed, control blank consisted of 350 μL PBS/2% chaps, and standard contained 10 μL standard provide in kit plus 690 μL PBS/2% chaps.\",\n \"Analysis of samples is carried out on a Packard Spectra Count at a wavelength of 550 nm.\",\n \"Protein analysis is carried out on 25 μL of each supernatant sample using the BCA protein assay (Pierce) following manufacturer's instructions.\",\n \"Analysis of samples is carried out on a Packard Spectra Count at a wavelength of 550 nm.\",\n \"Cellular Binding and Uptake of GSSP4 Polypeptides as Detected by Fluorescence Microscopy Fluorecein isothiocyanate (FITC) conjugation of GSSP4 polypeptides: Purified GSSP4 proteins at 1 mg/mL concentration are labeled with FITC using Sigma's FluoroTag FITC conjugation kit (Stock No.\",\n \"FITC-1).\",\n \"Protocol outlined in the Sigma Handbook for small scale conjugation is followed for GSSP4 protein labeling.\",\n \"Cell Culture: C2C12 mouse skeletal muscle cells (ATCC, Manassas, Va. CRL-1772) and Hepa-1-6 mouse hepatocytes (ATCC, Manassas, Va. CRL-1830) are seeded into 6 well plates at a cell density of 2×105 cells per well.\",\n \"C2C12 and Hepa-1-6 cells are cultured according to repository's instructions for 24-48 hours prior to analysis.\",\n \"Assay is performed when cells are 80% confluent.\",\n \"FITC labeled GSSP4 proteincellular binding and uptake using microscopy: C2C12 and Hepa 1-6 cells are incubated in the presence/absence of antibody directed against human LSR (81B: N-terminal sequence of human LSR; does not cross react with mouse LSR and 93A: c-terminal sequence, cross reacts with mouse LSR) or an antiserum directed against gC1qr (953) for 1 hour at 37° C., 5% CO2.LSR antibodies are added to the media at a concentration of 2 μg/mL.\",\n \"The anti-gC1qr antiserum is added to the media at a volume of 2.5 μL undiluted serum (high concentration) or 1:100 dilution (low concentration).\",\n \"Following incubation with specified antibody, FITC-GSSP4 polypeptide (50 nM/mL) is added to each cell culture well.\",\n \"Cells are again incubated for 1 hour at 37° C., 5% CO2.Cells are washed 2× with PBS, cells are scraped from well into 1 mL of PBS.\",\n \"Cell suspension is transferred to an eppendorf tube and centrifuged at 1000 rpm for 2 minutes.\",\n \"Supernatant is removed and cells resuspended in 200 μL of PBS.\",\n \"Binding and uptake of FITC-GSSP4 polypeptide is analyzed by fluorescence microscopy under 40× magnification.\",\n \"This assay may be useful for identifying agents that facilitate or prevent the uptake and/or binding of GSSP4 polypeptides to cells.\",\n \"Example 3 In Vivo Tests for Metabolic-Related Activity in Rodent Diabetes Models As metabolic profiles differ among various animal models of obesity and diabetes, analysis of multiple models is undertaken to separate the effects GSSP4 polypeptides on hyperglycemia, hyperinsulinemia, hyperlipidemia and obesity.\",\n \"Mutation in colonies of laboratory animals and different sensitivities to dietary regimens have made the development of animal models with non-insulin dependent diabetes associated with obesity and insulin resistance possible.\",\n \"Genetic models such as db/db and ob/ob (See Diabetes, (1982) 31(1): 1-6) in mice and fa/fa in zucker rats have been developed by the various laboratories for understanding the pathophysiology of disease and testing the efficacy of new antidiabetic compounds (Diabetes, (1983) 32: 830-838; Annu.\",\n \"Rep. Sankyo Res.\",\n \"Lab.\",\n \"(1994).\",\n \"46: 1-57).\",\n \"The homozygous animals, C57 BL/KsJ-db/db mice developed by Jackson Laboratory, US, are obese, hyperglycemic, hyperinsulinemic and insulin resistant (J. Clin.\",\n \"Invest., (1990) 85: 962-967), whereas heterozygous are lean and normoglycemic.\",\n \"In db/db model, mouse progressively develops insulinopenia with age, a feature commonly observed in late stages of human type II diabetes when blood sugar levels are insufficiently controlled.\",\n \"The state of pancreas and its course vary according to the models.\",\n \"Since this model resembles that of type II diabetes mellitus, the compounds of the present invention are tested for blood sugar and triglycerides lowering activities.\",\n \"Zucker (fa/fa) rats are severely obese, hyperinsulinemic, and insulin resistant (Coleman, Diabetes 31:1, 1982; E. Shafrir, in Diabetes Mellitus; H. Rifkin and D. Porte, Jr. Eds.\",\n \"(Elsevier Science Publishing Co., Inc., New York, ed.\",\n \"4, 1990), pp.\",\n \"299-340), and the fa/fa mutation may be the rat equivalent of the murine db mutation (Friedman et al., Cell 69:217-220, 1992; Truett et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 88:7806, 1991).\",\n \"Tubby (tub/tub) mice are characterized by obesity, moderate insulin resistance and hyperinsulinemia without significant hyperglycemia (Coleman et al., J. Heredity 81:424, 1990).\",\n \"Previously, leptin was reported to reverse insulin resistance and diabetes mellitus in mice with congenital lipodystrophy (Shimomura et al.\",\n \"Nature 401: 73-76 (1999).\",\n \"Leptin is found to be less effective in a different lipodystrophic mouse model of lipoatrophic diabetes (Gavrilova et al Nature 403: 850 (2000); hereby incorporated herein in its entirety including any drawings, figures, or tables).\",\n \"The streptozotocin (STZ) model for chemically-induced diabetes is tested to examine the effects of hyperglycemia in the absence of obesity.\",\n \"STZ-treated animals are deficient in insulin and severely hyperglycemic (Coleman, Diabetes 31:1, 1982; E. Shafrir, in Diabetes Mellitus; H. Rifkin and D. Porte, Jr. Eds.\",\n \"(Elsevier Science Publishing Co., Inc., New York, ed.\",\n \"4, 1990), pp.\",\n \"299-340).\",\n \"The monosodium glutamate (MSG) model for chemically-induced obesity (Olney, Science 164:719, 1969; Cameron et al., Cli.\",\n \"Exp.\",\n \"Pharmacol.\",\n \"Physiol.\",\n \"5:41, 1978), in which obesity is less severe than in the genetic models and develops without hyperphagia, hyperinsulinemia and insulin resistance, is also examined.\",\n \"Finally, a non-chemical, non-genetic model for induction of obesity includes feeding rodents a high fat/high carbohydrate (cafeteria diet) diet ad libitum.\",\n \"The instant invention encompasses the use of GSSP4 polypeptides for reducing the insulin resistance and hyperglycemia in any or all of the above rodent diabetes models or in humans with Type I or Type II diabetes or other prefered metabolic diseases described previously or models based on other mammals.\",\n \"In the compositions of the present invention the GSSP4 polypeptides may, if desired, be associated with other compatible pharmacologically-active antidiabetic agents such as insulin, leptin (U.S. provisional application No.\",\n \"60/155,506), or troglitazone, either alone or in combination.\",\n \"Assays include that described previously in Gavrilova et al.\",\n \"((2000) Diabetes November;49(11): 1910-6; (2000) Nature February 24;403(6772):850) using A-ZIP/F-1 mice, except that GSSP4 polypeptides are administered intraperotineally, subcutaneously, intramuscularly or intravenously.\",\n \"The glucose and insulin levels of the mice would be tested, and the food intake and liver weight monitored, as well as other factors, such as leptin, FFA, and TG levels, typically measured in our experiments.\",\n \"In Vivo Assay for Anti-Hyperglycemic Activity of GSSP4 Polypeptides Genetically altered obese diabetic mice (db/db) (male, 7-9 weeks old) are housed (7-9 mice/cage) under standard laboratory conditions at 22.degree.\",\n \"C. and 50% relative humidity, and maintained on a diet of Purina rodent chow and water ad libitum.\",\n \"Prior to treatment, blood is collected from the tail vein of each animal and blood glucose concentrations are determined using One Touch BasicGlucose Monitor System (Lifescan).\",\n \"Mice that have plasma glucose levels between 250 to 500 mg/dl are used.\",\n \"Each treatment group consists of seven mice that are distributed so that the mean glucose levels are equivalent in each group at the start of the study.\",\n \"db/db mice are dosed by micro-osmotic pumps, inserted using isoflurane anesthesia, to provide GSSP4 polypeptides, saline, and an irrelevant peptide to the mice subcutaneously (s.c.).\",\n \"Blood is sampled from the tail vein hourly for 4 hours and at 24, 30 h post-dosing and analyzed for blood glucose concentrations.\",\n \"Food is withdrawn from 0-4 h post dosing and reintroduced thereafter.\",\n \"Individual body weights and mean food consumption (each cage) are also measured after 24 h. Significant differences between groups (comparing GSSP4 treated to saline-treated) are evaluated using Student t-test.\",\n \"In Vivo Insulin Sensitivity Assay In vivo insulin sensitivity is examined by utilizing two-step hyperinsulinemic-euglycemic clamps according to the following protocol.\",\n \"Rodents from any or all of the various models described in Example 2 are housed for at least a week prior to experimental procedures.\",\n \"Surgeries for the placement of jugular vein and carotid artery catheters are performed under sterile conditions using ketamine and xylazine (i.m.)\",\n \"anesthesia.\",\n \"After surgery, all rodents are allowed to regain consciousness and placed in individual cages.\",\n \"GSSP4 polypeptides or vehicle is administered through the jugular vein after complete recovery and for the following two days.\",\n \"Sixteen hours after the last treatment, hyperinsulinemic-euglycemic clamps are performed.\",\n \"Rodents are placed in restrainers and a bolus of 4 .mu Ci [3-.sup.3 H] glucose (NEN) is administered, followed by a continuous infusion of the tracer at a dose of 0.2 mu.Ci/min (20 mu.1/min).\",\n \"Two hours after the start of the tracer infusion, 3 blood samples (0.3 ml each) are collected at 10 minute intervals (−20-0 min) for basal measurements.\",\n \"An insulin infusion is then started (5 mU/kg/min), and 100 .mu.1 blood samples are taken every 10 min.\",\n \"to monitor plasma glucose.\",\n \"A 30% glucose solution is infused using a second pump based on the plasma glucose levels in order to reach and maintain euglycemia.\",\n \"Once a steady state is established at 5 mU/kg/min insulin (stable glucose infusion rate and plasma glucose), 3 additional blood samples (0.3 ml each) are obtained for measurements of glucose, [3-.sup.3H] glucose and insulin (100-120 min.).\",\n \"A higher dose of insulin (25 mU/kg/min.)\",\n \"is then administered and glucose infusion rates are adjusted for the second euglycemic clamp and blood samples are taken at min.\",\n \"220-240.Glucose specific activity is determined in deproteinized plasma and the calculations of Rd and hepatic glucose output (HGO) are made, as described (Lang et al., Endocrinology 130:43, 1992).\",\n \"Plasma insulin levels at basal period and after 5 and 25 mU/kg/min.\",\n \"infusions are then determined and compared between GSSP4 treated and vehicle treated rodents.\",\n \"Insulin regulation of glucose homeostasis has two major components; stimulation of peripheral glucose uptake and suppression of hepatic glucose output.\",\n \"Using tracer studies in the glucose clamps, it is possible to determine which portion of the insulin response is affected by the GSSP4 polypeptides.\",\n \"Example 4 Effect of GSSP4 Polypeptides on Mice Fed a High-Fat Diet Experiments are performed using approximately 6 week old C57B1/6 mice (8 per group).\",\n \"All mice are housed individually.\",\n \"The mice are maintained on a high fat diet throughout each experiment.\",\n \"The high fat diet (cafeteria diet; D12331 from Research Diets, Inc.) has the following composition: protein kcal % 16, carbohydrate kcal % 26, and fat kcal % 58.The fat is primarily composed of coconut oil, hydrogenated.\",\n \"After the mice are fed a high fat diet for 6 days, micro-osmotic pumps are inserted using isoflurane anesthesia, and are used to provide full-length GSSP4 polypeptides, GSSP4 polypeptide fragments, saline, and an irrelevant peptide to the mice subcutaneously (s.c.) for 18 days.\",\n \"GSSP4 polypeptides are provided at doses of 100, 50, 25, and 2.5 μg/day and the irrelevant peptide is provided at 10 μg/day.\",\n \"Body weight is measured on the first, third and fifth day of the high fat diet, and then daily after the start of treatment.\",\n \"Final body weight and final blood samples are taken by cardiac puncture and are used to determine triglyceride (TG), total cholesterol (TC), glucose, leptin, and insulin levels.\",\n \"The amount of food consumed per day is also determined for each group.\",\n \"Plasma glucose is determined by a glucose oxidase procedure (Analox GM7) and plasma insulin determined by radioimmunoassay (Amerlex, Amersham).\",\n \"Example 5 Effect of GSSP4 Polypeptides on Plasma Free Fatty Acid in C57 BL/6 Mice The effect of GSSP4 polypeptides on postprandial lipemia (PPL) in normal C57BL6/J mice is tested.\",\n \"The mice used in this experiment are fasted for 2 hours prior to the experiment after which a baseline blood sample is taken.\",\n \"All blood samples are taken from the tail using EDTA coated capillary tubes (50 μL each time point).\",\n \"At time 0 (8:30 AM), a standard high fat meal (6 g butter, 6 g sunflower oil, 10 g nonfat dry milk, 10 g sucrose, 12 mL distilled water prepared fresh following Nb#6, JF, pg.1) is given by gavage (vol.=1% of body weight) to all animals.\",\n \"Immediately following the high fat meal, 25 μg a GSSP4 polypeptide is injected i.p.\",\n \"in 100 μL saline.\",\n \"The same dose (25 μg/mL in 100 μL) is again injected at 45 min and at 1 hr 45 min.\",\n \"Control animals are injected with saline (3×100 μL).\",\n \"Untreated and treated animals are handled in an alternating mode.\",\n \"Blood samples are taken in hourly intervals, and are immediately put on ice.\",\n \"Plasma is prepared by centrifugation following each time point.\",\n \"Plasma is kept at −20° C. and free fatty acids (FFA), triglycerides (TG) and glucose are determined within 24 hours using standard test kits (Sigma and Wako).\",\n \"Due to the limited amount of plasma available, glucose is determined in duplicate using pooled samples.\",\n \"For each time point, equal volumes of plasma from all 8 animals per treatment group are pooled.\",\n \"Example 6 Effect of GSSP4 Polypeptides on Plasma Leptin and Insulin in C57 BL/6 Mice The effect of GSSP4 polypeptides on plasma leptin and insulin levels during postprandial lipemia (PPL) in normal C57BL6/J mice is tested.\",\n \"The experimental procedure is the same as previously described, except that blood is drawn only at 0, 2 and 4 hours to allow for greater blood samples needed for the determination of leptin and insulin by RIA.\",\n \"Briefly, 16 mice are fasted for 2 hours prior to the experiment after which a baseline blood sample is taken.\",\n \"All blood samples are taken from the tail using EDTA coated capillary tubes (100 μL each time point).\",\n \"At time 0 (9:00 AM), a standard high fat meal is given by gavage (vol.=1% of body weight) to all animals.\",\n \"Immediately following the high fat meal, 25 μg of a GSSP4 polypeptide is injected i.p.\",\n \"in 100 μL saline.\",\n \"The same dose (25 μg in 100 μL) is again injected at 45 min and at 1 hr 45 min (treated group).\",\n \"Control animals are injected with saline (3×100 μL).\",\n \"Untreated and treated animals are handled in an alternating mode.\",\n \"Blood samples are immediately put on ice and plasma is prepared by centrifugation following each time point.\",\n \"Plasma is kept at −20° C. and free fatty acids (FFA) are determined within 24 hours using a standard test kit (Wako).\",\n \"Leptin and Insulin are determined by RIA (ML-82K and SRI-13K, LINCO Research, Inc., St. Charles, Mo.)\",\n \"following the manufacturer's protocol.\",\n \"However, only 20 μL plasma is used.\",\n \"Each determination is done in duplicate.\",\n \"Due to the limited amount of plasma available, leptin and insulin are determined in 4 pools of 2 animals each in both treatment groups.\",\n \"Example 7 Effect of GSSP4 Polypeptides on Plasma FFA, TG and Glucose in C57 BL/6 Mice The effect of GSSP4 polypeptides on plasma FFA, TG, glucose, leptin and insulin levels during postprandial lipemia (PPL) in normal C57BL6/J mice has been described.\",\n \"Weight loss resulting from GSSP4 polypeptides (2.5 μg/day) given to normal C57BL6/J mice on a high fat diet is shown.\",\n \"The experimental procedure is similar to described previously.\",\n \"Briefly, 14 mice are fasted for 2 hours prior to the experiment after which a baseline blood sample is taken.\",\n \"All blood samples are taken from the tail using EDTA coated capillary tubes (50 μL each time point).\",\n \"At time 0 (9:00 AM), a standard high fat meal is given by gavage (vol.=1% of body weight) to all animals.\",\n \"Immediately following the high fat meal, 4 mice are injected 25 μg of a GSSP4 polypeptide i.p.\",\n \"in 100 μL saline.\",\n \"The same dose (25 μg in 100 μL) is again injected at 45 min and at 1 hr 45 min.\",\n \"A second treatment group receives 3 times 50 μg GSSP4 polypeptide at the same intervals.\",\n \"Control animals are injected with saline (3×100 μL).\",\n \"Untreated and treated animals are handled in an alternating mode.\",\n \"Blood samples are immediately put on ice.\",\n \"Plasma is prepared by centrifugation following each time point.\",\n \"Plasma is kept at −20° C. and free fatty acids (FFA), triglycerides (TG) and glucose are determined within 24 hours using standard test kits (Sigma and Wako).\",\n \"Example 8 Effect of GSSP4 Polypeptides on FFA Following Epinephrine Injection In mice, plasma free fatty acids increase after intragastric administration of a high fat/carbohydrate test meal.\",\n \"These free fatty acids are mostly produced by the activity of lipolytic enzymes i.e.\",\n \"lipoprotein lipase (LPL) and hepatic lipase (HL).\",\n \"In this species, these enzymes are found in significant amounts both bound to endothelium and freely circulating in plasma.\",\n \"Another source of plasma free fatty acids is hormone sensitive lipase (HSL) that releases free fatty acids from adipose tissue after β-adrenergic stimulation.\",\n \"To test whether GSSP4 polypeptides also regulate the metabolism of free fatty acid released by HSL, mice are injected with epinephrine.\",\n \"Two groups of mice are given epinephrine (5 μg) by intraperitoneal injection.\",\n \"A treated group is injected with a GSSP4 polypeptide (25 μg) one hour before and again together with epinephrine, while control animals receive saline.\",\n \"Plasma is isolated and free fatty acids and glucose are measured as described above.\",\n \"Example 9 Effect of GSSP4 Polypeptides on Muscle FFA Oxidation To investigate the effect of GSSP4 polypeptides on muscle free fatty acid oxidation, intact hind limb muscles from C57BL/6J mice are isolated and FFA oxidation is measured using oleate as substrate (Clee et al (2000) J Lipid Res 41:521-531; Muoio et al (1999) Am J Physiol 276:E913-921).\",\n \"Oleate oxidation in isolated muscle is measured as previously described (Cuendet et al (1976) J Clin Invest 58:1078-1088; Le Marchand-Brustel (1978) Am J Physiol 234:E348-E358).\",\n \"Briefly, mice are sacrificed by cervical dislocation and soleus and EDL muscles are rapidly isolated from the hind limbs.\",\n \"The distal tendon of each muscle is tied to a piece of suture to facilitate transfer among different media.\",\n \"All incubations are carried out at 30° C. in 1.5 mL of Krebs-Henseleit bicarbonate buffer (118.6 mM NaCl, 4.76 mM KCl, 1.19 mM KH2PO4, 1.19 mM MgSO4, 2.54 mM CaCl2, 25 mM NaHCO3, 10 mM Hepes, pH 7.4) supplemented with 4% FFA free bovine serum albumin (fraction V, RIA grade, Sigma) and 5 mM glucose (Sigma).\",\n \"The total concentration of oleate (Sigma) throughout the experiment is 0.25 mM.\",\n \"All media are oxygenated (95% O2; 5% CO2) prior to incubation.\",\n \"The gas mixture is hydrated throughout the experiment by bubbling through a gas washer (Kontes Inc., Vineland, N.J.).\",\n \"Muscles are rinsed for 30 min in incubation media with oxygenation.\",\n \"The muscles are then transferred to fresh media (1.5 mL) and incubated at 30° C. in the presence of 1 μCi/mL [1-14C] oleic acid (American Radiolabeled Chemicals).\",\n \"The incubation vials containing this media are sealed with a rubber septum from which a center well carrying a piece of Whatman paper (1.5 cm×11.5 cm) is suspended.\",\n \"After an initial incubation period of 10 min with constant oxygenation, gas circulation is removed to close the system to the outside environment and the muscles are incubated for 90 min at 30° C. At the end of this period, 0.45 mL of Solvable (Packard Instruments, Meriden, Conn.) is injected onto the Whatman paper in the center well and oleate oxidation by the muscle is stopped by transferring the vial onto ice.\",\n \"After 5 min, the muscle is removed from the medium, and an aliquot of 0.5 mL medium is also removed.\",\n \"The vials are closed again and 1 mL of 35% perchloric acid is injected with a syringe into the media by piercing through the rubber septum.\",\n \"The CO2 released from the acidified media is collected by the Solvable in the center well.\",\n \"After a 90 min collection period at 30° C., the Whatman paper is removed from the center well and placed in scintillation vials containing 15 mL of scintillation fluid (HionicFlour, Packard Instruments, Meriden, Conn.).\",\n \"The amount of 14C radioactivity is quantitated by liquid scintillation counting.\",\n \"The rate of oleate oxidation is expressed as nmol oleate produced in 90 min/g muscle.\",\n \"To test the effect of gACRP30 or ACRP30 on oleate oxidation, these proteins are added to the media at a final concentration of 2.5 μg/mL and maintained in the media throughout the procedure.\",\n \"Example 10 Effect of GSSP4 Polypeptides on Triglyceride in Muscle & Liver Isolated from Mice To determine whether the increased FFA oxidation induced by GSSP4 polypeptides is also accompanied by increased FFA delivery into muscle or liver, the hindlimb muscle and liver triglyceride content is measured after the GSSP4 polypeptide treatment of mice.\",\n \"Hind limb muscles as well as liver samples are removed from treated and untreated animals and the triglyceride and free fatty acid concentration is determined following a standard lipid extraction method (Shimabukuro et al (1997) Proc Natl Acad Sci USA 94:4637-4641) followed by TG and FFA analysis using standard test kits.\",\n \"Example 11 Effect of GSSP4 Polypeptides on FFA following Intralipid Injection Two groups of mice are intravenously (tail vein) injected with 30 μL bolus of Intralipid-20% (Clintec) to generate a sudden rise in plasma FFAs, thus by-passing intestinal absorption.\",\n \"(Intralipid is an intravenous fat emulsion used in nutritional therapy).\",\n \"A treated group (GSSP4 polypeptide-treated) is injected with a GSSP4 polypeptide (25 μg) at 30 and 60 minutes before Intralipid is given, while control animals (▴ control) received saline.\",\n \"Plasma is isolated and FFAs are measured as described previously.\",\n \"The effect of GSSP4 polypeptides on the decay in plasma FFAs following the peak induced by Intralipid injection is then monitored.\",\n \"Example 12 Tests of Obesity-Related Activity in Humans Tests of the efficacy of in humans are performed in accordance with a physician's recommendations and with established guidelines.\",\n \"The parameters tested in mice are also tested in humans (e.g.\",\n \"food intake, weight, TG, TC, glucose, insulin, leptin, FFA).\",\n \"It is expected that the physiological factors would show changes over the short term.\",\n \"Changes in weight gain might require a longer period of time.\",\n \"In addition, the diet is carefully monitored.\",\n \"GSSP4 is given in daily doses of about 6 mg protein per 70 kg person or about 10 mg per day.\",\n \"Other doses are tested, for instance 1 mg or 5 mg per day up to 20 mg, 50 mg, or 100 mg per day.\",\n \"Example 13 Tests of Obesity-Related Activity in a Murine Lipoatrophic Diabetes Model Leptin was reported to reverse insulin resistance and diabetes mellitus in mice with congenital lipodystrophy (Shimomura et al.\",\n \"Nature 401: 73-76 (1999).\",\n \"Leptin is found to be less effective in a different lipodystrophic mouse model of lipoatrophic diabetes (Gavrilova et al Nature 403: 850 (2000); hereby incorporated herein in its entirety including any drawings, figures, or tables).\",\n \"The instant invention encompasses the use of GSSP4 or polypeptide fragments for reducing the insulin resistance and hyperglycaemia in this model either alone or in combination with leptin, the leptin peptide (U.S. provisional application No.\",\n \"60/155,506), or other compounds.\",\n \"Assays include that described previously in Gavrilova et al.\",\n \"((2000) Diabetes November;49(11): 1910-6; (2000) Nature February 24;403(6772):850) using A-ZIP/F-1 mice, except that would be administered using the methods previously described in Example 5 (or Examples 8-10).\",\n \"The glucose and insulin levels of the mice would be tested, and the food intake and liver weight monitored, as well as other factors, such as leptin, FFA, and TG levels, typically measured in our experiments (see Example 5, above, or Examples 8-10).\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10467554"}}},{"rowIdx":375,"cells":{"text":{"kind":"list like","value":[["Method, module, device and server for voice recognition","The invention concerns a voice recognition method implemented in at least a terminal, the voice recognition method using a language model, comprising the following steps: detecting at least a non-recognised expression in one of the terminals; recording in the terminal data representing the non-recognised expression; transmission by the terminal of the recorded data to a remote server; analysis, at the remote server, of said data and generation of data for correcting said language model taking into account at least part of the non-recognised expressions; and transmitting from the server to at least a terminal correction data, so as to enable subsequent recognition of at least some of the non-recognised expressions.","The invention also concerns corresponding modules, devices and remote server."],["1.Voice recognition process implemented in at least one terminal, the said voice recognition process using a language model, wherein it comprises the following steps: detection of at least one unrecognized expression in one of the said terminals; recording in the said terminal of data representative of the said unrecognized expression; transmission by the said terminal of the said recorded data to a remote server, via a first transmission channel; analysis, at the level of the said remote server, of the said data and generation of information for correcting the said language model taking account of at least one part of the said unrecognized expressions; and transmission via a second transmission channel from the said server to at least one terminal of the said correcting information, so as to allow future recognition of at least certain of the said unrecognized expressions.","2.Process according to claim 1, wherein the said data representative of the said unrecognized expressions comprise a compressed voice recording representative of parameters descriptive of the acoustic signal.","3.Process according to claim 1, wherein during the said step of transmission by the said terminal, the latter furthermore transmits to the said server at least one of the items of information forming part of the group comprising: information of context of use of the said voice recognition process when an expression has not been recognized; and information relating to the speaker who has uttered an unrecognized expression.","4.Process according to claim 1, wherein it implements an encryption and/or a scrambling of the said recorded data and/or of the said correcting information.","5.Voice recognition module using a language model, wherein it comprises: an analyser detecting unrecognized expressions; a recorder of data representative of at least one unrecognized expression; a transmitter transmitting the said recorded data to a remote server; and a receiver of correcting information allowing the correcting of the said language model transmitted to the said module allowing future recognition of at least certain of the said unrecognized expressions by the said module, the correcting information having been transmitted by the said remote server after analysis at the level of the said remote server of the said data, and after generation of information for correcting the said language model taking account of at least one part of the unrecognized expressions.","6.Voice recognition device using a language model, wherein it comprises: an analyser detecting unrecognized expressions; a recorder of data representative of at least one unrecognized expression; a transmitter transmitting the said recorded data to a remote server; and a receiver of correcting information allowing the correcting of the said language model transmitted to the said device allowing future recognition of at least certain of the said unrecognized expressions by the said device, the correcting information having been transmitted by the said remote server after analysis at the level of the said remote server of the said data, and after generation of information for correcting the said language model taking account of at least one part of the unrecognized expressions.","7.Voice recognition server, the said recognition being implemented in a set of at least one remote terminal, using a language model, wherein it comprises the following means: a receiver of data representative of at least one expression unrecognized by at least one terminal forming part of the said set and having detected the said unrecognized expression during a voice recognition operation; and a sender sending to the said set of at least one remote terminal correcting information obtained on the basis of an analysis of the said data received at the level of the said server, the said correcting information allowing the correcting by each of the terminals of the said set, of the said language model allowing future recognition of at least one part of the unrecognized expressions."],["The present invention concerns the field of voice interfaces.","More precisely, the invention relates to the optimization of language models and/or of phonetic units in terminals using voice recognition.","Information or control systems are making ever increasing use of a voice interface to make interaction with the user faster and/or more intuitive.","Since these systems are becoming ever more complex, the requirements in terms of voice recognition are ever more considerable, both as regards the extent of recognition (very large vocabulary) and the speed of recognition (real time).","Voice recognition processes based on the use of language models (probability that a given word of the vocabulary of the application follows another word or group of words in the chronological order of the sentence) and of phonetic units are known in the state of the art.","These techniques are in particular described in the work by Frederik Jelinek “Statistical methods for speech recognition” published by MIT Press in 1997.These techniques rely on language models and phonetic units which are produced from representative voice samples (emanating for example from a population of users of a terminal who are made to utter commands).","In practice, the language models must take account of the speaking style ordinarily employed by a user of the system, and in particular of his “defects”: hesitations, false starts, change of mind, etc.","The quality of a language model used greatly influences the reliability of the voice recognition.","This quality is most often measured by an index referred to as the perplexity of the language model, and which schematically represents the number of choices which the system must make for each decoded word.","The lower this perplexity, the better the quality.","The language model is necessary to translate the voice signal into a textual string of words, a step often used by dialogue systems.","It is then necessary to construct a comprehension logic which makes it possible to comprehend the query so as to reply to it.","There are two standard methods for producing large vocabulary language models: The so-called N-gram statistical method, most often employing a bigram or trigram, consists in assuming that the probability of occurrence of a word in the sentence depends solely on the N words which precede it, independently of the rest of its context in the sentence.","If one takes the example of the trigram for a vocabulary of 1000 words, it would be necessary to define 10003 probabilities to define the language model, this being impossible.","The words are therefore grouped into sets which are either defined explicitly by the model designer, or deduced by self-organizing methods.","This language model is therefore constructed from a text corpus automatically.","This type of language model is used mainly for voice dictation systems whose ultimate functionality is to translate the voice signal into a text, without any comprehension phase being necessary.","The second method consists in describing the syntax by means of a probabilistic grammar, typically a context-free grammar defined by virtue of a set of rules described in the so-called Backus Naur Form or BNF, or an extension of this form to contextual grammars.","The rules describing grammars are most often handwritten.","This type of language model is suitable for command and control applications, in which the recognition phase is followed by a phase of controlling an appliance or of searching for information in a database.","The language model of an application describes the set of expressions (for example sentences) that the application will be required to recognize.","A drawback of the prior art is that, if the language model is of poor quality, the recognition system, even if it performs extremely well at the acoustico-phonetic decoding level, will have mediocre performance for certain expressions.","The stochastic type language models do not have, properly speaking, a clear definition of the expressions which are in the language model, and of those which are outside.","Certain expressions simply have a higher a priori probability of occurrence than others.","The language models of probabilistic grammar type show a clear difference between expressions belonging to the language models, and expressions external to the language model.","In these models, expressions therefore exist which will never be able to be recognized, regardless of the quality of the phonetic models used.","These are generally expressions having no meaning or that carry a meaning outside of the field of application of the system developed.","It turns out that the language models of probabilistic type and their derivatives are more effective for command and control applications.","These grammars are often written by hand, and one of the main difficulties of the development of dialogue systems is to offer a language model of good quality.","In particular, as far as the models of grammar type are concerned, it is not possible to exhaustively define a language in particular if the latter is liable to be used by a large population (case for example of a remote control for mass-market appliances).","It is not possible to take account of all the possible expressions and turns of phrase (from formal language to slang), and/or of errors of grammar, etc.","The invention relates to a voice recognition process and system making it possible to modify and improve a language model remotely, on the basis of the recordings of expressions unrecognized by the system.","More precisely, the subject of the invention is a voice recognition process implemented in at least one terminal, the said voice recognition process using a language model, characterized in that it comprises the following steps: detection of at least one unrecognized expression in one of the terminals; recording in the terminal of data representative of the unrecognized expression; transmission by the terminal of the recorded data to a remote server, via a first transmission channel; analysis, at the level of the remote server, of the data and generation of information for correcting the language model taking account of at least one part of the unrecognized expressions; and transmission via a second transmission channel from the server to at least one terminal of the correcting information, so as to allow future recognition of at least certain of the unrecognized expressions.","Thus, the invention relies on an entirely novel and inventive approach to voice recognition, which makes it possible to update the various elements allowing voice recognition as a function of locally unrecognized expressions, a remote server being furnished with considerable resources (for example human and/or computational capabilities) generating correcting information.","It is noted that here the language models comprise: language models in the strict sense (this being the case, for example, when the data, the subject of the recognition, are of purely textual type); models formed of one or more language models in the strict sense and of one or more sets of phonetic units (this corresponding in particular to the general case of voice recognition applied to voice samples).","The invention goes well beyond the straightforward updating of a vocabulary.","Specifically, it is possible that, although all the words of an expression feature in the vocabulary used by the language model of the terminal, this expression may not be recognized.","Only the updating of the language model itself then makes it possible to have this expression recognized subsequently.","The updating of the vocabulary, which is one of the items of information from which the language model is derived, is not sufficient.","Here, the expressions are to be taken within the wide sense and relate to any vocal expression allowing interaction between a terminal and its user.","Expressions (or utterances) comprise in particular sentences, phrases, isolated or non-isolated words, code words specific to the terminal, instructions, commands, etc.","The correcting information may comprise in particular information allowing partial or complete modification of the language model and/or phonetic units present in each terminal by deleting, replacing or adding elements therein.","The server can receive data from each terminal allowing it to improve the language model and/or the phonetic units present in the data sending terminal and also in all other terminals, each of the terminals benefiting from the shared experience acquired by the server from all the terminals.","Thus, the invention makes it possible to take into account language styles or turns of phrase specific to certain users (for example, the expression “8 pm in the evening” (pleonasm which is hard to imagine a priori) instead of “8 pm” or “8 o'clock in the evening”) and for which provision had not been made in the course of the construction of the language model implemented.","Furthermore, the invention takes into account the evolution of living languages (new turns of phrase or expressions, etc).","It is noted that the invention applies equally well to language models of stochastic type and to language models of probabilistic grammar type.","When the invention is applied to language models of stochastic type, there are generally very many correcting data for influencing recognition, whereas the correcting data for a model of probabilistic grammar type may be scanty and have an appreciable influence on the effectiveness and reliability of recognition.","According to a particular characteristic, the process is noteworthy in that the data representative of the unrecognized expressions comprise a compressed voice recording representative of parameters descriptive of the acoustic signal.","Thus, the invention advantageously makes it possible to take into account the voice data sent to the source for fine analysis at the server level, while limiting the volume of data transmitted to the remote server.","According to a particular characteristic, the process is noteworthy in that during the step of transmission by the terminal, the latter furthermore transmits to the server at least one of the items of information forming part of the group comprising: information of context of use of the voice recognition process when an expression has not been recognized; and information relating to the speaker who has uttered an unrecognized expression.","Thus, voice recognition of the expressions unrecognized by the terminal, which may be performed remotely, is facilitated.","Furthermore, a check of the validity of the content of the unrecognized expressions may be performed as a function of the context.","(For example, a command “record the transmission” has a meaning and is therefore valid when the terminal to which it is addressed is a video recorder and has no meaning in respect of a mobile telephone).","According to a particular characteristic, the process is noteworthy in that it implements an encryption and/or a scrambling of the recorded data and/or of the correcting information.","Thus, the data are made secure effectively and remain confidential.","The information also relates to a voice recognition module using a language model, characterized in that it comprises: an analyser detecting unrecognized expressions; a recorder of data representative of at least one unrecognized expression; a transmitter transmitting the recorded data to a remote server; and a receiver of correcting information allowing the correcting of the language model transmitted to the module allowing future recognition of at least certain of the unrecognized expressions by the module, the correcting information having been transmitted by the remote server after analysis at the level of the remote server of the data, and after generation of information for correcting the language model taking account of at least one part of the unrecognized expressions.","The invention also relates to a voice recognition device using a language model, characterized in that it comprises: an analyser detecting unrecognized expressions; a recorder of data representative of at least one unrecognized expression; a transmitter transmitting the recorded data to a remote server; and a receiver of correcting information allowing the correcting of the language model transmitted to the device allowing future recognition of at least certain of the unrecognized expressions by the device, the correcting information having been transmitted by the remote server after analysis at the level of the remote server of the data, and after generation of information for correcting the language model taking account of at least one part of the unrecognized expressions.","The invention also relates to a voice recognition server, the recognition being implemented in a set of at least one remote terminal, using a language model, characterized in that it comprises the following means: a receiver of data representative of at least one expression unrecognized by at least one terminal forming part of the set of at least one remote terminal and having detected the unrecognized expression during a voice recognition operation; and a sender sending to the set of at least one remote terminal correcting information obtained on the basis of an analysis of the data received at the level of the server, the correcting information allowing the correcting, by each of the terminals of the set, of the language model allowing future recognition of at least one part of the unrecognized expressions.","The particular characteristics and the advantages of the module, of the device and of the server for voice recognition being similar to those of the voice recognition process, they are not recalled here.","Other characteristics and advantages of the invention will become more clearly apparent when reading the following description of a preferred embodiment given by way of straightforward non-limiting illustrative example, and of the appended drawings, among which: FIG.","1 depicts a general schematic of a system comprising a voice-controlled box, in which the technique of the invention may be implemented; FIG.","2 depicts a schematic of the voice recognition box of the system of FIG.","1; FIG.","3 describes an electronic diagram of a voice recognition box implementing the schematic of FIG.","2; FIG.","4 depicts a schematic of the server of the system of FIG.","1; FIG.","5 represents a flow chart of the process for testing an expression and for recording data relating to unrecognized expressions, as implemented by the recognition engine of FIG.","2; FIG.","6 represents a flow chart of the process for sending data relating to unrecognized expressions, as implemented by the rejection module of FIG.","2; FIG.","7 represents a flow chart of the process for receiving correcting data, as implemented by the module for loading the language models of FIG.","2; and FIG.","8 represents a flow chart of the process for receiving and for processing correcting data, as implemented within the remote server of FIG.","4.The general principle of the invention therefore relies on voice recognition implemented in terminals, the voice recognition process using a language model and/or a set of phonetic units that may be updated by a remote server when, in particular, the latter deems it necessary.","In a general manner each terminal can recognize expressions (for example sentence or command) formulated by a speaker and execute a corresponding action.","Nevertheless, it is often found that certain expressions that are entirely comprehensible to a human being are not recognized by the device or the module implementing the voice recognition.","The failure of recognition may have multiple reasons: vocabulary used by the speaker not forming part of the language model; particular pronunciation (with accent for example); particular turn of phrase not provided for by the voice recognition device or module; etc.","Specifically, the language models and the sets of phonetic units are often constructed on the basis of statistical data which take into account samples of expressions customarily used by a typical population, certain words of vocabulary, pronunciations, and/or turns of phrase then not being (and unable to be) taken into account.","The invention relies firstly on detecting expressions unrecognized by the voice recognition device or module.","When an expression has not been recognized, the terminal records data representative of the signal corresponding to the unrecognized expressions (such as, for example, a voice digital recording of the expression), with a view to sending them to a remote server.","At the level of the remote server centralizing the unrecognized expressions from a set of terminals, a human operator can then analyse the unrecognized expressions.","Certain of them may prove to be incomprehensible and/or unutilizable and will be discarded.","On the other hand, others will be entirely comprehensible to the operator who will be able (if he deems it useful) through a man/machine link up to “translate” these expressions hitherto unrecognized by the terminals into a code comprehensible to the server.","The server can then take these expressions into account together with their translation so as to generate information for correcting the language model and/or the set of phonetic units.","It will be noted that correction is understood here as: modification of the model; and/or supplementing the model.","The server then sends the correcting information to each of the terminals which can update its language model and/or set of phonetic units which are enriched with numerous expressions unrecognized by itself or by other terminals.","Thus, the voice recognition of each of the terminals is improved by benefitting from the experience shared by all the terminals.","According to a particular mode of the invention, the analysis is not performed by an operator but by the server itself which may have much more considerable resources at its disposal than a straightforward terminal.","According to particular embodiments, the terminals send the server context data (for example the time, the date, a control performed manually or vocally after the failure of a voice command, the location, the type of terminal, etc.)","together with the data representative of the signal corresponding to the unrecognized expressions.","This may facilitate the analysis work of the operator and/or of the server.","A general schematic of a system comprising a voice-controlled box, in which the technique of the invention may be implemented, is depicted in conjunction with FIG.","1.This system comprises in particular: a remote server 116 controlled by a human operator 122; and a plurality of user systems 114, 117 and 118.The remote server 116 is linked to each of the user systems 114, 117 and 118 via communication downlinks 115, 119 and 120 respectively.","These links may be permanent or temporary and be of any type well known to the person skilled in the art.","They may in particular be of broadcasting type and be based on RF, satellite or wire channels used by television or any other type such as, for example, an Internet type link.","FIG.","1 describes in particular the user system 114 which is linked via a communication uplink 121 to the server 116.This link can likewise be of any type well known to the person skilled in the art (in particular telephone, Internet, etc).","The user system 114 comprises in particular: a voice source 100 which may in particular consist of a microphone intended to pick up a voice signal produced by a speaker; a voice recognition box 102; a control box 105 intended to drive an appliance 107; a controlled appliance 107, for example of television, video recorder or mobile communication terminal type; a unit 109 for storing the expressions detected as unrecognized; an interface 112 allowing upward and downward communications to the server 116.The source 100 is linked to the voice recognition box 102, via a link 101 which allows it to transmit an analogue source wave representative of a voice signal to the box 102.The box 102 can retrieve context information 104 (such as for example the type of appliance 107 which can be controlled by the control box 105 or the list of control codes) via a link 104 and send commands to the control box 105 via a link 103.The control box 105 sends commands via a link 106, for example infrared, to the appliance 107, as a function of the information which it recognizes according to its language model and its dictionary.","The control box 105 detects the expressions which it does not recognize, and, instead of simply rejecting them, by sending a non-recognition signal, it performs a recording of these expressions to the storage unit 109 via a link 108.The unit 109 for storing the unrecognized expressions sends representative data to the interface 112 via a link 111, which relay them to the server 116 via the link 121.After correct transmission, the interface 110 can send a signal 110 to the storage unit 109 which can then erase the transmitted data.","The control box 105 receives, moreover, correcting data from the interface 112 via a link 113, that the interface 112 has itself received from the remote server via the link 115.These correcting data are taken into account by the control box 105 for the updating of language models and/or of sets of phonetic units.","According to the embodiment considered the source 100, the voice recognition box 102, the control box 105, the storage unit 109 and the interlace 112 form part of one and the same device and thus the links 101, 103, 104, 108, 111, 110 and 113 are links internal to the device.","The link 106 is typically a wireless link.","According to a first variant embodiment of the invention described in FIG.","1, the elements 100, 102, 105, 109 and 112 are partly or completely separate and do not form part of one and the same device.","In this case, the links 101, 103, 104, 108, 111, 110 and 113 are external wire or other links.","According to a second variant, the source 100, the boxes 102 and 105, the storage unit 109 and the interface 112 as well as the appliance 107 form part of one and the same device and are interlinked by internal buses (links 101, 103, 104, 108, 111, 110, 113 and 106).","This variant is particularly beneficial when the device is, for example, a mobile telephone or a portable communication terminal.","FIG.","2 depicts a schematic of a voice-controlled box such as the box 102 illustrated with regard to FIG.","2.It is noted that the box 102 receives from outside the analogue source wave 101 which is processed by an Acoustico-Phonetic Decoder 200 or APD (also called the “front end”).","The APD 200 samples the source wave 101 at regular intervals (typically every 10 ms) so as to produce real vectors or vectors belonging to code books, typically representing oral resonances that are sent via a link 201 to a recognition engine 203.The APD is for example based on a PLP (standing for “Perceptual Linear Prediction”) described in particular in the article “Perceptual Linear Prediction (PLP) analysis of speech” written by Hynek Hermansky and published in “Journal of the Acoustical Society of America”, Vol.","97, No4, 1990 on pages 1738-1752.With the aid of a dictionary 202, the recognition engine 203 analyses the real vectors that it receives, using in particular hidden Markov models or HMMs and language models (which represent the probability of one word following another word).","Recognition engines are in particular described in detail in the book “Statistical Methods for Speech Recognition” written by Frederick Jelinek, and published by MIT Press in 1997.The language model allows the recognition engine 203 (which may use in particular hidden Markov networks) to determine which words may follow a given word of any expression usable by the speaker in a given application, and to give the associated probability.","The words in question belong to the vocabulary of the application, which may be, independently of the language model, of small size (typically from 10 to 300 words), or of large size (for example of size greater than 300 000 words).","Patent application PCT/FR00/03329 dated 29 Nov. 1999 filed in the name of Thomson Multimedia describes a language model comprising a plurality of syntactic blocks.","The use of the invention which is the subject of the present patent application is particularly advantageous in conjunction with this type of modular language model, since the modules may be updated separately, thereby avoiding the downloading of overly large volume files.","The language models are transmitted by a language model loading module 207.The module 207 itself receives language models, updates or corrections of language models and/or of phonetic units transmitted from the server via the link 113.It is noted that the dictionary 202 belongs to the language model making reference to words from the dictionary.","Thus, the dictionary 202 itself can be updated and/or corrected via a language model loaded by the module 207.After implementation of a recognition operation based on the use of a Viterbi algorithm, the recognition engine 203 supplies the rejection module 211 with an ordered list of sequences of words in accordance with the language model, which exhibits the best score for the expression uttered.","The rejection module 211 works downstream of the recognition engine 203 and operates according to one or more of the following principles: Sometimes, for reasons specific to the Viterbi algorithm, the latter may not produce a consistent list because the scores are so low that the limit of acceptable accuracies of the machine in terms of arithmetic computation is overstepped.","There is therefore no consistent complete proposal.","Thus, when the rejection module 211 detects one or more scores below a predetermined acceptable limit, the expression is rejected.","Each element of the list calculated by the Viterbi algorithm has been retained because the associated score was among the highest relative scores of all the possible expressions, according to the language model.","Additionally, the Markov network associated with each of these expressions makes it possible to evaluate the intrinsic probability of the network in question producing the expression associated with the score observed.","The rejection module 211 analyses this probability and if it is less than a predetermined threshold of acceptability of probability, the expression is rejected.","According to another method, for the best proposals obtained via the Viterbi algorithm, the rejection module 211 performs a complementary processing of the expressions, using criteria which had not been taken into account in the course of the Viterbi development.","For example, it checks that those parts of the signal that have to be voiced because they are associated with vowels, are actually so.","If the expressions proposed do not fulfil these conditions, they are rejected.","When the rejection module 211 rejects an expression, as illustrated previously, the expression is said to be unrecognized and a signal indicating the rejected expression is sent to the recognition engine 203.In parallel, the rejection module transmits a recording of the unrecognized expression to the storage unit 109 via the link 108.The recognition engine 203 is responsible for recognizing expressions emanating from the APD 200 in the form of phonetic samples.","Thus, the recognition engine 203 uses: the phonetic units to construct the phonetic representation of a word in the form of a Markov model, each word of the dictionary 202 possibly possessing several “phonetizations”; and simultaneously the language model in the strict sense for recognizing expressions of greater or lesser complexity.","The recognition engine 203 supplies expressions which have been recognized (that is to say not rejected by the module 211) and which it has identified on the basis of the vectors received to a means 205 for translating these expressions into commands that can be understood by the appliance 107.This means 205 uses an artificial intelligence translation process which itself takes into account a context 104 supplied by the control box 105 before sending one or more commands 103 to the control box 105.FIG.","3 diagrammatically illustrates a voice recognition module or device 102 such as illustrated in conjunction with FIG.","1, and implementing the schematic of FIG.","2.The box 102 comprises interlinked by an address and data bus: a voice interface 301; an analogue-digital converter 302; a processor 304; a nonvolatile memory 305; a random access memory 306; a reception module 312; a transmission module 313; and an inputs/outputs interface 307.Each of the elements illustrated in FIG.","3 is well known to the person skilled in the art.","These commonplace elements are not described here.","It is observed moreover that the word “register” used throughout the description designates in each of the memories mentioned, both a memory area of small capacity (a few data bits) and a memory area of large capacity (making it possible to store an entire program or the whole of a sequence of transaction data).","The nonvolatile memory 305 (ROM) holds in particular the program for operating the processor 304 in a “prog” register 308.The random access memory 306 holds data, variables and intermediate results of processing in registers which for convenience possess the same names as the data that they hold and comprise in particular: a register 309 in which are held recordings of unrecognized expressions, “Exp_Not_Rec; a counter 310 of unrecognized sentences Nb_Exp_Not _Rec; and a language model in a register 311, Model_Language.","It is noted furthermore that the receive module 312 and send module 313 are modules that allow the transmission of data respectively from or to the remote server 116.The wire-based or wireless techniques for receiving and for sending are well known to the person skilled in the art of telecommunications and will not be detailed further.","FIG.","4 depicts the server 116 of the system illustrated with regard to FIG.","1.It is noted that the server 116 is controlled by a human operator 122 via any man/machine interface 404 (for example, of keyboard and screen type).","The server 116 itself comprises in particular: a receiver 400; an analyser 401; a module 402 for construction of correction of a language model and/or a set of phonetic units; and a sender 403.The receiver 400 is compatible with the sender 313 of a terminal and can receive from each terminal in particular data (for example recording) representative of unrecognized expressions via the link 121 and possibly of complementary data (for example contextual data).","The analyser 401 receives the set of data from the receiver 400 via a link 121 which it transmits to the operator 122 via an interface 404 which is for example a terminal furnished: with a screen and with a keyboard allowing dialogue with the server 116 and its control; with loudspeakers or an audio headset for listening to unrecognized recordings.","This interface 404 also allows the analyser 401 to receive information from the operator 122 indicating: that an unrecognized expression not covered by the language model remains incomprehensible, has no meaning within the application to the terminal and/or does not relate to the terminal (it should therefore not be included in the language model), this expression then being ignored for the correcting of the language model and discarded by the analyser 401; that an unrecognized expression nevertheless belongs to the language model in the strict sense (this then involves a pure recognition problem); in this case, this involves modifying the phonetic units and not the language model in the strict sense; or a translation into for example the form of a control code, after identification of the content of an expression by the operator, the unrecognized expression not belonging to the language model and having a meaning for the terminal for which it was intended; this then involves correcting the language model in the strict sense.","A combination of the second and third solutions is possible; in this case, this involves both modifying the phonetic units and the language model in the strict sense.","This embodiment corresponds to a manual processing of the unrecognized expressions.","According to this embodiment, the human operator 122 listens to the unrecognized expression and analyses the reasons for its rejection.","The operator 122 determines in particular whether the expression belongs to the language model or not.","In the case where the expression belongs to the language model, the operator analyses the expression to identify the intrinsic recognition problem (expression belonging to the language model and which ought to have been recognized and which was not for other reasons: noise, accent of the speaker, etc.).","According to a first variant, the processing is automatic and the intervention of a human operator becomes nil.","In this case, the server 116 and in particular the analyser 401 possess a relatively considerable computational power which may in particular be much larger than a terminal.","According to this variant, the analyser 401 analyses each unrecognized expression in a more appropriate manner than could be done by a terminal, using, for example, a richer language model and/or more complex phonetic models.","Not being subject to such strict real-time computational requirements as might be a terminal (which often requires a fast reaction time to a speaker command), the analyser 401 can also, for example, allow recognition demanding a longer processing time than in a terminal.","According to a second variant, the processing is semi-automatic and the intervention of a human operator remains limited to the cases that cannot be solved by the analyser.","The general structure of a server 116 is according to the preferred embodiment described here similar to that of a terminal such as described with regard to FIG.","3 and comprises in particular interlinked by an address and data bus: a processor; a random access memory; a nonvolatile memory; a suitable transmission module; a reception module; and a man/machine linkup interface.","According to FIG.","5 representing a flow chart of the testing of an expression and of the recording of data relating to unrecognized expressions, as implemented by the recognition engine 203 of FIG.","2, in the course of a first step of initialization 500, the microprocessor 304 commences the execution of the program 308 and initializes the variables of the random access memory 306.Then, in the course of a step 501 of waiting for an expression, it waits for and receives an expression sent by a speaker.","Next, in the course of a test 502, after having executed an operation of voice recognition for the expression received, it determines whether the expression has or has not been recognized according to one or more criteria illustrated with regard to the description of the rejection module 211 of FIG.","2.If it has, in the course of a control step 504, the terminal 102 takes into account the result of the voice recognition applied to the expression received and executes an appropriate action such as for example a command.","If it has not, in the course of a step 503 of recording an expression, the unrecognized expression is compressed and recorded in the storage unit 109 awaiting a transmission to the remote server 116 as illustrated with regard to FIG.","6.On completion of one of the steps 503 or 504, step 501 of waiting for an expression is repeated.","FIG.","6 representing a flow chart of the sending of data relating to unrecognized expressions, as implemented by the rejection module of FIG.","2, in the course of a first step of initialization 600, the microprocessor 304 commences the execution of the program 308 and initializes the variables of the random access memory 306.Then, in the course of a step 601 of waiting for expressions unrecognized by the voice recognition module 102, the microprocessor 304 waits for and then receives recordings of unrecognized expressions.","Then, in the course of a step 602, the terminal 114 connects up to the remote server 116 according to methods well known to the person skilled in the art of telecommunications.","Next, in the course of a step 603, the recordings of unrecognized expressions are shaped and sent to the remote server 116.Then, in the course of a step 604 of disconnection, the terminal disconnects from the remote server 116 and a signal is sent between the interface 112 with the remote server and the unit 109 for storing the data corresponding to the unrecognized expressions indicating the transmission of the recordings of expressions.","The data corresponding to these expressions are then erased from the storage unit 109.Next, step 601 is repeated.","FIG.","7 represents a flow chart of the receiving of correcting data, as implemented by the module 207 for loading the language models of FIG.","2.After a first step 700 of initialization, in the course of a step) 701, the terminal places itself on standby awaiting correcting data broadcast by a server 1116 to a plurality of terminals.","Next, during a step 702, the terminal takes into account the correcting data so as to update the language model and/or its set of phonetic units used by the voice recognition module.","According to the nature of the correcting data, these data will be able in particular: to substitute for existing data in the language model and/or its set of phonetic units; to modify existing data; to supplement existing data; and/or to bring about the deletion of existing data.","After the execution of step 702, step 703 is repeated.","FIG.","8 represents a flow chart of the receiving and processing of correcting data, as implemented within the remote server of FIG.","4.After a first step 800 of initialization of the parameters and of instigation of a program for management of the server, the server 116 places itself on standby waiting for a connection request originating from a terminal (which executes a step 602 illustrated with regard to FIG.","6) and establishes a connection with the terminal according to methods well known to the person skilled in the art of telecommunications.","Then, in the course of a step 802, the server 116 receives data originating from the connected terminal which executes a step 603 described previously.","These data contain in particular recordings of one or more expressions rejected by the terminal because they were not recognized by a voice recognition module implemented in the terminal.","When all the data have been received, the connection between the terminal and the server 116 is broken.","Next, in the course of a step 803 of processing the data received, the server 116 processes each of the received recordings of expressions either manually by the operator 122 or automatically or semi-automatically according to various alternatives illustrated with regard to FIG.","4.Then, in the course of a test 804, the server 116 determines whether in particular one or more expressions received were comprehensible and are relevant in respect of the terminal which transmitted this or these expressions.","An updating of the language models and/or of the phonetic units is then necessary.","If not, then the waiting step 801 is repeated.","In the converse case, the server 116 constructs a correction of the language model which may take several forms allowing a step 607 (illustrated previously) within the terminals after receipt of the correcting data.","These correcting data comprise in particular: an indicator specifying the nature of the correction (in particular substitution, modification, supplement, or deletion); and the correcting data themselves as a function of the indicator.","It is noted that if the language model comprises a plurality of syntactic blocks (case in particular of the language models such as described in patent PCT/FR00/03329 mentioned above), each module can be corrected separately.","In this case, the correction data also comprise an indicator of the module or modules to be corrected.","Then, in the course of a step 806, the server 116 broadcasts the correcting data to one or preferably to a set of terminals which will be able to update their language model and/or set of phonetic units according to a step 607.Step 801 is then repeated.","The procedure is thus iterative and may be repeated several times.","It also allows the application to be upgraded by adding new queries.","Of course, the invention is not limited to the exemplary embodiments mentioned hereinabove.","In particular, the person skilled in the art will be able to alter the definition of the terminals implementing the invention, the invention relating to any type of device and/or module using or capable of using a voice recognition process (being of the type for example of multimedia terminal, television, video recorder, a multimedia digital decoder (or set top box), audio or video equipment, fixed or mobile terminal, etc.).","Likewise, the invention relates to any type of remote server (for example Internet servers, equipment coupled to television program broadcasters, equipment coupled to mobile communication networks, service provider equipment, etc.).","Furthermore, according to the invention, the transmission channel for the data corresponding to the unrecognized sentences and the transmission channel for the data for correcting the language models and/or phonetic units are any whatsoever and include in particular: RF transmission pathways; satellite transmission pathways; the channels of television broadcasting networks; the channels of Internet type networks: the channels of telephone networks; the channels of mobile networks; and removable media.","Moreover, it is noted that the invention relates not only to unrecognized sentences but relates to any type of vocal expression such as for example one or more sentences, an isolated or unisolated word, a phrase, a voice code allowing dialogue between a machine and its user.","These oral expressions may be associated not only with commands, but with any type of data that can form the subject of a dialogue between a machine and its user, as, for example, information data that the user can transmit to the machine, configuration data, programming data etc.","It is also noted that the method of updating the language models which is described by the patent applies not only to processes of voice recognition in the strict sense, but applies also to processes for recognition of textual inputs supporting orthographic mistakes and/or typing mistakes, and also based on Markovian models or language models in the strict sense, as described in the patent.","It will be noted that the invention is not limited to a purely hardware installation but that it can also be implemented in the form of a sequence of instructions of a computer program or any form mixing a hardware part and a software part.","In the case where the invention is installed partially or totally in software form, the corresponding sequence of instructions may be stored in a removable storage means (such as for example a diskette, a CD-ROM or a DVD-ROM) or otherwise, this means of storage being readable partially or totally by a computer or a microprocessor."]],"string":"[\n [\n \"Method, module, device and server for voice recognition\",\n \"The invention concerns a voice recognition method implemented in at least a terminal, the voice recognition method using a language model, comprising the following steps: detecting at least a non-recognised expression in one of the terminals; recording in the terminal data representing the non-recognised expression; transmission by the terminal of the recorded data to a remote server; analysis, at the remote server, of said data and generation of data for correcting said language model taking into account at least part of the non-recognised expressions; and transmitting from the server to at least a terminal correction data, so as to enable subsequent recognition of at least some of the non-recognised expressions.\",\n \"The invention also concerns corresponding modules, devices and remote server.\"\n ],\n [\n \"1.Voice recognition process implemented in at least one terminal, the said voice recognition process using a language model, wherein it comprises the following steps: detection of at least one unrecognized expression in one of the said terminals; recording in the said terminal of data representative of the said unrecognized expression; transmission by the said terminal of the said recorded data to a remote server, via a first transmission channel; analysis, at the level of the said remote server, of the said data and generation of information for correcting the said language model taking account of at least one part of the said unrecognized expressions; and transmission via a second transmission channel from the said server to at least one terminal of the said correcting information, so as to allow future recognition of at least certain of the said unrecognized expressions.\",\n \"2.Process according to claim 1, wherein the said data representative of the said unrecognized expressions comprise a compressed voice recording representative of parameters descriptive of the acoustic signal.\",\n \"3.Process according to claim 1, wherein during the said step of transmission by the said terminal, the latter furthermore transmits to the said server at least one of the items of information forming part of the group comprising: information of context of use of the said voice recognition process when an expression has not been recognized; and information relating to the speaker who has uttered an unrecognized expression.\",\n \"4.Process according to claim 1, wherein it implements an encryption and/or a scrambling of the said recorded data and/or of the said correcting information.\",\n \"5.Voice recognition module using a language model, wherein it comprises: an analyser detecting unrecognized expressions; a recorder of data representative of at least one unrecognized expression; a transmitter transmitting the said recorded data to a remote server; and a receiver of correcting information allowing the correcting of the said language model transmitted to the said module allowing future recognition of at least certain of the said unrecognized expressions by the said module, the correcting information having been transmitted by the said remote server after analysis at the level of the said remote server of the said data, and after generation of information for correcting the said language model taking account of at least one part of the unrecognized expressions.\",\n \"6.Voice recognition device using a language model, wherein it comprises: an analyser detecting unrecognized expressions; a recorder of data representative of at least one unrecognized expression; a transmitter transmitting the said recorded data to a remote server; and a receiver of correcting information allowing the correcting of the said language model transmitted to the said device allowing future recognition of at least certain of the said unrecognized expressions by the said device, the correcting information having been transmitted by the said remote server after analysis at the level of the said remote server of the said data, and after generation of information for correcting the said language model taking account of at least one part of the unrecognized expressions.\",\n \"7.Voice recognition server, the said recognition being implemented in a set of at least one remote terminal, using a language model, wherein it comprises the following means: a receiver of data representative of at least one expression unrecognized by at least one terminal forming part of the said set and having detected the said unrecognized expression during a voice recognition operation; and a sender sending to the said set of at least one remote terminal correcting information obtained on the basis of an analysis of the said data received at the level of the said server, the said correcting information allowing the correcting by each of the terminals of the said set, of the said language model allowing future recognition of at least one part of the unrecognized expressions.\"\n ],\n [\n \"The present invention concerns the field of voice interfaces.\",\n \"More precisely, the invention relates to the optimization of language models and/or of phonetic units in terminals using voice recognition.\",\n \"Information or control systems are making ever increasing use of a voice interface to make interaction with the user faster and/or more intuitive.\",\n \"Since these systems are becoming ever more complex, the requirements in terms of voice recognition are ever more considerable, both as regards the extent of recognition (very large vocabulary) and the speed of recognition (real time).\",\n \"Voice recognition processes based on the use of language models (probability that a given word of the vocabulary of the application follows another word or group of words in the chronological order of the sentence) and of phonetic units are known in the state of the art.\",\n \"These techniques are in particular described in the work by Frederik Jelinek “Statistical methods for speech recognition” published by MIT Press in 1997.These techniques rely on language models and phonetic units which are produced from representative voice samples (emanating for example from a population of users of a terminal who are made to utter commands).\",\n \"In practice, the language models must take account of the speaking style ordinarily employed by a user of the system, and in particular of his “defects”: hesitations, false starts, change of mind, etc.\",\n \"The quality of a language model used greatly influences the reliability of the voice recognition.\",\n \"This quality is most often measured by an index referred to as the perplexity of the language model, and which schematically represents the number of choices which the system must make for each decoded word.\",\n \"The lower this perplexity, the better the quality.\",\n \"The language model is necessary to translate the voice signal into a textual string of words, a step often used by dialogue systems.\",\n \"It is then necessary to construct a comprehension logic which makes it possible to comprehend the query so as to reply to it.\",\n \"There are two standard methods for producing large vocabulary language models: The so-called N-gram statistical method, most often employing a bigram or trigram, consists in assuming that the probability of occurrence of a word in the sentence depends solely on the N words which precede it, independently of the rest of its context in the sentence.\",\n \"If one takes the example of the trigram for a vocabulary of 1000 words, it would be necessary to define 10003 probabilities to define the language model, this being impossible.\",\n \"The words are therefore grouped into sets which are either defined explicitly by the model designer, or deduced by self-organizing methods.\",\n \"This language model is therefore constructed from a text corpus automatically.\",\n \"This type of language model is used mainly for voice dictation systems whose ultimate functionality is to translate the voice signal into a text, without any comprehension phase being necessary.\",\n \"The second method consists in describing the syntax by means of a probabilistic grammar, typically a context-free grammar defined by virtue of a set of rules described in the so-called Backus Naur Form or BNF, or an extension of this form to contextual grammars.\",\n \"The rules describing grammars are most often handwritten.\",\n \"This type of language model is suitable for command and control applications, in which the recognition phase is followed by a phase of controlling an appliance or of searching for information in a database.\",\n \"The language model of an application describes the set of expressions (for example sentences) that the application will be required to recognize.\",\n \"A drawback of the prior art is that, if the language model is of poor quality, the recognition system, even if it performs extremely well at the acoustico-phonetic decoding level, will have mediocre performance for certain expressions.\",\n \"The stochastic type language models do not have, properly speaking, a clear definition of the expressions which are in the language model, and of those which are outside.\",\n \"Certain expressions simply have a higher a priori probability of occurrence than others.\",\n \"The language models of probabilistic grammar type show a clear difference between expressions belonging to the language models, and expressions external to the language model.\",\n \"In these models, expressions therefore exist which will never be able to be recognized, regardless of the quality of the phonetic models used.\",\n \"These are generally expressions having no meaning or that carry a meaning outside of the field of application of the system developed.\",\n \"It turns out that the language models of probabilistic type and their derivatives are more effective for command and control applications.\",\n \"These grammars are often written by hand, and one of the main difficulties of the development of dialogue systems is to offer a language model of good quality.\",\n \"In particular, as far as the models of grammar type are concerned, it is not possible to exhaustively define a language in particular if the latter is liable to be used by a large population (case for example of a remote control for mass-market appliances).\",\n \"It is not possible to take account of all the possible expressions and turns of phrase (from formal language to slang), and/or of errors of grammar, etc.\",\n \"The invention relates to a voice recognition process and system making it possible to modify and improve a language model remotely, on the basis of the recordings of expressions unrecognized by the system.\",\n \"More precisely, the subject of the invention is a voice recognition process implemented in at least one terminal, the said voice recognition process using a language model, characterized in that it comprises the following steps: detection of at least one unrecognized expression in one of the terminals; recording in the terminal of data representative of the unrecognized expression; transmission by the terminal of the recorded data to a remote server, via a first transmission channel; analysis, at the level of the remote server, of the data and generation of information for correcting the language model taking account of at least one part of the unrecognized expressions; and transmission via a second transmission channel from the server to at least one terminal of the correcting information, so as to allow future recognition of at least certain of the unrecognized expressions.\",\n \"Thus, the invention relies on an entirely novel and inventive approach to voice recognition, which makes it possible to update the various elements allowing voice recognition as a function of locally unrecognized expressions, a remote server being furnished with considerable resources (for example human and/or computational capabilities) generating correcting information.\",\n \"It is noted that here the language models comprise: language models in the strict sense (this being the case, for example, when the data, the subject of the recognition, are of purely textual type); models formed of one or more language models in the strict sense and of one or more sets of phonetic units (this corresponding in particular to the general case of voice recognition applied to voice samples).\",\n \"The invention goes well beyond the straightforward updating of a vocabulary.\",\n \"Specifically, it is possible that, although all the words of an expression feature in the vocabulary used by the language model of the terminal, this expression may not be recognized.\",\n \"Only the updating of the language model itself then makes it possible to have this expression recognized subsequently.\",\n \"The updating of the vocabulary, which is one of the items of information from which the language model is derived, is not sufficient.\",\n \"Here, the expressions are to be taken within the wide sense and relate to any vocal expression allowing interaction between a terminal and its user.\",\n \"Expressions (or utterances) comprise in particular sentences, phrases, isolated or non-isolated words, code words specific to the terminal, instructions, commands, etc.\",\n \"The correcting information may comprise in particular information allowing partial or complete modification of the language model and/or phonetic units present in each terminal by deleting, replacing or adding elements therein.\",\n \"The server can receive data from each terminal allowing it to improve the language model and/or the phonetic units present in the data sending terminal and also in all other terminals, each of the terminals benefiting from the shared experience acquired by the server from all the terminals.\",\n \"Thus, the invention makes it possible to take into account language styles or turns of phrase specific to certain users (for example, the expression “8 pm in the evening” (pleonasm which is hard to imagine a priori) instead of “8 pm” or “8 o'clock in the evening”) and for which provision had not been made in the course of the construction of the language model implemented.\",\n \"Furthermore, the invention takes into account the evolution of living languages (new turns of phrase or expressions, etc).\",\n \"It is noted that the invention applies equally well to language models of stochastic type and to language models of probabilistic grammar type.\",\n \"When the invention is applied to language models of stochastic type, there are generally very many correcting data for influencing recognition, whereas the correcting data for a model of probabilistic grammar type may be scanty and have an appreciable influence on the effectiveness and reliability of recognition.\",\n \"According to a particular characteristic, the process is noteworthy in that the data representative of the unrecognized expressions comprise a compressed voice recording representative of parameters descriptive of the acoustic signal.\",\n \"Thus, the invention advantageously makes it possible to take into account the voice data sent to the source for fine analysis at the server level, while limiting the volume of data transmitted to the remote server.\",\n \"According to a particular characteristic, the process is noteworthy in that during the step of transmission by the terminal, the latter furthermore transmits to the server at least one of the items of information forming part of the group comprising: information of context of use of the voice recognition process when an expression has not been recognized; and information relating to the speaker who has uttered an unrecognized expression.\",\n \"Thus, voice recognition of the expressions unrecognized by the terminal, which may be performed remotely, is facilitated.\",\n \"Furthermore, a check of the validity of the content of the unrecognized expressions may be performed as a function of the context.\",\n \"(For example, a command “record the transmission” has a meaning and is therefore valid when the terminal to which it is addressed is a video recorder and has no meaning in respect of a mobile telephone).\",\n \"According to a particular characteristic, the process is noteworthy in that it implements an encryption and/or a scrambling of the recorded data and/or of the correcting information.\",\n \"Thus, the data are made secure effectively and remain confidential.\",\n \"The information also relates to a voice recognition module using a language model, characterized in that it comprises: an analyser detecting unrecognized expressions; a recorder of data representative of at least one unrecognized expression; a transmitter transmitting the recorded data to a remote server; and a receiver of correcting information allowing the correcting of the language model transmitted to the module allowing future recognition of at least certain of the unrecognized expressions by the module, the correcting information having been transmitted by the remote server after analysis at the level of the remote server of the data, and after generation of information for correcting the language model taking account of at least one part of the unrecognized expressions.\",\n \"The invention also relates to a voice recognition device using a language model, characterized in that it comprises: an analyser detecting unrecognized expressions; a recorder of data representative of at least one unrecognized expression; a transmitter transmitting the recorded data to a remote server; and a receiver of correcting information allowing the correcting of the language model transmitted to the device allowing future recognition of at least certain of the unrecognized expressions by the device, the correcting information having been transmitted by the remote server after analysis at the level of the remote server of the data, and after generation of information for correcting the language model taking account of at least one part of the unrecognized expressions.\",\n \"The invention also relates to a voice recognition server, the recognition being implemented in a set of at least one remote terminal, using a language model, characterized in that it comprises the following means: a receiver of data representative of at least one expression unrecognized by at least one terminal forming part of the set of at least one remote terminal and having detected the unrecognized expression during a voice recognition operation; and a sender sending to the set of at least one remote terminal correcting information obtained on the basis of an analysis of the data received at the level of the server, the correcting information allowing the correcting, by each of the terminals of the set, of the language model allowing future recognition of at least one part of the unrecognized expressions.\",\n \"The particular characteristics and the advantages of the module, of the device and of the server for voice recognition being similar to those of the voice recognition process, they are not recalled here.\",\n \"Other characteristics and advantages of the invention will become more clearly apparent when reading the following description of a preferred embodiment given by way of straightforward non-limiting illustrative example, and of the appended drawings, among which: FIG.\",\n \"1 depicts a general schematic of a system comprising a voice-controlled box, in which the technique of the invention may be implemented; FIG.\",\n \"2 depicts a schematic of the voice recognition box of the system of FIG.\",\n \"1; FIG.\",\n \"3 describes an electronic diagram of a voice recognition box implementing the schematic of FIG.\",\n \"2; FIG.\",\n \"4 depicts a schematic of the server of the system of FIG.\",\n \"1; FIG.\",\n \"5 represents a flow chart of the process for testing an expression and for recording data relating to unrecognized expressions, as implemented by the recognition engine of FIG.\",\n \"2; FIG.\",\n \"6 represents a flow chart of the process for sending data relating to unrecognized expressions, as implemented by the rejection module of FIG.\",\n \"2; FIG.\",\n \"7 represents a flow chart of the process for receiving correcting data, as implemented by the module for loading the language models of FIG.\",\n \"2; and FIG.\",\n \"8 represents a flow chart of the process for receiving and for processing correcting data, as implemented within the remote server of FIG.\",\n \"4.The general principle of the invention therefore relies on voice recognition implemented in terminals, the voice recognition process using a language model and/or a set of phonetic units that may be updated by a remote server when, in particular, the latter deems it necessary.\",\n \"In a general manner each terminal can recognize expressions (for example sentence or command) formulated by a speaker and execute a corresponding action.\",\n \"Nevertheless, it is often found that certain expressions that are entirely comprehensible to a human being are not recognized by the device or the module implementing the voice recognition.\",\n \"The failure of recognition may have multiple reasons: vocabulary used by the speaker not forming part of the language model; particular pronunciation (with accent for example); particular turn of phrase not provided for by the voice recognition device or module; etc.\",\n \"Specifically, the language models and the sets of phonetic units are often constructed on the basis of statistical data which take into account samples of expressions customarily used by a typical population, certain words of vocabulary, pronunciations, and/or turns of phrase then not being (and unable to be) taken into account.\",\n \"The invention relies firstly on detecting expressions unrecognized by the voice recognition device or module.\",\n \"When an expression has not been recognized, the terminal records data representative of the signal corresponding to the unrecognized expressions (such as, for example, a voice digital recording of the expression), with a view to sending them to a remote server.\",\n \"At the level of the remote server centralizing the unrecognized expressions from a set of terminals, a human operator can then analyse the unrecognized expressions.\",\n \"Certain of them may prove to be incomprehensible and/or unutilizable and will be discarded.\",\n \"On the other hand, others will be entirely comprehensible to the operator who will be able (if he deems it useful) through a man/machine link up to “translate” these expressions hitherto unrecognized by the terminals into a code comprehensible to the server.\",\n \"The server can then take these expressions into account together with their translation so as to generate information for correcting the language model and/or the set of phonetic units.\",\n \"It will be noted that correction is understood here as: modification of the model; and/or supplementing the model.\",\n \"The server then sends the correcting information to each of the terminals which can update its language model and/or set of phonetic units which are enriched with numerous expressions unrecognized by itself or by other terminals.\",\n \"Thus, the voice recognition of each of the terminals is improved by benefitting from the experience shared by all the terminals.\",\n \"According to a particular mode of the invention, the analysis is not performed by an operator but by the server itself which may have much more considerable resources at its disposal than a straightforward terminal.\",\n \"According to particular embodiments, the terminals send the server context data (for example the time, the date, a control performed manually or vocally after the failure of a voice command, the location, the type of terminal, etc.)\",\n \"together with the data representative of the signal corresponding to the unrecognized expressions.\",\n \"This may facilitate the analysis work of the operator and/or of the server.\",\n \"A general schematic of a system comprising a voice-controlled box, in which the technique of the invention may be implemented, is depicted in conjunction with FIG.\",\n \"1.This system comprises in particular: a remote server 116 controlled by a human operator 122; and a plurality of user systems 114, 117 and 118.The remote server 116 is linked to each of the user systems 114, 117 and 118 via communication downlinks 115, 119 and 120 respectively.\",\n \"These links may be permanent or temporary and be of any type well known to the person skilled in the art.\",\n \"They may in particular be of broadcasting type and be based on RF, satellite or wire channels used by television or any other type such as, for example, an Internet type link.\",\n \"FIG.\",\n \"1 describes in particular the user system 114 which is linked via a communication uplink 121 to the server 116.This link can likewise be of any type well known to the person skilled in the art (in particular telephone, Internet, etc).\",\n \"The user system 114 comprises in particular: a voice source 100 which may in particular consist of a microphone intended to pick up a voice signal produced by a speaker; a voice recognition box 102; a control box 105 intended to drive an appliance 107; a controlled appliance 107, for example of television, video recorder or mobile communication terminal type; a unit 109 for storing the expressions detected as unrecognized; an interface 112 allowing upward and downward communications to the server 116.The source 100 is linked to the voice recognition box 102, via a link 101 which allows it to transmit an analogue source wave representative of a voice signal to the box 102.The box 102 can retrieve context information 104 (such as for example the type of appliance 107 which can be controlled by the control box 105 or the list of control codes) via a link 104 and send commands to the control box 105 via a link 103.The control box 105 sends commands via a link 106, for example infrared, to the appliance 107, as a function of the information which it recognizes according to its language model and its dictionary.\",\n \"The control box 105 detects the expressions which it does not recognize, and, instead of simply rejecting them, by sending a non-recognition signal, it performs a recording of these expressions to the storage unit 109 via a link 108.The unit 109 for storing the unrecognized expressions sends representative data to the interface 112 via a link 111, which relay them to the server 116 via the link 121.After correct transmission, the interface 110 can send a signal 110 to the storage unit 109 which can then erase the transmitted data.\",\n \"The control box 105 receives, moreover, correcting data from the interface 112 via a link 113, that the interface 112 has itself received from the remote server via the link 115.These correcting data are taken into account by the control box 105 for the updating of language models and/or of sets of phonetic units.\",\n \"According to the embodiment considered the source 100, the voice recognition box 102, the control box 105, the storage unit 109 and the interlace 112 form part of one and the same device and thus the links 101, 103, 104, 108, 111, 110 and 113 are links internal to the device.\",\n \"The link 106 is typically a wireless link.\",\n \"According to a first variant embodiment of the invention described in FIG.\",\n \"1, the elements 100, 102, 105, 109 and 112 are partly or completely separate and do not form part of one and the same device.\",\n \"In this case, the links 101, 103, 104, 108, 111, 110 and 113 are external wire or other links.\",\n \"According to a second variant, the source 100, the boxes 102 and 105, the storage unit 109 and the interface 112 as well as the appliance 107 form part of one and the same device and are interlinked by internal buses (links 101, 103, 104, 108, 111, 110, 113 and 106).\",\n \"This variant is particularly beneficial when the device is, for example, a mobile telephone or a portable communication terminal.\",\n \"FIG.\",\n \"2 depicts a schematic of a voice-controlled box such as the box 102 illustrated with regard to FIG.\",\n \"2.It is noted that the box 102 receives from outside the analogue source wave 101 which is processed by an Acoustico-Phonetic Decoder 200 or APD (also called the “front end”).\",\n \"The APD 200 samples the source wave 101 at regular intervals (typically every 10 ms) so as to produce real vectors or vectors belonging to code books, typically representing oral resonances that are sent via a link 201 to a recognition engine 203.The APD is for example based on a PLP (standing for “Perceptual Linear Prediction”) described in particular in the article “Perceptual Linear Prediction (PLP) analysis of speech” written by Hynek Hermansky and published in “Journal of the Acoustical Society of America”, Vol.\",\n \"97, No4, 1990 on pages 1738-1752.With the aid of a dictionary 202, the recognition engine 203 analyses the real vectors that it receives, using in particular hidden Markov models or HMMs and language models (which represent the probability of one word following another word).\",\n \"Recognition engines are in particular described in detail in the book “Statistical Methods for Speech Recognition” written by Frederick Jelinek, and published by MIT Press in 1997.The language model allows the recognition engine 203 (which may use in particular hidden Markov networks) to determine which words may follow a given word of any expression usable by the speaker in a given application, and to give the associated probability.\",\n \"The words in question belong to the vocabulary of the application, which may be, independently of the language model, of small size (typically from 10 to 300 words), or of large size (for example of size greater than 300 000 words).\",\n \"Patent application PCT/FR00/03329 dated 29 Nov. 1999 filed in the name of Thomson Multimedia describes a language model comprising a plurality of syntactic blocks.\",\n \"The use of the invention which is the subject of the present patent application is particularly advantageous in conjunction with this type of modular language model, since the modules may be updated separately, thereby avoiding the downloading of overly large volume files.\",\n \"The language models are transmitted by a language model loading module 207.The module 207 itself receives language models, updates or corrections of language models and/or of phonetic units transmitted from the server via the link 113.It is noted that the dictionary 202 belongs to the language model making reference to words from the dictionary.\",\n \"Thus, the dictionary 202 itself can be updated and/or corrected via a language model loaded by the module 207.After implementation of a recognition operation based on the use of a Viterbi algorithm, the recognition engine 203 supplies the rejection module 211 with an ordered list of sequences of words in accordance with the language model, which exhibits the best score for the expression uttered.\",\n \"The rejection module 211 works downstream of the recognition engine 203 and operates according to one or more of the following principles: Sometimes, for reasons specific to the Viterbi algorithm, the latter may not produce a consistent list because the scores are so low that the limit of acceptable accuracies of the machine in terms of arithmetic computation is overstepped.\",\n \"There is therefore no consistent complete proposal.\",\n \"Thus, when the rejection module 211 detects one or more scores below a predetermined acceptable limit, the expression is rejected.\",\n \"Each element of the list calculated by the Viterbi algorithm has been retained because the associated score was among the highest relative scores of all the possible expressions, according to the language model.\",\n \"Additionally, the Markov network associated with each of these expressions makes it possible to evaluate the intrinsic probability of the network in question producing the expression associated with the score observed.\",\n \"The rejection module 211 analyses this probability and if it is less than a predetermined threshold of acceptability of probability, the expression is rejected.\",\n \"According to another method, for the best proposals obtained via the Viterbi algorithm, the rejection module 211 performs a complementary processing of the expressions, using criteria which had not been taken into account in the course of the Viterbi development.\",\n \"For example, it checks that those parts of the signal that have to be voiced because they are associated with vowels, are actually so.\",\n \"If the expressions proposed do not fulfil these conditions, they are rejected.\",\n \"When the rejection module 211 rejects an expression, as illustrated previously, the expression is said to be unrecognized and a signal indicating the rejected expression is sent to the recognition engine 203.In parallel, the rejection module transmits a recording of the unrecognized expression to the storage unit 109 via the link 108.The recognition engine 203 is responsible for recognizing expressions emanating from the APD 200 in the form of phonetic samples.\",\n \"Thus, the recognition engine 203 uses: the phonetic units to construct the phonetic representation of a word in the form of a Markov model, each word of the dictionary 202 possibly possessing several “phonetizations”; and simultaneously the language model in the strict sense for recognizing expressions of greater or lesser complexity.\",\n \"The recognition engine 203 supplies expressions which have been recognized (that is to say not rejected by the module 211) and which it has identified on the basis of the vectors received to a means 205 for translating these expressions into commands that can be understood by the appliance 107.This means 205 uses an artificial intelligence translation process which itself takes into account a context 104 supplied by the control box 105 before sending one or more commands 103 to the control box 105.FIG.\",\n \"3 diagrammatically illustrates a voice recognition module or device 102 such as illustrated in conjunction with FIG.\",\n \"1, and implementing the schematic of FIG.\",\n \"2.The box 102 comprises interlinked by an address and data bus: a voice interface 301; an analogue-digital converter 302; a processor 304; a nonvolatile memory 305; a random access memory 306; a reception module 312; a transmission module 313; and an inputs/outputs interface 307.Each of the elements illustrated in FIG.\",\n \"3 is well known to the person skilled in the art.\",\n \"These commonplace elements are not described here.\",\n \"It is observed moreover that the word “register” used throughout the description designates in each of the memories mentioned, both a memory area of small capacity (a few data bits) and a memory area of large capacity (making it possible to store an entire program or the whole of a sequence of transaction data).\",\n \"The nonvolatile memory 305 (ROM) holds in particular the program for operating the processor 304 in a “prog” register 308.The random access memory 306 holds data, variables and intermediate results of processing in registers which for convenience possess the same names as the data that they hold and comprise in particular: a register 309 in which are held recordings of unrecognized expressions, “Exp_Not_Rec; a counter 310 of unrecognized sentences Nb_Exp_Not _Rec; and a language model in a register 311, Model_Language.\",\n \"It is noted furthermore that the receive module 312 and send module 313 are modules that allow the transmission of data respectively from or to the remote server 116.The wire-based or wireless techniques for receiving and for sending are well known to the person skilled in the art of telecommunications and will not be detailed further.\",\n \"FIG.\",\n \"4 depicts the server 116 of the system illustrated with regard to FIG.\",\n \"1.It is noted that the server 116 is controlled by a human operator 122 via any man/machine interface 404 (for example, of keyboard and screen type).\",\n \"The server 116 itself comprises in particular: a receiver 400; an analyser 401; a module 402 for construction of correction of a language model and/or a set of phonetic units; and a sender 403.The receiver 400 is compatible with the sender 313 of a terminal and can receive from each terminal in particular data (for example recording) representative of unrecognized expressions via the link 121 and possibly of complementary data (for example contextual data).\",\n \"The analyser 401 receives the set of data from the receiver 400 via a link 121 which it transmits to the operator 122 via an interface 404 which is for example a terminal furnished: with a screen and with a keyboard allowing dialogue with the server 116 and its control; with loudspeakers or an audio headset for listening to unrecognized recordings.\",\n \"This interface 404 also allows the analyser 401 to receive information from the operator 122 indicating: that an unrecognized expression not covered by the language model remains incomprehensible, has no meaning within the application to the terminal and/or does not relate to the terminal (it should therefore not be included in the language model), this expression then being ignored for the correcting of the language model and discarded by the analyser 401; that an unrecognized expression nevertheless belongs to the language model in the strict sense (this then involves a pure recognition problem); in this case, this involves modifying the phonetic units and not the language model in the strict sense; or a translation into for example the form of a control code, after identification of the content of an expression by the operator, the unrecognized expression not belonging to the language model and having a meaning for the terminal for which it was intended; this then involves correcting the language model in the strict sense.\",\n \"A combination of the second and third solutions is possible; in this case, this involves both modifying the phonetic units and the language model in the strict sense.\",\n \"This embodiment corresponds to a manual processing of the unrecognized expressions.\",\n \"According to this embodiment, the human operator 122 listens to the unrecognized expression and analyses the reasons for its rejection.\",\n \"The operator 122 determines in particular whether the expression belongs to the language model or not.\",\n \"In the case where the expression belongs to the language model, the operator analyses the expression to identify the intrinsic recognition problem (expression belonging to the language model and which ought to have been recognized and which was not for other reasons: noise, accent of the speaker, etc.).\",\n \"According to a first variant, the processing is automatic and the intervention of a human operator becomes nil.\",\n \"In this case, the server 116 and in particular the analyser 401 possess a relatively considerable computational power which may in particular be much larger than a terminal.\",\n \"According to this variant, the analyser 401 analyses each unrecognized expression in a more appropriate manner than could be done by a terminal, using, for example, a richer language model and/or more complex phonetic models.\",\n \"Not being subject to such strict real-time computational requirements as might be a terminal (which often requires a fast reaction time to a speaker command), the analyser 401 can also, for example, allow recognition demanding a longer processing time than in a terminal.\",\n \"According to a second variant, the processing is semi-automatic and the intervention of a human operator remains limited to the cases that cannot be solved by the analyser.\",\n \"The general structure of a server 116 is according to the preferred embodiment described here similar to that of a terminal such as described with regard to FIG.\",\n \"3 and comprises in particular interlinked by an address and data bus: a processor; a random access memory; a nonvolatile memory; a suitable transmission module; a reception module; and a man/machine linkup interface.\",\n \"According to FIG.\",\n \"5 representing a flow chart of the testing of an expression and of the recording of data relating to unrecognized expressions, as implemented by the recognition engine 203 of FIG.\",\n \"2, in the course of a first step of initialization 500, the microprocessor 304 commences the execution of the program 308 and initializes the variables of the random access memory 306.Then, in the course of a step 501 of waiting for an expression, it waits for and receives an expression sent by a speaker.\",\n \"Next, in the course of a test 502, after having executed an operation of voice recognition for the expression received, it determines whether the expression has or has not been recognized according to one or more criteria illustrated with regard to the description of the rejection module 211 of FIG.\",\n \"2.If it has, in the course of a control step 504, the terminal 102 takes into account the result of the voice recognition applied to the expression received and executes an appropriate action such as for example a command.\",\n \"If it has not, in the course of a step 503 of recording an expression, the unrecognized expression is compressed and recorded in the storage unit 109 awaiting a transmission to the remote server 116 as illustrated with regard to FIG.\",\n \"6.On completion of one of the steps 503 or 504, step 501 of waiting for an expression is repeated.\",\n \"FIG.\",\n \"6 representing a flow chart of the sending of data relating to unrecognized expressions, as implemented by the rejection module of FIG.\",\n \"2, in the course of a first step of initialization 600, the microprocessor 304 commences the execution of the program 308 and initializes the variables of the random access memory 306.Then, in the course of a step 601 of waiting for expressions unrecognized by the voice recognition module 102, the microprocessor 304 waits for and then receives recordings of unrecognized expressions.\",\n \"Then, in the course of a step 602, the terminal 114 connects up to the remote server 116 according to methods well known to the person skilled in the art of telecommunications.\",\n \"Next, in the course of a step 603, the recordings of unrecognized expressions are shaped and sent to the remote server 116.Then, in the course of a step 604 of disconnection, the terminal disconnects from the remote server 116 and a signal is sent between the interface 112 with the remote server and the unit 109 for storing the data corresponding to the unrecognized expressions indicating the transmission of the recordings of expressions.\",\n \"The data corresponding to these expressions are then erased from the storage unit 109.Next, step 601 is repeated.\",\n \"FIG.\",\n \"7 represents a flow chart of the receiving of correcting data, as implemented by the module 207 for loading the language models of FIG.\",\n \"2.After a first step 700 of initialization, in the course of a step) 701, the terminal places itself on standby awaiting correcting data broadcast by a server 1116 to a plurality of terminals.\",\n \"Next, during a step 702, the terminal takes into account the correcting data so as to update the language model and/or its set of phonetic units used by the voice recognition module.\",\n \"According to the nature of the correcting data, these data will be able in particular: to substitute for existing data in the language model and/or its set of phonetic units; to modify existing data; to supplement existing data; and/or to bring about the deletion of existing data.\",\n \"After the execution of step 702, step 703 is repeated.\",\n \"FIG.\",\n \"8 represents a flow chart of the receiving and processing of correcting data, as implemented within the remote server of FIG.\",\n \"4.After a first step 800 of initialization of the parameters and of instigation of a program for management of the server, the server 116 places itself on standby waiting for a connection request originating from a terminal (which executes a step 602 illustrated with regard to FIG.\",\n \"6) and establishes a connection with the terminal according to methods well known to the person skilled in the art of telecommunications.\",\n \"Then, in the course of a step 802, the server 116 receives data originating from the connected terminal which executes a step 603 described previously.\",\n \"These data contain in particular recordings of one or more expressions rejected by the terminal because they were not recognized by a voice recognition module implemented in the terminal.\",\n \"When all the data have been received, the connection between the terminal and the server 116 is broken.\",\n \"Next, in the course of a step 803 of processing the data received, the server 116 processes each of the received recordings of expressions either manually by the operator 122 or automatically or semi-automatically according to various alternatives illustrated with regard to FIG.\",\n \"4.Then, in the course of a test 804, the server 116 determines whether in particular one or more expressions received were comprehensible and are relevant in respect of the terminal which transmitted this or these expressions.\",\n \"An updating of the language models and/or of the phonetic units is then necessary.\",\n \"If not, then the waiting step 801 is repeated.\",\n \"In the converse case, the server 116 constructs a correction of the language model which may take several forms allowing a step 607 (illustrated previously) within the terminals after receipt of the correcting data.\",\n \"These correcting data comprise in particular: an indicator specifying the nature of the correction (in particular substitution, modification, supplement, or deletion); and the correcting data themselves as a function of the indicator.\",\n \"It is noted that if the language model comprises a plurality of syntactic blocks (case in particular of the language models such as described in patent PCT/FR00/03329 mentioned above), each module can be corrected separately.\",\n \"In this case, the correction data also comprise an indicator of the module or modules to be corrected.\",\n \"Then, in the course of a step 806, the server 116 broadcasts the correcting data to one or preferably to a set of terminals which will be able to update their language model and/or set of phonetic units according to a step 607.Step 801 is then repeated.\",\n \"The procedure is thus iterative and may be repeated several times.\",\n \"It also allows the application to be upgraded by adding new queries.\",\n \"Of course, the invention is not limited to the exemplary embodiments mentioned hereinabove.\",\n \"In particular, the person skilled in the art will be able to alter the definition of the terminals implementing the invention, the invention relating to any type of device and/or module using or capable of using a voice recognition process (being of the type for example of multimedia terminal, television, video recorder, a multimedia digital decoder (or set top box), audio or video equipment, fixed or mobile terminal, etc.).\",\n \"Likewise, the invention relates to any type of remote server (for example Internet servers, equipment coupled to television program broadcasters, equipment coupled to mobile communication networks, service provider equipment, etc.).\",\n \"Furthermore, according to the invention, the transmission channel for the data corresponding to the unrecognized sentences and the transmission channel for the data for correcting the language models and/or phonetic units are any whatsoever and include in particular: RF transmission pathways; satellite transmission pathways; the channels of television broadcasting networks; the channels of Internet type networks: the channels of telephone networks; the channels of mobile networks; and removable media.\",\n \"Moreover, it is noted that the invention relates not only to unrecognized sentences but relates to any type of vocal expression such as for example one or more sentences, an isolated or unisolated word, a phrase, a voice code allowing dialogue between a machine and its user.\",\n \"These oral expressions may be associated not only with commands, but with any type of data that can form the subject of a dialogue between a machine and its user, as, for example, information data that the user can transmit to the machine, configuration data, programming data etc.\",\n \"It is also noted that the method of updating the language models which is described by the patent applies not only to processes of voice recognition in the strict sense, but applies also to processes for recognition of textual inputs supporting orthographic mistakes and/or typing mistakes, and also based on Markovian models or language models in the strict sense, as described in the patent.\",\n \"It will be noted that the invention is not limited to a purely hardware installation but that it can also be implemented in the form of a sequence of instructions of a computer program or any form mixing a hardware part and a software part.\",\n \"In the case where the invention is installed partially or totally in software form, the corresponding sequence of instructions may be stored in a removable storage means (such as for example a diskette, a CD-ROM or a DVD-ROM) or otherwise, this means of storage being readable partially or totally by a computer or a microprocessor.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10467586"}}},{"rowIdx":376,"cells":{"text":{"kind":"list like","value":[["Tissue transglutaminase","The present invention relates to tissue transglutaminase (tTG), and more particularly to DNA coding for and polypeptides containing, the primary structural conformaion of one or more epitopes of tTG and to the use thereof in the detection of autoantibodies and/or lymphocytes produced in response to tTG."],["1.A DNA sequence encoding: a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes,: which DNA sequence comprises: (a) a DNA sequence as depicted in any of Seq.","Ids 1, 3, 5, 7, 9, 11, 13, 15, 17, 19; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of Seq.","Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, or one or more active fragments of amino acids as depicted in one or more of Seq.","Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes.","2.A DNA sequence encoding: a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes; which DNA sequence comprises: (a) a DNA sequence as depicted in any of Seq.","Ids 1, 3, 5, 7 and 9; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 1 to 89 of tTG, or one or more active fragments of amino acid numbers 1 to 89 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of Seq.","Ids 2, 4, 6, 8 and 10, or one or more active fragments of amino acids as depicted in one or more of Seq.","Ids 2 4, 6, 8 and 10, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes.","3.A DNA sequence encoding: a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes; which DNA sequence comprises: (a) a DNA sequence as depicted in any of Seq.","Ids 11, 13, 15, 17 or 19; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of Seq.","Ids 12, 14, 16 and 18, or one or more active fragments of amino acids as depicted in one or more of Seq.","Ids 12, 14, 16 and 18, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes.","4.A DNA sequence encoding: a polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies; which DNA sequence comprises: (a) a DNA sequence as depicted in any of Seq.","Ids 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of Seq.","Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, or one or more active fragments of amino acids as depicted in one or more of Seq.","Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies.","5.A DNA sequence encoding: a polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies; which DNA sequence comprises: (a) a DNA sequence as depicted in any of Seq.","Ids 1, 3, 5, 7 or 9; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 1 to 89 of tTG, or one or more active fragments of amino acid numbers 1 to 89 of tTG, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of Seq.","Ids 2, 4, 6, 8 and 10, or one or more active fragments of amino acids as depicted in one or more of Seq.","Ids 2, 4, 6, 8 and 10, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies.","6.A DNA sequence encoding: a polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies; which DNA sequence comprises: (a) a DNA sequence as depicted in any of Seq.","Ids 11, 13, 15, 17 or 19; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of Seq.","Ids 12, 14, 16, 18 and 20, or one or more active fragments of amino acids as depicted in one or more of FIGS.","18, 20, 22, 24 and 26, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies.","7.A DNA sequence as depicted in any of Seq.","Ids 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19, or a DNA sequence differing therefrom due to the degeneracy of the genetic code.","8.A biologically functional plasmid or DNA vector including a DNA sequence as defined in claim 1.9.A host cell which is transformed or transfected with a DNA sequence as defined in claim 1.10.A polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG.","11.A polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, or one or more active fragments of amino acid numbers 1 to 89 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG.","12.A polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, which polypeptide comprises the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes; with the exception of human, guinea pig, mouse, bovine or rat full length tTG.","13.A polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies; with the exception of human, guinea pig, mouse, bovine or rat full length tTG.","14.A polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, or one or more active fragments of amino acid numbers 1 to 89 of tTG, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG.","15.A polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, which polypeptide comprises the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies; with the exception of human, guinea pig, mouse, bovine or rat full length tTG.","16.A polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact,under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of Seq.","Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, or one or more active fragments of amino acids as depicted in one or more of Seq.","Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG.","17.A polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of Seq.","Ids 2, 4, 6, 8 and 10, or one or more active fragments of amino acids as depicted in one or more of Seq.","Ids 2, 4, 6, 8 and 10, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG.","18.A polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of Seq.","Ids 12, 14, 16, 18 and 20, or one or more active fragments of amino acids as depicted in one or more of Seq.","Ids 12, 14, 16, 18 and 20, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG.","19.A polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of Seq.","Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, or one or more active fragments of amino acids as depicted in one or more of Seq.","Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG.","20.A polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of Seq.","Ids 2, 4, 6, 8 and 10, or one or more active fragments of amino acids as depicted in one or more of Seq.","Ids 2, 4, 6, 8 and 10, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG.","21.A polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of Seq.","Ids 12, 14, 16, 18 and 20, or one or more active fragments of amino acids as depicted in one or more of Seq.","Ids 12, 14, 16, 18 and 20, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG.","22.A polypeptide as depicted in any of Seq.","Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20.23.A process of preparing a polypeptide as defined in claim 10, which process comprises: (i) providing a host cell which is transformed or transfected with a DNA sequence or a biologically functional plasmid or DNA vector; (ii) growing the host cell; and (iii) recovering a polypeptide as defined in claim 10.24.A method of screening for autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said method comprising: (a) providing either (i) said sample of body fluid from said subject or (ii) lymphocytes isolated from said sample; (b) contacting said sample or isolated lymphocytes with a polypeptide as defined in claim 10, under conditions that allow interaction of tTG with autoantibodies or lymphocytes produced in response to tTG, so as to permit said polypeptide to interact with autoantibodies, or lymphocytes, produced in response to tTG, and present in, or isolated from, said sample; and (c) monitoring the degree, or effect, of interaction of said polypeptide with either said autoantibodies, or said lymphocytes, produced in response to tTG and present in, or isolated from, said sample, thereby providing an indication of the presence of said autoantibodies, or said lymphocytes, in said sample, or isolated from said sample.","25.A method according to claim 24, for screening for autoantibodies produced in response to tTG and present in said sample.","26.A method of screening for autoantibodies produced in response to tTG in a sample of body fluid obtained from a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said method comprising: (a) providing said sample of body fluid from said subject; (b) contacting said sample with a polypeptide as defined in claim 10, under conditions that allow interaction of tTG with autoantibodies produced in response to tTG, so as to permit said polypeptide to interact with autoantibodies produced in response to tTG and present in said sample; and (c) monitoring the degree of interaction of said polypeptide with said autoantibodies produced in response to tTG and present in said sample, thereby providing an indication of the presence of said autoantibodies in said sample.","27.A method according to claim 24, which comprises directly monitoring interaction of (i) autoantibodies to tTG present in the sample of body fluid from the subject and (ii) a polypeptide as defined in any of claims 10 to 22.28.A method according to claim 24, which further comprises providing at least one competitor, whereby in step (b) a polypeptide as defined in any of claims 10 to 22, can interact with either the competitor or autoantibodies to tTG and present in said sample.","29.A method of screening for autoantibodies to tTG in a sample of body fluid obtained from a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said method comprising: (a) providing said sample of body fluid from said subject; (b) contacting said sample with (i) full length tTG, and (ii) at least one competitor capable of competing with autoantibodies to tTG in the interaction thereof with a polypeptide as defined in claim 10, under conditions that allow interaction of tTG with autoantibodies to tTG, so as to permit said full length tTG to interact with either autoantibodies to tTG present in said sample, or said competitor; and (c) monitoring the interaction of said full length tTG with said autoantibodies present in said sample, thereby providing an indication of the presence of said autoantibodies to tTG in said sample.","30.A kit for screening for autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said kit comprising: (a) a polypeptide as defined in claim 10; (b) means for contacting either (i) a sample of body fluid obtained from said subject, or (ii) lymphocytes isolated from a sample of body fluid obtained from said subject, with said polypeptide as defined in claims 10, under conditions that allow interaction of tTG with autoantibodies or lymphocytes produced in response to tTG, so as to permit said polypeptide to interact with autoantibodies, or lymphocytes, produced in response to tTG, and present in, or isolated from, said sample; and (c) means for monitoring the degree, or effect, of interaction of said polypeptide with either said autoantibodies, or said lymphocytes, produced in response to tTG and present in, or isolated from, said sample, thereby providing an indication of the presence of said autoantibodies, or lymphocytes, in said sample, or isolated from said sample.","31.A kit according to claim 30, for screening for autoantibodies produced in response to tTG in said sample of body fluid.","32.A kit for screening for autoantibodies produced in response to tTG in a sample of body fluid obtained from a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said kit comprising: (a) a polypeptide as defined in claim 10; (b) means for contacting a sample of body fluid obtained from said subject with said polypeptide as defined in claim 10, under conditions that allow interaction of tTG with autoantibodies produced in response to tTG, so as to permit said polypeptide to interact with autoantibodies produced in response to tTG and present in said sample; and (c) means for monitoring the degree of interaction of said polypeptide with said autoantibodies produced in response to tTG and present in said sample, thereby providing an indication of the presence of said autoantibodies in said sample.","33.A kit according to claim 30, which comprises means for directly monitoring interaction of (i) autoantibodies to tTG present in the sample of body fluid from the subject and (ii) a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG.","34.A kit according to of claim 30 to 32, which further comprises at least one competitor, whereby a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG, can interact with either the competitor or autoantibodies to tTG present in said sample of body fluid being screened.","35.A kit for screening for autoantibodies to tTG in a sample of body fluid obtained from a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said kit comprising: (a) full length tTG; (b) at least one competitor capable of competing with autoantibodies to tTG in the interaction thereof with a polypeptide as defined in claim 10; (c) means for contacting said sample of body fluid from said subject, said full length tTG and said competitor, under conditions that allow interaction of tTG with autoantibodies to tTG, so as to permit said full length tTG to interact with either autoantibodies to tTG present in said sample, or said competitor; and (d) means for monitoring the interaction of said full length tTG with said autoantibodies present in said sample, thereby providing an indication of the presence of said autoantibodies to tTG in said sample.","36.An antibody produced in response to one or more epitope regions of tTG, which epitope region comprises part or all of the primary structural conformation of amino acids 1 to 89 of tTG, or the primary structural conformation of amino acids 401 to 494 or 401 to 491 of tTG, or one or more fragments of an antibody produced in response to one or more epitope regions of tTG, which epitope region comprises part or all of the primary structural conformation of amino acids 1 to 89 of tTG, or the primary structural conformation of amino acids 401 to 494 or 401 to 491 of tTG.","37.An antibody according to claim 36, which is obtained by the Examples.","38.An antibody according to claim 36, which is monoclonal, recombinant or polyclonal.","39.An antibody according to claim 38, which is monoclonal.","40.A host cell containing an antibody as defined in claims 36.41.A hybridoma capable of secreting a monoclonal antibody as defined in claim 36.42.For use in diagnosis, an antibody as defined in claim 36.43.Use according to claim 42, in the diagnosis of autoimmune disease associated with an immune reaction to tTG.","44.For use in therapy, an antibody as defined in claim 36.45.Use according to claim 44, in the therapeutic treatment of autoimmune disease associated with an immune reaction to tTG.","46.Use according to claim 43, wherein the autoimmune disease is coeliac disease.","47.An antibody according to claim 36, in the manufacture of a medicament for the treatment of coeliac disease.","48.For use in diagnosis, a polypeptide as defined in claim 10.49.Use according to claim 48, in the diagnosis of autoimmune disease associated with an immune reaction to tTG.","50.For use in therapy, a polypeptide as defined in claim 10.51.Use according to claim 50, in the therapeutic treatment of autoimmune disease associated with an immune reaction to tTG.","52.Use according to claim 49, wherein the autoimmune disease is coeliac disease.","53.A pharmaceutical composition comprising a polypeptide as defined in claim 10, together with a pharmaceutically acceptable carrier, diluent or excipient therefor, wherein said polypeptide can interact with autoantibodies and/or lymphocytes produced in response to tTG.","54.A polypeptide as defined in claim 10, for use in the manufacture of a medicament for the treatment of coeliac disease.","55.A method of diagnosing the likely onset or presence of autoimmune disease associated with an immune reaction to tTG in a subject suspected of suffering from, susceptible to, having or recovering from, autoimmune disease associated with an immune reaction to tTG, the method comprising detecting autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid from the subject according to a method as defined in claim 24, and whereby the detected autoantibodies and/or lymphocytes can provide a diagnosis of the likely onset or presence of autoimmune disease associated with an immune reaction to tTG in the subject 56.A method of delaying or preventing the onset of autoimmune disease associated with an immune reaction to tTG in an animal subject suspected of suffering from, susceptible to or recovering from autoimmune disease associated with an immune reaction to tTG, which method comprises initially detecting autoantibodies or lymphocytes indicative of the onset or presence of autoimmune disease associated with an immune reaction to tTG in a sample of body fluid obtained from the subject according to a method as defined in claim 24, thereby providing a diagnosis of the likely onset of autoimmune disease associated with an immune reaction to tTG in the subject, and thereafter therapeutically treating the subject so as to delay the onset and/or prevent autoimmune disease associated with an immune reaction to tTG.","57.A method of monitoring dietary compliance by a subject having, suspected of suffering from, susceptible to or recovering from gluten sensitive enteropathy, which method comprises screening for autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from the subject according to a method as defined in claim 24, and whereby the detection of such autoantibodies and/or lymphocytes produced in response to tTG in the sample of body fluid provides an indication as to the dietary compliance of the subject to a gluten-free or substantially gluten free diet.","58.A method of treating autoimmune disease associated with an immune reaction to tTG in a subject, which method comprises initially detecting autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from the subject according to a method as defined in claim 24, thereby providing a diagnosis of autoimmune disease in the subject, and administering to the subject a therapeutically effective amount of at least one therapeutic agent effective in the treatment of such autoimmune disease.","59.A method of treating autoimmune disease associated with an immune reaction to tTG in a subject, which method comprises administering to the subject a therapeutically effective amount of a therapeutic agent identified as providing a therapeutic effect by interaction with amino acids 1 to 89 of tTG, and/or amino acids 401 to 494 of tTG, or amino acids 401 to 492 of tTG.","60.A therapeutic agent identified as providing a therapeutic effect by interaction with amino acids 1 to 89 of tTG, and/or amino acids 401 to 494 of tTG, or amino acids 401 to 491 of tTG.","61.A therapeutic agent according to claim 60, for use in the therapeutic treatment of an autoimmune disease associated with an immune reaction to tTG.","62.A method of cloning lymphocytes produced in response to tTG, which method comprises: providing a source of lymphocytes; contacting the lymphocytes with a polypeptide as defined in claim 10, so as to effect proliferation of said lymphocytes; and isolating and cloning the proliferating lymphocytes.","63.Use of lymphocytes prepared according to claim 62, in the therapeutic treatment of autoimmune disease associated with an immune reaction to tTG.","64.A pharmaceutical composition comprising lymphocytes prepared according to claim 62, together with a pharmaceutically acceptable carrier, diluent or excipient therefor.","65.Use of lymphocytes prepared according to claim 62, in the manufacture of a medicament for the treatment of autoimmune disease associated with an immune reaction to Ttg.","66.Use of lymphocytes prepared according to claim 62, in the manufacture of a medicament for the treatment of coeliac disease.","67.A biologically functional plasmid or DNA vector including a DNA sequence as defined in any of claim 2.68.A biologically functional plasmid or DNA vector including a DNA sequence as defined in any of claim 3 69.A biologically functional plasmid or DNA vector including a DNA sequence as defined in any of claim 4.70.A biologically functional plasmid or DNA vector including a DNA sequence as defined in any of claim 5.71.A biologically functional plasmid or DNA vector including a DNA sequence as defined in any of claim 6.72.A biologically functional plasmid or DNA vector including a DNA sequence as defined in any of claim 7.73.A host cell which is transformed or transfected with a plasmid or vector as defined in claim 67.74.A host cell which is transformed or transfected with a plasmid or vector as defined in claim 68.75.A host cell which is transformed or transfected with a plasmid or vector as defined in claim 69.76.A host cell which is transformed or transfected with a plasmid or vector as defined in claim 70.77.A host cell which is transformed or transfected with a plasmid or vector as defined in claim 71.78.A host cell which is transformed or transfected with a plasmid or vector as defined in claim 72."],["The present invention relates to tissue transglutaminase (tTG), and more particularly to DNA coding for and polypeptides containing, the primary structural conformation of one or more epitopes of tTG and to the use thereof in the detection of autoantibodies and/or lymphocytes produced in response to tTG.","tTG belongs to the class of transglutaminases (TGs).","The TGs are enzymes catalysing an acyl transfer depending on Ca2+, the γ-carboxamide groups of peptide-bonded glutamine residues acting as acyl donors.","Primarily, protein-bonded lysine residues function as acyl acceptors, so that the transfer results in an ε-(γ-glutamyl)lysine bond.","The substrate specificity of the TGs with respect to the acyl donors is very high (depending on the amino acid sequence), whereas an exceptionally wide spectrum of acceptors is available.","The covalent peptide bonds formed are highly stable and protease-resistant, resulting in an increased resistance of the crosslinked proteins to chemical, enzymatic or physical effects.","There is a widespread occurrence of various TGs in miscellaneous organs, tissues, in plasma and interstitial body fluids, correlating with the occurrence of transglutaminase-modified proteins in blood clots, on cell membranes, in the horny layer of the epidermis, in hair, in nails, and in the extracellular matrix.","The described TGs may be distinguished by their physical properties, their location in the body, and their primary structure.","tTG is also referred to as cellular, erythrocyte, endothelial, cytoplasmatic, liver, or type II TG, and is a monomer having a molecular weight of 75-85 kDa.","The complete amino acid sequence comprising 687 residues has been derived from cDNA.","At the protein level, there is an 84% homology between the human enzyme and the enzyme from mouse macrophages and an 81% homology between the human and guinea pig enzymes.","There are a number of diseases that may be associated with tTG and can include autoimmune diseases and inflammatory diseases.","In particular, autoimmune diseases associated with an immune reaction to tTG can be autoimmune diseases arising due to gluten sensitive enteropathy, such as coeliac disease or sprue.","Coeliac disease is a disease of the small intestine mucosa, the first manifestation predominantly occurring during the late infant and toddler ages.","If the corresponding clinical picture does not occur before the adult age, it is generally termed non-tropical sprue.","Thus, both of these terms describe the same disease, which is hereinafter referred to as coeliac disease.","Coeliac disease is estimated to effect about 1 in 300 individuals in Western countries.","The characteristic intolerance of dietary gluten present in wheat, barley, rye and related products results in an inflammatory disease of the upper small intestine in genetically susceptible individuals.","Typical symptoms include chronic diarrhoea, abdominal distension and failure to thrive.","In some patients symptoms of malabsorption such as anaemia and osteoporosis may manifest.","Other symptoms include inflammatory lesions in the small bowel, for example, villous atrophy, hypertrophic crypts and increased number of inter-epithelial lymphocytes.","Provided the disease is diagnosed in time the symptoms can generally be treated by adhering to a gluten-free diet.","However malabsorption may give rise to severe disease symptoms if the disease is not diagnosed and treated.","Ultimately, there is the increased risk of developing an intestinal lymphoma and also other gastrointestinal neoplasias.","Diagnosis of coeliac disease is currently based on clinical symptoms, histological identification of gluten sensitive enteropathy, and serological tests for observing any response to withdrawal of gluten from an individual's diet.","An example of a serological test used for diagnosis and monitoring of coeliac disease is an immunofluorescence test measuring endomysial antibody and measurement of autoantibodies to tTG.","WO98/03872 discloses that tTG is an autoantigen assocaited with coeliac disease and specifically discloses an ELISA based on guinea pig tTG of about 60% purity.","It would be advantageous to be able to non-invasively efficiently and reliably diagnose and monitor autoimmune diseases associated with tTG, such as coeliac disease as referred to above.","For example, the above described immunofluorescence test measuring endomysial antibody requires special expertise and equipment and is dependent on subjective assessment of fluorescence patterns.","It would, therefore, be advantageous to be able to replace the immunofluorescence test measuring endomysial antibody with a non-invasive sensitive and specific test for measuring autoantibodies to tTG.","It is, therefore, an aim of the present invention to provide an assay system, which alleviates some of the aforementioned problems arising in the diagnosis and monitoring of autoimmune diseases associated with an immune reaction to tTG.","It is a further aim of the present invention to provide an assay for measuring autoantibodies or lymphocytes produced in response to tTG, with improved sensitivity and specificity and also provide diagnostic kits for use in the simple and rapid detection of autoantibodies or lymphocytes produced in response to tTG substantially as referred to above.","A better understanding of tTG, and epitopes thereof, would be beneficial in achieving the above described aims of the present invention.","More particularly, we have now specifically identified that antigenic determinants (or epitopes) of tTG which interact with autoantibodies and/or lymphocytes produced in response to tTG, and present in sera from patients with coeliac disease, are located in the N-terminal and/or central part of the tTG molecule.","In particular, our studies have shown that the N-terminal region (typically amino acids 1 to 89) and/or the central region (typically amino acids 401 to 494 or amino acids 401 to 491) of the tTG molecule are particularly important for interaction with autoantibodies and/or lymphocytes produced in response to tTG and present in sera from coeliac patients.","There is provided by the present invention, therefore, a DNA sequence encoding: a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes); which DNA sequence comprises: (a) a DNA sequence as depicted in any of FIGS.","7, 9, 11, 13, 15, 17, 19, 21, 23 or 25; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of FIGS.","8, 10, 12, 14, 16, 18, 20, 22, 24 and 26, or one or more active fragments of amino acids as depicted in one or more of FIGS.","8, 10, 12, 14, 16, 18, 20, 22, 24 and 26, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes).","There is also provided by the present invention a DNA sequence encoding: a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes); which DNA sequence comprises: (a) a DNA sequence as depicted in any of FIGS.","7, 9, 11, 13 or 15; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 1 to 89 of tTG, or one or more active fragments of amino acid numbers 1 to 89 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of FIGS.","8, 10, 12, 14 and 16, or one or more active fragments of amino acids as depicted in one or more of FIGS.","8, 10, 12, 14 and 16, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes).","There is also provided by the present invention a DNA sequence encoding: a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes); which DNA sequence comprises: (a) a DNA sequence as depicted in any of FIGS.","17, 19, 21, 23 or 25; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of FIGS.","18, 20, 22, 24 and 26, or one or more active fragments of amino acids as depicted in one or more of FIGS.","18, 20, 22, 24 and 26, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes).","There is also provided by the present invention a DNA sequence encoding: a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies); which DNA sequence comprises: (a) a DNA sequence as depicted in any of FIGS.","7, 9, 11, 13, 15, 17, 19, 21, 23 or 25; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of FIGS.","8, 10, 12, 14, 16, 18, 20, 22, 24 and 26, or one or more active fragments of amino acids as depicted in one or more of FIGS.","8, 10, 12, 14, 16, 18, 20, 22, 24 and 26, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies).","There is also provided by the present invention a DNA sequence encoding: a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies); which DNA sequence comprises: (a) a DNA sequence as depicted in any of FIGS.","7, 9, 11, 13 or 15; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 1 to 89 of tTG, or one or more active fragments of amino acid numbers 1 to 89 of tTG, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of FIGS.","8, 10, 12, 14 and 16, or one or more active fragments of amino acids as depicted in one or more of FIGS.","8, 10, 12, 14 and 16, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies).","There is also provided by the present invention a DNA sequence encoding: a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies); which DNA sequence comprises: (a) a DNA sequence as depicted in any of FIGS.","17, 19, 21, 23 or 25; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of FIGS.","18, 20, 22, 24 and 26, or one or more active fragments of amino acids as depicted in one or more of FIGS.","18, 20, 22, 24 and 26, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies).","A DNA sequence as provided by the present invention can be of human origin (as depicted in FIGS.","7 and 77), of guinea pig origin (as depicted in FIGS.","9 and 99), of bovine origin (as depicted in FIGS.","11 and 11), of mouse origin (as depicted in FIGS.","13 and 33), or of rat origin (as depicted in FIGS.","15 and 55).","In particular, the present invention provides a DNA sequence as depicted in any of FIGS.","7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, or a DNA sequence differing therefrom due to the degeneracy of the genetic code.","More particularly, a DNA sequence as provided by the present invention can encode part or all of the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or part or all of the primary structural conformation of amino acid numbers 401 to 494 of tTG or part or all of the primary structural conformation of amino acid numbers 401 to 491 of tTG substantially as hereinafter described in greater detail.","A DNA sequence as provided by the present invention can be included in a biologically functional plasmid or DNA vector for expressing a polypeptide according to the present invention substantially as hereinafter described in greater detail and there is further provided by the present invention a biologically functional plasmid or DNA vector including a DNA sequence substantially as hereinbefore described.","Furthermore, there is still further provided by the present invention a host cell which is transformed or transfected with a DNA sequence substantially as hereinbefore described, or a plasmid or vector substantially as hereinbefore described.","A suitable host cell can be any prokaryote or eukaryote cell capable of replicating and expressing a DNA sequence substantially as hereinbefore described and suitable techniques are well known to one of ordinary skill in the art.","Polypeptides according to the present invention substantially as herein described can be expressed in various systems generating recombinant proteins.","For example, for expression in E coli, cDNA coding for the appropriate polypeptides according to the present invention can be cloned into a vector, such as pMEX8, pGEXcT or pQE-81L His or an equivalent.","In the case of expression in yeast (for example Saccharomyces cerevisiae or Schizosaccharomyces pombe), vectors such as pYES2, pESP2 or pYES2/CT or an equivalent, can be employed.","AcMNPV (Bac-N-Blue) vector or an equivalent can be used for expression in insect cells and pRC/CMV vector or an equivalent can be used for expression in mammalian cells, such as Chinese Hamster Ovary (CHO) cells.","A polypeptide according to the present invention can be expressed as a discrete protein, or as a fusion protein linked to, for example, glutathione S transferase (GST) or poly histidine linker.","For a discrete protein, affinity column chromatography purification using a mouse monoclonal antibody to the relevant part of tTG coupled to a Sepharose particle can be used.","If a tTG fragment is fused to GST, glutathione Sepharose chromatography purification can be used to isolate the fusion protein.","Specific proteases can be used to separate GST from tTG peptide and a second round of glutathione Sepharose chromatography can be used to separate GST from a tTG fragment.","In the case of peptides linked to poly histidine linker, the purification can be carried out using immobilised metal affinity chromatography.","There is also provided by the present invention a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes); with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length Ttg).","There is also provided by the present invention a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, or one or more active fragments of amino acid numbers 1 to 89 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).","There is also provided by the present invention a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), which polypeptide comprises the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes); with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).","There is also provided by the present invention a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies) and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies); with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).","There is also provided by the present invention a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies) and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, or one or more active fragments of amino acid numbers 1 to 89 of tTG, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).","There is also provided by the present invention a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies) and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), which polypeptide comprises the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies); with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).","The present invention further provides a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of FIGS.","8, 10, 12, 14, 16, 18, 20, 22, 24 and 26, or one or more active fragments of amino acids as depicted in one or more of FIGS.","8, 10, 12, 14, 16, 18, 20, 22, 24 and 26, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).","The present invention further provides a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of FIGS.","8, 10, 12, 14 and 16, or one or more active fragments of amino acids as depicted in one or more of FIGS.","8, 10, 12, 14 and 16, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).","The present invention further provides a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of FIGS.","18, 20, 22, 24 and 26, or one or more active fragments of amino acids as depicted in one or more of FIGS.","18, 20, 22, 24 and 26, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).","The present invention further provides a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of FIGS.","8, 10, 12, 14, 16, 18, 20, 22, 24 and 26, or one or more active fragments of amino acids as depicted in one or more of FIGS.","8, 10, 12, 14, 16, 18, 20, 22, 24 and 26, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).","The present invention further provides a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of FIGS.","8, 10, 12, 14 and 16, or one or more active fragments of amino acids as depicted in one or more of FIGS.","8, 10, 12, 14 and 16, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).","The present invention further provides a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of FIGS.","18, 20, 22, 24 and 26, or one or more active fragments of amino acids as depicted in one or more of FIGS.","18, 20, 22, 24 and 26, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).","A polypeptide as provided by the present invention can be of human origin (as depicted in FIGS.","8 and 88), of guinea pig origin (as depicted in FIGS.","10 and 20), of bovine origin (as depicted in FIGS.","12 and 22), of mouse origin (as depicted in FIGS.","14 and 44), or of rat origin (as depicted in FIGS.","16 and 66).","In particular, the present invention provides a polypeptide as depicted in any of FIGS.","8, 10, 12, 14, 16, 18, 20, 22, 24 or 26.Typically, a polypeptide according to the present invention is a synthetic polypeptide derived by recombinant techniques substantially as herein described and is most suitably derived from recombinant human tTG typically with at least 70% purity, more preferably at least 90% purity and even more preferably at least 95% purity.","Purity may be assessed by SDS PAGE analysis and Coomassie Blue staining as hereinafter discussed in connection with FIG.","1.The recombinant tTG (typically human) may be purified using affinity chromatography with tTG antibodies such as mouse tTG monoclonal antibody.","A polypeptide according to the present invention is preferably obtained by, or is obtainable by, expression of a DNA sequence according to the present invention substantially as hereinbefore described.","A polypeptide according to the present invention obtained by such expression can be advantageous in being free from association with other eukaryotic polypeptides or contaminants which might otherwise be associated with tTG in its natural environment.","The present invention further provides a process of preparing a polypeptide substantially as hereinbefore described, which process comprises: (i) providing a host cell substantially as hereinbefore described; (ii) growing the host cell; and (iii) recovering a polypeptide according to the present invention therefrom.","Recovery of a polypeptide according to the present invention can typically employ conventional isolation and purification techniques, such as chromatographic separations or immunological separations, known to one of ordinary skill in the art.","It is of course appreciated that there is provided within the scope of the present invention, one or more amino acid sequences or fragments thereof structurally distinct from the specific amino acid sequences of a polypeptide described herein (for example by the addition, deletion, substitution or insertion of one or more amino acids) but having substantially the same functional binding properties as the specific amino acid sequences of a polypeptide described herein.","In other words, the present invention clearly encompasses within its scope amino acid sequences or fragments thereof substantially homologous to specific amino acid sequences of a polypeptide described herein, or representing variants of specific amino acid sequences of a polypeptide described herein, but having substantially the same functional binding properties as the specific amino acid sequences of a polypeptide described herein.","A polypeptide according to the present invention can in a first preferred embodiment of the present invention be a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), which polypeptide consists essentially of (or consists of) the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or more particularly consists essentially of (or consists of) the primary structural conformation of amino acids as depicted in one or more of FIGS.","8, 10, 12, 14, 16, 18, 20, 22, 24 or 26, substantially as hereinbefore described.","Alternatively, in a second preferred embodiment of the present invention there is provided a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), which polypeptide consists essentially of (or consists of) one or more active fragments of amino acid numbers 1 to 89 of tTG, and/or one or more active fragments of amino acid numbers 401 to 494 of tTG or one or more active fragments of amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), or more particularly consists essentially of (or consists of) the primary structural conformation of one or more active fragments of amino acids as depicted in one or more of FIGS.","8, 10, 12, 14, 16, 18, 20, 22, 24 or 26, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) substantially as hereinbefore described.","For example, a polypeptide consisting essentially of (or consisting of) amino acid numbers 1 to 85 of tTG, amino acid numbers 5 to 89 of tTG, or the like, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes).","A still further alternative in a third preferred embodiment of the present invention is a polypeptide (in particular a synthetic polypeptide, such as a scaffold type polypeptide typically providing amino acids corresponding to the amino acids of tTG as described herein in a form specifically adapted for use in an assay method or kit according to the present invention substantially as hereinafter described in greater detail) with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which includes part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), which polypeptide includes amino acids comprising the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of amino acid numbers 1 to 89 of tTG, and/or one or more active fragments of amino acid numbers 401 to 494 of tTG or one or more active fragments of amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes); or more particularly which includes amino acids comprising the primary structural conformation of amino acids as depicted in one or more of FIGS.","8, 10, 12, 14, 16, 18, 20, 22, 24 or 26, or one or more active fragments of amino acids as depicted in one or more of FIGS.","8, 10, 12, 14, 16, 18, 20, 22, 24 or 26, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).","According to a particularly preferred embodiment of the present invention, there is also provided a method of screening for autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from a subject (in particular a human) suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said method comprising: (a) providing either (i) said sample of body fluid from said subject or (ii) lymphocytes isolated from said sample; (b) contacting said sample or isolated lymphocytes with a polypeptide according to the present invention substantially as hereinbefore described (under conditions that allow interaction of tTG with autoantibodies or lymphocytes produced in response to tTG) so as to permit said polypeptide to interact with autoantibodies, or lymphocytes, produced in response to tTG, and present in, or isolated from, said sample; and (c) monitoring the degree, or effect, of interaction of said polypeptide with either said autoantibodies, or said lymphocytes, produced in response to tTG and present in, or isolated from, said sample, thereby providing an indication of the presence of said autoantibodies, or said lymphocytes, in said sample, or isolated from said sample.","Substantially as described above, a method according to the present invention is suitable for screening for autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from a subject.","A method according to the present invention can, however, be particularly adapted for use in screening for autoantibodies produced in response to tTG in a sample of body fluid obtained from a subject substantially as hereinafter described in greater detail.","There is in particular provided by the present invention, therefore, a method of screening for autoantibodies produced in response to tTG in a sample of body fluid obtained from a subject (in particular a human) suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said method comprising: (a) providing said sample of body fluid from said subject; (b) contacting said sample with a polypeptide according to the present invention substantially as hereinbefore described (under conditions that allow interaction of tTG with autoantibodies produced in response to tTG) so as to permit said polypeptide to interact with autoantibodies produced in response to tTG and present in said sample; and (c) monitoring the degree of interaction of said polypeptide with said autoantibodies produced in response to tTG and present in said sample, thereby providing an indication of the presence of said autoantibodies in said sample.","A method according to the present invention may typically employ a control, such as a sample of body fluid from a normal subject, in other words a subject known to be without autoimmune disease associated with an immune reaction to tTG.","A method of screening for autoantibodies to tTG according to the present invention may comprise directly monitoring interaction of (i) autoantibodies to tTG present in the sample of body fluid from the subject and (ii) a polypeptide, as provided by the present invention substantially as hereinbefore described, typically by employing non-competitive sandwich type assay techniques known in the art.","Typically, in a method according to the present invention employing non-competitive techniques, monitoring of the degree of interaction of (i) autoantibodies to tTG present in the sample and (ii) a polypeptide according to the present invention substantially as hereinbefore described, can comprise providing labelling means either to a polypeptide according to the present invention substantially as hereinbefore described, or to a binding partner for autoantibodies to tTG, either of which technique would enable monitoring of the above described interaction.","For example, a method according to the present invention may comprise directly or indirectly labelling a polypeptide according to the present invention substantially as hereinbefore described; contacting the thus labelled polypeptide with a sample of body fluid being screened for tTG autoantibodies so as to provide a mixture thereof; and adding to the mixture a binding partner for autoantibodies to tTG (such as an anti-IgG reagent) present in the sample of body fluid, so as to cause precipitation of any complexes of labelled polypeptide and tTG autoantibodies present in the mixture.","Alternatively, it may be preferred that a method according to the present invention further comprises adding a labelled binding partner for tTG autoantibodies (such as a labelled anti-IgG reagent, for example protein A or anti-human IgG, or labelled full length tTG or an epitope thereof) to a mixture obtained by contacting (i) a polypeptide according to the present invention substantially as hereinbefore described immobilised to a support and (ii) a sample of body fluid being screened for autoantibodies to tTG.","It may alternatively be preferred that a method of screening for autoantibodies to tTG in the sample of body fluid according to the present invention, utilises the principles employed in known competitive assays.","For example, a method according to the present invention may employ at least one competitor capable of competing with autoantibodies to tTG in the interaction thereof with a polypeptide according to the present invention substantially as hereinbefore described.","Typically, a competitor as employed in a competitive assay method according to the present invention may comprise one or more antibodies, which may be natural or partly or wholly synthetically produced.","A competitor as employed in the present invention may alternatively comprise any other protein having a binding domain or region which is capable of competing with autoantibodies to tTG in the interaction thereof with a polypeptide according to the present invention substantially as hereinbefore described.","Preferably, however, a competitor as employed in the present invention comprises a monoclonal, recombinant or polyclonal antibody (especially a monoclonal antibody), capable of competing with tTG autoantibodies in the interaction thereof with a polypeptide according to the present invention substantially as hereinbefore described.","Typically, therefore, a competitive assay method according to the present invention may further comprise providing at least one competitor, such as a monoclonal or polyclonal antibody, whereby in step (b) of a method as herein described a polypeptide according to the present invention substantially as hereinbefore described can interact with either a competitor, such as a monoclonal or polyclonal antibody, or autoantibodies to tTG present in said sample.","Typically monitoring in a competitive assay method according to the present invention comprises comparing: (i) interaction of a polypeptide according to the present invention substantially as hereinbefore described and one or more competitors substantially as hereinbefore described (typically a monoclonal or polyclonal antibody), in the absence of said sample of body fluid being screened (that is a suspected disease sample), optionally in the presence of a sample of body fluid from a normal subject, typically a subject known to be without autoimmune disease associated with an immune reaction to tTG; with (ii) interaction of a polypeptide according to the present invention substantially as hereinbefore described and one or more competitors substantially as hereinbefore described (typically a monoclonal or polyclonal antibody), in the presence of said sample of body fluid being screened.","Typically, the comparison involves observing a decrease in interaction of a polypeptide according to the present invention substantially as hereinbefore described and the competitor in (ii) compared to (i) so as to provide an indication of the presence of autoantibodies to tTG in said sample.","Typically, the decrease in interaction can be observed by directly or indirectly labelling the competitor and monitoring any change in the interaction of the thus labelled competitor with a polypeptide according to the present invention substantially as hereinbefore described in the absence and in the presence of a sample of body fluid being screened for autoantibodies to tTG.","Suitably a polypeptide according to the present invention substantially as hereinbefore described may be immobilised to facilitate the above mentioned monitoring.","Alternatively, there is also provided by the present invention a method of screening for autoantibodies to tTG in a sample of body fluid obtained from a subject (in particular a human) suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said method comprising: (a) providing said sample of body fluid from said subject; (b) contacting said sample with (i) full length tTG (typically recombinantly obtained full length tTG), and (ii) at least one competitor capable of competing with autoantibodies to tTG in the interaction thereof with a polypeptide according to the present invention substantially as hereinbefore described, (under conditions that allow interaction of tTG with autoantibodies to tTG), so as to permit said full length tTG to interact with either autoantibodies to tTG present in said sample, or said competitor; and (c) monitoring the interaction of said full length tTG with said autoantibodies present in said sample, thereby providing an indication of the presence of said autoantibodies to tTG in said sample.","The full length tTG can typically be of human, guinea pig, bovine, mouse or rat origin, preferably human, and more preferably human recombinantly obtained full length tTG.","A competitor for use in such an assay typically comprises a monoclonal or polyclonal antibody (preferably monoclonal) substantially as hereinbefore described.","Suitably a detectable label that can be employed in a method according to the present invention can be selected from the group consisting of enzymic labels, isotopic labels, chemiluminescent labels, fluorescent labels, dyes and the like.","In the case where an isotopic label (such as 125I, 14C, 3H or 35S) is employed, monitoring may therefore comprise measuring radioactivity dependent on binding of a polypeptide according to the present invention substantially as hereinbefore described.","Radioactivity is generally measured using a gamma counter, or liquid scintillation counter.","In the case of a method of screening for lymphocytes according to the present invention, it is generally preferred that lymphocytes are initially isolated from a sample of body fluid from a subject using techniques well known to one of ordinary skill in the art, followed by contact with a polypeptide according to the present invention so as to stimulate the proliferation of the isolated lymphocytes.","Monitoring of the effect of interaction of a polypeptide according to the present invention and such proliferating lymphocytes, typically employs means known in the art for monitoring such proliferation of lymphocytes.","According to a further particularly preferred embodiment of the present invention, there is provided a kit for screening for autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from a subject (in particular a human) suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said kit comprising: (a) a polypeptide according to the present invention substantially as hereinbefore described; (b) means for contacting either (i) a sample of body fluid obtained from said subject, or (ii) lymphocytes isolated from a sample of body fluid obtained from said subject, with said polypeptide according to the present invention substantially as hereinbefore described (under conditions that allow interaction of tTG with autoantibodies or lymphocytes produced in response to tTG) so as to permit said polypeptide to interact with autoantibodies, or lymphocytes, produced in response to tTG, and present in, or isolated from, said sample; and (c) means for monitoring the degree, or effect, of interaction of said polypeptide with either said autoantibodies, or said lymphocytes, produced in response to tTG and present in, or isolated from, said sample, thereby providing an indication of the presence of said autoantibodies, or lymphocytes, in said sample or isolated from said sample.","Substantially as described above, a kit according to the present invention is suitable for screening for autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from a subject.","A kit according to the present invention can, however, be particularly adapted for use in screening for autoantibodies produced in response to tTG in a sample of body fluid obtained from a subject substantially as hereinafter described in greater detail.","There is in particular provided by the present invention, therefore, a kit for screening for autoantibodies produced in response to tTG in a sample of body fluid obtained from a subject (in particular a human) suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said kit comprising: (a) a polypeptide according to the present invention substantially as hereinbefore described; (b) means for contacting a sample of body fluid obtained from said subject with said polypeptide according to the present invention substantially as hereinbefore described (under conditions that allow interaction of tTG with autoantibodies produced in response to tTG) so as to permit said polypeptide to interact with autoantibodies produced in response to tTG and present in said sample; and (c) means for monitoring the degree of interaction of said polypeptide with said autoantibodies produced in response to tTG and present in said sample, thereby providing an indication of the presence of said autoantibodies in said sample.","A kit according to the present invention may typically further comprise control means, such as means for providing a sample of body fluid from a normal subject, in other words a subject known to be without autoimmune disease associated with an immune reaction to tTG.","A kit for screening for autoantibodies to tTG according to the present invention may comprise means for directly monitoring interaction of (i) autoantibodies to tTG present in the sample of body fluid from the subject and (ii) a polypeptide, as provided by the present invention substantially as hereinbefore described, typically comprising non-competitive sandwich type assay means known in the art.","Typically, in a kit according to the present invention comprising non-competitive assay means, means are provided for monitoring the degree of interaction of (i) autoantibodies to tTG present in the sample and (ii) a polypeptide according to the present invention substantially as hereinbefore described, and can comprise labelling means provided either to a polypeptide according to the present invention substantially as hereinbefore described, or to a binding partner for autoantibodies to tTG, either of which would enable monitoring of the above described interaction.","For example, a kit according to the present invention may comprise means for directly or indirectly labelling a polypeptide according to the present invention substantially as hereinbefore described; means for contacting the thus labelled polypeptide with a sample of body fluid being screened for tTG autoantibodies so as to provide a mixture thereof; a binding partner for autoantibodies to tTG (such as an anti-IgG reagent) present in the sample of body fluid; and means for adding the binding partner to the mixture so as to cause precipitation of any complexes of labelled polypeptide and tTG autoantibodies present in the mixture.","Alternatively, it may be preferred that a kit according to the present invention further comprises a labelled binding partner for tTG autoantibodies (such as a labelled anti-IgG reagent, for example protein A or anti-human IgG, or labelled full length tTG or an epitope thereof) and means for adding the labelled binding partner to a mixture obtained by contacting (i) a polypeptide according to the present invention substantially as hereinbefore described immobilised to a support and (ii) a sample of body fluid being screened for autoantibodies to tTG.","It may alternatively be preferred that a kit for screening for autoantibodies to tTG in the sample of body fluid according to the present invention, comprises known competitive assay means.","For example, a kit according to the present invention may further comprise at least one competitor capable of competing with autoantibodies to tTG in the interaction thereof with a polypeptide according to the present invention substantially as hereinbefore described.","Typically, a competitor as employed in a competitive assay kit according to the present invention may comprise one or more antibodies, which may be natural or partly or wholly synthetically produced.","A competitor as employed in the present invention may alternatively comprise any other protein having a binding domain or region which is capable of competing with autoantibodies to tTG in the interaction thereof with a polypeptide according to the present invention substantially as hereinbefore described.","Preferably, however, a competitor as employed in the present invention comprises a monoclonal or polyclonal antibody (especially a monoclonal antibody), capable of competing with tTG autoantibodies in the interaction thereof with a polypeptide according to the present invention substantially as hereinbefore described.","Typically, therefore, a competitive assay kit according to the present invention may further comprise at least one competitor, such as a monoclonal or polyclonal antibody, whereby a polypeptide according to the present invention substantially as hereinbefore described can interact with either a competitor, such as a monoclonal or polyclonal antibody, or autoantibodies to tTG present in a sample of body fluid being screened.","Typically monitoring means in a competitive assay kit according to the present invention comprise means for comparing: (i) interaction of a polypeptide according to the present invention substantially as hereinbefore described and one or more competitors substantially as hereinbefore described (typically a monoclonal or polyclonal antibody), in the absence of said sample of body fluid being screened (that is a suspected disease sample), optionally in the presence of a sample of body fluid from a normal subject, typically a subject known to be without autoimmune disease associated with an immune reaction to tTG; with (ii) interaction of a polypeptide according to the present invention substantially as hereinbefore described and one or more competitors substantially as hereinbefore described (typically a monoclonal or polyclonal antibody), in the presence of said sample of body fluid being screened.","Typically, the comparison involves observing a decrease in interaction of a polypeptide according to the present invention substantially as hereinbefore described and the competitor in (ii) compared to (i) so as to provide an indication of the presence of autoantibodies to tTG in said sample.","Typically, the decrease in interaction can be observed by directly or indirectly labelling the competitor and monitoring any change in the interaction of the thus labelled competitor with a polypeptide according to the present invention substantially as hereinbefore described in the absence and in the presence of a sample of body fluid being screened for autoantibodies to tTG.","Suitably a polypeptide according to the present invention substantially as hereinbefore described may be immobilised to facilitate the above mentioned monitoring.","Alternatively, there is also provided by the present invention a kit for screening for autoantibodies to tTG in a sample of body fluid obtained from a subject (in particular a human) suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said kit comprising: (a) full length tTG (typically recombinantly obtained full length tTG); (b) at least one competitor capable of competing with autoantibodies to tTG in the interaction thereof with a polypeptide according to the present invention substantially as hereinbefore described, (c) means for contacting said sample of body fluid from said subject, said full length tTG and said competitor (under conditions that allow interaction of tTG with autoantibodies to tTG), so as to permit said full length tTG to interact with either autoantibodies to tTG present in said sample, or said competitor; and (d) means for monitoring the interaction of said full length tTG with said autoantibodies present in said sample, thereby providing an indication of the presence of said autoantibodies to tTG in said sample.","The full length tTG can typically be of human, guinea pig, bovine, mouse or rat origin, preferably human, and more preferably human recombinantly obtained full length tTG.","A competitor for use in such an assay kit typically comprises a monoclonal or polyclonal antibody (preferably monoclonal) substantially as hereinbefore described.","Suitably a detectable label that can be employed in a kit according to the present invention can be selected from the group consisting of enzymic labels, isotopic labels, chemiluminescent labels, fluorescent labels, dyes and the like.","In the case where an isotopic label (such as 125I, 14C, 3H or 35S) is employed, monitoring means may therefore comprise means for measuring radioactivity dependent on binding of a polypeptide according to the present invention substantially as hereinbefore described.","Radioactivity is generally measured using a gamma counter, or liquid scintillation counter.","In the case of a kit for screening for lymphocytes according to the present invention, it is generally preferred that means are provided for initially isolating lymphocytes from a sample of body fluid from a subject, using techniques well known to one of ordinary skill in the art, and means are also provided for contacting a polypeptide according to the present invention to the present invention with such isolated lymphocytes so as to stimulate proliferation of the latter by the former.","Means (again known to one of ordinary skill in the art) for monitoring the effect of interaction of a polypeptide according to the present invention and such proliferating lymphocytes, are also provided in such a kit according to the present invention.","There is still further provided by the present invention an antibody produced in response to one or more epitope regions of tTG, which epitope region comprises part or all of the primary structural conformation of amino acids 1 to 89 of tTG, or part or all of the primary structural conformation of amino acids 401 to 494 or 401 to 491 of tTG, or one or more fragments of an antibody (such as Fab fragments) produced in response to one or more epitope regions of tTG, which epitope region comprises part or all of the primary structural conformation of amino acids 1 to 89 of tTG, or part or all of the primary structural conformation of amino acids 401 to 494 or 401 to 491 of tTG.","An antibody according to the present invention or fragment thereof can interact with a polypeptide according to the present invention substantially as hereinbefore described and preferably is obtainable by, or is obtained by, techniques described in the Examples.","Suitably, an antibody provided by the present invention can be monoclonal (preferred), recombinant or polyclonal.","Typically an antibody, such as a monoclonal antibody, as provided by the present invention is in substantially purified form.","More specifically, a monoclonal antibody as provided by the present invention can comprise a monoclonal antibody Mab A3-6B5, or a monoclonal antibody Mab A5-4E6, or one or more active fragments thereof (such as Fab fragments), as described in the Examples.","The present invention further provides a host cell containing an antibody substantially as described above and also a hybridoma capable of secreting a monoclonal antibody substantially as hereinbefore described.","There is still further provided by the present invention an antibody substantially as hereinbefore described, or one or more fragments of an antibody (such as Fab fragments) substantially as hereinbefore described, for use in diagnosis or therapy, in particular diagnosis or therapy of autoimmune disease associated with an immune reaction to tTG substantially as hereinbefore described.","Particularly the autoimmune disease can be coeliac disease (although other autoimmune diseases substantially as hereinafter described in greater detail can be diagnosed or treated by an antibody according to the present invention) and the present invention also provides an antibody substantially as hereinbefore described for use in the manufacture of a medicament for the treatment of coeliac disease.","There is still further provided by the present invention a polypeptide according to the present invention substantially as hereinbefore described, for use in diagnosis or therapy, in particular diagnosis or therapy of autoimmune disease associated with an immune reaction to tTG substantially as hereinbefore described.","In particular, autoimmune disease associated with an immune reaction to tTG as described herein comprises autoimmune disease arising due to gluten sensitive enteropathy in a subject, in particular a human subject, such as coeliac disease.","Other autoimmune diseases associated with an immune reaction to tTG that can be diagnosed or treated by the present invention include any of the following—dermatitis herpetiformis, Addison's disease, A1 (A1-autoimmune) haemolytic anaemia, A1 thrombocytopenic purpura, A1 thyroid diseases, A1 hyperthyroidism (Graves' disease), A1 hypothyroidism (including Hashimoto's thyroiditis), pernicious anaemia, myasthenia gravis, primary biliary cirrhosis, rheumatoid arthritis, Sjogren's syndrome, SLE, type 1 (insulin-dependent) diabetes mellitus, diabetes mellitus of other types, Wegener granulomatosis, and vitiligo.","Substantially as herein described autoimmune disease associated with an immune reaction to tTG can be screened for or diagnosed in a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG.","The patient group that can be classed as susceptible to an autoimmune disease associated with an immune reaction to tTG can typically include relatives of subjects previously diagnosed as having an autoimmune disease associated with an immune reaction to tTG, or subjects previously diagnosed with a primary or first autoimmune disease associated with an immune reaction to tTG and as such could be considered to be at risk of or prone to be suffering from a secondary or second autoimmune disease associated with an immune reaction to tTG.","For example, a subject known to be suffering from type 1 diabetes mellitus could be considered to be susceptible to suffering from coeliac disease.","It will be appreciated from the foregoing description that the present invention provides assay methods and kits for detecting autoantibodies (in particular) or lymphocytes produced in response to tTG in a sample of body fluid substantially as hereinbefore described.","The detection of such autoantibodies and/or lymphocytes produced in response to tTG in the sample of body fluid (or at least the level of such autoantibodies and/or lymphocytes in the sample) is indicative of the presence of autoimmune disease associated with an immune reaction to tTG in the subject from which the sample was obtained and can, therefore, enable the diagnosis of the likely onset or presence of autoimmune disease associated with an immune reaction to tTG.","There is, therefore, further provided by the present invention a method of diagnosing the likely onset or presence of autoimmune disease associated with an immune reaction to tTG in a subject (in particular a human) suspected of suffering from, susceptible to, having or recovering from, autoimmune disease associated with an immune reaction to tTG, the method comprising detecting autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid from the subject substantially as hereinbefore described, and whereby the detected autoantibodies and/or lymphocytes can provide a diagnosis of the likely onset or presence of autoimmune disease associated with an immune reaction to tTG in the subject.","There is still further provided by the present invention a method of delaying or preventing the onset of autoimmune disease associated with an immune reaction to tTG in an animal subject (in particular a human subject) suspected of suffering from, susceptible to or recovering from autoimmune disease associated with an immune reaction to tTG, which method comprises initially detecting autoantibodies or lymphocytes indicative of the onset or presence of autoimmune disease associated with an immune reaction to tTG in a sample of body fluid obtained from the subject substantially as hereinbefore described, thereby providing a diagnosis of the likely onset of autoimmune disease associated with an immune reaction to tTG in the subject, and thereafter therapeutically treating the subject so as to delay the onset and/or prevent autoimmune disease associated with an immune reaction to tTG.","Typically, such therapeutic treatment can, in the case of gluten sensitive enteropathy giving rise to coeliac disease, comprise compliance with a gluten-free or substantially gluten free diet.","The present invention can further provide a method of monitoring dietary compliance by a subject (in particular a human subject) having, suspected of suffering from, susceptible to or recovering from gluten sensitive enteropathy, which method comprises screening for autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from the subject substantially as hereinbefore described, and whereby the detection of such autoantibodies and/or lymphocytes produced in response to tTG in the sample of body fluid (or at least the level of such autoantibodies and/or lymphocytes in the sample) provides an indication as to the dietary compliance of the subject to a gluten-free or substantially gluten free diet.","A polypeptide according to the present invention substantially as hereinbefore described is particularly suitable for use in the therapeutic treatment of autoimmune disease associated with an immune reaction to tTG.","For example, tolerance can be achieved by administering a polypeptide according to the present invention substantially as hereinbefore described to a subject (in particular a human subject) suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG.","There is, therefore, further provided by the present invention a pharmaceutical composition comprising a polypeptide according to the present invention substantially as hereinbefore described, together with a pharmaceutically acceptable carrier, diluent or excipient therefor, wherein the polypeptide can interact with autoantibodies and/or lymphocytes produced in response to tTG.","The present invention further provides a polypeptide according to the present invention substantially as hereinbefore described for use in the manufacture of a medicament for the treatment of coeliac disease.","Compositions or medicaments according to the present invention should contain a therapeutic or prophylactic amount of at least one polypeptide according to the present invention in a pharmaceutically-acceptable carrier.","The pharmaceutical carrier can be any compatible, non-toxic substance suitable for delivery of the polypeptides to the patient.","Sterile water, alcohol, fats, waxes, and inert solids may be used as the carrier.","Pharmaceutically-acceptable adjuvants, buffering agents, dispersing agents and the like, may also be incorporated into the pharmaceutical compositions.","Such compositions can contain a single polypeptide or may contain two or more polypeptides according to the present invention.","It may be desirable to couple a polypeptide according to the present invention to immunoglobulins, e.g.","IgG, or to lymphoid cells from the patient being treated in order to promote tolerance.","Such an approach is described in Bradley-Mullen, Activation of Distinct Subsets of T Suppressor Cells with Type III Pneumococcal Polysaccharide Coupled to Syngeneic Spleen Cells, in: IMMUNOLOGICAL TOLERANCE TO SELF AND NON-SELF, Buttisto et al., eds., Annals N.Y. Acad.","Sci.","Vol.","392, pp 156-166, 1982.Alternatively, the polypeptides may be modified to maintain or enhance binding to the MHC while reducing or eliminating binding to the associated T-cell receptor.","In this way, the modified polypeptides may compete with natural tTG to inhibit helper T-cell activation and thus inhibit the immune response.","In all cases, care should be taken that administration of the pharmaceutical compositions of the present invention ameliorate but do not potentiate the autoimmune response.","Pharmaceutical compositions according to the present invention are useful for parenteral administration.","Preferably, the compositions will be administered parenterally, i.e.","subcutaneously, intramuscularly, or intravenously.","Thus, the invention provides compositions for parenteral administration to a patient, where the compositions comprise a solution or dispersion of the polypeptides in an acceptable carrier, as described above.","The concentration of the polypeptides in the pharmaceutical composition can vary widely, i.e.","from less than about 0.1% by weight, usually being at least about 1% by weight to as much as 20% by weight or more.","Typical pharmaceutical compositions for intramuscular injection would be made up to contain, for example, 1 ml of sterile buffered water and 1 to 100 μg of a purified polypeptide of the present invention.","A typical composition for intravenous infusion could be made up to contain 100 to 500 ml of sterile Ringer's solution and 100 to 500 mg of a purified polypeptide of the present invention.","Actual methods for preparing parenterally administrable compositions are well known in the art and described in more detail in various sources, including, for example, Remington's Pharmaceutical Science, 15th Edition, Mack Publishing Company, Easton, Pa. (1980).","In addition to using a polypeptide according to the present invention directly in pharmaceutical compositions, it is also possible to use a polypeptide according to the present invention to enhance tolerance to tTG in a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, employing the following principles.","More particularly, peripheral blood lymphocytes can be collected from the subject in a conventional manner and stimulated by exposure to a polypeptide according to the present invention, as defined above.","Usually, other mitogens and growth enhancers will be present, e.g., phytohemagglutinin, interleukin 2, and the like.","Proliferating T-helper cells may be isolated and cloned, also under the stimulation of a polypeptide according to the present invention.","Clones which continue to proliferate may then be used to prepare therapeutic compositions for the subject.","The cloned T-cells may be attenuated, e.g.","by exposure to radiation, and administered to the subject in order to induce tolerance.","Alternatively, the T-cell receptor or portions thereof may be isolated by conventional protein purification methods from the cloned T-cells and administered to the individual.","Such immunotherapy methods are described generally in Sinha et al.","(1990) Science 248:1380-1388.In some cases, after a T-helper cell has been cloned as described above, it may be possible to develop therapeutic peptides from the T-cell receptor, where the peptides would be beneficial for treating a patient population suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG.","In such cases, the T-cell receptor gene may be isolated and cloned by conventional techniques and peptides based on the receptor produced by recombinant techniques as described above.","The recombinantly-produced peptides may then be incorporated in pharmaceutical compositions as described above.","There is also provided by the present invention a method of cloning lymphocytes produced in response to tTG, which method comprises: providing a source of lymphocytes; contacting the lymphocytes with a polypeptide according to the present invention substantially as hereinbefore described, so as to effect proliferation of said lymphocytes; and isolating and cloning the proliferating lymphocytes.","The present invention also provides the use of cloned lymphocytes prepared as above, in the therapeutic treatment of autoimmune disease associated with an immune reaction to tTG.","There is provided, therefore, a pharmaceutical composition comprising cloned lymphocytes prepared as above, together with a pharmaceutically acceptable carrier, diluent or excipient therefor and the use of such cloned lymphocytes in the manufacture of a medicament for the treatment of autoimmune disease associated with an immune reaction to tTG, in particular coeliac disease.","There is also provided by the present invention one or more therapeutic agents identified as providing a therapeutic effect by interaction with amino acids 1 to 89 of tTG, and/or amino acids 401 to 494 of tTG, or amino acids 401 to 491 of tTG, and the present invention further provides one or more therapeutic agents for use in therapeutically interacting with amino acids 1 to 89 of tTG, and/or amino acids 401 to 494 of tTG or amino acids 401 to 491 of Ttg and as such for use in the therapeutic treatment of an autoimmune disease associated with an immune reaction to tTG.","There is, therefore, still further provided by the present invention a method of treating autoimmune disease associated with an immune reaction to tTG in a subject, which method comprises initially detecting autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from the subject substantially as hereinbefore described, thereby providing a diagnosis of autoimmune disease in the subject, and administering to the subject a therapeutically effective amount of at least one therapeutic agent effective in the treatment of such autoimmune disease, such as a polypeptide according to the present invention substantially as hereinbefore described.","The present invention also provides a method of treating autoimmune disease associated with an immune reaction to tTG in a subject (in particular a human subject), which method comprises administering to the subject a therapeutically effective amount of a therapeutic agent identified as providing a therapeutic effect by interaction with amino acids 1 to 89 of tTG, and/or amino acids 401 to 494 of tTG, or amino acids 401 to 491 of tTG.","The amount of therapeutic agent administered will depend on the specific autoimmune disease state being treated, possibly the age of the patient and will ultimately be at the discretion of an attendant physician.","There is still further provided by the present invention, in combination, a kit substantially as hereinbefore described, together with a therapeutically effective amount of at least one therapeutic agent effective in the treatment of autoimmune disease associated with an immune reaction to tTG substantially as hereinbefore described.","Substantially as hereinbefore described, the sample of body fluid being screened by the present invention will typically comprise blood samples or other fluid blood fractions, such as in particular serum samples or plasma samples, but the sample may in principle be another biological fluid, such as saliva or urine or solubilised tissue extracts.","The present invention will now be illustrated with reference to the accompanying Figures and Examples, which do not limit the invention in any way.","FIG.","1 shows results of SDS PAGE (9%) analysis of purified recombinant human tTG for use in the method and kit according to the invention.","FIG.","2 is a table showing binding of autoantibodies to tTG in sera from coeliac patients with full length and modified 35S-tTG proteins.","FIG.","3 is a table showing monoclonal antibody (namely monoclonal antibodies A3-6B5 and A5-4E6) binding to full length and modified 35S-tTG proteins.","FIG.","4 shows a schematic example of the method of the present invention when an antibody is employed.","FIG.","5 is a graph showing binding (absorbance at 450 nm in the vertical axis) of tTG with autoantibodies to tTG in different patients sera (horizontal axis).","The binding is measured in the presence or absence of mouse tTG monoclonal antibody A3-6B5.FIG.","6 is a graph showing binding (absorbance at 450 nm in the vertical axis) of tTG with autoantibodies to tTG in different patients sera (horizontal axis).","The binding is measured in the presence or absence of mouse tTG monoclonal antibody A5-4E6.FIG.","7 depicts cDNA derived from human tTG, encoding amino acids 1 to 89 of human tTG.","FIG.","8 depicts amino acids 1 to 89 of human tTG.","FIG.","9 depicts cDNA derived from guinea pig tTG, encoding amino acids 1 to 89 of guinea pig tTG.","FIG.","10 depicts amino acids 1 to 89 of guinea pig tTG.","FIG.","11 depicts CDNA derived from bovine tTG, encoding amino acids 1 to 89 of bovine tTG.","FIG.","12 depicts amino acids 1 to 89 of bovine tTG.","FIG.","13 depicts cDNA derived from mouse tTG, encoding amino acids 1 to 89 of mouse tTG.","FIG.","14 depicts amino acids 1 to 89 of mouse tTG.","FIG.","15 depicts cDNA derived from rat tTG, encoding amino acids 1 to 89 of rat tTG.","FIG.","16 depicts amino acids 1 to 89 of rat tTG.","FIG.","17 depicts cDNA derived from human tTG, encoding amino acids 401 to 491 of human tTG.","FIG.","18 depicts amino acids 401 to 491 of human tTG.","FIG.","19 depicts cDNA derived from guinea pig tTG, encoding amino acids 401 to 494 of guinea pig tTG.","FIG.","20 depicts amino acids 401 to 494 of guinea pig tTG.","FIG.","21 depicts cDNA derived from bovine tTG, encoding amino acids 401 to 491 of bovine tTG.","FIG.","22 depicts amino acids 401 to 491 of bovine tTG.","FIG.","23 depicts cDNA derived from mouse tTG, encoding amino acids 401 to 491 of mouse tTG.","FIG.","24 depicts amino acids 401 to 491 of mouse tTG.","FIG.","25 depicts cDNA derived from rat tTG, encoding amino acids 401 to 491 of rat tTG.","FIG.","26 depicts amino acids 401 to 491 of rat tTG.","FIG.","27 depicts the alignment of tTG sequences (human, guinea pig, bovine, mouse and rat) at the amino acid level.","Further illustration of the present invention is given in the following detailed worked Examples.","EXAMPLES Identification of Regions of tTG which are Recognised by tTG Autoantibodies Full length human tTG cDNA (endothelial cell isoform) was cloned into pTZ18R (Pharmacia) under control of the SP6 promoter.","This construct was used to produce a reference full length tTG protein.","Various in-frame deletions and truncations of the tTG gene were carried out using either naturally occurring restriction endonuclease sites or PCR mediated modifications to the sequence.","The modified tTG constructs were cloned into pTZ18R (as above).","Full length and modified tTG proteins were expressed in an in vitro transcription/translation rabbit reticulocyte system (Promega) incorporating 35S-methionine (Amersham Pharmacia Biotech) and purified as described before (Colls J, et al.","Clin.","Chem.","1995; 41/3:375-380).","Table 1 shows the vectors used and the corresponding tTG amino acid residues expressed following the in vitro transcription/translation described above.","TABLE 1 Non-expressed Vector Name Expressed residues (deleted) residues pTGM19 1-687 (full length) None pTGM25 1-628 629-687 pTGM22 1-491 492-687 pTGM24 1-400 401-687 pTGM26 90-687 1-89 pTGM27 172-687 1-171 pTGM28 207-687 1-206 pTGM29 281-687 1-280 pTGM30 356-687 1-355 pTGM31 399-687 1-398 Binding of tTG autoantibodies to the purified full length and modified tTG proteins was analysed using an immunoprecipitation assay (Nakachi K, et al Clin Chim Acta, Volume 304, pages 75 to 84, 2002).","Briefly, 10 μL of undiluted serum (in duplicate) was incubated with 50 μL of 35S-tTG (approx 15,000 dpm diluted in 10 mmol/L Tris HCl pH 7.6, 150 mmol/L NaCl, 2 mmol/L EDTA, 0.1% Tween 20, 10 g/L bovine serum albumin, 0.2 g/L NaN3; Assay Buffer) in Millipore filtration plates overnight at 4° C. Anti-human IgA agarose (50 μl) was then added and the plates incubated for 4 hours at 4° C. After incubation the reaction mixtures in the wells were washed 3 times.","The wells were then punched out into vials containing 1 mL of scintillation liquid and counted in a beta-counter.","FIG.","2 shows binding of tTG autoantibodies with different modified tTG proteins.","The results are expressed as % binding relative to binding to the full length tTG reference protein.","Serum Samples Serum from 34 patients with coeliac disease were used in the study.","The diagnosis was based on typical clinical symptoms and was confirmed by positive duodenal biopsy.","The sera tested positive for endomysial antibody and autoantibodies to tTG by standard ELISA.","In addition sera from healthy blood donors (n=10) negative for autoantibodies to tTG were used.","Production of Mouse Monoclonal Antibodies Both guinea pig tTG (Sigma, Poole, UK) and recombinant human tTG (rhtTG) were used as antigens to immunise mice.","Monoclonal antibodies (Mabs) were cloned using standard techniques.","Selected tTG Mabs were grown in culture and purified by protein A affinity chromatography.","The location of the binding sites of the mouse monoclonal antibodies was determined by an immunoprecipitation assay using the modified 35S-labelled tTG proteins as described above, except that anti-mouse IgG was added for precipitation.","When required, purified IgG was labelled with 125I using the chloramine T method.","Analysis of the mouse Mabs by immunoprecipitation with modified 35S-labelled tTG proteins indicated that the epitope for A3-6B5 Mab was contained within aa 1-89 of the human tTG protein and the epitope for A5-4E6 Mab was contained within aa 401-491 of the human tTG protein (FIG.","3).","Inhibition of Autoantibodies to tTG Binding with tTG by Mouse tTG Mabs The inhibition of autoantibodies to tTG binding with tTG by mouse tTG Mabs was investigated in a modified version of an ELISA described in Nakachi K, et al.","Clin Chim Acta (Volume 304, pages 75 to 84, 2002).","To streptavidin coated plate wells 50 μL of tTG-biotin was added plus 10μL of Mab (200 μ/ml) or Assay Buffer and incubated for 30 mins.","40 μL of patients' sera (typically diluted 1:100) was then added and incubated for 30 mins.","After this step all wells were aspirated, washed four times and then 100 μL of anti-human IgA-horseradish peroxidase (HRP) was added and incubated for 30 mins.","After aspiration and four washes, 100 μL of tetramethyl benzidine (TMB) solution was added and the plate was incubated for 15 mins in the dark.","50 μL of stop solution was then added and absorbance read at 450 nm in a plate reader.","All steps were performed at room temperature, all incubations except the final TMB incubation were done with 200 rpm shaking.","tTG-biotin, Mabs and human sera were diluted in Assay Buffer.","The results are shown in FIG.","5 which shows binding of tTG with autoantibodies to tTG in different patients sera in the presence or absence of mouse tTG monoclonal antibody A3-6B5 and FIG.","6 which shows binding of tTG with autoantibodies to tTG in different patients sera in the presence or absence of mouse tTG monoclonal antibody A5-4E6.Preparation of Purified Recombinant Human tTG Recombinant human tTG was expressed in the yeast Saccharomyces cerevisiae strain c13ABYS86 using an expression system and culture techniques described previously (Powell M, et al.","Clin Chim Acta 1996; 256: 175-188).","Recombinant human tTG extracted from the yeast was partially purified by anion exchange chromatography and preparations of tTG of greater than 95% purity were obtained by affinity chromatography using mouse tTG Mab (A5-6C7) coupled to CNBr Sepharose.","The purity of the purified tTG was assessed by SDS PAGE analysis on a 9% acrylamide gel followed by staining with Coomassie Blue as shown in FIG.","1.Assay for Screening a Sample of Body Fluid for Autoantibodies to tTG Based on the Inhibition of 125I-Mab Binding to Recombinant Human tTG An assay was set up based on the principles of the schematic example shown in FIG.","4.4 mL Nunc Maxisorp Startubes (Life Technologies) were coated overnight at 4° C. with 250 μL of 10 μg/mL recombinant human tTG (rhtTG).","Following aspiration and three washes with 500 μL Assay Buffer, tubes were incubated for 30 minutes at room temperature with 500 μL of Post-Coat Buffer containing bovine serum albumin.","After two washes with Assay Buffer, tubes were incubated with 100 μL of serum sample diluted 1:5 in Assay Buffer for 60 minutes at room temperature on a shaker.","Following aspiration and three washes with 500 μL of Assay Buffer, tubes were incubated on a shaker for 30 minutes at room temperature with 100 μL of 125I-labelled tTG Mab A3-6B5 (30,000 cpm; diluted in Assay Buffer).","After a final aspiration and 3 washes with 500 μL of Assay Buffer, tubes were counted for 1 minute in a gamma counter.","The results, measured in counts per minute (cpm), were used to calculate the % inhibition of 125I-Mab binding as follows: % inhibition = 100 - [ A B × 100 ] wherein, A=125I-Mab (cpm) bound to rhtTG in the presence of test sera as a % of total cpm of material added to the tube; and B=125I-Mab (cpm) bound to rhtTG in the presence of healthy blood donor sera (mean of 2 or 3 sera) as a % of total cpm of material added to the tube.","Results and Discussion FIG.","2 showed that C-terminal deletions of amino acids (aa) 492-687 of the tTG protein had no or little effect on tTG autoantibody binding.","More extensive C-terminal deletion (aa 401-687) resulted in lower tTG autoantibody binding in 14/15 sera studied.","N-terminal deletion of aa 1-89 of the tTG protein resulted in lower tTG autoantibody binding in all sera studied.","Therefore, the studies with modified tTG proteins described herein showed that tTG autoantibody binding sites on tTG are heterogenous and are dependent on the aa sequences in the N-terminal and central part of the molecule.","Furthermore, these studies showed that aa 1-89, and aa 401-491 of human tTG protein are important for binding of tTG autoantibodies present in the coeliac sera studied.","(aa 401-491 of human tTG correspond to aa 401-494 of tTG in other species, such as guinea pig tTG.)","Analysis of mouse Mabs by immunoprecipitation with modified 35S-labelled tTG proteins indicated that the epitope for A3-6B5 Mab was contained within aa 1-89 of the human tTG protein and the epitope for A5-4E6 Mab was contained within aa 401-491 of the human tTG protein (FIG.","3).","Furthermore, these Mabs, namely A3-6B5 Mab and A5-4E6 Mab, inhibited binding of tTG autoantibody positive sera with tTG in ELISA (see FIGS.","5 and 6).","Identification of amino acid sequences of the tTG molecule important for binding of autoantibodies to tTG tested, and production of mouse Mabs reactive with these amino acid sequences, allowed the development of a new assay to screen for the presence of autoantibodies to tTG in a sample of body fluid as described above.","The results of the assay based on autoantibodies to tTG in test sera inhibiting 125I-Mab A3-6B5 binding with recombinant human tTG are shown in Table 2.TABLE 2 Results of assay based on the inhibition of 125I-Mab binding to recombinant human tTG % Inhibition of tTG 35S rhtTG Serum 125I-A3-6B5 Mab Autoantibodies Autoantibodies Number binding ELISA u/mL IPA Index Coeliac 1 79 1000 100 2 35 110 52 3 91 42 7.7 4 49 150 21 5 89 1000 44 6 88 51 107 7 89 1000 122 8 42 102 107 9 48 45 3.3 Healthy blood donors 1 −11 Neg Neg 2 13 Neg Neg 3 −1 Neg Neg 4 3 Neg Neg 5 −3 Neg Neg Nine sera from coeliac patients which tested positive for tTG autoantibodies by immunoprecipitation assay (tTG autoantibodies index from 3.3-107) and ELISA (42-1000 u/mL) were analysed.","All these sera inhibited binding of 125I-Mab to rhtTG coated on the tube (35 to 91% inhibition).","In contrast, sera negative for autoantibodies to tTG only had a small effect on the binding of 125I-Mab to rhtTG (−11 to 13% inhibition)."]],"string":"[\n [\n \"Tissue transglutaminase\",\n \"The present invention relates to tissue transglutaminase (tTG), and more particularly to DNA coding for and polypeptides containing, the primary structural conformaion of one or more epitopes of tTG and to the use thereof in the detection of autoantibodies and/or lymphocytes produced in response to tTG.\"\n ],\n [\n \"1.A DNA sequence encoding: a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes,: which DNA sequence comprises: (a) a DNA sequence as depicted in any of Seq.\",\n \"Ids 1, 3, 5, 7, 9, 11, 13, 15, 17, 19; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of Seq.\",\n \"Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, or one or more active fragments of amino acids as depicted in one or more of Seq.\",\n \"Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes.\",\n \"2.A DNA sequence encoding: a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes; which DNA sequence comprises: (a) a DNA sequence as depicted in any of Seq.\",\n \"Ids 1, 3, 5, 7 and 9; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 1 to 89 of tTG, or one or more active fragments of amino acid numbers 1 to 89 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of Seq.\",\n \"Ids 2, 4, 6, 8 and 10, or one or more active fragments of amino acids as depicted in one or more of Seq.\",\n \"Ids 2 4, 6, 8 and 10, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes.\",\n \"3.A DNA sequence encoding: a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes; which DNA sequence comprises: (a) a DNA sequence as depicted in any of Seq.\",\n \"Ids 11, 13, 15, 17 or 19; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of Seq.\",\n \"Ids 12, 14, 16 and 18, or one or more active fragments of amino acids as depicted in one or more of Seq.\",\n \"Ids 12, 14, 16 and 18, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes.\",\n \"4.A DNA sequence encoding: a polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies; which DNA sequence comprises: (a) a DNA sequence as depicted in any of Seq.\",\n \"Ids 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of Seq.\",\n \"Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, or one or more active fragments of amino acids as depicted in one or more of Seq.\",\n \"Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies.\",\n \"5.A DNA sequence encoding: a polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies; which DNA sequence comprises: (a) a DNA sequence as depicted in any of Seq.\",\n \"Ids 1, 3, 5, 7 or 9; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 1 to 89 of tTG, or one or more active fragments of amino acid numbers 1 to 89 of tTG, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of Seq.\",\n \"Ids 2, 4, 6, 8 and 10, or one or more active fragments of amino acids as depicted in one or more of Seq.\",\n \"Ids 2, 4, 6, 8 and 10, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies.\",\n \"6.A DNA sequence encoding: a polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies; which DNA sequence comprises: (a) a DNA sequence as depicted in any of Seq.\",\n \"Ids 11, 13, 15, 17 or 19; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of Seq.\",\n \"Ids 12, 14, 16, 18 and 20, or one or more active fragments of amino acids as depicted in one or more of FIGS.\",\n \"18, 20, 22, 24 and 26, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG; (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies.\",\n \"7.A DNA sequence as depicted in any of Seq.\",\n \"Ids 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19, or a DNA sequence differing therefrom due to the degeneracy of the genetic code.\",\n \"8.A biologically functional plasmid or DNA vector including a DNA sequence as defined in claim 1.9.A host cell which is transformed or transfected with a DNA sequence as defined in claim 1.10.A polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG.\",\n \"11.A polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, or one or more active fragments of amino acid numbers 1 to 89 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG.\",\n \"12.A polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, which polypeptide comprises the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes; with the exception of human, guinea pig, mouse, bovine or rat full length tTG.\",\n \"13.A polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies; with the exception of human, guinea pig, mouse, bovine or rat full length tTG.\",\n \"14.A polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, or one or more active fragments of amino acid numbers 1 to 89 of tTG, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG.\",\n \"15.A polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, which polypeptide comprises the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies; with the exception of human, guinea pig, mouse, bovine or rat full length tTG.\",\n \"16.A polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact,under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of Seq.\",\n \"Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, or one or more active fragments of amino acids as depicted in one or more of Seq.\",\n \"Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG.\",\n \"17.A polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of Seq.\",\n \"Ids 2, 4, 6, 8 and 10, or one or more active fragments of amino acids as depicted in one or more of Seq.\",\n \"Ids 2, 4, 6, 8 and 10, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG.\",\n \"18.A polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of Seq.\",\n \"Ids 12, 14, 16, 18 and 20, or one or more active fragments of amino acids as depicted in one or more of Seq.\",\n \"Ids 12, 14, 16, 18 and 20, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG.\",\n \"19.A polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of Seq.\",\n \"Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, or one or more active fragments of amino acids as depicted in one or more of Seq.\",\n \"Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG.\",\n \"20.A polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of Seq.\",\n \"Ids 2, 4, 6, 8 and 10, or one or more active fragments of amino acids as depicted in one or more of Seq.\",\n \"Ids 2, 4, 6, 8 and 10, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG.\",\n \"21.A polypeptide with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of Seq.\",\n \"Ids 12, 14, 16, 18 and 20, or one or more active fragments of amino acids as depicted in one or more of Seq.\",\n \"Ids 12, 14, 16, 18 and 20, with which autoantibodies produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies, with the exception of human, guinea pig, mouse, bovine or rat full length tTG.\",\n \"22.A polypeptide as depicted in any of Seq.\",\n \"Ids 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20.23.A process of preparing a polypeptide as defined in claim 10, which process comprises: (i) providing a host cell which is transformed or transfected with a DNA sequence or a biologically functional plasmid or DNA vector; (ii) growing the host cell; and (iii) recovering a polypeptide as defined in claim 10.24.A method of screening for autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said method comprising: (a) providing either (i) said sample of body fluid from said subject or (ii) lymphocytes isolated from said sample; (b) contacting said sample or isolated lymphocytes with a polypeptide as defined in claim 10, under conditions that allow interaction of tTG with autoantibodies or lymphocytes produced in response to tTG, so as to permit said polypeptide to interact with autoantibodies, or lymphocytes, produced in response to tTG, and present in, or isolated from, said sample; and (c) monitoring the degree, or effect, of interaction of said polypeptide with either said autoantibodies, or said lymphocytes, produced in response to tTG and present in, or isolated from, said sample, thereby providing an indication of the presence of said autoantibodies, or said lymphocytes, in said sample, or isolated from said sample.\",\n \"25.A method according to claim 24, for screening for autoantibodies produced in response to tTG and present in said sample.\",\n \"26.A method of screening for autoantibodies produced in response to tTG in a sample of body fluid obtained from a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said method comprising: (a) providing said sample of body fluid from said subject; (b) contacting said sample with a polypeptide as defined in claim 10, under conditions that allow interaction of tTG with autoantibodies produced in response to tTG, so as to permit said polypeptide to interact with autoantibodies produced in response to tTG and present in said sample; and (c) monitoring the degree of interaction of said polypeptide with said autoantibodies produced in response to tTG and present in said sample, thereby providing an indication of the presence of said autoantibodies in said sample.\",\n \"27.A method according to claim 24, which comprises directly monitoring interaction of (i) autoantibodies to tTG present in the sample of body fluid from the subject and (ii) a polypeptide as defined in any of claims 10 to 22.28.A method according to claim 24, which further comprises providing at least one competitor, whereby in step (b) a polypeptide as defined in any of claims 10 to 22, can interact with either the competitor or autoantibodies to tTG and present in said sample.\",\n \"29.A method of screening for autoantibodies to tTG in a sample of body fluid obtained from a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said method comprising: (a) providing said sample of body fluid from said subject; (b) contacting said sample with (i) full length tTG, and (ii) at least one competitor capable of competing with autoantibodies to tTG in the interaction thereof with a polypeptide as defined in claim 10, under conditions that allow interaction of tTG with autoantibodies to tTG, so as to permit said full length tTG to interact with either autoantibodies to tTG present in said sample, or said competitor; and (c) monitoring the interaction of said full length tTG with said autoantibodies present in said sample, thereby providing an indication of the presence of said autoantibodies to tTG in said sample.\",\n \"30.A kit for screening for autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said kit comprising: (a) a polypeptide as defined in claim 10; (b) means for contacting either (i) a sample of body fluid obtained from said subject, or (ii) lymphocytes isolated from a sample of body fluid obtained from said subject, with said polypeptide as defined in claims 10, under conditions that allow interaction of tTG with autoantibodies or lymphocytes produced in response to tTG, so as to permit said polypeptide to interact with autoantibodies, or lymphocytes, produced in response to tTG, and present in, or isolated from, said sample; and (c) means for monitoring the degree, or effect, of interaction of said polypeptide with either said autoantibodies, or said lymphocytes, produced in response to tTG and present in, or isolated from, said sample, thereby providing an indication of the presence of said autoantibodies, or lymphocytes, in said sample, or isolated from said sample.\",\n \"31.A kit according to claim 30, for screening for autoantibodies produced in response to tTG in said sample of body fluid.\",\n \"32.A kit for screening for autoantibodies produced in response to tTG in a sample of body fluid obtained from a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said kit comprising: (a) a polypeptide as defined in claim 10; (b) means for contacting a sample of body fluid obtained from said subject with said polypeptide as defined in claim 10, under conditions that allow interaction of tTG with autoantibodies produced in response to tTG, so as to permit said polypeptide to interact with autoantibodies produced in response to tTG and present in said sample; and (c) means for monitoring the degree of interaction of said polypeptide with said autoantibodies produced in response to tTG and present in said sample, thereby providing an indication of the presence of said autoantibodies in said sample.\",\n \"33.A kit according to claim 30, which comprises means for directly monitoring interaction of (i) autoantibodies to tTG present in the sample of body fluid from the subject and (ii) a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG.\",\n \"34.A kit according to of claim 30 to 32, which further comprises at least one competitor, whereby a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact, under conditions that allow interaction of tTG with such autoantibodies or lymphocytes, with the exception of human, guinea pig, mouse, bovine or rat full length tTG, can interact with either the competitor or autoantibodies to tTG present in said sample of body fluid being screened.\",\n \"35.A kit for screening for autoantibodies to tTG in a sample of body fluid obtained from a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said kit comprising: (a) full length tTG; (b) at least one competitor capable of competing with autoantibodies to tTG in the interaction thereof with a polypeptide as defined in claim 10; (c) means for contacting said sample of body fluid from said subject, said full length tTG and said competitor, under conditions that allow interaction of tTG with autoantibodies to tTG, so as to permit said full length tTG to interact with either autoantibodies to tTG present in said sample, or said competitor; and (d) means for monitoring the interaction of said full length tTG with said autoantibodies present in said sample, thereby providing an indication of the presence of said autoantibodies to tTG in said sample.\",\n \"36.An antibody produced in response to one or more epitope regions of tTG, which epitope region comprises part or all of the primary structural conformation of amino acids 1 to 89 of tTG, or the primary structural conformation of amino acids 401 to 494 or 401 to 491 of tTG, or one or more fragments of an antibody produced in response to one or more epitope regions of tTG, which epitope region comprises part or all of the primary structural conformation of amino acids 1 to 89 of tTG, or the primary structural conformation of amino acids 401 to 494 or 401 to 491 of tTG.\",\n \"37.An antibody according to claim 36, which is obtained by the Examples.\",\n \"38.An antibody according to claim 36, which is monoclonal, recombinant or polyclonal.\",\n \"39.An antibody according to claim 38, which is monoclonal.\",\n \"40.A host cell containing an antibody as defined in claims 36.41.A hybridoma capable of secreting a monoclonal antibody as defined in claim 36.42.For use in diagnosis, an antibody as defined in claim 36.43.Use according to claim 42, in the diagnosis of autoimmune disease associated with an immune reaction to tTG.\",\n \"44.For use in therapy, an antibody as defined in claim 36.45.Use according to claim 44, in the therapeutic treatment of autoimmune disease associated with an immune reaction to tTG.\",\n \"46.Use according to claim 43, wherein the autoimmune disease is coeliac disease.\",\n \"47.An antibody according to claim 36, in the manufacture of a medicament for the treatment of coeliac disease.\",\n \"48.For use in diagnosis, a polypeptide as defined in claim 10.49.Use according to claim 48, in the diagnosis of autoimmune disease associated with an immune reaction to tTG.\",\n \"50.For use in therapy, a polypeptide as defined in claim 10.51.Use according to claim 50, in the therapeutic treatment of autoimmune disease associated with an immune reaction to tTG.\",\n \"52.Use according to claim 49, wherein the autoimmune disease is coeliac disease.\",\n \"53.A pharmaceutical composition comprising a polypeptide as defined in claim 10, together with a pharmaceutically acceptable carrier, diluent or excipient therefor, wherein said polypeptide can interact with autoantibodies and/or lymphocytes produced in response to tTG.\",\n \"54.A polypeptide as defined in claim 10, for use in the manufacture of a medicament for the treatment of coeliac disease.\",\n \"55.A method of diagnosing the likely onset or presence of autoimmune disease associated with an immune reaction to tTG in a subject suspected of suffering from, susceptible to, having or recovering from, autoimmune disease associated with an immune reaction to tTG, the method comprising detecting autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid from the subject according to a method as defined in claim 24, and whereby the detected autoantibodies and/or lymphocytes can provide a diagnosis of the likely onset or presence of autoimmune disease associated with an immune reaction to tTG in the subject 56.A method of delaying or preventing the onset of autoimmune disease associated with an immune reaction to tTG in an animal subject suspected of suffering from, susceptible to or recovering from autoimmune disease associated with an immune reaction to tTG, which method comprises initially detecting autoantibodies or lymphocytes indicative of the onset or presence of autoimmune disease associated with an immune reaction to tTG in a sample of body fluid obtained from the subject according to a method as defined in claim 24, thereby providing a diagnosis of the likely onset of autoimmune disease associated with an immune reaction to tTG in the subject, and thereafter therapeutically treating the subject so as to delay the onset and/or prevent autoimmune disease associated with an immune reaction to tTG.\",\n \"57.A method of monitoring dietary compliance by a subject having, suspected of suffering from, susceptible to or recovering from gluten sensitive enteropathy, which method comprises screening for autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from the subject according to a method as defined in claim 24, and whereby the detection of such autoantibodies and/or lymphocytes produced in response to tTG in the sample of body fluid provides an indication as to the dietary compliance of the subject to a gluten-free or substantially gluten free diet.\",\n \"58.A method of treating autoimmune disease associated with an immune reaction to tTG in a subject, which method comprises initially detecting autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from the subject according to a method as defined in claim 24, thereby providing a diagnosis of autoimmune disease in the subject, and administering to the subject a therapeutically effective amount of at least one therapeutic agent effective in the treatment of such autoimmune disease.\",\n \"59.A method of treating autoimmune disease associated with an immune reaction to tTG in a subject, which method comprises administering to the subject a therapeutically effective amount of a therapeutic agent identified as providing a therapeutic effect by interaction with amino acids 1 to 89 of tTG, and/or amino acids 401 to 494 of tTG, or amino acids 401 to 492 of tTG.\",\n \"60.A therapeutic agent identified as providing a therapeutic effect by interaction with amino acids 1 to 89 of tTG, and/or amino acids 401 to 494 of tTG, or amino acids 401 to 491 of tTG.\",\n \"61.A therapeutic agent according to claim 60, for use in the therapeutic treatment of an autoimmune disease associated with an immune reaction to tTG.\",\n \"62.A method of cloning lymphocytes produced in response to tTG, which method comprises: providing a source of lymphocytes; contacting the lymphocytes with a polypeptide as defined in claim 10, so as to effect proliferation of said lymphocytes; and isolating and cloning the proliferating lymphocytes.\",\n \"63.Use of lymphocytes prepared according to claim 62, in the therapeutic treatment of autoimmune disease associated with an immune reaction to tTG.\",\n \"64.A pharmaceutical composition comprising lymphocytes prepared according to claim 62, together with a pharmaceutically acceptable carrier, diluent or excipient therefor.\",\n \"65.Use of lymphocytes prepared according to claim 62, in the manufacture of a medicament for the treatment of autoimmune disease associated with an immune reaction to Ttg.\",\n \"66.Use of lymphocytes prepared according to claim 62, in the manufacture of a medicament for the treatment of coeliac disease.\",\n \"67.A biologically functional plasmid or DNA vector including a DNA sequence as defined in any of claim 2.68.A biologically functional plasmid or DNA vector including a DNA sequence as defined in any of claim 3 69.A biologically functional plasmid or DNA vector including a DNA sequence as defined in any of claim 4.70.A biologically functional plasmid or DNA vector including a DNA sequence as defined in any of claim 5.71.A biologically functional plasmid or DNA vector including a DNA sequence as defined in any of claim 6.72.A biologically functional plasmid or DNA vector including a DNA sequence as defined in any of claim 7.73.A host cell which is transformed or transfected with a plasmid or vector as defined in claim 67.74.A host cell which is transformed or transfected with a plasmid or vector as defined in claim 68.75.A host cell which is transformed or transfected with a plasmid or vector as defined in claim 69.76.A host cell which is transformed or transfected with a plasmid or vector as defined in claim 70.77.A host cell which is transformed or transfected with a plasmid or vector as defined in claim 71.78.A host cell which is transformed or transfected with a plasmid or vector as defined in claim 72.\"\n ],\n [\n \"The present invention relates to tissue transglutaminase (tTG), and more particularly to DNA coding for and polypeptides containing, the primary structural conformation of one or more epitopes of tTG and to the use thereof in the detection of autoantibodies and/or lymphocytes produced in response to tTG.\",\n \"tTG belongs to the class of transglutaminases (TGs).\",\n \"The TGs are enzymes catalysing an acyl transfer depending on Ca2+, the γ-carboxamide groups of peptide-bonded glutamine residues acting as acyl donors.\",\n \"Primarily, protein-bonded lysine residues function as acyl acceptors, so that the transfer results in an ε-(γ-glutamyl)lysine bond.\",\n \"The substrate specificity of the TGs with respect to the acyl donors is very high (depending on the amino acid sequence), whereas an exceptionally wide spectrum of acceptors is available.\",\n \"The covalent peptide bonds formed are highly stable and protease-resistant, resulting in an increased resistance of the crosslinked proteins to chemical, enzymatic or physical effects.\",\n \"There is a widespread occurrence of various TGs in miscellaneous organs, tissues, in plasma and interstitial body fluids, correlating with the occurrence of transglutaminase-modified proteins in blood clots, on cell membranes, in the horny layer of the epidermis, in hair, in nails, and in the extracellular matrix.\",\n \"The described TGs may be distinguished by their physical properties, their location in the body, and their primary structure.\",\n \"tTG is also referred to as cellular, erythrocyte, endothelial, cytoplasmatic, liver, or type II TG, and is a monomer having a molecular weight of 75-85 kDa.\",\n \"The complete amino acid sequence comprising 687 residues has been derived from cDNA.\",\n \"At the protein level, there is an 84% homology between the human enzyme and the enzyme from mouse macrophages and an 81% homology between the human and guinea pig enzymes.\",\n \"There are a number of diseases that may be associated with tTG and can include autoimmune diseases and inflammatory diseases.\",\n \"In particular, autoimmune diseases associated with an immune reaction to tTG can be autoimmune diseases arising due to gluten sensitive enteropathy, such as coeliac disease or sprue.\",\n \"Coeliac disease is a disease of the small intestine mucosa, the first manifestation predominantly occurring during the late infant and toddler ages.\",\n \"If the corresponding clinical picture does not occur before the adult age, it is generally termed non-tropical sprue.\",\n \"Thus, both of these terms describe the same disease, which is hereinafter referred to as coeliac disease.\",\n \"Coeliac disease is estimated to effect about 1 in 300 individuals in Western countries.\",\n \"The characteristic intolerance of dietary gluten present in wheat, barley, rye and related products results in an inflammatory disease of the upper small intestine in genetically susceptible individuals.\",\n \"Typical symptoms include chronic diarrhoea, abdominal distension and failure to thrive.\",\n \"In some patients symptoms of malabsorption such as anaemia and osteoporosis may manifest.\",\n \"Other symptoms include inflammatory lesions in the small bowel, for example, villous atrophy, hypertrophic crypts and increased number of inter-epithelial lymphocytes.\",\n \"Provided the disease is diagnosed in time the symptoms can generally be treated by adhering to a gluten-free diet.\",\n \"However malabsorption may give rise to severe disease symptoms if the disease is not diagnosed and treated.\",\n \"Ultimately, there is the increased risk of developing an intestinal lymphoma and also other gastrointestinal neoplasias.\",\n \"Diagnosis of coeliac disease is currently based on clinical symptoms, histological identification of gluten sensitive enteropathy, and serological tests for observing any response to withdrawal of gluten from an individual's diet.\",\n \"An example of a serological test used for diagnosis and monitoring of coeliac disease is an immunofluorescence test measuring endomysial antibody and measurement of autoantibodies to tTG.\",\n \"WO98/03872 discloses that tTG is an autoantigen assocaited with coeliac disease and specifically discloses an ELISA based on guinea pig tTG of about 60% purity.\",\n \"It would be advantageous to be able to non-invasively efficiently and reliably diagnose and monitor autoimmune diseases associated with tTG, such as coeliac disease as referred to above.\",\n \"For example, the above described immunofluorescence test measuring endomysial antibody requires special expertise and equipment and is dependent on subjective assessment of fluorescence patterns.\",\n \"It would, therefore, be advantageous to be able to replace the immunofluorescence test measuring endomysial antibody with a non-invasive sensitive and specific test for measuring autoantibodies to tTG.\",\n \"It is, therefore, an aim of the present invention to provide an assay system, which alleviates some of the aforementioned problems arising in the diagnosis and monitoring of autoimmune diseases associated with an immune reaction to tTG.\",\n \"It is a further aim of the present invention to provide an assay for measuring autoantibodies or lymphocytes produced in response to tTG, with improved sensitivity and specificity and also provide diagnostic kits for use in the simple and rapid detection of autoantibodies or lymphocytes produced in response to tTG substantially as referred to above.\",\n \"A better understanding of tTG, and epitopes thereof, would be beneficial in achieving the above described aims of the present invention.\",\n \"More particularly, we have now specifically identified that antigenic determinants (or epitopes) of tTG which interact with autoantibodies and/or lymphocytes produced in response to tTG, and present in sera from patients with coeliac disease, are located in the N-terminal and/or central part of the tTG molecule.\",\n \"In particular, our studies have shown that the N-terminal region (typically amino acids 1 to 89) and/or the central region (typically amino acids 401 to 494 or amino acids 401 to 491) of the tTG molecule are particularly important for interaction with autoantibodies and/or lymphocytes produced in response to tTG and present in sera from coeliac patients.\",\n \"There is provided by the present invention, therefore, a DNA sequence encoding: a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes); which DNA sequence comprises: (a) a DNA sequence as depicted in any of FIGS.\",\n \"7, 9, 11, 13, 15, 17, 19, 21, 23 or 25; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of FIGS.\",\n \"8, 10, 12, 14, 16, 18, 20, 22, 24 and 26, or one or more active fragments of amino acids as depicted in one or more of FIGS.\",\n \"8, 10, 12, 14, 16, 18, 20, 22, 24 and 26, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes).\",\n \"There is also provided by the present invention a DNA sequence encoding: a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes); which DNA sequence comprises: (a) a DNA sequence as depicted in any of FIGS.\",\n \"7, 9, 11, 13 or 15; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 1 to 89 of tTG, or one or more active fragments of amino acid numbers 1 to 89 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of FIGS.\",\n \"8, 10, 12, 14 and 16, or one or more active fragments of amino acids as depicted in one or more of FIGS.\",\n \"8, 10, 12, 14 and 16, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes).\",\n \"There is also provided by the present invention a DNA sequence encoding: a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes); which DNA sequence comprises: (a) a DNA sequence as depicted in any of FIGS.\",\n \"17, 19, 21, 23 or 25; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of FIGS.\",\n \"18, 20, 22, 24 and 26, or one or more active fragments of amino acids as depicted in one or more of FIGS.\",\n \"18, 20, 22, 24 and 26, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes).\",\n \"There is also provided by the present invention a DNA sequence encoding: a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies); which DNA sequence comprises: (a) a DNA sequence as depicted in any of FIGS.\",\n \"7, 9, 11, 13, 15, 17, 19, 21, 23 or 25; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of FIGS.\",\n \"8, 10, 12, 14, 16, 18, 20, 22, 24 and 26, or one or more active fragments of amino acids as depicted in one or more of FIGS.\",\n \"8, 10, 12, 14, 16, 18, 20, 22, 24 and 26, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies).\",\n \"There is also provided by the present invention a DNA sequence encoding: a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies); which DNA sequence comprises: (a) a DNA sequence as depicted in any of FIGS.\",\n \"7, 9, 11, 13 or 15; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 1 to 89 of tTG, or one or more active fragments of amino acid numbers 1 to 89 of tTG, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of FIGS.\",\n \"8, 10, 12, 14 and 16, or one or more active fragments of amino acids as depicted in one or more of FIGS.\",\n \"8, 10, 12, 14 and 16, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies).\",\n \"There is also provided by the present invention a DNA sequence encoding: a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies); which DNA sequence comprises: (a) a DNA sequence as depicted in any of FIGS.\",\n \"17, 19, 21, 23 or 25; (b) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (c) a DNA sequence encoding a polypeptide comprising the primary structural conformation of amino acids as depicted in one or more of FIGS.\",\n \"18, 20, 22, 24 and 26, or one or more active fragments of amino acids as depicted in one or more of FIGS.\",\n \"18, 20, 22, 24 and 26, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG); (d) a DNA sequence differing from the DNA sequence of (a) in codon sequence due to the degeneracy of the genetic code; (e) a DNA sequence comprising a fragment or an allelic variation of the DNA sequence of (a); and (f) a DNA sequence which hybridizes to any of DNA sequences (a), (b), (c), (d) or (e) and encodes a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies).\",\n \"A DNA sequence as provided by the present invention can be of human origin (as depicted in FIGS.\",\n \"7 and 77), of guinea pig origin (as depicted in FIGS.\",\n \"9 and 99), of bovine origin (as depicted in FIGS.\",\n \"11 and 11), of mouse origin (as depicted in FIGS.\",\n \"13 and 33), or of rat origin (as depicted in FIGS.\",\n \"15 and 55).\",\n \"In particular, the present invention provides a DNA sequence as depicted in any of FIGS.\",\n \"7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, or a DNA sequence differing therefrom due to the degeneracy of the genetic code.\",\n \"More particularly, a DNA sequence as provided by the present invention can encode part or all of the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or part or all of the primary structural conformation of amino acid numbers 401 to 494 of tTG or part or all of the primary structural conformation of amino acid numbers 401 to 491 of tTG substantially as hereinafter described in greater detail.\",\n \"A DNA sequence as provided by the present invention can be included in a biologically functional plasmid or DNA vector for expressing a polypeptide according to the present invention substantially as hereinafter described in greater detail and there is further provided by the present invention a biologically functional plasmid or DNA vector including a DNA sequence substantially as hereinbefore described.\",\n \"Furthermore, there is still further provided by the present invention a host cell which is transformed or transfected with a DNA sequence substantially as hereinbefore described, or a plasmid or vector substantially as hereinbefore described.\",\n \"A suitable host cell can be any prokaryote or eukaryote cell capable of replicating and expressing a DNA sequence substantially as hereinbefore described and suitable techniques are well known to one of ordinary skill in the art.\",\n \"Polypeptides according to the present invention substantially as herein described can be expressed in various systems generating recombinant proteins.\",\n \"For example, for expression in E coli, cDNA coding for the appropriate polypeptides according to the present invention can be cloned into a vector, such as pMEX8, pGEXcT or pQE-81L His or an equivalent.\",\n \"In the case of expression in yeast (for example Saccharomyces cerevisiae or Schizosaccharomyces pombe), vectors such as pYES2, pESP2 or pYES2/CT or an equivalent, can be employed.\",\n \"AcMNPV (Bac-N-Blue) vector or an equivalent can be used for expression in insect cells and pRC/CMV vector or an equivalent can be used for expression in mammalian cells, such as Chinese Hamster Ovary (CHO) cells.\",\n \"A polypeptide according to the present invention can be expressed as a discrete protein, or as a fusion protein linked to, for example, glutathione S transferase (GST) or poly histidine linker.\",\n \"For a discrete protein, affinity column chromatography purification using a mouse monoclonal antibody to the relevant part of tTG coupled to a Sepharose particle can be used.\",\n \"If a tTG fragment is fused to GST, glutathione Sepharose chromatography purification can be used to isolate the fusion protein.\",\n \"Specific proteases can be used to separate GST from tTG peptide and a second round of glutathione Sepharose chromatography can be used to separate GST from a tTG fragment.\",\n \"In the case of peptides linked to poly histidine linker, the purification can be carried out using immobilised metal affinity chromatography.\",\n \"There is also provided by the present invention a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes); with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length Ttg).\",\n \"There is also provided by the present invention a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, or one or more active fragments of amino acid numbers 1 to 89 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).\",\n \"There is also provided by the present invention a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), which polypeptide comprises the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes); with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).\",\n \"There is also provided by the present invention a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies) and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 1 to 89 of tTG, and/or amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies); with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).\",\n \"There is also provided by the present invention a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies) and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), which polypeptide comprises the primary structural conformation of amino acid numbers 1 to 89 of tTG, or one or more active fragments of amino acid numbers 1 to 89 of tTG, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).\",\n \"There is also provided by the present invention a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies) and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), which polypeptide comprises the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of: amino acid numbers 401 to 494 of tTG or amino acid numbers 401 to 491 of tTG, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies); with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).\",\n \"The present invention further provides a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of FIGS.\",\n \"8, 10, 12, 14, 16, 18, 20, 22, 24 and 26, or one or more active fragments of amino acids as depicted in one or more of FIGS.\",\n \"8, 10, 12, 14, 16, 18, 20, 22, 24 and 26, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).\",\n \"The present invention further provides a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of FIGS.\",\n \"8, 10, 12, 14 and 16, or one or more active fragments of amino acids as depicted in one or more of FIGS.\",\n \"8, 10, 12, 14 and 16, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).\",\n \"The present invention further provides a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of FIGS.\",\n \"18, 20, 22, 24 and 26, or one or more active fragments of amino acids as depicted in one or more of FIGS.\",\n \"18, 20, 22, 24 and 26, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).\",\n \"The present invention further provides a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of FIGS.\",\n \"8, 10, 12, 14, 16, 18, 20, 22, 24 and 26, or one or more active fragments of amino acids as depicted in one or more of FIGS.\",\n \"8, 10, 12, 14, 16, 18, 20, 22, 24 and 26, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).\",\n \"The present invention further provides a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of FIGS.\",\n \"8, 10, 12, 14 and 16, or one or more active fragments of amino acids as depicted in one or more of FIGS.\",\n \"8, 10, 12, 14 and 16, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).\",\n \"The present invention further provides a polypeptide with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies) and which comprises part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), which polypeptide comprises the primary structural conformation of amino acids as depicted in one or more of FIGS.\",\n \"18, 20, 22, 24 and 26, or one or more active fragments of amino acids as depicted in one or more of FIGS.\",\n \"18, 20, 22, 24 and 26, with which autoantibodies produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).\",\n \"A polypeptide as provided by the present invention can be of human origin (as depicted in FIGS.\",\n \"8 and 88), of guinea pig origin (as depicted in FIGS.\",\n \"10 and 20), of bovine origin (as depicted in FIGS.\",\n \"12 and 22), of mouse origin (as depicted in FIGS.\",\n \"14 and 44), or of rat origin (as depicted in FIGS.\",\n \"16 and 66).\",\n \"In particular, the present invention provides a polypeptide as depicted in any of FIGS.\",\n \"8, 10, 12, 14, 16, 18, 20, 22, 24 or 26.Typically, a polypeptide according to the present invention is a synthetic polypeptide derived by recombinant techniques substantially as herein described and is most suitably derived from recombinant human tTG typically with at least 70% purity, more preferably at least 90% purity and even more preferably at least 95% purity.\",\n \"Purity may be assessed by SDS PAGE analysis and Coomassie Blue staining as hereinafter discussed in connection with FIG.\",\n \"1.The recombinant tTG (typically human) may be purified using affinity chromatography with tTG antibodies such as mouse tTG monoclonal antibody.\",\n \"A polypeptide according to the present invention is preferably obtained by, or is obtainable by, expression of a DNA sequence according to the present invention substantially as hereinbefore described.\",\n \"A polypeptide according to the present invention obtained by such expression can be advantageous in being free from association with other eukaryotic polypeptides or contaminants which might otherwise be associated with tTG in its natural environment.\",\n \"The present invention further provides a process of preparing a polypeptide substantially as hereinbefore described, which process comprises: (i) providing a host cell substantially as hereinbefore described; (ii) growing the host cell; and (iii) recovering a polypeptide according to the present invention therefrom.\",\n \"Recovery of a polypeptide according to the present invention can typically employ conventional isolation and purification techniques, such as chromatographic separations or immunological separations, known to one of ordinary skill in the art.\",\n \"It is of course appreciated that there is provided within the scope of the present invention, one or more amino acid sequences or fragments thereof structurally distinct from the specific amino acid sequences of a polypeptide described herein (for example by the addition, deletion, substitution or insertion of one or more amino acids) but having substantially the same functional binding properties as the specific amino acid sequences of a polypeptide described herein.\",\n \"In other words, the present invention clearly encompasses within its scope amino acid sequences or fragments thereof substantially homologous to specific amino acid sequences of a polypeptide described herein, or representing variants of specific amino acid sequences of a polypeptide described herein, but having substantially the same functional binding properties as the specific amino acid sequences of a polypeptide described herein.\",\n \"A polypeptide according to the present invention can in a first preferred embodiment of the present invention be a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), which polypeptide consists essentially of (or consists of) the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or more particularly consists essentially of (or consists of) the primary structural conformation of amino acids as depicted in one or more of FIGS.\",\n \"8, 10, 12, 14, 16, 18, 20, 22, 24 or 26, substantially as hereinbefore described.\",\n \"Alternatively, in a second preferred embodiment of the present invention there is provided a polypeptide with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), which polypeptide consists essentially of (or consists of) one or more active fragments of amino acid numbers 1 to 89 of tTG, and/or one or more active fragments of amino acid numbers 401 to 494 of tTG or one or more active fragments of amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), or more particularly consists essentially of (or consists of) the primary structural conformation of one or more active fragments of amino acids as depicted in one or more of FIGS.\",\n \"8, 10, 12, 14, 16, 18, 20, 22, 24 or 26, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) substantially as hereinbefore described.\",\n \"For example, a polypeptide consisting essentially of (or consisting of) amino acid numbers 1 to 85 of tTG, amino acid numbers 5 to 89 of tTG, or the like, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes).\",\n \"A still further alternative in a third preferred embodiment of the present invention is a polypeptide (in particular a synthetic polypeptide, such as a scaffold type polypeptide typically providing amino acids corresponding to the amino acids of tTG as described herein in a form specifically adapted for use in an assay method or kit according to the present invention substantially as hereinafter described in greater detail) with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes) and which includes part or all of the primary structural conformation of one or more epitopes of tTG with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), which polypeptide includes amino acids comprising the primary structural conformation of amino acid numbers 1 to 89 of tTG, and/or the primary structural conformation of amino acid numbers 401 to 494 of tTG or the primary structural conformation of amino acid numbers 401 to 491 of tTG, or one or more active fragments of amino acid numbers 1 to 89 of tTG, and/or one or more active fragments of amino acid numbers 401 to 494 of tTG or one or more active fragments of amino acid numbers 401 to 491 of tTG, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes); or more particularly which includes amino acids comprising the primary structural conformation of amino acids as depicted in one or more of FIGS.\",\n \"8, 10, 12, 14, 16, 18, 20, 22, 24 or 26, or one or more active fragments of amino acids as depicted in one or more of FIGS.\",\n \"8, 10, 12, 14, 16, 18, 20, 22, 24 or 26, with which autoantibodies and/or lymphocytes produced in response to tTG can interact (under conditions that allow interaction of tTG with such autoantibodies or lymphocytes), with the exception of human, guinea pig, mouse, bovine or rat full length tTG (or more generally full length tTG).\",\n \"According to a particularly preferred embodiment of the present invention, there is also provided a method of screening for autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from a subject (in particular a human) suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said method comprising: (a) providing either (i) said sample of body fluid from said subject or (ii) lymphocytes isolated from said sample; (b) contacting said sample or isolated lymphocytes with a polypeptide according to the present invention substantially as hereinbefore described (under conditions that allow interaction of tTG with autoantibodies or lymphocytes produced in response to tTG) so as to permit said polypeptide to interact with autoantibodies, or lymphocytes, produced in response to tTG, and present in, or isolated from, said sample; and (c) monitoring the degree, or effect, of interaction of said polypeptide with either said autoantibodies, or said lymphocytes, produced in response to tTG and present in, or isolated from, said sample, thereby providing an indication of the presence of said autoantibodies, or said lymphocytes, in said sample, or isolated from said sample.\",\n \"Substantially as described above, a method according to the present invention is suitable for screening for autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from a subject.\",\n \"A method according to the present invention can, however, be particularly adapted for use in screening for autoantibodies produced in response to tTG in a sample of body fluid obtained from a subject substantially as hereinafter described in greater detail.\",\n \"There is in particular provided by the present invention, therefore, a method of screening for autoantibodies produced in response to tTG in a sample of body fluid obtained from a subject (in particular a human) suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said method comprising: (a) providing said sample of body fluid from said subject; (b) contacting said sample with a polypeptide according to the present invention substantially as hereinbefore described (under conditions that allow interaction of tTG with autoantibodies produced in response to tTG) so as to permit said polypeptide to interact with autoantibodies produced in response to tTG and present in said sample; and (c) monitoring the degree of interaction of said polypeptide with said autoantibodies produced in response to tTG and present in said sample, thereby providing an indication of the presence of said autoantibodies in said sample.\",\n \"A method according to the present invention may typically employ a control, such as a sample of body fluid from a normal subject, in other words a subject known to be without autoimmune disease associated with an immune reaction to tTG.\",\n \"A method of screening for autoantibodies to tTG according to the present invention may comprise directly monitoring interaction of (i) autoantibodies to tTG present in the sample of body fluid from the subject and (ii) a polypeptide, as provided by the present invention substantially as hereinbefore described, typically by employing non-competitive sandwich type assay techniques known in the art.\",\n \"Typically, in a method according to the present invention employing non-competitive techniques, monitoring of the degree of interaction of (i) autoantibodies to tTG present in the sample and (ii) a polypeptide according to the present invention substantially as hereinbefore described, can comprise providing labelling means either to a polypeptide according to the present invention substantially as hereinbefore described, or to a binding partner for autoantibodies to tTG, either of which technique would enable monitoring of the above described interaction.\",\n \"For example, a method according to the present invention may comprise directly or indirectly labelling a polypeptide according to the present invention substantially as hereinbefore described; contacting the thus labelled polypeptide with a sample of body fluid being screened for tTG autoantibodies so as to provide a mixture thereof; and adding to the mixture a binding partner for autoantibodies to tTG (such as an anti-IgG reagent) present in the sample of body fluid, so as to cause precipitation of any complexes of labelled polypeptide and tTG autoantibodies present in the mixture.\",\n \"Alternatively, it may be preferred that a method according to the present invention further comprises adding a labelled binding partner for tTG autoantibodies (such as a labelled anti-IgG reagent, for example protein A or anti-human IgG, or labelled full length tTG or an epitope thereof) to a mixture obtained by contacting (i) a polypeptide according to the present invention substantially as hereinbefore described immobilised to a support and (ii) a sample of body fluid being screened for autoantibodies to tTG.\",\n \"It may alternatively be preferred that a method of screening for autoantibodies to tTG in the sample of body fluid according to the present invention, utilises the principles employed in known competitive assays.\",\n \"For example, a method according to the present invention may employ at least one competitor capable of competing with autoantibodies to tTG in the interaction thereof with a polypeptide according to the present invention substantially as hereinbefore described.\",\n \"Typically, a competitor as employed in a competitive assay method according to the present invention may comprise one or more antibodies, which may be natural or partly or wholly synthetically produced.\",\n \"A competitor as employed in the present invention may alternatively comprise any other protein having a binding domain or region which is capable of competing with autoantibodies to tTG in the interaction thereof with a polypeptide according to the present invention substantially as hereinbefore described.\",\n \"Preferably, however, a competitor as employed in the present invention comprises a monoclonal, recombinant or polyclonal antibody (especially a monoclonal antibody), capable of competing with tTG autoantibodies in the interaction thereof with a polypeptide according to the present invention substantially as hereinbefore described.\",\n \"Typically, therefore, a competitive assay method according to the present invention may further comprise providing at least one competitor, such as a monoclonal or polyclonal antibody, whereby in step (b) of a method as herein described a polypeptide according to the present invention substantially as hereinbefore described can interact with either a competitor, such as a monoclonal or polyclonal antibody, or autoantibodies to tTG present in said sample.\",\n \"Typically monitoring in a competitive assay method according to the present invention comprises comparing: (i) interaction of a polypeptide according to the present invention substantially as hereinbefore described and one or more competitors substantially as hereinbefore described (typically a monoclonal or polyclonal antibody), in the absence of said sample of body fluid being screened (that is a suspected disease sample), optionally in the presence of a sample of body fluid from a normal subject, typically a subject known to be without autoimmune disease associated with an immune reaction to tTG; with (ii) interaction of a polypeptide according to the present invention substantially as hereinbefore described and one or more competitors substantially as hereinbefore described (typically a monoclonal or polyclonal antibody), in the presence of said sample of body fluid being screened.\",\n \"Typically, the comparison involves observing a decrease in interaction of a polypeptide according to the present invention substantially as hereinbefore described and the competitor in (ii) compared to (i) so as to provide an indication of the presence of autoantibodies to tTG in said sample.\",\n \"Typically, the decrease in interaction can be observed by directly or indirectly labelling the competitor and monitoring any change in the interaction of the thus labelled competitor with a polypeptide according to the present invention substantially as hereinbefore described in the absence and in the presence of a sample of body fluid being screened for autoantibodies to tTG.\",\n \"Suitably a polypeptide according to the present invention substantially as hereinbefore described may be immobilised to facilitate the above mentioned monitoring.\",\n \"Alternatively, there is also provided by the present invention a method of screening for autoantibodies to tTG in a sample of body fluid obtained from a subject (in particular a human) suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said method comprising: (a) providing said sample of body fluid from said subject; (b) contacting said sample with (i) full length tTG (typically recombinantly obtained full length tTG), and (ii) at least one competitor capable of competing with autoantibodies to tTG in the interaction thereof with a polypeptide according to the present invention substantially as hereinbefore described, (under conditions that allow interaction of tTG with autoantibodies to tTG), so as to permit said full length tTG to interact with either autoantibodies to tTG present in said sample, or said competitor; and (c) monitoring the interaction of said full length tTG with said autoantibodies present in said sample, thereby providing an indication of the presence of said autoantibodies to tTG in said sample.\",\n \"The full length tTG can typically be of human, guinea pig, bovine, mouse or rat origin, preferably human, and more preferably human recombinantly obtained full length tTG.\",\n \"A competitor for use in such an assay typically comprises a monoclonal or polyclonal antibody (preferably monoclonal) substantially as hereinbefore described.\",\n \"Suitably a detectable label that can be employed in a method according to the present invention can be selected from the group consisting of enzymic labels, isotopic labels, chemiluminescent labels, fluorescent labels, dyes and the like.\",\n \"In the case where an isotopic label (such as 125I, 14C, 3H or 35S) is employed, monitoring may therefore comprise measuring radioactivity dependent on binding of a polypeptide according to the present invention substantially as hereinbefore described.\",\n \"Radioactivity is generally measured using a gamma counter, or liquid scintillation counter.\",\n \"In the case of a method of screening for lymphocytes according to the present invention, it is generally preferred that lymphocytes are initially isolated from a sample of body fluid from a subject using techniques well known to one of ordinary skill in the art, followed by contact with a polypeptide according to the present invention so as to stimulate the proliferation of the isolated lymphocytes.\",\n \"Monitoring of the effect of interaction of a polypeptide according to the present invention and such proliferating lymphocytes, typically employs means known in the art for monitoring such proliferation of lymphocytes.\",\n \"According to a further particularly preferred embodiment of the present invention, there is provided a kit for screening for autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from a subject (in particular a human) suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said kit comprising: (a) a polypeptide according to the present invention substantially as hereinbefore described; (b) means for contacting either (i) a sample of body fluid obtained from said subject, or (ii) lymphocytes isolated from a sample of body fluid obtained from said subject, with said polypeptide according to the present invention substantially as hereinbefore described (under conditions that allow interaction of tTG with autoantibodies or lymphocytes produced in response to tTG) so as to permit said polypeptide to interact with autoantibodies, or lymphocytes, produced in response to tTG, and present in, or isolated from, said sample; and (c) means for monitoring the degree, or effect, of interaction of said polypeptide with either said autoantibodies, or said lymphocytes, produced in response to tTG and present in, or isolated from, said sample, thereby providing an indication of the presence of said autoantibodies, or lymphocytes, in said sample or isolated from said sample.\",\n \"Substantially as described above, a kit according to the present invention is suitable for screening for autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from a subject.\",\n \"A kit according to the present invention can, however, be particularly adapted for use in screening for autoantibodies produced in response to tTG in a sample of body fluid obtained from a subject substantially as hereinafter described in greater detail.\",\n \"There is in particular provided by the present invention, therefore, a kit for screening for autoantibodies produced in response to tTG in a sample of body fluid obtained from a subject (in particular a human) suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said kit comprising: (a) a polypeptide according to the present invention substantially as hereinbefore described; (b) means for contacting a sample of body fluid obtained from said subject with said polypeptide according to the present invention substantially as hereinbefore described (under conditions that allow interaction of tTG with autoantibodies produced in response to tTG) so as to permit said polypeptide to interact with autoantibodies produced in response to tTG and present in said sample; and (c) means for monitoring the degree of interaction of said polypeptide with said autoantibodies produced in response to tTG and present in said sample, thereby providing an indication of the presence of said autoantibodies in said sample.\",\n \"A kit according to the present invention may typically further comprise control means, such as means for providing a sample of body fluid from a normal subject, in other words a subject known to be without autoimmune disease associated with an immune reaction to tTG.\",\n \"A kit for screening for autoantibodies to tTG according to the present invention may comprise means for directly monitoring interaction of (i) autoantibodies to tTG present in the sample of body fluid from the subject and (ii) a polypeptide, as provided by the present invention substantially as hereinbefore described, typically comprising non-competitive sandwich type assay means known in the art.\",\n \"Typically, in a kit according to the present invention comprising non-competitive assay means, means are provided for monitoring the degree of interaction of (i) autoantibodies to tTG present in the sample and (ii) a polypeptide according to the present invention substantially as hereinbefore described, and can comprise labelling means provided either to a polypeptide according to the present invention substantially as hereinbefore described, or to a binding partner for autoantibodies to tTG, either of which would enable monitoring of the above described interaction.\",\n \"For example, a kit according to the present invention may comprise means for directly or indirectly labelling a polypeptide according to the present invention substantially as hereinbefore described; means for contacting the thus labelled polypeptide with a sample of body fluid being screened for tTG autoantibodies so as to provide a mixture thereof; a binding partner for autoantibodies to tTG (such as an anti-IgG reagent) present in the sample of body fluid; and means for adding the binding partner to the mixture so as to cause precipitation of any complexes of labelled polypeptide and tTG autoantibodies present in the mixture.\",\n \"Alternatively, it may be preferred that a kit according to the present invention further comprises a labelled binding partner for tTG autoantibodies (such as a labelled anti-IgG reagent, for example protein A or anti-human IgG, or labelled full length tTG or an epitope thereof) and means for adding the labelled binding partner to a mixture obtained by contacting (i) a polypeptide according to the present invention substantially as hereinbefore described immobilised to a support and (ii) a sample of body fluid being screened for autoantibodies to tTG.\",\n \"It may alternatively be preferred that a kit for screening for autoantibodies to tTG in the sample of body fluid according to the present invention, comprises known competitive assay means.\",\n \"For example, a kit according to the present invention may further comprise at least one competitor capable of competing with autoantibodies to tTG in the interaction thereof with a polypeptide according to the present invention substantially as hereinbefore described.\",\n \"Typically, a competitor as employed in a competitive assay kit according to the present invention may comprise one or more antibodies, which may be natural or partly or wholly synthetically produced.\",\n \"A competitor as employed in the present invention may alternatively comprise any other protein having a binding domain or region which is capable of competing with autoantibodies to tTG in the interaction thereof with a polypeptide according to the present invention substantially as hereinbefore described.\",\n \"Preferably, however, a competitor as employed in the present invention comprises a monoclonal or polyclonal antibody (especially a monoclonal antibody), capable of competing with tTG autoantibodies in the interaction thereof with a polypeptide according to the present invention substantially as hereinbefore described.\",\n \"Typically, therefore, a competitive assay kit according to the present invention may further comprise at least one competitor, such as a monoclonal or polyclonal antibody, whereby a polypeptide according to the present invention substantially as hereinbefore described can interact with either a competitor, such as a monoclonal or polyclonal antibody, or autoantibodies to tTG present in a sample of body fluid being screened.\",\n \"Typically monitoring means in a competitive assay kit according to the present invention comprise means for comparing: (i) interaction of a polypeptide according to the present invention substantially as hereinbefore described and one or more competitors substantially as hereinbefore described (typically a monoclonal or polyclonal antibody), in the absence of said sample of body fluid being screened (that is a suspected disease sample), optionally in the presence of a sample of body fluid from a normal subject, typically a subject known to be without autoimmune disease associated with an immune reaction to tTG; with (ii) interaction of a polypeptide according to the present invention substantially as hereinbefore described and one or more competitors substantially as hereinbefore described (typically a monoclonal or polyclonal antibody), in the presence of said sample of body fluid being screened.\",\n \"Typically, the comparison involves observing a decrease in interaction of a polypeptide according to the present invention substantially as hereinbefore described and the competitor in (ii) compared to (i) so as to provide an indication of the presence of autoantibodies to tTG in said sample.\",\n \"Typically, the decrease in interaction can be observed by directly or indirectly labelling the competitor and monitoring any change in the interaction of the thus labelled competitor with a polypeptide according to the present invention substantially as hereinbefore described in the absence and in the presence of a sample of body fluid being screened for autoantibodies to tTG.\",\n \"Suitably a polypeptide according to the present invention substantially as hereinbefore described may be immobilised to facilitate the above mentioned monitoring.\",\n \"Alternatively, there is also provided by the present invention a kit for screening for autoantibodies to tTG in a sample of body fluid obtained from a subject (in particular a human) suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, said kit comprising: (a) full length tTG (typically recombinantly obtained full length tTG); (b) at least one competitor capable of competing with autoantibodies to tTG in the interaction thereof with a polypeptide according to the present invention substantially as hereinbefore described, (c) means for contacting said sample of body fluid from said subject, said full length tTG and said competitor (under conditions that allow interaction of tTG with autoantibodies to tTG), so as to permit said full length tTG to interact with either autoantibodies to tTG present in said sample, or said competitor; and (d) means for monitoring the interaction of said full length tTG with said autoantibodies present in said sample, thereby providing an indication of the presence of said autoantibodies to tTG in said sample.\",\n \"The full length tTG can typically be of human, guinea pig, bovine, mouse or rat origin, preferably human, and more preferably human recombinantly obtained full length tTG.\",\n \"A competitor for use in such an assay kit typically comprises a monoclonal or polyclonal antibody (preferably monoclonal) substantially as hereinbefore described.\",\n \"Suitably a detectable label that can be employed in a kit according to the present invention can be selected from the group consisting of enzymic labels, isotopic labels, chemiluminescent labels, fluorescent labels, dyes and the like.\",\n \"In the case where an isotopic label (such as 125I, 14C, 3H or 35S) is employed, monitoring means may therefore comprise means for measuring radioactivity dependent on binding of a polypeptide according to the present invention substantially as hereinbefore described.\",\n \"Radioactivity is generally measured using a gamma counter, or liquid scintillation counter.\",\n \"In the case of a kit for screening for lymphocytes according to the present invention, it is generally preferred that means are provided for initially isolating lymphocytes from a sample of body fluid from a subject, using techniques well known to one of ordinary skill in the art, and means are also provided for contacting a polypeptide according to the present invention to the present invention with such isolated lymphocytes so as to stimulate proliferation of the latter by the former.\",\n \"Means (again known to one of ordinary skill in the art) for monitoring the effect of interaction of a polypeptide according to the present invention and such proliferating lymphocytes, are also provided in such a kit according to the present invention.\",\n \"There is still further provided by the present invention an antibody produced in response to one or more epitope regions of tTG, which epitope region comprises part or all of the primary structural conformation of amino acids 1 to 89 of tTG, or part or all of the primary structural conformation of amino acids 401 to 494 or 401 to 491 of tTG, or one or more fragments of an antibody (such as Fab fragments) produced in response to one or more epitope regions of tTG, which epitope region comprises part or all of the primary structural conformation of amino acids 1 to 89 of tTG, or part or all of the primary structural conformation of amino acids 401 to 494 or 401 to 491 of tTG.\",\n \"An antibody according to the present invention or fragment thereof can interact with a polypeptide according to the present invention substantially as hereinbefore described and preferably is obtainable by, or is obtained by, techniques described in the Examples.\",\n \"Suitably, an antibody provided by the present invention can be monoclonal (preferred), recombinant or polyclonal.\",\n \"Typically an antibody, such as a monoclonal antibody, as provided by the present invention is in substantially purified form.\",\n \"More specifically, a monoclonal antibody as provided by the present invention can comprise a monoclonal antibody Mab A3-6B5, or a monoclonal antibody Mab A5-4E6, or one or more active fragments thereof (such as Fab fragments), as described in the Examples.\",\n \"The present invention further provides a host cell containing an antibody substantially as described above and also a hybridoma capable of secreting a monoclonal antibody substantially as hereinbefore described.\",\n \"There is still further provided by the present invention an antibody substantially as hereinbefore described, or one or more fragments of an antibody (such as Fab fragments) substantially as hereinbefore described, for use in diagnosis or therapy, in particular diagnosis or therapy of autoimmune disease associated with an immune reaction to tTG substantially as hereinbefore described.\",\n \"Particularly the autoimmune disease can be coeliac disease (although other autoimmune diseases substantially as hereinafter described in greater detail can be diagnosed or treated by an antibody according to the present invention) and the present invention also provides an antibody substantially as hereinbefore described for use in the manufacture of a medicament for the treatment of coeliac disease.\",\n \"There is still further provided by the present invention a polypeptide according to the present invention substantially as hereinbefore described, for use in diagnosis or therapy, in particular diagnosis or therapy of autoimmune disease associated with an immune reaction to tTG substantially as hereinbefore described.\",\n \"In particular, autoimmune disease associated with an immune reaction to tTG as described herein comprises autoimmune disease arising due to gluten sensitive enteropathy in a subject, in particular a human subject, such as coeliac disease.\",\n \"Other autoimmune diseases associated with an immune reaction to tTG that can be diagnosed or treated by the present invention include any of the following—dermatitis herpetiformis, Addison's disease, A1 (A1-autoimmune) haemolytic anaemia, A1 thrombocytopenic purpura, A1 thyroid diseases, A1 hyperthyroidism (Graves' disease), A1 hypothyroidism (including Hashimoto's thyroiditis), pernicious anaemia, myasthenia gravis, primary biliary cirrhosis, rheumatoid arthritis, Sjogren's syndrome, SLE, type 1 (insulin-dependent) diabetes mellitus, diabetes mellitus of other types, Wegener granulomatosis, and vitiligo.\",\n \"Substantially as herein described autoimmune disease associated with an immune reaction to tTG can be screened for or diagnosed in a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG.\",\n \"The patient group that can be classed as susceptible to an autoimmune disease associated with an immune reaction to tTG can typically include relatives of subjects previously diagnosed as having an autoimmune disease associated with an immune reaction to tTG, or subjects previously diagnosed with a primary or first autoimmune disease associated with an immune reaction to tTG and as such could be considered to be at risk of or prone to be suffering from a secondary or second autoimmune disease associated with an immune reaction to tTG.\",\n \"For example, a subject known to be suffering from type 1 diabetes mellitus could be considered to be susceptible to suffering from coeliac disease.\",\n \"It will be appreciated from the foregoing description that the present invention provides assay methods and kits for detecting autoantibodies (in particular) or lymphocytes produced in response to tTG in a sample of body fluid substantially as hereinbefore described.\",\n \"The detection of such autoantibodies and/or lymphocytes produced in response to tTG in the sample of body fluid (or at least the level of such autoantibodies and/or lymphocytes in the sample) is indicative of the presence of autoimmune disease associated with an immune reaction to tTG in the subject from which the sample was obtained and can, therefore, enable the diagnosis of the likely onset or presence of autoimmune disease associated with an immune reaction to tTG.\",\n \"There is, therefore, further provided by the present invention a method of diagnosing the likely onset or presence of autoimmune disease associated with an immune reaction to tTG in a subject (in particular a human) suspected of suffering from, susceptible to, having or recovering from, autoimmune disease associated with an immune reaction to tTG, the method comprising detecting autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid from the subject substantially as hereinbefore described, and whereby the detected autoantibodies and/or lymphocytes can provide a diagnosis of the likely onset or presence of autoimmune disease associated with an immune reaction to tTG in the subject.\",\n \"There is still further provided by the present invention a method of delaying or preventing the onset of autoimmune disease associated with an immune reaction to tTG in an animal subject (in particular a human subject) suspected of suffering from, susceptible to or recovering from autoimmune disease associated with an immune reaction to tTG, which method comprises initially detecting autoantibodies or lymphocytes indicative of the onset or presence of autoimmune disease associated with an immune reaction to tTG in a sample of body fluid obtained from the subject substantially as hereinbefore described, thereby providing a diagnosis of the likely onset of autoimmune disease associated with an immune reaction to tTG in the subject, and thereafter therapeutically treating the subject so as to delay the onset and/or prevent autoimmune disease associated with an immune reaction to tTG.\",\n \"Typically, such therapeutic treatment can, in the case of gluten sensitive enteropathy giving rise to coeliac disease, comprise compliance with a gluten-free or substantially gluten free diet.\",\n \"The present invention can further provide a method of monitoring dietary compliance by a subject (in particular a human subject) having, suspected of suffering from, susceptible to or recovering from gluten sensitive enteropathy, which method comprises screening for autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from the subject substantially as hereinbefore described, and whereby the detection of such autoantibodies and/or lymphocytes produced in response to tTG in the sample of body fluid (or at least the level of such autoantibodies and/or lymphocytes in the sample) provides an indication as to the dietary compliance of the subject to a gluten-free or substantially gluten free diet.\",\n \"A polypeptide according to the present invention substantially as hereinbefore described is particularly suitable for use in the therapeutic treatment of autoimmune disease associated with an immune reaction to tTG.\",\n \"For example, tolerance can be achieved by administering a polypeptide according to the present invention substantially as hereinbefore described to a subject (in particular a human subject) suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG.\",\n \"There is, therefore, further provided by the present invention a pharmaceutical composition comprising a polypeptide according to the present invention substantially as hereinbefore described, together with a pharmaceutically acceptable carrier, diluent or excipient therefor, wherein the polypeptide can interact with autoantibodies and/or lymphocytes produced in response to tTG.\",\n \"The present invention further provides a polypeptide according to the present invention substantially as hereinbefore described for use in the manufacture of a medicament for the treatment of coeliac disease.\",\n \"Compositions or medicaments according to the present invention should contain a therapeutic or prophylactic amount of at least one polypeptide according to the present invention in a pharmaceutically-acceptable carrier.\",\n \"The pharmaceutical carrier can be any compatible, non-toxic substance suitable for delivery of the polypeptides to the patient.\",\n \"Sterile water, alcohol, fats, waxes, and inert solids may be used as the carrier.\",\n \"Pharmaceutically-acceptable adjuvants, buffering agents, dispersing agents and the like, may also be incorporated into the pharmaceutical compositions.\",\n \"Such compositions can contain a single polypeptide or may contain two or more polypeptides according to the present invention.\",\n \"It may be desirable to couple a polypeptide according to the present invention to immunoglobulins, e.g.\",\n \"IgG, or to lymphoid cells from the patient being treated in order to promote tolerance.\",\n \"Such an approach is described in Bradley-Mullen, Activation of Distinct Subsets of T Suppressor Cells with Type III Pneumococcal Polysaccharide Coupled to Syngeneic Spleen Cells, in: IMMUNOLOGICAL TOLERANCE TO SELF AND NON-SELF, Buttisto et al., eds., Annals N.Y. Acad.\",\n \"Sci.\",\n \"Vol.\",\n \"392, pp 156-166, 1982.Alternatively, the polypeptides may be modified to maintain or enhance binding to the MHC while reducing or eliminating binding to the associated T-cell receptor.\",\n \"In this way, the modified polypeptides may compete with natural tTG to inhibit helper T-cell activation and thus inhibit the immune response.\",\n \"In all cases, care should be taken that administration of the pharmaceutical compositions of the present invention ameliorate but do not potentiate the autoimmune response.\",\n \"Pharmaceutical compositions according to the present invention are useful for parenteral administration.\",\n \"Preferably, the compositions will be administered parenterally, i.e.\",\n \"subcutaneously, intramuscularly, or intravenously.\",\n \"Thus, the invention provides compositions for parenteral administration to a patient, where the compositions comprise a solution or dispersion of the polypeptides in an acceptable carrier, as described above.\",\n \"The concentration of the polypeptides in the pharmaceutical composition can vary widely, i.e.\",\n \"from less than about 0.1% by weight, usually being at least about 1% by weight to as much as 20% by weight or more.\",\n \"Typical pharmaceutical compositions for intramuscular injection would be made up to contain, for example, 1 ml of sterile buffered water and 1 to 100 μg of a purified polypeptide of the present invention.\",\n \"A typical composition for intravenous infusion could be made up to contain 100 to 500 ml of sterile Ringer's solution and 100 to 500 mg of a purified polypeptide of the present invention.\",\n \"Actual methods for preparing parenterally administrable compositions are well known in the art and described in more detail in various sources, including, for example, Remington's Pharmaceutical Science, 15th Edition, Mack Publishing Company, Easton, Pa. (1980).\",\n \"In addition to using a polypeptide according to the present invention directly in pharmaceutical compositions, it is also possible to use a polypeptide according to the present invention to enhance tolerance to tTG in a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG, employing the following principles.\",\n \"More particularly, peripheral blood lymphocytes can be collected from the subject in a conventional manner and stimulated by exposure to a polypeptide according to the present invention, as defined above.\",\n \"Usually, other mitogens and growth enhancers will be present, e.g., phytohemagglutinin, interleukin 2, and the like.\",\n \"Proliferating T-helper cells may be isolated and cloned, also under the stimulation of a polypeptide according to the present invention.\",\n \"Clones which continue to proliferate may then be used to prepare therapeutic compositions for the subject.\",\n \"The cloned T-cells may be attenuated, e.g.\",\n \"by exposure to radiation, and administered to the subject in order to induce tolerance.\",\n \"Alternatively, the T-cell receptor or portions thereof may be isolated by conventional protein purification methods from the cloned T-cells and administered to the individual.\",\n \"Such immunotherapy methods are described generally in Sinha et al.\",\n \"(1990) Science 248:1380-1388.In some cases, after a T-helper cell has been cloned as described above, it may be possible to develop therapeutic peptides from the T-cell receptor, where the peptides would be beneficial for treating a patient population suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to tTG.\",\n \"In such cases, the T-cell receptor gene may be isolated and cloned by conventional techniques and peptides based on the receptor produced by recombinant techniques as described above.\",\n \"The recombinantly-produced peptides may then be incorporated in pharmaceutical compositions as described above.\",\n \"There is also provided by the present invention a method of cloning lymphocytes produced in response to tTG, which method comprises: providing a source of lymphocytes; contacting the lymphocytes with a polypeptide according to the present invention substantially as hereinbefore described, so as to effect proliferation of said lymphocytes; and isolating and cloning the proliferating lymphocytes.\",\n \"The present invention also provides the use of cloned lymphocytes prepared as above, in the therapeutic treatment of autoimmune disease associated with an immune reaction to tTG.\",\n \"There is provided, therefore, a pharmaceutical composition comprising cloned lymphocytes prepared as above, together with a pharmaceutically acceptable carrier, diluent or excipient therefor and the use of such cloned lymphocytes in the manufacture of a medicament for the treatment of autoimmune disease associated with an immune reaction to tTG, in particular coeliac disease.\",\n \"There is also provided by the present invention one or more therapeutic agents identified as providing a therapeutic effect by interaction with amino acids 1 to 89 of tTG, and/or amino acids 401 to 494 of tTG, or amino acids 401 to 491 of tTG, and the present invention further provides one or more therapeutic agents for use in therapeutically interacting with amino acids 1 to 89 of tTG, and/or amino acids 401 to 494 of tTG or amino acids 401 to 491 of Ttg and as such for use in the therapeutic treatment of an autoimmune disease associated with an immune reaction to tTG.\",\n \"There is, therefore, still further provided by the present invention a method of treating autoimmune disease associated with an immune reaction to tTG in a subject, which method comprises initially detecting autoantibodies or lymphocytes produced in response to tTG in a sample of body fluid obtained from the subject substantially as hereinbefore described, thereby providing a diagnosis of autoimmune disease in the subject, and administering to the subject a therapeutically effective amount of at least one therapeutic agent effective in the treatment of such autoimmune disease, such as a polypeptide according to the present invention substantially as hereinbefore described.\",\n \"The present invention also provides a method of treating autoimmune disease associated with an immune reaction to tTG in a subject (in particular a human subject), which method comprises administering to the subject a therapeutically effective amount of a therapeutic agent identified as providing a therapeutic effect by interaction with amino acids 1 to 89 of tTG, and/or amino acids 401 to 494 of tTG, or amino acids 401 to 491 of tTG.\",\n \"The amount of therapeutic agent administered will depend on the specific autoimmune disease state being treated, possibly the age of the patient and will ultimately be at the discretion of an attendant physician.\",\n \"There is still further provided by the present invention, in combination, a kit substantially as hereinbefore described, together with a therapeutically effective amount of at least one therapeutic agent effective in the treatment of autoimmune disease associated with an immune reaction to tTG substantially as hereinbefore described.\",\n \"Substantially as hereinbefore described, the sample of body fluid being screened by the present invention will typically comprise blood samples or other fluid blood fractions, such as in particular serum samples or plasma samples, but the sample may in principle be another biological fluid, such as saliva or urine or solubilised tissue extracts.\",\n \"The present invention will now be illustrated with reference to the accompanying Figures and Examples, which do not limit the invention in any way.\",\n \"FIG.\",\n \"1 shows results of SDS PAGE (9%) analysis of purified recombinant human tTG for use in the method and kit according to the invention.\",\n \"FIG.\",\n \"2 is a table showing binding of autoantibodies to tTG in sera from coeliac patients with full length and modified 35S-tTG proteins.\",\n \"FIG.\",\n \"3 is a table showing monoclonal antibody (namely monoclonal antibodies A3-6B5 and A5-4E6) binding to full length and modified 35S-tTG proteins.\",\n \"FIG.\",\n \"4 shows a schematic example of the method of the present invention when an antibody is employed.\",\n \"FIG.\",\n \"5 is a graph showing binding (absorbance at 450 nm in the vertical axis) of tTG with autoantibodies to tTG in different patients sera (horizontal axis).\",\n \"The binding is measured in the presence or absence of mouse tTG monoclonal antibody A3-6B5.FIG.\",\n \"6 is a graph showing binding (absorbance at 450 nm in the vertical axis) of tTG with autoantibodies to tTG in different patients sera (horizontal axis).\",\n \"The binding is measured in the presence or absence of mouse tTG monoclonal antibody A5-4E6.FIG.\",\n \"7 depicts cDNA derived from human tTG, encoding amino acids 1 to 89 of human tTG.\",\n \"FIG.\",\n \"8 depicts amino acids 1 to 89 of human tTG.\",\n \"FIG.\",\n \"9 depicts cDNA derived from guinea pig tTG, encoding amino acids 1 to 89 of guinea pig tTG.\",\n \"FIG.\",\n \"10 depicts amino acids 1 to 89 of guinea pig tTG.\",\n \"FIG.\",\n \"11 depicts CDNA derived from bovine tTG, encoding amino acids 1 to 89 of bovine tTG.\",\n \"FIG.\",\n \"12 depicts amino acids 1 to 89 of bovine tTG.\",\n \"FIG.\",\n \"13 depicts cDNA derived from mouse tTG, encoding amino acids 1 to 89 of mouse tTG.\",\n \"FIG.\",\n \"14 depicts amino acids 1 to 89 of mouse tTG.\",\n \"FIG.\",\n \"15 depicts cDNA derived from rat tTG, encoding amino acids 1 to 89 of rat tTG.\",\n \"FIG.\",\n \"16 depicts amino acids 1 to 89 of rat tTG.\",\n \"FIG.\",\n \"17 depicts cDNA derived from human tTG, encoding amino acids 401 to 491 of human tTG.\",\n \"FIG.\",\n \"18 depicts amino acids 401 to 491 of human tTG.\",\n \"FIG.\",\n \"19 depicts cDNA derived from guinea pig tTG, encoding amino acids 401 to 494 of guinea pig tTG.\",\n \"FIG.\",\n \"20 depicts amino acids 401 to 494 of guinea pig tTG.\",\n \"FIG.\",\n \"21 depicts cDNA derived from bovine tTG, encoding amino acids 401 to 491 of bovine tTG.\",\n \"FIG.\",\n \"22 depicts amino acids 401 to 491 of bovine tTG.\",\n \"FIG.\",\n \"23 depicts cDNA derived from mouse tTG, encoding amino acids 401 to 491 of mouse tTG.\",\n \"FIG.\",\n \"24 depicts amino acids 401 to 491 of mouse tTG.\",\n \"FIG.\",\n \"25 depicts cDNA derived from rat tTG, encoding amino acids 401 to 491 of rat tTG.\",\n \"FIG.\",\n \"26 depicts amino acids 401 to 491 of rat tTG.\",\n \"FIG.\",\n \"27 depicts the alignment of tTG sequences (human, guinea pig, bovine, mouse and rat) at the amino acid level.\",\n \"Further illustration of the present invention is given in the following detailed worked Examples.\",\n \"EXAMPLES Identification of Regions of tTG which are Recognised by tTG Autoantibodies Full length human tTG cDNA (endothelial cell isoform) was cloned into pTZ18R (Pharmacia) under control of the SP6 promoter.\",\n \"This construct was used to produce a reference full length tTG protein.\",\n \"Various in-frame deletions and truncations of the tTG gene were carried out using either naturally occurring restriction endonuclease sites or PCR mediated modifications to the sequence.\",\n \"The modified tTG constructs were cloned into pTZ18R (as above).\",\n \"Full length and modified tTG proteins were expressed in an in vitro transcription/translation rabbit reticulocyte system (Promega) incorporating 35S-methionine (Amersham Pharmacia Biotech) and purified as described before (Colls J, et al.\",\n \"Clin.\",\n \"Chem.\",\n \"1995; 41/3:375-380).\",\n \"Table 1 shows the vectors used and the corresponding tTG amino acid residues expressed following the in vitro transcription/translation described above.\",\n \"TABLE 1 Non-expressed Vector Name Expressed residues (deleted) residues pTGM19 1-687 (full length) None pTGM25 1-628 629-687 pTGM22 1-491 492-687 pTGM24 1-400 401-687 pTGM26 90-687 1-89 pTGM27 172-687 1-171 pTGM28 207-687 1-206 pTGM29 281-687 1-280 pTGM30 356-687 1-355 pTGM31 399-687 1-398 Binding of tTG autoantibodies to the purified full length and modified tTG proteins was analysed using an immunoprecipitation assay (Nakachi K, et al Clin Chim Acta, Volume 304, pages 75 to 84, 2002).\",\n \"Briefly, 10 μL of undiluted serum (in duplicate) was incubated with 50 μL of 35S-tTG (approx 15,000 dpm diluted in 10 mmol/L Tris HCl pH 7.6, 150 mmol/L NaCl, 2 mmol/L EDTA, 0.1% Tween 20, 10 g/L bovine serum albumin, 0.2 g/L NaN3; Assay Buffer) in Millipore filtration plates overnight at 4° C. Anti-human IgA agarose (50 μl) was then added and the plates incubated for 4 hours at 4° C. After incubation the reaction mixtures in the wells were washed 3 times.\",\n \"The wells were then punched out into vials containing 1 mL of scintillation liquid and counted in a beta-counter.\",\n \"FIG.\",\n \"2 shows binding of tTG autoantibodies with different modified tTG proteins.\",\n \"The results are expressed as % binding relative to binding to the full length tTG reference protein.\",\n \"Serum Samples Serum from 34 patients with coeliac disease were used in the study.\",\n \"The diagnosis was based on typical clinical symptoms and was confirmed by positive duodenal biopsy.\",\n \"The sera tested positive for endomysial antibody and autoantibodies to tTG by standard ELISA.\",\n \"In addition sera from healthy blood donors (n=10) negative for autoantibodies to tTG were used.\",\n \"Production of Mouse Monoclonal Antibodies Both guinea pig tTG (Sigma, Poole, UK) and recombinant human tTG (rhtTG) were used as antigens to immunise mice.\",\n \"Monoclonal antibodies (Mabs) were cloned using standard techniques.\",\n \"Selected tTG Mabs were grown in culture and purified by protein A affinity chromatography.\",\n \"The location of the binding sites of the mouse monoclonal antibodies was determined by an immunoprecipitation assay using the modified 35S-labelled tTG proteins as described above, except that anti-mouse IgG was added for precipitation.\",\n \"When required, purified IgG was labelled with 125I using the chloramine T method.\",\n \"Analysis of the mouse Mabs by immunoprecipitation with modified 35S-labelled tTG proteins indicated that the epitope for A3-6B5 Mab was contained within aa 1-89 of the human tTG protein and the epitope for A5-4E6 Mab was contained within aa 401-491 of the human tTG protein (FIG.\",\n \"3).\",\n \"Inhibition of Autoantibodies to tTG Binding with tTG by Mouse tTG Mabs The inhibition of autoantibodies to tTG binding with tTG by mouse tTG Mabs was investigated in a modified version of an ELISA described in Nakachi K, et al.\",\n \"Clin Chim Acta (Volume 304, pages 75 to 84, 2002).\",\n \"To streptavidin coated plate wells 50 μL of tTG-biotin was added plus 10μL of Mab (200 μ/ml) or Assay Buffer and incubated for 30 mins.\",\n \"40 μL of patients' sera (typically diluted 1:100) was then added and incubated for 30 mins.\",\n \"After this step all wells were aspirated, washed four times and then 100 μL of anti-human IgA-horseradish peroxidase (HRP) was added and incubated for 30 mins.\",\n \"After aspiration and four washes, 100 μL of tetramethyl benzidine (TMB) solution was added and the plate was incubated for 15 mins in the dark.\",\n \"50 μL of stop solution was then added and absorbance read at 450 nm in a plate reader.\",\n \"All steps were performed at room temperature, all incubations except the final TMB incubation were done with 200 rpm shaking.\",\n \"tTG-biotin, Mabs and human sera were diluted in Assay Buffer.\",\n \"The results are shown in FIG.\",\n \"5 which shows binding of tTG with autoantibodies to tTG in different patients sera in the presence or absence of mouse tTG monoclonal antibody A3-6B5 and FIG.\",\n \"6 which shows binding of tTG with autoantibodies to tTG in different patients sera in the presence or absence of mouse tTG monoclonal antibody A5-4E6.Preparation of Purified Recombinant Human tTG Recombinant human tTG was expressed in the yeast Saccharomyces cerevisiae strain c13ABYS86 using an expression system and culture techniques described previously (Powell M, et al.\",\n \"Clin Chim Acta 1996; 256: 175-188).\",\n \"Recombinant human tTG extracted from the yeast was partially purified by anion exchange chromatography and preparations of tTG of greater than 95% purity were obtained by affinity chromatography using mouse tTG Mab (A5-6C7) coupled to CNBr Sepharose.\",\n \"The purity of the purified tTG was assessed by SDS PAGE analysis on a 9% acrylamide gel followed by staining with Coomassie Blue as shown in FIG.\",\n \"1.Assay for Screening a Sample of Body Fluid for Autoantibodies to tTG Based on the Inhibition of 125I-Mab Binding to Recombinant Human tTG An assay was set up based on the principles of the schematic example shown in FIG.\",\n \"4.4 mL Nunc Maxisorp Startubes (Life Technologies) were coated overnight at 4° C. with 250 μL of 10 μg/mL recombinant human tTG (rhtTG).\",\n \"Following aspiration and three washes with 500 μL Assay Buffer, tubes were incubated for 30 minutes at room temperature with 500 μL of Post-Coat Buffer containing bovine serum albumin.\",\n \"After two washes with Assay Buffer, tubes were incubated with 100 μL of serum sample diluted 1:5 in Assay Buffer for 60 minutes at room temperature on a shaker.\",\n \"Following aspiration and three washes with 500 μL of Assay Buffer, tubes were incubated on a shaker for 30 minutes at room temperature with 100 μL of 125I-labelled tTG Mab A3-6B5 (30,000 cpm; diluted in Assay Buffer).\",\n \"After a final aspiration and 3 washes with 500 μL of Assay Buffer, tubes were counted for 1 minute in a gamma counter.\",\n \"The results, measured in counts per minute (cpm), were used to calculate the % inhibition of 125I-Mab binding as follows: % inhibition = 100 - [ A B × 100 ] wherein, A=125I-Mab (cpm) bound to rhtTG in the presence of test sera as a % of total cpm of material added to the tube; and B=125I-Mab (cpm) bound to rhtTG in the presence of healthy blood donor sera (mean of 2 or 3 sera) as a % of total cpm of material added to the tube.\",\n \"Results and Discussion FIG.\",\n \"2 showed that C-terminal deletions of amino acids (aa) 492-687 of the tTG protein had no or little effect on tTG autoantibody binding.\",\n \"More extensive C-terminal deletion (aa 401-687) resulted in lower tTG autoantibody binding in 14/15 sera studied.\",\n \"N-terminal deletion of aa 1-89 of the tTG protein resulted in lower tTG autoantibody binding in all sera studied.\",\n \"Therefore, the studies with modified tTG proteins described herein showed that tTG autoantibody binding sites on tTG are heterogenous and are dependent on the aa sequences in the N-terminal and central part of the molecule.\",\n \"Furthermore, these studies showed that aa 1-89, and aa 401-491 of human tTG protein are important for binding of tTG autoantibodies present in the coeliac sera studied.\",\n \"(aa 401-491 of human tTG correspond to aa 401-494 of tTG in other species, such as guinea pig tTG.)\",\n \"Analysis of mouse Mabs by immunoprecipitation with modified 35S-labelled tTG proteins indicated that the epitope for A3-6B5 Mab was contained within aa 1-89 of the human tTG protein and the epitope for A5-4E6 Mab was contained within aa 401-491 of the human tTG protein (FIG.\",\n \"3).\",\n \"Furthermore, these Mabs, namely A3-6B5 Mab and A5-4E6 Mab, inhibited binding of tTG autoantibody positive sera with tTG in ELISA (see FIGS.\",\n \"5 and 6).\",\n \"Identification of amino acid sequences of the tTG molecule important for binding of autoantibodies to tTG tested, and production of mouse Mabs reactive with these amino acid sequences, allowed the development of a new assay to screen for the presence of autoantibodies to tTG in a sample of body fluid as described above.\",\n \"The results of the assay based on autoantibodies to tTG in test sera inhibiting 125I-Mab A3-6B5 binding with recombinant human tTG are shown in Table 2.TABLE 2 Results of assay based on the inhibition of 125I-Mab binding to recombinant human tTG % Inhibition of tTG 35S rhtTG Serum 125I-A3-6B5 Mab Autoantibodies Autoantibodies Number binding ELISA u/mL IPA Index Coeliac 1 79 1000 100 2 35 110 52 3 91 42 7.7 4 49 150 21 5 89 1000 44 6 88 51 107 7 89 1000 122 8 42 102 107 9 48 45 3.3 Healthy blood donors 1 −11 Neg Neg 2 13 Neg Neg 3 −1 Neg Neg 4 3 Neg Neg 5 −3 Neg Neg Nine sera from coeliac patients which tested positive for tTG autoantibodies by immunoprecipitation assay (tTG autoantibodies index from 3.3-107) and ELISA (42-1000 u/mL) were analysed.\",\n \"All these sera inhibited binding of 125I-Mab to rhtTG coated on the tube (35 to 91% inhibition).\",\n \"In contrast, sera negative for autoantibodies to tTG only had a small effect on the binding of 125I-Mab to rhtTG (−11 to 13% inhibition).\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10467587"}}},{"rowIdx":377,"cells":{"text":{"kind":"list like","value":[["Dispensing device","The invention relates to a dispensing device for dispensing particle suspensions (12), especially cell suspensions, comprising a reservoir (10) for receiving the particle suspension (12).","A suspending agent (26) is provided in the reservoir (10) that influences the particle suspension (12).","The suspending agent (26) serves to preserve homogenous particle distribution in the particle suspension (12).","A microdispenser (22) is connected to the reservoir (10) by a fluid element (18).","The microdispenser serves for delivering microdroplets on a titer plate."],["1.A dispensing device for the dispensing of particle suspensions (12), particularly of cell suspensions, comprising a reservoir (10) for receiving the particle suspension (12), a suspension means (26) acting on the particle suspension (12) for maintaining a substantially homogeneous particle distribution in the particle suspension (12), a microdispenser (22) for dispensing microdroplets of a volume smaller than 5 μl, and a fluid element (18) connecting the reservoir (10) to the microdispenser (22) for continuous provision of particle suspension (12) at the microdispenser (22), the suspension means being provided as a stirrer (26).","2.The dispensing device according to claim 1, characterized in that the suspension means (26) is configured to the effect that substantially no damage is caused to the particles, particularly the cells.","3.The dispensing device according to claim 1 or 2, characterized in that the stirrer comprises at least three, preferably at least four stirring blades.","4.The dispensing device according to claim 3, characterized in that the stirring blades include an angle of 50 to 85°, preferably 60 to 70° relative to a longitudinal axis of the stirrer (26).","5.The dispensing device according to claim 3 or 4, characterized in that the stirring blades comprise mixing faces of a slightly concave shape.","6.The dispensing device according to any one of claims 1-5, characterized in that the reservoir (10) is connected to a gas supply device for gas supply to the particle suspensions.","7.The dispensing device according to any one of claims 1-6, characterized in that the microdispenser (22) is piezoelectrically activated.","8.The dispensing device according to any one of claims 1-7, characterized in that the microdispenser comprises a pump chamber having a volume between 100 nl and 5 ml, more preferably between 100 nl and 10 μl and most preferably between 500 nl and 2 μl.","9.The dispensing device according to any one of claims 1-8, characterized in that the microdispenser comprises a dispensing opening (24) having a diameter smaller than 500 μm, preferably smaller than 200 μm and most preferably smaller than 100 μm.","10.The dispensing device according to any one of claims 1-9, characterized in that the fluid element (18) has a shut-off device (20) connected thereto for interrupting the supply of particle suspension (12) to the microdispenser (22).","11.The dispensing device according to claim 10, characterized in that the shut-off device is provided as a tube squeeze valve (20).","12.The dispensing device according to any one of claims 1-11, characterized in that the microdispenser (22) is connected, via a second fluid element (56), to a supply container (46) for rinsing liquid (48) for cleaning the microdispenser (22).","13.The dispensing device according to claim 12, characterized in that the second fluid element (56) is connected to a shut-off device (58)."],["The invention relates to a dispensing device for the dispensing of particle suspensions, particularly of cell suspensions.","Particularly when conducting research in the field of active substances, a large number of active substances is tested nowadays by means of high-throughput screening.","In doing so, a large number of samples is tested in a short time in an automated process.","In high-throughput screening, the active substances are inserted into miniaturized microtitration plates or other suitable sample carriers with wells provided therein.","Since the costs for the active substances, the storage of these substances in substance libraries and the disposal of the active substances after the test process are extremely expensive, the volumes of the individual wells are as small as possible.","In modern titration plates, the volumes of the individual wells are often in the microliter and nanoliter range.","Thus, it is required that minimum quantities be inserted into the wells by pipetting or dispensing.","A suitable pipetting device for active substances without sensitive particles is described e.g.","in WO 96/24040.Since the examination of particle suspensions, particularly of cell suspensions, has gained increased importance, it has become desirable to develop a device adapted to supply even minimum quantities of particle suspension into wells.","In this regard, a particular problem resides in that e.g.","cells and other particles which have low mechanical stability may be easily damaged when introduced into the wells.","Especially, pipetting of liquid samples cannot be performed if the samples are particle suspensions since the particles have to pass the very narrow pipette opening both during the suctional intake into the pipette and during ejection from the pipette.","In this regard, for instance, a considerable danger exists that the cells are affected by shearing forces and that the surface function of the cells is impaired.","Thus, the pipetting device described in WO 96/24040 is not useful for particle suspensions.","Further, a system for the dispensing of liquids is known from WO 98/06821.In this system, one individual particle or one individual cell is dispensed per dispensing process.","This known device is extremely complex and expensive.","Such a device is particularly ill-suited for high-throughput screening since the filling of the individual wells which shall take up a specific number of particles or cells, is too time-consuming to allow e.g.","10,000 tests to be carried out per day in effective systems.","It is an object of the invention to provide a dispensing device for the dispensation of particle suspensions, particularly of cell suspensions, which is adapted to dispense minimum quantities over periods of several hours.","According to the invention, the above object is achieved by a dispensing device having the features mentioned in claim 1.The dispensing device according to the invention comprises a reservoir for receiving a particle suspension, particularly a cell suspension.","The particle suspension can further comprise synthetic microparticles such as beads, particularly polymeric beads.","Particularly when dispensing cells or particles, the problem exists that these are not distributed homogeneously in the liquid, thus precluding the possibility to fill e.g.","the wells of a titration plate with a reproducible number of cells or particles with small variance because, due to the occurrence of sedimentation effects and the like reasons, the concentration of the cells or particles in the suspensions will be locally altered.","Thereby, the number of the dosed cells or particles is changed in each dosing process to such an extent that the measurement results are rendered unreliable.","According to the invention, for homogenizing the particle distribution in the particle suspension, the dispensing device is provided with a suspension means acting on the particle suspension.","The suspension means effects a mixing of the particle suspension so that a substantially homogeneous particle distribution in the particle suspension will be obtained.","The variance of the number of the individual particles or cells in the wells will thus be extremely small.","Preferably, the variance between the individual wells is smaller than 15%, and more preferably smaller than 10%.","Such a small variance can also be realized during continuous dispensation over a period of several hours.","For instance, the cell concentration in the suspension is between 1×105 and 2×106 per ml.","This will result in a number of 100-2,000 cells per well in case of a filling quantity of 1 μl per well.","Even with such high cell concentrations, the above mentioned low variance can be obtained through the provision of a suspension means in the reservoir.","Further, the dispensing device comprises a microdispenser for the dispensing of microdroplets.","The microdroplets, being of a volume smaller than 5 μl, preferably smaller than 100 nl, more preferably smaller than 10 nl, still more preferably smaller than 3 nl and most preferably smaller than 0.5 nl, are dispensed by the microdispenser into the wells or recesses of a sample carrier.","For this purpose, the microdispenser is connected to the reservoir via a fluid element for continuous supply of particle suspension to the microdispenser.","The inventive dispensing device makes it possible, due to the provision of a suspension means, to dispense a particle suspension, particularly a cell suspension, over a period of several hours with only low variance of the number of particles or cells in the individual dispensing processes.","Thus, with the inventive dispensing device, one can generate a uniform and reproducible number of particles or cells in dependence from the supplied volume.","Particularly, the suspension means is of a suitable type to substantially preclude damage to the particles, especially the cells, during the mixing of the particle suspension.","To obtain the above effect, use can be made of a rod-type stirrer, for instance.","This rod-type stirrer, which can be e.g.","a rod-type stirrer with triangular stirring element, is driven e.g.","by magnetic forces generated by a motor arranged below the bottom of the reservoir.","In this manner, an extremely effective suspension can be obtained in the particle suspension.","Since, however, in such a rod-type stirrer, friction will occur between the stirring element and the bottom of the reservoir, the resultant high shear forces may cause destruction of the cells.","Thus, the suspension means is preferably provided as a suspended stirring system, particularly a suspended rod-type stirrer.","This stirrer is installed in the reservoir, if possible in a centered position, via a rotatable axis connected e.g.","to the cover of the reservoir.","The driving motion can be imparted by a motor connected to the axis and arranged externally of the reservoir.","The motor can also be arranged below the bottom of the reservoir and drive the rod-type stirrer by magnetic forces.","To allow for the best possible driving of the stirrer by use of magnetic forces, the stirrer preferably comprises rare-earth metals.","By the provision of the rare-earth metals, the driving force can be imparted to the stirrer also through thick reservoir walls, particularly through thick reservoir bottoms.","A rod-type stirrer arranged in free suspended attachment in the particle suspension offers the advantage that the stirring can be performed without the stirrer touching the bottom or a wall of the reservoir.","This will minimize the mechanical stresses acting on the cells or other particles included in the particle suspension while obtaining a good homogeneity within the particle suspension.","The stirrer of the preferably used type comprises at least two, preferably four stirring blades.","These are arranged preferably at uniform distances on the circumference of the stirrer.","The individual stirring blades are preferably arranged at an angle of 50 to 85°, and more preferably 60 to 70° relative to the longitudinal axis of the stirrer.","This angle will be determined to the effect that the stirring blades are arranged at an angle of 40 to 5° and 30 to 20°, respectively, relative to a plane extending horizontally when the stirrer is arranged vertically.","As compared to this horizontal plane, the inclination of the stirring blades is thus relatively flat.","This has the advantage that the thrust between the particles in the particle suspension and the stirring blades is reduced.","This thrust would be largest if the stirring blades were arranged in parallel to the longitudinal axis.","Preferably, the stirring blades have rounded edges.","To further reduce the thrust between the particles and the stirring blades, the mixing faces of the mixing blades are of a slightly convex shape.","The mixing faces are defined as those faces of the stirring blades onto which the particles impinge during the mixing.","The stirrer is preferably made from inert biocompatible materials or is coated with such materials.","Preferably, the stirrer is provided with a PTFE coating.","Further, the surface of the stirring blades is preferably as smooth as possible to avoid damage to the particles.","Particularly in case of dispensing cell particles over a period of several hours, the gas supply to the cells, particularly the supply with oxygen and carbon dioxide, is critical.","Further, the supply of carbon dioxide serves for the pH regulation of the liquid.","According to a preferred embodiment, the dispensing device of the invention comprises, in addition to the suspension means which does already enhance the gas infeed to the cells or particles, with a gas supply device.","For this purpose, the reservoir is connected to the a gas supply device e.g.","via a gas conduit.","Preferably, the gas supply device is of a type which can be controlled to supply gas to the reservoir in dependence from e.g.","the particle type.","To this end, the reservoir is preferably configured such that it is not filled completely with the particle suspension and a gas space is formed in the upper region of the reservoir.","To further improve the supply of particles, especially the cells, with oxygen, carbon dioxide or other gases, the gas can also be introduced into the liquid so that the gas will form bubbles which will rise in the liquid, thus safeguarding the gas supply to the particles.","However, it has been found that the introduction of gases into the liquid disadvantageously entails the risk of shear effects on the cells caused by the rising and possibly bursting gas bubbles.","Preferably, in the inventive device, the gas to be supplied to the particles, especially the cells, is not introduced directly into the particle suspension but only into the reservoir so that the corresponding gases will be introduced into the space between the surface of the liquid and the cover.","The movement of the particle suspension effected by the suspension means will bring about a sufficient intake of the required gases into the suspension.","To guarantee the dispensing of the particle suspension over a period of several hours, the reservoir preferably has a volume of 1-500 ml, with the size of the reservoir being adapted to the volumes of the wells provided e.g.","in titration plates.","The microdispenser provided for the dispensation of the particle suspension is preferably activated piezoelectrically.","This offers the advantage that very small microdroplets can be generated whose volume is smaller than 10 nl, preferably smaller than 3 nl and most preferably smaller than 0.5 nl.","Further, the microdispenser can be configured to the effect that one piezoelement acts onto a flexible channel, which channel preferably is a round hose closed in itself, or the like.","Thus, advantageously, the microdispenser can be cleaned in a simple manner and thereby kept in a sterile condition.","The microdispenser is provided e.g.","as a glass-silicon structure having a pump chamber arranged therein.","The pump chamber comprises an elastic chamber wall which is connected to an electrically controllable actuator provided to generate pressure in the pump chamber and to reduce the chamber volume, respectively.","When the actuator is suitably controlled, the microdispenser will dispense a droplet.","The pump chamber is preferably delimited by an inlet opening and an outlet opening which forms the outlet opening of the microdispenser or is connected thereto.","The inlet opening is connected to the reservoir via a fluid element such as a hose.","By the provision of an inlet opening, it is prevented that the pressure generated by the actuator in the pump chamber causes a displacement of liquid in the direction of the reservoir instead of the direction of the outlet opening.","Via the inlet opening, a continuous inflow of the to-be-dispensed liquid is safeguarded.","The pump chamber has a volume preferably between 100 nl and 5 ml, more preferably between 100 nl and 10 μl and most preferably between 500 nl and 2 μl.","The nozzle width of the outlet opening is preferably smaller than 500 μm, more preferably smaller than 200 μm and most preferably smaller than 100 μm.","Thus, with the preferred dispensing device, one can generate a drop size of a volume smaller than 10 nl, preferably smaller than 3 nl and most preferred smaller than 0.5 nl.","Further, the microdispenser can be a piston-type dispensing unit with valve and nozzle, or the like.","By suitable control of the piston and the valve, it is likewise possible to generate very small droplets.","In this arrangement, the piston space corresponding to the above pump chamber also has the above described dimensions so that droplet sizes corresponding to those mentioned above can be generated also here.","According to a preferred embodiment, the fluid element arranged between the reservoir and the microdispenser is provided with a shut-off unit.","The shut-off unit serves for the interruption of the supply of the particle suspension to the microdispenser.","Thus, it is possible to clean the microdispenser.","Cleaning is performed e.g.","at fixed time intervals.","Preferably, the shut-off unit is a component which does not immediately interfere with the fluid system.","This has the advantage that the sterility demands required for the handling of particle suspensions are fulfilled.","The provision of a contactless shut-off unit further has the advantage that the shut-off unit is biocompatible.","This means that no valve material, especially no metal particles, can come into contact e.g.","with the cell suspension and affect the same.","A particularly suited shut-off unit is a tube squeeze valve.","To facilitate the cleaning of the microdispenser, the microdispenser is preferably connected, via a second fluid element, to a supply container having a rinsing liquid provided therein.","Thus, an automatic cleaning of the microdispenser can be performed.","For this purpose, preferably, the second fluid element has a second shut-off unit connected thereto which can have the same configuration as the above described first shut-off unit.","Also, a conventional valve can be provided in the second fluid unit or at the outlet of the supply container for the rinsing liquid because lower sterility demands have to be kept for the rinsing liquid.","Preferably, the inner diameters of the fluid elements and the shut-off devices do not or only slightly differ from each other.","Thereby, a sedimentation of the particles and thus a de-homogenizing of the particle suspension are avoided.","The diameters of the whole fluid system which preferably do not or only slightly differ from each other, are preferably in the range between 45 mm and 500 μm.","Most preferred are diameters between 1.5 mm and 200 μm.","Preferably, the diameters of the individual channels provided in the device, particularly in dependence from the dispensed quantity of liquid, are selected to the effect that the dwelling time of the particle suspension, particularly the cell suspension, in the system is shorter than 10 min., preferably shorter than 5 min.","and most preferably shorter than 2 min.","Thus, sedimentation effects in the system are substantially avoided.","These dwelling times are of course not valid for the reservoir in which the sedimentation is avoided by the inventive provision of a suspension agent.","A preferred embodiment of the invention will be explained in greater detail hereunder with reference to the accompanying drawing.","In the drawing: FIG.","1 is a schematic representation of an embodiment of the dispensing device according to the invention.","The dispensing device comprises a reservoir 10 in which the particle suspension 12 is received.","The reservoir 10 has an outlet 16 provided with an outlet connector piece 14.The connector piece 14 has a hose 18 connected thereto as a fluid element.","The hose is connected to a microdispenser 22 with a tube squeeze valve 20 interposed along the hose.","The inner diameters of hose 18 as well as of outlet connector piece 14 are preferably identical.","Thus, the contact element is free of a dead volume.","An identical connection exists also to the tube squeeze valve 20.By the provision of substantially constant inner diameters of the fluid elements, the occurrence of restriction regions which would form a flow resistance to the particles of the suspensions, is prevented.","In case of such resistance, shear effects would occur, and the number of particles transported in the particle suspension would vary over time.","This would lead to the so-called keystone effect, causing an non-reproducible number of cells or particles in the wells.","The microdispenser 22 has an outlet opening 24 provided to eject the microdroplets for filling the wells of a titration plate.","The suspension means of reservoir 10 is arranged as rod-type stirrer 26 by which the particle distribution in particle suspension 12 is kept homogenous throughout an extended period of preferably more than 6 hours, more preferably more than 10 hours.","Instead of the suspended rod-type stirrer 26 illustrated according to the instant embodiment, also a plurality of suspension means can be provided.","Preferably, these are also located internally of reservoir 10 and therein are either supported on the reservoir bottom 28 or are mounted in a suspended arrangement within particle suspension 12.The driving of the suspension means can be performed, as in the case of the rod-type stirrer 26 in the illustrated embodiment, by means of a motor 30.Motor 30 is arranged externally of reservoir 10.In the illustrated embodiment, motor 30 is arranged externally on a cover 32 of reservoir 10.Further, it is possible to drive the suspension means in a contactless manner or through magnetic actuation, for instance.","A further possibility for the intermixing of the particle suspension and thus for the maintenance of a homogeneous particle distribution resides in generating a flow within the reservoir 10.This can be accomplished e.g.","by the provision of circulating pumps.","For this purpose, preferably two reservoirs are provided between which the particle suspension is moved by a circulating pump.","The reservoir 10 is connected to a gas supply device 34 for supplying gas to the particle suspension 12, particularly the cell suspension.","By means of the gas supply device 34, gas, e.g.","oxygen or carbon monoxide, is fed via conduits 36,38 into a gas chamber 40.The gas chamber 40 is arranged internally of reservoir 10 and is provided above the particle suspension 12.For control of the quantity of gas in the gas chamber 40 and of the different gas components, the gas supply device 34 is provided with a suitable control means.","Further, a pressure control element 42 is arranged on the cover 32 for measuring the gas pressure in the gas chamber 40 and to control the pressure through the gas supply device 34.Preferably, for tempering the particle suspension, a tempering element 44 is provided which in the illustrated embodiment is arranged on an outer wall of reservoir 10.The tempering element 44 serves for the heating and/or cooling of the particle suspension 12.Further, the illustrated embodiment of the dispensing device of the invention comprises a supply container 46 containing a rinsing liquid 48.The pressure container 46 is also connected to a pressure control element 50 for pressure control.","The pressure control element 50 makes it possible to generate an underpressure or overpressure in supply container 46.An outlet 52 of supply container 46 is provided with an outlet connecting piece 54 which is connected to a tube 56.Tube 56 is connected, via a second tube squeeze valve 58, also with microdispenser 22.In the illustrated embodiment, the connection is effected via a Y-type tube connection member 60 so that the two tubes 18,56 are joined upstream of the microdispenser 22 and only one tube is connected to microdispenser 22."]],"string":"[\n [\n \"Dispensing device\",\n \"The invention relates to a dispensing device for dispensing particle suspensions (12), especially cell suspensions, comprising a reservoir (10) for receiving the particle suspension (12).\",\n \"A suspending agent (26) is provided in the reservoir (10) that influences the particle suspension (12).\",\n \"The suspending agent (26) serves to preserve homogenous particle distribution in the particle suspension (12).\",\n \"A microdispenser (22) is connected to the reservoir (10) by a fluid element (18).\",\n \"The microdispenser serves for delivering microdroplets on a titer plate.\"\n ],\n [\n \"1.A dispensing device for the dispensing of particle suspensions (12), particularly of cell suspensions, comprising a reservoir (10) for receiving the particle suspension (12), a suspension means (26) acting on the particle suspension (12) for maintaining a substantially homogeneous particle distribution in the particle suspension (12), a microdispenser (22) for dispensing microdroplets of a volume smaller than 5 μl, and a fluid element (18) connecting the reservoir (10) to the microdispenser (22) for continuous provision of particle suspension (12) at the microdispenser (22), the suspension means being provided as a stirrer (26).\",\n \"2.The dispensing device according to claim 1, characterized in that the suspension means (26) is configured to the effect that substantially no damage is caused to the particles, particularly the cells.\",\n \"3.The dispensing device according to claim 1 or 2, characterized in that the stirrer comprises at least three, preferably at least four stirring blades.\",\n \"4.The dispensing device according to claim 3, characterized in that the stirring blades include an angle of 50 to 85°, preferably 60 to 70° relative to a longitudinal axis of the stirrer (26).\",\n \"5.The dispensing device according to claim 3 or 4, characterized in that the stirring blades comprise mixing faces of a slightly concave shape.\",\n \"6.The dispensing device according to any one of claims 1-5, characterized in that the reservoir (10) is connected to a gas supply device for gas supply to the particle suspensions.\",\n \"7.The dispensing device according to any one of claims 1-6, characterized in that the microdispenser (22) is piezoelectrically activated.\",\n \"8.The dispensing device according to any one of claims 1-7, characterized in that the microdispenser comprises a pump chamber having a volume between 100 nl and 5 ml, more preferably between 100 nl and 10 μl and most preferably between 500 nl and 2 μl.\",\n \"9.The dispensing device according to any one of claims 1-8, characterized in that the microdispenser comprises a dispensing opening (24) having a diameter smaller than 500 μm, preferably smaller than 200 μm and most preferably smaller than 100 μm.\",\n \"10.The dispensing device according to any one of claims 1-9, characterized in that the fluid element (18) has a shut-off device (20) connected thereto for interrupting the supply of particle suspension (12) to the microdispenser (22).\",\n \"11.The dispensing device according to claim 10, characterized in that the shut-off device is provided as a tube squeeze valve (20).\",\n \"12.The dispensing device according to any one of claims 1-11, characterized in that the microdispenser (22) is connected, via a second fluid element (56), to a supply container (46) for rinsing liquid (48) for cleaning the microdispenser (22).\",\n \"13.The dispensing device according to claim 12, characterized in that the second fluid element (56) is connected to a shut-off device (58).\"\n ],\n [\n \"The invention relates to a dispensing device for the dispensing of particle suspensions, particularly of cell suspensions.\",\n \"Particularly when conducting research in the field of active substances, a large number of active substances is tested nowadays by means of high-throughput screening.\",\n \"In doing so, a large number of samples is tested in a short time in an automated process.\",\n \"In high-throughput screening, the active substances are inserted into miniaturized microtitration plates or other suitable sample carriers with wells provided therein.\",\n \"Since the costs for the active substances, the storage of these substances in substance libraries and the disposal of the active substances after the test process are extremely expensive, the volumes of the individual wells are as small as possible.\",\n \"In modern titration plates, the volumes of the individual wells are often in the microliter and nanoliter range.\",\n \"Thus, it is required that minimum quantities be inserted into the wells by pipetting or dispensing.\",\n \"A suitable pipetting device for active substances without sensitive particles is described e.g.\",\n \"in WO 96/24040.Since the examination of particle suspensions, particularly of cell suspensions, has gained increased importance, it has become desirable to develop a device adapted to supply even minimum quantities of particle suspension into wells.\",\n \"In this regard, a particular problem resides in that e.g.\",\n \"cells and other particles which have low mechanical stability may be easily damaged when introduced into the wells.\",\n \"Especially, pipetting of liquid samples cannot be performed if the samples are particle suspensions since the particles have to pass the very narrow pipette opening both during the suctional intake into the pipette and during ejection from the pipette.\",\n \"In this regard, for instance, a considerable danger exists that the cells are affected by shearing forces and that the surface function of the cells is impaired.\",\n \"Thus, the pipetting device described in WO 96/24040 is not useful for particle suspensions.\",\n \"Further, a system for the dispensing of liquids is known from WO 98/06821.In this system, one individual particle or one individual cell is dispensed per dispensing process.\",\n \"This known device is extremely complex and expensive.\",\n \"Such a device is particularly ill-suited for high-throughput screening since the filling of the individual wells which shall take up a specific number of particles or cells, is too time-consuming to allow e.g.\",\n \"10,000 tests to be carried out per day in effective systems.\",\n \"It is an object of the invention to provide a dispensing device for the dispensation of particle suspensions, particularly of cell suspensions, which is adapted to dispense minimum quantities over periods of several hours.\",\n \"According to the invention, the above object is achieved by a dispensing device having the features mentioned in claim 1.The dispensing device according to the invention comprises a reservoir for receiving a particle suspension, particularly a cell suspension.\",\n \"The particle suspension can further comprise synthetic microparticles such as beads, particularly polymeric beads.\",\n \"Particularly when dispensing cells or particles, the problem exists that these are not distributed homogeneously in the liquid, thus precluding the possibility to fill e.g.\",\n \"the wells of a titration plate with a reproducible number of cells or particles with small variance because, due to the occurrence of sedimentation effects and the like reasons, the concentration of the cells or particles in the suspensions will be locally altered.\",\n \"Thereby, the number of the dosed cells or particles is changed in each dosing process to such an extent that the measurement results are rendered unreliable.\",\n \"According to the invention, for homogenizing the particle distribution in the particle suspension, the dispensing device is provided with a suspension means acting on the particle suspension.\",\n \"The suspension means effects a mixing of the particle suspension so that a substantially homogeneous particle distribution in the particle suspension will be obtained.\",\n \"The variance of the number of the individual particles or cells in the wells will thus be extremely small.\",\n \"Preferably, the variance between the individual wells is smaller than 15%, and more preferably smaller than 10%.\",\n \"Such a small variance can also be realized during continuous dispensation over a period of several hours.\",\n \"For instance, the cell concentration in the suspension is between 1×105 and 2×106 per ml.\",\n \"This will result in a number of 100-2,000 cells per well in case of a filling quantity of 1 μl per well.\",\n \"Even with such high cell concentrations, the above mentioned low variance can be obtained through the provision of a suspension means in the reservoir.\",\n \"Further, the dispensing device comprises a microdispenser for the dispensing of microdroplets.\",\n \"The microdroplets, being of a volume smaller than 5 μl, preferably smaller than 100 nl, more preferably smaller than 10 nl, still more preferably smaller than 3 nl and most preferably smaller than 0.5 nl, are dispensed by the microdispenser into the wells or recesses of a sample carrier.\",\n \"For this purpose, the microdispenser is connected to the reservoir via a fluid element for continuous supply of particle suspension to the microdispenser.\",\n \"The inventive dispensing device makes it possible, due to the provision of a suspension means, to dispense a particle suspension, particularly a cell suspension, over a period of several hours with only low variance of the number of particles or cells in the individual dispensing processes.\",\n \"Thus, with the inventive dispensing device, one can generate a uniform and reproducible number of particles or cells in dependence from the supplied volume.\",\n \"Particularly, the suspension means is of a suitable type to substantially preclude damage to the particles, especially the cells, during the mixing of the particle suspension.\",\n \"To obtain the above effect, use can be made of a rod-type stirrer, for instance.\",\n \"This rod-type stirrer, which can be e.g.\",\n \"a rod-type stirrer with triangular stirring element, is driven e.g.\",\n \"by magnetic forces generated by a motor arranged below the bottom of the reservoir.\",\n \"In this manner, an extremely effective suspension can be obtained in the particle suspension.\",\n \"Since, however, in such a rod-type stirrer, friction will occur between the stirring element and the bottom of the reservoir, the resultant high shear forces may cause destruction of the cells.\",\n \"Thus, the suspension means is preferably provided as a suspended stirring system, particularly a suspended rod-type stirrer.\",\n \"This stirrer is installed in the reservoir, if possible in a centered position, via a rotatable axis connected e.g.\",\n \"to the cover of the reservoir.\",\n \"The driving motion can be imparted by a motor connected to the axis and arranged externally of the reservoir.\",\n \"The motor can also be arranged below the bottom of the reservoir and drive the rod-type stirrer by magnetic forces.\",\n \"To allow for the best possible driving of the stirrer by use of magnetic forces, the stirrer preferably comprises rare-earth metals.\",\n \"By the provision of the rare-earth metals, the driving force can be imparted to the stirrer also through thick reservoir walls, particularly through thick reservoir bottoms.\",\n \"A rod-type stirrer arranged in free suspended attachment in the particle suspension offers the advantage that the stirring can be performed without the stirrer touching the bottom or a wall of the reservoir.\",\n \"This will minimize the mechanical stresses acting on the cells or other particles included in the particle suspension while obtaining a good homogeneity within the particle suspension.\",\n \"The stirrer of the preferably used type comprises at least two, preferably four stirring blades.\",\n \"These are arranged preferably at uniform distances on the circumference of the stirrer.\",\n \"The individual stirring blades are preferably arranged at an angle of 50 to 85°, and more preferably 60 to 70° relative to the longitudinal axis of the stirrer.\",\n \"This angle will be determined to the effect that the stirring blades are arranged at an angle of 40 to 5° and 30 to 20°, respectively, relative to a plane extending horizontally when the stirrer is arranged vertically.\",\n \"As compared to this horizontal plane, the inclination of the stirring blades is thus relatively flat.\",\n \"This has the advantage that the thrust between the particles in the particle suspension and the stirring blades is reduced.\",\n \"This thrust would be largest if the stirring blades were arranged in parallel to the longitudinal axis.\",\n \"Preferably, the stirring blades have rounded edges.\",\n \"To further reduce the thrust between the particles and the stirring blades, the mixing faces of the mixing blades are of a slightly convex shape.\",\n \"The mixing faces are defined as those faces of the stirring blades onto which the particles impinge during the mixing.\",\n \"The stirrer is preferably made from inert biocompatible materials or is coated with such materials.\",\n \"Preferably, the stirrer is provided with a PTFE coating.\",\n \"Further, the surface of the stirring blades is preferably as smooth as possible to avoid damage to the particles.\",\n \"Particularly in case of dispensing cell particles over a period of several hours, the gas supply to the cells, particularly the supply with oxygen and carbon dioxide, is critical.\",\n \"Further, the supply of carbon dioxide serves for the pH regulation of the liquid.\",\n \"According to a preferred embodiment, the dispensing device of the invention comprises, in addition to the suspension means which does already enhance the gas infeed to the cells or particles, with a gas supply device.\",\n \"For this purpose, the reservoir is connected to the a gas supply device e.g.\",\n \"via a gas conduit.\",\n \"Preferably, the gas supply device is of a type which can be controlled to supply gas to the reservoir in dependence from e.g.\",\n \"the particle type.\",\n \"To this end, the reservoir is preferably configured such that it is not filled completely with the particle suspension and a gas space is formed in the upper region of the reservoir.\",\n \"To further improve the supply of particles, especially the cells, with oxygen, carbon dioxide or other gases, the gas can also be introduced into the liquid so that the gas will form bubbles which will rise in the liquid, thus safeguarding the gas supply to the particles.\",\n \"However, it has been found that the introduction of gases into the liquid disadvantageously entails the risk of shear effects on the cells caused by the rising and possibly bursting gas bubbles.\",\n \"Preferably, in the inventive device, the gas to be supplied to the particles, especially the cells, is not introduced directly into the particle suspension but only into the reservoir so that the corresponding gases will be introduced into the space between the surface of the liquid and the cover.\",\n \"The movement of the particle suspension effected by the suspension means will bring about a sufficient intake of the required gases into the suspension.\",\n \"To guarantee the dispensing of the particle suspension over a period of several hours, the reservoir preferably has a volume of 1-500 ml, with the size of the reservoir being adapted to the volumes of the wells provided e.g.\",\n \"in titration plates.\",\n \"The microdispenser provided for the dispensation of the particle suspension is preferably activated piezoelectrically.\",\n \"This offers the advantage that very small microdroplets can be generated whose volume is smaller than 10 nl, preferably smaller than 3 nl and most preferably smaller than 0.5 nl.\",\n \"Further, the microdispenser can be configured to the effect that one piezoelement acts onto a flexible channel, which channel preferably is a round hose closed in itself, or the like.\",\n \"Thus, advantageously, the microdispenser can be cleaned in a simple manner and thereby kept in a sterile condition.\",\n \"The microdispenser is provided e.g.\",\n \"as a glass-silicon structure having a pump chamber arranged therein.\",\n \"The pump chamber comprises an elastic chamber wall which is connected to an electrically controllable actuator provided to generate pressure in the pump chamber and to reduce the chamber volume, respectively.\",\n \"When the actuator is suitably controlled, the microdispenser will dispense a droplet.\",\n \"The pump chamber is preferably delimited by an inlet opening and an outlet opening which forms the outlet opening of the microdispenser or is connected thereto.\",\n \"The inlet opening is connected to the reservoir via a fluid element such as a hose.\",\n \"By the provision of an inlet opening, it is prevented that the pressure generated by the actuator in the pump chamber causes a displacement of liquid in the direction of the reservoir instead of the direction of the outlet opening.\",\n \"Via the inlet opening, a continuous inflow of the to-be-dispensed liquid is safeguarded.\",\n \"The pump chamber has a volume preferably between 100 nl and 5 ml, more preferably between 100 nl and 10 μl and most preferably between 500 nl and 2 μl.\",\n \"The nozzle width of the outlet opening is preferably smaller than 500 μm, more preferably smaller than 200 μm and most preferably smaller than 100 μm.\",\n \"Thus, with the preferred dispensing device, one can generate a drop size of a volume smaller than 10 nl, preferably smaller than 3 nl and most preferred smaller than 0.5 nl.\",\n \"Further, the microdispenser can be a piston-type dispensing unit with valve and nozzle, or the like.\",\n \"By suitable control of the piston and the valve, it is likewise possible to generate very small droplets.\",\n \"In this arrangement, the piston space corresponding to the above pump chamber also has the above described dimensions so that droplet sizes corresponding to those mentioned above can be generated also here.\",\n \"According to a preferred embodiment, the fluid element arranged between the reservoir and the microdispenser is provided with a shut-off unit.\",\n \"The shut-off unit serves for the interruption of the supply of the particle suspension to the microdispenser.\",\n \"Thus, it is possible to clean the microdispenser.\",\n \"Cleaning is performed e.g.\",\n \"at fixed time intervals.\",\n \"Preferably, the shut-off unit is a component which does not immediately interfere with the fluid system.\",\n \"This has the advantage that the sterility demands required for the handling of particle suspensions are fulfilled.\",\n \"The provision of a contactless shut-off unit further has the advantage that the shut-off unit is biocompatible.\",\n \"This means that no valve material, especially no metal particles, can come into contact e.g.\",\n \"with the cell suspension and affect the same.\",\n \"A particularly suited shut-off unit is a tube squeeze valve.\",\n \"To facilitate the cleaning of the microdispenser, the microdispenser is preferably connected, via a second fluid element, to a supply container having a rinsing liquid provided therein.\",\n \"Thus, an automatic cleaning of the microdispenser can be performed.\",\n \"For this purpose, preferably, the second fluid element has a second shut-off unit connected thereto which can have the same configuration as the above described first shut-off unit.\",\n \"Also, a conventional valve can be provided in the second fluid unit or at the outlet of the supply container for the rinsing liquid because lower sterility demands have to be kept for the rinsing liquid.\",\n \"Preferably, the inner diameters of the fluid elements and the shut-off devices do not or only slightly differ from each other.\",\n \"Thereby, a sedimentation of the particles and thus a de-homogenizing of the particle suspension are avoided.\",\n \"The diameters of the whole fluid system which preferably do not or only slightly differ from each other, are preferably in the range between 45 mm and 500 μm.\",\n \"Most preferred are diameters between 1.5 mm and 200 μm.\",\n \"Preferably, the diameters of the individual channels provided in the device, particularly in dependence from the dispensed quantity of liquid, are selected to the effect that the dwelling time of the particle suspension, particularly the cell suspension, in the system is shorter than 10 min., preferably shorter than 5 min.\",\n \"and most preferably shorter than 2 min.\",\n \"Thus, sedimentation effects in the system are substantially avoided.\",\n \"These dwelling times are of course not valid for the reservoir in which the sedimentation is avoided by the inventive provision of a suspension agent.\",\n \"A preferred embodiment of the invention will be explained in greater detail hereunder with reference to the accompanying drawing.\",\n \"In the drawing: FIG.\",\n \"1 is a schematic representation of an embodiment of the dispensing device according to the invention.\",\n \"The dispensing device comprises a reservoir 10 in which the particle suspension 12 is received.\",\n \"The reservoir 10 has an outlet 16 provided with an outlet connector piece 14.The connector piece 14 has a hose 18 connected thereto as a fluid element.\",\n \"The hose is connected to a microdispenser 22 with a tube squeeze valve 20 interposed along the hose.\",\n \"The inner diameters of hose 18 as well as of outlet connector piece 14 are preferably identical.\",\n \"Thus, the contact element is free of a dead volume.\",\n \"An identical connection exists also to the tube squeeze valve 20.By the provision of substantially constant inner diameters of the fluid elements, the occurrence of restriction regions which would form a flow resistance to the particles of the suspensions, is prevented.\",\n \"In case of such resistance, shear effects would occur, and the number of particles transported in the particle suspension would vary over time.\",\n \"This would lead to the so-called keystone effect, causing an non-reproducible number of cells or particles in the wells.\",\n \"The microdispenser 22 has an outlet opening 24 provided to eject the microdroplets for filling the wells of a titration plate.\",\n \"The suspension means of reservoir 10 is arranged as rod-type stirrer 26 by which the particle distribution in particle suspension 12 is kept homogenous throughout an extended period of preferably more than 6 hours, more preferably more than 10 hours.\",\n \"Instead of the suspended rod-type stirrer 26 illustrated according to the instant embodiment, also a plurality of suspension means can be provided.\",\n \"Preferably, these are also located internally of reservoir 10 and therein are either supported on the reservoir bottom 28 or are mounted in a suspended arrangement within particle suspension 12.The driving of the suspension means can be performed, as in the case of the rod-type stirrer 26 in the illustrated embodiment, by means of a motor 30.Motor 30 is arranged externally of reservoir 10.In the illustrated embodiment, motor 30 is arranged externally on a cover 32 of reservoir 10.Further, it is possible to drive the suspension means in a contactless manner or through magnetic actuation, for instance.\",\n \"A further possibility for the intermixing of the particle suspension and thus for the maintenance of a homogeneous particle distribution resides in generating a flow within the reservoir 10.This can be accomplished e.g.\",\n \"by the provision of circulating pumps.\",\n \"For this purpose, preferably two reservoirs are provided between which the particle suspension is moved by a circulating pump.\",\n \"The reservoir 10 is connected to a gas supply device 34 for supplying gas to the particle suspension 12, particularly the cell suspension.\",\n \"By means of the gas supply device 34, gas, e.g.\",\n \"oxygen or carbon monoxide, is fed via conduits 36,38 into a gas chamber 40.The gas chamber 40 is arranged internally of reservoir 10 and is provided above the particle suspension 12.For control of the quantity of gas in the gas chamber 40 and of the different gas components, the gas supply device 34 is provided with a suitable control means.\",\n \"Further, a pressure control element 42 is arranged on the cover 32 for measuring the gas pressure in the gas chamber 40 and to control the pressure through the gas supply device 34.Preferably, for tempering the particle suspension, a tempering element 44 is provided which in the illustrated embodiment is arranged on an outer wall of reservoir 10.The tempering element 44 serves for the heating and/or cooling of the particle suspension 12.Further, the illustrated embodiment of the dispensing device of the invention comprises a supply container 46 containing a rinsing liquid 48.The pressure container 46 is also connected to a pressure control element 50 for pressure control.\",\n \"The pressure control element 50 makes it possible to generate an underpressure or overpressure in supply container 46.An outlet 52 of supply container 46 is provided with an outlet connecting piece 54 which is connected to a tube 56.Tube 56 is connected, via a second tube squeeze valve 58, also with microdispenser 22.In the illustrated embodiment, the connection is effected via a Y-type tube connection member 60 so that the two tubes 18,56 are joined upstream of the microdispenser 22 and only one tube is connected to microdispenser 22.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10467596"}}},{"rowIdx":378,"cells":{"text":{"kind":"list like","value":[["Information processing apparatus and method","When reproducing content, a client reads attribute information defined in the header of content data corresponding to a content ID specified by a user.","When the read attribute information meets an attribute condition defined in a license stored in a storage unit defining the attribute condition, the encrypted content data is decrypted and output.","After the content is distributed, a collected edition or a best edition can be newly released without creating new content by issuing the license having an attribute condition that limits, for example, a release data and an artist.","Defining a license with a specific subscription ID as the attribute condition allows the user with the license to use new release content with the subscription ID without additional purchase of a license."],["1.An information processing apparatus comprising: content receiving means for receiving content including encrypted content data and attribute information; content storage means for storing the content; license receiving means for receiving a license including an attribute condition defining a condition regarding the attribute information on a piece of the content that can be used; license storage means for storing the license; determining means for determining whether the attribute information on the piece of the content meets the attribute condition of the license stored in the license storage unit; decrypting means for decrypting the encrypted content data of the piece of the content based on the determination of the determining means that the attribute information on the piece of the content meets the attribute condition of the license; and outputting means for outputting the content data decrypted by the decrypting means.","2.An information processing apparatus according to claim 1, wherein the content further includes a content key for decrypting the content data.","3.An information processing apparatus according to claim 1, wherein the attribute information includes a combination of an attribute item and an attribute value.","4.An information processing apparatus according to claim 1, wherein the attribute item includes information on a record company, an artist, a release date, a content provider, a genre, a subscription, and a label.","5.An information processing apparatus according to claim 1, wherein the attribute condition includes a combination of an attribute item, an attribute value, and an operator.","6.An information processing apparatus comprising: receiving means for receiving a license request including a license ID for uniquely identifying the license including the attribute condition defining the condition regarding the attribute information included in the content; storage means for storing the license along with the license ID; obtaining means for obtaining the license corresponding to the license ID included in the license request; signature means for adding a digital signature to the license; and sending means for sending the license with the signature added thereto by the signature means.","7.An information processing apparatus according to claim 6, further comprising license processing means for attaching a terminal ID to the license obtained by the obtaining means.","8.An information processing apparatus comprising: storing means for storing content including encrypted content data and attribute information; receiving means for receiving a content request including a content ID for uniquely identifying the content; and sending means for sending a piece of content corresponding to the content ID included in the content request, wherein: the attribute information included in the piece of the content is information used for determining whether an attribute condition of the license is met when the piece of the content is used; and the attribute condition of the license is information defining a condition regarding the attribute information on the piece of the content that can be used.","9.An information processing method comprising: a content receiving step of receiving content including encrypted content data and attribute information; a content storing step of storing the content; a license receiving step of receiving a license including an attribute condition defining a condition regarding the attribute information on a piece of the content that can be used; a license storing step of storing the license; a determining step of determining whether the attribute information on the piece of the content meets the attribute condition of the license stored at the license storing step; a decrypting step of decrypting the encrypted content data of the piece of the content based on the determination at the determining step that the attribute information on the piece of the content meets the attribute condition of the license; and an outputting step of outputting the content data decrypted at the decrypting step.","10.A program for causing a computer to execute: a content receiving step of receiving content including encrypted content data and attribute information; a content storing step of storing the content; a license receiving step of receiving a license including an attribute condition defining a condition regarding the attribute information on a piece of the content that can be used; a license storing step of storing the license; a determining step of determining whether the attribute information on the piece of the content meets the attribute condition of the license stored at the license storing step; a decrypting step of decrypting the encrypted content data of the piece of the content based on the determination at the determining step that the attribute information on the piece of the content meets the attribute condition of the license; and an outputting step of outputting the content data decrypted at the decrypting step.","11.A program storage medium containing a program to cause a computer to execute: a content receiving step of receiving content including encrypted content data and attribute information; a content storing step of storing the content; a license receiving step of receiving a license including an attribute condition defining a condition regarding the attribute information on a piece of the content that can be used; a license storing step of storing the license; a determining step of determining whether the attribute information on the piece of the content meets the attribute condition of the license stored at the license storing step; a decrypting step of decrypting the encrypted content data of the piece of the content based on the determination at the determining step that the attribute information on the piece of the content meets the attribute condition of the license; and an outputting step of outputting the content data decrypted at the decrypting step."],["<SOH> BACKGROUND ART <EOH>Recently, systems have been realized for exchanging music data for free among a plurality of users in which a user provides his/her own music data to other users via the Internet and receives other users' music data that the user does not have.","Theoretically, only with the presence of one piece of music data and another piece of content, such systems allow the other users to take advantage of them.","Because of this, many users do not purchase the content, causing a copyright holder to lose their chance to obtain royalties that the copyright holder deserves.","Accordingly, there are systems in which the content to be distributed is encrypted and the license for using the content is separately provided, so that the encrypted content cannot be decrypted and reproduced without the license corresponding to the encrypted content.","This enables the content to be freely distributed while the copyright of the copyright holder is protected.","However, the above system has difficulties in flexibly establishing a correspondence between the license and the content, and difficulties in newly distributing the content to which the already-distributed license can be applied."],["<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.","1 is a block diagram showing a construction of a content providing system to which the present invention is applied.","FIG.","2 is a block diagram showing a construction of a client in FIG.","1 .","FIG.","3 is a flowchart illustrating content download processing by the client in FIG.","1 .","FIG.","4 is a flowchart illustrating content providing processing by a content server in FIG.","1 .","FIG.","5 is a diagram showing an example data format.","FIG.","6 is a diagram illustrating types of attribute items.","FIG.","7 is a diagram showing a license structure.","FIG.","8 is a flowchart illustrating reproduction processing by the client.","FIG.","9 is a flowchart illustrating license acquisition processing.","FIG.","10 is a flowchart illustrating license acquisition processing.","FIG.","11 is a flowchart illustrating license acquisition processing.","FIG.","12 is a flowchart illustrating details of the license acquisition processing.","FIG.","13 is a flowchart illustrating content data obtaining processing.","FIG.","14 is a diagram illustrating a key structure.","FIG.","15 is a diagram illustrating a relationship between the key structure and a license.","FIG.","16 is a diagram illustrating license granting processing by a license server.","detailed-description description=\"Detailed Description\" end=\"lead\"?"],["TECHNICAL FIELD The present invention relates to information processing apparatuses and methods, and more particularly to an information processing apparatus and a method for preventing unauthorized copying and using of content that is not licensed from a copyright holder.","BACKGROUND ART Recently, systems have been realized for exchanging music data for free among a plurality of users in which a user provides his/her own music data to other users via the Internet and receives other users' music data that the user does not have.","Theoretically, only with the presence of one piece of music data and another piece of content, such systems allow the other users to take advantage of them.","Because of this, many users do not purchase the content, causing a copyright holder to lose their chance to obtain royalties that the copyright holder deserves.","Accordingly, there are systems in which the content to be distributed is encrypted and the license for using the content is separately provided, so that the encrypted content cannot be decrypted and reproduced without the license corresponding to the encrypted content.","This enables the content to be freely distributed while the copyright of the copyright holder is protected.","However, the above system has difficulties in flexibly establishing a correspondence between the license and the content, and difficulties in newly distributing the content to which the already-distributed license can be applied.","DISCLOSURE OF INVENTION In view of such cases, the present invention is made in order to allow content to be freely distributed and circulated and in order to freely establish a set of pieces of content that can be used in accordance with a license.","A first information processing apparatus of the present invention includes content receiving means for receiving content including encrypted content data and attribute information; content storage means for storing the content; license receiving means for receiving a license including an attribute condition defining a condition regarding the attribute information on a piece of the content that can be used; license storage means for storing the license; determining means for determining whether the attribute information on the piece of the content meets the attribute condition of the license stored in the license storage unit; decrypting means for decrypting the encrypted content data of the piece of the content based on the determination of the determining means that the attribute information on the piece of the content meets the attribute condition of the license; and outputting means for outputting the content data decrypted by the decrypting means.","The content can further include a content key for decrypting the content data.","The attribute information can include a combination of an attribute item and an attribute value.","The attribute item can include information on a record company, an artist, a release date, a content provider, a genre, a subscription, and a label.","The attribute condition can include a combination of an attribute item, an attribute value, and an operator.","A second information processing apparatus of the present invention includes receiving means for receiving a license request including a license ID for uniquely identifying the license including the attribute condition defining the condition regarding the attribute information included in the content; storage means for storing the license along with the license ID; obtaining means for obtaining the license corresponding to the license ID included in the license request; signature means for adding a digital signature to the license; and sending means for sending the license with the signature added thereto by the signature means.","The information processing apparatus can further include license processing means for attaching a terminal ID to the license obtained by the obtaining means.","A third information processing apparatus of the present invention includes storing means for storing content including encrypted content data and attribute information; receiving means for receiving a content request including a content ID for uniquely identifying the content; and sending means for sending a piece of content corresponding to the content ID included in the content request, wherein the attribute information included in the piece of the content is information used for determining whether an attribute condition of the license is met when the piece of the content is used and the attribute condition of the license is information defining a condition regarding the attribute information on the piece of the content that can be used.","An information processing method of the present invention includes a content receiving step of receiving content including encrypted content data and attribute information; a content storing step of storing the content; a license receiving step of receiving a license including an attribute condition defining a condition regarding the attribute information on a piece of the content that can be used; a license storing step of storing the license; a determining step of determining whether the attribute information on the piece of the content meets the attribute condition of the license stored at the license storing step; a decrypting step of decrypting the encrypted content data of the piece of the content based on the determination at the determining step that the attribute information on the piece of the content meets the attribute condition of the license; and an outputting step of outputting the content data decrypted at the decrypting step.","A program of the present invention is a program which causes a computer to execute a content receiving step of receiving content including encrypted content data and attribute information; a content storing step of storing the content; a license receiving step of receiving a license including an attribute condition defining a condition regarding the attribute information on a piece of the content that can be used; a license storing step of storing the license; a determining step of determining whether the attribute information on the piece of the content meets the attribute condition of the license stored at the license storing step; a decrypting step of decrypting the encrypted content data of the piece of the content based on the determination at the determining step that the attribute information on the piece of the content meets the attribute condition of the license; and an outputting step of outputting the content data decrypted at the decrypting step.","A program storage medium of the present invention contains a program to cause a computer to execute a content receiving step of receiving content including encrypted content data and attribute information; a content storing step of storing the content; a license receiving step of receiving a license including an attribute condition defining a condition regarding the attribute information on a piece of the content that can be used; a license storing step of storing the license; a determining step of determining whether the attribute information on the piece of the content meets the attribute condition of the license stored at the license storing step; a decrypting step of decrypting the encrypted content data of the piece of the content based on the determination at the determining step that the attribute information on the piece of the content meets the attribute condition of the license; and an outputting step of outputting the content data decrypted at the decrypting step.","BRIEF DESCRIPTION OF THE DRAWINGS FIG.","1 is a block diagram showing a construction of a content providing system to which the present invention is applied.","FIG.","2 is a block diagram showing a construction of a client in FIG.","1.FIG.","3 is a flowchart illustrating content download processing by the client in FIG.","1.FIG.","4 is a flowchart illustrating content providing processing by a content server in FIG.","1.FIG.","5 is a diagram showing an example data format.","FIG.","6 is a diagram illustrating types of attribute items.","FIG.","7 is a diagram showing a license structure.","FIG.","8 is a flowchart illustrating reproduction processing by the client.","FIG.","9 is a flowchart illustrating license acquisition processing.","FIG.","10 is a flowchart illustrating license acquisition processing.","FIG.","11 is a flowchart illustrating license acquisition processing.","FIG.","12 is a flowchart illustrating details of the license acquisition processing.","FIG.","13 is a flowchart illustrating content data obtaining processing.","FIG.","14 is a diagram illustrating a key structure.","FIG.","15 is a diagram illustrating a relationship between the key structure and a license.","FIG.","16 is a diagram illustrating license granting processing by a license server.","BEST MODE FOR CARRYING OUT THE INVENTION FIG.","1 shows a construction of a content providing system to which the present invention is applied.","Clients 1-1 and 1-2 (hereinafter, referred to simply as client 1 when each of the clients does not have to be distinguished from one another) are linked to the Internet 2.Although only two clients are shown in this example, an arbitrary number of clients are linked to the Internet 2.The Internet 2 is also linked to at least one content server 3 providing content to the client 1; at least one license server 4 granting the client 1 a license required for using the content provided from the content server 3; and at least one accounting server 5 performing an accounting process for the client 1 when the license is granted to the client 1.The numbers of these at least one content servers 3, at least one license servers 4, and at least one accounting servers 5 linked to the Internet 2 are also arbitrary.","FIG.","2 shows a construction of the client 1.In FIG.","2, a CPU (Central Processing Unit) 21 executes various processes in accordance with the programs stored in a ROM (Read Only Memory) 22 or loaded from a storage unit 28 to a RAM (Random Access Memory) 23.A timer 20 performs clocking to provide time information to the CPU 21.The RAM 23 also appropriately stores data and the like required for causing the CPU 21 to execute various processes.","An encryption/decryption unit 24 encrypts content data and decrypts already-encrypted content data.","A codec unit 25 uses, for example, ATRAC (Adaptive Transform Acoustic Coding) 3 mode to encode the content data, which is provided via an input/output interface 32 to and stored in a semiconductor memory 44 that is connected to a drive 30.The semiconductor memory 44 includes, for example, a memory stick (Trade Mark).","The CPU 21, ROM 22, RAM 23, encryption/decryption unit 24, and codec unit 25 are interconnected via a bus 31.The input/output interface 32 is also connected to this bus 31.The input/output interface 32 includes an input unit 26 including a keyboard and a mouse; an output unit 27 including a display such as a CRT and a LCD and a speaker; a storage unit 28 including a hard disk; and a communication unit 29 including a modem and a terminal adapter.","The communication unit 29 performs a communication process via the Internet 2.The communication unit 29 also performs analog signal or digital signal communication processing with another client.","If necessary, the input/output interface 32 is also connected to the drive 30 on which a magnetic disk 41, optical disk 42, magneto-optic disk 43, or semiconductor memory 44 is appropriately mounted.","If necessary, a computer program read from them is installed in the storage unit 28.Although not shown, the content server 3, the license server 4, and the accounting server 5 each have primarily the same construction as that of the client 1 shown in FIG.","2.Next, processing will be described with reference to the flowchart in FIG.","3 in which the client 1 receives the content from the content server 3.When a user operates the input unit 26 to request access to the content server 3, the CPU 21 controls the communication unit 29 to have access to the content server 3 via the Internet 2.At step S2, when the user operates the input unit 26 to specify content to be provided, the CPU 21 receives specification information and sends the content ID of the specified content from the communication unit 29 to the content server 3 via the Internet 2.As described below with reference to the flowchart in FIG.","4, the content server 3 that receives this sends the content including the encrypted content data corresponding to the sent content ID.","Accordingly, the CPU 21 receives this content via the communication unit 29 at step S3, and the content is provided to the storage unit 28 including a hard disk where it is stored.","Next, content providing processing by the content server 3 that is the counterpart of the above-described processing by the client 1 will be described with reference to the flowchart in FIG.","4.The construction of the client 1 in FIG.","2 is also applied to that of the content server 3.At step S21, the CPU 21 of the content server 3 waits for access from the client 1 via the communication unit 29 from the Internet 2.When determining the occurrence of the access, the CPU 21 proceeds to step S22 where the content ID specifying the content sent from the client 1 is obtained.","The content specification information is the content ID sent at step S2 in FIG.","3.At step S23, the CPU 21 of the content server 3 reads the content specified with the information obtained by the process at step S22 from among the pieces of content data stored in the storage unit 28.The CPU 21 provides the content data read from the storage unit 28 at step S24 to the encryption/decryption unit 24 where the content data is encrypted.","Since the codec unit 25 has already encoded the content data stored in the storage unit 28 by means of the ATRAC3 mode, this encoded content data is to be encrypted.","Alternatively, the content data that is encrypted in advance can be stored in the storage unit 28.In this case, the process at step 24 can be skipped.","Next, at step S25, the CPU 21 of the content server 3 adds a key required for decrypting the encrypted content data and attribute information representing various information regarding the content to a header that includes a format for sending the encrypted content data.","At step S26, the CPU 21 of the content server 3 sends the content, which is obtained by formatting the content data encrypted by the process at step S24 and the header having the attribute information and the digital signature attached thereto by the process at step S25, to the client 1 that has access via the Internet 2 from communication unit 29.FIG.","5 shows the format configuration when the content server 3 provides the content to the client 1 in this manner.","As shown in the figure, this format includes the header and data.","In the header, there are disposed attribute information (attribute list), a digital signature which is added to the attribute information with the encryption key of the license server, an enabling key block (EKB), and a content key Kc (KR (Kc)) encrypted with a root key KR obtained by decrypting the EKB with the DNK.","In the attribute information, there is a plurality of descriptions of attribute entries each including a combination of an attribute item and the corresponding attribute value.","FIG.","6 shows types of the attribute items.","CID, RCID, CIID, AID, GID, and LID are IDs uniquely identifying content, a record company, a content issuer, an artist, a genre, and a label.","RelDate represents the release date of the content; the subscription ID is an attribute item used for the subscription license described below.","The URL represents address information that is accessed when the license is obtained to use the content.","Specifically, in the case of the system in FIG.","1, it is the address of the license server 4 required for receiving the license.","The data includes an arbitrary number of encryption blocks; the encryption blocks each include an initial vector (IV (Initial vector)), a Seed, and data EK′c(data) obtained by encrypting the content data with a key K′c.","The key K′c is a value computed by applying the content key Kc and a Seed value determined with a random number to a hash function, as shown in the following expression: K′c=Hash(Kc, Seed).","The initial vector IV and the seed of each encryption block are determined to take different values.","This encryption is performed by dividing the content data in units of eight bytes to encrypt the content data in units of eight bytes.","Encryption of subsequent eight-byte data is performed using a CBC (Cypher Block Chaning) mode which is performed by utilizing the result of encryption of the previous eight-byte data.","In the case of the CBC mode, when the first eight bytes of the content data are encrypted, there is no previous eight-byte data.","The encryption is therefore performed using the initial vector IV as the initial value when the first eight bytes are encrypted.","Even though one encryption block is broken, the effects are prevented from reaching other encryption blocks because of the encryption using the CBC mode.","This encryption will be described below with reference to FIGS.","14 and 15.Thus, the client 1 can receive the content from the content server 3 without charge and restriction.","However, each client 1 needs to acquire the license when using the obtained content.","Acquiring the license, the client 1 registers online or offline in advance at the license server to obtain service data.","The service data includes a device node key (DNK) and a terminal ID, which serve to decrypt the EKB.","The service data and the license obtained from the license server are securely stored in the storage unit 28 of the client 1.FIG.","7 shows the configuration of the license.","The license includes a license ID, a timestamp, an expiration date, an attribute condition, a usage rule, and the digital signature, whereby the digital signature is added to these with the secret key of the license server; the timestamp represents the day of issue of the license; the expiration date represents the time limit up to when the license can be used and after which the license is expired; the attribute condition is the attribute condition of the content that the client 1 can utilize with the corresponding license, attribute condition which is represented with a conditional expression including a combination of an attribute item, the value corresponding to the attribute item, a comparison operator, and a logic operator; the usage rule represents a rule description for using the content to which the license can be applied, and it includes the same terminal ID as the one included in the service data.","Hereinafter, there will be shown an example license configuration that can be realized with a combination of the attribute information included in the content and the attribute condition included in the license.","The attribute information of the content c1 is defined as follows: c1:cid={1}, aid={0,1}, reldate=year 2000 Nov. 10th This means that the content ID is 1, the artist IDs are 0 and 1 (i.e., a collaboration of the artists of the artist ID 0 and the artist ID 1), and the release date is year 2000, Nov. 10th.","Likewise, the attribute information of content c2, c3, and c4 will be defined as follows: c2: cid={2}, aid={0}, reldate=year 2000 Dec. 20th c3: cid={3}, aid={0}, reldate=year 2001 Mar.","1st c4: cid={4}, aid={0}, reldate=year 2001 Oct. 21st On the other hand, the attribute condition of the license 11 is defined as follows: 11: 1 ε cid v 2 ε cid The right of use r1 corresponds to the content c1 and c2.In other words, the terminal with the right of use r1 allows the content c1 and c2 to be used.","The attribute condition of the license 12 is defined as follows: 12: 0 ε aid {circumflex over ( )} (year 2001 Jan. 1st<reldate<year 2001 Dec. 31st) This means a condition that represents the content released by the artist of the ID 0 during year 2001.The license 12 corresponds to the content c3 and c4.At this point in time, the content c3 and c4 can be used at the terminal acquiring the license 12.It is assumed that content c5 with the following attribute information is provided afterwards.","c5: cid={5}, aid={0}, reldate={year 2001 Dec. 1st} When this content is obtained by means of download or the like from the content server 3, the client 1 that has already acquired the license 12 can use the content c5 without another access to the license server or the like.","When a distributor attempts to newly provide a combination of the content c1, c2, and c5 as a best edition, which is followed by the distribution thereof, issuing the following license 13 accommodating this situation enables the best edition to be provided without the creation of new content and with direct use of the content already distributed or in circulation.","13: 1 ε cid v 2 ε cid v 5 ε cid Thus, the right of use obtained by combining the content already distributed and the content in circulation can be newly provided with ease.","For example, by issuing a license with an attribute condition that limits the release date and the artist, a collected edition including works during a specific period by a certain artist can be newly provided.","By issuing a license with an attribute condition limiting the artist, a collected edition of a certain group (including works by the group and solo works of members of the group) can be newly provided.","Next, there will be described an example that defines, as a subscription service, a license that permits additional use of some of the new releases every month.","The attribute condition of the license 14 is defined as follows: 14: 3 ε cid v 4 ε cid v 1 ε sid For the client 1 acquiring this license 14, the content c3 and c4 that are already provided can be used.","It is assumed that content c6 and c7 with the following attribute information are provided as new releases the next month.","C6: cid={6}, sid={1} C7: cid={7}, sid={1} In this case, the client 1 with the license 14 can use the content c6 and c7 without the necessity of purchasing a new license.","Likewise, by providing the content with the subscription ID of 1 each month, without purchasing another license, the client 1 with the license 14 can add the content that can be used.","Thus, by representing the attribute condition as a combination of the attribute item, the attribute value, and the operator, such as the logic operator and a relational operator, a set of the content that can be used is able to be flexibly set.","The operators included in the attribute condition are not limited to the ones described here, and thus various types of other operators can be used.","Processing will be described with reference to FIG.","8 when the client 1 reproduces the content.","At step S41, the CPU 21 of the client 1 obtains the content ID that is specified by the user through the operation of the input unit 26.The CPU 21 reads the attribute information defined in the header of the content data corresponding to the obtained content ID.","Next, the CPU 21 proceeds to step S42 where the CPU 21 determines whether any license whose attribute condition defined therein is satisfied with the attribute information read at step S41 is already acquired and stored in the storage unit 28 by the client 1.When such a license cannot be found, the CPU 21 proceeds to step S43 where the CPU 21 causes a message prompting the acquisition of the license to be shown via the output unit 27 on the display.","When it is determined at step S42 that the license is already acquired, the CPU 21 proceeds to step S44 where the CPU 21 determines whether the acquired license is expired.","Whether the license is expired is determined by comparing the expiration date specified as a description of the license and the present time clocked by the timer 20.When it is determined that the expiration date is already reached, the CPU 21 proceeds to step S45 where license update processing is performed.","The details of this license update processing will be described with reference to the flowchart in FIG.","8.When it is determined at step 44 that the license is still unexpired, or when the license is updated at step S45, the CPU 21 proceeds to step S45 where the CPU 21 verifies the digital signature included in the header of the content and the digital signature in the license with the public key of the license server 4.When the verification result of the digital signature proves that the digital signature is valid, the CPU 21 proceeds to step S46 where the CPU 21 reads the encrypted content data from the storage unit 28 and stores it in the RAM 23.At step S47, the CPU 21 provides the encryption block data stored in the RAM 23 in units of encryption blocks disposed in the Data in FIG.","5 to the encryption/decryption unit 24 where the encryption block data is decrypted.","Furthermore, the CPU 21 provides the content data decrypted by the encryption/decryption unit 24 at step S48 to the codec unit 25 where the decrypted data is decoded.","The CPU 21 provides the data decoded by the codec unit 25 from the input/output interface 32 to the output unit 27 where the decoded data is D-A converted for output from a loudspeaker.","Processing will be described with reference to FIGS.","9 to 11 in which the client acquires the license from the license server 4.FIG.","9 shows license acquisition processing when a user of the client 1 determines the content to be used.","When issuing of a license list is requested to the license server 4 in response to the specification of the content through the operation of the input unit 26 by the user, the CPU 21 controls the communication unit 29 to send the request of the license list including the content ID of the specified content to the content server 3 via the Internet 2.When receiving the license list request, the license server 4 extracts the licenses that can be applied to the content corresponding to the content ID included in the received license list and sends the client 1 the license list in which the license ID, the license name, the condition of the content to be used, the list of content can be used currently, the working conditions of the content, and the like of each of the licenses are defined.","When the client 1 receives the license list from the license server 4, the CPU 21 displays information on each license included in the license list on the output unit 27.When the user selects a desired license by referring to the information, the CPU 21 controls the communication unit 29 to establish a session by means of a two-way authentication, such as SSL.","Subsequently, a license request including the license ID, the terminal ID, the user ID for accounting, and the password of the selected license is encrypted to be sent to the content server 3 via the Internet 2.When receiving the license request sent from the client 1, the license server 4 performs license issuing processing, described later, and sends the client 1 the license corresponding to the license ID included in the license request.","When receiving the license sent from the license server 4, the client 1 encrypts the received license and so on to be stored in a secure state in the storage unit 28.As described above, the user can acquire the license for using the content which the client 1 already has obtained.","Alternatively, when the user performs an operation of reproducing the content that the client obtains and when the license for reproducing the content is not acquired, the above license acquisition processing may be automatically started.","Next, processing is shown in FIG.","16 in which the user specifies various search conditions to search for the license and acquire it.","Initially, in order to search for a user's desired license, the user operates the input unit 26 to specify the search condition, such as the license name, the license type, the title of the content to which the license can be applied, the album name, the genre, the artist name, and the release date.","The CPU 21 controls the communication unit to send the content server 3 the license request including data obtained by formatting the input search condition.","When receiving the license list request sent from the client 1, the content server searches for the licenses satisfying the search condition included in the license list request from the storage unit 28, and the license list including information on each of the licenses, such as the license ID, is sent to the client 1.When the client 1 receives the license list from the license server 4, the CPU 21 causes information on each of the licenses included in the license list to be shown on the output unit 27.When the user selects a desired license by referring to the information, the CPU 21 controls the communication unit 29 to establish a session by means of the two-way authentication, such as the SSL.","Subsequently, the license request including the license ID, the terminal ID, the user ID for accounting, and the password of the selected license is encrypted to be sent via Internet 2 to the content server 3.Receiving the license request sent from the client 1, the license server 4 performs the license issuing processing, described later, and then sends the client 1 the license corresponding to the license ID included in the license request.","When receiving the license sent from the license server 4, the client 1 encrypts the received license to be stored in a secure state in the storage unit 28.As described above, the user can search for the desired license and acquire it.","Next, the license acquisition processing is shown in FIG.","11 in a case in which the user knows the license ID of the desired license.","When the user operates the input unit 26 to specify the license ID of the desired license, the CPU 21 controls the communication unit 29 to establish a session by means of the two-way authentication, such as SSL.","Subsequently, the license request including the license ID, the terminal ID, the user ID for accounting, and the password of the selected license is encrypted and sent via the Internet 2 to the content server 3.When receiving the license request sent from the client 1, the license server 4 performs the license issuing processing, described later, and then sends the client 1 the license corresponding to the license ID included in the license request.","When receiving the license sent from the license server 4, the client 1 encrypts the received license or the like to be stored in a secure state in the storage unit 28.The user knows the license ID from license advertisements inserted in magazines or the like and can acquire the desired license in the above-described manner by specifying the license ID.","Alternatively, the license acquisition processing may start by allowing the user to perform selection or the like by clicking to URL link information on the license server including the license IDs inserted in HTML files of Web sites, e-mail, and the like.","The details of the license issuing processing in FIGS.","9 to 11 will be described with reference to the flowchart in FIG.","12.In this case again, the construction of the client 1 in FIG.","2 is applied to the construction of the license server 4.Initially, at step S102, the CPU 21 obtains the license ID, the terminal ID, the user ID, and the password included in the license request.","The CPU 21 of the license server 4 has access to the accounting server 5 via the communication unit 29 to request authorization processing of the user corresponding to the user ID and the password.","When receiving a request of the authorization processing via the Internet 2 from the license server 4, the accounting server 5 checks the track record of the past payments of the user corresponding to the user ID and the password to determine whether the user has a record of no payment for the value of the license in the past.","When the user does not have such a record, the result of the authorization that permits the grant of the license is sent.","When the user has the record of “no payment”, the result of the authorization that does not permit the grant of the license is sent.","At step S104, the CPU 21 of the license server 4 determines whether the result of the authorization from the accounting server 5 permits the grant of the license.","When the grant of the license is permitted, the CPU 21 proceeds to step S105 where the license corresponding to the license ID is acquired from a database.","The terminal ID is inserted into the usage rule field of the license and the digital signature is generated with the private key of the license server 4 to be attached.","At step S107, the CPU 21 of the license server 4 sends the license with the terminal ID and the digital signature attached from the communication unit 29 via the Internet 2 to the client 1.At step S108, the CPU 21 of the license server 4 stores the license just sent at step S107 in the storage unit 28 in such a manner that the license is associated with the user ID and the password obtained by the process at step S102.In addition, the CPU 21 executes the accounting processing at step S109.To be specific, the CPU 21 requests the accounting server 5 via the communication unit 29 to perform the accounting processing on the user corresponding to the user ID and the password.","The accounting server 5 performs the accounting processing on the user based on this accounting request.","As described above, when the user does not pay in response to this accounting processing, the user is not able to receive the license afterwards even though the grant of the license is requested.","In other words, since the authorization result that does not permit the grant of the license is sent from the accounting server 5 in this case, the CPU 21 proceeds from step S104 to step S110 where error processing is performed.","To be specific, the CPU 21 of the license server 4 displays a message notifying the client 1, controlling the communication unit 29 to have access, that the grant of the license is unable to be permitted, and then the process is terminated.","In this case, as described above, being unable to receive the license, the client 1 cannot use the content.","Next, processing will be described with a reference to FIG.","13 in which the content data of the content to which the license can be applied is obtained.","When the user operates the input unit 26 to select the license, the CPU controls the communication unit 29 to send the content list request including the license ID of the selected license to the content server 3 via the Internet 2.When receiving the content list request, the license server 4 obtains the license ID included in the content list request.","The license server 4 can extract the content to which the corresponding license can be applied, with the license ID as the key, from a license database.","Subsequently, the license server sends the client 1 the content list including content information, such as the content ID, the URL for downloading the content, the content name, the artist name, and the genre of each piece of the extracted content.","Controlling the output unit to receive the content list, the client 1 displays the content information of each piece of the content included in the content list.","When the user selects the content to be downloaded by referring to the displayed content information, the client 1 sends the content request to the content server 3 in accordance with the URL of the content.","When receiving the content request, the content server sends the client 1 the content with the content ID included in the content request.","The client 1 receives the content from the content server 3 and stores the received content in the storage unit 28.As described above, the user retrieves the content to which the license is capable of being applied and causes the client to download from the content server 3.FIG.","14 shows a key constructing method when Broadcast Encryption is adopted as a key management system.","As shown in FIG.","14, keys take a hierarchical tree structure whose lowest layer leaves each correspond to devices.","In an example in FIG.","14, keys corresponding to sixteen devices, numbered 0 to 15, are produced.","Keys are each specified so as to correspond to the node indicated with a circle in the figure.","In this example, the root node at the top layer corresponds to a key KR, the nodes at the second layer correspond to keys K0 and K1, the nodes at the third layer correspond to keys K00 to K11, and the nodes at the fourth layer correspond to keys K000 to K111.The leaves(device nodes) which serve as the nodes at the bottom layer correspond to keys K0000 to K1111.Because of the hierarchical structure, for example, the key K001 is regarded as the upper-layer key of the keys K0010 and K0011, and the key K00 is regarded as the upper-layer key of the keys K000 and K001.Likewise, the K0 is regarded as the upper-layer key of the keys K00 and K01, and the key KR is regarded as the upper-layer key of the keys K0 and K1.The keys for utilizing the content are formed using the keys corresponding to the nodes of a path from a device node (leaf) at the bottom layer to the root node at the top layer.","For example, the keys for utilizing the content of number 3 are formed using the keys of the path from the leaf to the root including the keys K0011, K0001, K00, K0, and KR.","In the system of the present invention, as shown in, for example, FIG.","15, the hierarchical tree structure including keys corresponding to 8×24×32 stages of nodes are used.","In this key system, categories correspond to the keys corresponding to nodes down to the lower eight stages from the root node.","The category here indicates the category, such as the category of devices using a semiconductor memory, for example the memory stick, and the category of devices receiving a digital broadcast.","In the example in FIG.","15, a system of the present invention is applied to one node among the nodes from the root node down to the ones at the eighth stage.","The licenses correspond to the keys that correspond to another twenty-four stages of lower layer nodes.","This enables approximately 16 mega (=224=approximately one million and six hundred thousand) of licenses to be defined.","In addition, the lowest thirty-two stage layers enable approximately four Giga (=232=approximately four billion) users to be defined.","Keys corresponding to the nodes of the lowest thirty-two stages constitute DNK.","Each piece of the content corresponds to one of the paths constituted by nodes of sixty-four (=8+24+32) stages.","In other words, encrypting each piece of the content uses the keys corresponding to the nodes constituting the assigned path.","The key at an upper layer is encrypted using the key at a lower proximate layer and are disposed in the EKB in FIG.","5.Not being disposed in the EKB, the DNK at the bottom stage is disposed in the service data obtained at registration of the client to the license server and is provided to the user's client 1 as shown in FIG.","16.The client 1 uses the DNK defined in the service data to decrypt the key at an upper proximate layer defined in the EKB distributed along with the content data.","The client 1 uses the key obtained through the description to decrypt a key at a further upper layer included in the EKB.","By sequentially performing the above-described process, the client 1 can obtain every key belonging to the path of the content.","The client 1 uses the KR obtained after the above EKB decryption processing to decrypt the encrypted content key KR(KC) and the content key KC can be obtained.","Alternatively, the keys in the present invention can be formed using keys other than the key system employing the Broadcast Encryption as shown in FIGS.","14 and 15.In place of so-called a personal computer, the client to which the present invention is applied can be a PDA (Personal Digital Assistants), a cellular phone, a game terminal, or the like.","When a series of processes is performed using software, the programs that constitute the software are installed from a network or storage media to a computer incorporated in dedicated hardware, a universal personal computer that can execute various features by installing various programs thereon, etc.","These storage media, which are distributed, as shown in FIG.","2, for providing the programs to the user, are formed using not only package medium involving the magnetic disk 41 (including a floppy disk), the optical disk 42 (including a CD-ROM (Compact Disk-Read Only Memory) and a DVD (Digital Versatile Disk)), a magneto-optic disk 43 (including an MD (Mini-Disk)), and a semiconductor memory 44, which are independent of the device itself and store the programs, but also the ROM 22 and the hard disk included in the storage unit 28, which are incorporated in the device beforehand to be provided to the user and store the programs.","In the present specification, steps defining the programs stored in the storage media, steps which are sequentially performed in accordance with the described order, may not be necessarily performed sequentially.","The steps may include a process executed parallel or separately.","The system in the present specification represents the entirety of an apparatus including a plurality of the devices.","INDUSTRIAL APPLICABILITY As described above, an information processing apparatus, a method, a program storage medium, and a program according to the present invention format encrypted data and attribute information on content in accordance with a predetermined format to be output and causes a license to include an attribute condition.","When the attribute information of the content meets the attribute condition of the license, since the encrypted data can be decrypted, the data is prevented from being used in an authorized manner while the license can be flexibly issued."]],"string":"[\n [\n \"Information processing apparatus and method\",\n \"When reproducing content, a client reads attribute information defined in the header of content data corresponding to a content ID specified by a user.\",\n \"When the read attribute information meets an attribute condition defined in a license stored in a storage unit defining the attribute condition, the encrypted content data is decrypted and output.\",\n \"After the content is distributed, a collected edition or a best edition can be newly released without creating new content by issuing the license having an attribute condition that limits, for example, a release data and an artist.\",\n \"Defining a license with a specific subscription ID as the attribute condition allows the user with the license to use new release content with the subscription ID without additional purchase of a license.\"\n ],\n [\n \"1.An information processing apparatus comprising: content receiving means for receiving content including encrypted content data and attribute information; content storage means for storing the content; license receiving means for receiving a license including an attribute condition defining a condition regarding the attribute information on a piece of the content that can be used; license storage means for storing the license; determining means for determining whether the attribute information on the piece of the content meets the attribute condition of the license stored in the license storage unit; decrypting means for decrypting the encrypted content data of the piece of the content based on the determination of the determining means that the attribute information on the piece of the content meets the attribute condition of the license; and outputting means for outputting the content data decrypted by the decrypting means.\",\n \"2.An information processing apparatus according to claim 1, wherein the content further includes a content key for decrypting the content data.\",\n \"3.An information processing apparatus according to claim 1, wherein the attribute information includes a combination of an attribute item and an attribute value.\",\n \"4.An information processing apparatus according to claim 1, wherein the attribute item includes information on a record company, an artist, a release date, a content provider, a genre, a subscription, and a label.\",\n \"5.An information processing apparatus according to claim 1, wherein the attribute condition includes a combination of an attribute item, an attribute value, and an operator.\",\n \"6.An information processing apparatus comprising: receiving means for receiving a license request including a license ID for uniquely identifying the license including the attribute condition defining the condition regarding the attribute information included in the content; storage means for storing the license along with the license ID; obtaining means for obtaining the license corresponding to the license ID included in the license request; signature means for adding a digital signature to the license; and sending means for sending the license with the signature added thereto by the signature means.\",\n \"7.An information processing apparatus according to claim 6, further comprising license processing means for attaching a terminal ID to the license obtained by the obtaining means.\",\n \"8.An information processing apparatus comprising: storing means for storing content including encrypted content data and attribute information; receiving means for receiving a content request including a content ID for uniquely identifying the content; and sending means for sending a piece of content corresponding to the content ID included in the content request, wherein: the attribute information included in the piece of the content is information used for determining whether an attribute condition of the license is met when the piece of the content is used; and the attribute condition of the license is information defining a condition regarding the attribute information on the piece of the content that can be used.\",\n \"9.An information processing method comprising: a content receiving step of receiving content including encrypted content data and attribute information; a content storing step of storing the content; a license receiving step of receiving a license including an attribute condition defining a condition regarding the attribute information on a piece of the content that can be used; a license storing step of storing the license; a determining step of determining whether the attribute information on the piece of the content meets the attribute condition of the license stored at the license storing step; a decrypting step of decrypting the encrypted content data of the piece of the content based on the determination at the determining step that the attribute information on the piece of the content meets the attribute condition of the license; and an outputting step of outputting the content data decrypted at the decrypting step.\",\n \"10.A program for causing a computer to execute: a content receiving step of receiving content including encrypted content data and attribute information; a content storing step of storing the content; a license receiving step of receiving a license including an attribute condition defining a condition regarding the attribute information on a piece of the content that can be used; a license storing step of storing the license; a determining step of determining whether the attribute information on the piece of the content meets the attribute condition of the license stored at the license storing step; a decrypting step of decrypting the encrypted content data of the piece of the content based on the determination at the determining step that the attribute information on the piece of the content meets the attribute condition of the license; and an outputting step of outputting the content data decrypted at the decrypting step.\",\n \"11.A program storage medium containing a program to cause a computer to execute: a content receiving step of receiving content including encrypted content data and attribute information; a content storing step of storing the content; a license receiving step of receiving a license including an attribute condition defining a condition regarding the attribute information on a piece of the content that can be used; a license storing step of storing the license; a determining step of determining whether the attribute information on the piece of the content meets the attribute condition of the license stored at the license storing step; a decrypting step of decrypting the encrypted content data of the piece of the content based on the determination at the determining step that the attribute information on the piece of the content meets the attribute condition of the license; and an outputting step of outputting the content data decrypted at the decrypting step.\"\n ],\n [\n \"<SOH> BACKGROUND ART <EOH>Recently, systems have been realized for exchanging music data for free among a plurality of users in which a user provides his/her own music data to other users via the Internet and receives other users' music data that the user does not have.\",\n \"Theoretically, only with the presence of one piece of music data and another piece of content, such systems allow the other users to take advantage of them.\",\n \"Because of this, many users do not purchase the content, causing a copyright holder to lose their chance to obtain royalties that the copyright holder deserves.\",\n \"Accordingly, there are systems in which the content to be distributed is encrypted and the license for using the content is separately provided, so that the encrypted content cannot be decrypted and reproduced without the license corresponding to the encrypted content.\",\n \"This enables the content to be freely distributed while the copyright of the copyright holder is protected.\",\n \"However, the above system has difficulties in flexibly establishing a correspondence between the license and the content, and difficulties in newly distributing the content to which the already-distributed license can be applied.\"\n ],\n [\n \"<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.\",\n \"1 is a block diagram showing a construction of a content providing system to which the present invention is applied.\",\n \"FIG.\",\n \"2 is a block diagram showing a construction of a client in FIG.\",\n \"1 .\",\n \"FIG.\",\n \"3 is a flowchart illustrating content download processing by the client in FIG.\",\n \"1 .\",\n \"FIG.\",\n \"4 is a flowchart illustrating content providing processing by a content server in FIG.\",\n \"1 .\",\n \"FIG.\",\n \"5 is a diagram showing an example data format.\",\n \"FIG.\",\n \"6 is a diagram illustrating types of attribute items.\",\n \"FIG.\",\n \"7 is a diagram showing a license structure.\",\n \"FIG.\",\n \"8 is a flowchart illustrating reproduction processing by the client.\",\n \"FIG.\",\n \"9 is a flowchart illustrating license acquisition processing.\",\n \"FIG.\",\n \"10 is a flowchart illustrating license acquisition processing.\",\n \"FIG.\",\n \"11 is a flowchart illustrating license acquisition processing.\",\n \"FIG.\",\n \"12 is a flowchart illustrating details of the license acquisition processing.\",\n \"FIG.\",\n \"13 is a flowchart illustrating content data obtaining processing.\",\n \"FIG.\",\n \"14 is a diagram illustrating a key structure.\",\n \"FIG.\",\n \"15 is a diagram illustrating a relationship between the key structure and a license.\",\n \"FIG.\",\n \"16 is a diagram illustrating license granting processing by a license server.\",\n \"detailed-description description=\\\"Detailed Description\\\" end=\\\"lead\\\"?\"\n ],\n [\n \"TECHNICAL FIELD The present invention relates to information processing apparatuses and methods, and more particularly to an information processing apparatus and a method for preventing unauthorized copying and using of content that is not licensed from a copyright holder.\",\n \"BACKGROUND ART Recently, systems have been realized for exchanging music data for free among a plurality of users in which a user provides his/her own music data to other users via the Internet and receives other users' music data that the user does not have.\",\n \"Theoretically, only with the presence of one piece of music data and another piece of content, such systems allow the other users to take advantage of them.\",\n \"Because of this, many users do not purchase the content, causing a copyright holder to lose their chance to obtain royalties that the copyright holder deserves.\",\n \"Accordingly, there are systems in which the content to be distributed is encrypted and the license for using the content is separately provided, so that the encrypted content cannot be decrypted and reproduced without the license corresponding to the encrypted content.\",\n \"This enables the content to be freely distributed while the copyright of the copyright holder is protected.\",\n \"However, the above system has difficulties in flexibly establishing a correspondence between the license and the content, and difficulties in newly distributing the content to which the already-distributed license can be applied.\",\n \"DISCLOSURE OF INVENTION In view of such cases, the present invention is made in order to allow content to be freely distributed and circulated and in order to freely establish a set of pieces of content that can be used in accordance with a license.\",\n \"A first information processing apparatus of the present invention includes content receiving means for receiving content including encrypted content data and attribute information; content storage means for storing the content; license receiving means for receiving a license including an attribute condition defining a condition regarding the attribute information on a piece of the content that can be used; license storage means for storing the license; determining means for determining whether the attribute information on the piece of the content meets the attribute condition of the license stored in the license storage unit; decrypting means for decrypting the encrypted content data of the piece of the content based on the determination of the determining means that the attribute information on the piece of the content meets the attribute condition of the license; and outputting means for outputting the content data decrypted by the decrypting means.\",\n \"The content can further include a content key for decrypting the content data.\",\n \"The attribute information can include a combination of an attribute item and an attribute value.\",\n \"The attribute item can include information on a record company, an artist, a release date, a content provider, a genre, a subscription, and a label.\",\n \"The attribute condition can include a combination of an attribute item, an attribute value, and an operator.\",\n \"A second information processing apparatus of the present invention includes receiving means for receiving a license request including a license ID for uniquely identifying the license including the attribute condition defining the condition regarding the attribute information included in the content; storage means for storing the license along with the license ID; obtaining means for obtaining the license corresponding to the license ID included in the license request; signature means for adding a digital signature to the license; and sending means for sending the license with the signature added thereto by the signature means.\",\n \"The information processing apparatus can further include license processing means for attaching a terminal ID to the license obtained by the obtaining means.\",\n \"A third information processing apparatus of the present invention includes storing means for storing content including encrypted content data and attribute information; receiving means for receiving a content request including a content ID for uniquely identifying the content; and sending means for sending a piece of content corresponding to the content ID included in the content request, wherein the attribute information included in the piece of the content is information used for determining whether an attribute condition of the license is met when the piece of the content is used and the attribute condition of the license is information defining a condition regarding the attribute information on the piece of the content that can be used.\",\n \"An information processing method of the present invention includes a content receiving step of receiving content including encrypted content data and attribute information; a content storing step of storing the content; a license receiving step of receiving a license including an attribute condition defining a condition regarding the attribute information on a piece of the content that can be used; a license storing step of storing the license; a determining step of determining whether the attribute information on the piece of the content meets the attribute condition of the license stored at the license storing step; a decrypting step of decrypting the encrypted content data of the piece of the content based on the determination at the determining step that the attribute information on the piece of the content meets the attribute condition of the license; and an outputting step of outputting the content data decrypted at the decrypting step.\",\n \"A program of the present invention is a program which causes a computer to execute a content receiving step of receiving content including encrypted content data and attribute information; a content storing step of storing the content; a license receiving step of receiving a license including an attribute condition defining a condition regarding the attribute information on a piece of the content that can be used; a license storing step of storing the license; a determining step of determining whether the attribute information on the piece of the content meets the attribute condition of the license stored at the license storing step; a decrypting step of decrypting the encrypted content data of the piece of the content based on the determination at the determining step that the attribute information on the piece of the content meets the attribute condition of the license; and an outputting step of outputting the content data decrypted at the decrypting step.\",\n \"A program storage medium of the present invention contains a program to cause a computer to execute a content receiving step of receiving content including encrypted content data and attribute information; a content storing step of storing the content; a license receiving step of receiving a license including an attribute condition defining a condition regarding the attribute information on a piece of the content that can be used; a license storing step of storing the license; a determining step of determining whether the attribute information on the piece of the content meets the attribute condition of the license stored at the license storing step; a decrypting step of decrypting the encrypted content data of the piece of the content based on the determination at the determining step that the attribute information on the piece of the content meets the attribute condition of the license; and an outputting step of outputting the content data decrypted at the decrypting step.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS FIG.\",\n \"1 is a block diagram showing a construction of a content providing system to which the present invention is applied.\",\n \"FIG.\",\n \"2 is a block diagram showing a construction of a client in FIG.\",\n \"1.FIG.\",\n \"3 is a flowchart illustrating content download processing by the client in FIG.\",\n \"1.FIG.\",\n \"4 is a flowchart illustrating content providing processing by a content server in FIG.\",\n \"1.FIG.\",\n \"5 is a diagram showing an example data format.\",\n \"FIG.\",\n \"6 is a diagram illustrating types of attribute items.\",\n \"FIG.\",\n \"7 is a diagram showing a license structure.\",\n \"FIG.\",\n \"8 is a flowchart illustrating reproduction processing by the client.\",\n \"FIG.\",\n \"9 is a flowchart illustrating license acquisition processing.\",\n \"FIG.\",\n \"10 is a flowchart illustrating license acquisition processing.\",\n \"FIG.\",\n \"11 is a flowchart illustrating license acquisition processing.\",\n \"FIG.\",\n \"12 is a flowchart illustrating details of the license acquisition processing.\",\n \"FIG.\",\n \"13 is a flowchart illustrating content data obtaining processing.\",\n \"FIG.\",\n \"14 is a diagram illustrating a key structure.\",\n \"FIG.\",\n \"15 is a diagram illustrating a relationship between the key structure and a license.\",\n \"FIG.\",\n \"16 is a diagram illustrating license granting processing by a license server.\",\n \"BEST MODE FOR CARRYING OUT THE INVENTION FIG.\",\n \"1 shows a construction of a content providing system to which the present invention is applied.\",\n \"Clients 1-1 and 1-2 (hereinafter, referred to simply as client 1 when each of the clients does not have to be distinguished from one another) are linked to the Internet 2.Although only two clients are shown in this example, an arbitrary number of clients are linked to the Internet 2.The Internet 2 is also linked to at least one content server 3 providing content to the client 1; at least one license server 4 granting the client 1 a license required for using the content provided from the content server 3; and at least one accounting server 5 performing an accounting process for the client 1 when the license is granted to the client 1.The numbers of these at least one content servers 3, at least one license servers 4, and at least one accounting servers 5 linked to the Internet 2 are also arbitrary.\",\n \"FIG.\",\n \"2 shows a construction of the client 1.In FIG.\",\n \"2, a CPU (Central Processing Unit) 21 executes various processes in accordance with the programs stored in a ROM (Read Only Memory) 22 or loaded from a storage unit 28 to a RAM (Random Access Memory) 23.A timer 20 performs clocking to provide time information to the CPU 21.The RAM 23 also appropriately stores data and the like required for causing the CPU 21 to execute various processes.\",\n \"An encryption/decryption unit 24 encrypts content data and decrypts already-encrypted content data.\",\n \"A codec unit 25 uses, for example, ATRAC (Adaptive Transform Acoustic Coding) 3 mode to encode the content data, which is provided via an input/output interface 32 to and stored in a semiconductor memory 44 that is connected to a drive 30.The semiconductor memory 44 includes, for example, a memory stick (Trade Mark).\",\n \"The CPU 21, ROM 22, RAM 23, encryption/decryption unit 24, and codec unit 25 are interconnected via a bus 31.The input/output interface 32 is also connected to this bus 31.The input/output interface 32 includes an input unit 26 including a keyboard and a mouse; an output unit 27 including a display such as a CRT and a LCD and a speaker; a storage unit 28 including a hard disk; and a communication unit 29 including a modem and a terminal adapter.\",\n \"The communication unit 29 performs a communication process via the Internet 2.The communication unit 29 also performs analog signal or digital signal communication processing with another client.\",\n \"If necessary, the input/output interface 32 is also connected to the drive 30 on which a magnetic disk 41, optical disk 42, magneto-optic disk 43, or semiconductor memory 44 is appropriately mounted.\",\n \"If necessary, a computer program read from them is installed in the storage unit 28.Although not shown, the content server 3, the license server 4, and the accounting server 5 each have primarily the same construction as that of the client 1 shown in FIG.\",\n \"2.Next, processing will be described with reference to the flowchart in FIG.\",\n \"3 in which the client 1 receives the content from the content server 3.When a user operates the input unit 26 to request access to the content server 3, the CPU 21 controls the communication unit 29 to have access to the content server 3 via the Internet 2.At step S2, when the user operates the input unit 26 to specify content to be provided, the CPU 21 receives specification information and sends the content ID of the specified content from the communication unit 29 to the content server 3 via the Internet 2.As described below with reference to the flowchart in FIG.\",\n \"4, the content server 3 that receives this sends the content including the encrypted content data corresponding to the sent content ID.\",\n \"Accordingly, the CPU 21 receives this content via the communication unit 29 at step S3, and the content is provided to the storage unit 28 including a hard disk where it is stored.\",\n \"Next, content providing processing by the content server 3 that is the counterpart of the above-described processing by the client 1 will be described with reference to the flowchart in FIG.\",\n \"4.The construction of the client 1 in FIG.\",\n \"2 is also applied to that of the content server 3.At step S21, the CPU 21 of the content server 3 waits for access from the client 1 via the communication unit 29 from the Internet 2.When determining the occurrence of the access, the CPU 21 proceeds to step S22 where the content ID specifying the content sent from the client 1 is obtained.\",\n \"The content specification information is the content ID sent at step S2 in FIG.\",\n \"3.At step S23, the CPU 21 of the content server 3 reads the content specified with the information obtained by the process at step S22 from among the pieces of content data stored in the storage unit 28.The CPU 21 provides the content data read from the storage unit 28 at step S24 to the encryption/decryption unit 24 where the content data is encrypted.\",\n \"Since the codec unit 25 has already encoded the content data stored in the storage unit 28 by means of the ATRAC3 mode, this encoded content data is to be encrypted.\",\n \"Alternatively, the content data that is encrypted in advance can be stored in the storage unit 28.In this case, the process at step 24 can be skipped.\",\n \"Next, at step S25, the CPU 21 of the content server 3 adds a key required for decrypting the encrypted content data and attribute information representing various information regarding the content to a header that includes a format for sending the encrypted content data.\",\n \"At step S26, the CPU 21 of the content server 3 sends the content, which is obtained by formatting the content data encrypted by the process at step S24 and the header having the attribute information and the digital signature attached thereto by the process at step S25, to the client 1 that has access via the Internet 2 from communication unit 29.FIG.\",\n \"5 shows the format configuration when the content server 3 provides the content to the client 1 in this manner.\",\n \"As shown in the figure, this format includes the header and data.\",\n \"In the header, there are disposed attribute information (attribute list), a digital signature which is added to the attribute information with the encryption key of the license server, an enabling key block (EKB), and a content key Kc (KR (Kc)) encrypted with a root key KR obtained by decrypting the EKB with the DNK.\",\n \"In the attribute information, there is a plurality of descriptions of attribute entries each including a combination of an attribute item and the corresponding attribute value.\",\n \"FIG.\",\n \"6 shows types of the attribute items.\",\n \"CID, RCID, CIID, AID, GID, and LID are IDs uniquely identifying content, a record company, a content issuer, an artist, a genre, and a label.\",\n \"RelDate represents the release date of the content; the subscription ID is an attribute item used for the subscription license described below.\",\n \"The URL represents address information that is accessed when the license is obtained to use the content.\",\n \"Specifically, in the case of the system in FIG.\",\n \"1, it is the address of the license server 4 required for receiving the license.\",\n \"The data includes an arbitrary number of encryption blocks; the encryption blocks each include an initial vector (IV (Initial vector)), a Seed, and data EK′c(data) obtained by encrypting the content data with a key K′c.\",\n \"The key K′c is a value computed by applying the content key Kc and a Seed value determined with a random number to a hash function, as shown in the following expression: K′c=Hash(Kc, Seed).\",\n \"The initial vector IV and the seed of each encryption block are determined to take different values.\",\n \"This encryption is performed by dividing the content data in units of eight bytes to encrypt the content data in units of eight bytes.\",\n \"Encryption of subsequent eight-byte data is performed using a CBC (Cypher Block Chaning) mode which is performed by utilizing the result of encryption of the previous eight-byte data.\",\n \"In the case of the CBC mode, when the first eight bytes of the content data are encrypted, there is no previous eight-byte data.\",\n \"The encryption is therefore performed using the initial vector IV as the initial value when the first eight bytes are encrypted.\",\n \"Even though one encryption block is broken, the effects are prevented from reaching other encryption blocks because of the encryption using the CBC mode.\",\n \"This encryption will be described below with reference to FIGS.\",\n \"14 and 15.Thus, the client 1 can receive the content from the content server 3 without charge and restriction.\",\n \"However, each client 1 needs to acquire the license when using the obtained content.\",\n \"Acquiring the license, the client 1 registers online or offline in advance at the license server to obtain service data.\",\n \"The service data includes a device node key (DNK) and a terminal ID, which serve to decrypt the EKB.\",\n \"The service data and the license obtained from the license server are securely stored in the storage unit 28 of the client 1.FIG.\",\n \"7 shows the configuration of the license.\",\n \"The license includes a license ID, a timestamp, an expiration date, an attribute condition, a usage rule, and the digital signature, whereby the digital signature is added to these with the secret key of the license server; the timestamp represents the day of issue of the license; the expiration date represents the time limit up to when the license can be used and after which the license is expired; the attribute condition is the attribute condition of the content that the client 1 can utilize with the corresponding license, attribute condition which is represented with a conditional expression including a combination of an attribute item, the value corresponding to the attribute item, a comparison operator, and a logic operator; the usage rule represents a rule description for using the content to which the license can be applied, and it includes the same terminal ID as the one included in the service data.\",\n \"Hereinafter, there will be shown an example license configuration that can be realized with a combination of the attribute information included in the content and the attribute condition included in the license.\",\n \"The attribute information of the content c1 is defined as follows: c1:cid={1}, aid={0,1}, reldate=year 2000 Nov. 10th This means that the content ID is 1, the artist IDs are 0 and 1 (i.e., a collaboration of the artists of the artist ID 0 and the artist ID 1), and the release date is year 2000, Nov. 10th.\",\n \"Likewise, the attribute information of content c2, c3, and c4 will be defined as follows: c2: cid={2}, aid={0}, reldate=year 2000 Dec. 20th c3: cid={3}, aid={0}, reldate=year 2001 Mar.\",\n \"1st c4: cid={4}, aid={0}, reldate=year 2001 Oct. 21st On the other hand, the attribute condition of the license 11 is defined as follows: 11: 1 ε cid v 2 ε cid The right of use r1 corresponds to the content c1 and c2.In other words, the terminal with the right of use r1 allows the content c1 and c2 to be used.\",\n \"The attribute condition of the license 12 is defined as follows: 12: 0 ε aid {circumflex over ( )} (year 2001 Jan. 1st<reldate<year 2001 Dec. 31st) This means a condition that represents the content released by the artist of the ID 0 during year 2001.The license 12 corresponds to the content c3 and c4.At this point in time, the content c3 and c4 can be used at the terminal acquiring the license 12.It is assumed that content c5 with the following attribute information is provided afterwards.\",\n \"c5: cid={5}, aid={0}, reldate={year 2001 Dec. 1st} When this content is obtained by means of download or the like from the content server 3, the client 1 that has already acquired the license 12 can use the content c5 without another access to the license server or the like.\",\n \"When a distributor attempts to newly provide a combination of the content c1, c2, and c5 as a best edition, which is followed by the distribution thereof, issuing the following license 13 accommodating this situation enables the best edition to be provided without the creation of new content and with direct use of the content already distributed or in circulation.\",\n \"13: 1 ε cid v 2 ε cid v 5 ε cid Thus, the right of use obtained by combining the content already distributed and the content in circulation can be newly provided with ease.\",\n \"For example, by issuing a license with an attribute condition that limits the release date and the artist, a collected edition including works during a specific period by a certain artist can be newly provided.\",\n \"By issuing a license with an attribute condition limiting the artist, a collected edition of a certain group (including works by the group and solo works of members of the group) can be newly provided.\",\n \"Next, there will be described an example that defines, as a subscription service, a license that permits additional use of some of the new releases every month.\",\n \"The attribute condition of the license 14 is defined as follows: 14: 3 ε cid v 4 ε cid v 1 ε sid For the client 1 acquiring this license 14, the content c3 and c4 that are already provided can be used.\",\n \"It is assumed that content c6 and c7 with the following attribute information are provided as new releases the next month.\",\n \"C6: cid={6}, sid={1} C7: cid={7}, sid={1} In this case, the client 1 with the license 14 can use the content c6 and c7 without the necessity of purchasing a new license.\",\n \"Likewise, by providing the content with the subscription ID of 1 each month, without purchasing another license, the client 1 with the license 14 can add the content that can be used.\",\n \"Thus, by representing the attribute condition as a combination of the attribute item, the attribute value, and the operator, such as the logic operator and a relational operator, a set of the content that can be used is able to be flexibly set.\",\n \"The operators included in the attribute condition are not limited to the ones described here, and thus various types of other operators can be used.\",\n \"Processing will be described with reference to FIG.\",\n \"8 when the client 1 reproduces the content.\",\n \"At step S41, the CPU 21 of the client 1 obtains the content ID that is specified by the user through the operation of the input unit 26.The CPU 21 reads the attribute information defined in the header of the content data corresponding to the obtained content ID.\",\n \"Next, the CPU 21 proceeds to step S42 where the CPU 21 determines whether any license whose attribute condition defined therein is satisfied with the attribute information read at step S41 is already acquired and stored in the storage unit 28 by the client 1.When such a license cannot be found, the CPU 21 proceeds to step S43 where the CPU 21 causes a message prompting the acquisition of the license to be shown via the output unit 27 on the display.\",\n \"When it is determined at step S42 that the license is already acquired, the CPU 21 proceeds to step S44 where the CPU 21 determines whether the acquired license is expired.\",\n \"Whether the license is expired is determined by comparing the expiration date specified as a description of the license and the present time clocked by the timer 20.When it is determined that the expiration date is already reached, the CPU 21 proceeds to step S45 where license update processing is performed.\",\n \"The details of this license update processing will be described with reference to the flowchart in FIG.\",\n \"8.When it is determined at step 44 that the license is still unexpired, or when the license is updated at step S45, the CPU 21 proceeds to step S45 where the CPU 21 verifies the digital signature included in the header of the content and the digital signature in the license with the public key of the license server 4.When the verification result of the digital signature proves that the digital signature is valid, the CPU 21 proceeds to step S46 where the CPU 21 reads the encrypted content data from the storage unit 28 and stores it in the RAM 23.At step S47, the CPU 21 provides the encryption block data stored in the RAM 23 in units of encryption blocks disposed in the Data in FIG.\",\n \"5 to the encryption/decryption unit 24 where the encryption block data is decrypted.\",\n \"Furthermore, the CPU 21 provides the content data decrypted by the encryption/decryption unit 24 at step S48 to the codec unit 25 where the decrypted data is decoded.\",\n \"The CPU 21 provides the data decoded by the codec unit 25 from the input/output interface 32 to the output unit 27 where the decoded data is D-A converted for output from a loudspeaker.\",\n \"Processing will be described with reference to FIGS.\",\n \"9 to 11 in which the client acquires the license from the license server 4.FIG.\",\n \"9 shows license acquisition processing when a user of the client 1 determines the content to be used.\",\n \"When issuing of a license list is requested to the license server 4 in response to the specification of the content through the operation of the input unit 26 by the user, the CPU 21 controls the communication unit 29 to send the request of the license list including the content ID of the specified content to the content server 3 via the Internet 2.When receiving the license list request, the license server 4 extracts the licenses that can be applied to the content corresponding to the content ID included in the received license list and sends the client 1 the license list in which the license ID, the license name, the condition of the content to be used, the list of content can be used currently, the working conditions of the content, and the like of each of the licenses are defined.\",\n \"When the client 1 receives the license list from the license server 4, the CPU 21 displays information on each license included in the license list on the output unit 27.When the user selects a desired license by referring to the information, the CPU 21 controls the communication unit 29 to establish a session by means of a two-way authentication, such as SSL.\",\n \"Subsequently, a license request including the license ID, the terminal ID, the user ID for accounting, and the password of the selected license is encrypted to be sent to the content server 3 via the Internet 2.When receiving the license request sent from the client 1, the license server 4 performs license issuing processing, described later, and sends the client 1 the license corresponding to the license ID included in the license request.\",\n \"When receiving the license sent from the license server 4, the client 1 encrypts the received license and so on to be stored in a secure state in the storage unit 28.As described above, the user can acquire the license for using the content which the client 1 already has obtained.\",\n \"Alternatively, when the user performs an operation of reproducing the content that the client obtains and when the license for reproducing the content is not acquired, the above license acquisition processing may be automatically started.\",\n \"Next, processing is shown in FIG.\",\n \"16 in which the user specifies various search conditions to search for the license and acquire it.\",\n \"Initially, in order to search for a user's desired license, the user operates the input unit 26 to specify the search condition, such as the license name, the license type, the title of the content to which the license can be applied, the album name, the genre, the artist name, and the release date.\",\n \"The CPU 21 controls the communication unit to send the content server 3 the license request including data obtained by formatting the input search condition.\",\n \"When receiving the license list request sent from the client 1, the content server searches for the licenses satisfying the search condition included in the license list request from the storage unit 28, and the license list including information on each of the licenses, such as the license ID, is sent to the client 1.When the client 1 receives the license list from the license server 4, the CPU 21 causes information on each of the licenses included in the license list to be shown on the output unit 27.When the user selects a desired license by referring to the information, the CPU 21 controls the communication unit 29 to establish a session by means of the two-way authentication, such as the SSL.\",\n \"Subsequently, the license request including the license ID, the terminal ID, the user ID for accounting, and the password of the selected license is encrypted to be sent via Internet 2 to the content server 3.Receiving the license request sent from the client 1, the license server 4 performs the license issuing processing, described later, and then sends the client 1 the license corresponding to the license ID included in the license request.\",\n \"When receiving the license sent from the license server 4, the client 1 encrypts the received license to be stored in a secure state in the storage unit 28.As described above, the user can search for the desired license and acquire it.\",\n \"Next, the license acquisition processing is shown in FIG.\",\n \"11 in a case in which the user knows the license ID of the desired license.\",\n \"When the user operates the input unit 26 to specify the license ID of the desired license, the CPU 21 controls the communication unit 29 to establish a session by means of the two-way authentication, such as SSL.\",\n \"Subsequently, the license request including the license ID, the terminal ID, the user ID for accounting, and the password of the selected license is encrypted and sent via the Internet 2 to the content server 3.When receiving the license request sent from the client 1, the license server 4 performs the license issuing processing, described later, and then sends the client 1 the license corresponding to the license ID included in the license request.\",\n \"When receiving the license sent from the license server 4, the client 1 encrypts the received license or the like to be stored in a secure state in the storage unit 28.The user knows the license ID from license advertisements inserted in magazines or the like and can acquire the desired license in the above-described manner by specifying the license ID.\",\n \"Alternatively, the license acquisition processing may start by allowing the user to perform selection or the like by clicking to URL link information on the license server including the license IDs inserted in HTML files of Web sites, e-mail, and the like.\",\n \"The details of the license issuing processing in FIGS.\",\n \"9 to 11 will be described with reference to the flowchart in FIG.\",\n \"12.In this case again, the construction of the client 1 in FIG.\",\n \"2 is applied to the construction of the license server 4.Initially, at step S102, the CPU 21 obtains the license ID, the terminal ID, the user ID, and the password included in the license request.\",\n \"The CPU 21 of the license server 4 has access to the accounting server 5 via the communication unit 29 to request authorization processing of the user corresponding to the user ID and the password.\",\n \"When receiving a request of the authorization processing via the Internet 2 from the license server 4, the accounting server 5 checks the track record of the past payments of the user corresponding to the user ID and the password to determine whether the user has a record of no payment for the value of the license in the past.\",\n \"When the user does not have such a record, the result of the authorization that permits the grant of the license is sent.\",\n \"When the user has the record of “no payment”, the result of the authorization that does not permit the grant of the license is sent.\",\n \"At step S104, the CPU 21 of the license server 4 determines whether the result of the authorization from the accounting server 5 permits the grant of the license.\",\n \"When the grant of the license is permitted, the CPU 21 proceeds to step S105 where the license corresponding to the license ID is acquired from a database.\",\n \"The terminal ID is inserted into the usage rule field of the license and the digital signature is generated with the private key of the license server 4 to be attached.\",\n \"At step S107, the CPU 21 of the license server 4 sends the license with the terminal ID and the digital signature attached from the communication unit 29 via the Internet 2 to the client 1.At step S108, the CPU 21 of the license server 4 stores the license just sent at step S107 in the storage unit 28 in such a manner that the license is associated with the user ID and the password obtained by the process at step S102.In addition, the CPU 21 executes the accounting processing at step S109.To be specific, the CPU 21 requests the accounting server 5 via the communication unit 29 to perform the accounting processing on the user corresponding to the user ID and the password.\",\n \"The accounting server 5 performs the accounting processing on the user based on this accounting request.\",\n \"As described above, when the user does not pay in response to this accounting processing, the user is not able to receive the license afterwards even though the grant of the license is requested.\",\n \"In other words, since the authorization result that does not permit the grant of the license is sent from the accounting server 5 in this case, the CPU 21 proceeds from step S104 to step S110 where error processing is performed.\",\n \"To be specific, the CPU 21 of the license server 4 displays a message notifying the client 1, controlling the communication unit 29 to have access, that the grant of the license is unable to be permitted, and then the process is terminated.\",\n \"In this case, as described above, being unable to receive the license, the client 1 cannot use the content.\",\n \"Next, processing will be described with a reference to FIG.\",\n \"13 in which the content data of the content to which the license can be applied is obtained.\",\n \"When the user operates the input unit 26 to select the license, the CPU controls the communication unit 29 to send the content list request including the license ID of the selected license to the content server 3 via the Internet 2.When receiving the content list request, the license server 4 obtains the license ID included in the content list request.\",\n \"The license server 4 can extract the content to which the corresponding license can be applied, with the license ID as the key, from a license database.\",\n \"Subsequently, the license server sends the client 1 the content list including content information, such as the content ID, the URL for downloading the content, the content name, the artist name, and the genre of each piece of the extracted content.\",\n \"Controlling the output unit to receive the content list, the client 1 displays the content information of each piece of the content included in the content list.\",\n \"When the user selects the content to be downloaded by referring to the displayed content information, the client 1 sends the content request to the content server 3 in accordance with the URL of the content.\",\n \"When receiving the content request, the content server sends the client 1 the content with the content ID included in the content request.\",\n \"The client 1 receives the content from the content server 3 and stores the received content in the storage unit 28.As described above, the user retrieves the content to which the license is capable of being applied and causes the client to download from the content server 3.FIG.\",\n \"14 shows a key constructing method when Broadcast Encryption is adopted as a key management system.\",\n \"As shown in FIG.\",\n \"14, keys take a hierarchical tree structure whose lowest layer leaves each correspond to devices.\",\n \"In an example in FIG.\",\n \"14, keys corresponding to sixteen devices, numbered 0 to 15, are produced.\",\n \"Keys are each specified so as to correspond to the node indicated with a circle in the figure.\",\n \"In this example, the root node at the top layer corresponds to a key KR, the nodes at the second layer correspond to keys K0 and K1, the nodes at the third layer correspond to keys K00 to K11, and the nodes at the fourth layer correspond to keys K000 to K111.The leaves(device nodes) which serve as the nodes at the bottom layer correspond to keys K0000 to K1111.Because of the hierarchical structure, for example, the key K001 is regarded as the upper-layer key of the keys K0010 and K0011, and the key K00 is regarded as the upper-layer key of the keys K000 and K001.Likewise, the K0 is regarded as the upper-layer key of the keys K00 and K01, and the key KR is regarded as the upper-layer key of the keys K0 and K1.The keys for utilizing the content are formed using the keys corresponding to the nodes of a path from a device node (leaf) at the bottom layer to the root node at the top layer.\",\n \"For example, the keys for utilizing the content of number 3 are formed using the keys of the path from the leaf to the root including the keys K0011, K0001, K00, K0, and KR.\",\n \"In the system of the present invention, as shown in, for example, FIG.\",\n \"15, the hierarchical tree structure including keys corresponding to 8×24×32 stages of nodes are used.\",\n \"In this key system, categories correspond to the keys corresponding to nodes down to the lower eight stages from the root node.\",\n \"The category here indicates the category, such as the category of devices using a semiconductor memory, for example the memory stick, and the category of devices receiving a digital broadcast.\",\n \"In the example in FIG.\",\n \"15, a system of the present invention is applied to one node among the nodes from the root node down to the ones at the eighth stage.\",\n \"The licenses correspond to the keys that correspond to another twenty-four stages of lower layer nodes.\",\n \"This enables approximately 16 mega (=224=approximately one million and six hundred thousand) of licenses to be defined.\",\n \"In addition, the lowest thirty-two stage layers enable approximately four Giga (=232=approximately four billion) users to be defined.\",\n \"Keys corresponding to the nodes of the lowest thirty-two stages constitute DNK.\",\n \"Each piece of the content corresponds to one of the paths constituted by nodes of sixty-four (=8+24+32) stages.\",\n \"In other words, encrypting each piece of the content uses the keys corresponding to the nodes constituting the assigned path.\",\n \"The key at an upper layer is encrypted using the key at a lower proximate layer and are disposed in the EKB in FIG.\",\n \"5.Not being disposed in the EKB, the DNK at the bottom stage is disposed in the service data obtained at registration of the client to the license server and is provided to the user's client 1 as shown in FIG.\",\n \"16.The client 1 uses the DNK defined in the service data to decrypt the key at an upper proximate layer defined in the EKB distributed along with the content data.\",\n \"The client 1 uses the key obtained through the description to decrypt a key at a further upper layer included in the EKB.\",\n \"By sequentially performing the above-described process, the client 1 can obtain every key belonging to the path of the content.\",\n \"The client 1 uses the KR obtained after the above EKB decryption processing to decrypt the encrypted content key KR(KC) and the content key KC can be obtained.\",\n \"Alternatively, the keys in the present invention can be formed using keys other than the key system employing the Broadcast Encryption as shown in FIGS.\",\n \"14 and 15.In place of so-called a personal computer, the client to which the present invention is applied can be a PDA (Personal Digital Assistants), a cellular phone, a game terminal, or the like.\",\n \"When a series of processes is performed using software, the programs that constitute the software are installed from a network or storage media to a computer incorporated in dedicated hardware, a universal personal computer that can execute various features by installing various programs thereon, etc.\",\n \"These storage media, which are distributed, as shown in FIG.\",\n \"2, for providing the programs to the user, are formed using not only package medium involving the magnetic disk 41 (including a floppy disk), the optical disk 42 (including a CD-ROM (Compact Disk-Read Only Memory) and a DVD (Digital Versatile Disk)), a magneto-optic disk 43 (including an MD (Mini-Disk)), and a semiconductor memory 44, which are independent of the device itself and store the programs, but also the ROM 22 and the hard disk included in the storage unit 28, which are incorporated in the device beforehand to be provided to the user and store the programs.\",\n \"In the present specification, steps defining the programs stored in the storage media, steps which are sequentially performed in accordance with the described order, may not be necessarily performed sequentially.\",\n \"The steps may include a process executed parallel or separately.\",\n \"The system in the present specification represents the entirety of an apparatus including a plurality of the devices.\",\n \"INDUSTRIAL APPLICABILITY As described above, an information processing apparatus, a method, a program storage medium, and a program according to the present invention format encrypted data and attribute information on content in accordance with a predetermined format to be output and causes a license to include an attribute condition.\",\n \"When the attribute information of the content meets the attribute condition of the license, since the encrypted data can be decrypted, the data is prevented from being used in an authorized manner while the license can be flexibly issued.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10467603"}}},{"rowIdx":379,"cells":{"text":{"kind":"list like","value":[["Gonococcal proteins and nucleic acids","The invention provides proteins from gonococcus (Neisseria gonorrhoeae), including amino acid sequences, the corresponding nucleotide sequences, expression data, and serological data.","The proteins are useful antigens for vaccines, immunogenic compositions, and/or diagnostics.","They are also useful for distinguishing between gonococcus and meningococcus and, in particular, between gonococcus and serogroup B meningococcus."],["1.A protein comprising an amino acid sequence selected from the group consisting of SEQ IDs 26, 72, 230, 984, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314 and 316: 2.A protein having 50% or greater sequence identity to a protein according to claim 1.3.A protein comprising a fragment of an amino acid sequence selected from the group consisting of 26, 72, 230, 984, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204,206,208, 210, 212,214, 216, 218,220,222, 224, 226, 228, 232,234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314 and 316.4.An antibody which specifically binds to a protein according to any one of claims 1 to 3.5.A nucleic acid molecule which encodes a protein according to any one of claims 1 to 3.6.A nucleic acid molecule according to claim 5, comprising a nucleotide sequence selected from the group consisting of SEQ IDs 25, 71, 229, 983, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313 and 315.7.A nucleic acid molecule comprising a fragment of a nucleotide sequence selected from the group consisting of SEQ IDs 25, 71, 229, 983, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313 and 315.8.A nucleic acid molecule comprising a nucleotide sequence complementary to a nucleic acid molecule according to any one of claims 5 to 7.9.A nucleic acid molecule comprising a nucleotide sequences having 50% or greater sequence identity to a nucleic acid molecule according to any one of claims 5 to 8.10.A nucleic acid molecule which can hybridise to a nucleic acid molecule according to any one of claims 5 to 9 under high stringency conditions.","11.A composition comprising a protein, a nucleic acid molecule, or an antibody according to any preceding claim.","12.A composition according to claim 11 being a vaccine composition or a diagnostic composition.","13.A composition according to claim 11 or claim 12 for use as a pharmaceutical.","14.The use of a composition according to claim 13 in the manufacture of a medicament for the treatment or prevention of infection due to streptococcus bacteria, particularly N.gonorrhoeae.","15.A process for distinguishing N.gonorrhoeae from N.meningitidis, comprising the steps of: (a) contacting a protein, a nucleic acid molecule, or an antibody according to any one of claims 1 to 10 with a biological sample under conditions suitable for the formation of an antibody-antigen complexes; and (b) detecting said complexes."],["<SOH> BACKGROUND ART <EOH>Neisseria gonorrhoeae is a bacterial pathogen.","There is currently no effective vaccine against N.gonorrhoeae infection.","It is an object of the invention to provide proteins and nucleic acid useful in vaccine study and/or manufacture.","N.gonorrhoeae is related to N.meningitidis.","Sequence data are now available for serogroup B of meningococcus [e.g.","WO99/24578; WO99/36544; WO99/57280; WO00/22430; WO00/66791; Tettelin et al.","(2000) Science 287:1809-1815] and also for serogroup A [Parkhill et al.","(2000) Nature 404:502-506].","It is a further object of the invention to provide proteins and nucleic acid useful in distinguishing between gonococcus and meningococcus and, in particular, between gonococcus and serogroup B meningococcus."],["<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>There are no drawings.","detailed-description description=\"Detailed Description\" end=\"lead\"?"],["TECHNICAL FIELD This invention relates to proteins from the bacterium Neisseria gonorrhoeae, and more particularly to such proteins which do not have corresponding homologs or orthologs in serogroup B N.meningitidis.","BACKGROUND ART Neisseria gonorrhoeae is a bacterial pathogen.","There is currently no effective vaccine against N.gonorrhoeae infection.","It is an object of the invention to provide proteins and nucleic acid useful in vaccine study and/or manufacture.","N.gonorrhoeae is related to N.meningitidis.","Sequence data are now available for serogroup B of meningococcus [e.g.","WO99/24578; WO99/36544; WO99/57280; WO00/22430; WO00/66791; Tettelin et al.","(2000) Science 287:1809-1815] and also for serogroup A [Parkhill et al.","(2000) Nature 404:502-506].","It is a further object of the invention to provide proteins and nucleic acid useful in distinguishing between gonococcus and meningococcus and, in particular, between gonococcus and serogroup B meningococcus.","DISCLOSURE OF THE INVENTION The invention provides proteins comprising the N.gonorrhoeae amino acid sequences disclosed in the examples (the even-numbered SEQ IDS 2 to 8622).","159 of these have no homolog in serogroup B meningococcus and these have been given a name in the form “NGSn”.","It also provides proteins comprising amino acid sequences having sequence identity to the N.gonorrhoeae amino acid sequences disclosed in the examples.","Depending on the particular sequence, the degree of sequence identity is preferably greater than 50% (e.g.","60%, 70%, 80%, 90%, 95%, 99% or more).","These proteins include homologs, orthologs, allelic variants and functional mutants.","Typically, 50% identity or more between two proteins is considered to be an indication of functional equivalence.","Identity between proteins is preferably determined by the Smith-Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affine gap search with parameters gap open penalty=12 and gap extension penalty=1.The invention further provides proteins comprising fragmnents of the N.gonorrhoeae amino acid sequences disclosed in the examples.","The fragments should comprise at least n consecutive amino acids from the sequences and, depending on the particular sequence, n is 7 or more (e.g.","8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more).","Preferably the fragments comprise one or more epitopes from the sequence.","Other preferred fragments are (a) the N-terminal signal peptides of the proteins disclosed in the examples, and (b) the proteins disclosed in the examples, but without their N-terminal signal peptides.","The proteins of the invention can, of course, be prepared by various means (e.g.","recombinant expression, purification from Neisseria, chemical synthesis etc.)","and in various forms (e.g.","native, fusions etc.).","They are preferably prepared in substantially pure form (i.e.","substantially free from other N.gonorrhoeae or host cell proteins).","The proteins of the invention are preferably Neisserial proteins, more preferably N.gonorrhoeae proteins.","The invention provides antibodies which bind to these proteins.","These may be polyclonal or monoclonal and may be produced by any suitable means.","The antibodies may include a detectable label.","The invention provides nucleic acid comprising the N.gonorrhoeae nucleotide sequences disclosed in the examples.","In addition, the invention provides nucleic acid comprising nucleotide sequences having sequence identity to the N.gonorrhoeae nucleotide sequences disclosed in the examples.","Furthermore, the invention provides nucleic acid which can hybridise to the N.gonorrhoeae nucleic acid disclosed in the examples, preferably under “high stringency” conditions (e.g.","65° C. in a 0.1×SSC, 0.5% SDS solution).","Nucleic acid comprising fragments of these sequences are also provided.","These should comprise at least n consecutive nucleotides from the N.gonorrhoeae sequences and, depending on the particular sequence, n is 10 or more (e.g.","12, 14, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more).","The invention also provides nucleic acid encoding the proteins and protein fragments of the invention.","The invention includes nucleic acid comprising sequences complementary to those described above (e.g.","for antisense or probing purposes).","Nucleic acid according to the invention can be prepared in many ways (e.g.","by chemical synthesis, from genomic or cDNA libraries, from the organism itself etc.)","and can take various forms (e.g.","single stranded, double stranded, vectors, probes etc.).","In addition, the term “nucleic acid” includes DNA and RNA, and also their analogues, such as those containing modified backbones, and also peptide nucleic acids (PNA) etc.","The invention provides vectors comprising nucleotide sequences of the invention (e.g.","cloning or expression vectors) and host cells transformed with such vectors.","The invention provides compositions comprising protein, antibody, and/or nucleic acid according to the invention.","These compositions may be suitable as immunogenic compositions, for instance, or as diagnostic reagents, or as vaccines.","The invention also provides nucleic acid, protein, or antibody according to the invention for use as medicaments (e.g.","as vaccines) or as diagnostic reagents.","It also provides the use of nucleic acid, protein, or antibody according to the invention in the manufacture of: (i) a medicament for treating or preventing infection due to Neisseria; (ii) a diagnostic reagent for detecting the presence of Neisseria or of antibodies raised against Neisseria; and/or (iii) a reagent which can raise antibodies against Neisseria.","Said Neisseria may be any species, but is preferably N.gonorrhoeae.","The invention also provides a method of treating a patient, comprising administering to the patient a therapeutically effective amount of nucleic acid, protein, and/or antibody of the invention.","According to further aspects, the invention provides various processes.","A process for producing proteins of the invention is provided, comprising the step of culturing a host cell of to the invention under conditions which induce protein expression.","A process for producing protein or nucleic acid of the invention is provided, wherein the protein or nucleic acid is synthesised in part or in whole using chemical means.","A process for detecting polynucleotides of the invention is provided, comprising the steps of: (a) contacting a nucleic probe according to the invention with a biological sample under hybridising conditions to form duplexes; and (b) detecting said duplexes.","A process for detecting proteins of the invention is provided, comprising the steps of: (a) contacting an antibody of the invention with a biological sample under conditions suitable for the formation of an antibody-antigen complexes; and (b) detecting said complexes.","A process for distinguishing N.gonorrhoeae from N.meningitidis is provided, comprising the steps of: (a) contacting an antibody of the invention with a biological sample under conditions suitable for the formation of an antibody-antigen complexes; and (b) detecting said complexes.","Alternatively, the steps may be (a) contacting nucleic acid of the invention with a biological sample under conditions suitable for the nucleic acid hybridisation; and (b) detecting any such hybridisation.","Alternatively, the steps may be (a) contacting a protein of the invention with a biological sample (e.g.","blood or serum) under conditions suitable for the formation of an antibody-antigen complexes; and (b) detecting said complexes.","A summary of standard techniques and procedures which may be employed in order to perform the invention (e.g.","to utilise the disclosed sequences for vaccination or diagnostic purposes) follows.","This summary is not a limitation on the invention, but gives examples that may be used, but are not required.","General The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art.","Such techniques are explained fully in the literature eg.","Sambrook Molecular Cloning; A Laboratory Manual, Second Edition (1989) or Third Edition (2000); DNA Cloning, Volumes I and II (D. N Glover ed.","1985); Oligonucleotide Synthesis (M. J. Gait ed, 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds.","1984); Transcription and Translation (B. D. Hames & S. J. Higgins eds.","1984); Animal Cell Culture (R. I. Freshney ed.","1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the Methods in Enzymology series (Academic Press, Inc.), especially volumes 154 & 155; Gene Transfer Vectors for Mammalian Cells (J. H. Miller and M. P. Calos eds.","1987, Cold Spring Harbor Laboratory); Mayer and Walker, eds.","(1987), Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Scopes, (1987) Protein Purification: Principles and Practice, Second Edition (Springer-Verlag, N.Y.), and Handbook of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell eds 1986).","Standard abbreviations for nucleotides and amino acids are used in this specification.","Definitions A composition containing X is “substantially free of” Y when at least 85% by weight of the total X+Y in the composition is X. Preferably, X comprises at least about 90% by weight of the total of X+Y in the composition, more preferably at least about 95% or even 99% by weight.","The term “comprising” means “including” as well as “consisting” e.g.","a composition “comprising” X may consist exclusively of X or may include something additional e.g.","X+Y.","The term “heterologous” refers to two biological components that are not found together in nature; The components may be host cells, genes, or regulatory regions, such as promoters.","Although the heterologous components are not found together in nature, they can function together, as when a promoter heterologous to a gene is operably linked to the gene.","Another example is where a Neisseria sequence is heterologous to a mouse host cell.","A further examples would be two epitopes from the same or different proteins which have been assembled in a single protein in an arrangement not found in nature.","An “origin of replication” is a polynucleotide sequence that initiates and regulates replication of polynucleotides, such as an expression vector.","The origin of replication behaves as an autonomous unit of polynucleotide replication within a cell, capable of replication under its own control.","An origin of replication may be needed for a vector to replicate in a particular host cell.","With certain origins of replication, an expression vector can be reproduced at a high copy number in the presence of the appropriate proteins within the cell, Examples of origins are the autonomously replicating sequences, which are effective in yeast; and the viral T-antigen, effective in COS-7 cells.","A “mutant” sequence is defined as DNA, RNA or amino acid sequence differing from but having sequence identity with the native or disclosed sequence.","Depending on the particular sequence, the degree of sequence identity between the native or disclosed sequence and the mutant sequence is preferably greater than 50% (eg.","60%, 70%, 80%, 90%, 95%, 99% or more, calculated using the Smith-Waterman algorithm as described above).","As used herein, an “allelic variant” of a nucleic acid molecule, or region, for which nucleic acid sequence is provided herein is a nucleic acid molecule, or region, that occurs essentially at the same locus in the genome of another or second isolate, and that, due to natural variation caused by, for example, mutation or recombination, has a similar but not identical nucleic acid sequence.","A coding region allelic variant typically encodes a protein having similar activity to that of the protein encoded by the gene to which it is being compared.","An allelic variant can also comprise an alteration in the 5′ or 3′ untranslated regions of the gene, such as in regulatory control regions (eg.","see U.S. Pat.","No.","5,753,235).","Expression Systems The Neisseria nucleotide sequences can be expressed in a variety of different expression systems; for example those used with mammalian cells, baculoviruses, plants, bacteria, and yeast.","i. Mammalian Systems Mammalian expression systems are known in the art.","A mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3′) transcription of a coding sequence (eg.","structural gene) into mRNA.","A promoter will have a transcription initiating region, which is usually placed proximal to the 5′ end of the coding sequence, and a TATA box, usually located 25-30 base pairs (bp) upstream of the transcription initiation site.","The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site.","A mammalian promoter will also contain an upstream promoter element, usually located within 100 to 200 bp upstream of the TATA box.","An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation [Sambrook et al.","(1989) “Expression of Cloned Genes in Mammalian Cells.” In Molecular Cloning: A Laboratory Manual, 2nd ed.].","Mammalian viral genes are often highly expressed and have a broad host range; therefore sequences encoding mammalian viral genes provide particularly useful promoter sequences.","Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), and herpes simplex virus promoter.","In addition, sequences derived from non-viral genes, such as the murine metallotheionein gene, also provide useful promoter sequences.","Expression may be either constitutive or regulated (inducible), depending on the promoter can be induced with glucocorticoid in hormone-responsive cells.","The presence of an enhancer element (enhancer), combined with the promoter elements described above, will usually increase expression levels.","An enhancer is a regulatory DNA sequence that can stimulate transcription up to 1000-fold when linked to homologous or beterologous promoters, with synthesis beginning at the normal RNA start site.","Enhancers are also active when they are placed upstream or downstream from the transcription initiation site, in either normal or flipped orientation, or at a distance of more than 1000 nucleotides from the promoter [Maniatis et al.","(1987) Science 236:1237; Alberts et al.","(1989) Molecular Biology of the Cell, 2nd ed.].","Enhancer elements derived from viruses may be particularly useful, because they usually have a broader host range.","Examples include the SV40 early gene enhancer [Dijkema et al (1985) EMBO J.","4:7611 and the enhancer/promoters derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus [Gorman et al.","(1982b) Proc.","Natl.","Acad.","Sci.","79:6777] and from human cytomegalovirus [Boshart et al.","(1985) Cell 41:521].","Additionally, some enhancers are regulatable and become active only in the presence of an inducer, such as a hormone or metal ion [Sassone-Corsi and Borelli (1986) Trends Genet.","2:215; Maniatis et al.","(1987) Science 236:1237].","A DNA molecule may be expressed intracellularly in mammalian cells.","A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon.","If desired, the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.","Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in mammalian cel Whitelaw (1988) “Termination and 3′ end processing of eukaryotic RNA, In Transcription and splicing (ed.","B. D. Hames and D. M. Glover); Proudfoot (1989) Trends Biochem.","Sci.","14:105].","These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA.","Examples of transcription terminater/polyadenylation signals include those derived from SV40 [Sambrook et al (1989) “Expression of cloned genes in cultured mammalian cells.” In Molecular Cloning: A Laboratory Manual].","Usually, the above described components, comprising a promoter, polyadenylation signal, and transcription termination sequence are put together into expression constructs.","Enhancers, introns with functional splice donor and acceptor sites, and leader sequences may also be included in an expression construct, if desired.","Expression constructs are often maintained in a replicon, such as an extrachromosomal element (eg.","plasmids) capable of stable maintenance in a host, such as mammalian cells or bacteria.","Mammalian replication systems include those derived from animal viruses, which require trans-acting factors to replicate.","For example, plasmids containing the replication systems of papovaviruses, such as SV40 [Gluzman (1981) Cell 23:175] or polyomavirus, replicate to extremely high copy number in the presence of the appropriate viral T antigen.","Additional examples of mammalian replicons include those derived from bovine papillomavirus and Epstein-Barr virus.","Additionally, the replicon may have two replicaton systems, thus allowing it to be maintained, for example, in mammalian cells for expression and in a prokaryotic host for cloning and amplification.","Examples of such mammalian-bacteria shuttle vectors include pMT2 [Kaufman et al.","(1989) Mol.","Cell.","Biol.","9:946] and pHEBO [Shimizu et al.","(1986) Mol.","Cell.","Biol.","6:1074].","The transformation procedure used depends upon the host to be transformed.","Methods for introduction of heterologous polynucleotides into mammalian cells are known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.","Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg.","Hep G2), and a number of other cell lines.","ii.","Baculovirus Systems The polynucleotide encoding the protein can also be inserted into a suitable insect expression vector, and is operably linked to the control elements within that vector.","Vector construction employs techniques which are known in the art.","Generally, the components of the expression system include a transfer vector, usually a bacterial plasmid, which contains both a fragment of the baculovirus genome, and a convenient restriction site for insertion of the heterologous gene or genes to be expressed; a wild type baculovirus with a sequence homologous to the baculovirus-specific fragment in the transfer vector (this allows for the homologous recombination of the heterologous gene in to the baculovirus genome); and appropriate insect host cells and growth media.","After inserting the DNA sequence encoding the protein into the transfer vector, the vector and the wild type viral genome are transfected into an insect host cell where the vector and viral genome are allowed to recombine.","The packaged recombinant virus is expressed and recombinant plaques are identified and purified.","Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego Calif. (“MaxBac” kit).","These techniques are generally known to those skilled in the art and fully described in Summers & Smith, Texas Agricultural Experiment Station Bulletin No.1555(1987) (“Summers & Smith”).","Prior to inserting the DNA sequence encoding the protein into the baculovirus genome, the above described components, comprising a promoter, leader (if desired), coding sequence, and transcription termination sequence, are usually assembled into an intermediate transplacement construct (transfer vector).","This may contain a single gene and operably linked regulatory elements; multiple genes, each with its owned set of operably linked regulatory elements; or multiple genes, regulated by the same set of regulatory elements.","Intermediate transplacement constructs are often maintained in a replicon, such as an extra-chromosomal element (e.g.","plasmids) capable of stable maintenance in a host, such as a bacterium.","The replicon will have a replication system, thus allowing it to be maintained in a suitable host for cloning and amplification.","Currently, the most commonly used transfer vector for introducing foreign genes into AcNPV is pAc373.Many other vectors, known to those of skill in the art, have also been designed.","These include, for example, pVL985 (which alters the polyhedrin start codon from ATG to ATT, and which introduces a BamH1 cloning site 32 basepairs downstream from the ATT; see Luckow and Summers, Virology (1989)17:31.The plasmid usually also contains the polyhedrin polyadenylation signal (Miller et al.","(1988) Ann.","Rev.","Microbiol., 42:177) and a prokaryotic ampicillin-resistance (amp) gene and origin of replication for selection and propagation in E. coli.","Baculovirus transfer vectors usually contain a baculovirus promoter.","A baculovirus promoter is any DNA sequence capable of binding a baculovirus RNA polymerase and initiating the downstream (5′to 3′) transcription of a coding sequence (eg.","structural gene) into mRNA.","A promoter will have a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence.","This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site.","A baculovirus transfer vector may also have a second domain called an enhancer, which, if present, is usually distal to the structural gene.","Expression may be either regulated or constitutive.","Structural genes, abundantly transcribed at late times in a viral infection cycle, provide particularly useful promoter sequences.","Examples include sequences derived from the gene encoding the viral polyhedron protein, Friesen et al., (1986) “The Regulation of Baculovirus Gene Expression,” in: The Molecular Biology of Baculoviruses (ed.","Walter Doerfler); EPO Publ.","Nos.","127 839 and 155 476; and the gene encoding the p10 protein, Vlak et al., (1988), J. Gen. Virol.","69:765.DNA encoding suitable signal sequences can be derived from genes for secreted insect or baculovirus proteins, such as the baculovirus polyhedrin gene (Carbonell et al.","(1988) Gene, 73:409).","Alternatively, since the signals for mammalian cell posttranslational modifications (such as signal peptide cleavage, proteolytic cleavage, and phosphorylation) appear to be recognized by insect cells, and the signals required for secretion and nuclear accumulation also appear to be conserved between the invertebrate cells and vertebrate cells, leaders of non-insect origin, such as those derived from genes encoding human α-interferon, Maeda et al., (1985), Nature 315:592; human gastrin-releasing peptide, Lebacq-Verheyden et al., (1988), Molec.","Cell.","Biol.","8:3129; human IL-2, Smith et al., (1985) Proc.","Nat'l Acad.","Sci.","USA, 82:8404; mouse IL-3, (Miyajima et al., (1987) Gene 58:273; and human glucocerebrosidase, Martin et al.","(1988) DNA, 7:99, can also be used to provide for secretion in insects.","A recombinant polypeptide or polyprotein may be expressed intracellularly or, if it is expressed with the proper regulatory sequences, it can be secreted.","Good intracellular expression of nonfused foreign proteins usually requires heterologous genes that ideally have a short leader sequence containing suitable translation initiation signals preceding an ATG start signal.","If desired, methionine at the N-terminus may be cleaved from the mature protein by in vitro incubation with cyanogen bromide.","Alternatively, recombinant polyproteins or proteins which are not naturally secreted can be secreted from the insect cell by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in insects.","The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the translocation of the protein into the endoplasmic reticulum.","After insertion of the DNA sequence and/or the gene encoding the expression product precursor of the protein, an insect cell host is co-transformed with the heterologous DNA of the transfer vector and the genomic DNA of wild type baculovirus—usually by co-transfection.","The promoter and transcription termination sequence of the construct will usually comprise a 2-5 kb section of the baculovirus genome.","Methods for introducing heterologous DNA into the desired site in the baculovirus virus are known in the art.","(See Summers & Smith supra; Ju et al.","(1987); Smith et al., Mol.","Cell.","Biol.","(1983) 3:2156; and Luckow and Summers (1989)).","For example, the insertion can be into a gene such as the polyhedrin gene, by homologous double crossover recombination; insertion can also be into a restriction enzyme site engineered into the desired baculovirus gene.","Miller et al., (1989), Bioessays 4:91.The DNA sequence, when cloned in place of the polyhedrin gene in the expression vector, is flanked both 5′ and 3′ by polyhedrin-specific sequences and is positioned downstream of the polyhedrin promoter.","The newly formed baculovirus expression vector is subsequently packaged into an infectious recombinant baculovirus.","Homologous recombination occurs at low frequency (between about 1% and about 5%); thus, the majority of the virus produced after cotransfection is still wild-type virus.","Therefore, a method is necessary to identify recombinant viruses.","An advantage of the expression system is a visual screen allowing recombinant viruses to be distinguished.","The polyhedrin protein, which is produced by the native virus, is produced at very high levels in the nuclei of infected cells at late times after viral infection.","Accumulated polyhedrin protein forms occlusion bodies that also contain embedded particles.","These occlusion bodies, up to 15 μm in size, are highly refractile, giving them a bright shiny appearance that is readily visualized under the light microscope.","Cells infected with recombinant viruses lack occlusion bodies.","To distinguish recombinant virus from wild-type virus, the transfection supernatant is plaqued onto a monolayer of insect cells by techniques known to those skilled in the art.","Namely, the plaques are screened under the light microscope for the presence (indicative of wild-type virus) or absence (indicative of recombinant virus) of occlusion bodies.","“Current Protocols in Microbiology” Vol.","2 (Ausubel et al.","eds) at 16.8 (Supp.","10, 1990); Summers & Smith, supra; Miller et al.","(1989).","Recombinant baculovirus expression vectors have been developed for infection into several insect cells.","For example, recombinant baculoviruses have been developed for, inter alia: Aedes aegypti , Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni (WO 89/046699; Carbonell et al., (1985) J. Virol.","56:153; Wright (1986) Nature 321:718; Smith et al., (1983) Mol.","Cell.","Biol.","3:2156; and see generally, Fraser, et al.","(1989) In Vitro Cell.","Dev.","Biol.","25:225).","Cells and cell culture media are commercially available for both direct and fusion expression of heterologous polypeptides in a baculovirus/expression system; cell culture technology is generally known to those skilled in the art.","See, eg.","Summers & Smith supra.","The modified insect cells may then be grown in an appropriate nutrient medium, which allows for stable maintenance of the plasmid(s) present in the modified insect host.","Where the expression product gene is under inducible control, the host may be grown to high density, and expression induced.","Alternatively, where expression is constitutive, the product will be continuously expressed into the medium and the nutrient medium must be continuously circulated, while removing the product of interest and augmenting depleted nutrients.","The product may be purified by such techniques as chromatography, eg.","HPLC, affinity chromatography, ion exchange chromatography, etc.","; electrophoresis; density gradient centrifugation; solvent extraction, etc.","As appropriate, the product may be further purified, as required, so as to remove substantially any insect proteins which are also present in the medium, so as to provide a product which is at least substantially free of host debris, eg.","proteins, lipids and polysaccharides.","In order to obtain protein expression, recombinant host cells derived from the transformants are incubated under conditions which allow expression of the recombinant protein encoding sequence.","These conditions will vary, dependent upon the host cell selected.","However, the conditions are readily ascertainable to those of ordinary skill in the art, based upon what is known in the art.","iii.","Plant Systems There are many plant cell culture and whole plant genetic expression systems known in the art.","Exemplary plant cellular genetic expression systems include those described in patents, such as: U.S. Pat.","Nos.","5,693,506; 5,659,122; and 5,608,143.Additional examples of genetic expression in plant cell culture has been described by Zenk, Phytochemistry 30:3861-3863 (1991).","Descriptions of plant protein signal peptides may be found in addition to the references described above in Vaulcombe et al., Mol.","Gen. Genet.","209:3340 (1987); Chandler et al., Plant Molecular Biology 3:407-418 (1984); Rogers, J. Biol.","Chem.","260:3731-3738 (1985); Rothstein et al., Gene 55:353-356 (1987); Whittier et al., Nucleic Acids Research 15:2515-2535 (1987); Wirsel et al., Molecular Microbiology 3:3-14 (1989); Yu et al., Gene 122:247-253 (1992).","A description of the regulation of plant gene expression by the phytohormone, gibberellic acid and secreted enzymes induced by gibberellic acid can be found in R. L. Jones and J. MacMillin, Gibberellins: in: Advanced Plant Physiology,.","Malcolm B. Wilkins, ed., 1984 Pitman Publishing Limited, London, pp.","21-52.References that describe other metabolically-regulated genes: Sheen, Plant Cell, 2:1027-1038(1990); Maas et al., EMBO J.","9:3447-3452 (1990); Benkel and Hickey, Proc.","Natl.","Acad.","Sci.","84:1337-1339 (1987).","Typically, using techniques known in the art, a desired polynucleotide sequence is inserted into an expression cassette comprising genetic regulatory elements designed for operation in plants.","The expression cassette is inserted into a desired expression vector with companion sequences upstream and downstream from the expression cassette suitable for expression in a plant host.","The companion sequences will be of plasmid or viral origin and provide necessary characteristics to the vector to permit the vectors to move DNA from an original cloning host, such as bacteria, to the desired plant host.","The basic bacterial/plant vector construct will preferably provide a broad host range prokaryote replication origin; a prokaryote selectable marker; and, for Agrobacterium transformations, T DNA sequences for Agrobacterium-mediated transfer to plant chromosomes.","Where the heterologous gene is not readily amenable to detection, the construct will preferably also have a selectable marker gene suitable for determining if a plant cell has been transformed.","A general review of suitable markers, for example for the members of the grass family, is found in Wilmink and Dons, 1993, Plant Mol.","Biol.","Reptr, 11(2):165-185.Sequences suitable for permitting integration of the heterologous sequence into the plant genome are also recommended.","These might include transposon sequences and the like for homologous recombination as well as Ti sequences which permit random insertion of a beterologous expression cassette into a plant genome.","Suitable prokaryote selectable markers include resistance toward antibiotics such as ampicillin or tetracycline.","Other DNA sequences encoding additional functions may also be present in the vector, as is known in the art.","The nucleic acid molecules of the subject invention may be included into an expression cassette for expression of the protein(s) of interest.","Usually, there will be only one expression cassette, although two or more are feasible.","The recombinant expression cassette will contain in addition to the heterologous protein encoding sequence the following elements, a promoter region, plant 5′ untranslated sequences, initiation codon depending upon whether or not the structural gene comes equipped with one, and a transcription and translation termination sequence.","Unique restriction enzyme sites at the 5′ and 3′ ends of the cassette allow for easy insertion into a pre-existing vector.","A heterologous coding sequence may be for any protein relating to the present invention.","The sequence encoding the protein of interest will encode a signal peptide which allows processing and translocation of the protein, as appropriate, and will usually lack any sequence which might result in the binding of the desired protein of the invention to a membrane.","Since, for the most part, the transcriptional initiation region will be for a gene which is expressed and translocated during germination, by employing the signal peptide which provides for translocation, one may also provide for translocation of the protein of interest.","In this way, the protein(s) of interest will be translocated from the cells in which they are expressed and may be efficiently harvested.","Typically secretion in seeds are across the aleurone or scutellar epithelium layer into the endosperm of the seed.","While it is not required that the protein be secreted from the cells in which the protein is produced, this facilitates the isolation and purification of the recombinant protein.","Since the ultimate expression of the desired gene product will be in a eucaryotic cell it is desirable to determine whether any portion of the cloned gene contains sequences which will be processed out as introns by the host's splicosome machinery.","If so, site-directed mutagenesis of the “intron” region may be conducted to prevent losing a portion of the genetic message as a false intron code, Reed and Maniatis, Cell 41:95-105, 1985.The vector can be microinjected directly into plant cells by use of micropipettes to mechanically transfer the recombinant DNA.","Crossway, Mol.","Gen. Genet, 202:179-185, 1985.The genetic material may also be transferred into the plant cell by using polyethylene glycol, Krens, et al., Nature, 296, 72-74, 1982.Another method of introduction of nucleic acid segments is high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface, Klein, et al., Nature, 327,70-73, 1987 and Knudsen and Muller, 1991, Planta, 185:330-336 teaching particle bombardment of barley endosperm to create transgenic barley.","Yet another method of introduction would be fusion of protoplasts with other entities, either minicells, cells, lysosomes or other fusible lipid-surfaced bodies, Fraley, et al., Proc.","Natl.","Acad.","Sci.","USA, 79,1859-1863, 1982.The vector may also be introduced into the plant cells by electroporation.","(Fromm et al., Proc.","Natl Acad.","Sci.","USA 82:5824, 1985).","In this technique, plant protoplasts are electroporated in the presence of plasmids containing the gene construct.","Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids.","Electroporated plant protoplasts reform the cell wall, divide, and form plant callus, All plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be transformed by the present invention so that whole plants are recovered which contain the transferred gene.","It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to all major species of sugarcane, sugar beet, cotton, fruit and other trees, legumes and vegetables.","Some suitable plants include, for example, species from the genera Fragaria, Lotus, Medicago, Onobryclis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersion, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hererocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucum is, Browaalia, Glycine, Lolium, Zea, Triticum, Sorghum, and Datura.","Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts containing copies of the heterologous gene is first provided.","Callus tissue is formed and shoots may be induced from callus and subsequently rooted.","Alternatively, embryo formation can be induced from the protoplast suspension.","These embryos germinate as natural embryos to form plants.","The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins.","It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa.","Shoots and roots normally develop simultaneously.","Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture.","If these three variables are controlled, then regeneration is fully reproducible and repeatable.","In some plant cell culture systems, the desired protein of the invention may be excreted or alternatively, the protein may be extracted from the whole plant.","Where the desired protein of the invention is secreted into the medium, it may be collected.","Alternatively, the embryos and embryoless-half seeds or other plant tissue may be mechanically disrupted to release any secreted protein between cells and tissues.","The mixture may be suspended in a buffer solution to retrieve soluble proteins.","Conventional protein isolation and purification methods will be then used to purify the recombinant protein.","Parameters of time, temperature pH, oxygen, and volumes will be adjusted through routine methods to optimize expression and recovery of heterologous protein.","iv.","Bacterial Systems Bacterial expression techniques are known in the art.","A bacterial promoter is any DNA sequence capable of binding bacterial RNA polymerase and initiating the downstream (3′) transcription of a coding sequence (eg.","structural gene) into mRNA.","A promoter will have a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence.","This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site.","A bacterial promoter may also have a second domain called an operator, that may overlap an adjacent RNA polymerase binding site at which RNA synthesis begins.","The operator permits negative regulated (inducible) transcription, as a gene repressor protein may bind the operator and thereby inhibit transcription of a specific gene.","Constitutive expression may occur in the absence of negative regulatory elements, such as the operator.","In addition, positive regulation may be achieved bya gene activator protein binding sequence, which, if present is usually proximal (5′) to the RNA polymerase binding sequence.","An example of a gene activator protein is the catabolite activator protein (CAP), which helps initiate transcription of the lac operon in Escherichia coli (E. coli) [Raibaud et al.","(1984) Annu.","Rev.","Genet.","18:173].","Regulated expression may therefore be either positive or negative, thereby either enhancing or reducing transcription.","Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences.","Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) [Chang et al.","(1977) Nature 198:1056], and maltose.","Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp) [Goeddel et al.","(1980) Nuc.","Acids Res.","8:4057; Yelverton et al.","(1981) Nucl.","Acids Res.","9:731; U.S. Pat.","No.","4,738,921; EP-A-0036776 and EP-A-0121775].","The g-laotamase (bla) promoter system [Weissmann (1981) “The cloning of interferon and other mistakes.” In Interferon 3 (ed.","1.Gresser)], bacteriophage lambda P L [Shimatake et al.","(1981) Nature 292:128] and T5 [U.S. Pat.","No.","4,689,406] promoter systems also provide useful promoter sequences.","In addition, synthetic promoters which do not occur in nature also function as bacterial promoters.","For example, transcription activation sequences of one bacterial or bacteriophage promoter may be joined with the operon sequences of another bacterial or bacteriophage promoter, creating a synthetic hybrid promoter [U.S. Pat.","No.","4,551,433].","For example, the tac promoter is a hybrid trp-lac promoter comprised of both trp promoter and lac operon sequences that is regulated by the lac repressor [Amann et al.","(1983) Gene 25:167; de Boer et al.","(1983) Proc.","Natl.","Acad.","Sci.","80:21].","Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription.","A naturally occurring promoter of non-bacterial origin can also be coupled with a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes.","The bacteriophage T7 RNA polymeraselpromoter system is an example of a coupled promoter system [Studier et al.","(1986) J. Mol.","Biol.","189:113; Tabor et al.","(1985) Proc Natl.","Acad.","Sci.","82:1074].","In addition, a hybrid promoter can also be comprised of a bacteriophage promoter and an E. coli operator region (EPO-A-0 267 851).","In addition to a functioning promoter sequence, an efficient ribosome binding site is also useful for the expression of foreign genes in prokaryotes.","In E. coli, the ribosome binding site is called the Shine-Dalgarno (SD) sequence and includes an initiation codon (ATG) and a sequence 3-9 nucleotides in length located 3-11 nucleotides upstream of the initiation codon [Shine et al.","(1975) Nature 254:34].","The SD sequence is thought to promote binding of mRNA to the ribosome by the pairing of bases between the SD sequence and the 3′ and of E. coli 16S rRNA [Steitz et al.","(1979) “Genetic signals and nucleotide sequences in messenger RNA.” In Biological Regulation and Development: Gene Expression (ed.","R. F. Goldberger)].","To express eukaryotic genes and prokaryotic genes with weak ribosome-binding site [Sambrook et al.","(1989) “Expression of cloned genes in Escherichia coli.” In Molecular Cloning: A Laboratory Manual].","A DNA molecule may be expressed intracellularly.","A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus will always be a methionine, which is encoded by the ATG start codon.","If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide or by either in vivo on in vitro incubation with a bacterial methionine N-terminal peptidase (EPO-A-O 219 237).","Fusion proteins provide an alternative to direct expression.","Usually, a DNA sequence encoding the N-terminal portion of an endogenous bacterial protein, or other stable protein, is fused to the 5′ end of heterologous coding sequences.","Upon expression, this construct will provide a fusion of the two amino acid sequences.","For example, the bacteriophage lambda cell gene can be linked at the 5′ terminus of a foreign gene and expressed in bacteria.","The resulting fusion protein preferably retains a site for a processing enzyme (factor Xa) to cleave the bacteriophage protein from the foreign gene [Nagai et al.","(1984) Nature 309:810].","Fusion proteins can also be made with sequences from the lacZ [Jia et al.","(1987) Gene 60:197], trpE [Allen et al.","(1987) J. Biotechnol.","5:93; Makoff et al.","(1989) J. Gen. Microbiol.","135:11], and Chey [EP-A-0 324 647] genes.","The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site.","Another example is a ubiquitin fusion protein.","Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (eg.","ubiquitin specific processing-protease) to cleave the ubiquitin from the foreign protein.","Through this method, native foreign protein can be isolated [Miller et al.","(1989) Bio/Technology 7:698].","Alternatively, foreign proteins can also be secreted from the cell by creating chimeric DNA molecules that encode a fusion protein comprised of a signal peptide sequence fragment that provides for secretion of the foreign protein in bacteria [U.S. Pat.","No.","4,336,336].","The signal sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell.","The protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria).","Preferably there are processing sites, which can be cleaved either in vivo or in vitro encoded between the signal peptide fragment and the foreign gene.","DNA encoding suitable signal sequences can be derived from genes for secreted bacterial proteins, such as the E. coli outer membrane protein gene (ompA) [Masui et al.","(1983), in: Experimental Manipulation of Gene Expression; Ghrayeb et al.","(1984) EMBO J.","3:2437] and the E. coli alkaline phosphatase signal sequence (phoA) [Oka et al.","(1985) Proc.","Natl.","Acad.","Sci.","82:7212].","As an additional example, the signal sequence of the alpha-amylase gene from various Bacillus strains can be used to secrete heterologous proteins from B. subtilis [Palva et al.","(1982) Proc.","Natl.","Acad.","Sci.","USA 79:5582; EP-A-0 244 042].","Usually, transcription termination sequences recognized by bacteria are regulatory regions located 3′ to the translation stop codon, and thus together with the promoter flank the coding sequence.","These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA.","Transcription termination sequences frequently include DNA sequences of about 50 nucleotides capable of forming stem loop structures that aid in terminating transcription.","Examples include transcription termination sequences derived from genes with strong promoters, such as the trp gene in E. coli as well as other biosynthetic genes.","Usually, the above described components, comprising a promoter, signal sequence (if desired), coding sequence of interest, and transcription termination sequence, are put together into expression constructs.","Expression constructs are often maintained in a replicon, such as an extracbromosomal element (eg.","plasmids) capable of stable maintenance in a host, such as bacteria.","The replicon will have a replication system, thus allowing it to be maintained in a prokaryotic host either for expression or for cloning and amplification.","In addition, a replicon may be either a high or low copy number plasmid.","A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and usually about 10 to about 150.A host containing a high copy number plasmid will preferably contain at least about 10, and more preferably at least about 20 plasmids.","Either a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host.","Alternatively, the expression constructs can be integrated into the bacterial genome with an integrating vector.","Integrating vectors usually contain at least one sequence homologous to the bacterial chromosome that allows the vector to integrate.","Integrations appear to result from recombinations between homologous DNA in the vector and the bacterial chromosome.","For example, integrating vectors constructed with DNA from various Bacillus strains integrate into the Bacillus chromosome (EP-A-0 127 328).","Integrating vectors may also be comprised of bacteriophage or transposon sequences.","Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of bacterial strains that have been transformed.","Selectable markers can be expressed in the bacterial host and may include genes which render bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin), and tetracycline [Davies et al.","(1978) Annu.","Rev.","Microbiol.","32:469].","Selectable markers may also include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways.","Alternatively, some of the above described components can be put together in transformation vectors.","Transformation vectors are usually comprised of a selectable market that is either maintained in a replicon or developed into an integrating vector, as described above.","Expression and transformation vectors, either extra-chromosomal replicons or integrating vectors, have been developed for transformation into many bacteria.","For example, expression vectors have been developed for, inter alia, the following bacteria: Bacillus subtilis [Palva et al.","(1982) Proc.","Natl.","Acad.","Sci.","USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953; WO 84/04541], Escherichia coli [Shimatake et al.","(1981) Nature 292:128; Amann et al.","(1985) Gene 40:183; Studier et al.","(1986) J. Mol.","Biol.","189:113; EP-A-0 036 776,EP-A-0 136 829 and EP-A-0 136 907], Streptococcus cremoris [Powell et al.","(1988) Appl.","Environ.","Microbiol.","54:655]; Streptococcus lividans [Powell et al.","(1988) Appl.","Environ.","Microbiol.","54:655], Streptomyces lividans [U.S. Pat.","No.","4,745,056].","Methods of introducing exogenous DNA into bacterial hosts are well-known in the art, and usually include either the transformation of bacteria treated with CaCl2 or other agents, such as divalent cations and DMSO.","DNA can also be introduced into bacterial cells by electroporation.","Transformation procedures usually vary with the bacterial species to be transformed.","See eg.","[Masson et al.","(1989) FEMS Microbiol.","Lett.","60:273; Palva et al.","(1982) Proc.","Natl.","Acad.","Sci.","USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953; WO 84/04541, Bacillus], [Miller et al.","(1988) Proc.","Natl.","Acad.","Sci.","85:856; Wang et al.","(1990) J. Bacteriol.","172:949, Campylobacter], [Cohen et al.","(1973) Proc.","Natl.","Acad.","Sci.","69:2110; Dower et al.","(1988) Nucleic Acids Res.","16:6127; Kushner (1978) “An improved method for transformation of Escherichia coli with ColE1-derived plasmids.","In Genetic Engineering: Proceedings of the International Symposium on Genetic Engineering (eds.","H. W. Boyer and S. Nicosia); Mandel et al.","(1970) J. Mol.","Biol.","53:159; Taketo (1988) Biochim.","Biophys.","Acta 949:318; Escherichia], [Chassy et al.","(1987) FEMS Microbiol.","Lett.","44:173 Lactobacillus]; [Fiedler et al.","(1988) Anal.","Biochem 170:38, Pseudomonas]; [Augustin et al.","(1990) FEMS Microbiol.","Lett.","66:203, Staphylococcus], [Barany et al.","(1980) J. Bacterial.","144:698; Harlander (1987) “Transformation of Streptococcus lactis by electroporation, in: Streptococcal Genetics (ed.","J. Ferretti and R. Curtiss III); Perry et al.","(1981) Infect.","Immun.","32:1295; Powell et al.","(1988) Appl.","Environ.","Microbial.","54:655; Somkuti et al.","(1987) Proc.","4th Evr.","Cong.","Biotechnology 1:412, Streptococcus].","v. Yeast Expression Yeast expression systems are also known to one of ordinary skill in the art.","A yeast promoter is any DNA sequence capable of binding yeast RNA polymerase and initiating the downstream (3′) transcription of a coding sequence (eg.","structural gene) into mRNA.","A promoter will have a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence.","This transcription initiation region usually includes an RNA polymerase binding site (the “TATA Box”) and a transcription initiation site.","A yeast promoter may also have a second domain called an upstream activator sequence (UAS), which, if present, is usually distal to the structural gene.","The UAS permits regulated (inducible) expression.","Constitutive expression occurs in the absence of a UAS.","Regulated expression may be either positive or negative, thereby either enhancing or reducing transcription.","Yeast is a fermenting organism with an active metabolic pathway, therefore sequences encoding enzymes in the metabolic pathway provide particularly useful promoter sequences.","Examples include alcohol dehydrogenase (ADH) (EP-A-0 284 044), enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, and pyruvate kinase (PyK) (EPO-A-0 329 203).","The yeast PH05 gene, encoding acid phosphatase, also provides useful promoter sequences [Myanohara et al.","(1983) Proc.","Natl.","Acad.","Sci.","USA 80:1].","In addition, synthetic promoters which do not occur in nature also function as yeast promoters.","For example, UAS sequences of one yeast promoter maybe joined with the transcription activation region of another yeast promoter, creating a synthetic hybrid promoter.","Examples of such hybrid promoters include the ADH regulatory sequence linked to the GAP transcription activation region (U.S. Pat.","Nos.","4,876,197 and 4,880,734).","Other examples of hybrid promoters include promoters which consist of the regulatory sequences of either the ADH2, GAL4, GAL10, OR PH05 genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK (EP-A-0 164 556).","Furthermore, a yeast promoter can include naturally occurring promoters of non-yeast origin that have the ability to bind yeast RNA polymerase and initiate transcription.","Examples of such promoters include, inter alia, [Cohen et al.","(1980) Proc.","Natl.","Acad.","Sci.","USA 77:1078; Henikoff et al.","(1981) Nature 283:835; Hollenberg et al.","(1981) Curr.","Topics Microbiol.","Immunol.","96:119; Hollenberg et al.","(1979) “The Expression of Bacterial Antibiotic Resistance Genes in the Yeast Saccharomyces cerevisiae,” in: Plasmids of Medical, Environmental and Commercial Importance (eds.","K. N. Timmis and A. Publer); Mercerau-Puigalon et al.","(1980) Gene 11:163; Panthier et al.","(1980) Curr.","Genet.","2:109;].","A DNA molecule may be expressed intracellularly in yeast.","A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon.","If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.","Fusion proteins provide an alternative for yeast expression systems, as well as in mammalian, baculovirus, and bacterial expression systems.","Usually, a DNA sequence encoding the N-terminal portion of an endogenous yeast protein, or other stable protein, is fused to the 5′ end of heterologous coding sequences.","Upon expression, this construct will provide a fusion of the two amino acid sequences.","For example, the yeast or human superoxide dismutase (SOD) gene, can be linked at the 5′ terminus of a foreign gene and expressed in yeast.","The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site.","See eg.","EP-A-0 196 056.Another example is a ubiquitin fusion protein.","Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (eg.","ubiquitin-specific processing protease) to cleave the ubiquitin from the foreign protein.","Through this method, therefore, native foreign protein can be isolated (eg.","WO88/024066).","Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provide for secretion in yeast of the foreign protein.","Preferably, there are processing sites encoded between the leader fragment and the foreign gene that can be cleaved either in vivo or in vitro.","The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell.","DNA encoding suitable signal sequences can be derived from genes for secreted yeast proteins, such as the yeast invertase gene (EP-A-0 012 873; JPO.","62,096,086) and the A-factor gene (U.S. Pat.","No.","4,588,684).","Alternatively, leaders of non-yeast origin, such as an interferon leader, exist that also provide for secretion in yeast (EP-A-0 060 057).","A preferred class of secretion leaders are those that employ a fragment of the yeast alpha-factor gene, which contains both a “pre” signal sequence, and a “pro” region.","The types of alpha-factor fragments that can be employed include the full-length pre-pro alpha factor leader (about 83 amino acid residues) as well as truncated alpha-factor leaders (usually about 25 to about 50 amino acid residues) (U.S. Pat.","Nos.","4,546,083 and 4,870,008; EP-A-0 324 274).","Additional leaders employing an alpha-factor leader fragment that provides for secretion include hybrid alpha-factor leaders made with a presequence of a first yeast, but a pro-region from a second yeast alphafactor.","(eg.","see WO 89/02463.)","Usually, transcription termination sequences recognized by yeast are regulatory regions located 3′ to the translation stop codon, and thus together with the promoter flank the coding sequence.","These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA.","Examples of transcription terminator sequence and other yeast-recognized termination sequences, such as those coding for glycolytic enzymes.","Usually, the above described components, comprising a promoter, leader (if desired), coding sequence of interest, and transcription termination sequence, are put together into expression constructs.","Expression constructs are often maintained in a replicon, such as an extrachromosomal element (eg.","plasmids) capable of stable maintenance in a host, such as yeast or bacteria.","The replicon may have two replication systems, thus allowing it to be maintained, for example, in yeast for expression and in a prokaryotic host for cloning and amplification.","Examples of such yeast-bacteria shuttle vectors include YEp24 [Botstein et al.","(1979) Gene 8:17-24], pCl/1 [Brake et al.","(1984) Proc.","Natl.","Acad.","Sci USA 81:4642-4646], and YRp17 (Stinchcomb et al.","(1982) J. Mol.","Biol.","158:157].","In addition, a replicon may be either a high or low copy number plasmid.","A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and usually about 10 to about 150.A host containing a high copy number plasmid will preferably have at least about 10, and more preferably at least about 20.Enter a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host.","See eg.","Brake et al., supra.","Alternatively, the expression constructs can be integrated into the yeast genome with an integrating vector.","Integrating vectors usually contain at least one sequence homologous to a yeast chromosome that allows the vector to integrate, and preferably contain two homologous sequences flanking the expression construct.","Integrations appear to result from recombinations between homologous DNA in the vector and the yeast chromosome [Orr-Weaver et al.","(1983) Methods in Enzymol.","101:228-2451.An integrating vector may be directed to a specific locus in yeast by selecting the appropriate homologous sequence for inclusion in the vector.","See Orr-Weaver et al., supra.","One or more expression construct may integrate, possibly affecting levels of recombinant protein produced [Rine et al.","(1983) Proc.","Natl.","Acad.","Sci.","USA 80:6750].","The chromosomal sequences included in the vector can occur either as a single segment in the vector, which results in the integration of the entire vector, or two segments homologous to adjacent segments in the chromosome and flanking the expression construct in the vector, which can result in the stable integration of only the expression construct.","Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of yeast strains that have been transformed.","Selectable markers may include biosynthetic genes that can be expressed in the yeast host, such as ADE2, HIS4, LEU2, TRP1, and ALG7, and the G418 resistance gene, which confer resistance in yeast cells to tunicamycin and G418, respectively.","In addition, a suitable selectable marker may also provide yeast with the ability to grow in the presence of toxic compounds, such as metal.","For example, the presence of CUP1 allows yeast to grow in the presence of copper ions [Butt et al.","(1987) Microbiol, Rev.","51:351].","Alternatively, some of the above described components can be put together into transformation vectors.","Transformation vectors are usually comprised of a selectable marker that is either maintained in a replicon or developed into an integrating vector, as described above.","Expression and transformation vectors, either extrachromosomal replicons or integrating vectors, have been developed for transformation into many yeasts.","For example, expression vectors have been developed for, inter alia, the following yeasts:Candida albicans [Kurtz, et at.","(1986) Mol.","Cell.","Biol.","6:142], Candida maltosa [Kunze, et at.","(1985) J.","Basic Microbiol, 25:141].","Hansenula polymorpha [Gleeson, et al.","(1986) J. Gen. Microbiol.","132:3459; Roggenkamp et al.","(1986) Mol.","Gen. Genet.","202:302], Kluyveromyces fragilis [Das, et al.","(1984) J. Bacteriol.","158:1165], Kluyveromyces lactis [De Louvencourt et al.","(1983) J. Bacteriol.","154:737; Van den Berg et al.","(1990) Bio/Technology 8:135], Pichia guillerimondii [Kunze et al.","(1985) J.","Basic Microbiol.","25:141], Pichia pastoris [Cregg, et al.","(1985) Mol.","Cell.","Biol.","5:3376; U.S. Pat.","Nos.","4,837,148 and 4,929,555], Saccharomyces cerevisiae [Hinnen et al.","(1978) Proc.","Natl.","Acad.","Sci.","USA 75:1929; Ito et al.","(1983) J. Bacteriol.","153:163], Schizosaccharomyces pombe [Beach and Nurse (1981) Nature 300:706], and Yarrowia lipolytica [Davidow, et al.","(1985) Curr.","Genet.","10:380471 Gaillardin, et al.","(1985) Curr.","Genet.","10:49].","Methods of introducing exogenous DNA into yeast hosts are well-known in the art, and usually include either the transformation of spheroplasts or of intact yeast cells treated with alkali cations.","Transformation procedures usually vary with the yeast species to be transformed.","See eg.","[Kurtz et al.","(1986) Mol.","Cell.","Biol.","6:142; Kunze et al.","(1985) J.","Basic Microbiol.","25:141; Candida]; [Gleeson et al.","(1986) J. Gen. Microbiol.","132:3459; Roggenkamp et al.","(1986) Mol.","Gen. Genet.","202:302; Hansenula]; [Das et al.","(1984) J. Bacterial.","158:1165; De Louvencourt et al.","(1983) J. Bacteriol.","154:1165; Van den Berg et al.","(1990) Bio/Technology 8:135; Kluyveromyces]; [Cregg et at.","(1985) Mol.","Cell.","Biol.","5:3376; Kunze et al.","(1985) J.","Basic Microbiol.","25:141; U.S. Pat.","Nos.","4,837,148 and 4,929,555; Pichia]; [Hinnen et al.","(1978) Proc.","Natl.","Acad.","Sci.","USA 75;1929; Ito et al.","(1983) J. Bacteriol.","153:163 Saccharomyces]; [Beach and Nurse (1981) Nature 300:706; Schizosaccbaromyces]; [Davidow et al.","(1985) Curr.","Genet.","10:39; Gaillardin et al.","(1985) Curr.","Genet.","10:49; Yarrowia].","Antibodies As used herein, the term “antibody” refers to a polypeptide or group of polypeptides composed of at least one antibody combining site.","An “antibody combining site” is the three-dimensional binding space with an internal surface shape and charge distribution complementary to the features of an epitope of an antigen, which allows a binding of the antibody with the antigen.","“Antibody” includes, for example, vertebrate antibodies, hybrid antibodies, chimeric antibodies, humanised antibodies, altered antibodies, univalent antibodies, Fab proteins, and single domain antibodies.","Antibodies against the proteins of the invention are useful for affinity chromatography, immunoassays, and distinguishing/identifying Neisseria proteins.","Antibodies to the proteins of the invention, both polyclonal and monoclonal, may be prepared by conventional methods.","In general, the protein is first used to immunize a suitable animal, preferably a mouse, rat, rabbit or goat.","Rabbits and goats are preferred for the preparation of polyclonal sera due to the volume of serum obtainable, and the availability of labeled anti-rabbit and anti-goat antibodies.","Immunization is generally performed by mixing or emulsifying the protein in saline, preferably in an adjuvant such as Freund's complete adjuvant, and injecting the mixture or emulsion parenterally (generally subcutaneously or intramuscularly).","A dose of 50-200 μg/injection is typically sufficient.","Immunization is generally boosted 2-6 weeks later with one or more injections of the protein in saline, preferably using Freund's incomplete adjuvant.","One may alternatively generate antibodies by in vitro immunization using methods known in the art, which for the purposes of this invention is considered equivalent to in vivo immunization.","Polyclonal antisera is obtained by bleeding the immunized animal into a glass or plastic container, incubating the blood at 25° C. for one hour, followed by incubating at 4° C. for 2-18 hours.","The serum is recovered by centrifugation (eg.","1,000g for 10 minutes).","About 20-50 ml per bleed may be obtained from rabbits.","Monoclonal antibodies are prepared using the standard method of Kohler & Milstein [Nature (1975) 256:495-96], or a modification thereof.","Typically, a mouse or rat is immunized as described above.","However, rather than bleeding the animal to extract serum, the spleen (and optionally several large lymph nodes) is removed and dissociated into single cells.","If desired, the spleen cells may be screened (after removal of nonspecifically adherent cells) by applying a cell suspension to a plate or well coated with the protein antigen.","B-cells expressing membrane-bound immunoglobulin specific for the antigen bind to the plate, and are not rinsed away with the rest of the suspension.","Resulting B-cells, or all dissociated spleen cells, are then induced to fuse with myeloma cells to form hybridomas, and are cultured in a selective medium (eg.","hypoxanthine, aminopterin, thymidine medium, “HAT”).","The resulting hybridomas are plated by limiting dilution, and are assayed for production of antibodies which bind specifically to the immunizing antigen (and which do not bind to unrelated antigens).","The selected MAb-secreting hybridomas are then cultured either in vitro (eg.","in tissue culture bottles or hollow fiber reactors), or in vivo (as ascites in mice).","If desired, the antibodies (whether polyclonal or monoclonal) may be labeled using conventional techniques.","Suitable labels include fluorophores, chromophores, radioactive atoms (particularly 32P and 125I), electron-dense reagents, enzymes, and ligands having specific binding partners.","Enzymes are typically detected by their activity.","For example, horseradish peroxidase is usually detected by its ability to convert 3,3′,5,5′-tetramethylbenzidine (TMB) to a blue pigment, quantifiable with a spectrophotometer.","“Specific binding partner” refers to a protein capable of binding a ligand molecule with high specificity, as for example in the case of an antigen and a monoclonal antibody specific therefor.","Other specific binding partners include biotin and avidin or streptavidin, IgG and protein A, and the numerous receptor-ligand couples known in the art.","It should be understood that the above description is not meant to categorize the various labels into distinct classes, as the same label may serve in several different modes.","For example, 125I may serve as a radioactive label or as an electron-dense reagent.","HRP may serve as enzyme or as antigen for a MAb.","Further, one may combine various labels for desired effect.","For example, MAbs and avidin also require labels in the practice of this invention: thus, one might label a MAb with biotin, and detect its presence with avidin labeled with 125I, or with an anti-biotin MAb labeled with HRP.","Other permutations and possibilities will be readily apparent to those of ordinary skill in the art, and are considered as equivalents within the scope of the instant invention.","Pharmaceutical Compositions Pharmaceutical compositions can comprise either polypeptides, antibodies, or nucleic acid of the invention.","The pharmaceutical compositions will comprise a therapeutically effective amount of either polypeptides, antibodies, or polynucleotides of the claimed invention.","The term “therapeutically effective amount” as used herein refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect.","The effect can be detected by, for example, chemical markers or antigen levels.","Therapeutic effects also include reduction in physical symptoms, such as decreased body temperature.","The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration.","Thus, it is not useful to specify an exact effective amount in advance.","However, the effective amount for a given situation can be determined by routine experimentation and is within the judgement of the clinician.","For purposes of the present invention, an effective dose will be from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA constructs in the individual to which it is administered.","A pharmaceutical composition can also contain a pharmaceutically acceptable carrier.","The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents.","The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.","Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles.","Such carriers are well known to those of ordinary skill in the art.","Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.","A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub.","Co., N.J. 1991).","Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol.","Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.","Typically, the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.","Liposomes are included within the definition of a pharmaceutically acceptable carrier.","Delivery Methods Once formulated, the compositions of the invention can be administered directly to the subject.","The subjects to be treated can be animals; in particular, human subjects can be treated.","Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue.","The compositions can also be administered into a lesion.","Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications (eg.","see WO98/20734), needles, and gene guns or hyposprays.","Dosage treatment may be a single dose schedule or a multiple dose schedule.","Vaccines Vaccines according to the invention may either be prophylactic (ie.","to prevent infection) or therapeutic (ie.","to treat disease after infection).","Such vaccines comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid, usually in combination with “pharmaceutically acceptable carriers,” which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition.","Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles.","Such carriers are well known to those of ordinary skill in the art.","Additionally, these carriers may function as immunostimulating agents (“adjuvants”).","Furthermore, the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, etc.","pathogens.","Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59™ (WO 90/14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds.","Powell & Newman, Plenum Press 1995), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE (see below), although not required) formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton, Mass.","), (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below) either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) Ribi™ adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.)","containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox™); (3) saponin adjuvants, such as Stimulon™ (Cambridge Bioscience, Worcester, Mass.)","may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes); (4) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (5) cytokines, such as interleukins (eg.","IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.","), interferons (eg.","gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc; and (6) other substances that act as immunostimulating agents to enhance the effectiveness of the composition.","Alum and MF59™ are preferred.","As mentioned above, muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuram yl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.","The immunogenic compositions (eg.","the immunising antigen/immunogen/polypeptide/protein/nucleic acid, pharmaceutically acceptable carrier, and adjuvant) typically will contain diluents, such as water, saline, glycerol, ethanol, etc.","Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.","Typically, the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.","The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above under pharmaceutically acceptable carriers.","Immunogenic compositions used as vaccines comprise an immunologically effective amount of the antigenic or immunogenic polypeptides, as well as any other of the above-mentioned components, as needed.","By “immunologically effective amount”, it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention.","This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (eg.","nonhuman primate, primate, etc.","), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors.","It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.","The immunogenic compositions are conventionally administered parenterally, eg.","by injection, either subcutaneously, intramuscularly, or transdermally/transcutaneously (eg.","WO98/20734).","Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications.","Dosage treatment may be a single dose schedule or a multiple dose schedule.","The vaccine may be administered in conjunction with other immunoregulatory agents.","As an alternative to protein-based vaccines, DNA vaccination may be used [eg.","Robinson & Torres (1997) Seminars in Immunol 9:271-283; Donnelly et al.","(1997) Annu Rev Immunol 15:617-648; later herein].","Gene Delivery Vehicles Gene therapy vehicles for delivery of constructs including a coding sequence of a therapeutic of the invention, to be delivered to the mammal for expression in the mammal, can be administered either locally or systemically.","These constructs can utilize viral or non-viral vector approaches in in vivo or ex vivo modality.","Expression of such coding sequence can be induced using endogenous mammalian or heterologous promoters.","Expression of the coding sequence in vivo can be either constitutive or regulated.","The invention includes gene delivery vehicles capable of expressing the contemplated nucleic acid sequences.","The gene delivery vehicle is preferably a viral vector and, more preferably, a retroviral, adenoviral, adeno-associated viral (AAV), herpes viral, or alphavirus vector.","The viral vector can also be an astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, or togavirus viral vector.","See generally, Jolly (1994) Cancer Gene Therapy 1:51-64; Kimura (1994) Human Gene Therapy 5:845-852; Connelly (1995) Human Gene Therapy 6:185-193; and Kaplitt (1994) Nature Genetics 6:148-153.Retroviral vectors are well known in the art and we contemplate that any retroviral gene therapy vector is employable in the invention, including B, C and D type retroviruses, xenotropic retroviruses (for example, NZB-X1, NZB-X2 and NZB9-1 (see O'Neill (1985) J. Virol.","53:160) polytropic retroviruses eg.","MCF and MCF-MLV (see Kelly (1983) J. Virol.","45:291), spumaviruses and lentiviruses.","See RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985.Portions of the retroviral gene therapy vector may be derived from different retroviruses.","For example, retrovector LTRs may be derived from a Murine Sarcoma Virus, a TRNA binding site from a Rous Sarcoma Virus, a packaging signal from a Murine Leukemia Virus, and an origin of second strand synthesis from an Avian Leukosis Virus.","These recombinant retroviral vectors may be used to generate transduction competent retroviral vector particles by introducing them into appropriate packaging cell lines (see U.S. Pat.","No.","5,591,624).","Retrovirus vectors can be constructed for site-specific integration into host cell DNA by incorporation of a chimeric integrase enzyme into the retroviral particle (see WO96/37626).","It is preferable that the recombinant viral vector is a replication defective recombinant virus.","Packaging cell lines suitable for use with the above-described retrovirus vectors are well known in the art, are readily prepared (see WO95/30763 and WO92/05266), and can be used to create producer cell lines (also termed vector cell lines or “VCLs”) for the production of recombinant vector particles.","Preferably, the packaging cell lines are made from human parent cells (eg.","HT1080 cells) or mink parent cell lines, which eliminates inactivation in human serum.","Preferred retroviruses for the construction of retroviral gene therapy vectors include Avian Leukosis Virus, Bovine Leukemia, Virus, Murine Leukemia Virus, Mink-Cell Focus-Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis Virus and Rous Sarcoma Virus.","Particularly preferred Murine Leukemia Viruses include 4070A and 1504A (Hartley and Rowe (1976) J Virol 19:19-25), Abelson (ATCC No.","VR-999), Friend (ATCC No.","VR-245), Graffi, Gross (ATCC Nol VR-590), Kirsten, Harvey Sarcoma Virus and Rauscher (ATCC No.","VR-998) and Moloney Murine Leukemia Virus (ATCC No.","VR-190).","Such retroviruses may be obtained from depositories or collections such as the American Type Culture Collection (“ATCC”) in Rockville, Md.","or isolated from known sources using commonly available techniques.","Exemplary known retroviral gene therapy vectors employable in this invention include those described in patent applications GB2200651, EP0415731, EP0345242, EP0334301, WO89/02468; WO89/05349, WO89/09271, WO90/02806, WO90/07936, WO94/03622, WO93/25698, WO93/25234, WO93/11230, WO93/10218, WO91/02805, WO91/02825, WO95/07994, U.S. Pat.","Nos.","5,219,740, 4,405,712, 4,861,719, 4,980,289, 4,777,127, 5,591,624.See also Vile (1993) Cancer Res 53:3860-3864; Vile (1993) Cancer Res 53:962-967; Ram (1993) Cancer Res 53 (1993) 83-88; Takamiya (1992) J Neurosci Res 33:493-503; Baba (1993) J Neurosurg 79:729-735; Mann (1983) Cell 33:153; Cane (1984) Proc Natl Acad Sci 81:6349; and Miller (1990) Human Gene Therapy 1.Human adenoviral gene therapy vectors are also known in the art and employable in this invention, See, for example, Berkner (1988) Biotechniques 6:616 and Rosenfeld (1991) Science 252:431, and WO93/07283, WO93/06223, and WO93/07282.Exemplary known adenoviral gene therapy vectors employable in this invention include those described in the above referenced documents and in WO94/12649, WO93/03769, WO93/19191, WO94/28938, WO95/11984, WO95/00655, WO95/27071, WO95/29993, WO95/34671, WO96/05320, WO94/08026, WO94/11506, WO93/06223, WO94/24299, WO95/14102, WO95/24297, WO95/02697, WO94/28152, WO94/24299, WO95/09241, WO95/25807, WO95/05835, WO94/18922 and WO95/09654.Alternatively, administration of DNA linked to killed adenovirus as described in Curiel (1992) Hum.","Gene Ther.","3:147-154 may be employed.","The gene delivery vehicles of the invention also include adenovirus associated virus (AAV) vectors.","Leading and preferred examples of such vectors for use in this invention are the AAV-2 based vectors disclosed in Srivastava, WO93/09239.Most preferred AAV vectors comprise the two AAV inverted terminal repeats in which the native D-sequences are modified by substitution of nucleotides, such that at least 5 native nucleotides and up to 18 native nucleotides, preferably at least 10 native nucleotides up to 18 native nucleotides, most preferably 10 native nucleotides are retained and the remaining nucleotides of the D-sequence are deleted or replaced with non-native nucleotides.","The native D-sequences of the AAV inverted terminal repeats are sequences of 20 consecutive nucleotides in each AAV inverted terminal repeat (ie.","there is one sequence at each end) which are not involved in HP formation.","The non-native replacement nucleotide may be any nucleotide other than the nucleotide found in the native D-sequence in the same position.","Other employable exemplary AAV vectors are pWP-19, pWN-1, both of which are disclosed in Nahreini (1993) Gene 124:257-262.Another example of such an AAV vector is psub201 (see Samulski (1987) J. Virol.","61:3096).","Another exemplary AAV vector is the Double-D ITR vector.","Construction of the Double-D ITR vector is disclosed in U.S. Pat.","No.","5,478,745.Still other vectors are those disclosed in Carter U.S. Pat.","No.","4,797,368 and Muzyczka U.S. Pat.","No.","5,139,941, Chartejee U.S. Pat.","No.","5,474,935, and Kotin WO94/288157.Yet a further example of an AAV vector employable in this invention is SSV9AFABTKneo, which contains the AFP enhancer and albumin promoter and directs expression predominantly in the liver.","Its structure and construction are disclosed in Su (1996) Human Gene Therapy 7:463-470.Additional AAV gene therapy vectors are described in U.S. Pat.","Nos.","5,354,678, 5,173,414, 5,139,941, and 5,252,479.The gene therapy vectors of the invention also include herpes vectors.","Leading and preferred examples are herpes simplex virus vectors containing a sequence encoding a thymidine kinase polypeptide such as those disclosed in U.S. Pat.","No.","5,288,641 and EP0176170 (Roizman).","Additional exemplary herpes simplex virus vectors include HFEM/ICP6-LacZ disclosed in WO95/04139 (Wistar Institute), pHSVlac described in Geller (1988) Science 241:1667-1669 and in WO90/09441 and WO92/07945, HSV Us3::pgC-lacZ described in Fink (1992) Human Gene Therapy 3:11-19 and HSV 7134, 2 RH 105 and GAL4 described in EP 0453242 (Breakefield), and those deposited with the ATCC with accession numbers VR-977 and VR-260.Also contemplated are alpha virus gene therapy vectors that can be employed in this invention.","Preferred alpha virus vectors are Sindbis viruses vectors.","Togaviruses, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Middleberg virus (ATCC VR-370), Ross River virus (ATCC VR-373; ATCC VR-1246), Venezuelan equine encephalitis virus (ATCC VR923; ATCC VR-1250; ATCC VR-1249; ATCC VR-532), and those described in U.S. Pat.","Nos.","5,091,309, 5,217,879, and WO92/10578.More particularly, those alpha virus vectors described in U.S. Ser.","No.","08/405,627, filed Mar.","15, 1995, WO94/21792, WO92/10578, WO95/07994, U.S. Pat.","Nos.","5,091,309 and 5,217,879 are employable.","Such alpha viruses may be obtained from depositories or collections such as the ATCC in Rockville, Md.","or isolated from known sources using commonly available techniques.","Preferably, alphavirus vectors with reduced cytotoxicity are used (see U.S. Ser.","No.","08/679640).","DNA vector systems such as eukaryotic layered expression systems are also useful for expressing the nucleic acids of the invention.","See WO95/07994 for a detailed description of eukaryotic layered expression systems.","Preferably, the eukaryotic layered expression systems of the invention are derived from alphavirus vectors and most preferably from Sindbis viral vectors.","Other viral vectors suitable for use in the present invention include those derived from poliovirus, for example ATCC VR-58 and those described in Evans, Nature 339 (1989) 385 and Sabin (1973) J. Biol.","Standardization 1:115; rhinovirus, for example ATCC VR-11 10 and those described in Arnold (1990) J Cell Biochem L401; pox viruses such as canary pox virus or vaccinia virus, for example ATCC VR-111 and ATCC VR-2010 and those described in Fisher-Hoch (1989) Proc Natl Acad Sci 86:317; Flexner (1989) Ann NY Acad Sci 569:86, Flexner (1990) Vaccine 8:17; in U.S. Pat.","Nos.","4,603,112 and 4,769,330 and WO89/01973; SV40 virus, for example ATCC VR-305 and those described in Mulligan (1979) Nature 277:108 and Madzak (1992) J Gen Virol 73:1533; influenza virus, for example ATCC VR-797 and recombinant influenza viruses made employing reverse genetics techniques as described in U.S. Pat.","No.","5,166,057 and in Enami (1990) Proc Natl Acad Sci 87:3802-3805; Enami & Palese (1991) J Virol 65:2711-2713 and Luytjes (1989) Cell 59:110, (see also McMichael (1983) NEI Med 309:13, and Yap (1978) Nature 273:238 and Nature (1979) 277:108); human immunodeficiency virus as described in EP-0386882 and in Buchschacher (1992) J. Virol.","66:2731; measles virus, for example ATCC VR-67 and VR-1247 and those described in EP-0440219; Aura virus, for example ATCC VR-368; Bebaru virus, for example ATCC VR-600 and ATCC VR-1240; Cabassou virus, for example ATCC VR-922; Chikungunya virus, for example ATCC VR-64 and ATCC VR-1241; Fort Morgan Virus, for example ATCC VR-924; Getah virus, for example ATCC VR-369 and ATCC VR-1243; Kyzylagach virus, for example ATCC VR-927; Mayaro virus, for example ATCC VR-66; Mucambo virus, for example ATCC VR-580 and ATCC VR-1244; Ndumu virus, for example ATCC VR-371; Pixuna virus, for example ATCC VR-372 and ATCC VR-1245; Tonate virus, for example ATCC VR-925; Triniti virus, for example ATCC VR-469; Una virus, for example ATCC VR-374; Whataroa virus, for example ATCC VR-926; Y-62-33 virus, for example ATCC VR-375; O'Nyong virus, Eastern encephalitis virus, for example ATCC VR-65 and ATCC VR-1242; Western encephalitis virus, for example ATCC VR-70, ATCC VR-1251, ATCC VR-622 and ATCC VR-1252; and coronavirus, for example ATCC VR-740 and those described in Hamre (1966) Proc Soc Exp Biol Med 121:190.Delivery of the compositions of this invention into cells is not limited to the above mentioned viral vectors.","Other delivery methods and media may be employed such as, for example, nucleic acid expression vectors, polycationic condensed DNA linked or unlinked to killed adenovirus alone, for example see U.S. Ser.","No.","08/366,787, filed Dec. 30, 1994 and Curiel (1992) Hum Gene Ther 3:147-154 ligand linked DNA, for example see Wu (1989) J Biol Chem 264:16985-16987, eucaryotic cell delivery vehicles cells, for example see U.S. Ser.","No.","08/240,030, filed May 9, 1994, and U.S. Ser.","No.","08/404,796, deposition of photopolymerized hydrogel materials, hand-held gene transfer particle gun, as described in U.S. Pat.","No.","5,149,655, ionizing radiation as described in U.S. Pat.","No.","5,206,152 and in WO92/11033, nucleic charge neutralization or fusion with cell membranes.","Additional approaches are described in Philip (1994) Mol Cell Biol 14:2411-2418 and in Woffendin (1994) Proc Natl Acad Sci 91:1581-1585.Particle mediated gene transfer may be employed, for example see U.S. Ser.","No.","60/023,867.Briefly, the sequence can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, as described in Wu & Wu (1987) J. Biol.","Chem.","262:4429-4432, insulin as described in Hucked (1990) Biochem Pharmacol 40:253-263, galactose as described in Plank (1992) Bioconjugate Chem 3:533-539, lactose or transferrin.","Naked DNA may also be employed.","Exemplary naked DNA introduction methods are described in WO 90/11092 and U.S. Pat.","No.","5,580,859.Uptake efficiency may be improved using biodegradable latex beads.","DNA coated latex beads are efficiently transported into cells after endocytosis initiation by the beads.","The method may be improved further by treatment of the beads to increase hydrophobicity and thereby facilitate disruption of the endosome and release of the DNA into the cytoplasm.","Liposomes that can act as gene delivery vehicles are described in U.S. Pat.","No.","5,422,120, WO95/13796, WO94/23697, WO91/14445 and EP-524,968.As described in U.S. Ser.","No.","60/023,867, on non-viral delivery, the nucleic acid sequences encoding a polypeptide can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then be incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, insulin, galactose, lactose, or transferrin.","Other delivery systems include the use of liposomes to encapsulate DNA comprising the gene under the control of a variety of tissue-specific or ubiquitously-active promoters.","Further non-viral delivery suitable for use includes mechanical delivery systems such as the approach described in Woffendin et al (1994) Proc.","Natl.","Acad.","Sci.","USA 91(24):11581-11585.Moreover, the coding sequence and the product of expression of such can be delivered through deposition of photopolymerized hydrogel materials.","Other conventional methods for gene delivery that can be used for delivery of the coding sequence include, for example, use of hand-held gene transfer particle gun, as described in U.S. Pat.","No.","5,149,655; use of ionizing radiation for activating transferred gene, as described in U.S. Pat.","No.","5,206,152 and WO92/11033.Exemplary liposome and polycationic gene delivery vehicles are those described in U.S. Pat.","Nos.","5,422,120 and 4,762,915; in WO 95/13796; WO94/23697; and WO91/14445; in EP-0524968; and in Stryer, Biochemistry, pages 236-240 (1975) W.H.","Freeman, San Francisco; Szoka (1980) Biochem Biophys Acta 600:1; Bayer (1979) Biochem Biophys Acta 550:464; Rivnay (1987) Meth Enzymol 149:119; Wang (1987) Proc Natl Acad Sci 84:7851; Plant (1989) Anal Biochem 176:420.A polynucleotide composition can comprises therapeutically effective amount of a gene therapy vehicle, as the term is defined above.","For purposes of the present invention, an effective dose will be from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA constructs in the individual to which it is administered.","Delivery Methods Once formulated, the polynucleotide compositions of the invention can be administered (1) directly to the subject; (2) delivered ex vivo, to cells derived from the subject; or (3) in vitro for expression of recombinant proteins.","The subjects to be treated can be mammals or birds.","Also, human subjects can be treated.","Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue.","The compositions can also be administered into a lesion.","Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications (eg.","see WO98/20734), needles, and gene guns or hyposprays.","Dosage treatment may be a single dose schedule or a multiple dose schedule.","Methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and described in eg.","WO93/14778.Examples of cells useful in ex vivo applications include, for example, stem cells, particularly hem atopoetic, lymph cells, macrophages, dendritic cells, or tumor cells.","Generally, delivery of nucleic acids for both ex vivo and in vitro applications can be accomplished by the following procedures, for example, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei, all well known in the art.","Polynucleotide and Polypeptide Pharmaceutical Compositions In addition to the pharmaceutically acceptable carriers and salts described above, the following additional agents can be used with polynucleotide and/or polypeptide compositions.","A. Polypeptides One example are polypeptides which include, without limitation: asioloorosomucoid (ASOR); transferrin; asialoglycoproteins; antibodies; antibody fragments; ferritin; interleukins; interferons, granulocyte, macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), stem cell factor and erythropoietin.","Viral antigens, such as envelope proteins, can also be used.","Also, proteins from other invasive organisms, such as the 17 amino acid peptide from the circumsporozoite protein of plasmodium falciparum known as RII.","B. Hormones, Vitamins, etc.","Other groups that can be included are, for example: hormones, steroids, androgens, estrogens, thyroid hormone, or vitamins, folic acid.","C. Polyalkylenes, Polysaccharides, etc.","Also, polyalkylene glycol can be included with the desired polynucleotides/polypeptides.","In a preferred embodiment, the polyalkylene glycol is polyethlylene glycol.","In addition, mono-, di-, or polysaccharides can be included.","In a preferred embodiment of this aspect, the polysaccharide is dextran or DEAE-dextran.","Also, chitosan and poly(lactide-co-glycolide) D. Lipids, and Liposomes The desired polynucleotidelpolypeptide can also be encapsulated in lipids or packaged in liposomes prior to delivery to the subject or to cells derived therefrom.","Lipid encapsulation is generally accomplished using liposomes which are able to stably bind or entrap and retain nucleic acid.","The ratio of condensed polynucleotide to lipid preparation can vary but will generally be around 1:1 (mg DNA:micromoles lipid), or more of lipid.","For a review of the use of liposomes as carriers for delivery of nucleic acids, see, Hug and Sleight (1991) Biochim.","Biophys.","Acta.","1097:1-17; Straubinger (1983) Meth.","Enzymol.","101:512-527.Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations.","Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Feigner (1987) Proc.","Natl.","Acad.","Sci.","USA 84:7413-7416); mRNA (Malone (1989) Proc.","Natl.","Acad.","Sci.","USA 86:6077-6081); and purified transcription factors (Debs (1990) J. Biol.","Chem.","265:10189-10192), in functional form.","Cationic liposomes are readily available.","For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Feigner supra).","Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boerhinger).","Other cationic liposomes can be prepared from readily available materials using techniques well known in the art.","See, eg.","Szoka (1978) Proc.","Natl.","Acad.","Sci.","USA 75:4194-4198; WO90/11092 for a description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.","Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials.","Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others.","These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios.","Methods for making liposomes using these materials are well known in the art.","The liposomes can comprise multilammelar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs).","The various liposome-nucleic acid complexes are prepared using methods known in the art.","See eg.","Straubinger (1983) Meth.","Immunol.","101:512-527; Szoka (1978) Proc.","Natl.","Acad.","Sci.","USA 75:4194-4198; Papahadjopoulos (1975) Biochim.","Biophys.","Acta 394:483; Wilson (1979) Cell 17:77); Deamer & Bangham (1976) Biochim.","Biophys.","Acta 443:629; Ostro (1977) Biochem.","Biophys.","Res.","Commun.","76:836; Fraley (1979) Proc.","Natl.","Acad.","Sci.","USA 76:3348); Enoch & Strittmatter (1979) Proc.","Natl.","Acad.","Sci.","USA 76:145; Fraley (1980) J. Biol.","Chem.","(1980) 255:10431; Szoka & Papahadjopoulos (1978) Proc.","Natl.","Acad.","Sci.","USA 75:145; and Schaefer-Ridder (1982) Science 215:166.E.","Lipoproteins In addition, lipoproteins can be included with the polynucleotide/polypeptide to be delivered.","Examples of lipoproteins to be utilized include: chylomicrons, HDL, IDL, LDL, and VLDL.","Mutants, fragments, or fusions of these proteins can also be used.","Also, modifications of naturally occurring lipoproteins can be used, such as acetylated LDL.","These lipoproteins can target the delivery of polynucleotides to cells expressing lipoprotein receptors.","Preferably, if lipoproteins are including with the polynucleotide to be delivered, no other targeting ligand is included in the composition.","Naturally occurring lipoproteins comprise a lipid and a protein portion.","The protein portion are known as apoproteins.","At the present, apoproteins A, B, C, D, and E have been isolated and identified.","At least two of these contain several proteins, designated by Roman numerals, AI, AII, AIV; CI, CII, CIII.","A lipoprotein can comprise more than one apoprotein.","For example, naturally occurring chylomicrons comprises of A, B, C & E, over time these lipoproteins lose A and acquire C & E. VLDL comprises A, B, C & E apoproteins, LDL comprises apoprotein B; and HDL comprises apoproteins A, C, & E. The amino acid of these apoproteins are known and are described in, for example, Breslow (1985) Annu Rev.","Biochem 54:699; Law (1986) Adv.","Exp Med.","Biol.","151:162; Chen (1986) J Biol Chem 261:12918; Kane (1980) Proc Natl Acad Sci USA 77:2465; and Utermann (1984) Hum Genet 65:232.Lipoproteins contain a variety of lipids including, triglycerides, cholesterol (free and esters), and phospholipids, The composition of the lipids varies in naturally occurring lipoproteins.","For example, chylomicrons comprise mainly triglycerides.","A more detailed description of the lipid content of naturally occurring lipoproteins can be found, for example, in Meth.","Enzymol.","128 (1986).","The composition of the lipids are chosen to aid in conformation of the apoprotein for receptor binding activity.","The composition of lipids can also be chosen to facilitate hydrophobic interaction and association with the polynucleotide binding molecule.","Naturally occurring lipoproteins can be isolated from serum by ultracentrifugation, for instance.","Such methods are described in Meth.","Enzymol.","(supra); Pitas (1980) J. Biochem.","255:5454-5460 and Mahey (1979) J Clin.","Invest 64:743-750.Lipoproteins can also be produced by in vitro or recombinant methods by expression of the apoprotein genes in a desired host cell.","See, for example, Atkinson (1986) Annu Rev Biophys Chem 15:403 and Radding (1958) Biochim Biophys Acta 30: 443.Lipoproteins can also be purchased from commercial suppliers, such as Biomedical Techniologies, Inc., Stoughton, Mass., USA.","Further description of lipoproteins can be found in Zuckermann et al.","PCT/US97/14465.F.","Polycationic Agents Polycationic agents can be included, with or without lipoprotein, in a composition with the desired polynucleotide/polypeptide to be delivered.","Polycationic agents, typically, exhibit a net positive charge at physiological relevant pH and are capable of neutralizing the electrical charge of nucleic acids to facilitate delivery to a desired location.","These agents have both in vitro, ex vivo, and in vivo applications.","Polycationic agents can be used to deliver nucleic acids to a living subject either intramuscularly, subcutaneously, etc.","The following are examples of useful polypeptides as polycationic agents: polylysine, polyarginine, polyornithine, and protamine.","Other examples include histones, protamines, human serum albumin, DNA binding proteins, non-histone chromosomal proteins, coat proteins from DNA viruses, such as (X174, transcriptional factors also contain domains that bind DNA and therefore may be useful as nucleic aid condensing agents.","Briefly, transcriptional factors such as C/CEBP, cjun, c-fos, AP-1, AP-2, AP-3, CPF, Prot-1, Sp-1, Oct-1, Oct-2, CREP, and TFIID contain basic domains that bind DNA sequences.","Organic polycationic agents include: spermine, spermidine, and purtrescine.","The dimensions and of the physical properties of a polycationic agent can be extrapolated from the list above, to construct other polypeptide polycationic agents or to produce synthetic polycationic agents.","Synthetic polycationic agents which are useful include, for example, DEAE-dextran, polybrene.","Lipofectin™, and lipofectAMINE™ are monomers that form polycationic complexes when combined with polynucleotides/polypeptides.","Immunodiagnostic Assays Neisseria antigens of the invention can be used in immunoassays to detect antibody levels (or, conversely, anti-Neisseria antibodies can be used to detect antigen levels).","Immunoassays based on well defined, recombinant antigens can be developed to replace invasive diagnostics methods.","Antibodies to Neisseria proteins within biological samples, including for example, blood or serum samples, can be detected.","Design of the immunoassays is subject to a great deal of variation, and a variety of these are known in the art.","Protocols for the immunoassay may be based, for example, upon competition, or direct reaction, or sandwich type assays.","Protocols may also, for example, use solid supports, or may be by immunoprecipitation.","Most assays involve the use of labeled antibody or polypeptide; the labels may be, for example, fluorescent, chemiluminescent, radioactive, or dye molecules.","Assays which amplify the signals from the probe are also known; examples of which are assays which utilize biotin and avidin, and enzyme-labeled and mediated immunoassays, such as ELISA assays.","Kits suitable for immunodiagnosis and containing the appropriate labeled reagents are constructed by packaging the appropriate materials, including the compositions of the invention, in suitable containers, along with the remaining reagents and materials (for example, suitable buffers, salt solutions, etc.)","required for the conduct of the assay, as well as suitable set of assay instructions.","Nucleic Acid Hybridisation “Hybridization” refers to the association of two nucleic acid sequences to one another by hydrogen bonding.","Typically, one sequence will be fixed to a solid support and the other will be free in solution.","Then, the two sequences will be placed in contact with one another under conditions that favor hydrogen bonding.","Factors that affect this bonding include: the type and volume of solvent; reaction temperature; time of hybridization; agitation; agents to block the non-specific attachment of the liquid phase sequence to the solid support (Denhardt's reagent or BLOTTO); concentration of the sequences; use of compounds to increase the rate of association of sequences (dextran sulfate or polyethylene glycol); and the stringency of the washing conditions following hybridization.","See Sambrook et al.","[supral Volume 2, chapter 9, pages 9.47 to 9.57.“Stringency” refers to conditions in a hybridization reaction that favor association of very similar sequences over sequences that differ.","For example, the combination of temperature and salt concentration should be chosen that is approximately 120 to 200° C. below the calculated Tm of the hybrid under study.","The temperature and salt conditions can often be determined empirically in preliminary experiments in which samples of genomic DNA immobilized on filters are hybridized to the sequence of interest and then washed under conditions of different stringencies.","See Sambrook et al.","at page 9.50.Variables to consider when performing, for example, a Southern blot are (1) the complexity of the DNA being blotted and (2) the homology between the probe and the sequences being detected.","The total amount of the fragment(s) to be studied can vary a magnitude of 10, from 0.1 to lug for a plasmid or phage digest to 10−9 to 10−8 g for a single copy gene in a highly complex eukaryotic genome.","For lower complexity polynucleotides, substantially shorter blotting, hybridization, and exposure times, a smaller amount of starting polynucleotides, and lower specific activity of probes an be used.","For example, a single-copy yeast gene can be detected with an exposure time of only 1 hour starting with 1 μg of yeast DNA, blotting for two hours, and hybridizing for 4-8 hours with a probe of 108 cpm/μg.","For a single-copy mammalian gene a conservative approach would start with 10 μg of DNA, blot overnight, and hybridize overnight in the presence of 10% dextran sulfate using a probe of greater than 108 cpm/μg, resulting in an exposure time of ˜24 hours.","Several factors can affect the melting temperature (Tm) of a DNA-DNA hybrid between the probe and the fragment of interest, and consequently, the appropriate conditions for hybridization and washing.","In many cases the probe is not 100% homologous to the fragment.","Other commonly encountered variables include the length and total G+C content of the hybridizing sequences and the ionic strength and form amide content of the hybridization buffer.","The effects of all of these factors can be approximated by a single equation: Tm=81+16.6(log10Ci)+0.4[%(G+C)]0.6(% formamide)−600/n-1.5(% mismatch).","where Ci is the salt concentration (monovalent ions) and n is the length of the hybrid in base pairs (slightly modified from Meinkoth & Wahl (1984) Anal.","Biochem.","138: 267-284).","In designing a hybridization experiment, some factors affecting nucleic acid hybridization can be conveniently altered.","The temperature of the hybridization and washes and the salt concentration during the washes are the simplest to adjust.","As the temperature of the hybridization increases (ie.","stringency), it becomes less likely for hybridization to occur between strands that are nonhomologous, and as a result, background decreases.","If the radiolabeled probe is not completely homologous with the immobilized fragment (as is frequently the case in gene family and interspecies hybridization experiments), the hybridization temperature must be reduced, and background will increase.","The temperature of the washes affects the intensity of the hybridizing band and the degree of background in a similar manner.","The stringency of the washes is also increased with decreasing salt concentrations.","In general, convenient hybridization temperatures in the presence of 50% formamide are 42° C. for a probe with is 95% to 100% homologous to the target fragment, 37° C for 90% to 95% homology, and 32° C. for 85% to 90% homology.","For lower homologies, formamide content should be lowered and temperature adjusted accordingly, using the equation above.","If the homology between the probe and the target fragment are not known, the simplest approach is to start with both hybridization and wash conditions which are nonstringent.","If non-specific bands or high background are observed after autoradiography, the filter can be washed at high stringency and reexposed.","If the time required for exposure makes this approach impractical, several hybridization and/or washing stringencies should be tested in parallel.","Nucleic Acid Probe Assays Methods such as PCR, branched DNA probe assays, or blotting techniques utilizing nucleic acid probes according to the invention can determine the presence of cDNA or mRNA.","A probe is said to “hybridize” with a sequence of the invention if it can form a duplex or double stranded complex, which is stable enough to be detected.","The nucleic acid probes will hybridize to the Neisseria nucleotide sequences of the invention (including both sense and antisense strands).","Though many different nucleotide sequences will encode the amino acid sequence, the native Neisseria sequence is preferred because it is the actual sequence present in cells.","mRNA represents a coding sequence and so a probe should be complementary to the coding sequence; single-stranded cDNA is complementary to mRNA, and so a cDNA probe should be complementary to the non-coding sequence.","The probe sequence need not be identical to the Neisseria sequence (or its complement)—some variation in the sequence and length can lead to increased assay sensitivity if the nucleic acid probe can form a duplex with target nucleotides, which can be detected.","Also, the nucleic acid probe can include additional nucleotides to stabilize the formed duplex, Additional Neisseria sequence may also be helpful as a label to detect the formed duplex.","For example, a non-complementary nucleotide sequence may be attached to the 5′ end of the probe, with the remainder of the probe sequence being complementary to a Neisseria sequence.","Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the a Neisseria sequence in order to hybridize therewith and thereby form a duplex which can be detected.","The exact length and sequence of the probe will depend on the hybridization conditions (e.g.","temperature, salt condition etc.).","For example, for diagnostic applications, depending on the complexity of the analyte sequence, the nucleic acid probe typically contains at least 10-20 nucleotides, preferably 15-25, and more preferably at least 30 nucleotides, although it may be shorter than this.","Short primers generally require cooler temperatures to form sufficiently stable hybrid complexes with the template.","Probes may be produced by synthetic procedures, such as the triester method of Matteucci et al.","[J.","Am.","Chem.","Soc.","(1981) 103:31853, or according to Urdea et al.","[Proc.","Natl.","Acad.","Sci.","USA (1983) 80: 7461], or using commercially available automated oligonucleotide synthesizers.","The chemical nature of the probe can be selected according to preference.","For certain applications, DNA or RNA are appropriate.","For other applications, modifications may be incorporated eg.","backbone modifications, such as phosphorothioates or methylphosphonates, can be used to increase in vivo half-life, alter RNA affinity, increase nuclease resistance etc.","[eg.","see Agrawal & Jyer (1995) Curr Opin Biotechnol 6:12-19; Agrawal (1996) TIBTECH 14:376-387]; analogues such as peptide nucleic acids may also be used [eg.","see Corey (1997) TIBTECH 15:224-229; Buchardt et al.","(1993) TIBTECH 11:384-386].","Alternatively, the polymerase chain reaction (PCR) is another well-known means for detecting small amounts of target nucleic acid.","The assay is described in Mullis et al.","[Meth.","Enzymol.","(1987) 155:335-350] & U.S. Pat.","Nos.","4,683,195 & 4,683,202.Two “primer” nucleotides hybridize with the target nucleic acids and are used to prime the reaction.","The primers can comprise sequence that does not hybridize to the sequence of the amplification target (or its complement) to aid with duplex stability or, for example, to incorporate a convenient restriction site.","Typically, such sequence will flank the desired Neisseria sequence.","A thermostable polymerase creates copies of target nucleic acids from the primers using the original target nucleic acids as a template.","After a threshold amount of target nucleic acids are generated by the polymerase, they can be detected by more traditional methods, such as Southern blots.","When using the Southern blot method, the labelled probe will hybridize to the Neisseria sequence (or its complement).","Also, mRNA or cDNA can be detected by traditional blotting techniques described in Sambrook et al [supra].","mRNA, or cDNA generated from mRNA using a polymerase enzyme, can be purified and separated using gel electrophoresis.","The nucleic acids on the gel are then blotted onto a solid support, such as nitrocellulose.","The solid support is exposed to a labelled probe and then washed to remove any unhybridized probe.","Next, the duplexes containing the labeled probe are detected.","Typically, the probe is labelled with a radioactive moiety.","BRIEF DESCRIPTION OF DRAWINGS There are no drawings.","MODES FOR CARRYING OUT THE INVENTION The following examples describe nucleic acid sequences which have been identified in N.gonorrhoeae, along with their inferred translation products.","The examples are generally in the following format: a nucleotide sequence which has been identified in N.gonorrhoeae.","The strain used is FA1090 [Dempsey et al.","(1991) J. Bacteriol.","173:5476-5486] the inferred translation product of this sequence a computer analysis (e.g.","PSORT output) of the translation product, indicating antigenicity homologous sequences (where relevant) results of expression and purification (where relevant) These examples typically include details of sequence homology between species and strains.","Proteins that are similar in sequence are generally similar in both structure and function, and the homology often indicates a common evolutionary origin.","Comparison with sequences of proteins of known function is widely used as a guide for the assignment of putative protein function to a new sequence and has proved particularly useful in whole-genome analyses.","Open reading frames (ORFs) within nucleotide sequences were predicted using the GLIMMER program [Salzberg et al.","(1998) Nucleic Acids Res 26:544-8).","All predicted open-reading frames longer than 60 aa were screened against the meningococcus serotype B (‘MenB’) ORFs (accession NC002183) using the BLASTP algorithm [Altschul et al.","(1990) J. Mol.","Biol.","215:403-410].","ORFs were considered to be gonococcus-specific if they showed sequence identity to a MenB ORF lower than 60% over the whole protein length, or matching the MenB ORF over less than 30% of the length.","Open reading frames are usually shown with a N-terminal methionine.","Where this is not the case (e.g.","SEQ IDs 12, 18, 20, 32, 54, 62, 66, 84, 98, 102, 104, 112, 116, 118, 126, 128, 130, 134, 136, 138, 146, 152, 162, 186, 228, 238, 240, 278, 280, 288, 290, 298, 300, 308, 314), a non-ATG start codon is present, but the N-terminus amino acid will be methionine when translated using this start codon.","If an upstream start codon is used, however, the “native” amino acid will be translated (e.g.","if the sequence is expressed with N-terminus fusion sequences).","Even where the first amino acid is not shown as methionine, the invention encompasses sequences in which the first amino acid is methionine.","Various tests can be used to assess the in vivo immunogenicity of the proteins identified in the examples.","For example, the proteins can be expressed recombinantly and used to screen patient sera by immunoblot.","A positive reaction between the protein and patient serum indicates that the patient has previously mounted an immune response to the protein in question ie.","the protein is an immunogen.","This method can also be used to identify immunodominant proteins.","The recombinant protein can also be conveniently used to prepare antibodies e.g.","in a mouse.","These can be used for direct confirmation that a protein is located on the cell-surface.","Labelled antibody (e.g.","fluorescent labelling for FACS) can be incubated with intact bacteria and the presence of label on the bacterial surface confirms the location of the protein.","For protein expression of 14 antigens, sequences were amplified using the following primers: Restriction Sequences site NGS5 Fwd CGCGGATCCCATATG-TGGGCAGAACAACCGGC NdeI Rev CCCGCTCGAG-GTTTTCAGCAGGGGGATTG XhoI NGS7 Fwd CGCGGATCCCATATG-GCCGGTAAAGAGCAATTTAC NdeI Rev CCCGCTCGAG-AGCCAAGAAGAACCCGTTAT XhoI NGS13 Fwd CGCGGATCCGCTAGCTGCGTTGCCGACCCCG NheI Rev CCCGCTCGAG-CATGTGCCGTGCGGCGT XhoI NGS36 Fwd CGCGGATCCGCTAGC-GACACCCCGAACAATACC NheI Rev CCCGCTCGAG-AAACCTGCCCTTGATGCC XhoI NGS37 Fwd CGCGGATCCCATATG-GTAGAAGTTAAAGGCGGGG NdeI Rev CCCGCTCGAG-TTTTTTCGCGCCGCCGAA XhoI NGS38 Fwd CGCGGATCCCATATG-GCCGACGAACGCCGCC NdeI Rev CCCGCTCGAG-AAACCGATATTTAAAACCCAACAGCC XhoI NGS39 Fwd CGCGGATCCGCTAGCAACCAAGAAGGGATTACCG NheI Rev CCCGCTCGAG-TTTTTGAGCATAATGACTTTTGCCCT XhoI NGS67 Fwd CGCGGATCCCATATG-CGTGCGCACGGACACG NdeI Rev CCCGCTCGAG-GGCGGCGAGTTTTTCGC XhoI NGS106 Fwd CGCGGATCCCATATG-GCAAACAGCGGAACGATAG NdeI Rev CCCGCTCGAG-AAAATCCTGCGGGATCGGT XhoI NGS115 Fwd CGCGGATCCCATATG-GGGGGCGGCTCCGGC NdeI Rev CCCGCTCGAG-TTCGGCCAACAATGCTTCC XhoI NGSΔG115 Fwd CGCGGATCCCATATG-GATGCCCAATCTTCACAAAG NdeI Rev CCCGCTCGAG-TTCGGCCAACAATGCTTCC XhoI NGS118 Fwd CGCGGATCCCATATG-ACCGCCCTTCCCTCTGA NdeI Rev CCCGCTCGAG-CGGCTGCCATTCGCGTT XhoI NGS122 Fwd CGCGGATCCCATATG-AACCCGAACGATGCGTTTT NdeI Rev CCCGCTCGAG-AGGGTAAAACTTATTCAAATCGGCAA XhoI NGS144 Fwd CGCGGATCCCATATGGCTTCTGAAAATTCTGTAGC NdeI Rev CCCGCTCGAG-GAACACGCTTTTCATTACACCCA XhoI NGS151 Fwd CGCGGATCCCATATGCACGGTATGCATAAGAGCA NdeI Rev CCCGCTCGAG-TTGCTGATGCGGCTTTATTCG XhoI EXAMPLE 1 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 1> which encodes amino acid sequence <SEQ ID 2; NGS1>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.47 Possible cleavage site: 36 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 4.72 threshold: 0.0 PERIPHERAL Likelihood = 4.72 modified ALOM score: −1.44 Rule: cytoplasmic protein * * * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.326(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >sp|P45941|LYQC_BACSU HYPOTHETICAL 21.5 KD PROTEIN IN CWLA-CISA INTERGENIC REGION pir||E69949 hypothetical protein yqcF-Bacillus subtilis dbj|BAA06963.1| (D32216) ORF95 [Bacillus subtilis] dbj|BAA12427.1| (P84432) YqcF [Bacillus subtilis] emb|CAB14528.1| (Z99117) yqcF [Bacillus subtilis] Length = 192 Score = 35.5 bits (81), Expect = 0.45 Identities = 36/162 (22%), Positives 77/162 (47%), Gaps 5/162 (3%) Query: 19 DSGSQYKLNIAAIPSSPNRDLKTYITLGLSKHDLHYK---SRFEILFVCSLKYDENQIFP 75 D ++I ++ P + +Y TLGLS H +NY+ + I V +++ + Sbjct: 29 DDNKSSIDILSVSDQPQEGITSYSTLGLSDHSIEGTPLRIEIV~AAMESASDIYAN 88 Query: 76 FLRWLAETIIENKKILLRGQVVYLPRSIVNS-TKMDALYVSAPFYFDDDFQVCYGEHYNI 134 L A II + G + S+ + T M + PF +++D ++ + N+ Sbjct: 89 VLSTCAFNIINSNFTCAPGVIFKNVISMYDQETDMKHIMFVPPFLWEEDLELLEFSNKNV 148 Query: 135 VFPLLVPLYKQEAELVEKKGWNAFEQFLLDNEVGNLSDMNRK 176 + + +P+ + E ++ EK G + + Q LL+++ ++ D+ R+ Sbjct: 149 TWLMALPISEGELQVAEKHG-SDYLQDLLESKQIDIFDIKRE 189 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 2 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 3> which encodes amino acid sequence <SEQ ID 4; NGS2>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −7.2 Possible cleavage site: 18 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 5.89 threshold: 0.0 PERIPHERAL Likelihood = 5.89 modified ALOM score: −1.68 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.367(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >emb|CAC01359.1|(AL390975) hypothetical protein SCP8.21 [Streptomyces coelicolor A3(2)] Length = 198 Score = 37.2.bits (85), Expect = 0.15 Identities = 29/107 (27%), Positives = 51/107 (47%), Gaps = 3/107 (2%) Query: 73 ETPEHIETLAMLASASYPDQFQLGKNIGRPWVEQSSFRHFLISLPYPYGQELEY 130 +T + + LA+LA++ G ++++G P + F L++ P ++LE Sbjct: 88 DTDKVLRPLAVLAASPQVEGVIVAPGASLDVGEPLWPGAPFTSVLVAEPGGLVEDLELDA 147 Query: 131 -MDNRFFWLLPITQTERLFLNTHSVEELETKFDEAGIDYLDINRAS 176 +D VRF LLP+T E + H L+ ++ G D D +R S Sbjct: 148 PLDPVFLPLLPMTPNEAAWKRVHGAPALQERWLNHGTDLRDPSRRS 194 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 3 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 5> which encodes amino acid sequence <SEQ ID 6; NGS3>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −1.69 Possible cleavage site: 32 >>> Seems to have a cleavable N-term signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 33 ALOM: Finding transmembrane regions (Klein et al.)","count: 5 value: −10.56 threshold: 0.0 INTEGRAL Likelihood = −10.56 Transmembrane 182-198 (171-201) INTEGRAL Likelihood = −7.86 Transmembrane 251-267 (244-273) INTEGRAL Likelihood = −7.48 Transmembrane 142-158 (136-167) INTEGRAL Likelihood = −6.32 Transmembrane 55-71 (50-82) INTEGRAL Likelihood = −2.97 Transmembrane 100-116 (99-117) PERIPHERAL Likelihood = 4.72 modified ALOM score: 2.61 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.522(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >sp|P19845|NOSY_PSEST MEMBRANE PROTEIN NOSY PRECURSOR pir||S13585 nosY protein precursor-Pseudomonas stutzeri emb|CAA37717.1|(X53676) nosY [Pseudomonas stutzeri] Length = 276 Score = 163 bits (413), Expect = 2e−39 Identities = 117/275 (42%), Positives = 174/275 (62%), Gaps = 2/275 (0%) Query: 1 MNPVWIITGKEARDSLRNRWVLAAVLLLAALALSLGFLGSSPTGSVKVDPLTVTVVSLSS 60 MN VW I KE D LRNRW+LA LL A LA+ + +LG++ +G + + T+ SL+S Sbjct: 1 MNQVWNIARKELSDGLRNRWLLAISLLFAVLAGIAWLGAAASGQLGFTSIPATIASLAS 60 Query: 61 LLSIFLIPLIAMLLSYDALIGEIERGTMALLLSYPIWRNQILAGKFVGHLIILALATTAGY 120 L+ FL+PLIA+LL+YDA++GE E GT+ LLL+YP+ R QIL GKFVGH +ILALA G+ Sbjct: 61 LATFLMPLIALLLAYDAIVGEDEGGTLMLLLTYPLGRGQILLGKFVGHGLILALAVLIGF 120 Query: 121 GLAGITLQLANGGFDIAA-WKPFALLAASVILGAAFLSMGYLISAKVKERGTAAGISIG 179 G A + + L G ++ + F + +S +LG FL+ Y++S KV E+ +AAG+++G Sbjct: 121 GCAALAIALLVEGVELGMLFWAFGRFHISSTLLGWVFLAFAYVLSGKNEKSSAAGLALG 180 Query: 180 VWLFFVVIFDMALLGILVADSKQVITAPVVETVLLFNPTDIYRLLNLTGYENTAMYAGMA 239 VW F V+ +L + S+ ++ +LL NPTDIYRL+NL+G+E + G+ Sbjct: 181 VW-FLFVLVFDLVLLALLVLSEGKFNPELLPWLLLLNPTDIYRLINLSGFEGSGSAMGVL 239 Query: 240 GLSGQIGLTVPVLLTAQVLWVIIPLVLAAGIFRKR 274 L + + VL + W+ + L+LA IFR+R Sbjct: 240 SLGADLPVPAAVLCLLAWIGVSLLLAYAIFRRR 274 A homolog (amino acids 226-276) was found in serogroup A N.meninigitidis but not in serogroup B, so NGS3 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 4 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 7> which encodes amino acid sequence <SEQ ID 8; NGS4>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 1.53 Possible cleavage site: 58 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 0.63 threshold: 0.0 PERIPHERAL Likelihood = 0.63 modified ALOM score: −0.63 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.103(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >sp|Q59746|NOSZ_RHIME NITROUS-OXIDE REDUCTASE PRECURSOR (N(2)OR) (N2O REDUCTASE) gb|AAC44023.1|(U47133) nitrous-oxide reductase [Sinorhizobium meliloti] prf||2209347B nitrous-oxide reductase [Rhizobium meliloti] Length = 639 Score = 660 bits (1704), Expect = 0.0 Identities = 344/536 (64%), Positives = 407/536 (75%), Gaps = 23/536 (4%) Query: 1 MSDEKLEQNGLSRRSFLGTAA--ASGAGIAGAGLIGLAGCSKDGEQAAANASGAAPVAKA 58 MS+E+ + L+RR LGT A A+ + G L L+G G A A+A Sbjct: 1 MSNEETKMR-LNRRQMLGTTAFMAAAGAVGAGGALTLSG-------------GTATPARA 46 Query 59 QGESKPGQLSSEVGPGELDQYYGFLSGGQSGEMRLIGLPSMRELMRIPVFNMDSATGWGR 118 Q S S EV PGELD+YY F S GQSGE+R++G PSMRE+MRIPVFN SATGWG+ Sbjct: 47 QETSGS---SYEVKPGELDEYYVFFSSGQSGEIRIVGAPSMREMMRIPVFNRCSATGWGQ 103 Query: 119 TNESLKVLNGNITEETRKFLKDSGLRCYPNGDLHHPHLSFTDQTYDGRYAYANDKANNRV 178 TNES KV+ + ET +FLKD G Y NGDLHHPH SFTD TYDGRY YANDK+N+RV Sbjct: 104 TNESRKVMTEGLLPETVEFLKDQG-GLYLNGDLHHPHPSFTDGTYDGRYLYANDKSNSRV 162 Query: 179 CRVRLDVMKADIIDIPNDSGIHGIHGLRPQRYPKTGYVFANGEHITPVSGVGK-LDDAKTWN 237 CR+RLDVMK DKII +PN +HGLR Q+YPKTGYVF NGE PV GK + D ++ Sbjct: 163 CRIRLDVMKCDKIIQLPNQHTVHGLRVQKYPKTGYVFCNGEDAVPVPNDGKTMGDKNSYQ 222 Query: 238 AVYTAIDGETMEIAWQVLVDGNLDNGDADYQGKYSFATCYNSERALTVQGASSNEQDWCV 297 A++TA+DGETME+AWQV+VDGNLDN DADYQGKY FATCYNSE T+ ++EQDW V Sbjct: 223 AIFTAVDGETMEVAWQVMVDGNLDNVDADYQGKYCFATCYNSEEGFTLADMMASEQDWVV 282 Query: 298 VFDLKAIEEGIKAGDFKEVNGVLDGRABAKSKYTRYIPVFNSPHGCNASPDGKYIMPN 357 +F+LK IEE + GD+KE+ GV +LDGR S YTRY+PVPNSPHG N +PDG +++ N Sbjct: 283 IFNLKRIEEAVAKGDYKEIGGVPVLDGR--KGSPYTRYVPVPNSPHGINTAPDGIHVVAN 340 Query: 358 GKLPPTVTVLDVSKLDDLFAGKIKERDVVAEPQLGLGPLHTAFDGRGNAYTTLFIDSQM 417 GKL PTVTV DV K DDLF KI+ RD VVAEP+LGLGPLHTA+DG+GNAYTTLFIDSQ+ Sbjct: 341 GKLSPTVTVFDVRKFDDLFDDKIQARDTVVAEPELGLGPLHTAYDGKGNAYTTLFIDSQV 400 Query: 418 VKWNIDDAIKAYGEKIDPIKQKLDVHYQPGHNHTTMGETKEADGQWLVSLNKFSKDRFL 477 KWNI+DA +AY GEK+DPI+ KLDVHYQPGHNHT+MG+TKEADG+WL+SLNKFSKDR+L Sbjct: 401 CKWNIEDAKRAYAGEKVDPIRHKLDVHYQPGHTSMGQTKEADGKWLISLNKFSKDRYL 460 Query: 478 NAGPLKPECDQLIGISGDEMRLVHDNPTFAEPHDLCLVAASKLNPGKTWDRKDPWF 533 N GPLKPE DQLI ISGDEM LVHDNPTFAEPHD +V ASK+NP W+R DP+F Sbjct: 461 NVGPLKPENDQLIDISGDEMVLVHDNPTFAEPHDATIVHASKINPVHVWNRDDPFF 516 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 5 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 9> which encodes amino acid sequence <SEQ ID 10; NGS5>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 1.09 Possible cleavage site: 19 >>> Seems to have a cleavable N-term signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 20 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 7.43 threshold: 0.0 PERIPHERAL Likelihood = 7.43 modified ALOM score: −1.99 Score for OM-PP discrimination: 4.97 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: 4.97 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Outer membrane?","score: 0.496525 ----- Final Results ----- bacterial outer membrane --- Certainty= 0.781(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.138(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has no homology with sequences in the databases.","The protein was expressed in E.coli as an insoluble 43.56 kDa His-fusion product and then purified.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 6 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 11> which encodes amino acid sequence <SEQ ID 12; NGS6>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.93 Possible cleavage site: 36 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 6.42 threshold: 0.0 PERIPHERAL Likelihood = 6.42 modified ALOM score: −1.78 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.447(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir|F83173 outer membrane protein OprC PA3790 [imported]-Pseudomonas aeruginosa (strain PAO1) dbj|BAA05664.1|(D28119) outer membrane protein C [Pseudomonas aeruginosa] gb|AAG07177.1|AE004797_12 (AE004797) outer membrane protein OprC [Pseudomonas aeruginosa] Length = 723 Score = 77.9 bits (191), Expect = 1e−13 Identities = 58/188 (30%), Positives = 89/188 (46%), Gaps = 13/188 (6%) Query: 49 VKDLIIFDRAHGQSGTASKGGIITRNVDAILFTAQAYARYNFNPHWAAGIKAAYNYGHN 108 +TL, V+D I+F G G++++ NVDAR+ + A Y +W AY +G N Sbjct: 546 VQDFILFSYREGMMGSSTQ-----ATNVDARIMGGELGASYQLTGNWKTDASLAYAWGKN 600 Query: 109 ETDGRPPYQIRPFEAAVQADYKNYFAHGSYNIGAATRFVAKQTRGDFDMASGLGIDKREA 168 +D R QI P EA Y+ G ++ G+ R VA Q R D + +G D ++ Sbjct: 601 SSDDRALPQIPPLEARFGLTYE----EGDWSAGSLWRVVAPQNRIARDQGNVVGKDFDKS 656 Query: 169 AKGFTVADVYAGVNIKDKYGLRLGVNNVFNKKYVEYI--SGDHVLALSPS-VVYAPGRTY 225 A GF V + + L GV+N+F+K Y E++ +GD S + V PGRT+ Sbjct: 657 A-GFGVFSLNGAYRVTRNVKLSAGVDNLFDKDYTEHLNKAGDAGFGFSANETVPEPGRTF 715 Query: 226 WLSLHAAF 233 W + +F Sbjct: 716 WTKVDFSF 723 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 7 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 13> which encodes amino acid sequence <SEQ ID 14; NGS7>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Hejine) Signal Score (−7.5): 4.94 Possible cleavage site: 26 >>> Seems to have a cleavable N-term signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 27 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 0.79 threshold: 0.0 PERIPHERAL Likelihood = 0.79 modified ALOM score: −0.66 Score for OM-PP discrimination: −18.85 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: −18.85 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Periplasmic space?‘0 score: 1.8846 ----- Final Results ----- bacterial periplasmic space --- Certainty= 0.929(Affirmative) < succ> bacterial outer membrane --- Certainty= 0.211(Affimative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir||D72405 hypothetical protein-Thermotoga maritima (strain MSB8) gb|AAD35294.1|AE001705_5 (AE001705) hypothetical protein [Thermotoga maritima] Length = 300 Score = 81.8 bits (201), Expect = 1e−14 Identities = 72/289 (24%), Positives = 124/289 (41%), Gaps = 17/289 (5%) Query: 38 PAMPSVTIAVAALQGKLAKQADVSLKIWRSPDQLRAGVASGQFKVMMSPSNVGVNLRNQG 97 +TL, P P++ V + GK+ DV ++IW++P++ A + S + + P VG NL +G Sbjct: 24 PLGPALIPVVPIMDGKIP--TDVKIEIWKNPEEAVAKIVSKEVDFAVLPVTVGANLYGKG 81 Query: 98 QKVGMNILTNGITQLVCKGSAIASP-QDLVGKKILVPF-KNDMPDIVLQALLKKLKIDA 155 ++ +V + +LV A + L G+++ P + D++++ L K + Sbjct: 82 VRIKLVGVHEWKVFYLVASDDATFDGWESLRGQEVYTPHGRGQTVDVLMRYFLSKAGLTL 141 Query: 156 HK-VSITYAATPPEAVGLFPSKGYHAVILPEPMATASLLKGKTIGINVVHGFDLVKAWGQ 214 + V I YA P E V LF S LPEP + L +GK + D K WG+ Sbjct: 142 DRDVKILYAP-PQEIVALFKSGKVKYAALPEPFVSMCLDRGKVV-------LDFQKEWGK 193 Query: 215 AFDTKPLIPMAGIIANEEYFHAHKAQFDIFHQDLKNALNWILANRQNAAKIGKNYLPAPE 274 IP+AG+ E K + + L +++ W+ N ++ L P Sbjct: 194 ELGVPGRIPIAGLFVRE---GVDKETVEKVKALIDSIRWMKENLDETVQLSSEKLGIPA 250 Query: 275 PALVHGLDGAPLTVSKGSEVKNEILKFYEILMQFNPELLGGKLPDNGFF 323 L L+ + + E+ F + L + PE K+PD GF+ Sbjct: 251 KILKSSLERIEFEYVPVEKCREEVETFLKKLNELYPEGF-EKIPDEGFY 298 The protein was expressed in E.coli as an insoluble 32.89 kDa His-fusion product and then purified.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 8 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 15> which encodes amino acid sequence <SEQ ID 16; NGS8>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 2.39 Possible cleavage site: 15 >>> Seems to have a cleavable N-term signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 16 ALOM: Finding transmembrane regions (Klein et al.)","count: 4 value: −8.23 threshold: 0.0 INTEGRAL Likelihood = −8.23 Transmembrane 49-65 (41-73) INTEGRAL Likelihood = −7.38 Transmembrane 83-99 (75-106) INTEGRAL Likelihood = −7.06 Transmembrane 110-126 (100-133) INTEGRAL Likelihood = −4.41 Tranamembrane 164-180 (163-187) PERIPHERAL Likelihood = 5.89 modified ALOM score: 2.15 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.429(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >sp|P38044|NRTB_SYNP7 NITRATE TRANSPORT PERMEASE PROTEIN NRTB pir||S30892 nitrate transport protein nrtB-Synechococcus sp.","(strain PCC 7942) emb|CAA43810.1|(X61625) nitrate transporter [Synechococcus sp.]","prf||1908370A nitrate transporter [Synechococcus sp.]","Length = 279 Score = 67.5 bits (164), Expect = 1e−10 Identities = 54/202 (26%), Positives = 96/202 (46%), Gaps = 7/202 (3%) Query: 4 VALWAWGSAVFGEFMLPAPVEFQKSL--DLLKHFQEN-----EIGISLWRSVVGISVAL 56 +A+W SA+ G+ LP P+ V + +++ F +N +G+ + S+ +++ Sbjct: 36 LAIWQVISAILGQDRLPGPINVVANTWMPYIVEPFFDNGGTSKGLGLQILISLQRVAIGY 95 Query: 57 IAGLAAGLVAGLVAGSFKTAMALLKPVITILLAMPPIIWVVMALFWFGFGNPSVLFTIIV 116 + G++ G V G K L PVI +L +PP+ W ++L F N S +F I + Sbjct: 96 LLAACTGILVGGVLGMSKFLGKGLDPVIQVLRTVPPLAWFPISLMVFQDANTSAIFVIFI 155 Query: 117 LVAPLTFASAAVGMASVNKQHEELFDAYKLGRLKKIRYLYIPHLTGYVISSVGVAVAMGV 176 + AVG+ + + + KL + I + IP YV + + +AV + Sbjct: 156 TAIWPIIINTAVGINQIPDDYNNVARVLKLSKKDYILNILIPSTVPYVFAGLRIAVGLAW 215 Query: 177 KAVIMAELLGASKGVGARIADA 198 A++ AE+L A G+G I DA Sbjct: 216 LAIVAAEMLKADGGIGYFIWDA 237 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 9 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 17> which encodes amino acid sequence <SEQ ID 18; NGS9>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.07 Possible cleavage site: 29 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 1 value: −1.81 threshold: 0.0 INTEGRAL Likelihood = −1.81 Transmembrane 97-113 (96-113) PERIPHERAL Likelihood = 4.24 modified ALOM score: 0.86 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.172(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >sp|P97027|SSUB_BACSU PUTATIVE ALIPHATIC SULFONATES THANSPORT ATP-BINDING PROTEIN SSUB pir||G69816 nitrate ABC transporter (binding protein) homolog ygaL- B. subtilis emb|CAB07520.1|(Z93102) hypothetical 30.6 kd protein [Bacillus subtilis] emb|CAB12711.1|(Z99108) similar to nitrate ABC transporter (binding protein) [Bacillus subtilis] Length = 274 Score = 99.5 bits (247), Expect = 3e−20 Identities = 68/181 (37%), Positives 102/181 (55%), Gaps = 9/181 (4%) Query: 4 LFGPSGCGKTTVLRLIAGLETPKSGTIRNTFH-------KTGFLFQENRLPENLTAMQNI 56 L GPSGCGK+T+L++IAGL++ G++ + GF+FQE+RL LT QNI Sbjct: 56 LIGPSGCGKSTLLKIIAGLDSEYDGSVEINGRSVTAPGIQQGFIFQEHRLFPWLTVEQNI 115 Query: 57 A--IFMDNPDEGEIVALAAKVGLTAGDLNKYPTELSGGMAKRVAFLRLLLCGCDLALLDE 114 A + + +P + V ++ G YP ELSGGM++RVA R LL ++ LLDE Sbjct: 116 AADLNLKDPKVKQKVDELIEIVRLKGSEKAYPRELSGGMSQRVAITRALLREPELLLDE 175 Query: 115 PFVGLDRDLRDILVAMLVEKIERQGMACILVTHDRFEAARLSHEIMLLSAKGMNVQNVIT 174 PF LD R L +L++ ++ ILVTHD E+ L +E+ +L AK + ++ Sbjct: 176 PFGALDAFTRKHLQDVLLDIWRKKTTNILVTHDIDESVYLGNELAILKAKPGKIHKLMP 235 Query: 175 L 175 + Sbjct: 236 I 236 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 10 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 19> which encodes amino acid sequence <SEQ ID 20; NGS10>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal score (−7.5): 2.27 Possible cleavage site: 26 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 5.14 threshold: 0.0 PERIPHERAL Likelihood = 5.14 modified ALOM score: −1.53 *** Reasoning Step: 2 imb2 HYPID: 2 CFP: 0.1 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.100(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir||A82615 surface protein XF1981 [imported]-Xylella fastidiosa (strain 9a5c) gb|AAF84783.1|AE004017_6 (AE004017) surface protein [Xylella fastidiosa] Length = 1190 Score = 50.2 bits (119), Expect = 2e−05 Identities = 59/210 (28%), Positives = 92/210 (43%), Gaps = 5/210 (2%) Query: 17 SIGTSAEANAPGAALGGSSEASKFSIAEGYLASSDGYGAIAIGSAAKI-KQLEKGTIN 75 ++G A+A GA A+G + AS K S A G A + G++A+G AK + + Sbjct: 876 AVGVGTLASAEGATAVGSGAAASGKGSTAIGRNAVASADGSVALGDGARGAESYTG 935 Query: 76 HIVGNDNKGLYVDADGNVTKITVRTESEKDILSRYGQTYGAVALGFRSSSHNLFA----S 131 G N + + G+ +K RT S L + N + Sbjct: 936 KYSGLQNNTVGTVSVGDASKGETRTVSNVADAKEATDAVNLRQLDRVAQDANRYVDNKIE 995 Query: 132 SFGAFSTATAIESLAVGDSSQSTGYRSATFGSHSRALAEESLALGYETRANYGSVALGA 191 S T + SL + + G + G + A +S+A+G + A+A +VA+G Sbjct: 996 SLSEGQTFVKVNSLNNSATPIAAGVDATAIGVGATASGADSIAMGNKASADNAVAIGN 1055 Query: 192 ESVANEENTVSVSSDTLKRKIVNVIADGTED 221 SVA+ NTVSV S +R++ NVA GT D Sbjct: 1056 HSVADRANTVSVGSAGSERQVTNVAAGTAD 1085 >sp|P10858|YADA_YERPS INVASIN PRECURSOR (OUTER MEMBRANE ADHESIN) pir||S04534 invasin precursor-Yersinia pseudotuberculosis plasmid pIBI emb|CAA32088.1|(X13883) Yop1 preprotein (AA 1-434) [Yersinia pseudotuberculosis] prf||1411295A invasin [Yersinia pseudotuberculosis[ Length = 434 Score = 42.1 bits (98), Expect = 0.006 Identities = 35/134 (26%), Positives = 68/134 (50%), Gaps = 28/134 (20%) Query: 116 AVALGFRSSSHLFASSFGFSTATAIESDSSQSTGYRSATFGSHSRA-------- 167 ++A+G + + A + G+ S AT + S+A+G S++ G + T+G+ S A Sbjct: 107 SIAIGATAEAAKPAAVAVGSGSIATGVNSVAIGPLSKALGDSAVTYGASSTAQKDGVAIG 166 Query: 168 ----LAEESLALGYETRANAYGSVALGA----------------ESVANEENTVSVSSDT 207 ++ +A+G+ ++ +A SVA+G S + EN+VS+ ++ Sbjct: 167 ASDTGVAVGFNSKDAQNSVAIGHSSHVAADHGYSIAIGDHSKTDRENSVSIGHES 226 Query: 208 LKRKIVNVADGTED 221 L R++ ++A GTED Sbjct: 227 LNRQLTHLAAGTED 240 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 11 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 21> which encodes amino acid sequence <SEQ ID 22; NGS11>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −0.16 Possible cleavage site: 60 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 4.67 threshold: 0.0 PERIPHERAL Likelihood = 4.67 modified ALOM score: −1.43 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.297(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >sp|P10858|YADA_YERPS INVASIN PRECURSOR (OUTER MEMBRANE ADHESIN) pir||S04534 invasin precursor-Yersinia pseudotuberculosis plasmid pIBI emb|CAA32088.1|(X13883) Yop1 preprotein (AA 1-434) [Yersinia pseudotuberculosis] prf||1411295A invasin [Yersinia pseudotuberculosis] Length = 434 Score = 41.3 bits (96), Expect = 0.007 Identities = 27/71 (38%), Positives = 48/71 (67%), Gaps = 4/71 (5%) Query: 16 QLNRLSKRTNRVGASAAALASL-KPAQLGKNDKFAFSLGFGSYKNAQAVAMGAVFKPAEN 74 +L++L KR ++ AS+AAL SL +P +GK + F+ G G Y+++QA+A+G+ ++ E+ Sbjct: 353 RLDKLDKRVDKGLASSAALNSLFQPYGVGKVN---FTAGVGGYRSSQALAIGSGYRVNES 409 Query: 75 VLLNVAGSFAG 85 V L ++AG Sbjct: 410 VALKAGVAYAG 420 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 12 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 23> which encodes amino acid sequence <SEQ ID 24; NGS12>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −1.29 Possible cleavage site: 61 >>> Seems to have a cleavable N-term signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 62 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 6.36 threshold: 0.0 PERIPHERAL Likelihood = 6.36 modified ALOM score: −1.77 Score for OM-PP discrimination: 6.03 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: 6.03 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Outer membrane?","score: 0.602784 ----- Final Results ----- bacterial outer membrane --- Certainty= 0.867(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.158(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has no homology with sequences in the databases.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 13 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 25> which encodes amino acid sequence <SEQ ID 26; NGS13>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.64 Possible cleavage site: 51 >>> May be a lipoprotein Amino Acid Composition of Predicted Mature Form: calculated from 21 ALOM: Finding transmembrane regions (Klein et al.)","count: 1 value: −1.01 threshold: 0.0 INTEGRAL Likelihood = −1.01 Transmembrane 36-52 (36-52) PERIPHERAL Likelihood = 5.14 modified ALOM score: 0.70 Rule: inner or outer membrane protein Rule: inner or outer membrane protein Rule: cytoplasmic membrane protein *** Reasoning Step: 2 Lipoprotein?","Inner membrane?","----- Final Results ----- bacterial outer membrane --- Certainty= 0.790(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.742(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gb|AAC33455.1|(AF067083) outer membrane protein homolog [Vitreoscilla sp.]","Length = 217 Score = 236 bits (602), Expect = 2e−61 Identities = 134/217 (61%), Positives = 166/217 (75%) Query: 1 MTFFKPSTVVLTASALALSGCVADPVTGQQSPNKSAMYGLGGAAVCGWGALTHSGKGAR 60 M +K +++ T +A+ALS C DP+TGQ N + + LGGAA CGIVGALTH KGAR Sbjct: 1 WKAWKKFSLMATVAAVALSACATDPMTGQVDRNNTVLGALGGAATCGIGALTHGSKGAR 60 Query: 61 NSALACGAIGAGVGGYMDYQEQRLRQNLAGTQIEIQRQGNQIRLVMPESVTFATGSAALG 120 NSALACGAIGAGVG YMD+QE++LRQ+LA TQ+E+ R G++IRLVMPES+TFATGS L Sbjct: 61 NSALACGAIGAGVGAYMDHQERQLRQSLANTQVEVNRVGDEILVMPESITFATGSYQLN 120 Query: 121 GSAQYALNTAAQTLVQYPDTTLTINGHTDNTGSDAVNNPLSQHRAQAVAYYLQTRGVAAS 180 SA +LN+ + L QY DTT+ I GHTD+TGSDA+N PLS++RA AVA YL +R VA++ Sbjct: 121 SSASTSLNSVSSVLAQYTDTTINIVGHTDSTGSDAINEPLSRNRASAVANYLVSRNVASN 180 Query: 181 RLTVYGYGSHMPVASNATVEGRAQNRRVEILINPDQR 217 R+T G G PVASN TV GRA+NRRVEI +NP QR Sbjct: 181 RITTVGAGCRQPVASNNTVAGRAENRRVEITVNPIQR 217 >gb|AAD40344.1|U88088_22 (U88088) OmpA [Pseudomonas alcaligenes] Length = 220 Score = 130 bits (328), Expect = 1e−29 Identities = 90/219 (41%), Positives = 127/219 (57%), Gaps = 6/219 (2%) Query 7 STVVLTASALALSGCVA---DPVTGQQSPNKSAMYGLGGAAVCGIVGALTHSGKGARNSA 63 S + + L+GC + + T + + A L GA ++G + +GA A Sbjct: 3 SVIAASLVIFTLTGCASIQNEDGTTKNTALYGAGGALAGAVAGALIGK-ENRAQGALIGA 61 Query: 64 LACGAIGAGVGGYMDYQEQRLRQNLAGTQIEIQRQGNQIRLVMPESVTFATGSALLLGGSA 123 G++GAG G Y D QE LR+ + G+ ++++RQG++I +VMP ++TFATG A + + Sbjct: 62 AVAGSLGAGYGYYADKQEAELREQMKGSGVQVERQGDEIVIVMPGAITFATGKAEIQPNF 121 Query: 124 QYALNTAAQTLVQYPDTTLTINGHTDNTGSDAVNNPLSQHRAQAVAYYLQTRGVAASRLT 183 LN A + YPD+ L + GHTD+ GS N LSQ RAQ+VA +L+ GV R+ Sbjct: 122 ANTLNQLAGSFPNYPDSRLIVTGHTDSVGSYEANELLSQRRAQSVAQFLRGNGVQTDRIE 181 Query: 184 VGYGHMPVASNATEGRAQNRRVEILINPDQRAVNAA 222 V G G + PVASNAT EGRAQNRRVEI + P RAV A Sbjct: 182 VIGAGPNQPVASNATAEGRAQNRRVEIKLAP--RAVQQA 218 The protein was expressed in E.coli as a soluble 22.55 kDa His-fusion product and then purified.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 14 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 27> which encodes amino acid sequence <SEQ ID 28; NOS14>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.32 Possible cleavage site: 40 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 3.39 threshold: 0.0 PERIPHERAL Likelihood = 3.39 modified ALOM score: −1.18 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.254(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has no homology with sequences in the databases.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 15 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 29> which encodes amino acid sequence <SEQ ID 30; NGS15>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −1.75 Possible cleavage site: 45 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 5.89 threshold: 0.0 PERIPHERAL Likelihood = 5.89 modified ALOM score: −1.68 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.232(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >sp|P10487|RCI1_ECOLI SHUFFLON-SPECIFIC DNA RECOMBINASE pir||S03815 probable integrase-Escherichia coli dbj|BAA77989.1|(AB027308) shufflon-specific DNA recoinbinase [Plasmid R64] Length = 384 Score = 67.1 bits (163), Expect = 3e−10 Identities = 75/301 (24%), Positives = 125/301 (40%), Gaps = 34/301 (11%) Query: 68 KVKMMTLSEAMRKYLNETLGAGRSKKMGL---RFLMEFPIGGIGIDKLKRSDFAEHVMQR 124 +++ M+LS A+ KYL + + + PI +D++ D A + R Sbjct: 5 RIRKMSLSRALDKYLKTVSVHKKGHQQEFYRSNVIKRYPIALRNMDEITTVDIATYRDVR 64 Query: 125 RRGIPELDIAPIAASTALQELQYIRSVLKHAFYVWGLEIGWQELDFAANGLKRSNMVAKS 184 I PI +T EL + S+ A WG N ++ S Sbjct: 65 LAEINPRTGKPITGNTELELALLSSLFNIARVEWG--------TCRTNPVELVEKPKVS 116 Query: 185 AIRDRLPTTEELQTLTTYFLRQWQSRKSSIPMHLIMWLAIYTSRRQDEICRLLFDDWHKN 244 + RDR T+E + L+YF R+ ++ +++I LA+ T+ RQ EI L W Sbjct: 117 SGRDRRLTSSEBRRLSRYF------REKNLMLYVIFHLALETAMRQGEILAL---RWEHI 167 Query: 245 DCTRPVRDLKNPNGSTGNNKEFDILPMALPVIDELPEESVRKRMLANKGIADSLVPCNGK 304 D V L P G++++ + A + +P + ++ Sbjct: 168 DLRHGVAL--PETNGHSRDVPLSRRARNFLQMMP-----------VNLHGNVFDYTAS 214 Query: 305 SVSAAWTRACKTLGIKDLRFHULREEAATRMAEDG-FTIPQNQRVTLHDGWNSLQRYVSVR 364 AW A + L I+DL FHDLRHEA +R E G + ++ ++ H N L+RY +R Sbjct: 215 GFKNAWRIATQRLRIEDLHRHDLRHEAISRFFELGSLNVMEIAAISGHRSMNMLKRYTHLR 275 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 16 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 31> which encodes amino acid sequence <SEQ ID 32; NGS16>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.64 Possible cleavage site: 20 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 4.67 threshold: 0.0 PERIPHERAL Likelihood = 4.67 modified ALOM score: −1.43 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.262(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >sp|P10484|T1M1_ECOLI TYPE I RESTRICTION ENZYME ECOR124II M PROTEIN (M.ECOR124II) pir||S02166 type I site-specific deoxyribonuclease (EC 3.1.21.3) EcoR124/3 chain hsdM-Escherichia coli plasmid R124/3 emb|CAA31541.1|(X13145) hsdM protein (AA 1-520) [Escherichia coli] Length = 520 Score = 44.4 bits (104), Expect = 0.002 Identities = 65/235 (27%), Positives = 99/235 (41%), Gaps = 55/235 (23%) Query: 107 NRKKAGGYAEYITGGSLRRLVAARRYCGEHPGVFDGAAGSG--------QLEQYIEPS 158 N K+GG E+ T + +L+A ++D AAGSG Q + +I Sbjct: 191 NAGKSGG--EFFTPQHVSKLIAQLAMHGQTHVNKIYDPAAGSGSLLLQAKKQFDNHIIEE 248 Query: 159 DFRAVEIQAEACRALLQNYPAAKVYNTSLFL--------------------YTDGEPQDC 198 F EI N+ + ++FL + D +P D Sbjct: 249 GFFGQEI----------NHTTYNLARMNMFLHNINYDKRDIKLGNTLTEPHFRDEKPFDA 298 Query: 199 TVMNPPFSIKLKDLSEDEKSRIAQEYPWKKSGV------ADEIFVLKGLE--NARRFGFF 250 V NPP+S+K + D+ + I E + +GV AD FVL L +A+ Sbjct: 299 IVSNPPYSVKW--IGSDDPTLINDER-FAPAGVLAPKSKADFAFVLHALNYLSAKGRAAI 355 Query: 251 ILFPGIAYR-KSEQRFRE-IIGNRLAE--LNRIQNAFEDTPIEVLLLVIDKDKTD 301 + FPGI YR +EQ+ R+ ++ N E ++ N F T I V +LV+ K KTD Sbjct: 356 VCFPGIFYRGGAEQKIRQYLVDNNYVETVISLAPNLFFGTTIAVNILVLSKHKTD 410 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 17 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 33> which encodes amino acid sequence <SEQ ID 34; NGS17>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.29 Possible cleavage site: 16 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 2.60 threshold: 0.0 PERIPHERAL Likelihood = 2.60 modified ALOM score: −1.02 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.284(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >ref|NP_052389.1|translocator YopD [Yersinia enterocolitica] sp|P37132|YOPD_YEREN YOPD PROTEIN gb|AAD16812.1|(AF102990) translocator YopD [Yersinia enterocolitica] Length = 306 Score = 32.1 bits (72), Expect = 8.2 Identities = 29/93 (31%), Positives = 43/93 (46%), Gaps = 17/93 (18%) Query: 13 MLAAKRAAKESTRQERAVKRAGTVRNVDRNRLSARSKAQKENIARMLSGAKVSEDEALTC 72 +L R A+E Q+R ++ T+ AQKE +A M+SGAK+ A+ Sbjct: 89 LLELARKAREMGLQQRDIENKATI------------SAQKEQVAEMVSGAKLMIANAVVS 136 Query: 73 GIMMRLSLQDMRYACNQELINFAEHIVKQVQRL 105 GIM S ++ +E+ IVKQ Q L Sbjct: 137 GIMAATSTVASAFSIAKEV-----KIVKQEQIL 164 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 18 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 35> which encodes amino acid sequence <SEQ ID 36; NGS18>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −2.56 Possible cleavage site: 38 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 4.56 threshold: 0.0 PERIPHERAL Likelihood = 4.56 modified ALOM score: −1.41 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.397(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has no homology with sequences in the databases.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 19 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 37> which encodes amino acid sequence <SEQ ID 38; NGS19>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.12 Possible cleavage site: 43 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 8.49 threshold: 0.0 PERIPHERAL Likelihood = 8.49 modified ALOM score: −2.20 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.250(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >ref|NP_043483.1|orf14 [Bacteriophage HP1] sp|P51716|YO14_BPHP1 HYPOTHETICAL 14.9 KD PROTEIN IN REP-HOL INTERGENIC REGION (ORF14) pir||S69520 hypothetical protein 14-phage HP1 gb|AAB09199.1|(U24159) orf14 [Bacteriophage HP1] Length = 133 Score = 73.3 bits (179), Expect = 1e−12 Identities = 44/129 (34%), Positives = 74/129 (57%), Gaps = 6/129 (4%) Query: 1 MFIPAALHKDEHSAYGVTIPDLPGCFSCGDTVEEAVANARSAAYMHIDGMIEDGGFKNLA 60 M P + K + Y V++PD+PGCFS GDT+ EA+ NA+ A HI+GM+ED + L Sbjct: 1 NLYPICIEK-VNDGYVVSVPDVPGCFSAGDTLSEAMLNAKEAISFHIEGMLEDD--EELP 57 Query: 61 VSS-IADLSQEPDYHGATWVMIEIDPAKISRQQIRFNVSWPQYLLDRVDEY--TSANHET 117 S+ I +P+Y ++++D + + + N++ P LL R+D++ T ++ Sbjct: 58 KSNPIEQYINQPEYKDFIVTVVDVDLTHLMGKAEKINITVPALLLHRIDQFIATHPEYKN 117 Query: 118 RSGFLAKAA 126 RS FL++ A Sbjct: 118 RSNFLSQLA 126 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 20 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 39> which encodes amino acid sequence <SEQ ID 40; NGS20>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −0.1 Possible cleavage site: 19 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 7.58 threshold: 0.0 PERIPHERAL Likelihood = 7.58 modified ALOM score: −2.02 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.057(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has no homology with sequences in the databases.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 21 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 41> which encodes amino acid sequence <SEQ ID 42; NGS21>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.52 Possible cleavage site: 52 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 5.83 threshold: 0.0 PERIPHERAL Likelihood = 5.83 modified ALOM score: −1.67 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.311(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >ref|NP_040628.1|cI (repressor; 237) [bacteriophage lambda] ref|NP_061378.1|phage lambda repressor protein CI [Escherichia coil] sp|P03034|RPC1_LAMBD REPRESSOR PROTEIN CI pir||RPBPL repressor protein cI-phage lambda emb|CAA24991.1|(X00166) coding sequence cI gene [bacteriophage lambda] gb|AAA96581.1|(J02459) cI (repressor; 237) [bacteriophage lambda] emb|CAB96428.1|(AJ277653) phage lambda repressor protein CI [Escherichia coli] Length = 237 Score = 62.5 bits (151), Expect = 5e−09 Identities = 36/85 (42%), Positives = 51/85 (59%) Query: 2 KKRELNEIETAECAELKRIFNSKKEELKLTQYKLAEAVGVTQSAVNHYLNGTNALNASIA 61 KK+ L + + + LK I+ KK EL L+Q +A+ +G+ QS V NG NALNA A Sbjct: 4 KKKPLTQEQLEDARRKKAIYEKKKELGLSQESVADKMGMGQSGVGALFNGINALNAYNA 63 Query: 62 SQFAKILQIPVSDFSLRLAEEISSM 86 + AKIL++ V +FS +A EI M Sbjct: 64 ALLAKILKVSVEEFSPSIAREIYEM 88 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 22 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 43> which encodes amino acid sequence <SEQ ID 44; NGS22>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −2.6 Possible cleavage site: 43 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 7.74 threshold: 0.0 PERIPHERAL Likelihood = 7.74 modified ALOM score: −2.05 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.072(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir||S30432 hypothetical protein-Streptomyces clavuligerus plasmid pSCL Length = 307 Score = 43.6 bits (102), Expect = 0.002 Identities = 25/86 (29%), Positives = 49/86 (56%), Gaps = 2/86 (2%) Query: 6 MGMAFKT-GIPRGRFVLVLCDCANDDGLCYPSQETLAEDTGFAETAVRQHIKWLKDNN 64 MGM F G+ ++ +L+ + + G C+PS++ L +D G + + V++ +L N Sbjct: 1 MGMVFAAEGLDGSEKLLLLGYTNWTDPYGYCWPSEDRLVDDCGTSRSTVQRTKRKLVKKN 60 Query: 65 FIKSARRQRGR-ERKSDIYRINVALL 89 ++S RR+ + E S++ R+N+ LL Sbjct: 61 LLRSVRRKNSKGEPISNLSRVNLPLL 86 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 23 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 45> which encodes amino acid sequence <SEQ ID 46; NGS23>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −2.8 Possible cleavage site: 59 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 0.05 threshold: 0.0 PERIPHERAL Likelihood = 0.05 modified ALOM score: −0.51 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Result ----- bacterial cytoplasm --- Certainty= 0.195(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >sp|P07905|DNAC_ECOLI DNA REPLICATION PROTEIN DNAC pir||XMECNC DNA replication protein dnaC-Escherichia coli (strain K-12) Length = 245 Score = 110 bits (275), Expect = 2e−23 Identities = 75/224 (33%), Positives = 116/224 (51%), Gaps = 23/224 (10%) Query: 50 EAADEMAAYAETLRRGAMRDA----------LEKRIGRSGIAPRFRNCRIENYAV--SDS 97 + +E+ A+ + +GA+R A +++ RSGI P +NC ENY V Sbjct: 24 KTGEELLAWQK--EQGAIRSAALERENRAMKMQRTFNRSGIRPLHQNCSFENYRVECEGQ 81 Query: 98 IPGMARAKAAAAEYAANFADVLQTGRSMIFSGRRGTGKNHLACGIAREVIAAGKSALVIT 157 + +++A+ E+ N A S IFSG+ GTGKNHLA I E++ GKS L+IT Sbjct: 82 MNALSKARQYVEEFDGNIA-------SFIFSGKPGTGKNHLAAAICNELLLRGKSVLIIT 134 Query: 158 VGDMLRTVKDSF--GGGGEAGAVGIFVKPDLLVLDEFGAGSLSETDGRILFSVVNARYER 215 V D++ +KD+F G E + DLLV+DE G + S+ + I+ +V+ R Sbjct: 135 VADIMSAMKDTFRNSGTSEEQLLNDLSNVDLLVIDEIGVQTESKYEKVIINQIVDRRSSS 194 Query: 216 LMPMLVLTNLTAEAFRENTDARIRDRLRDGGGKLIPFDWESTRA 259 P +LTN E + R+ DR+R G + F+W+SYR+ Sbjct: 195 KRPTGHLTNSNMEEMTKLLGERVMDRMRLGNSLWVIFNWDSYRS 238 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 24 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 47> which encodes amino acid sequence <SEQ ID 48; NGS24>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.76 Possible cleavage site: 26 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 1.43 threshold: 0.0 PERIPHERAL Likelihood = 1.43 modified ALOM score: −0.79 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.112(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >ref|NP_053228.1|pXO2-73 [Bacillus anthracis] gb|AAF13678.1|AF188935_76 (AF188935) pXO2-73 [Bacillus anthracis] Length = 541 Score = 125 bits (315), Expect = 9e−28 Identities = 139/535 (25%), Positives = 254/535 (46%), Gaps = 70/535 (13%) Query: 14 PVLFIGTGMSLRYLDNSYTWDGLLSKIAIDLFGDDREYLNIKSRYCEDGRFQYEEIAEEL 73 P LFIG+G S RYL N W GL+ K + +L + EY Y + E+AE + Sbjct: 19 PFLFIGSGFSKRYL-NLEDWAGMKKFS-NLMPYEFEY------YSSTANKDWAEVAELM 70 Query: 74 QSKFDKVL--ENDPDGEFKEINDKFFENMRAGNTLSRFKIYISTLLSQLNYK----DNSN 127 F + E + KE D R + S K+ ++ L+ + YK + ++ Sbjct: 71 AKDFHPIWWKEQQFENNRKEFKD------RISSKQSPLKVEVAKYIiNSIEYKYGLDEKND 124 Query: 128 TELSELKKARKNVGSIITTNYDKLAQDIFEFNPL---IGN-DILLSNPY--GSVYKIHGC 181 E°+ LKK + IITTN+D L + IFE + IG ++L S+P +YKIHGC Sbjct: 125 KEIAALKKIV--IDGIITTNWDLLLEQIFEEQEMQVYIGQKELLFSHPLEINEIYKIHGC 182 Query: 182 VDDPSKIIITKKDYEKFKEKYELIRAQLLSLFIHNPIIFLGYNVGDENIKEILKTIFTYV 241 P +++T DY+ + EK + A+LL++FI +P+IFLGY++ D+NI++ILK I + Sbjct: 183 SSIPDSLVLTTSDYKGYNEKNAYLAAKLLTVFIEHPVIFLGYSISDDNIQQIILKAITRCL 242 Query: 242 EQNSPSANKIRRNFLLVEYEPESNNEDIVEDIDIT-GFSTIRINKIKTDNFSQIYKALA 300 +Q++ K R L+ E ED E++ +T G T+ I ++KT+++ +IY ALA Sbjct: 243 DQDNIHKLKDR----LIFVERAGQEEDSFENNSSLTIGKITVPITRVKTNDYEKIYNALA 298 Query: 301 ELTLPISAMDVRKFQSIAKEIYTGGNIKVSF---TEDMDNLNNSDKVVAIGSTKTISYNF 357 + S +R+ +S E+ + + + D+ + + V+ +G K + Sbjct: 299 QNKRKFSMKMMRQMKSQIYELVKTNDPEEKIYVVDGEYDDTQDIEFVIGLG-VKNVVEEM 357 Query: 358 QTTSEMMSN----------------YFKIIEEENS----QLLKLIDKHSIASTQYFPI-- 395 Q+ E+ ++ + +++ +E ++K+ + S QY P+ Sbjct: 358 QSNHEISADKELSEHGYGGISDIELFNELLSDEPKYDYDSIVKISLPQILRSNQYVPLFR 417 Query: 396 YGFSRICSDIHKEAVLKRQQKEKLDHFIEEINRRCKNNHSSIQSILDDENISDTYKNDAI 455 Y D ++ +K + K + F+ E ++ N S + + + + + + Sbjct: 418 YVLESSVEDELLDSKIKNKLKMRYTDFIITETQKNIKLDWQFKNLDEVLKGFPDIKV 477 Query: 456 AWG----IWNNQLSEDEVENYL---KNFVNKKN----THYKRLLCMFDYKKYADT 499 A + L+ D+++++L FV +KN T +RL ++D+ KY + Sbjct: 478 AIEQIPLLGQKNLNNCDDLDFLIKNSKFVKEKNTPERTGIRRLFRIYDWLKYGQS 532 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 25 A DNA sequence was identified in N,gonorrhoeae <SEQ ED 49> which encodes amino acid sequence <SEQ ID 50; NGS25>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 4.76837e−07 Possible cleavage site: 56 >>> May be a lipoprotein Amino Acid Composition of Predicted Mature Form: calculated from 21 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 6.68 threshold: 0.0 PERIPHERAL Likelihood = 6.68 modified ALOM score: −1.84 Rule: inner or outer membrane protein Rule: inner or outer membrane protein *** Reasoning Step: 2 Lipoprotein?","Inner membrane?","----- Final Results ----- bacterial outer membrane --- Certainty= 0.790(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.700(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|7433O05|pir||B70313 cytochrome-C peroxidase (EC 1.11.1.5)- Aquifex aeolicus gi|2982865|gb|AAC06485.1|(AE000675) cytochrome C peroxidase [Aquifex aeolicus] Length = 355 Score = 345 bits (885), Expect = 7e−94 Identities = 180/336 (53%), Positives = 237/336 (69%), Gaps = 12/336 (3%) Query: 59 EDQDLLKRAQGVFQPLPTVEEMQKIRPFTEEQVKLGHQLWYEPRLSKGNTVSCNSCHNLA 118 +D++LLK A+ F+PLP V E + P T E+VKLG L+Y+PRLSK +SCN+CHNLA Sbjct: 22 DDKELLKKMARQYFKPLPKVAENPQ-NPVTPEKVKLGKMLYYDPRLSKSGLISHCNTCHNLA 80 Query: 119 SAGVDNMPTSQGHKGQFGGRNSPTALNAALLGSQFWDGRAADVEEQAGGPLVNPVEMAND 178 GVDN+PTS GH+ G RN+PT NAA+ +QFWDGRA DVEEQA GP+VNP+EMAN Sbjct: 81 RYGVDNLPTSIGHRWAIGPRNAPTVYNAAIHIAQFWDGRAKDVEEQALGPIVNPIEMAN- 139 Query: 179 SQEAAAAKIAKVPEYQEFKAFP-EDGAVSFKNITTALGAFERTLLTPTKWDEYLKGNV 237 ++E A + +PEY E+FKKAFP E V ++NI A+GAFERTL+TP+++DE+LKGN Sbjct: 140 TEENAVKTLKSIPEYVELFKKAFPNEKDPVKYENIGKAIGAFERTLMTPSRFDEFLKGNT 199 Query: 238 NALSEQERKGRAFDNGCIACHNGNLGGTTFQKRGLVQGPYWK------FIEDP--KR 289 AL+EQE++G++ F++ GC+ACHNG +GG F KFG++ YWK + P K Sbjct: 200 KALTEQEKRGLKTFIEVGCVACHNGPGVGGNMFAKFGMIT-EYWKVTYPYVLVGKPAIKV 258 Query: 290 DKGEADVTKKTEDEFFFRVPGLRNVAKTYPFHNGSVWELDKAVTIMGKAQLGKDIPKED 349 D GR VTKK ED F F+VP LRN+ TYPYFH+GSVW L+ AV IM K QLGK++ + Sbjct: 259 DFGRFGVTKKEEDMFVFKVPSLRNIEHTYPYFHDGSVWSLEDAVRIMAKTQLGKELTDQQ 318 Query: 350 VDNIVVFLNALSGNVSESARTHPELPLTAPMESKPD 385 V +IV FL AL+G + + A +PELP + KP+ Sbjct: 319 VKDIVAFLKALTGKIPKHALEVPELPPSTDKTPKPE 354 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 26 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 51> which encodes amino acid sequence <SEQ ID 52; NGS26>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 0.610001 Possible cleavage site: 15 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 5.04 threshold: 0.0 PERIPHERAL Likelihood = 5.04 modified ALOM score: −1.51 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.127(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir||T13296 hypothetical protein 8-Streptococcus phage phi-O1205 gb|AAC79524.1|(U88974) ORF8 [Streptococcus thermophilus temperate bacteriophage O1205] Length = 157 Score = 62.5 bits (151), Expect = 2e−09 Identities = 53/161 (32%), Positives = 86/161 (52%), Gaps = 8/161 (4%) Query: 5 TLYRCDVQAALDYYFDSETEREDTLEAV--IGQFEVKAQSVIAYIKNQEITEKMLEGH 62 TLY + + D ET + DTLEA+ +E K + + IK+ E + + Sbjct: 3 TLYELTDQLLEIYNMDVDDET-KLDTLEAIDWTTDYENKVEGYVKVIKSLEADIEARKNE 6 Query: 63 IRQMTGKLKAAKARNQSLKDYLARNMQAAGITEIKADDGTFKASFRKSEAVVILDEAQIP 122 +++ G K+ +++ LK LA +M G T + D FK FRKSEAVV+ +E ++P Sbjct: 62 KKRLDGLNKSDQSKIDKLKTALAVSMAETGQTRV--DTTLFKVGFRKSEAVVV-NEEKLP 118 Query: 123 AEFMREAVKTEPDKTAIRXAIESGRQVAGAKIEGRKNLQIR 163 E+ K PDK +++ ++SG+ + GA +E R+NL IR Sbjct: 119 KEYQIATYK--PDKKTLKLKSGKHIEGATLEERRNLNIR 157 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 27 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 53> which encodes amino acid sequence <SEQ ID 54; NGS27>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Hejine) Signal Score (−7.5): −5.45 Possible cleavage site: 49 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 1.80 threshold: 0.0 PERIPHERAL Likelihood = 1.80 modified ALOM score: −0.86 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty = 0.559 (Affirmative) < succ> motifs: Subtilase_Asp (S, T, A, I, V)X(L, I, V, M, F)(L, I, V, M)D(D, S, T, A)G(L, I, V, M, F, C)x) (A)x(L)(I)D(D)G(I)x{2}(D) 79: DDDFL AALIDDGIVFD V A homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS27 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 28 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 55> which encodes amino acid sequence <SEQ ID 56; NGS28>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −0.19 Possible cleavage site: 61 >>> Seems to have a cleavable N-term signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 62 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 0.69 threshold: 0.0 PERIPHERAL Likelihood = 0.69 modified ALOM score: −0.64 Score for OM-PP discrimination: −24.78 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: −24.78 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Periplasmic space?","score: 2.47798 Periplasmic space?","score: 2.47798 ----- Final Results ----- bacterial periplasmic space --- Certainty= 0.916(Affirmative) < succ> The protein has homology with the following sequences in the databases: >prf||1306286A mobilization protein B [Escherichia coli] Length = 529 Score = 34.7 bits (78), Expect = 2.4 Identities = 24/69 (34%), Positives = 31/69 (44%), Gaps = 12/69 (17%) Query: 344 QLRARQQEIPVDYARTAVCGRIPFRRHSRPTLRSRTLGAQRRRIVPNVGQAGGIRAD--- 400 +LRA Q++P D+ +T V P R R + GA GQ G IR D Sbjct: 440 RLRAAGQDLPADFVKTTVLDNTPIRWFYRAASQESRSGA---------GQTGEIRVDVER 490 Query: 401 RTPNTQRGT 409 R P +RGT Sbjct: 491 RGPAGRRGT 499 A homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS28 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 29 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 57> which encodes amino acid sequence <SEQ ID 58; NGS29>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.61 Possible cleavage site: 31 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 4.03 threshold: 0.0 PERIPHERAL Likelihood = 4.03 modified ALOM score: −1.31 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.106(Affirmative) < succ> The protein has homology with the following sequences in the databases: emb|CAB83930.1|(AL162753) hypothetical protein NMA0640 [Neisseria meningitidis Z2491] Length = 387 Score = 653 bits (1685), Expect = 0.0 Identities = 324/388 (83%), Positives = 351/388 (89%), Gaps = 1/388 (0%) Query: 1 MNITIAAPYCSLPSEPHFNRFWYLAELLSQSHDVLLITSNFKHYDKSFRRPEDAKAASQG 60 MNITI APYCSLPSEP+FNRFWYLAE LSQSHDVLLITS F+HYDKSFRR EDA A S G Sbjct: 1 MNITIVAPYCSLPSEPYFNRFWYLAERLSQSHDVLLITSRFRHYDKSFRRHEDAAATSNG 60 Query: 61 RLKVMLLEESGYSKNVSLGRVTSHHRFVKHFEKWLENCRPGEQDVVYSAYPLIATNLLLG 120 RL+V LL+E GY KNVSL RV SH FV++ +WL + + EQD+VYSAYPL+ATNLLLG Sbjct: 61 RLRVKLLDEPGYRKNVSLARVASHRVFVRNLARWLHSPQAAEQDIVYSAYPLMATNLLLG 120 Query: 121 KHKARLGYKLIVDVQDVWPESFSSVVPFLKKIPHNLLPFASRANRAYRYADALVAVSQTY 180 KHKARLGYKLIVDVQDVWPESFSSVVPFLKK+PH LLPFASRANRAYR ADAL+AVSQTY Sbjct: 121 KHKARLGYKLIVDVQDVWPESFSSVVPFLKKVPHKLLPFASRANRAYRCADALIAVSQTY 180 Query: 181 LDRAKEANPNVPGEVVYIGADFAAIAPPPRFRSKTVRFFYLGTLSYNYDVETVCKGVRKL 240 LDRAKEANPNVPGE VYIG DFAAIA PPRFRSKTVR FYLGTLSY+YDVETVCKGVRKL Sbjct: 181 LDRAKEANPNVPGETVYIGTDFAAIA-PPRFRSKTVRLFYLGTLSYSYDVETVCKGVRKL 239 Query: 241 LDDGENVELHIMGGGPDLDRLKQYACDGIKFYGYIPYAEMMSVAKGCDIAVNAIHSYAMQ 300 LDDGENVELHIMGGGPDL++LKQY IKFYGY+PY+EMMS+AK CDIAVNAIHS+AMQ Sbjct: 240 LDDGENVELHIMGGGPDLEKLKQYENRAIKFYGYLPYSEMMSIAKACDIAVNAIHSHAMQ 299 Query: 301 SITNKLSDYMALQKPILNSQVHDEVAEVLTLLPHENYRSGDVDGFVQAAKDILKRKNDPV 360 S+TNKLSDYMALQKPILNSQ + EV ++L LLPHENYRSGDVD FVQAAK+ILKRK+DPV Sbjct: 300 SVTNKLSDYMALQKPILNSQNNAEVLDLLNLLPHENYRSGDVDSFVQAAKNILKRKDDPV 359 Query: 361 QSDEIVRRFRHDISYRKIVNLIERLANE 388 QSDEIVRRFR DISYRKIVNLIERLA+E Sbjct: 360 QSDEIVRRFRRDISYRKIVNLIERLAHE 387 >emb|CAB58324.1|(AL121855) hypothetical protein SCF62.09 [Streptomyces coelicolor A3 (2)] Length = 407 Score = 54.7 bits (130), Expect = 2e-06 Identities = 57/243 (23%), Positives = 105/243 (42%), Gaps = 24/243 (9%) Query: 99 RPGEQDVVYSAYP---LIATNLLLGKHKARLGYKLIVDVQDVWPESFSSVVPFLKKIPHN 155 R G DVV++ P L L L R G + + D D+ PE + S K + + Sbjct: 81 RVGPVDVVHACNPPDLLFLPALWL----KRRGARFVFDQHDLIPELYLSRFGRGKDLLYR 136 Query: 156 LLPFASANRAYRYADALVAVSQTYLDRAKEANPNVPGEVVYIGA-----DFAAIAPPPR 210 + R YR AD ++A +++Y D A P +V ++ F + P P Sbjct: 137 AVCALERWT--YRAADVVLATNESYKDVAIRRGGRRPDDVFVVRSAPATDRFQPVPPEPE 194 Query: 211 F-RSKTVRFFYLGTLSYNYDVETVCKGVRKLLDDGENVELH--IMGGGPDLDRLKQYA-- 265 R K YLG + V+ + + KL D+ + H +G G D + + + Sbjct: 195 LKRGKPHLLCYLGVMGPQDGVDYALRALAKLRDEVGRTDWHAVFVGSGDAFDAMVELSRS 254 Query: 266 ---CDGIKFYGYIPYAEMMSVAKGCDIAVNAIHSYAMQSIT--NKLSDYMALQKPILNSQ 320 + ++F G IP A++++ D+ ++ + ++ NK+ +YMA+ +PI++ Sbjct: 255 LGLDEQVRFTGRIPDADLVRHLSTADVCLSPDPRNPLNDVSTMNKVLEYMAMGRPIVSFD 314 Query: 321 VHD 323 + + Sbjct: 315 LRE 317 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, NGS29 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 30 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 59> which encodes amino acid sequence <SEQ ID 60; NGS30>.","Analysis of this protein sequence reveals the following: Signal Score (−7.5): −4.8 Possible cleavage site: 46 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 1.54 threshold: 0.0 PERIPHERAL Likelihood = 1.54 modified ALOM score: −0.81 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.113(Affirmative) < succ> The protein has homology with the following sequences in the databases: fastidiosa (strain 9a5c) gb|AAF84279.1|AE003977_2 (AB003977) conserved hypothetical protein [Xylella fastidiosa] Length = 376 Score = 73.6 bits (179), Expect = 3e−12 Identities = 82/354 (23%), Positives = 143/354 (40%), Gaps = 35/354 (9%) Query: 1 MKIILTTSMSGLGGTETATVRLGRLLKRHGHDIILASSDG-PFVGEAQASGIRWQPVDFY 59 MKI+ T + +G GG E R ++ GH + L G P A+ +G+ ++ + Sbjct: 1 MKILHTEAATGCGGEEIYIYRHMLSMQAQGHHMALLCQPGAPLSTMARNAGLPVYHINMH 60 Query: 60 RGGLAGYLKSTFAYARMLRREQPDIIDCQMARVVPACALAAKIVSPKTKIICHSHGLDAA 119 G L +L+RE D+++ A AA++ +T++I S L A Sbjct: 61 --GPWRVLNGIHTVQHLLQRETFDVVNTTSHVDTLIAAAAARLT--RTRLIVRSRHLMAP 116 Query: 120 TYPKTAKLFDKLGAYIIGNCICHEREKLIRHGFPAGRIAYA---------YNTPPEFHFRK 170 K+ + L +I +H R+ LI+ G RI +T PE +++ Sbjct: 117 I--KSQLTYTYLPHRVITVSQHRDLLIKQGIQPTRIGIVPPITA-QPPWMDTDPEHAWQR 174 Query: 171 TEK-------------ECAVLGTLSRLDTRAVHLMLDILKKMVGRNIPVRLNMAGIGEE 217 ++ ++G ++ L + +LD + + N + L +AG GE Sbjct: 175 LQQTRHVVRTELGFNDNDIIVGCVAVLREAKGHRELLDAIAPLCQANPRLHLVIAGDGEP 234 Query: 218 -MDNLKAQAKRLGIEDKVTFLGGVRDLTGYFKEVDILVNTPHCVGDHGAGVGNNILEAGL 276 M +L A K L +E ++ LG D DI + G LEA Sbjct: 235 VMQHLLAHRKTLTLETQIHLLGYRHDAPRLMSGFDIFA-----LATQKEAAGTVFLEAAQ 289 Query: 277 YDTPVVTYNMAGISEMVITGQTGYCIPFGDDEAFIEAVDTLIKHPELRSQNGKA 330 P++ + G+ EM+ G + G+ A A+ TL+ + + R MG+A Sbjct: 290 AGIPIIATRVGGVPEMLQEGTNAILVTPGNQTALTNALHTLVTNNQQRHSMGRA 343 A homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS30 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 31 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 61> which encodes amino acid sequence <SEQ ID 62; NGS31>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.36 Possible cleavage site: 16 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 3.50 threshold: 0.0 PERIPHERAL Likelihood = 3.50 modified ALOM score: −1.20 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.299(Affirmative) < succ> The protein has homology with the following sequences in the databases: gb|AAB49297.1|(U84350) hypothetical hydroxylase a [Amycolatopsis orientalis] Length = 491 Score = 111 bits (278), Expect = 1e−23 Identities = 87/269 (32%), Positives = 123/269 (45%), Gaps = 15/269 (5%) Query: 1 LKNGAAFSWGSRYTEFDF----TDKFSDGPGTVYQVRRAVRDKILIEEAAKQGVEVRFGH 56 +K G F WG+R + F + K + YQV RA FD IL++ A +GV VR G Sbjct: 73 IKRGGTFRWGARPEPWTFHFGISAKMAGSTSHAYQVERAKFDDILLKNAKSKGVVVREGC 132 Query: 57 GVTAFDNSGDFARLNIETDT-GESYELTAKFVLDASGY-GRVLPRLLNLETPSHLPPRQT 114 V G+ TD G ++E++A+FV+DASG R+ ++ S Sbjct: 133 SVNDVVEDGERVTGARYTDADGNAHEVSARFVIDASGNKSRLYTKVNGRNYSEFFRSLA 192 Query: 115 HFTHIDDNITHPKFDRNKILITTHPQHRDVWIWLIPFGDNRCSVGVV---GTPKLAGES 171 F + + P+ IL W W I P D SVG V DK+ G+ Sbjct: 193 LFGYFEGGKRLPEPVSGNILSVAFDSG---WFWYIPLSDTLTSVGAVVRREDADKIQGDR 249 Query: 172 ETVLKKFVYECPHLSEILDKAVWENDFPFRSIQ---GYSANVKSLHGEHFALLGNAAEFL 228 E L + ECP++SE L A + ++ YS S L+G+AA F+ Sbjct: 250 EKALNTLIAECPLISEYLSNATRVTRYGELRVRKDYSYQQDSYWRPGMVLVGDAACFV 309 Query: 229 DPVFSSGVTIALHSAELAADLLTKQLKGE 257 DPVFSSGV +A +SA LAA + L G+ Sbjct: 310 DPVFSSGVHLATYSALLAARSINSVLAGD 338 A homolog (amino acids 280-341) was found in serogroup A N.meningitidis but not in serogroup B, so NGS31 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 32 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 63> which encodes amino acid sequence <SEQ ID 64; NGS32>.","Analysis of this protein sequence reveals the following: Signal Score (−7.5): −3.49 Possible cleavage site: 38 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 7.80 threshold: 0.0 PERIPHERAL Likelihood = 7.80 modified ALOM score: −2.06 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.278(Affirmative) < succ> The protein has homology with the following sequences in the databases: fastidiosa (strain 9a5c) gb|AAF83310.1|AE003899_2 (AE003899) phage-related repressor protein [Xylella fastidiosa] Length = 143 Score = 87.0 bits (214), Expect = 2e−16 Identities = 40/71 (56%), Positives = 54/71 (75%) Query: 1 MFSGEQLGQAISEAIKRKNVSQKEVADHFGVKQPSVSGWIKNGRIDKKHLDKLIDYFSDV 60 M +GEQLG+AI +A++ K V+ ++A+HFGVK PSV GWIK GRI K+ L L YFSDV Sbjct: 1 MLTGEQLGRAIKQAMQLKGVTPTKMAEHFGVKAPSSVYGWIKEGRISKEKLPSLWSYFSDV 60 Query: 61 VTPSHFGIETF 71 V P+H+G+E + Sbjct: 61 VGPTHWGLEAW 71 >sp|P18680|RPC1_BPHK0 26 KD REPRESSOR PROTEIN (REGULATORY PROTEIN CI) emb|CAA34222.1|(X16093) cI gene product (AA 1-208) [Bacteriophage HK022] Length = 235 Score = 80.5 bits (197), Expect = 2e−14 Identities = 60/200 (30%), Positives = 99/200 (49%), Gaps = 15/200 (7%) Query: 22 QKEVADHFGVKQPSVSGWIKNGRIDKKHLDKLIDYFSDVVTPSHF--------GIETFRV 73 Q ++A V ++S W 1 +K DK+ S + T + + GI + Sbjct: 29 QADLARLKVTPKAISKFNGESIPRK--DESLASVLGTTAAYLHGYADDDGITVNHL 86 Query: 74 LKSNEQSSIRFPRLNAEATCGAGT-INDHYIEVVDYVTVAAAWAREKLGGNLNK-IQVIT 131 +SN+ R L+ +A+ G GT +++ +IE + + AR G + ++VIT Sbjct: 87 SRSNDY--YRVDVLDVQASAGPGTMVSNEFIEKIRAIEYTTEQARILFNGRPQESVKVIT 144 Query: 132 ARGDSMEPTIENGDVMFVDTAVEAFDGDGLYLLWYIDGLKAKRLQSTVGGGLMIISDNSS 191 RGDSME TI GD +FVD ++ FDGDG+Y+ Y + KRLQ L +ISDN++ Sbjct: 145 VRGDSMEGTINPGDEIFVDVSITCFDGDGIYVFVYGKTMHVKRLQMQ-KNRLAVISDNAA 203 Query: 192 YRTETVRGEDLNAVRIIGRI 211 Y + + + I+ ++ Sbjct: 204 YDRWYIEEGEEEQLHILAKV 223 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 33 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 65> which encodes amino acid sequence <SEQ ID 66; NGS33>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.87 Possible cleavage site: 31 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 4.88 threshold: 0.0 PERIPHERAL Likelihood = 4.88 modified ALOM score: −1.48 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.313(Affirmative) < succ> The protein has homology with the following sequences in the databases: gb|AAF31132.1|(AF069529) Gp54 [Bacteriophage HK97] Length = 273 Score = 47.4 bits (111), Expect = 3e−04 Identities = 33/123 (26%), Positives = 52/123 (41%), Gaps = 20/123 (16%) Query: 221 NGGLSGKPKNANVPRRRKTHGVPLQEIADLYNEVLGGRLPSVQVLNDTRKRAIANRWCEM 280 NGG G+ K P RRK + + + YN +G RLP +N+ RKR + + Sbjct: 160 NGGGDGQVK----PERRKAERIDYESFLNAYNTEVGDRPHAVAVNEKRKRRL-KKIIPQ 214 Query: 281 LGTAAPNGKVRFGDKETGIAFAGFFRKVA--MNPFWMGENQTGFAVGFDWIFKAGNFVK 338 L T +G F + R PF+ G+N TG+ FD++ + + Sbjct: 215 LKTPNVDG-------------FRAYVRAFVHQAKPFYFGDNDTGWTADFDYLLREDSLTG 261 Query: 339 ILE 341 + E Sbjct: 262 VRE 264 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 34 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 67> which encodes amino acid sequence <SEQ ID 68; NGS34>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.65 Possible cleavage site: 50 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 3.76 threshold: 0.0 PERIPHERAL Likelihood = 3.76 modified ALOM score: −1.25 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.310(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir||H82649 hypothetical protein XF1674 XF1569 [imported]- Xylella fastidiosa (strain 9a5c) gb|AAF84378.1|AE003986_8 (AE003986) hypothetical protein [Xylella fastidiosa] gg|AAF84483.1|AE003993_2 (AE003993) hypothetical protein [Xylella fastidiosa] Length = 316 Score = 167 bits (424), Expect = 2e−40 Identities = 108/308 (35%), Positives = 152/308 (49%), Gaps = 30/308 (9%) Query: 10 ETSVIRSLSSASLYMFTRRMFYQRRGYVWQRANHHAPICNLERVFTIGETKRLIINIPPR 69 E +VI++ A FTR F QR+ ++ HH I ++ V G K ++IN+PP Sbjct: 10 EQAVIKARCEADHLFFTRYFFKQRQQLRFRVNWHHHVIAGVVDDVIAGRRKDVVINVPPG 69 Query: 70 YSKTEIAVVNFIAWAMGRVPDCEFIHASYSAALAVNNSVQIRNLVQHEEYRAIFP-DLAL 128 SKTE+ +N +A + P F+H SYS LA+ NS R +VQ +EYRA++P ++A Sbjct: 70 SSKTELVAINVMARGLALNPYARFLHISYSDDLALLSETAREIVQSDEYRALWPLEIAD 129 Query: 129 AGESGHHWKTT-----AGGVMYXXXXXXXXXXXXXXRHREGFGGCIIIDDPHKADEARSE 183 +S W AGGV Y G+ G IIIDDP K ++A S+ Sbjct: 130 DAKSKKRWVVDGKKAGGV-YAVSLGGQVTGFRAGHAPGWQGAIIIDDPLKVEDAYSK 188 Query: 184 VRRQNVIDWFQNTVESRKNSPDTPIILIMQRLHEKDLAGWLLDGGNGEEWEHLCLPAIQE 243 R +TV+SRK SPDTPII+IMQRL + D G++ GG WE + +PA+ + Sbjct: 189 TGRSKANRKLVSTKSRASPDTPIIVIMQRLAQDDPTGFIQSGGFPGAWECIEIPALID 248 Query: 244 DG-----------------------TALWPEKHDIETLRREQAAPWFAGQYLQKPAPP 280 D + WP K + L +E YVF+GQY Q+P+P Sbjct: 249 DAYVSRLPEHVQGQVVRDAQDQDGRYSYWPYKEPLAELLALEATDRYVFSGQYQQRPSPL 308 Query: 281 DGGTFKPD 288 GG K D Sbjct: 309 GGGIIKGD 316 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 35 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 69> which encodes amino acid sequence <SEQ ID 70; NGS35>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −1.07 Possible cleavage site: 40 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 1.64 threshold: 0.0 PERIPHERAL Likelihood = 1.64 modified ALOM score: −0.83 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.020(Affirmative) < succ> The protein has homology with the following sequences in the databases: >ref|NP_047925.1|gp34 [Bacteriophage phi-C31] emb|CAA07104.1|(AJ006589) gp34 [Bacteriophage phi-C31] Length = 457 Score = 59.7 bits (143), Expect = 1e−07 Identities = 68/272 (25%), Positives = 117/272 (43%), Gaps = 49/272 (18%) Query: 226 GYSPVEQIIMTVNIALKRQVHALEYYTAGSVPDALVGVPETWSADDIRRFQEYWDLLLSG 285 G SP+ +++ AL Q + +++ G++P A+V VP T S + + R +E W SG Sbjct: 192 GCSPISYARESIGLALAAQKYGSKFFANGGAMPGAVVEVPGTMSKEGLARAREAWRAANSG 251 Query: 286 -----------ETAQRRKMRFVPGELSRNFRETRQPPLKDVYDEWLARVVCFAFSVEPTP 334 E A+ K+ P E F +T+Q + ++ AR+ F V P Sbjct: 252 VDNAHRVALLTEGAKFSKVAMSPDEAQ--FLQTRQFQVPEI-----ARI----FGVPPH- 299 Query: 335 FVAQVNRSVAETS--REQSLSDGMGSLKNWVKALIDDVLARYMDMAA--YEFVWKGEESL 390 ++ S + S EQ+++ M SL+ W++ + A + FV + + Sbjct: 300 LISDATNSTSWGSGLAEQNIAFTMFSLRPWLERIEAGFNRLLFAETADRFRFVKFNLDEI 359 Query: 391 N---PKEQAEIYAIYKNAGILTADEIRAELGKEPLP-GQG--------------QPEPDK 432 PKE+ E++++ GI + DE+RA PLP G G +PEP+ Sbjct: 360 KRGAPKERMELWSLGLQNGIYSIDEVRAAEDMTPLPDGLGEKYRVPLNLGEVGEEPEPEP 419 Query: 433 QDG----RKPEEPPNQGAEKLGKSESPMSEDE 460 P E P++ E GK + + +E Sbjct: 420 APAPPAIEPPAEEPDEEPEPEGKPDDEGATEE 451 A homolog (amino acids 641-700) was found in serogroup A N.meningitidis but not in serogroup B, so NGS35 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 36 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 71> which encodes amino acid sequence <SEQ ID 72; NGS36>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 4.3 Possible cleavage site: 26 >>> Seems to have a cleavable N-term signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 27 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 1.70 threshold: 0.0 PERIPHERAL Likelihood = 1.70 modified ALOM score: −0.84 Score for OM-PP discrimination: 0.02 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: 0.02 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Outer membrane?","score: 0.00213559 Outer membrane?","score: 0.00213559 ----- Final Results ----- bacterial outer membrane --- Certainty= 0.232(Affirmative) < succ> The protein has homology with the following sequences in the databases: >pir||D82437 TonB receptor-related protein VCA0625 [imported]-Vibrio cholerae (group O1 strain N16961) gb|AAF96526.1|(AE004392) TonB receptor-related protein [Vibrio cholerae] Length = 784 Score = 103 bits (256), Expect = 2e−20 Identities = 104/427 (24%), Positives = 162/427 (37%), Gaps = 100/427 (23%) Query: 31 NTEQQKELNTIVVHGKRS-ADQKGADDVYYKNVSNAYVGKEYLERYRVQSAGDVLKGLNG 89 NTEQ + T+ VHG+ DQ+ D L++ R + D+ G+ Sbjct: 57 NTEQAVD-ETVTVHGQSILTDQRTRSD---------------LDkVRGIANADIFSGITS 100 Query: 90 VYNMNTRTAGGAITPNIRGITGKGRIPVTIDGTEQTIDVWMNNYGVGDRNYLDPALFRSI 149 V + N GA+ IRG+ G+GR+P+ IDG+ Q+ GV DR Y+D L S+ Sbjct: 101 VQSNNMHNEAGALDIGIRGVQGEGRVIFIDGSLQSTHTSRGYQGVSDRTYIDTDLLSSL 160 Query: 150 AVEKSPALTRG--VKSGVGGAMSIRTIEPSDIIPEGRNWGIEVKTEFSGNTVAQKNDLRQ 207 V K + VGG ++ T+ DII + + +G+ +K Sbjct: 161 TVNKGATIESSPYASGAVGGVVNATTLGIKDIIKDDQAFGVVLK---------------- 204 Query: 208 FLGRDYRTLSPIGATADGVSGMPDVLTGYTGKPSPTALLLDEGIADTKFSGGKSHTNFKD 267 A A+ + PDV Y+ + LDE + F G Sbjct: 205 -------------ARANNHNRTPDVSGDYSEQGQ---YALDERGEHSAFKHG-------- 240 Query: 268 DRQLMLSAAFKTDITDGLAAYSHRQKGNYYAGKRGYQSYLNNPI--YGADACYDQYPDKS 325 LML ++ + + + AYS R KGN++AGK+GY+ Y P+ G + + S Sbjct: 241 --SLMLGLGYQAESFTVLAYSKRSKGNHFAGKKGYEEY-QEPVVGQGQEVVNTSFESDS 297 Query: 326 WREKDILCKSASLVPNMAVLFRPGEEIMNSHTDTKILLLKNNWYLPDNQKISLQYMDNK 385 W K S N +R H +L WY Y D K Sbjct: 298 WLFK---LASDTGTAHNADFNYR--------HHAQKAGEVLMAYWYKSSEDWEGNPYPDGK 347 Query: 386 IGFGEINPLITAWILGFAEQSLNEPVQQAPGIGTKIDSKTYKIGYEWKPQNNKWIDLQAD 445 + W LG A+ + TY Y ++P ++ W++L A+ Sbjct: 348 -------DRMPQWGLGTAKVN------------------TYSANYYYQP-DHPWLNLNAN 381 Query: 446 MWRVKTD 452 W + D Sbjct: 382 FWYTEAD 388 Score = 94.7 bits (234), Expect = 5e−18 Identities = 80/290 (27%), Positives = 126/290 (42%), Gaps = 37/290 (12%) Query: 929 SYDLDNHLFARYASRFPSLYELTAATGSGGLYGSETVAEYS----LKPEKSTNWEV 984 +Y L + +LF + +R R PSLYE T S V Y+ +KPE++ N EV Sbjct: 514 TYALTPSTQLFLKSSRTYRMPSLYETTL---------SNEVFSYNPYNPIKPEQAWNNEV 564 Query: 985 GYNFNFAPHFAKLRQGDLRLTYYSNKIKNQIDTSN--EDGGMIQ---------YDKAVSK 1033 G F + + + +L ++Y+ N IK+ I + GM + YDK Sbjct: 565 GVQFMASNSVLQDDRLNLSVSYFRNSIKDFISGGRLAKTPGMSEWQANFTFTNYDKLQLS 624 Query: 1034 GVELQSRLDSGRFFASFGGTYRLKHMVCDKGIAFKFDYYLQRVPECLEGGFGLSRFFQSL 1093 G EL + + F T + +C A C GF + Sbjct: 625 GWELGAHYQYAWILYTHPAATLYSETKICSVQQA-----QYAESDTCNSLGFAWGLTPTRI 679 Query: 1094 QPKYSLTLDVGTRFFNEKLELGMRAIHHSKAERRNYDKLIADGAGQVYARNGKPYGWHAA 1153 PK +L L+VGT+FFN+ L+ G++ +HS + N +A A Y Sbjct: 680 PPKQNLYLNVGTKFFNDTLDSGVSYHSG--KSNPSDWLAGTAANPILEIPSDY----- 732 Query: 1154 TLLDAYARYRIGKHIDLNFSVTNLANRYYLDPMSSTPVPGPGRTITFGIK 1203 +D Y++Y + + L F++ N+ +RY + P S +P PGRTIT G + Sbjct: 733 -TIDLYSQYELNANTQIFFAINNVTDRYQVRPGSVVSMPDPGRTITLGFE 781 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 37 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 73> which encodes amino acid sequence <SEQ ID 74; NGS37>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 4.47 Possible cleavage site: 21 >>> Seems to have a cleavable N-term signal seq.","Amino Acid composition of Predicted Mature Form: calculated from 22 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 7.21 threshold: 0.0 PERIPHERAL Likelihood = 7.21 modified ALOM score: −1.94 Score for OM-PP discrimination: 16.42 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: 16.42 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Outer membrane?","score: 1.64214 Outer membrane?","score: 1.64214 ----- Final Results ----- bacterial outer membrane --- Certainty= 0.938(Affirmative) < succ> The protein has homology with the following sequences in the databases: >sp|Q03155|AIDA_ECOLI ADHESIN AIDA-I PRECURSOR pir||S28634 adhesin AIDA-I precursor-Escherichia coli plasmid pIB6 emb|CAA46156.1|(X65022) AIDA-I [Escherichia coli] Length = 1286 Score = 35.8 bits (81), Expect = 0.67 Identities = 34/138 (24%), Positives = 62/138 (44%), Gaps = 16/138 (11%) Query: 3 ASQLTLAVLLAAAFGSAYAVEVKGGDSSKGQLIQAAESDFLPFGSGAADIKVSTGNGLSK 62 A L + + + G+A+AV + G SS G + + E+ + G G ++ V++G ++ Sbjct: 31 AKNTLLVLAWSTIGNAPAVNISGTVSS-GGTVSSGETQIVYSGRGNSNATVNSGG-TQ 87 Query: 63 SINLEAGPAQRIRNKYGNAPINGGNQNTNVNGAANSRYLQPGDINPIA--GWFSKTRLA- 119 +N N + G+QN +GA S + G I ++ G S T L+ Sbjct: 88 IVNNGGKTTATTVN-------SSGSQNVGTSGATISTIVNSGGIQRVSSGGVASATNLSG 140 Query: 120 ---QVWYEKRANNTEVFS 134 ++ A+NT +FS Sbjct: 141 GAQNIYNLGHASNTVIFS 158 The protein was expressed in E.coli as an insoluble 32.45 kDa His-fusion product and then purified.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 38 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 75> which encodes amino acid sequence <SEQ ID 76; NGS38>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 0.34 Possible cleavage site: 24 >>> Seems to have a cleavable N-term signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 25 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 3.98 threshold: 0.0 PERIPHERAL Likelihood = 3.98 modified ALOM score: −1.30 Score for OM-PP discrimination: 2.87 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: 2.87 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Outer membrane?","score: 0.287446 Outer membrane?","score: 0.287446 ----- Final Results ----- bacterial outer membrane --- Certainty= 0.607(Affirmative) < succ> The protein has homology with the following sequences in the databases: >sp|Q03155|AIDA_ECOLI ADHESIN AIDA-I PRECURSOR pir||S28634 adhesin AIDA-I precursor-Escherichia coli plasmid pIB6 emb|CAA461S6.1|(X65022) AIDA-I [Escherichia coli] Length = 1286 Score = 35.8 bits (81), Expect = 0.67 Identities = 34/138 (24%), Positives = 62/138 (44%), Gaps = 16/138 (11%) Query: 3 ASQLTLAVLLAAAFGSAYAVKGGDSSKGQLIQAAESDFLPFGSGAADIKVSTGNGLSK 62 A L + + + G+A+A+V + G SS G + + E+ + G G ++ V++G ++ Sbjct: 31 AKNTLLVLAVVSTIGNAFAVNISGTVSS-GGTVSSGETQIVYSGRGNSNATVNSGG--TQ 87 Query: 63 SINLEAGPAQRIRKYGNAPINQGNQNTNVNGAANSRYLQPGDINPIA--GWFSKRLA- 119 +N N + G+QN +GA S + G I ++ G S T L+ Sbjct: 88 IVNNGGKTTATTVN-------SSGSQNVGTSGATISTIVNSGGIQRVSSGGVASATNLSG 140 Query: 120 ----QVWYEKRANNTEVFS 134 ++ A+NT +FS Sbjct: 141 GAQNIYNLGHASNTVIFS 158 >pir||G81213 conserved hypothetical protein NMB0313 [imported]-Neisseria meningitidis (group B strain MD58) gb|AAF40758.1|(AE002388) conserved hypothetical protein [Neisseria meningitidis MC58] Length = 488 Score = 84.3 bits (207), Expect = 3e−15 Identities = 111/498 (22%), Positives = 185/498 (36%), Gaps = 35/498 (7%) Query: 7 LLFLPLCTVCLAAPSNDAADERRRLLDEGSRQTQQYRESGW--LDTEQARGEVEENDGY 64 +L LPL S A+E R D SR + E+ +D E+ G+V E + Sbjct: 19 MLLLPLLA------SAAYAEETPREPDLRSRPEFRLHEAEKPIDREKPGQVREKGKVL 72 Query: 65 SIGGEIYQVGDTAEELESAIYHALNARQWHKVRQFAARYAKLPRHKPALIHLADALQKRD 124 I GE E L A+Y A+ + +R Y + + L A + + Sbjct: 73 QIDGETLL--KNPELLSRAMYSAVVSNNIAGIRVILPIYLQQAQQDKMLALYAQGILAQA 130 Query: 125 EGDFRAAGNSFQTALEAEPDNPRLLLEAGRFYAEDNQNKESAAAFEKVLKTDIPAETRPI 184 +G + A + ++ + A+PD P + + E+ QN+ +A F+++ ++P + Sbjct: 131 DGRVKEAISHYRELIAAQPDAPAVRMRLAAALFENRQNEAAADQFDRLKAENLPPQLMEQ 190 Query: 185 VENYLSELGKRRRWHGQISLGYGYNSNVNQGNGINQCVWEIAGMCLMERTLPAPTDSTFS 244 VE Y L +R W N+NQ Q + T P D T Sbjct: 191 VELYRKALRERDAWKVNGGFSVTREHNINQAPKRQQ---------YGKWTFPKQVDGTAV 241 Query: 245 SYSATAEKTVPLKGNHGVQVRGVLYGNRYTEKDKDSAAMPDYGYNGSLYAGYAYADARS 304 +Y AEK LK G + G Y K + + G +AD R Sbjct: 242 NYRLGAEKKWSLKNGWYTTAGGDVSGRVYPGNKK-------FNDMTAGVSGGIGFADRRK 294 Query: 305 SFSLLPYFEYDFRNRHTHYRAWGADADWSRTLSPHNRINSHAGAKKTGYGGQSKTYFADF 364 L + E + GA ++R +P W+ S A + G ++ +D Sbjct: 295 DAGLAVFHERRTYGNDAYSYTNGARLYFNRWQTPKWQTLSSA---EWGRLKNTRRARSDN 351 Query: 365 KQYELGAGAEFSITLKSGLLVNFDAAPKAYP-EKSSSSKEYTARLGAYRLFSGGTYLNAV 423 ++ F + + D R+ P ++ + Y R A+ GG+ L+++ Sbjct: 352 THLQISNSLVFYRNARQYMGGLDFYRERNPADRGDNFNRYGLRF-AWGQEWGGSGLSSL 410 Query: 424 LLY--RRSLYDAASFVSDNK--RRRDKQYIMMAAAGFPQWNIKGVYPELRFRRTIAHSNA 479 L + Y+ F S K RRRDK+ + + KG+ P L SN Sbjct: 411 LRLGAAKRHYEKPGFFSGFKGERRRDKELNTSLSLWHRALHFKGITPRLTLSHRETRSND 470 Query: 480 VYYRYRQNEWLLGFKYRF 497 V+ Y +N +F F Sbjct: 471 VFNEYEKNRAFVEFNKTF 488 >pir||C81790 conserved hypothetical protein NMA2174 [imported]- Neisseria meningitidis (group A strain Z2491) emb|CAB85386.1|(AL162758) conserved hypothetical protein [Neisseria meningitidis Z2491] Length = 490 Score = 84.0 bits (206), Expect = 4e−15 Identities = 111/498 (22%), Positives = 185/498 (36%), Gaps = 35/498 (7%) Query: 7 LLFLPLCTVCLAAPSNDAADERRRELLDEGSRQTQQYRESGW--LDTEQARGEVEENDGYI 64 +L LPL S A+E R D SR + E+ +D E+ G+V E + Sbjct: 23 MLLLPLLA------SAAYAEETPREPDLRSRPEFRLHEAEVKPIDREKVPGQVREKGKVL 74 Query: 65 SIGGEIYQVGDTAEELESAIYHALNARQWHKVRQFAARYAKLPRHKPALIHLADALQKRD 124 I GE E L A+Y A+ + +R Y + + L A + + Sbjct: 75 QIDGETLL--KNPELLSRAMYSAVVSNNIAGIRVILPIYLQQAQQDKMLALYAQGILAQA 132 Query: 125 EGDFRAAGNSFQTALEAEPDNPRLLLEAGRFYAEDNQNKESAAAFEKVLKTDIPAETRPI 184 +G + A + ++ + A+PD P + + E+ QN+ +A F+++ ++P + Sbjct: 133 DGRVKEAISHYRELIVAQPDAPAVRMRLAAALFENRQNEAAADQFDRLKAENLPPQLMEQ 192 Query: 185 VENYLSELGKRRWHGQISLGYGYNSNVNQGNGINQCVWEIAGMCLMERTLPAPTDSTFS 244 VE Y L +R W N+NQ Q + T P D T Sbjct: 193 VELYRRKALRERDAWKVNGGFSVTREHNINQAPKRQQ---------YGKWTFPKQVDGTAV 243 Query: 245 SYSATAEKTVPIKGNHGVQVGVLYGNRYTEKSDYGYRNGSLYAGYAYADARS 304 +Y AEK LK G + G Y K + + G +AD R Sbjct: 244 NYRLGAEKKWSLKNGWYTTAGGDVSGRVYPGNKK-------FNDMTAGVSGGIGFADRRK 296 Query: 305 SFSLLPYFEYDFRNRHTHYRAWGADADWSRTLSPHRINSHAGAKKTGYGGQSKTYFADF 364 L + E + GA ++R +P W+ S A +G ++ +D Sbjct: 297 DAGLAVFHERRTYGNDAYSYTNGARLYFNRWQTPKWQTLSSA---EWGRLKNTRRARSDN 353 Query: 365 KQYELGAGAEFSITLKSGLLVNFDAARKAYP-EKSSSSKEYTARLGAYRLFSGGTYLNAV 423 ++ F + + D R+ P ++ + Y R A+ GG+ L+++ Sbjct: 354 THLQISNSLVFYRNARQYWMGGLDFYRERNPADRGDNFNRYGLRF-AWGQEWGGSGLSSL 412 Query: 424 LLY--RRSLYDAASFVSDNK--RRRDKQYIMMAAAGFPQWNIKGVYPELRFRRTIAHSNA 479 L + Y+ F S K RRRDK+ + + KG+ P L SN Sbjct: 413 LRLGAAKRHYEKPGFFSGFKGERRRDKELNTSLSLWHRALHFKGITPRLTLSHRETRSND 472 Query: 480 VYYRYRQNEWLLGFKYRF 497 V+ Y +N + F F Sbjct: 473 VFNEYEKNRAFVEFNKTF 490 The protein was expressed in E.coli as an insoluble 52.03 kDa His-fusion product and then purified.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 39 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 77> which encodes amino acid sequence <SEQ ID 78; NGS39>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.38 Possible cleavage site: 18 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 7.16 threshold: 0.0 PERIPHERAL Likelihood = 7.16 modified ALOM score: −1.93 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.325(Affirmative) < succ> The protein has homology with the following sequences in the databases: >ref|NP_052685.1|serine protease EspP [Eseherichia coli] pir||T00317 probable serine proteinase espP, extracellular-Escherichia coli plasmid pO157 pir||T42120 probable serine proteinase espP, extracellular-Escherichia coli plasmid pO157 emb|CAA66144.1|(X97542) putative exoprotein-precursor [Escherichia coli] dbj|BAA31836.1|(AB011549) serine protease EspP [Escherichia coli] gb|AAC70088.1|(AF074613) putative exoprotein-precursor [Escherichia coli O157:H7] Length = 1300 Score = 58.9 bits (141), Expect = 2e−07 Identities = 153/687 (22%), Positives = 248/687 (35%), Gaps = 106/687 (15%) Query: 194 DLTVENKTLSDA---EFGVYALNTSMVNLSSKDNNEVKSTQVGLYSQDGGSINVDR--- 247 D +NT +DA Y N ++ +LS D E + + G + +V R Sbjct: 595 DYVAGMQNTEADAVKQNGNAYKTNNAVSDLSQPDW-ETGTFRFGTLHLENSDFSVGRNAN 653 Query: 248 --------KDNIIEGDAVALVGKGGSQNIRAS----RTLISSKSLGIHAEQAAKIAITG 295 K NI GD A + +NI R N++ S G E IT Sbjct: 654 VIGDIQASKSNITIGDTTAYIDLHAGKITGDGFGFRQNIVRGNSQG---ETLFTGGITA 710 Query: 296 ASNTIHASNAAIRSLDKSEVKIDGQITIDSNVANLARQDGSIH---LNYKDDTRITGATV 352 +TI + A ++ + TI+ N A++ Q G ++ + +TG Sbjct: 711 EDSTIVIKDKAKALFSNYVYLLNTKATIE-NGADVTTQSGMFSTSDISISGNLSMTGNPD 769 Query: 353 SDKGLVAIKPLNNTNIVADTIHYKGDVLAVNKGKVELDF----TPNILLAGRLDNFSGLT 408 D LN+ + + + ++A NK V D + +I+ + S L+ Sbjct: 770 KDNKFEPSIYLNDASYLLTPDSAR--LVMASWGPIHSTKSASIMPGHDESPLSQLS 827 Query: 409 DSKHKNLFENYVANLDSKSAGEINFNLAKDAL----WTMTGQSWLDKLEGQGTIDFNNDA 464 D K L + D G +N A + W +TG S L L+ ++ + D+ Sbjct: 828 DRTSKGLALGLLGGFDVSYRGSVNAPSASATMNNTWWQLTGDSALKTLKSTNSMVYFTDS 887 Query: 465 KTSGR--ALHIGELAGANK-FLMHLNKDGIHSDMLYVKKGTSTPQEVVVKNLSEVLDSMN 521 + + L + ELA +N + M N SD L VKK S ++ L + L Sbjct: 888 ANNKKFHTLTVDELATSNSAYAMRTNLS--ESDKLEVKKHLSGENNIL---LVDFLQKPT 942 Query: 522 YGERLRFATVTNSKNEFVNGKKYIDDTHLMEDALTVEYSAHNGXXXXXXXXXXSFNGSEM 581 ++L V+ K+ N K T D V Sbjct: 943 PEKQLNIELVSAPKTNENFKASKQTIGFSDVTPV------------------------ 978 Query: 582 TAEKAGDPYVNKTYTDNRQNVYLVKQATGNPSRNVKNINDMFDSTAHYAFT--LDTYAKR 639 + DD + T++ N K+AT N + S + AF ++ KR Sbjct: 979 ITTRETDDKI--TWSLTGYNTVANKEATRNAAA--------LFSVPYKAFLNEVNNLNKR 1028 Query: 640 EGERAFSTLDKKEGDWIRLTHTRVIQSNAFRFHNNDFEIGYDRFSLNEQEKKRKWGISLD 699 G+ ++ + G W R+ S F + ++G D+ K G+ L Sbjct: 1029 MGD--LRDINGEAGAWARIMSGTGSASGGFSDNYTHVQVGVDK-------KHELDGLDLF 1079 Query: 700 YGHGRTSLWNTFGKD----KIRKYELALYNTTQYIDKEGDETGYIDNVLKIGKLRNRVIA 755 G T ++ D K + LY + + D YID + K N A Sbjct: 1080 TGFTVTHTDSSASAFSGKTKSVGAGLYASAMF-----DSGAYIDLIGKYVHHDNEYTA 1134 Query: 756 RNHMGQLWGKGKYSNTLFSISTEYGRRKFLDDDKLWRITPQVQLQYSYLRGTGYRI-DNG 814 G G YS + E G R + +D W I PQ +L Y + G + D G Sbjct: 1135 -TFAGL--GTRDYSTHSWYAGAEAGYRYHVTEDA-W-IEPQAELVYGSVSGKQFAWKDQG 1189 Query: 815 INVNLSHA--NSLIGRLGLDVVRKFDG 839 +++++ N LIGR G+DV + F G Sbjct: 1190 MHLSMKDKDYNPLIGRTGVDVGKSFSG 1216 Score = 36.6 bits (83), Expect = 1.2 Identities = 97/412 (23%), Positives = 164/412 (39%), Gaps = 83/412 (20%) Query: 63 DNIVTMKSGDADADYVNNSKVLTETPYYKSKRGSNGIFAYGDKSLVKLIGEIVK--SE 120 D V G + ++ SK Y + +G + A+ S V + +N + +E Sbjct: 163 DKFVVETRGATEGADISLSKQQALERYGVNYKGEKKLIAFRAGSGVVSVKKNGRITPFNE 222 Query: 121 ISEKSKALNGGFRHIGIYS-W---QNAKVE----LSSKSDN--------------IVQGG 158 +S K + LNG F HI +S W N + + ++++ D+ +V G Sbjct: 223 VSYKPEMLNGSFVHIDDWSGWLILTNNQFDEFNNIASQGDSGSALFVYDNQKKKWVVAGT 282 Query: 159 IWGLYS----NNSSISLKGKNNVISNPKYNVFAYKKAKVDLTVENKNTLSDAEFGVYALN 214 +WG+Y+ N + K I N K N ++Y VD++ T+ + + + Sbjct: 283 VWGIYNYANGKNHAAYSKWNQTTIDNLK-NKYSY---NVDMSGAQVATIENGK--LTGTG 336 Query: 215 TSMVNLSSKDNNEVKSTQVGLYSQ----DGGSINVDRKDNIIEGDAVALVGKG-----GS 265 + ++ +KD + L S GG + D+K + GD G G GS Sbjct: 337 SDTTDIKNKDLIFTGGGDILLKSSFDNGAGGLVFNDKKTYRVNGDDFTFKGAGVDTRNGS 396 Query: 266 Q---NIR-ASRTNL--ISSKSLGIHAEQAAK------IAITGASNTIHASNAAIRSLDKS 313 NIR ++ NL I +L + Q + I GA T +N I S D Sbjct: 397 TVEWNIRYDNKDNLHKIGDGTLDVKTQNTNLKTGEGLVILGAEKTF--NNIYITSGD-G 453 Query: 314 EVKIDGQITIDSNVAN---LARQDGSIHLN-YKDDTRITGATSDKGLV--------AIK 361 V+++ + + N A+ G++ LN Y AT D G V +I Sbjct: 454 TVRLNAENALSGGEYNGIFFAKNGGTLDLNGYNQSFNKIAAT--DSGAVITNTSTKKSIL 511 Query: 362 PLNNTNIVADTIHYKG-----DVLAVNKGKVELDFTPNILLAGRLDNFSGLT 408 LNNT AD I++ DVL ++ K E ++L G +D + ++ Sbjct: 512 SLNNT---ADYIYHGNINGNLDVLQHHETKKE---NRRLILDGGVDTTNDIS 557 The protein was expressed in E.coli as an insoluble 95.92 kDa His-fusion product and then purified.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 40 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 79> which encodes amino acid sequence <SEQ ID 80; NGS40>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.18 Possible cleavage site: 17 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 7.05 threshold: 0.0 PERIPHERAL Likelihood = 7.05 modified ALOM score: −1.91 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.108(Affirmative) < succ> The protein has no homology with sequences in the databases.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 41 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 81> which encodes amino acid sequence <SEQ ID 82; NGS41>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) signal Score (−7.5): −2.47 Possible cleavage site: 17 >>> May be a lipoprotein Amino Acid Composition of Predicted Mature Form: calculated from 16 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 7.37 threshold: 0.0 PERIPHERAL Likelihood = 7.37 modified ALOM score: −1.97 Rule: inner or outer membrane protein Rule: inner or outer membrane protein *** Reasoning Step: 2 Lipoprotein?","Inner membrane?","----- Final Results ----- bacterial outer membrane --- Certainty= 0.790(Affirmative) < succ> The protein has no homology sequences in the databases.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 42 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 83> which encodes amino acid sequence <SEQ ID 84; NGS42>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.2 Possible cleavage site: 14 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 6.58 threshold: 0.0 PERIPHERAL Likelihood = 6.58 modified ALOM score: −1.82 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.514(Affirmative) < succ> The protein has no homology with sequences in the databases.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 43 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 85> which encodes amino acid sequence <SEQ ID 86; NGS43>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.34 Possible cleavage site: 39 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 2 value: −4.78 threshold: 0.0 INTEGRAL Likelihood = −4.78 Transmembrane 1881-1897 (1876-1898) INTEGRAL Likelihood = −1.01 Transmembrane 1966-1982 (1966-1982) PERIPHERAL Likelihood = 1.91 modified ALOM score: 1.46 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.291(Affirmative) < succ> The protein has no homology with sequences in the databases.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 44 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 87> which encodes amino acid sequence <SEQ ID 88; NGS44>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.49 Possible cleavage site: 58 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 1 value: −1.33 threshold: 0.0 INTEGRAL Likelihood = −1.33 Transmembrane 141-157 (140-157) PERIPHERAL Likelihood = 2.54 modified ALOM score: 0.77 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.153(Affirmative) < succ> The protein has no homology with sequences in the databases.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 45 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 89> which encodes amino acid sequence <SEQ ID 90; NGS45>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.07 Possible cleavage site: 46 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 2.07 threshold: 0.0 PERIPHERAL Likelihood = 2.07 modified ALOM score: −0.91 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.333(Affirmative) < succ> The protein has homology with the following sequences in the databases: >ref|NP_049512.1|putative portal protein [Bacteriophage 933W] ref|NP_050550.1|hypothetical protein [Bacteriophage VT2-Sa] gb|AAD25457.1|AF125520_52 (AF125520) putative portal protein [Bacteriophage 933W] dbj|BAA84334.1|(AP000363) hypothetical protein [Bacteriophage VT2-Sa] dbj|BAA94158.1|(AP000422) portal protein [Escherichia coli] O157:H7] Length = 714 Score = 314 bits (805), Expect = 2e−84 Identities = 213/658 (32%), Positives = 327/658 (49%), Gaps = 22/658 (3%) Query: 7 ETGVLPDKNGEPLTIG---EYRLFVGEMMNQPAWRAVADKEMDYADGRQLDNELLQKQR 62 ET + KN T + + ++ +QP WR A+K Y DG QL E+LQ + Sbjct: 4 ETNTMATKNDNGATPRFSQRQLQALCSDIDSQPKWRDAANKACAYYDGDQLPPEVLQVLK 63 Query: 63 ELGLPPAVENLITPTLLSVQGYEATIRTDWRVTADGETGGRD-VADALNFKLNRAERQSR 121 + G P + NLI PT+ V G EA RTD V +D + +A+A+N + A R Sbjct: 64 DRGQPMTIHNLIAPTVDGVLGMAKTRTDLVVMSDEPDDETEKLAEAINAEFADACRLGN 123 Query: 122 ADKACSDAFRGQIACGIGWVTRNPNPFEFPYECGVIHRNAIHWDMKSYKYDLSDARWL 181 +KA SDA+ QI G+ WVEV RN +PF ++ + RN + WD S + DLSD RWL Sbjct: 124 MNKARSDAYAEQIKAGLSWVEVRRNSDPFGPEFKVSTVSRNEVFWLSREADLSDCRWL 183 Query: 182 IRRWLLPERLAQFFPEYAGHFKAMGRGGSDWR-ISGELDGGGNTGLADAWGISGRNTV 240 +RRRW+ + FP G + + DWR + G +L AW Sbjct: 184 MRRRWMDTDEAKATFP---GMAQVIDYAIDDWRGFVDTTVTEOQPSPLMSAWEEYQSWDR 240 Query: 241 SEEFWFNETTRELAVAEVWYRRWVTADCLRDKKTGRTVEFDGANPNHREMAANGAV-LFA 299 + W R + + V+YR + + + GR V FD N A+G V + Sbjct: 241 QQNEWLQRERRRVLLQVVYYRTFERLPVI-ELSNQRVVAFDKNNLMQAVAVASGRVQVKV 299 Query: 300 ASVPRMRRAFVVGDLVVRDEPTPYPHQKFPYPFFGFREDNTGIPYVRNMKYAQDNLN 359 V R+R A+ VG + D P P FP VPF+G++R+D TG PYG + AQD +N Sbjct: 300 GRVSRIREAWFVGPHFIVDRPCSAPQGMFPLVPFWGYRKDKTGEPYGLISEAIPAQDEVN 359 Query: 360 STNSKLRWGLSAIRTVRTKGIVDMSDEQFRRNIARVDADIVLNKIEAAQPGAR--FDVSR 417 KL W L A R + + +SD I R D I LN + Q F V + Sbjct: 360 FRRIKLTWLLQAKRVIMDEDATQLSDNDLMEQIERPDGIIKLNPVRKNQKSVADVFRVEQ 419 Query: 418 DFELSAQHWQMLQDSRATIRQISGITPSVGNRGNATSGRQESIQVEQSNQSLGLVMDNF 477 DF++++Q +Q++Q+S I+ G+ +F+G ATSG S VEQ +L + DN+ Sbjct: 420 DFQVASQQFQVMQESEKLIQDTMGVYSAFLGQDSGATSGVAISNLVEQGAPLAEINDNY 479 Query: 478 RQSRSLVGELLLAMIIEDLGS-DEQTVVIEGDAVTQGRTVVINRPETDPVTGKAYLSNDL 536 + + VG LLLA +++DL VVI D + +T+V+N E D L+ND+ Sbjct: 480 QFACQQVGRLLLAYLLDDLKKRRNHAVVINRDDRQRRQTIVLN-AEGD----NGELTNDI 534 Query: 537 QNIRLKVALEDVPSTNSYRSQQLGAMSEAVKSLPPEYQAAVLPFMVSLMDIPFKDKVIEK 596 + +AL V T ++++Q MSE ++ LPP+ QA VL V+L+D+P K + +E+ Sbjct: 535 SRLNTHIALAPVQQTPAFKAQLAQHMSEVIQGLPPQVQAVVLDLWVNLLDVPQKQEFVER 594 Query: 597 IK-EVRVQETPEQI--EARIAQAVQDALAKSGNDIKRRELALKEQRTASEIKEIEARA 651 I+ + ++P+++ E + A Q AL + +++ RE+A + + ++ A A Sbjct: 595 IRAALGTPKSPDEMTPEEQEVAAQQQALQQQQAELQMREMAGRVAKLEADAARAHAAA 652 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 46 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 91> which encodes amino acid sequence <SEQ ID 92; NGS46>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.25 Possible cleavage site: 37 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 4.77 threshold: 0.0 PERIPHERAL Likelihood = 4.77 modified ALOM score: −1.45 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.281(Affirmative) < succ> The protein has homology with the following sequences in the databases: >sp|P44184|YE10_HAEIN HYPOTHETICAL PROTEIN HI1410 pir||E64028 hypothetical protein HI1410-Haemophilus influenzae (strain Rd KW20) gb|AAC23058.1|(U32820) H. influenzae predicted coding region HI1410 [Haemophilus influenzae Rd] Length = 394 Score = 150 bits (379), Expect = 3e−35 Identities = 75/168 (44%), Positives = 114/168 (67%), Gaps = 2/168 (1%) Query: 57 REIQKSMRDSVHRLLKDIWAQLGLGHYEITDFEIRGANGTLFVFSGLQSHTVDSIKSFE 116 REIQKS+ DSV ++L D++ L L F+++ +I G NG+ F F+GL+++ + SIKS Sbjct: 3 REIQKSISDSVIQMLADQIEMLSLQAFVQKTQIIGQNGSRFTFAGLKTN-ITSSIKSMT 61 Query: 117 GIDIVWVEEGHGVSKKSWDVLTPTIRKEGSEIWITLWPDMETDETYRRFIAMPSEDTWLC 176 GID+VWVEEG VSK+SWD+L PTIR++GS+I ++NP D+TY+RF+ P E Sbjct: 62 GIDVVWVEEGENVSESWDILIPTIREDGSQIIVSFNPKNILDDTYQRFVIHPPERCKSV 121 Query: 177 EINWRDNPWFPEALNRERLKAQRSMNKEDYGNIWEGRPRNVSEGAVYR 224 +NW+DNP+FP+L E ++ R + E Y +++EG P S+ A+ + Sbjct: 122 LVNWQDNPYFPKEL-MEDMEQMRERDYELYRHVYEGEPVADSDLAIIK 168 >ref|NP_050979.1|P18 [Bacteriophage APSE-1] gb|AAF03961.1|AF157835_18 (AF157835) P18 [Bacteriophage APSE-1] Length = 469 Score = 117 bits (294), Expect = 2e−25 Identities = 72/233 (30%), Positives = 110/233 (46%), Gaps = 13/233 (5%) Query: 17 LFKPCRYKVMYXXXXXXXXXXXXXXXXXXXXQRPLRILCAREIQKSMRDSVHRLLKDKVA 76 +FKP R KV + R LC RE S+ DS H +L+ +V Sbjct: 1 MFKPKRIKVYFGGRGGMKTVSFAKIALITASMHKRRFLCLREFMNSIEDSGHAVLQAEVE 60 Query: 77 QLGLGHFYEITDEEIRGANGTLFVFSGLQSHTVDSIKSFEGIDIVWVEEGHGVSKKSWDV 136 LGL + + I + I G N ++F + L + + SIKS D+ WVEE VS+KS D Sbjct: 61 TLGLQNRFRILNTYIEGINDSIFKYGQL-ARNIASIKSKHDFDVAWVEEAETVSEKSLDS 119 Query: 137 LTPTIRKEGSEIWITLNPDMETDETYRRFIA-----------MPSEDTWLCEINWRDNPW 185 L PTIRK GSE+W + NP E Y+RF+ +D ++ ++++ DNPW Sbjct: 120 LIPTIRKPGSELWFSFNPAEEDGAVYKRFVKPYKELIDTQGYYEDDDLYVGKVSYLDNPW 179 Query: 186 FPEALNRERLKAQRSMNKEDYGNIWEGRPMVSEGAVYRHEIQDAFHSGRVTL 238 P L + K +R N + + +++ G E A+ + E +A + L Sbjct: 180 LPAELKNDAQKMKRE-NYKKWRHVYGGECDANYEDALIQPEWVEAAIDAHIKL 231 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 47 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 93> which encodes amino acid sequence <SEQ ID 94; NGS47>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.87 Possible cleavage site: 31 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 4.88 threshold: 0.0 PERIPHERAL Likelihood = 4.88 modified ALOM score: −1.48 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.313(Affirmative) < succ> The protein has homology with the following sequences in the databases: >ref|NP_037739.1|Gp54 [Bacteriophage HK97] gb|AAF31132.1|(AF069529) Gp54 [Bacteriophage HK97] Length = 273 Score = 47.4 bits (111), Expect = 3e−04 Identities = 33/123 (26%), Positives = 52/123 (41%), Gaps = 20/123 (16%) Query: 242 NGGLSGKPKNANVPRRRKTHGVPLQEIADLYNEVLGGRLPSVQVLNDTRKRAIANRWCEM 301 NGG G+ K P RRK + + + YN +G RLP +N+ RKR + + Sbjct: 160 NGGGDGQVK----PERRKAERIDYESFLNAYNTEVGDRLPHAVAVNEKRKRRL-KKIIPQ 214 Query: 302 LGTAAPNGKVRFGDKETGLAWFAGFFEKVA--MNPFHGENQTGPAVGFDWIFKAGNFVK 359 L T +G F + R PF+ G+N TG+ FD++ + ++ Sbjct: 215 LKTPNVDG-------------FRAYVRAFHQAKPFFGDNDTGWTADFDYLLREDSLTG 261 Query: 360 ILE 362 + E Sbjct: 262 VRE 264 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 48 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 95> which encodes amino acid sequence <SEQ ID 96; NGS48>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −6.85 Possible cleavage site: 15 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 7.16 threshold: 0.0 PERIPHERAL Likelihood = 7.16 modified ALOM score: −1.93 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.379(Affirmative) < succ> The protein has homology with the following sequences in the databases: >dbj|BAA36059.1|(D90754) Outer membrane protein P.69 precursor [Escherichia coli] Length = 762 Score = 64.7 bits (156), Expect = 1e−09 Identities = 79/292 (27%), Positives = 121/292 (41%), Gaps = 55/292 (18%) Query: 3 NGARWTVTNDSMLKELDLSEDAQVEFSDNNK----FVKVSVSKLKGDGGVFKMYGDIV-- 56 N + W VT++S L L LS V+F+ + F ++V L G+ F M D+V Sbjct: 289 NNSVWNVTSNSNLDTLALSHST-VDFASHGSTAGTFATLNVENLSGNS-TFIMRADVVGE 346 Query: 57 ----KGESDKLITRKGSEGTHIIEYMDDAKAKTTGREYLKLVENKGNQEDNKASNKASYK 112 + D L S G H++ + TTG E L +V+ D AS AS + Sbjct: 347 GNGVNNKGDLLNISGSSAGNHVLAIRNQGSEATTGNEVLTVVKTT----DGAASFSASSQ 402 Query: 113 LNVRCTEQGGWCFALGESG------ASKKVNISTDGKRDF-------YLYPD-------- 151 + E GG+ + + ++G AS V T + PD Sbjct: 403 V-----ELGGYLYDVRKNGTNWELYASGTVPEPTPNPEPTPAPAQPPIVNPDPTPEPAPT 457 Query: 152 ---TLTPGASSSVLFGEALYQLNAVSDETLVQRMGEIHADGMPQEDNNVWIKRVGGKFSG 208 T T A + L Y LN V +TL+QRMG++ +D N+W++ GG Sbjct: 458 PKPTTTADAGGNYL--NVGYLLNYVENRTLMQRMGDLRNQ---SKDGNIWLRSYGGSLDS 512 Query: 209 SRSDYRVGGYGNRYWGFAGGFNRTGFGDKWIHYKGLMLRHLQSSYASEDYVG 260 S ++ G+ Y G G ++ D Y GL ++ S++AS DY G Sbjct: 513 FASG-KLSGFDMGYSGIQFGGDKR-LSDVMPLYVGL---YIGSTHASPDYSG 559 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 49 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 97> which encodes amino acid sequence <SEQ ID 98; NGS49>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −8.37 Possible cleavage site: 15 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 4.93 threshold: 0.0 PERIPHERAL Likelihood = 4.93 modified ALOM score: −1.49 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.355(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: gi|11282647|pir|H81959 patch repair protein (EC 3.1.-.-) NMA0429 [imported]-Neisseria meningitidis (group A strain Z2491) gi|7379179|emb|CAB83728.1|(AL162753) patch repair protein [Neisseria meningitidis Z2491] Length = 140 Score = 256 bits (628), Expect = 8e−68 Identities = 131/140 (93%), Positives = 132/140 (93%) Query: 1 MTDIFTPSKRSFVMSKIHSKETKPEVLVRKFLFSQGFRYRKNDKRYAGKPDIVLPKYKTV 60 MTDIFT SKRSFVM KIHSKETKPEVLVRKFLF QGFRYRKNDKRY GKPDIVL KYKTV Sbjct: 1 MTDIFTTSKRSFVMLKIHSKETKPEVLVRFLFFQGFRYRKNDKRYVGKPDIVLSKYKTV 60 Query: 61 VFIHGCFWHGHSCNKGHIPKSNMDFWLEKITKNRERDIKNETELEKIGFKVIVVWECELK 120 VFIHGCFW+GHSCNKGHIPKSN DFWLEKITKN ERDIKNETELEKIGFKVIVVWECELK Sbjct: 61 VFIHGCFWYGHSCNKGHIPKSNTDFWLEKITKNCERDIKNETELEKIGFKVIVVWECELK 120 Query: 121 NKAICRERLNRLVEEIKDAV 140 NKAICRERLNRLV EIKDAV Sbjct: 121 NKAICRERLNRLVREIKDAV 140 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 50 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 99> which encodes amino acid sequence <SEQ ID 100; NGS50>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −6.6 Possible cleavage site: 50 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 7.80 threshold: 0.0 PERIPHERAL Likelihood = 7.80 modified ALOM score: −2.06 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.398(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|11352963|pir||G81959 conserved hypothetical protein NMA0428 [imported]-Neisseria meningitidis (group A strain Z2491) gi|7379178|emb|CAB83727.1|(AL162753) conserved hypothetical protein [Neisseria meningitidis Z2491] Length = 548 Score = 371 bits (954), Expect = e−102 Identities = 189/197 (95%), Positives = 194/197 (97%) Query: 1 VKGESGVDIENWKNKLPEKEREPVEVILNRLEDSELTNKEQAEVISALHSIIPEYPYYHW 60 VKGESGVDIE+WKNKLPEKEREPVEVILNRLEDSELTNKEQAEVISALHSIIPEYPYYHW Sbjct: 350 VKGESGVDIEDWKNKLPEKEREPVEVILNRLEDSELTNKEQAEVISALHSIIPEYPYYHW 409 Query: 61 RHLHQDLHTACNDFYNEKKDYLSAAIEAVKVFEDKVQKQTGLHSIDGRELIEKAFGSKKS 120 RHLHQDLHTACNDFYNEKKDYLSAAIEAVKVFEDKVQKQTGLHSIDGRELIE+AFGSK S Sbjct: 410 RHLHQDLHTACNDFYNEKKDYLSAAIEAVKVFEDKVQKQTGLHSIDGRELIEQAFGSKKS 469 Query: 121 MLLLTNNKTQAEQNLEDGLEQLACGTWTGFRNPVQHELRANLSPSIFNDKDALDLISLVS 180 +LLLTNNKT+AEQNLEDGLEQLACGTWTGFRNPVQHELRANLSPSIFNDKDALDLISLVS Sbjct: 470 ILLLTNNKTKAEQNLEDGLEQLACGTWTGFRNPVQHELRANLSPSIFNDKDALDLISLVS 529 Query: 181 YLLRKVEQTKKRAKPTS 197 YLLRKVEQTKKR+K S Sbjct: 530 YLLRKVEQTKKRSKVVS 546 >gi|10955124|ref|NP_059780.1|ymh [Agrobacterium tumefaciens] gi|5738274|gb|AAB91582.2|(AF242881) ymh [Agrobacterium tumefaciens] Length = 266 Score = 58.7 bits (141), Expect = 5e−08 Identities = 40/127 (31%), Positives = 69/127 (53%), Gaps = 5/127 (3%) Query: 61 RHLHQDLHTACNDFYNEKKDYLSAAIEAVKVFEDKVQKQTGLHSIDGRELIEKAFGSKKS 120 R +H D+ C + +Y A +EAVK DK++++TGL + DG L+++AF Sbjct: 137 RGVHPDVLRFCREEL-LVDNYFHAVLEAVKSVADKIRQTGL-TDDGAVLVDRAFSGDAP 194 Query: 121 MLLLTNNTQAEQNEDGLEQLACGTWTGFRNPVQHELRANLSPSIFNDKDALDLISLVS 180 ML + ++++E+ + G L GT++ FRN H R + S +DA DL S+ S Sbjct: 195 MLAINELQSESEKGEQRGFSNLVKGTFSMFRNTTAHAPRIHWQMS---KEDAEDLFSMFS 251 Query: 181 YLLRKVE 187 + R+++ Sbjct: 252 LMHRRID 258 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, NGS50 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 51 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 101> which encodes amino acid sequence <SEQ ID 102; NGS51>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 0.14 Possible cleavage site: 42 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 5.67 threshold: 0.0 PERIPHERAL Likelihood = 5.67 modified ALOM score: −1.63 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.145(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir||G81959 conserved hypothetical protein NMA0428 [imported]-Neisseria meningitidis (group A strain Z2491) emb|CAB83727.1|(AL162753) conserved hypothetical protein [Neisseria meningitidis Z2491] Length = 548 Score = 532 bits (1371), Expect = e−150 Identities = 272/285 (95%), Positives = 280/285 (97%) Query: 1 MSEEKLKMSFEPTVIEHLGVKMYSHTVPAIAELIANAYDACATEVEVRLFDKPEHKIVIK 60 MSEEKLKMSFEPTVIEHLGVKMYSHTVPAIAELIANAYDACATEVEVRLFDKPEHKIVIK Sbjct: 1 MSEEKLKMSFEPTVIEHLGVKMYSHTVPAIAELIANAYDACATEVEVRLFDKPEHKIVIK 60 Query: 61 DNGIGMSFDEINDFYLRIGRNRREEKQASPCGRIPTGKKGLGKLALFRLGNKIEISTIQG 120 DNGIGMSFDEINDFYLRIGRNRREEKQASPCGRIPTGKKGLGKLALF LGNKIEISTIQG Sbjct: 61 DNGIGMSFDEINDFYLRIGRNRREEKQASPCGRIPTGKKGLGKLALFRLGNKIEISTIQG 120 Query: 121 NERVTFTLDYAEIKKSERIYQPEFQKESVKPNTENGTTITLTELTKKQGYPLDNYVGHLS 180 NERVTFTLDYAEI++S+ IYQPEF+KESV+ N E+GTTITLTELTKKQGYPLDNYV HLS Sbjct: 121 NERVTFTIDYAEIRRSKGIYQPEFRKESVESNIESGTTITLTELTKKQGYPLDNYVEHLS 180 Query: 181 RLFDFPAQDFKIKVSLNGSEPRIIDGNLKYNLVTPQFEWEYQDLATNISSLSSKFEQYEY 240 RLFDFPAQDFKIKVSLNGSEP+IIDGNLKY+LVTPQFEWEYQDLATNISSLSSKFEQYEY Sbjct: 181 RLFDFPAQDFKIKVSLNGSEPKIIDGNLKYDLVTPQFEWEYQDLATNISSLSSKFEQYEY 240 Query: 241 SGLIQGKFITTEKPLKNNMKGITLFANGRMVNMPEFFTDSESSHF 285 SGLIQGKFITTEKPLKNNMKGITLFANGRMVNMPEFFTDSESSHF Sbjct: 241 SGLIQGKFITTEKPLKNNMKGITLFANGRMVNMPEFFTDSESSHF 285 >emb|CAC22276.1|(AJ302030) putative heat shock protein [Listeria monocytogenes] Length = 181 Score = 70.2 bits (171), Expect = 2e−11 Identities = 57/173 (32%), Positives = 90/173 (51%) , Gaps = 10/173 (5%) Query: 1 MSEEKLKMSFEPTVIEHLGVKMYSHTVPAIAELIANAYDACATEVEVRLFDKPEHKIVIK 60 MSE++ + +P ++E LG +Y++ + ELIANAYDA A V V E+K++++ Sbjct: 1 MSEKEYNLDIDPRILELLGPHLYTNIYYILGELIANAYDADAKNVYVIDRIDEENKLIVE 60 Query: 61 DNGIGMSFD--EINDFYLRIGRNRREEKQASPC---GRIPTGKKGLGKLALFRLGNKIEI 115 D+G GMS++ ++ +F L + + R S R G+KG+GKLA + + I Sbjct: 61 DDGSGMSYENKDVKNF-LSVAKESRTNAINSYTKLNNRRKMGRKGVGKLASLSVSENVNI 119 Query: 116 STIQGNERVFTLDYAEI-KKSERIYQPEFQKESVKPNTENGTTITLTELTKK 167 TI+ E+ F L I KK E I + +K +GT I +T T K Sbjct: 120 KTIKDGEKSGFVLSRKVINKKLEAINEDTISFIKIK---NHGTAIEMTNPTYK 169 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, NGS51 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 52 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 103> which encodes amino acid sequence <SEQ ID 104; NGS52>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.5 Possible cleavage site: 49 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 7.64 threshold: 0.0 PERIPHERAL Likelihood = 7.64 modified ALOM score: −2.03 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.213(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: 2.1.1.73) NMA0427 [imported]-Neisseria meningitidis (group A strain Z2491) gi|7379177|emb|CAB83726.1|(AL162753) modification methylase (cytosine- specific DNA methylase) [Neisseria meningitidis Z2491] Length = 351 Score = 310 bits (794), Expect = 8e−84 Identities = 152/154 (98%), Positives = 153/154 (98%) Query: 1 LGMENGFPKIMAGHQDETDFMHSCAGLSDINLKRLALIPKNGGNRLAFAHIPELQLECFI 60 LGMENGFPKI+AGHQDETDFMHSCAGLSDINLKRLALIPKNGGNRLAFAHIPELQLECFI Sbjct: 198 LGMENGFPKIIAGHQDETDFMHSCAGLSDINLKRLALIPKNGGNRLAFAHIPELQLECFI 257 Query: 61 GKDNSFKDTFGRLWWDKPAPTITTKFFSISNGRAFAHPEEDRALSLREGATLQSFPRNYVF 120 GKDNSFKDTFGRLWWDKPAPTITTKFFSISNGRAFAHPEEDRALSLREGATLQSFPRNYVF Sbjct: 258 GKDNSFKDTFGRLWWDKPAPTITTKFFSISNGRAFAHPEEDRALSLREGATLQSFPRNYVF 317 Query: 121 KAGSRDKIARLIGNAVPPMYTEKIGRAIVDNIEC 154 KAGSRDKIARLIGNAVPPMY EKIGRAIVDNIEC Sbjct: 318 KAGSRDKIARLIGNAVPPMYAEKIGRAIVDNIEC 351 >gi|1274411|sp|P25265|MTD2_HERAU MODIFICATION METHYLASE HGIDII (CYTOSINE-SPECIFIC METHYLTRANSFERASE HGIDII) (M.HGIDII) gi|538661|pir||JT0594 site-specific DNA-methyltransferase (cytosine-specific) (EC 2.1.1.73)-Herpetosiphon aurantiacus gi|487731|emb|CAA38941.1|(X55141) methyltransferase [Herpetosiphon aurantiacus] Length = 354 Score = 95.6 bits (237), Expect = 3e−19 Identities = 62/142 (43%), Positives = 82/142 (57%), Gaps = 9/142 (6%) Query: 12 AGHQDETDFM HSCAGLSDINLKRLALIPKNGGNRLAFAHIP-ELQLECFIGKD-NSFKDT 69 +G E D MH+ + L DINL+R+ G +A P EL EC + S+ Sbjct: 200 SGGHWEGDSMHAASRLEDINLRRIQHSVPGG----TWADWPEELIAECHKKESGESYGSV 255 Query: 70 FGRLWWDKPAPTITTKFFSISNGEFAHPEEDRALSLREGATLQSFPRNYVFKAGSRDK-- 127 +GR+ WDK APTITT+ NGRF HPE+DRA+SLRE A LQ+FPR+Y F + K Sbjct: 256 YGRMEWDKVAPTITTQCNGYGNGRFGHPEQDRAISLRALLQTFPRSYQFAPEGQLKFK 315 Query: 128 -IARLIGNAVPPMYTEKIGRAI 148 ++R IGNAVP I ++I Sbjct: 316 TVSRQIGNAVPVALGRVIAKSI 337 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, NGS52 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 53 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 105> which encodes amino acid sequence <SEQ ID 106; NGS53>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −7.56 Possible cleavage site: 31 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 5.36 threshold: 0.0 PERIPHERAL Likelihood = 5.36 modified ALOM score: −1.57 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.189(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|11256915|pir||F81959 site-specific DNA-methyltransferase (cytosine- specific) (EC2.1.1.73) NMA0427 [imported]-Neisseria meningitidis (group A strain Z2491) gi|7379177|emb|CAB83726.1|(AL162753) modification methylase (cytosine- specific DNA methylase) [Neisseria meningitidis Z2491] Length = 351 Score = 247 bits (606), Expect = 5e−65 Identities = 124/149 (83%), Positives = 127/149 (85%) Query: 1 LQPETLEKELGLKKNDDDLILIGCSPCQYWSVIQTDKRKSEKSKSLLLEFQRFVEYFNPG 60 LQPETLEKELGLKKNDDDLILIGCSPCQYWSVIQTDKRKSEKSKSLLLEFQRFVEYFNPG Sbjct: 59 LQPETLEKELGLKKNDDDLILIGCSPCQYWSVIQTDKRKSEKSKSLLLEFQRFVEYFNPG 118 Query: 61 YVVVENVPGILSRMKESGLDNFIKLLEEKGFTVHFGIHNTADYGIPQSRKRFTLIANRIT 120 YVVVENVPGILSRMKESGLDNFIKLLEEKGFTVHFGIHNTADYGIPQSRKRFTLIANRIT Sbjct: 119 YVVVENVPGILSRMKESGLDNFIKLLEEKGFTVHFGIHNTADYGIPQSRKRFTLIANRIT 178 Query: 121 KKSWNQSSIRANGLRYAMFWEWKTAFPKL 149 K+ L + FPK+ Sbjct: 179 KEKLEPVKYSGKRLTVRDVLGMENGFPKI 207 >gi|127441|sp|P25265|MTD2_HERAU MODIFICATION METHYLASE HGIDII (CYTOSINE- SPECIFIC METHYLTRANSFERASE HGIDII) (M.HGIDII) gi|538661|pir||JT0594 site-specific DNA-methyltransferase (cytosine- specific) (EC 2.1.1.73)-Herpetosiphon aurantiacus gi|48773|emb|CAA38941.1|(X55141) methyltransferase [Herpetosiphon aurantiacus] Length = 354 Score = 71.9 bits (169), Expect = 4e−12 Identities = 39/105 (37%), Positives = 57/105 (54%), Gaps = 1/105 (0%) Query: 12 LKKNDDDLILIGCSPCQYWSVIQTDKRKSEKSKSLLLEFQRFVEYFNPGYVVVENVPGIL 71 L N+IL+GC+PCQ +S T K ++ LL EF R + P + +ENVP + Sbjct: 64 LYPNNQHKILVGCAPCQDFSQY-TKKSRTGTKWQLLTEFSRLIREIEPDIISMENVPEVR 122 Query: 72 SRMKESGLDNFIKLLEEKGFTHFGIHNTADYGIPQSSRKRFTLIA 116 + + +NFI+ LE+ G+ V + + DYGIPQ R R L A Sbjct: 123 TFNRGEVFNFIQSLEQLGYWSHSVVHCPDYGIPQQRDRLVLFA 167 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, NGS53 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 54 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 107> which encodes amino acid sequence <SEQ ID 108; NGS54>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −6.82 Possible cleavage site: 50 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 6.89 threshold: 0.0 PERIPHERAL Likelihood = 6.89 modified ALOM score: −1.88 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.253(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: gi|1074456|pir||D64155 hypothetical protein HI0597-Haemophilus influenzae (strain Rd KW20) gi|1573586|gb|AAC22254.1|(U32741) conserved hypothetical protein [Haemophilus influenzae Rd] Length = 272 Score = 188 bits (459), Expect = 2e−47 Identities = 95/100 (95%), Positives = 97/100 (97%) Query: 1 MNLPFRAMVSDLGGTLLTPEHLVGDLTIDTLRVLEQKGVDIILATGRNHTDMSSILGKIG 60 MNLPFRAMVSDL GTLLTPEHLVGDLTIDTLR LEQKGVDIILATGRNHTD+SSILGKIG Sbjct: 1 MNLPFRAMVSDLDGTLLTPEHLVGDLTIDTLRALEQKGVDIILATGRNHTDVSSILGKIG 60 Query: 61 AERAVMITSNGARVRDLQGNLLYSNSLPEELVLELYKTSY 100 AERAVMITSNGARVRDLQGNLLYSNSLPEELVLELYKT + Sbjct: 61 AERAVMITSNGARVRDLQGNLLYSNSLPEELVLELYKTPF 100 A homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS54 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 55 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 109> which encodes amino acid sequence <SEQ ID 110; NGS55>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.46 Possible cleavage site: 37 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 3.02 threshold: 0.0 PERIPHERAL Likelihood = 3.02 modified ALOM score: −1.10 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.311(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: aeruginosa (strain PAO1) gi|9948791|gb|AAG06104.1|AEOO4699_9 (AE004699) probable FMN oxidoreductase [Pseudomonas aeruginosa] Length = 411 Score = 279 bits (686), Expect = 2e−74 Identities = 157/375 (41%), Positives 228/375 (59%), Gaps 10/375 (2%) Query: 1 MEEQLAQNDQ-PSEKLVRLYGAWAEGGAGVLVTGNVMVAESGKGSINDVLISDDRALKLM 59 MEE +4 Q PSE4+L+RLY AWA+GGAG+L++GNVMV V++ DD LE Sbjct: 24 MEENMADAAQAPSERLMRLYQAWADGGAGLLISGNVMVDSRAMTGPGGVVLEDDAQLEKF 83 Query: 60 KKWAKARTQNDTLLIMQINKAGKQSPAVKTPLAPSAVPL--GMNGFINPPRELSADE 117 ++WA+ +QINH G+Q A + + APSAVPL GM+ P+ + Sbjct: 84 RRWARIGRSAGAGFWLGINHPGRQMQANLGQQAWAPSAVPLELGGMSRHFATPKAMDEAM 143 Query: 118 INGLIQQFVQTAKIAEQAGFSGVQIYAVHGYLISQFLSPHHNRRQDQWGGSLENRMRFLL 177 I +IQ+F ++ +AE+AGFSGV+I+A HGYL+SQFLSP NRR D WGGSLENR R LL Sbjct: 144 IAEVIQRFARSAGLAERAGFSGVEIHHGYLLSQFLSPLSNRRSDAWGGSLENRARLLL 203 Query: 178 ETYTAIRAAGKDFLVGVXLNSADFQKGGFDESESVQVVQKLSEMGIDFIEVSGGNYESP 237 E A+RA F V VKLNSADFQ+GGF ++ +VV+ L +G+D +E+SGG+YE+P Sbjct: 204 EIVRAVRAEVAPGFAVAVKLNSADFQRGGFSADDAREVVRMLDGLGVDLVELSGGSYEAP 263 Query: 238 QMLA-AKDS-TRKREAFFIDYAEKARAASQAPLIITGGFRSQTAMEDALSSGHLDLVGIA 295 M A+D T REA+F+++A RAA++ P+++TGG R + E L+SG +D+VGI Sbjct: 264 AMQGEARDGRTLAREAYFVEFARDIRAAARMPVMVTGGIRRRVAEQVLASG-VDMVGIG 322 Query: 296 RPFALVPDLANKMQNRTYQTVQADRIQTGVDKKAGAMLEMNWYMTQMDLIGQGKQSN 355 A+ P+L + Q I + +K ++ M Q+ + +G+ +N Sbject: 323 TALAIEPNLPRDWRAGKDSAPQLRPI----TWENKPLASLANMAVKFQLRKLSRGRATN 378 Query: 356 PKIVGVESIAENFAG 370 P++ ++ AG Sbjct: 379 PRVSPLCALLAQQAG 393 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 56 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 111> which encodes amino acid sequence <SEQ ID 112; NGS56>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.69 Possible cleavage site: 54 >>> Seems to have an uncleavable N-term signal seq Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 4.29 threshold: 0.0 PERIPHERAL Likelihood = 4.29 modified ALOM score: −1.36 *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.042(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|7444004|pir||D70029 transcription regulator ArsR family homolog yvbA- Bacillus subtilis gi|2635892|emb|CAB15384.1|(Z99121) similar to transcriptional regulator (ArsR family) [Bacillus subtilis] Length = 90 Score = 51.3 bits (118), Expect = 3e−06 Identities = 24/65 (36%), Positives = 42/65 (63%), Gaps = 1/65 (1%) Query: 15 IFTVLSDENRHQILHVLWKHGRMNVNELTEHLHLSRPAVSHHLKIMLQAGAVAVEQVGKE 74 +F +SD R +IL +L K G M ++ EH ++S+P++SHHL I+QA ++ + G+ Sbjct: 4 VFKAISDPTRRKILDLL-KGGDMTAGDIAEHFNISHPSISHHLNILKQAEVISDHRKGQF 62 Query: 75 RFYSI 79 +YS+ Sbjct: 63 IYYSL 67 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 57 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 113> which encodes amino acid sequence <SEQ ID 114; NGS57>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −2.57 Possible cleavage site: 55 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 5.67 threshold: 0.0 PERIPHERAL Likelihood = 5.67 modified ALOM score: −1.63 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.160(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|10444407|gb|AAG17897.1|AF297971_1 (AF297971) restriction endonuclease R.NgoMIII [Neisseria gonorrhoeae] Length = 213 Score = 319 bits (818), Expect = 1e−86 Identities = 156/156 (100%), Positives = 156/156 (100%) Query: 1 LYKQYADWNRLSYNAPIYVGKAVPKGWRQARNSDNALNQSTELFHRLKEHSRSIAAVSDL 60 LYKQYADWNRLSYNAPIYVGKAVPKGWRQARNSDNALNQSTELFHRLKEHSRSIAAVSDL Sbjct: 58 LYKQYADWNRLSYNAPIYVGKAVPKGWRQARNSDNALNQSTELFHRLKEHSRSIAAVSDL 117 Query: 61 DPSDFMCRFVIFEGAGSDMIGTIEAALIKLHKPLWNSCDGFGNHDPGKGRYEQAKSDWD 120 DPSDFMCRFVIFEGAGSDMIGTIEAALIKLHKPLWNSCDGFGNHDPGKGRYEQAKSDWD Sbjct: 118 DPSDFMCRFVIFEGAGSDMIGTIEAALIKLHKPLWNSCDGFGNHDPGKGRYEQAKSDWD 177 Query: 121 VLHSGRVWADRLNGIPNSYESILENINTHLEIIKRK 156 VLHSGRVWADRLNGIPNSYESILENINTHLEIIKRK Sbjct: 178 VLHSGRVWADRLNGIPNSYESILENINTHLEIIKRK 213 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 58 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 115> which encodes amino acid sequence <SEQ ID 116; NGS58>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −1.92 Possible cleavage site: 16 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 5.41 threshold: 0.0 PERIPHERAL Likelihood = 5.41 modified ALOM score: −1.58 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.107(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|10444408|gb|AAG17898.1|AF29797_2 (AF297971) DNA cytosine methyltransferase M.NgoMIII [Neisseria gonorrhoeae] Length = 377 Score = 759 bits (1960), Expect = 0.0 Identities = 377/377 (100%), Positives = 377/377 (100%) Query: 1 MKSLEIFSGAGGLAKGLELAGFQHASFIELNKDACNSLRSNFNPKLVYQGDVADFDLSSQ 60 MKSLEIFSGAGGLAKGLELAGFQHASFIELNKDACNSLRSNFNPKLVYQGDVADFDLSSQ Sbjct: 1 MKSLEIFSGAGGLAKGLELAGFQHASFIELNKDACNSLRSNFNPKLVYQGDVADFDLSSQ 60 Query: 61 EGIEVIAGGPPCQPFSLGGKHLAHEDRRDMFPHAVRYVEYYRPKAFIFENVKGLLRKSFA 120 EGIEVIAGGPPCQPFSLGGKHLAHEDRRDMFPHAVRYVEYYRPKAFIFENVKGLLRKSFA Sbjct: 61 EGIEVIAGGPPCQPFSLGGKHLAHEDRRDMFPHAVRYVEYYRPKAFIFENVKGLLRKSFA 120 Query: 121 DYFEYILLRLTYPNLGILQNEDWKGHLTRLKEIEFNLYKGIKYKVSYQLLNAADYGVPQK 180 DYFEYILLRLTYPNLGILQNEDWKGHLTRLKEIEFNLYKGIKYKVSYQLLNAADYGVPQK Sbjct: 121 DYFEYILLRLTYPNLGILQNEDWKGHLTRLKEIEFNLYKGIKYKVSYQLLNAADYGVPQK 180 Query: 181 RERVVIVGIRADLDIDWKFPKRTHSEDRLNWEKYVTGEYWEKHNEPKRFNKDIAEKLQKK 240 RERVVIVGIRADLDIDWKFPKRTHSEDRLNWEKYVTGEYWEKHNEPKRFNKDIAEKLQKK Sbjct: 181 RERVVIVGIRADLDIDWKFPKRTHSEDRLNWEKYVTGEYWEKHNEPKRFNKDIAEKLQKK 240 Query: 241 YGIFEPEKKPWQTVRDTLSDIPHPLGNHKITGHEYRDGARIYPGHTGSGIDEPSKTIKAG 300 YGIFEPEKKPWQTVRDTLSDIPHPLGNHKITGHEYRDGARIYPGHTGSGIDEPSKTIKAG Sbjct: 241 YGIFEPEKKPWQTVRDTLSDIPHPLGNHKITGHEYRDGARIYPGHTGSGIDEPSKTIKAG 300 Query: 301 GHGVPGGENMIRYDDGTVRYFTSYEAKLLQTFPEEFVISGAWGEAMRQIGNAVPVKLSEI 360 GHGVPGGENMIRYDDGTVRYFTSYEAKLLQTFPEEFVISGAWGEAMRQIGNAVPVKLSEI Sbjct: 301 GHGVPGGENMIRYDDGTVRYFTSYEAKLLQTFPEEFVISGAWGEAMRQIGNAVPVKLSEI 360 Query: 361 LGKHLMGVLSEKSSLHN 377 LGKHLMGVLSEKSSLHN Sbjct: 361 LGKHLMGVLSEKSSLHN 377 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 59 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 117> which encodes amino acid sequence <SEQ ID 118; NGS59>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.82 Possible cleavage site: 60 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 2.86 threshold: 0.0 PERIPHERAL Likelihood = 2.86 modified ALOM score: −1.07 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.197(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|11353338|pir||F81882 hypothetical protein NMA1155 [imported] Neisseria meningitidis (group A strain Z2491) gi|7379848|emb|CABB4417.1|(AL162755) hypothetical protein NMA1155 [Neisseria meningitidis Z2491] Length = 120 Score = 131 bits (329), Expect = 2e−30 Identities = 64/68 (94%), Positives = 67/68 (98%) Query: 1 LSDISASRAAYMDVQKQYPFETVAVCVLPNHIHAIWTLPPDDADYSLLRRLIKTKFSAYS 60 +S+ISASRAAYMDVQKQYPFETVAVCVLPNHIHAIWTLPPDDADYSLLRRLIKTKFSAYS Sbjct: 1 MSNISASRAAYMDVQKQYPFETVAVCVLPNHIHAIWTLPPDDADYSLLRRLIKTKFSAYS 60 Query: 61 PHTKNLGA 68 P+TKNL A Sbjct: 61 PYTKNLSA 68 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 60 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 119> which encodes amino acid sequence <SEQ ID 120; NGS60>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.14 Possible cleavage site: 16 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 2.76 threshold: 0.0 PERIPHERAL Likelihood = 2.76 modified ALOM score: −1.05 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.330(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|11281269|pir||D81804 hypothetical protein NMA1789 [imported]-Neisseria meningitidis (group A strain Z2491) gi|7380430|emb|CAB85016.1|(AL162757) hypothetical protein [Neisseria meningitidis Z2491] Length = 243 Score = 154 bits (389), Expect 5e−37 Identities = 82/85 (96%), Positives = 82/85 (96%) Query: 12 MNTKTELQKLLEEDISTLKETLIRVDALPPRYVRSIATPIVRRWLIDKQLNILAKEIGLT 71 MNTKTELQKLLEEDISTL ETLI DALPPRYVRSIATPIVRRWLIDKQLNILAKEIGLT Sbjct: 1 MNTKTELQKLLEEDISTLTETLICADALPPRYVRSIATPIVRRWLIDKQLNILAKEIGLT 60 Query: 72 IELPILDTSLVFEKLSTLENKVNFY 96 IELPILDTSLVFEKLSTLENKVNFY Sbjct: 61 IELPILDTSLVFEKLSTLENKVNFY 85 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, NGS60 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 61 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 121> which encodes amino acid sequence <SEQ ID 122; NGS61>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −6.88 Possible cleavage site: 32 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 5.52 threshold: 0.0 PERIPHERAL Likelihood = 5.52 modified ALOM score: −1.60 Rule: cytoplasmic protein *** Reasoning step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.300(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|11281269|pir||D81804 hypothetical protein NMA1789 [imported]-Neisseria meningitidis (group A strain Z2491) gi|7380430|emb|CAB5016.1|(AL162757) hypothetical protein [Neisseria meningitidis Z2491] Length = 243 Score = 193 bits (491), Expect = 5e−49 Identities = 96/101 (95%), Positives = 97/101 (95%) Query: 1 MAGGVYLGGKIISPIYHSSQEFSGEPIIYAETNIILCPAEKFLTLKRVFHNGNIFNMNQI 60 MAGGVYLGG+ IS IYHSSQEFSGEPIIYAETNIILCPAEKFLTLKRVFHNGNIFNMNQI Sbjct: 86 MAGGVYLGGEFISSIYHSSQEFSGEPIIYAEPNIILCPAEKFLTLKRVFHNGNIFNMNQI 145 Query: 61 ITFLSNKQGGVRFDKNYDKYKTWQVAIEKAANFLKLGNPYN 101 ITFLSNKQGGV FDKNYDKYKTWQVAIEKAANFLKLGNPYN Sbjct: 146 ITFLSNKQGGVHFDKNYDKYKTWQVAIEKAANFLKLGNPYN 186) As a homolog (amino acids 1-96) was found in serogroup A N.meningitidis but not in serogroup B, NGS61 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 62 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 123> which encodes amino acid sequence <SEQ ID 124; NGS62>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −2.43 Possible cleavage site: 44 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 4.72 threshold: 0.0 PERIPHERAL Likelihood = 4.72 modified ALOM score: −1.44 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.324(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> A homolog was found in serogroup A N.meninigitidis but not in serogroup B, so NGS62 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 63 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 125> which encodes amino acid sequence <SEQ ID 126; NGS63>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 0.74 Possible cleavage site: 24 >>> Seems to have a cleavable N-term signal seq.","Amino Acid Composition of predicted Mature Form: calculated from 25 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 10.72 threshold: 0.0 PERIPHERAL Likelihood = 10.72 modified ALOM score: −2.64 Score for OM-PP discrimination: −22.14 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: −22.14 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Periplasmic space?","Score: 2.21378 Periplasmic space?","Score: 2.21378 ----- Final Results ----- bacterial periplasmic space --- Certainty= 0.931(Affirmative) < succ> bacterial outer membrane --- Certainty= 0.237(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|11284146|pir||B81142 hypothetical protein NMB0912 [imported]-Neisseria meningitidis (group B strain MD5B) gi|7226150|gb|AAF41320.1|(AE002443) hypothetical protein [Neisseria meningitidis MC58] Length = 208 Score = 51.7 bits (119), expect = 3e−06 Identities = 30/72 (41%), Positives = 40/72 (54%) Query: 5 LLKNWKPLLILSAIAFFAVSWQLDRAAQYRRGYGAAVSEVSERLKAAAVEHAEHARKSSA 64 LLK WKP+ +L I +W DRA +YR G AA +E+S RLK +E A+ AR + Sbjct: 43 LLKYWKPVGVLLLIVLIFTAWHFDRAEKYRMGREAAAAEISNRLKDGYIEQAKQARSAEQ 102 Query: 65 AYQAQKAAEEEK 76 A A R+ K Sbjct: 103 KAAAAPAERQTK 114 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 64 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 127> which encodes amino acid sequence <SEQ ID 128; NGS64>.","Analysis of this protein sequence reveals the following: McG: Examining signal sequence (McGeoch) Length of UR: 0 Peak Value of UR: 2.99 Net Charge of CR: 4 Discriminant Score: 5.35 GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −2.53 Possible cleavage site: 33 >>> Seems to have an uncleavable N-term signal seq Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 3.23 threshold: 0.0 PERIPHERAL Likelihood = 3.23 modified ALOM score: −1.15 *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.054(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has no homology with sequences in the databases.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 65 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 129> which encodes amino acid sequence <SEQ ID 130; NGS65>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.61 Possible cleavage site: 61 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 3.34 threshold: 0.0 PERIPHERAL Likelihood = 3.34 modified ALOM score: −1.17 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.236(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|1175791|sp|P44189|YE18_HAEIN HYPOTHETICAL PROTEIN HI1418 gi|1074769|pir||A64029 hypothetica1 protein HI1418-Haemophilus influenzae (strain Rd KW20) gi|1574254|gb|AAC23068.1|(U32821) H. influenzae predicted coding region HI1418 [Haemophilus influenzae Rd] Length = 201 Score = 104 bits (251), Expect = 1e−21 Identities = 58/119 (48%), Positives = 72/119 (59%), Gaps = 4/119 (3%) Query: 51 LKMQNTISFSFKSQNRTQI-LGAEPFCLGDVAEILQIQNAR---QLPLKDQGIQKSS 106 +K Q S F+FK VR + E WFC DV IL N+R Q K G+ K Sbjct: 14 MKNQIQFSTFFKDLPVRTILDPKGEFFCGTDVCHILGYTNSRKALQDHCKQGGVTKRY 73 Query: 107 VATKKGNQELLFINEPNLYRVIFRSRKAEAVFQDWIFEEVIPQIRKTGGYQITPKTTA 165 TK +QE+ FINEPNLYR+I +SRK EA F+ W+FEEV+PQIRKTG YQ+ P+ A Sbjct: 74 TPTKSADQEMTFINEPNLYRLIIKSRKPEAEPFEAWVFEEVLPQIRKTGKYQLQPQQLA 132 >gi|11281012|pir||A81144 hypothetical protein NMB0900 [imported]-Neisseria meningitidis (group B strain MD58) gi|7226137|gb|AAF41308.1|(AE002442) hypothetical protein [Neisseria meningitidis MC58] Length = 305 Score = 104 bits (249), Expect = 2e−21 Identities = 73/137 (53%), Positives = 93/137 (67%), Gaps = 2/137 (1%) Query: 190 YSMIHQRFNVEAVEGIPADKLPEAVAYHALTLHTG-LAGEVPDREPLPAPQPALPISGN 248 +S + +F E +PA++ PE ++ + + + G L GEV DREPLPAPQPALPISGN Sbjct: 164 WSAVKSKFGCSYKE-VPAEQFPELSMGRVAVENGVLYGEVLDREPLPAPQPALPISGN 222 Query: 249 ALADIAAMVYYGTRHIELGKDVSAPLKQLGCKQAVTMWTVWHETRSILKRSVAALEVLRG 308 AL D+A V YG I++G+DVS PLKQLGCRQAVTMWTVW ETRS LK + ALE L Sbjct: 223 ALYDLAVAVRYGAWAIQMGRDVSLPLKQLGCKQAVTMWTVWAETRSRLKAAANALEALNA 282 Query: 309 YADKDASGRIAACLEGI 325 +AD + + +I L I Sbjct: 283 HADAEHAAKIRPMLPEI 299 >gi|7460273|pir||T13267 hypothetical protein-Lactococcus lactis phage BK5-T gi|928839|gb|AAA98590.1|(L44593) ORF266; putative [Lactococcus phage BK5-T] Length = 266 Score = 75.9 bits (179), Expect = 6e−13 Identities = 42/111 (37%), Positives = 63/111 (55%), Gaps = 3/111 (2%) Query: 55 NTISVFSFKSQNVRTQILGAEPWFCLGDVAEILQIQNAR---QLPLKDQGIQKSSVATKK 111 N + F+F + VRT ++ EPWF DVA + +N R + +KD+ ++S + T Sbjct: 2 NELQNFNFNNLPVRTVLINDEPWFVGKDVAIAIGYKNFRDALKSHVKDKYKRESRITTPS 61 Query: 112 GNQELLFINEPNLYRVIFRSRKAEAVKFQDWIFEBVIPQIRKTGGYQITPK 162 G Q + I+EP LY++ S+ A FQDW++EEV+P IRK G Y K Sbjct: 62 GVQSVTVISEPGLYQLAGESKLPSAEPFQDWEELPTIRKHGAYMTDAK 112 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 66 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 131> which encodes amino acid sequence <SEQ ID 132; NGS66>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.12 Possible cleavage site: 53 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 8.96 threshold: 0.0 PERIPHERAL Likelihood = 8.96 modified ALOM score: −2.29 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.402(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has no homology with sequences in the databases.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 67 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 133> which encodes amino acid sequence <SEQ ID 134; NGS67>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 5.71 Possible cleavage site: 22 >>> Seems to have a cleavable N-term signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 23 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 2.81 threshold: 0.0 PERIPHERAL Likelihood = 2.81 modified ALOM score: −1.06 Score for OM-PP discrimination: −32.34 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: −32.34 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Periplasmic space?","Score: 3.23391 Periplasmic space?","Score: 3.23391 ----- Final Results ----- bacterial periplasmic space --- Certainty= 0.928(Affirmative) < succ> bacterial outer membrane --- Certainty= 0.199(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|7475078|pir||H69834 hypothetical protein yhjQ-Bacillus subtilis gi|2226189|emb|CAA74479.1|(Y14081) hypothetical protein [Bacillus subtilis] gi|2633396|emb|CAB12900.1|(Z99109) yhjQ [Bacillus subtilis] Length = 108 Score = 32.9 bits (74), Expect = 2.1 Identities = 27/98 (27%), Positives = 44/98 (44%), Gaps = 4/98 (4%) Query: 54 CLDAGQVCLTHCLSLLTQGDTSMSDCAVAVRQMLALCGAVHDLAAQNSPLTRDAAKVCLE 113 C+ A C T CL Q +S C R+ +C +SP ++ +C+ Sbjct: 15 CMKACNHCFTKCLEESVQ--HHLSGCIRLDRECADICALAVKAMQTDSPFMKEICALCAD 72 Query: 114 ACKQCAKACKEHSAHHAECKACYESCLDCIKECEKLAA 151 C+ C C +H H C+AC ++C C ++C +AA Sbjct: 73 ICEACGTECGKHD--HDHCQACAKACFTCAEQCRSMAA 108 >gi|7479923|pir||T36241 hypothetical protein SCE39.31c-Streptomyces coelicolor gi|4582392|emb|CAB40339.1|(AL049573) hypothetical protein [Streptomyces coelicolor A3(2)] Length = 136 Score = 30.9 bits (69), Expect = 7.7 Identities = 27/102 (26%), Positives = 43/102 (41%), Gaps = 6/102 (5%) Query: 54 CLDAGQVCLTHCLSLLTQGDTSMSDCAVAVRQMLALCGAVHDLAAQ----NSPLTRDAAK 109 C A C CLS T D ++C +C A + ++ ++ +TR + Sbjct: 34 CAQACTACADACLSEPTVAD--LTKCIRTDMDCADVCTATAAVLSRHTGYDANVTRAVLQ 91 Query: 110 VCLEACKQCAKACKEHSAHHAECKACYESCLDCIKECEKLAA 151 C C C C H+ H C+ C E+C C + C++L A Sbjct: 92 ACATVCAACGDECARHAGMHEHCRVCAEACRSCEQACQELLA 133 The protein was expressed in E.coli as a soluble 14.19 kDa His-fusion product and then purified.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 68 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 135> which encodes amino acid sequence <SEQ ID 136; NGS68>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.05 Possible cleavage site: 38 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 5.25 threshold: 0.0 PERIPHERAL Likelihood = 5.25 modified ALOM score: −1.55 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.220(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|11353493|pir||A81795 hypothetical protein NMA2214 [imported]-Neisseria meningitidis (group A strain Z2491) gi|7380833|emb|CAB85425.1|(AL162758) hypothetical protein [Neisseria meningitidis Z2491] Length = 208 Score = 263 bits (673), Expect = 3e−69 Identities = 140/145 (96%), Positives = 143/145 (98%) Query: 1 LDWRGNKPLGAAELADLKPLYKDFMYWERGLHMYKASAVVPTGYVRVGNTAPLCGEDTQR 60 LDWRGNKPLGAAELADLKPLYKDFMYWERGLHMYKASAVVPTGYVRVGNTAPL GEDTQR Sbjct: 44 LDWQGNKPLGAAELADLKPLYKDFMYWERGLHMYKASAVVPTGYVRVGNTAPLVGEDTQR 103 Query 63 YASFWGDGYDVYRQLRWRQIPEKQRKAFKKAAKSKNTVMFAGREYGISKQNLSDVWDDFE 120 YASFWGDGYDVYRQLRW+QIPEKQRKAFKKAAKSK TVMFAGREYGISKQNLSDVWDDFE Sbjct: 104 YASFWGDGYDVYRQLRWQQIPEKQRKAFKKAAKSKKTVMFAGREYGISKQNLSDVWDDFE 163 Query: 121 DAMELAKAFPCLSSLFLTKWHKNLYE 145 DAMELAKAFPCLSSLFLTKWHKNLY+ Sbjct: 164 DAMELAKAFPCLSSLFLTKWHKNLYD 188 >gi|11280955|pir|B81219 hypothetical protein NMB0273 [imported]-Neisseria meningitidis (group B strain MD58) gi|7225497|gb|AAF40727.1|(AE002383) hypothetical protein [Neisseria meningitidis MC58] Length = 141 Score = 216 bits (550), Expect = 5e−55 Identities = 117/121 (96%), Positives = 119/121 (97%) Query: 25 MYWERGLHMYKASAVVPTGYVRVGNTAPLCGEDTQRYASFWGDGYDVYRQLRWRQIPEKQ 84 MYWERGLHMYKASAVVPTGYVRVGNTAP CGEDTQRYASFWGDGYDVYRQLRW+QIPEKQ Sbjct: 1 MYWERGLHMYKASAVVPTGYVRVGNTAPLVGEDTQRYASFWGDGYDVYRQLRWQQIPEKQ 60 Query: 85 RKAFKKAAKSKNTVMFAGREYGISKQNLSDVWDDFEDAMELKAFPCLSSLFLTKWHKNLY 144 RKAFKKAAKSK TVMFAGREYGISKQNLSDVWDDFEDAMELKAFPCLSSLFLTKWHKNLY Sbjct: 61 RKAFKKAAKSKKTVMFAGREYGISKQNLSDVWDDFEDAMELKAFPCLSSLFLTKWHKNLY 120 Query: 145 E 145 + Sbjct: 121 D 121 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 69 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 137> which encodes amino acid sequence <SEQ ID 138; NGS69>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.63 Possible cleavage site: 43 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 6.84 threshold: 0.0 PERIPHERAL Likelihood = 6.84 modified ALOM score: −1.87 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.361(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|7464552|pir||C64707 hypothetical protein HP1499-Helicobacter pylori (strain 26695) gi|2314683|gb|AAD08545.1|(AE000648) H. pylori predicted coding region HP1499 [Helicobacter pylori 26695] Length = 272 Score = 38.2 bits (88), Expect = 0.13 Identities = 34/123 (27%), Positives = 58/123 (46%), Gaps = 10/123 (8%) Query: 3 EFKPIFGQDFGLSKKEAIRKVLKWLPSHLKFTLMVAQGIQG------FHPKAVFWKNDKN 56 EF+ I G DF + ++IR +L ++ K + FHPK + N K Sbjct: 54 EFEIIVGLDFKTTDSKSIRFLLDLNKTYKKLRFYCYGDKENNKTDIVFHPKIYMFDNGK- 112 Query: 57 EYYALIGSSNLTHAAFNSNYEAN-ILTKISEQDFIKVKSWADEI--AMKSIPVSEDWLEE 113 E ++IGS+NLT +N+E N I T+ + ++ + + I A +E++L+ Sbjct: 113 EKTSIIGSTNLTKGGLENNFEVNTIFTEKKPLYYTQLNAIYNSIKYADSLFTPNEEYLQN 172 Query: 114 YQE 116 Y E Sbjct: 173 YNE 175 A homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS69 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 70 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 139> which encodes amino acid sequence <SEQ ID 140; NGS70>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −1.18 Possible cleavage site: 22 >>> Seems to have a cleavable N-term signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 23 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 4.19 threshold: 0.0 PERIPHERAL Likelihood = 4.19 modified ALOM score: −1.34 Score for OM-PP discrimination: −5.89 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: −5.89 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Periplasmic space?","Score: 0.588927 Periplasmic space?","Score: 0.588927 ----- Final Results ----- bacterial periplasmic space --- Certainty= 0.849(Affirmative) < succ> bacterial outer membrane --- Certainty= 0.106(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|11353344|pir||A81886 hypothetical protein NMAl183 [imported]-Neisseria meningitidis (group A strain Z2491) gi|7379875|emb|CAB84445.1|(AL162755) hypothetical protein NMA1183 [Neisseria meningitidis Z2491] Length = 74 Score = 58.7 bits (141), Expect = 2e−08 Identities = 30/43 (69%), Positives = 32/43 (73%) Query: 62 IGGFGGVGGFGGLKPALVYRNFRIIATNRPAATRARPRQTTVA 104 +G|||G+|G|GGLKP|LVY|N||IIATNRPAATRA|PR|TTVA Sbjct: 32 MGNIDGIDGSGGLKPTLVYWNHCIIATNRPAATRAHPRHTTVA 74 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 71 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 141> which encodes amino acid sequence <SEQ ID 142; NGS71>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −1.98 Possible cleavage site: 28 >>> Seems to have a cleavable N-term signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 29 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 5.04 threshold: 0.0 PERIPHERAL Likelihood = 5.04 modified ALOM score: −1.51 Score for OM-PP discrimination: −9.17 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: −9.17 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Periplasmic space?","Score: 0.916744 Periplasmic space?","Score: 0.916744 ----- Final Results ----- bacterial periplasmic space --- Certainty= 0.923(Affirmative) < succ> bacterial outer membrane --- Certainty= 0.146(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has no homology with sequences in the databases.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 72 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 143> which encodes amino acid sequence <SEQ ID 144; NGS72>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.18 Possible cleavage site: 42 >>> Seems to have an uncleavable N-term signal seq Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 4 value: −8.76 threshold: 0.0 INTEGRAL Likelihood = −8.76 Transmembrane 11-27 (8-37) INTEGRAL Likelihood = −6.90 Transmembrane 80-96 (75-102) INTEGRAL Likelihood = −2.39 Transmembrane 98-114 (98-114) INTEGRAL Likelihood = −1.12 Transmembrane 47-63 (47-64) PERIPHERAL Likelihood = 3.55 modified ALOM score: 2.25 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.450(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|11354095|pir||H81995 probable transmembrane transport protein NMA0047 [imported]-Neisseria meningitidis (group A strain Z2491) gi|7378822|emb|CAB83364.1|(AL162752) putative transmembrane transport protein [Neisseria meningitidis Z2491] Length = 405 Score = 257 bits (658), Expect = 5e−68 Identities = 152/162 (93%), Positives = 154/162 (94%) Query: 1 MTHTASKTPKLWAVIAAAAFILLITIGMRMTLGLFVQPVVNTTELNIAQFSLIITVFQLM 60 MTHTASKTPK W I AAAFILLITIGMRMTLGLFVQPVVNTTEL+IAQFSLII VFQLM Sbjct: 1 MTHTASKTPKFWLTITAAAFILLITIGMRMTLGLFVQPVVNTTELNIAQFSLIITVFQLM 60 Query: 61 WGVLQPLSGALADRFGAFRVLSGGALLLVCACLIASNIPTYWGLMIAVGLLLAFGTGSGG 120 WGV QPLSGALADRFGAFRVLSGGA+LLVCACLIA NIPTYWGLMIAVGLLLAFGTGSGG Sbjct: 61 WGVSQPLSGALADRFGAFRVLSGGAVLLVCACLIAPNIPTYWGLMIAVGLLLAFGTGSGG 120 Query: 121 FSIIMGQVAAQVPTHKRGLASGLVNAGGSAGQFLFAPLVQGL 162 FSIIMGQVAAQVP HKRGLASGLVNAGGSAGQFLFAPLVQGL Sbjct: 121 FSIIMGQVAAQVPAHKRGLASGLVNAGGSAGQFLFAPLVQGL 162 >gi|11351469|pir||F83484 probable MFS transporter PA1286 [imported]- Pseudomonas aeruginosa (strain PAO1) gi|9947221|gb|AAG04675.1|AE004558_4 (AE004558) probable MFS transporter [Pseudomonas aeruginosa] Length = 399 Score = 72.5 bits (177), Expect = 3e−12 Identities = 53/149 (35%), Positives = 81/149 (53%) Query: 14 VIAAAAFILLITIGMRMTLGLFVQPVVNTTELNIAQFSLIITVFQLMWGVLQPLSGALAD 73 ++ + A IL +++G+R GLF+ P+ F+ I + L+WG+ QP +GALAD Sbjct: 8 ILLSGALILALSLGVRHGFGLFLAPMSADFGWGREVFAFAIALQNLVWGLAQPFTGALAD 67 Query: 74 RFGAFRVLSGGALLLVCACLIASNIPTYWGIMIAVGLLLAPGTGSGGFSIIMGQVAAQVP 133 R+GA R + G LL ++ + GL ++ GLL+ G FS+I+G V VP Sbjct: 68 RYGAARAVLVGGLLYALGLVLMGLSQSASGLSLSAGLLIGLGLSGTSFSVILGAVGRAVP 127 Query: 134 THKRGLASGLVNAGGSAGQFLPAPLVQGL 162 +R +A G+ +A GS GQF P GL Sbjct: 128 AEQRSMAMGISSAAGSFGQFAMLPGTLGL 156 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, NGS72 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 73 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 145> which encodes amino acid sequence <SEQ ID 146; NGS73>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.26 Possible cleavage site: 52 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 3 value: −3.72 threshold: 0.0 INTEGRAL Likelihood = −3.72 Transmembrane 172-188 (171-190) INTEGRAL Likelihood = −2.97 Transmembrane 119-135 (114-137) INTEGRAL Likelihood = −1.86 Transmembrane 209-225 (205-225) PERIPHERAL Likelihood = 4.88 modified ALOM score: 1.24 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.249(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|11354095|pir||H81995 probable transmembrane transport protein NMA0047 [imported]-Neisseria meningitidis (group A strain Z2491) gi|7378822|emb|CAB83364.1|(AL162752) putative transmembrane transport protein [Neisseria meningitidis Z2491] Length = 405 Score = 222 bits (567), Expect = 3e−57 Identities = 116/123 (94%), Positives = 117/123 (94%), Gaps = 4/123 (3%) Query: 103 QGLVVLPEVGWTGTFYVWGAIALLILPVSWWLAGGNNGGNNAAHTQHTQATHGQSLGEAV 162 QGLVVLPEVGWTGTFYVWGAIALL LPVSWWLA GGNNAAHTQH QATHGQSLGEAV Sbjct: 160 QGLVVLPEVGWTGTFYVWGAIALLTLPVSWWLA----GGNNAAHTQHAQATHGQSLGEAV 215 Query: 163 KTAFKTPSYILLHLSFFACGFHIAFLVTHLPTEVALCGLPATVASTSIAIIGLANIAGCV 222 KTAFKTPSYILLHLSFFACGFHIAFLVTHLPTEVALCGLPATVASTSIAIIGLANIAGC+ Sbjct: 216 KTAFKTPSYILLHLSFFACGFHIAFLVTHLPTEVALCGLPATVASTSIAIIGLANIAGCI 275 Query: 223 FSG 225 FSG Sbjct: 276 FSG 278 >gi|11351469|pir||F83484 probable MFS transporter PA1286 [imported]-Pseudomonas aeruginosa (strain PAO1) gi|9947221|gb|AAG04675.1|AE004558_4 (AE004558) probable MFS transporter [Pseudomonas aeruginosa] Length = 399 Score = 54.4 bits (130), Expect = 1e−06 Identities = 37/115 (32%), Positives = 56/115 (48%), Gaps = 10/115 (8%) Query: 111 VGWTGTFYVWGAIALLILPVSWWLAGGNNGGNNAAHTQHTQATHGQSLGEAVKTAFKTPS 170 +GW+ G + LI+P++ + H QSLGEA++ A Sbjct: 160 LGWSSALLALGLLVALIVPLAGLM----------KDRPLPPQGHEQSLGEALREACAHSG 209 Query: 171 YILLHLSFFACGFHIAFLVTHLPTEVALCGLPATVASTSIAIIGLANIAGCVFSG 225 + LL L FF CGF + F+ HLP + LPA V +T +A++GL N+ G +G Sbjct: 210 FWLLALGFFVCGFQVVFIGVHLPAYLVDQHLPAQVGTTVLALVGLFNVFGTYIAG 264 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS73 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 74 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 147> which encodes amino acid sequence <SEQ ID 148; NGS74>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 0.41 Possible cleavage site: 30 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 2 value: −1.49 threshold: 0.0 INTEGRAL Likelihood = −1.49 Transmembrane 15-31 (15-31) INTEGRAL Likelihood = −1.28 Transmembrane 8-99 (83-99) PERIPHERAL Likelihood = 1.59 modified ALOM score: 0.80 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.160(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir||H81995 probable transmembrane transport protein NMA0047 [imported]- Neisseria meningitidis (group A strain Z2491) emb|CAB3364.1|(AL162752) putative transmembrane transport protein [Neisseria meningitidis Z2491] Length = 405 Score = 148 bits (374), Expect = 2e−35 Identities = 97/106 (91%), Positives = 103/106 (96%) Query: 1 MVLIYIFSPKTDLNFYIFAAALGFTWLATVAPTAAVTGKLFGTRYLATLFGLVMLTHQIG 60 M+LIYIFSPKTDLNFYIFAAALGFTWLATV PTA++TGKLFGTRYLATLFGL ML+HQIG Sbjct: 300 MILIYIFSPKTDLNFYIFAAALGFTWLATVTPTASITGKLFGTRYLATLFGLTMLSHQIG 359 Query: 61 GFLGSYIGGIVITQFGDYGWMWYADAVLAGTAALLVLPVREPRTAA 106 GFLGSYIGGIVITQFGDYGWMWYADA+LAGTAALL LP+REPRTAA Sbjct: 360 GFLGSYIGGIVITQFGDYGWMWYADALLAGTAALLNLPIREPRTAA 405 >pir||F83484 probable MFS transporter PA1286 [imported]-Pseudomonas aeruginosa (strain PAO1) gb|AAGO4675.1|AE004558_4 (AE004558) probable MFS transporter [Pseudomonas aeruginosa Length = 399 Score = 59.0 bits (142), Expect = 2e−08 Identities = 40/101 (39%), Positives = 61/101 (59%) Query: 1 MVLIYIFSPKTDLFYIFAAALGFTWLATVAPTAAVGKLFGTRYLATLFGLVMLTHQIG 60 +++++++ P + + Y F A+G WL+TV T LFG R L+ L G+V L HQ+G Sbjct: 286 VIVLFLWLPLSVYSAYAFGVAMGLLWLSTVPLTNGTTLFGVRNLSMLGGIVFLFHQLG 345 Query: 61 GFLGSYIGGIVITQFGDYGWMWYADAVLAGTAALLVLPVRE 101 FLG ++GG+V + G Y +W +L+ AALL PVRE Sbjct: 346 AFLGGWLGGVVYDRTGSYDLVWQLSILLSLLAALLNWPVRE 386 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, NGS74 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 75 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 149> which encodes amino acid sequence <SEQ ID 150; NGS75>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.2 Possible cleavage site: 22 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 4.45 threshold: 0.0 PERIPHERAL Likelihood = 4.45 modified ALOM score: −1.39 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.237(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: pir||S04419 type II site-specific deoxyribonuclease (EC 3.1.21.4) NgoPII- Neisseria gonorrhoeae emb|CAA368B7.1|(X52661) NgoPII restriction and modification [N. gonorrhoeae] prf||1613419A NgoPII endonuclease [Neisseria gonorrhoeae] Length = 278 Score = 240 bits (614), Expect = 4e−63 Identities = 128/129 (99%), Positives = 128/129 (99%) Query: 1 MNIIDAIINLANNPVVGVNSHSQSNNRANQAGDALEEYVKDLFSGSFNLNETQRIARHAK 60 MNIIDAIINLANNPVVGV SHSQSNNRANQAGDALEEYVKDLFSGSFNLNETQRIARHAK Sbjct: 1 MNIIDAIINLANNPVVGVESHSQSNNRANQAGDALEEYVKDLFSGSFNLNETQRIARHAK 60 Query: 61 VFSYLGNNSNPPDAMLRNGDAIEVKKIESKDSALALNSSHPKSKLSVDDSMLTKACKDAE 120 VFSYLGNNSNPPDAMLRNGDAIEVKKIESKDSALALNSSHPKSKLSVDDSMLTKACKDAE Sbjct: 61 VFSYLGNNSNPPDAMLRNGDAIEVKKIESKDSALALNSSHPKSKLSVDDSMLTKACKDAE 120 Query: 121 KWEEKDIIY 129 KWEEKDIIY Sbjct: 121 KWEEKDIIY 129 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 76 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 151> which encodes amino acid sequence <SEQ ID 152; NGS76>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −1.73 Possible cleavage site: 13 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 9.39 threshold: 0.0 PERIPHERAL Likelihood = 9.39 modified ALOM score: −2.38 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.272(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: pir||S04419 type II site-specific deoxyribonuclease (EC 3.1.21.4) NgoPII- Neisseria gonorrhoeae emb|CAA36887.1|(X52661) NgoPII restriction and modification [N. gonorrhoeae] prf||1613419A NgoPII endonuclease [Neisseria gonorrhoeae] Length = 278 Score = 268 bits (687), Expect = 2e−71 Identities = 136/136 (100%), Positives = 136/136 (100%) Query: 1 LAMVYGIDYCADAECYLKIKNQIKEGIGNIGGIQFAETKELGRVNRIDPLNITYLRVRGM 60 LAMVYGIDYCADAECYLKIKNQIKEGIGNIGGIQFAETKELGRVNRIDPLNITYLRVRGM Sbjct: 143 LAMVYGIDYCADAECYLKIKNQIKEGIGNIGGIQFAETKELGRVNRIDPLNITYLRVRGM 202 Query: 61 WGIENPWFVFNYIYQRNMEKSFNFMAIINEDKWNSFNNTDKLLAIQDSKLAISDIKIKNP 120 WGIENPWFVFNYIYQRNMEKSFNFMAIINEDKWNSFNNTDKLLAIQDSKLAISDIKIKNP Sbjct: 203 WGIENPWFVFNYIYQRNMEKSFNFMAIINEDKWNSFNNTDKLLAIQDSKLAISDIKIKNP 262 Query: 121 NNPARLRNAKLITYHL 136 NNPARLRNAKLITYHL Sbjct: 263 NNPARLRNAKLITYHL 278 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 77 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 153> which encodes amino acid sequence <SEQ ID 154; NGS77>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.51 Possible cleavage site: 58 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 3.18 threshold: 0.0 PERIPHERAL Likelihood = 3.18 modified ALOM score: −1.14 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.127(Affirmative) <succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir|||CTNHP2 site-specific DNA-methyltransferase (cytosine-specific) (EC 2.1.1.73) NgoPII-Neisseria gonorrhoeae emb|CAA30038.1|(X06965) NgoPII methylase (AA 1-341) [Neisseria gonorrhoeae] emb|CAA3688B.1|(K52661) NgoPII restriction and modification [Neisseria gonorrhoeae] gb|AAA170419.1|(L14564) cytosine methylase [Neisseria gonorrhoeae] prf||1613419B NgoPII methylase [Neisseria gonorrhoeae] Length = 341 Score = 682 bits (1761), Expect = 0.0 Identities = 341/341 (100%), Positives = 341/341 (100%) Query: 1 MQNSSPTTYNPMKIISLFSGCGGLDLGFEKAGFEIPAANEYDKTIWATFKANHPKTHLIE 60 MQNSSPTTYNPMKIISLFSGCGGLDLGFEKAGFEIPAANEYDKTIWATFKANHPKTHLIE Sbjct: 1 MQNSSPTTYNPMKIISLFSGCGGLDLGFEKAGFEIPAANEYDKTIWATFKANHPKTHLIE 60 Query: 61 GDIRKIKEEDFPEEIDGIIGGPPCQSWSEAGALRGIDDARGQLFFDYIRILKSKQPKFFL 120 GDIRKIKEEDFPEEIDGIIGGPPCQSWSEAGALRGIDDARGQLFFDYIRILKSKQPKFFL Sbjct: 61 GDIRKIKEEDFPEEIDGIIGGPPCQSWSEAGALRGIDDARGQLFFDYIRILKSKQPKFFL 120 Query: 121 AENVSGMLANRHNGAVQNLLKMFDGCGYDVTLTMANAKDYGVAQERKRVFYIGFRKDLEI 180 AENVSGMLANRHNGAVQNLLKMFDGCGYDVTLTMANAKDYGVAQERKRVFYIGFRKDLEI Sbjct: 121 AENVSGMLANRHNGAVQNLLKMFDGCGYDVTLTMANAKDYGVAQERKRVFYIGFRKDLEI 180 Query: 181 KFSFPKGSTVEDKDKITLKDVIWDLQDTAVPSAPQNKTNPDAVNNNEYFTGSFSPIFMSR 240 KFSFPKGSTVEDKDKITLKDVIWDLQDTAVPSAPQNKTNPDAVNNNEYFTGSFSPIFMSR Sbjct: 181 KFSFPKGSTVEDKDKITLKDVIWDLQDTAVPSAPQNKTNPDAVNNNEYFTGSFSPIFMSR 240 Query: 241 NRVKAWDEQGFTVQASGRQCQLHPQAPKMEKHGANDYRFAAGKETLYRRMTVREVARIQG 300 NRVKAWDEQGFTVQASGRQCQLHPQAPKMEKHGANDYRFAAGKETLYRRMTVREVARIQG Sbjct: 241 NRVKAWDEQGFTVQASGRQCQLHPQAPKMEKHGANDYRFAAGKETLYRRMTVREVARIQG 300 Query: 301 FPDNFKFIYQNVNDAYKMIGNAVPVNLAYEIAAAIKKTLER 341 FPDNFKFIYQNVNDAYKMIGNAVPVNLAYEIAAAIKKTLER Sbjct: 301 FPDNFKFIYQNVNDAYKMIGNAVPVNLAYEIAAAIKKTLER 341 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 78 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 155> which encodes amino acid sequence <SEQ ID 156; NGS78>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −8.33 Possible cleavage site: 24 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 2.17 threshold: 0.0 PERIPHERAL Likelihood = 2.17 modified ALOM score: −0.93 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.220(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir||H82000 hypothetical protein NMA0089 [imported]-Neisseria meningitidis (group A strain Z2491) emb|CAB83405.1|(AL162752) hypothetical protein NMA0089 [Neisseria meningitidis Z2491] Length = 226 Score = 422 bits (1085), Expect = e−117 Identities = 217/228 (95%), Positives = 220/228 (96%), Gaps = 2/228 (0%) Query: 1 MERYKNAVRKDKAAELYLLNLSLSRELFHVVSIFEIVLRNKIDICFRQEFKDRNRLYDSI 60 MERYKNAV KDKAAELYLLNLSLSRELFHVVSIFEIVLRNKIDIC +Q FKD N LY+SI Sbjct: 1 MERYKNAVGKDKAAELYLLNLSLSRELFHVVSIFEIVLRNKIDICLQQAFKDGNWLYNSI 60 Query: 61 QPQTNPALKYQGCFLRNGTKESAELIKVALSKIQNNSGGKFDHNQLVAGLGFGFWRYLFA 120 QPQTNPALKYQGCFLRNGTKESAELIKVALSKIQNNSGGKFDHNQLVAGLGFGFWRYLFA Sbjct: 61 QPQTNPALKYQGCFLRNGTKESAELIKVALSKIQNNSGGKFDHNQLVAGLGFGFWRYLFA 120 Query: 121 GGKDAQFDAAGKVLMKVFPKKPKSTPSVQYNQKWIFRELSNINNFRNRLAHHEPICFSFK 180 GGKDAQFDA GKVLMKVFPKKPKSTPSVQ+NQKWIFRELSNINNFRNRLAHHEPIC FK Sbjct: 121 GGKDAQFDATGKVLMKVFPKKPKSTPSVQHNQKWIFRELSNINNFRNRLAHHEPIC--FK 178 Query: 181 GAIKDTGYARNIHQSIFELLNYMDVDTASVFSHFSDQVIAVCDEIDKL 228 GAIKDTGYARNIHQSIFELLNYMDVDTASVFSHFSDQVIAVCDEIDKL Sbjct: 179 GAIKDTGYARNIHQSIFELLNYMDVDTASVFSHFSDQVIAVCDEIDKL 226 >ref|NP_053274.1|Hypothetical gene [Agrobacterium tumefaciens] dbj|BAA87659.1|(AB016260) Hypothetical gene [Agrobacterium tumefaciens] Length = 380 Score = 43.6 bits (102), Expect = 0.002 Identities = 53/215 (24%), Positives = 86/215 (39%), Gaps = 42/215 (19%) Query: 5 KNAVRKDKAAELYLLNLSLSRELFHVVSIFEIVLRNKIDICFRQEFKDRNRLYDSIQPQT 64 K ++ A LYL N +++ + +++ E+ LRN +D F Sbjct: 55 KGGNHEEYAMALYLYNARVAKAFLYPLNVAEVTLRNAVDGILVARFG------------- 101 Query: 65 NPALKYQGCFLRNGTKESAELIKVALSKIQNNSGGKFDHNQLVAGLGFGFWRYLFAGGKD 124 A +Q R+ T L L K +G +Q+VA L F FW LF Sbjct: 102 --ANWHQDATFRDQTLTGNGL--ATLDKAIQRAGAGAARDQIVATLTFDFWSNLFR---- 153 Query: 125 AQFDAAGKVLMKVPKKPKSTPSVQYNQKWIFRELSN----INNFRNRLAHHEPICFSFK 180 ++ + + + + P +Q+ + +E+ N IN FRNR+AHHEP+ Sbjct: 154 PEYGGLWRTTVNI------AFPHLQHGESR--QEIQNINKPINFRNRVAHHEPVL---- 201 Query: 181 GAIKDTGYARNIHQSIFELLNYMDVDTASVFSSFS 215 D +IH I L+ +TA+ H S Sbjct: 202 ----DLNVT-DIHAKIVRLIELRCAETATWMKHHS 231 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS78 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 79 A DNA sequence was identified in N.gonorrhoeae <SEQ D 157> which encodes amino acid sequence <SEQ ID 158; NGS79>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 2.07 Possible cleavage site: 17 >>> Seems to have a cleavable N-term signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 18 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 9.49 threshold: 0.0 PERIPHERAL Likelihood = 9.49 modified ALOM score: −2.40 Score for OM-PP discrimination: −11.72 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: −11.72 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Periplasmic space?","Score: 1.17242 Periplasmic space?","Score: 1.17242 ----- Final Results ----- bacterial periplasmic space --- Certainty= 0.932(Affirmative) < succ> bacterial outer membrane --- Certainty= 0.240(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> A homolog (amino acids 23-85) was found in serogroup A N.meningitidis but not in serogroup B, so NGS79 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 80 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 159> which encodes amino acid sequence <SEQ ID 160; NGS80>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −8.49 Possible cleavage site: 57 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 4.98 threshold: 0.0 PERIPHERAL Likelihood = 4.98 modified ALOM score: −1.50 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.428(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir||E81080 conserved hypothetical protein NMB1466 [imported]-Neisseria meningitidis (group B strain MD58) gb|AAF41823.1|(AE002496) conserved hypothetical protein [Neisseria meningitidis MC58] Length = 243 Score = 148 bits (375), Expect = 7e−35 Identities = 82/189 (43%), Positives = 109/189 (57%) Query: 120 VDRMFNMAGNHFARLGISSGSGVHYWNARDFSEQAFHAEVGYRYRNSRLEWGFRPFVKQNR 179 + R N +HF GISG GVHYW+ +DFSEQ+ GY+ R+ +G PFV+QN Sbjct: 1 MSREINAGRHHFLYGGISGGGVHYWDNKDFSEQSLRLSFGYKNRSVTRSFGIVPFVEQNL 60 Query: 180 LGNNRYTANTGIVLDYSRRLNEKWRSTQSFQYGRKQYHDEYIAKRYNSKTISVSGTFSYY 239 LG +RY G D+S+RL+E+WR T + K Y ++ A RY+S T Y Sbjct: 61 LGGSRYNFVGGFNADFSQRLSERWRLTLNAGNMWKHYQEDRTAARYDSHMPLAGATLMYS 120 Query: 240 AMSAWQLYGGISGNFDNTVEKEQASBRYGVSLGTVKILDGGLGLKLGAGYTKRIFKAPAT 299 A W LYGG + T E EQAS R G+ +G VK DGGLGL+ YT+R+F AP T Sbjct: 121 APKDWLLYGGADWSHNITKEAEQASIRKGLRVGAVKTFDGGLGLRANLRYTRRMFDAPGT 180 Query: 300 LIYNFTRRD 308 ++Y F R+D Sbjct: 181 IVYRFPRKD 189 >gb|AAD11779.1|(AF118122) putative outer membrane protein OmpU [Neisseria meningitidis] Length = 488 Score = 72.1 bits (176), Expect = 7e−12 Identities = 71/300 (23%), Positives = 128/300 (42%), Gaps = 17/300 (5%) Query: 3 EAADLYRELLSERPDLVYPRFDLGVMLFEDKQYREALVQLHRAE-EVLPPDMRQLAREYI 61 EA YREL++ +PD R L LF+++Q A Q R + E LPP + + Y Sbjct: 136 EAISHYRELIAAQPDAPAVRMRLAAALFDNRQNEAAADQFDRLKAENLPPQLMEQVELYR 195 Query: 62 RQAEAVQAWHPSFNMNYEQTDNVNNASLSRDIVINGRKWIKSEDSLPKRANT--IRYELG 119 + AW + + + N+N A + KW + PK+ +G + Y LG Sbjct: 196 KALRERDAWKVNGGFSVTREHNINQAPKRQQR----GLW-----TFPKQVDGTAVNYRLG 246 Query: 120 VDRMFNMAGNHFARLGISGSGVHYWNARDFSEQAFHAEVGYRYRNSRLEWGFRPFVKQNR 179 ++ +++ + G SG Y + F++ G + + R + G F ++ Sbjct: 247 AEKKWSLKNGWYTTAGGDVSGRVYPGNKKFNDMTAGVSGGIGFADRRKDAGLAVFHERRT 306 Query: 180 LGNNRYTANTGIVLDYSRRLNEKWRSTQSFQYGRKQYHDEYLAKRYNSKTISVSGTFSYY 239 GN+ Y+ G L ++R KW++ S ++GR + R ++ + +S + +Y Sbjct: 307 YGNDAYSYTNGARLYFNRWQTPKWQTLSSAEWGRLK---NTRRARSDNTHLQISNSLVFY 363 Query: 240 AMSAWQLYGGISGMFD-NTVEKEQASRRYGVSLGTVKILDG-GLGLKLGAGYTKRIFKAP 297 + GG+ + N ++ RYG+ + G GL L G KR ++ P Sbjct: 364 RNARQYWMGGLDFYRERNPADRGDNFNRYGLRFAWGQEWGGSGLSSLLRLGAAKRHYEKP 423 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 81 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 161> which encodes amino acid sequence <SEQ ID 162; NGS81>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −6.25 Possible cleavage site: 15 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 7.05 threshold: 0.0 PERIPHERAL Likelihood = 7.05 modified ALOM score: −1.91 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.232(Affirmative) < succ> The protein has homology with the following sequences in the databases: gi|10803654|ref|NP_046052.1|putative ISH4 transposase [Halobacterium sp.","NRC-1] gi|7484109|pir||T08324 probable transposase H1306-Halobacterium sp.","(strain NRC-1) insertion sequence ISH4 plasmid pNRC100 gi|2822385|gb|AAC82891.1|(AF016485) putative ISH4 transposase [Halobacterium sp.","NRC-1] gi|10580476|gb|AAG19350.1|(AE005029) Vng0918h [Halobacterium sp.","NRC-1] Length = 294 Score = 52.1 bits (124), Expect = 4e−06 Identities = 36/139 (25%), Positives = 63/139 (44%), Gaps = 7/139 (5%) Query: 31 CPHCQSTHFVKNGKDCGNQRFLCRDCKKSFVEQTGTILYNTQKDIEVWEKYIHCMIE-KY 89 CP C++ ++ G QR+LC+DC ++F +QTGT+ ++ + W ++ I Sbjct: 28 CPSCRAESVIRYGSYRVFQRYLCKDCDRTFNDQTGTVFEHSAVALRKWFLAVYTYIRLNT 87 Query: 90 PLRKCAEICKINLATAFTWRHKILDALQNMMNEVELDGIVQADETYSTISYKGHHKNFNL 149 +R+ ++ T + + L AL L+G V+ DE Y KG ++ Sbjct: 88 SIRQLDAEIDVSYKTVYRRVQRFLRALD--APRPHLEGPVEIDEFYVKAGLKGRERD--- 142 Query: 150 PRPAHKRGTRATKRGISKE 168 +P+ RG RG E Sbjct: 143 -QPSRSRGLSTRGRGTYAE 160 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 82 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 163> which encodes amino acid sequence <SEQ ID 164; NGS82>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.66 Possible cleavage site: 57 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 1 value: −0.85 threshold: 0.0 INTEGRAL Likelihood = −0.85 Transmembrane 76-92 (76-92) PERIPHERAL Likelihood = 1.75 modified ALOM score: 0.67 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.134(Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi|586070|sp|Q07605|T4BA_BACCO RESTRICTION ENZYME BGCI ALPHA SUBUNIT [INCLUDES: ADENINE-SPECIFIC METHYLTRANSFERASE ACTIVITY] gi|1075788|pir||A53125 restriction enzyme BcgI alpha chain-Bacillus coagulans gi|304140|gb|AAA16626.1|(L17341) restriction endonuclease alpha subunit [Bacillus coagulans] Length = 637 Score = 91.4 bits (226), Expect = 1e−17 Identities = 78/256 (30%), Positives = 123/256 (47%), Gaps = 42/256 (16%) Query: 1 MFALAASNMILRGDGKANLHQSSCFMTDFQDLIKNPKPETGLKRPNVGFLNPPYAQSKSD 60 +F +A +NMILRGDGK+NL + +C F + I N G+ N +NPPY+Q+K+D Sbjct: 394 LFTIATTNMILRGDGKSNLIRDNCLT--FDNTIMN---GYGI---NKILMNPPYSQAKND 445 Query: 61 AELH--ELYFVKEMLKMLAEGGTGIAIIPVSCVIAPSK----AKSEIVKYHRLKAVMSMP 114 H EL F+++ L+ML GG AI+P S ++ ++ K +I+K H L+ V+++ Sbjct: 446 QTQHLSELSFIQQALEMLVVGGKLCAIVPQSTMVGKNRHDKARKKQILKQHTLETVITLN 505 Query: 115 SELFYPVGTVTCIVVFEAHKPHFQTVVIDPDTQEEISTKKACENTWFGYWRDDGFEKTKH 174 + F+ VG CIV+F+A H + ++ F + DDG KH Sbjct: 506 KDTFHGVGVNPCIVIFKAGIKHPEN-----------------KRVSFVNFEDDGHVVRKH 548 Query: 175 LGRIDLYDRWQGIKARWLEHYL-----NNEVHTGESVTAFVTDNDEWVAEAYLETDYSKI 229 +G + G + EH L + + T V + D DEW+ Y D Sbjct: 549 VGLVG-----DGTEKGKREHLLAVLAGDEDDGTDLIVKTAIKDTDEWLHSFYYFND-GIP 602 Query: 230 TRADFEQVVREFALFQ 245 + DF + V + FQ Sbjct: 603 SEDDFYKTVANYLTFQ 618 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 83 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 165> which encodes amino acid sequence <SEQ ID 166; NGS83>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −8.04 Possible cleavage site: 43 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 1 value: −1.44 threshold: 0.0 INTEGRAL Likelihood = −1.44 Transmembrane 55-71 (55-71) PERIPHERAL Likelihood = 4.03 modified ALOM score: 0.79 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.157 (Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi|586071|sp|Q07606|T4BB_BACCO RESTRICTION ENZYNE BGCI BETA SUBUNIT gi|1075789|pir||B53125 restriction enzyme BcgI beta chain-Bacillus coagulans gi|304141|gb|AAA16627.1|(L17341) restriction endonuclease beta subunit [Bacillus coagulans] Length = 341 Score = 44.0 bits (103), Expect = 0.002 Identities = 46/195 (23%), Positives = 79/195 (39%), Gaps = 23/195 (11%) Query: 4 LQEIFDVSYGSKLDLNKMSSFNPTINFVGRSGKNNGVTASVDLLKNTKPYPAGLLTVALG 63 + ++FDV G +D NK ++R NG +D K K Y L + +G Sbjct: 12 ISDLFDVVIGKTIDGNKAQRNENGTPYITRKATRNGFEFMIDGEKE-KLYSGKLPVITIG 70 Query: 64 GSVLSTFLQNKPFYTAQNVAVLNPKTEMTEQQKLFYCAAIFANAYRFSACGREANRT-LR 122 F+Q F+T V + PK ++ L Y + NA + + N T L+ Sbjct: 71 NETSKPFVQEFHFFTGTKVNICIPKLDLNRNH-LLYITTMIENATKMFSYSYTINSTRLK 129 Query: 123 QL--FVPSLDEIPSW--------------VESVNLNPSAGVTEPKLKESLDLPVVRQSKR 166 L +P E P W ++ ++ + GV++ + + L + Sbjct: 130 SLKILLPIKGEEPDWDYMNTYISKILSNMEKNFDVQQNDGVSDLRSLKDLSW----SQFK 185 Query: 167 LPEIFTIQNGIAATK 181 +DEIF+I +G+ TK Sbjct: 186 MDEIFSINSGVRLTK 200 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 84 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 167> which encodes amino acid sequence <SEQ ID 168; NGS84>.","Analysis of this protein sequence reveals the following: Signal Score (−7.5): 3.15 Possible cleavage site: 33 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 1.22 threshold: 0.0 PERIPHERAL Likelihood = 1.22 modified ALOM score: −0.74 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.072(Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi|2495432|sp|P55409|Y4DJ_RHISN HYPOTHETICAL TRANSCRIPTIONAL REGULATOR Y4DJ gi|7465604|pir||T02773 y4dJ protein-Rhizobium sp.","plasmid pNGR234a gi|2182353|gb|AAB91639.1|(AE000069) Y4dJ [Rhizobium sp.","NGR234] Length = 77 Score = 44.4 bits (104), Expect = 7e−04 Identities = 25/61 (40%), Positives = 36/61 (58%) Query: 92 KAGGETFVSLRKGFTQSELATAAGLPQPYLSRIENSKQSLQDKTVQKLANALGVSPLE 151 K G F LR +KG TQ E+ +G Q YLS +E +++ T+ +LA ALGVS +E Sbjct: 5 KLVGSNFARLREEKGLTQEEEARSGFSQQYLSSLERGRRNPTVITLYELAQALGVSHVE 64 Query: 152 V 152 + Sbjct: 65 L 65 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 85 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 169> which encodes amino acid sequence <SEQ ID 170; NGS85>.","Analysis of this protein sequence reveals the following: Signal Score (−7.5): −6.09 Possible cleavage site: 15 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 2.92 threshold: 0.0 PERIPHERAL Likelihood = 2.92 modified ALOM score: −1.08 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.480(Affirmative) < succ> The protein has no homology with sequences in the databases.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 86 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 171> which encodes amino acid sequence <SEQ ID 172; NGS86>.","Analysis of this protein sequence reveals the following: Signal Score (−7.5): −2.92 Possible cleavage site: 21 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 1 value: −2.76 threshold: 0.0 INTEGRAL Likelihood = −2.76 Transmembrane 179-195 (179-195) PERIPHERAL Likelihood = 2.17 modified ALOM score: 1.05 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.210 (Affirmative) < succ> The protein has homology with the following sequences in the databases: >sp|Q05205|PPB_LYSEN ALKALINE PHOSPHATASE PRECURSOR (APASE) pir||A42467 alkaline phosphatase (EC 3.1.3.1) phoA precursor-Lysobacter enzymogenes emb|CAA39978.1|(X56656) alkaline phosphatase [Lysobacter enzymogenes] Length = 539 Score = 37.5 bits (86), Expect = 0.40 Identities = 28/82 (34%), Positives = 43/82 (52%), Gaps = 8/82 (9%) Query: 189 VALGLQAYWDVAGANNGATGQSPNIKTAQVPAKITRRNADGTTDTFGGGSARKSAAASVS 248 V GL A W+V+ A + AQV +++ R+ GT D + G+A A AS S Sbjct: 458 VLRGLMA-WNVSSA------AGKTLTGAQVKLQVSDRST-GTYDLYRAGAAWTEANASYS 509 Query: 249 GIEAGKKVTAVIPAVRGAVAYA 270 G+ G K+ +V+P+ GA + A Sbjct: 510 GVSLGSKIGSVVPSATGAQSIA 531 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 87 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 173> which encodes amino acid sequence <SEQ ID 174; NGS97>.","Analysis of this protein sequence reveals the following: Signal Score (−7.5): 0.18 Possible cleavage site: 35 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 1.70 threshold: 0.0 PERIPHERAL Likelihood = 1.70 modified ALOM score: −0.84 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.138(Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi|12514207|gb|AAG55499.1|AE005289_17 (AE005289) unknown protein encoded by cryptic prophage CP-933M [Escherichia coli O157:H7] gi|12514720|gb|AAG55907.1|AE005324_10 (AE005324) unknown protein encoded by prophage CP-933N [Escherichia coli O157:H7] Length = 108 Score = 30.9 bits (69), Expect = 9.1 Identities = 21/55 (38%), Positives = 28/55 (50%), Gaps = 3/55 (5%) Query: 1 MAAPVSLEEFKQRIGVEHDRRDDFFLSVIDGVSAAAEAYIGRSLLAADYVGRYDG 55 M A ++LEE K + V+HD DD + + +A AYI S D V R DG Sbjct: 1 MTALLTLEEIKAHLRVDHDADDDMLMDKVRQATAVLLAYIQGS---RDKVIREDG 52 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 88 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 175> which encodes amino acid sequence <SEQ ID 176; NGS88>.","Analysis of this protein sequence reveals the following: Signal Score (−7.5): −3.69 Possible cleavage site: 43 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 6.05 threshold: 0.0 PERIPHERAL Likelihood = 6.05 modified ALOM score: −1.71 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.227(Affirmative) < succ> The protein has no homology with sequences in the databases.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 89 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 177> which encodes amino acid sequence <SEQ ID 178; NGS89>.","Analysis of this protein sequence reveals the following: Signal Score (−7.5): −4.77 Possible cleavage site: 26 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 1.38 threshold: 0.0 PERIPHERAL Likelihood = 1.38 modified ALOM score: −0.78 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.284(Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi|9634142|ref|NP_037684.1|gp24 [Enterobacteria phage HK022] gi|6863134|gb|AAF30375.1|AF069308_23 (AF069308) gp24 [Enterobacteria phage HK022] Length = 1183 Score = 44.9 bits (102), Expect = 0.006 Identities = 38/127 (29%), Positives = 64/127 (49%), Gaps = 11/127 (8%) Query: 851 NKALRDKINLIDGNGAGSVNERVEAVRSTADGNAAAVQTHARSI---NG-LEAQYTVK-- 904 NKA + +N + + ++ + +T +GN +A+ T+A++I NG L A Y +K Sbjct: 989 NKASINSLNQTFSDYQQATATQINGITATVNGNTSAITTNAQAIANVNGDLSAMYNIKVG 1048 Query: 905 VDANGK--VAGFGLATTPKNGTPESKFIVNADRFGI-GAAGKADVFPFVVDTQKNRVGIN 961 V +NG+ AG G+ +S+ I ADRF + AAG + PFV+ Q + I Sbjct: 1049 VSSNGQYYAAGMGIGVENTPSGMQSQVIFLADRFAVTTAAGNSVALPFVI--QNGQTFIR 1106 Query: 962 GELVVNG 968 + +G Sbjct: 1107 ASFIQDG 1113 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 90 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 179> which encodes amino acid sequence <SEQ ID 180; NGS90>.","Analysis of this protein sequence reveals the following: Signal Score (−7.5): −2.82 Possible cleavage site: 24 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 4 value: −9.66 threshold: 0.0 INTEGRAL Likelihood = −9.66 Transmembrane 321-337 (317-349) INTEGRAL Likelihood = −6.48 Transmembrane 351-367 (340-371) INTEGRAL Likelihood = −5.73 Transmembrane 907-923 (903-926) INTEGRAL Likelihood = −0.00 Transmembrane 430-446 (430-446) PERIPHERAL Likelihood = 2.17 modified ALOM score: 2.43 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.486 (Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi|12514839|gb|AAG56002.1|AE005332_9 (AE005332) putative tail component of prophage CP-933X [Escherichia coli O157:H7] Length = 1026 Score = 111 bits (279), Expect = 3e−23 Identities = 78/274 (28%), Positives = 146/274 (52%), Gaps = 10/274 (3%) Query: 69 AAGNQAQQASEKVRAEVGKIGSGLSGLTKLLAGLATADFAKSVLDTADAMQSINSQVRQV 128 AA + ++A ++ +++ +I + G+T AG A ++ AD S+N++++Q Sbjct: 45 AAAREQRRALAELHSQLTEIRASAVGMTGAFAG---AFATGHLISLADEWSSVNARLKQA 101 Query: 129 TSSETEYLAVQQQLLDTANRTRASLESTANLYVSTSRALKDYGYTQQEILKFTEAANNAM 188 + S E+ + Q+ L+D + RT + A L+ ++ ++++YGY+ ++LK TEA + + Sbjct: 102 SQSSDEFASSQKVLMDISQRTGTAFSDNAALFARSAASMREYGYSADDVLKVTEAISTGL 161 Query: 189 TIGGVGAQQQAAALMQLSQALGSGVLQGDEFKSISEAAPILLDTIAEYMGKSRDEIKKLG 248 I G + + + Q SQAL GVL+G+EF S++E+ ++ +A MG +R ++K + Sbjct: 162 KISGASTAEAGSVITQFSQALAQGVLRGEEFNSVNESGDRIVRALAAGMGVARKDLKAMA 221 Query: 249 SEGKLTADVIFKAISGASEKFGEQAAKMPVTMGQALTVFSNNWQSMVSKLLNDSGTMSGI 308 +GKLTAD + A+ ++ A MP T+ ++T N + + V G + Sbjct: 222 DDGKLTADKVVPALISQLGILRDEYAAMPETVSSSITKVENAFMAWV-------GGANEA 274 Query: 309 AAVIKLIADNLNLVVPIVAGFAVAVAAAVAPTLA 342 + V K ++ LN V + A AV A VA +A Sbjct: 275 SGVTKTLSGMLNGVAGQIDNVATAVGALVAVGVA 308 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 91 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 181> which encodes amino acid sequence <SEQ ID 182; NGS91>.","Analysis of this protein sequence reveals the following: Signal Score (−7.5): −0.63 Possible cleavage site: 36 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 0.16 threshold: 0.0 PERIPHERAL Likelihood = 0.16 modified ALOM score: −0.53 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.250(Affirmative) < succ> The protein has homology with the following sequences in the databases: (AF237934) putative integrase/recombinase [Pasteurella multocida] Length = 329 Score = 449 (206.9 bits), Expect = 4.4e−91, Sum P(2) = 4.4e−91 Identities = 93/196 (47%), Positives = 129/196 (65%) Query: 56 IFADLIRRYLSEVTPSKRGAREESYRIGRALKTPLAKVRLADLRPQDFADWRDQRLQEVS 115 IF D+I RY +EV+ +K+GAR E R+ R L+ ++ + + DLR +DF +W RL EVS Sbjct: 55 IFRDVIERYQNEVSITKKGARNEIIRLNRFLRYDISNLYIRDLRKEDFEEWIRIRLTEVS 114 Query: 116 PTSVGRELTTLSAVCEHAMKEWGLLRENPVRKISKPKKSRARTRRPTEQEIADICAALLY 175 SV REL T+S+V A+ +WG + +P+ I KPK S R R +EQ+I I Y Sbjct: 115 DASVRRELVTISSVLTTAINKWGYISRHPMTGIEKPKNSAERKERYSEQDIKTILETARY 174 Query: 176 RPNEKPKMAVQRVAVAVLFAIETAMRAGEICGLKWADVNMRRRIAHLPITKNGDSRDVPL 235 ++ P QRVA+A+LFAIETAMRAGEI +KW +V + +RI HLP TKNG SRDVPL Sbjct: 175 CEDKLPITLKQRVAIAMLFAIETAMRAGEIASIKWDNVFLEKRIVHLPTTKNGHSRDVPL 234 Query: 236 SLRAAELIEQLRGIDD 251 S RA LI +++ +++ Sbjct: 235 SQRAVALILKMKEVEN 250 Score = 248 (114.3 bits), Expect = 4.4e−91, Sum P(2) = 4.4e−91 Identities = 48/76 (63%), Positives = 57/76 (75%) Query: 254 VFSLDAKSLDVLFRRARDNCGIQGLHFHDTRREALTRLSKKVPVEVLAKISGHRDLRILL 313 VF +SL FR + CG++ LHFHDTRREALTRLSKKV V LAKISGHRDLRIL Sbjct: 254 VFQTTPESLSTTFRVLKKECGLEHLHFHDTRREALTRLSKKVDVMTLAKISGHRDLRILQ 313 Query: 314 NVYYRPDMADIAKMLD 329 N YY P+M+++A +LD Sbjct: 314 NTYYAPNMSEVANLLD 329 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 92 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 183> which encodes amino acid sequence <SEQ ID 184; NGS92>.","Analysis of this protein sequence reveals the following: Signal Score (−7.5): −7.85 Possible cleavage site: 25 >>> Seems to have an uncleavable N-term signal seq Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 1 value: −8.33 threshold: 0.0 INTEGRAL Likelihood = −8.33 Transmembrane 6-22 (1-25) PERIPHERAL Likelihood = 5.99 modified ALOM score: 2.17 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.433 (Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi|963205|ref|NP_048840.1|A484L [Paramecium bursaria Chlorella virus 1] gi|7461623|pir|T17986 hypothetical protein A484L-Chlorella virus PBCV-1 gi|1620155|gb|AAC96851.1|(U42580) A484L [Paramecium bursaria Chlorella virus 1] Length = 155 Score = 31.6 bits (70), Expect = 3.5 Identities = 20/72 (27%), Positives = 36/72 (49%) Frame = +1 Query: 52 LQINLKMLEKRIDFLVENIDKYYQQYGSYPNNFDFISTKTDFTTESYCDFWDKNIAGYGN 231 + +NLKM I F +DKY +QY +Y N F T+ + + ++ + +I N Sbjct: 23 IAVNLKMNGVSIPF----VDKYSKQYPTYTKNALFHVTRFNNAYQKTFEYKNISIDTINN 78 Query: 232 CYFVKNDKDYTI 267 + +++D Y I Sbjct: 79 LFSIRDDVLYNI 90 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 93 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 185> which encodes amino acid sequence <SEQ ID 186; NGS93>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.08 Possible cleavage site: 14 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 0.79 threshold: 0.0 PERIPHERAL Likelihood = 0.79 modified ALOM score: −0.66 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.320(Affirmative) < succ> The protein has homology with the following sequences in the databases: >gb|AAG22017.1|AF288038_2 (AF288038) putative HsdR ]Streptococcus thermophilus] Length = 740 Score = 674 bits (1738), Expect = 0.0 Identities = 364/746 (48%), Positives = 489/746 (64%), Gaps = 40/746 (5%) Query: 14 NENSRVKIPAVLHLMRLGYDYLSLKNANW---DRQTNIFPEIFVDSLCRINPDLPPDDAR 70 +E +RV+IPA HLMRLGY YL D +TNI IF + + N D Sbjct: 8 SELTRVQIPAAFHLMRLGYTYLPHNGKEIMGRDPETNILISIFREQFLKFNNYATDLDVE 67 Query: 71 RLLADIRLELDNEDLGQKFYERLTNQSGGKKLIDFQNFDNNSFHVVTELPCINGDEAFRP 130 R L +I++ELD DLG+ FY R+ + SG +D++N +NN+FH+ E+ C NG + FRP Sbjct: 68 RELNNIKIELDQNDLGRAFYNRIVSDSG-PTYVDWENPENNTFHLALEVTCQNGGDEFRP 126 Query: 131 DIALLVNGMPLVFIEVKKPN----NKGGIGEERERMGKRAKNPKFRRFINITQFMIFSNN 186 DI + +NG+PL +IEVK+PN K I E+ R R +N +FRRF NITQ + FS+N Sbjct: 127 DIVIFINGLPLSYIEVKQPNAIRDGKTAIQSEQSRTAVRFENRRFRRFNNITQLISFSDN 186 Query: 187 MEYDDGATEPAQGAFYASSACGKPVFNYFREEHKXXXXXXXXXXXXXXXXXVLQDNNLPV 246 + Y G + QG+FY S+A K FN F+EE + VL+D N Sbjct: 187 LPYISGQGQQKQGSFYCSNAFSKTKFNAFKEEREEELIYSIRSLGEEEIDAVLKDVNRFA 246 Query: 247 IKHSPEFISNKSPDTPTNRILTSLLCRERLSFLLQHGLTYVK--ASQGLVQ--KHIMRYP 302 +K PEF +N+ P TP N ++SL ++RL FLL++GL YV+ + G +Q KH+MRYP Sbjct: 247 LKSQPEFKTNQDPSTPCNTFISSLYQKKRLLFLLRYGLAYVEEHSKDGTIQLQKHVMRYP 306 Query: 303 QLFATLAIEKHLANGGKKGVIWHTQGSGKTALAYYNTRYLTHYYAKQGIVPKFYFIVDRL 362 Q FAT AIE + G +KGVIWHTQGSGKTAL+Y+N RYLT+Y++KQGIVP+FYF+VDRL Sbjct: 307 QFFATKAIEDAIGKGVRKGVIWHTQGSGKTALSYFNIRYLTNYFSKQGIVPQFYFVVDRL 366 Query: 363 DLLKQAQREFTARDLVVHTIDSREAFAADIKSAQTLHNHAGKAEITVVNIQKFQDDPDVV 422 DL QA REFT R L V I+S Q L+ ++ VVNIQKF+D+ D+ Sbjct: 367 DLADQATREFTKRGLKVKRINS----------PQELNEKHDAYQVAVVNIQKFKDNSDLT 416 Query: 423 ARNDYDLAIQRVYFLDEVHRSYNPKGSFLANLNQSDVNAVKIGLTGTPLI-----GVTA- 476 + YDL Q +YF+DE HRSYN KGS+L NL +D NA+KI LTGTPLI G T Sbjct: 417 DHSGYDLNRQNIYFIDEAHRSYNEKGSYLPNLYNADKNAIKIALTGTPLITYKKDGKTKE 476 Query: 477 GNVNTRELFGDYIHKYYYNASIADGYTLRLIREEIGSRYKAQLQEALAQLEIEKGSFDRK 536 + TR++FGDYIHKYYYN SI DG+TLRL+RE+I + YK LQ EI +G + Sbjct: 477 SHATTRDIFGDYIHKYYYNQSIDDGFTLRLMREDIETSYKETLQTI--NEEILRGDLSKD 534 Query: 537 EIYAHPHFVHPMLDYILDDFAKFRKTN-QDESLGAMVVCDSAEQARQL---FEHFQTASD 592 +I+AHP +V PMLG+IL+DF + R D+S+G M+VCDS++QAR++ E ++ + Sbjct: 535 DIFAHPRYVSPMLDFILEDFNRARDVVFDDDSIGGMIVCDSSKQAREIEKQLEERRSRGE 594 Query: 593 HNFTAALILHDVGTKEERDQWVKDFKAGKIDILFVYNMLLTGFDAPRLKKLYLGRLIKAH 652 N T+ALILHD G KE + V+ ++ GKID++ VY+MLLTGFDAPRLK+LYLGR IKAH Sbjct: 595 TNITSALILHDEGDKEYKKDRVESYREGKIDLVIVYSMLLTGFDAPRLKRLYLGRKIKAH 654 Query: 653 NLLQTLTRVNRTYKSYRYGYVVDFADIEREFDKTNRAYWDELSEN-----LGDEIGS-YS 706 NLLQTLTRVNR YK Y++GYV+DFADI +EFDKTNRAY +EL+ E G+++ + + Sbjct: 655 NLLQTLTRVNRPYKDYQFGYVIDFADISKEFDKTNRAYLEELNQEYDPKNTGEDVENVFG 714 Query: 707 QLFKTAEEIEQEIADIKNALFDFDTE 732 LF +A+EI +++ + L ++ TE Sbjct: 715 SLFVSADEISKQLEKSETILMNPTE 740 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 94 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 187> which encodes amino acid sequence <SEQ ID 188; NGS94>.","Analysis of this protein sequence reveals the following: Signal Score (−7.5): −3.19 Possible cleavage site: 35 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 5.73 threshold: 0.0 PERIPHERAL Likelihood = 5.73 modified ALOM score: −1.65 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.302(Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi|10717100|gb|AAG22014.1|AF288037_3 (AF288037) putative HsdS [Streptococcus thermophilus] Length = 402 Score = 154 bits (389), Expect = 2e−36 Identities = 123/348 (35%), Positives = 168/348 (47%), Gaps = 48/348 (13%) Query: 73 GKTAFVDILDDGEVAFGSTEFIVLRAKNET--NPEFLYYFAISPDFRKRAIECMEGTSGR 130 GKT ++ DGE ++ IV +E+ +FLYYF + F G++ + Sbjct: 74 GKT----VIFDGEDSYFQDSNIVWIENDESKVTNQFLYYFLQTNPFIT-----TNGSTIK 124 Query: 131 QRVNENALKTLELPIPEPQIQQSIAAVLSALDKKIALNKQINARLEEMAKTLYDYWFVQF 190 + N+N T +P Q Q I +L LDKKI +N QIN LE MAKTLYDYWFVQF Sbjct: 125 RLYNDNLRDTKIPNVPSIQQQNQITDILGTLDKKIQINNQINQELEAMAKTLYDYWFVQF 184 Query: 191 DFPDANGKPYKSSGGDMVFDETLKREIPKGWGSIELQSCL---AKIPNTTKILNKDIKDF 247 DFPD NGKPYKSSGG MV++ LKREIP+GWG+ +L S L + N K N++ K + Sbjct: 185 DFPDQNGKPYKSSGGKMVYNPELKREIPEGWGAEKLSSLLKIGKETTNPKKFPNEEFKYY 244 Query: 248 --------GKYPVVD----QSQDFICGFTNKEKSILNPQDAHIIFGDHTRIVKLVNFQYA 295 G Y + +S F + S LNP +I+ + F Sbjct: 245 SIPEFDTTGTYSLERGESIKSNKFKFKVEKNDLLVSKLNPWFNRVIYNLEENAIASTEF--- 301 Query: 296 RGADGTQVILSNNERMPNYLFYQIINQIDLSSY------GYARHFK-----FLKEFKIIL 344 ++ R YQ+ + Y G + K + F+I Sbjct: 302 -------IVWKTFNRFEKNFLYQVATGKEFIEYCTRFATGTSNSHKRVSPDIMVGFQIPF 354 Query: 345 PSKDISQKYNEIANTFFVKVRNNLKQNHHLTQLRDFLLPMLMNGQVSV 392 I QK+ EI ++ +V N +QN LTQLRD++LPMLMNGQV V Sbjct: 355 EKTHI-QKFGEIIDSIRTQVLQNNEQNQELTQLRDWILPMLMNGQVKV 401 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 95 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 189> which encodes amino acid sequence <SEQ ID 190; NGS95>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −6.12 Possible cleavage site: 19 >>> Seems to have an uncleavable N-term signal seq Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 3 value: −10.51 threshold: 0.0 INTEGRAL Likelihood = −10.51 Transmembrane 112-128 (109-132) INTEGRAL Likelihood = −4.46 Transmembrane 50-66 (46-70) INTEGRAL Likelihood = −2.23 Transmembrane 7-23 (7-23) PERIPHERAL Likelihood = 4.19 modified ALOM score: 2.60 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.520 (Affirmative) < succ> The protein has homology with the following sequences in the databases: >pir||G69096 hypothetical protein MTH1717-Methanobacterium thermoautotrophicum (strain Delta H) gb|AAB86189.1|(AE000928) unknown [Methanothermobacter thermautotrophicus] Length = 557 Score = 35.4 bits (80), Expect = 0.50 Identities = 25/80 (31%), Positives = 47/80 (58%), Gaps = 5/80 (6%) Query: 52 LLFYFLIPFIATATVLWLSKYLGKDEFKQGEVKELEYVNDNFLPSYLGYFFVALSIPDNN 111 L+F+F+ P + TATVL + K + ++ F++ EV L + +PS++ ++ IP++ Sbjct: 92 LVFFFISPLLGTATVLVIYK-VARETFEREEVALLSAFLFSMVPSFVAR--TSVFIPESM 148 Query: 112 LFLLFVMYGIIFLLVSCSKS 131 LL GI+++LV K+ Sbjct: 149 GLLL--TSGILYMLVKYLKT 166 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 96 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 191> which encodes amino acid sequence <SEQ ID 192; NGS96>.","Analysis of this protein sequence reveals the following: GyH: Examining signal sequence (von Heijne) Signal Score (−7.5): −7.76 Possible cleavage site: 28 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 6.15 threshold: 0.0 PERIPHERAL Likelihood = 6.15 modified ALOM score: −1.73 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.362(Affirmative) < succ> The protein has no homology with sequences in the databases.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 97 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 193> which encodes amino acid sequence <SEQ ID 194; NGS97>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −0.48 Possible cleavage site: 13 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 8.86 threshold: 0.0 PERIPHERAL Likelihood = 8.86 modified ALOM score: −2.27 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.127(Affirmative) < succ> The protein has homology with the following sequences in the databases: >ref|NP_052265.1|P2 J homolog; baseplate or base of tail fibre [Enterobacteria phage 186] gb|AAC34162.1|(U32222) P2 J homolog; baseplate or base of tail fibre [Enterobacteria phage 186] Length = 302 Score = 112 bits (280), Expect = 3e−24 Identities = 65/151 (43%), Positives = 85/151 (56%), Gaps = 1/151 (0%) Query: 1 MGNSRLSQLPAPAAIEETDFEGIFARKKAALTALCPESIRETVAQTLELESEPLTIDLQQ 60 M LS LP P +EE DFE I A + A L +L PE +E VA+TL LESEP+ LQ+ Sbjct: 1 MATVDLSLLPVPDVVEELDFETILAERIATLISLYPEDQQEAVARTLALESEPIVKLLQE 60 Query: 61 QAYQELLVRNRINEAVKANLLAYAQGSDLDHIAAQYGLSRKTIRXXXXXXXXXXXXEYET 120 AY+E++ R R+NEA +A +LAYA+ SDLD++ A + + R +R E E Sbjct: 61 NAYREVIWRQRVNEAARAGMLAYARDSDLDNLGANFNVERLVVRPADDTTIPPTPAEMEL 120 Query: 121 DDAFRARV-QAHPEKYAAGPRTAYEAHAIDA 150 D FR R+ QA AG AYE H A Sbjct: 121 DADFRLRIQQAFEGMSVAGSTGAYEFHGRSA 151 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 98 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 195> which encodes amino acid sequence <SEQ ID 196; NGS98>.","Analysis of this protein sequence reveals the following: Signal Score (−7.5): −3.68 Possible cleavage site: 33 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 4.61 threshold: 0.0 PERIPHERAL Likelihood = 4.61 modified ALOM score: −1.42 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.182(Affirmative) < succ> The protein has no homology with sequences in the databases.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 99 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 197> which encodes amino acid sequence <SEQ ID 198; NGS99>.","Analysis of this protein sequence reveals the following: Signal Score (−7.5): −4.87 Possible cleavage site: 19 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 4.93 threshold: 0.0 PERIPHERAL Likelihood = 4.93 modified ALOM score: −1.49 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.189(Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi|10172952|dbj|BAB04058.1|(AP001508) BH0339-unknown conserved protein in others [Bacillus halodurans] Length = 283 Score = 83.7 bits (206), Expect = 1e−15 Identities = 59/156 (37%), Positives = 87/156 (54%), Gaps = 8/156 (5%) Query: 10 VRGPVQLAFAQSIDPIVPPEVSITRMAVTNEKDLEKERTMGRKYIVPYVVYRVHGFISAN 69 VRGPV + A SIDPI IT+ + D TMG K+ V + VY G I+ Sbjct: 129 VRGPVSIHTATSIDPIDIVSTQITKSVNSVTGDKRSSDTMGMKHRVDFGVYVFKGSINTQ 188 Query: 70 LAAKTGFSDDDLAKLWQALTLMFEHDRSAAR--GEMARKLVFKHDSALGSQPAHKLFD 127 LA KTGF+++D K+ +AL +FE+D S+AR G M K+ ++H S LG + K+ Sbjct: 189 LAEKTGFTNEDAEKIKRALITLFENDSSSARPDGSMEVHKVYWWEHSSKLGQYSSAKVHR 248 Query: 128 AVKVERVNGESGTPASGFGDYKISVVSDGLNGVSVE 163 ++K+E ++ TP S F DY + + L+G+ VE Sbjct: 249 SLKIE---SKTDTPKS-FDDYAVELYE--LDGLGVE 278 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 100 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 199> which encodes amino acid sequence <SEQ ID 200; NGS100>.","Analysis of this protein sequence reveals the following: Signal Score (−7.5): −3.03 Possible cleavage site: 18 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 6.63 threshold: 0.0 PERIPHERAL Likelihood = 6.63 modified ALOM score: −1.83 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.185(Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi|1175791|sp|P44189|YE18_HAEIN HYPOTHETICAL PROTEIN HI1418 gi|1074769|pir||A64029 hypothetical protein HI1418-Haemophilus influenzae (strain Rd KW20) gi|1574254|gb|AAC23068.1|(U32821) H. influenzae predicted coding region HI1418 [Haemophilus influenzae Rd] Length = 201 Score = 144 bits (364), Expect = 1e−33 Identities = 71/109 (65%), Positives = 79/109 (72%) Query: 8 NFQQNSVRTVADNKGELWFLANDVCEILGYTNPRRTVDLHCKSRGVTKRYTPTTSGEQEM 67 NF+ VR + D KGE WF DVC ILGYTN R+ + HCK GVTKRYTPT S +QEM Sbjct: 24 NFKDLPVRVILDPKGEFWFCGTDVCHILGYTNSRKALQDHCKQGGVTKRYTPTKSADQEM 83 Query: 68 TYINEPNLYRLIIKSRKPAAEAFEEWVMETVLPAIRKTGGCQVGPKTTA 116 T+INEPNLYRLIIKSRKP AE FE WV E VLP IRKTG Q+ P+ A Sbjct: 84 TFINEPNLYRLIIKSEKPEAEPFEAWVFEEVLPQIRKTGKYQLQPQQLA 132 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 101 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 201> which encodes amino acid sequence <SEQ ID 202; NGS101>.","Analysis of this protein sequence reveals the following: Signal Score (−7.5): −1.23 Possible cleavage site: 47 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 3.55 threshold: 0.0 PERIPHERAL Likelihood = 3.55 modified ALOM score: −1.21 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.126(Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi|9632520|ref|NP_049514.1|hypothetical protein [Bacteriophage 933W] gi|9633449|ref|NP_050552.1|hypothetical protein [Bacteriophage VT2-Sa] gi|4585431|gb|AAD25459.1|AF125520_54 (AF125520) hypothetical protein [Bacteriophage 933W] gi|5881645|dbj|BAA84336.1|(AP000363) hypothetical protein [Bacteriophage VT2-Sa] gi|7649882|dbj|BAA94l60.1|(AP000422) hypothetical protein [Escherichia coli O157:H7] Length = 404 Score = 177 bits (449), Expect = 3e−43 Identities = 130/425 (30%), Positives = 204/425 (47%), Gaps = 27/425 (6%) Query: 7 TAYGDPQAMMKQAAGLFAMHMQRNSTLNRLAGKMPAGTA-GAEATLRKQTTQHMPVVRCQ 65 T QA LF + S +N L + A A + KQT+ PVVR Sbjct: 2 TTVTSAQANKLYQVALFTAANRNRSMVNILTEQQEAPKAVSPDKKSTKQTSAGAPVVRIT 61 Query: 66 DLTRGMGDEIRFNLVNPVSALPIMGDNTAEGRGVGMSLSEAGLRVNQARFPVDGGGTMTN 125 DL + GDE+ F++++ +S P MGD EGRG +S ++ L++NQ R VD GG M+ Sbjct: 62 DLNKQAGDEVTFSIMHKLSKRPTMGDERVEGRGEDLSHADFSLKINQGRHLVDAGGRMSQ 121 Query: 126 QRSPADYRALIRPAAQSLMDRYADQTLLVHMAGARGFHDNIEWGVPLAGDPKFNDYAVNP 185 QR+ + + R + + DQ +VH+AGARG + +P A P+F +N Sbjct: 122 QRTKFNLASSARTLLGTYFNDLQDQCAIVHLAGARGDFVADDTILPTAEHPEFKKIMIND 181 Query: 186 VKAPSKNRHFTASGDAVTGVGDNGGELKIASTDLFTMDTVDSMRTVLDQIPLPPPIVKFE 245 V P+ +RHF GD +I + D+F++ VD++ +D++ P V+ Sbjct: 182 VLPPTHDRHFFG--------GDATSFEQIEAADIFSIGLVDNLSLFIDEMAHPLQPVRLS 233 Query: 246 GDKAAGDSPLRVWLLSPAQYNRF---AADPKFRQLQASAIARASQANQNPLFLGDAGLWN 302 GD+ G+ P V ++P Q+N + + + Q+ A+ RA N +PLF G+ +W Sbjct: 234 GDELHGEDPYYVLYVTPRQWNDWYTSTSGKDWNQMMVRAVNRAKGFN-HPLFKGECAMWR 292 Query: 303 GFILVKMP-RPIRFYAGDEMKYCADKFSEAESGLKIPASFADKFAVDRSVILGGQAVLEA 361 ++ K PIRFY G ++ + + A +DR++++LG QA+ A Sbjct: 293 NILVRKYAGMPIRFYQGSKVLVSENNLTATTK------EVAAATNIDRAMLLGAQALANA 346 Query: 362 FANTGKHGGMPFFWSEKELDHGNRVETLVGTIRGVAKTRFAVDVGGGAKEITDYGVTVVD 421 + G+ G F EK+ D NR E + I G+ K RF G ++ D+GV VD Sbjct: 347 Y---GQKAGGHFNMVEKKTDMDNRTEIAISWINGLKKIRFPEKSG----KMQDHGVIAVD 399 Query: 422 TVVPL 426 T V L Sbjct: 400 TAVKL 404 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 102 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 203> which encodes amino acid sequence <SEQ ID 204; NGS 102>.","Analysis of this protein sequence reveals the following: Signal Score (−7.5): −6.09 Possible cleavage site: 15 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 2.92 threshold: 0.0 PERIPHERAL Likelihood = 2.92 modified ALOM score: −1.08 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.480(Affirmative) < succ The protein has no homology with sequences in the databases.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 103 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 205> which encodes amino acid sequence <SEQ ID 206; NGS 103>.","Analysis of this protein sequence reveals the following: Signal Score (−7.5): −1.29 Possible cleavage site: 34 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 1 value: −0.00 threshold: 0.0 INTEGRAL Likelihood = −0.00 Transmembrane 22-38 (22-38) PERIPHERAL Likelihood = 4.88 modified ALOM score: 0.50 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.100(Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi|11277848|pir||E81145 replicative DNA helicase NMB0885 [imported]-Neisseria meningitidis (group B strain MD58) gi|7226124|gb|AAF41296.1|(AE002441) replicative DNA helicase [Neisseria meningitidis MC58] Length = 468 Score = 233 bits (594), Expect = 5e−60 Identities = 158/456 (34%), Positives = 245/456 (53%), Gaps = 34/456 (7%) Query: 15 SVGAEQNILGGILIEPTAIARCA-ILTPEKFYQAQHRIIFRALLDMAAANEPIDIITLND 73 S+ AEQ++LGG+++E A R A +++ E FY+ +HR+IFR++ + + P D+IT+ + Sbjct: 23 SMEAEQSVLGGLMLENPAWDRIADVVSGEDFYRHEHRLIFRSIAKLINESRPADVITVQE 82 Query: 74 KLEARGEAENAGGLAYLIDLNQNTPSAKNISRYVGIVNDRFVERGLLKASAAIEKIAVSK 133 L+ E E AGG YLI L QNTPSA NI RY IV +R + R L + I + A + Sbjct: 83 DLQRNEELEAAGGFEYLITLAWNTPSAANIRRYAEIVRERSIMRQLAEVGTEIARSAYNP 142 Query: 134 DGGTVAEKLSKAADELAAVGKDAVKRETKTFGQTVEDLIGGLDKRLDGVR--------FG 185 G + L +A +++ + + K +K + DL+ + +R+D + G Sbjct: 143 QGRDAGQLLDEAENKVFQIAESTAK--SKQGFLEMPDLLKEVVQRIDMLYSRDNPDEVTG 200 Query: 186 LPTGLMKLDGMTGGLPDGNLIVIAARPSMGKTVLAENIARFALKQGK-AVHFQSYEMSAV 244 +PTG + LD T GL G+LI++A RPSMGKT + NIA +G+ V S EM Sbjct: 201 VPTGFIDLDKKTSGLQPGDLIIVAGRPSMGKTAFSINIAEHVAVEGRLPVAVFSMEMGGA 260 Query: 245 ELARRGMAAECNIPMQNLKTGNLTQSDYANM----------PIYVSQAKEWKFDVNCDLL 294 +L R + + + LKTG L + + P+Y+ + Sbjct: 261 QLVMRMLGSVGRLDQSVLKTGRLEDEHWGRLNEAVVKLSDAPVYIDETPGLTALELRARA 320 Query: 295 NVDELCFLAKEKKLTTGLDLLVVDHLHIMPRAGRDE--VAELGNISRRLKNLAAELNTPV 352 F K L L+V+D+L +M +GR + +ELG ISR LK LA EL P+ Sbjct: 321 RRLARQFNNK-------LGLIVIDYLQLMAGSGRSDNRASELGEISRSLKALAKELQVPI 373 Query: 353 VLVAQLNRGNTKQADKRPNMADIRGSGAIEQDANIIIMPHRESYYDGNENP--SIAELII 410 + ++QL+R + DKRP M+D+R SGAIEQDA++I+ +R+ YY+ ++P +AE II Sbjct: 374 IALSQLSRTVESRTDKRPMMSDLRESGAIEQDADLIMFLMYRDEYYN-QDSPMKGLAECII 432 Query: 411 AKNRDGEMGTVVCGWKGQFMKFEEEPDLAWQAPKHD 446 K+R+G +G + W GQF KF+ + +A D Sbjct: 433 GKHRNGPVGKIFLTWTGQFTKFDNAAYIPEEAKIED 468 >gi|11277846|pir||E81876 probable replicative DNA helicase (EC 3.6.1.-) NMA1105 [imported]- Neisseria meningitidis (group A strain Z2491) gi|7379799|emb|CAB84367.1|(AL162755) putative replicative DNA helicase [Neisseria meningitidis Z2491] Length = 468 Score = 230 bits (588), Expect = 2e−59 Identities = 158/456 (34%), Positives = 244/456 (52%), Gaps = 34/456 (7%) Query: 15 SVGAEQNILGGILIEPTAIARCA-ILTPEKFYQAQHRIIFRALLDMAAANEPIDIITLND 73 S+ AEQ++LGG+++E A R A +++ E FY+ +HR+IFR++ + + P D+IT+ + Sbjct: 23 SMEAEQSVLGGLMLENPAWDRIADVVSGEDFYRHEHRLIFRSIAKLINESRPADVITVQE 82 Query: 74 KLEARGEAENAGGLAYLIDLNQNTPSAKNISRYVGIVNDRFVERGLLKASAAIEKIAVSK 133 L+ E E AGG YLI L QNTPSA NI RY IV +R + R L + I + A + Sbjct: 83 DLQRNEELEAAGGFEYLITLAQNTPSAANIRRYAEIVRERSIMQLAEVGTEIARSAYNP 142 Query: 134 DGGTVAEKLSKAADELAAVGKDAVKRETKTFGQTVEDLIGGLDKRLDGVR--------FG 185 G + L +A +++ + + K +K + DL+ + +R+D + G Sbjct: 143 QGRDAGQLLDEAENKVFQIAESTAK--SKQGFLEMPDLLKEVVQRIDMLYSRDNPDEVTG 200 Query: 186 LPTGLMKLDGMTGGLPDGNLIVIAARPSMGKTVLAENIARFALKQGK-AVHFQSYEMSAV 244 + TG + LD T GL G+LI++A RPSMGKT + NIA +GK V S EM Sbjct: 201 VSTGFIDLDKKTSGLQPGDLIIVAGRPSMGKTAFSINIAEHVAVEGKLPVAVFSMEMGGA 260 Query: 245 ELARRGMAAECNIPMQNLKTGNLTQSDYANM----------PIYVSQAKEWKFDVNCDLL 294 +L R + + + LKTG L + + P+Y+ + Sbjct: 261 QLVMRMLGSVGRLDQSVLKTGRLEDEHWGRLNEAVVKLSDAPVYIDETPGLTALELRARA 320 Query: 295 NVDELCFLAKEKKLTTGLDLLVVDHLHIMPRAGRDE--VAELGNISRRLKNLAAELNTPV 352 F K L L+V+D+L +M +GR + +ELG ISR LK LA EL P+ Sbjct: 321 RRLARQFNNK-------LGLIVIDYLQLMAGSGRSDNRASELGEISRSLKALAKELQVPI 373 Query: 353 VLVAQLNRGNTKQADKRPNMADIRGSGAIEQDANIIIMPHRESYYDGNENP--SIAELII 410 + ++QL+R + DKRP M+D+R SGAIEQDA++I+ +R+ YY+ ++P +AE II Sbjct: 374 IALSQLSRTVESRTDKRPMMSDLRESGAIEQDADLIMFMYRDEYYN-QDSPMKGLAECII 432 Query: 411 AKNRDGEMGTVVCGWKGQFMKFEEEPDLAWQAPKHD 446 K+R+G +G + W GQF KF+ + +A D Sbjct: 433 GKHRNGPVGKIFLTWTGQFTKFDNAAYIPEEAKIED 468 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 104 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 207> which encodes amino acid sequence <SEQ ID 208; NGS104>.","Analysis of this protein sequence reveals the following: Signal Score (−7.5): −2.11 Possible cleavage site: 15 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 5.04 threshold: 0.0 PERIPHERAL Likelihood = 5.04 modified ALOM score: −1.51 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.220(Affirmative) < succ The protein has homology with the following sequences in the databases: >gi|7515458|pir||T13296 hypothetical protein 8-Streptococcus phage phi- O1205 gi|2444088|gb|AAC79524.1|(U88974) ORF8 [Streptococcus thermophilus temperate bacteriophage O1205] Length = 157 Score = 62.1 bits (150), Expect = 3e−09 Identities = 53/161 (32%), Positives = 86/161 (52%), Gaps = 8/161 (4%) Query: 5 TLYRCAADVQAGLDYYFDSETEREDTLEAV--IGQFEVKAQSVIAYIKNQEITEKMLEGH 62 TLY + + D ET + DTLEA+ +E K + + IK+ E + + Sbjct: 3 TLYELTDQLLEIYNMDVDDET-KLDTLEAIDWTTDYENKVEGYVKVIKSLEADIEARKNE 61 Query: 63 IRQMTGKLKAAKARNQSLKDYLARNMQAAGITEIKADDGTFKASFRKSEAVVILDEAQIP 122 +++ G K+ +++ LK LA +M G T + D FK FRKSEAVV+ +E ++P Sbjct: 62 KKRLDGLNKSDQSKIDKLKTALAVSMAETGQTRV--DTTLFKVGFRKSEAVVV-NEEEKLP 118 Query: 123 AEFMREAVKTEPDKTAIRKAIESGRQVAGAKIEGRKNLQIR 163 E+ K PDK +++ ++SG+ + GA +E R+NL IR Sbjct: 119 KEYQIATYK--PDKKTLKBLLKSGKHIEGATLEERRNLNIR 157 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 105 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 209> which encodes amino acid sequence <SEQ ID 210; NGS 105>.","Analysis of this protein sequence reveals the following: Signal Score (−7.5): −5.52 Possible cleavage site: 31 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 2.60 threshold: 0.0 PERIPHERAL Likelihood = 2.60 modified ALOM score: −1.02 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.135(Affirmative) < succ> The protein has no homology with sequences in the databases.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 106 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 211> which encodes amino acid sequence <SEQ ID 212; NGS 106>.","Analysis of this protein sequence reveals the following: Signal Score (−7.5): 4.8 Possible cleavage site: 26 >>> Seems to have a cleavable N-term signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 27 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 7.80 threshold: 0.0 PERIPHERAL Likelihood = 7.80 modified ALOM score: −2.06 Score for OM-PP discrimination: 4.38 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: 4.38 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Outer membrane?","Score: 0.437687 Outer membrane?","Score: 0.437687 ----- Final Results ----- bacterial outer membrane --- Certainty= 0.768(Affirmative) < succ> The protein has no homology with sequences in the databases, although it is similar to HMW1 from Haemophilus influenzae.","The protein was expressed in E.coli as an insoluble 43.56 kDa His-fusion product and then purified.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 107 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 213> which encodes amino acid sequence <SEQ ID 214; NGS 107>.","Analysis of this protein sequence reveals the following: Signal Score (−7.5): −3.83 Possible cleavage site: 51 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 4.61 threshold: 0.0 PERIPHERAL Likelihood = 4.61 modified ALOM score: −1.42 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.146(Affirmative) < succ> The protein has no homology with sequences in the databases.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 108 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 215> which encodes amino acid sequence <SEQ ID 216; NGS108>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −6.14 Possible cleavage site: 19 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 8.43 threshold: 0.0 PERIPHERAL Likelihood = 8.43 modified ALOM score: −2.19 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.574(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir||G81977 probable lipoprotein NMA0586 [imported]-Neisseria meningitidis (group A strain Z2491) emb|CAB83877.1|(AL162753) putative lipoprotein [Neisseria meningitidis Z2491] Length = 280 Score = 52.9 bits (126), Expect = 5e−06 Identities = 43/134 (32%), Positives = 63/134 (46%), Gaps = 23/134 (17%) Query: 174 LGDIRGVATDEDKLPKAGSFQYEGRAFGGNGVLSKESLDNHNGVFRYTIDFDRRKGSGSI 233 +GDI G T DKLP+ G Y G AFG D+ +G YTIDF ++G G I Sbjct: 156 IGDIAGEHTSFDKLPEGGRATYRGTAFGS---------DDASGKLTYTIDFAAKQGHGKI 206 Query: 234 EGMEQYGKIKLEEAAIERIPYRESGSSLGLKDRVSYFGVNEGVAMLEKDNEIKKYHLGIF 293 E ++ ++ ++ AA + P ++ + + ++L E Y LGIF Sbjct: 207 EHLKS-PELNVDLAASDIKPDKKRHAVI-------------SGSVLYNQAEKGSYSLGIF 252 Query: 294 GEAANEVAGAVSQE 307 G A EVAG+ E Sbjct: 253 GGQAQEVAGSAEVE 266 >pir||D81032 hypothetical protein NMB1870 [imported]-Neisseria meningitidis (group B strain MD58) gb|AAF42204.1|(AE002537) hypothetical protein [Neisseria meningitidis MC58] Length = 320 Score = 50.6 bits (120), Expect = 3e−05 Identities = 50/168 (29%), Positives 76/168 (44%), Gaps = 28/168 (16%) Query: 136 VYEQPYSVVRGYFGYSRKDGNPIEGDGQNPEEIPFDLYLGDIRGVATDEDKLPKAGSFQY 195 VY+Q +S + + +D E G+ + F +GDI G T DKLP+ G Y Sbjct: 163 VYKQSHSALTAFQTEQIQDS---EHSGKMVAKRQFR--IGDIAGEHTSFDKLPEGGRATY 217 Query: 196 EGRAFGGNGVLSKESLDNHNGVFRYTIDFDRRKGSGSIEGMEQYGKIKLEEAAIERIPYR 255 G AFG D+ G YTIDF ++G+G IE ++ ++ ++ AA + P Sbjct: 218 RGTAFGS---------DDAGGKLTYTIDFAAKQGNGKIEHLKS-PELNVDLAAADIKPDG 267 Query: 256 ESGSSLGLKDRVSYFGVNEGVAMLEKDNEIKKYHLGIFGEAANEVAGA 303 + + + ++L E Y LGIFG A EVAG+ Sbjct: 268 KRHAVI-------------SGSVLYNQAEKGSYSLGIFGGKAQEVAGS 302 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 109 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 217> which encodes amino acid sequence <SEQ ID 218; NGS109>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.39 Possible cleavage site: 25 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 7.00 threshold: 0.0 PERIPHERAL Likelihood = 7.00 modified ALOM score: −1.90 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.353(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir||A82012 hypothetical protein NMA0179 [imported]-Neisseria meningitidis (group A strain Z2491) emb|CAB83494.1|(AL162752) hypothetical protein NMA0179 [Neisseria meningitidis Z2491] Length = 97 Score = 183 bits (464), Expect = 1e−45 Identities = 92/97 (94%), Positives = 95/97 (97%) Query: 44 MKANDKLNRQIDVLQKQSAAIHNEAYIEMNTLLYRHREVVSIHNRKADYAEKGKERIALF 103 MK NDKLNRQIDVLQKQSAAIHNEAYIEMNTLLYRHREVVS+HNRKADYAEKGKE+IALF Sbjct: 1 MKTNDKLNRQIDVLQKQSAAIHNEAYIEMNTLLYRHREVVSVHNRKADYAEKGKEQIALF 60 Query: 104 PRGLNGITKLPAAVLLPERPYHFDMKEVLYIFSRIPR 140 PRGLNGITKLPAAVLLPERPYHFDMKEVL+IFS IPR Sbjct: 61 PRGLNGITKLPAAVLLPERPYHFDMKEVLHIFSWIPR 97 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, NGS109 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 110 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 219> which encodes amino acid sequence <SEQ ID 220; NGS110>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −2.76 Possible cleavage site: 41 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 1 value: −0.00 threshold: 0.0 INTEGRAL Likelihood = −0.00 Transmembrane 88-104 (88-104) PERIPHERAL Likelihood = 7.69 modified ALOM score: 0.50 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.100(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has no homology with sequences in the databases.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 111 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 221> which encodes amino acid sequence <SEQ ID 222; NGS111>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.89 Possible cleavage site: 21 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 2.44 threshold: 0.0 PERIPHERAL Likelihood = 2.44 modified ALOM score: −0.99 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.293(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gb|AAC45840.1|(AF001598) restriction endonuclease [Neisseria gonorrhoeae] Length = 374 Score = 539 bits (1390), Expect = e−152 Identities = 285/285 (100%), Positives = 285/285 (100%) Query: 1 MGFIEPFLSSYTPLSRDYVQARTNRKRQTLLSKIVYTHSGFQRSVTENSNIRQINFLIKT 60 MGFIEPFLSSYTPLSRDYVQARTNRKRQTLLSKIVYTHSGFQRSVTENSNIRQINFLIKT Sbjct: 90 MGFIEPFLSSYTPLSRDYVQARTNRKRQTLLSKIVYTHSGFQRSVTENSNIRQINFLIKT 149 Query: 61 LVEHPQGKLNKKEIAAMMLVDLKTFQQDYLTETELNDYFQQGIESGFIERKYNQISYLWN 120 LVEHPQGKLNKKEIAAMMLVDLKTFQQDYLTETELNDYFQQGIESGFIERKYNQISYLWN Sbjct: 150 LVEHPQGKLNKKEIAAMMLVDLKTFQQDYLTETELNDYFQQGIESGFIERKYNQISYLWN 209 Query: 121 LLDKLDDLKRVGDDLYFAEDAQRIFGNLDEITVRKRDPYLHRLYKNQLQEESEEHYGNVK 180 LLDKLDDLKRVGDDLYFAEDAQRIFGNLDEITVRKRDPYLHRLYKNQLQEESEEHYGNVK Sbjct: 210 LLDKLDDLKRVGDDLYFAEDAQRIFGNLDEITVRKRDPYLHRLYKNQLQEESEEHYGNVK 269 Query: 181 CMLEKLAYPVLIASHIKPFILSDDTEAYDPNNGLLLSRTLDSLFDLKYISFDDEGNMVKS 240 CMLEKLAYPVLIASHIKPFILSDDTEAYDPNNGLLLSRTLDSLFDLKYISFDDEGNMVKS Sbjct: 270 CMLEKLAYPVLIASHIKPFILSDDTEAYDPNNGLLLSRTLDSLFDLKYISFDDEGNMVKS 329 Query: 241 KRLSDDVWRRWCDVKLDNNLLNDKRKSYLAYHRELMLQEDQEFHI 285 KRLSDDVWRRWCDVKLDNNLLNDKRKSYLAYHRELMLQEDQEFHI Sbjct: 330 KRLSDDVWRRWCDVKLDNNLLNDKRKSYLAYHRELMLQEDQEFHI 374 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 112 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 223> which encodes amino acid sequence <SEQ ID 224; NGS112>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −9.08 Possible cleavage site: 54 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 1 value: −1.22 threshold: 0.0 INTEGRAL Likelihood = −1.22 Transmembrane 160-176 (160-177) PERIPHERAL Likelihood = 0.58 modified ALOM score: 0.74 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.149(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_12644572 gi|12644572|sp|Q50973|T2B1_NEIGO TYPE II RESTRICTION ENZYME NGOBI (ENDONUCLEASE NGOBI) (R.NGOBI) (R.NGOI) gb|AAB03207.2|(U42459) NgoI restriction endonuclease R.NgoI [N. gonorrhoeae] Length = 350 Score = 694 bits (1791), Expect = 0.0 Identities = 349/350 (99%), Positives = 349/350 (99%) Query: 1 MTLEEQQAKEALDGIIKKSRVHLYKPIQIAEILYHDRCIKQLDFLNLDTYRNQSKRWRDE 60 MTLEEQQAKEALDGIIKKSRVHLYKPIQIAEILYHDRCIKQLDFLNLDTYRNQSKRWRDE Sbjct: 1 MTLEEQQAKEALDGIIKKSRVHLYKPIQIAEILYHDRCIKQLDFLNLDTYRNQSKRWRDE 60 Query: 61 ICRRFLGISTSSAKFQDNLFEKNAIPPEKLAVLGTLNRQSDGGVESTITKQFFNRFSQM 120 ICRRFLGISTSSAKFQDNLFEKNAIPPEKLAVLGTLNRQSDGGVESTITKQFFNRFSQM Sbjct: 61 ICRRFLGISTSSAKFQDNLFEKNAIPPEKLAVLGTLNRQSDGGVESTITKQFFNRFSQM 120 Query: 121 SEALAYVGNTDRYSFQLSEFLNLFWLEPGLKRSIDKIYEIVVYALFDALVSELGITVSID 180 SEALAYVGNTDRYSFQLSEFLNLFWLEPGLKRSIDKIYEIVVYALFDALVSELGITVSID Sbjct: 121 SEALAYVGNTDRYSFQLSEFLNLFWLEPGLKRSIDKIYEIVVYALFDALVSELGITVSID 180 Query: 181 FPKENLFLWEEYQDFAEKIITMPKNEHLKLPAKIHRVGVTNAADRGLDMWSNFGLAIQVK 240 FPKENLFLWEEYQDFAEKIITMPKNEHLKLPAKIHRVGVTNAADRGLDMWSNFGLAIQVK Sbjct: 181 FPKENLFLWEEYQDFAEKIITMPKNEHLKLPAKIHRVGVTNAADRGLDMWSNFGLAIQVK 240 Query: 241 HLSLDEELAEDIVSSISADRIVIVCKKAEQSVIVSLLTQIGWKSRIQNIVTEDDLISWYE 300 HLSLDEELAEDIVSSISADRIVIVCKKAEQSVIVSLLTQIGWKSRIQNIVTEDDLISWYE Sbjct: 241 HLSLDEELAEDIVSSISADRIVIVCKKAEQSVIVSLLTQIGWKSRIQNIVTEDDLISWYE 300 Query: 301 KALRQQYPIAEALLENIKTEIMREFPAVNEANEFLDFAQNRGYDITVTHF 350 KALRQQYPIAEALLENIKTEIMREFPAVNEANEFLDFAQNRGYDITVTHF Sbjct: 301 KALRQQYPIAEALLENIKTEIMREFPAVNEANEFLDFAQNRGYDITVTHF 350 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 113 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 225> which encodes amino acid sequence <SEQ ID 226; NGS113>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −1.7 Possible cleavage site: 43 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 4 value: −9.77 threshold: 0.0 INTEGRAL Likelihood = −9.77 Transmembrane 187-203 (183-208) INTEGRAL Likelihood = −7.22 Transmembrane 25-41 (19-46) INTEGRAL Likelihood = −4.14 Transmembrane 139-155 (138-155) INTEGRAL Likelihood = −2.87 Transmembrane 86-102 (85-102) PERIPHERAL Likelihood = 1.27 modified ALOM score: 2.45 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.491(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_15902668 gi|15902668|ref|NP_358218.1|\\(NC_003098) ABC transporter membrane-spanning permease-glutamine transport [Streptococcus pneumoniae R6] gb|AAK99428.1|(AE008440) ABC transporter membrane-spanning permease- glutamine transport [Streptococcus pneumoniae R6] Length = 226 Score = 218 bits (556), Expect = 7e−56 Identities = 113/218 (51%), Positives = 155/218 (70%) Query: 1 MNWPYLIDAVPKFADAAKLTLELSVYGVVLSLLFLPVAVVTAYRIRPFYALARAYIELS 60 M+W + +P + A LTL ++V+G++ S L GL V+++ YRI +A AYIEILS Sbjct: 1 MNWPYLIDAVPKFADAAKLTLELSVYGVVLSLLFGLPVAVVTAYRIRPFYALARAYIELS 60 RNTPLLIQLFFLY+GLP++GI C + LVFLG SYMAE+ R+G+ A+ + Q G Query: 61 RNTPLLIQLFFLYYGLPKMGIKWDGFTCGVIALVFLGASYMAEAVRAGILAVPKGQVGAG 120 RNTPLLIQLFFLY+GLP++GI C +LVFLG SYMAE+ R+G+ A+ + G G Sbjct: 61 RNTPLLIQLFFLYFGLPRIGIVLSSEVCATLGLVFLGGSYMAESFRSGLEAISQTQQEIG 120 Query: 121 KAIGLSRFQVFRYVELPQVWAVAVPAIGANILFLMKETSVVSTVGIAELLFVTKDVIGMD 180 AIGL+ QVFRYV LPQ AVA+P+ AN++FL+KETSV S V +A+L++V KD+IG+ Sbjct: 121 LAIGLTPLQVFRYVVLPQATAVALPSFSANVIFLIKETSVFSAVALADLMYVAKDLIGLY 180 Query: 181 YKTNEALFLLFAAYLIILLPVSLLARRIENRVRSAKYG 218 Y+T+ AL +L AYLI+LLP+SL+ IE R+R A +G Sbjct: 181 YETDIALAMLVVAYLIMLLPISLVFSWIERRIRHAGFG 218 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 114 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 227> which encodes amino acid sequence <SEQ ID 228; NGS 114>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −0.46 Possible cleavage site: 17 >>> Seems to have a cleavable N-term signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 18 ALOM: Finding transmembrane regions (Klein et al.)","count: 3 value: −5.36 threshold: 0.0 INTEGRAL Likelihood = −5.36 Transmembrane 50-66 (47-67) INTEGRAL Likelihood = −4.83 Transmembrane 183-199 (176-200) INTEGRAL Likelihood = −1.81 Transmembrane 72-88 (72-88) PERIPHERAL Likelihood = 0.26 modified ALOM score: 1.57 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.314(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_15902667 gi|15902667|ref|NP_358217.1|\\(NC_003098) ABC transporter membrane-spanning permease-glutamine transport [Streptococcus pneumoniae R6] gb|AAK99427.1|(AE008440) ABC transporter membrane-spanning permease- glutamine transport [Streptococcus pneumoniae R6] Length = 225 Score = 218 bits (555), Expect = 9e−56 Identities = 111/206 (53%), Positives = 151/206 (72%) Query: 3 EGLLLTAQISLISVAASCVLGTLFGLVLRSRNRLVRFVGRFYLETIRIVPILVWLFGLYF 62 +GL +T IS++SV S + GT+ G+++ S +R++RF+ R YLE IRI+P LV LF +YF Sbjct: 20 QGLGVTIGISILSVLLSMMFGTVMGIIMTSHSRIIRFLTRLYLEFIRIMPQLVLLFIVYF 79 Query: 63 GLSVWTGIHIGGFWVCVWVFSLWGVAEMGKLVRGALESIEKHQVESGLAPGLSRGQVFRC 122 GL+ I+I G + VF+LWG AEMGDLVRGA+ S+ KHQ ESG A GL+ Q++ Sbjct: 80 GLARNFNINISGETSAIIVFTLWGTAEMGDLVRGAITSLPKHQFESGQALGLTNVQLYYH 139 Query: 123 IELPQSIRRVLPGAVNLFTRMIKTSSLAWLIGVIEVVKVGQQIIENSLLTQPNASFWVYG 182 I +PQ +RR+LP A+NL TRMIKT+SL LIGV+EV KVGQQII+++ LT P ASFW+YG Sbjct: 140 IIIPQVLRRLLPQAINLVTRMIKTTSLVVLIGVVEVTKVGQQIIDSNRLTIPTASFWIYG 199 Query: 183 LIFMLYFFCCWPLSLLAAKLEQKWEH 208 I +LYF C+P+S L+ LE+ W + Sbjct: 200 TILVLYFAVCYPISKLSTHLEKHWRN 225 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 115 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 229> which encodes amino acid sequence <SEQ ID 230; NGS 115>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −0.639999 Possible cleavage site: 38 >>> May be a lipoprotein Amino Acid Composition of Predicted Mature Form: calculated from 23 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 5.25 threshold: 0.0 PERIPHERAL Likelihood = 5.25 modified ALOM score: −1.55 Rule: inner or outer membrane protein Rule: inner or outer membrane protein *** Reasoning Step: 2 Lipoprotein?","Inner membrane?","----- Final Results ----- bacterial outer membrane --- Certainty= 0.790(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.700(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_4588485 gi|4588485|gb|AAD26123.1|\\(AF109148) antigenic protein [Actinobacillus pleuropneumoniae] Length = 278 Score = 407 bits (1045), Expect = e−112 Identities = 212/282 (75%), Positives = 242/282 (85%), Gaps = 7/282 (2%) Query: 1 MKLNAKLKALLASAAIAVGLTACGGGSGDAQSSQSSGAA-TVAAIKEKGVIRIGVFGDKP 59 MKL+ LK LLA+A A LTAC +A ++QSS A +VA IKEKGVIRIGVFGDKP Sbjct: 1 MKLSTTLKTLLATAITAFALTACD----NANNAQSSTAKDSVAQIKEKGVIRIGVFGDKP 56 Query: 60 PFGYVDANGKNQGFDVEIAKDLAKDLLGSPDKVEFVLTEAANRVEYVRSGKVDLILANFT 119 PFGYVDANGK+QGFDVEIAK++A DLLGS DKVEFVLTEAANRVEY++S KVDLILANFT Sbjct: 57 PFGYVDANGKSQGFDVEIAKEIANDLLGSSDKVEFVLTEAANRVEYLKSNKVDLILANFT 116 Query: 120 QTPERAEAVDFADPYMKVALGVVSPKNKPITDMAQLKDQTLLVNKGTTADAFFTKSHPEV 179 +TPERAE VDFA PYM VALGVVSPK + I+D+ QL+ +TLLVNKGTTADA+FTK+HPE+ Sbjct: 117 KTPERAEVVDFAAPYMNVALGVVSPKVRLISDLKQLEGKTLLVNKGTTADAYFTKNHPEI 176 Query: 180 KLLKFDQNTETFDALKDGRGVALAHDNALLWAWAKENPNFEVAIGNLGPAEFIAPAVQKG 239 LLKFDQNTETFDALKDGRGVALAHDNAL+WAWAKENP F+VAIG++GPAE IAPVQKG Sbjct: 177 NLLKFDQNTETFDALKDGRGVALAHDNALVWAWAKENTFDVAIGSVGPAEQIAPAVQKG 236 Query: 240 NADLLNWVNGEIAAMKKDGRLKAAYEKTLLPVYGEKVKPEAL 281 N LL+ +N EIA K +G+LKAAYEKTL+PVYG+ KPE L Sbjct: 237 NQALLDVINKEIAEFKTNGKLKAAYERKTLVPVYGD--KPELL 276 The protein was expressed in E.coli as a soluble 28.16 kDa His-fusion product, lacking its leader peptide and its poly-glycine sequence (GGGSG), and then purified.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 116 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 231> which encodes amino acid sequence <SEQ ID 232; NGS116>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −7.13 Possible cleavage site: 61 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 1 value: −1.86 threshold: 0.0 INTEGRAL Likelihood = −1.86 Transmembrane 51-67 (51-67) PERIPHERAL Likelihood = 1.54 modified ALOM score: 0.87 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.174(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has no homology to sequences in the databases.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 117 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 233> which encodes amino acid sequence <SEQ ID 234; NGS117>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): 0.25 Possible cleavage site: 40 >>> Seems to have a cleavable N-term signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 41 ALOM: Finding transmembrane regions (Klein et al.)","count: 2 value: −4.57 threshold: 0.0 INTEGRAL Likelihood = −4.57 Transmembrane 100-116 (99-118) INTEGRAL Likelihood = −1.59 Transmembrane 54-70 (54-70) PERIPHERAL Likelihood = 0.53 modified ALOM score: 1.41 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.283(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_15793413 gi|15793413|ref|NP_283235.1|\\(NC_003116) putative integral membrane protein [Neisseria meningitidis Z2491] pir||C81957 probable integral membrane protein NMA0408 [imported]- Neisseria meningitidis (group A strain Z2491) emb|CAB83707.1|(AL162753) putative integral membrane protein [Neisseria meningitidis Z2491] Length = 550 Score = 1115 bits (2885), Expect = 0.0 Identities = 539/550 (98%), Positives = 545/550 (99%) Query: 1 MVAYAFLFLFVTAAVLLIVRSHYRWTYFFASALFVFLAGGMLMLTAQWQRALNFASVWFV 60 MVAY FLFLFVTAA++LI+RSHYRWTYFFASALFVFLAGGMLMLTAQWQRALNFASVWFV Sbjct: 1 MVAYVFLFLFVTAALVLIIRSHYRWTYFFASALFVFLAGGMLMLTAQWQRALNFASVWFV 60 Query: 61 VLILFHRLKIHYYKQPLLISDFLLIADWRNWETLFHYKEAVIGMAGLLALAGYAVFGWSG 120 VLILFHRLKIHYYKQPLLISDFLLIADWRNWETLFHYKEAVIGMAGLLALA YAVFGWSG Sbjct: 61 VLILFHRLKIHYYKQPLLISDFLLIADWRNWETLFHYKEAVIGMAGLLALAAYAVFGWSG 120 Query: 121 ADSLGMPWRWAGAVLFAAAFVSVRHFSKHPGAVKTWLDSLPDDGRDVFLNLPMSCRAVFF 180 ADSL +PWRWAGAVLFAAAFVS+RHFSKHPGAVKTWLDSLPDDGRDVFLNLPMSCRAVFF Sbjct: 121 ADSLDVPWRWAGAVLFAAAFVSMRHFSKHPGAVKTWLDSLPDDGRDVFLNLPMSCRAVFF 180 Query: 181 QVPVFEGDGEAFARQMPSETRPYGMSDEKPDIVVTLMESTLDPHCFDFAAAKIPDLKMFG 240 QVPVFEGDGEAFARQMPSETRP GMSDEKPDIVVTLMESTLDPHCFDFAAAKIPDLKMFG Sbjct: 181 QVPVFEGDGEAFARQMPSETRPCGMSDEKPDIVVTLMESTLDPHCFDFAAAKIPDLKMFG 240 Query: 241 RQEDTVFSSPLRVHTFGGATWKSEFAFLAGVPSTDFGALASGVFYSVVPHLQTGFVRNLR 300 RQEDTVFSSPLRVHTFGGATWKSEFAFLAGVPSTDFGALASGVFYSVVPHLQTGFVRNLR Sbjct: 241 RQEDTVFSSPLRVHTFGGATWKSEFAFLAGVPSTDFGALASGVFYSVVPHLQTGFVRNLR 300 Query: 301 EHGYFCVALSPFTKGNYNAKAAYDHFGFNLMFQPQDLGYPAPMGKNLWHISSEEMMQYAR 360 EHGYFCVALSPFTKGNYNAKAAYDHFGFNLMFQPQDLGYPAPMGKNLWHISSEEMMQYAR Sbjct: 301 EHGYFCVALSPFTKGNYNAKAAYDHFGFNLMFQPQDLGYPAPMGKNLWHISSEEMMQYAR 360 Query: 361 MILEKRHPDLENVRQPMFVYVLTMKEHGPYRTDTDNVFDLDAPDLNAKTVSALNDYIGRI 420 MILEKRHPDLENVRQPMFVYVLTMKEHGPYRTDTDNVFDLDAPDLNAKTVSALNDYIGRI Sbjct: 361 MILEKRHPDLENVRQPMFVYVLTMKEHGPYRTDTDNVFDLDAPDLNAKTVSALNDYIGRI 420 Query: 421 ADLDKAVESFDRYLHERGKPFVFGYFGDHQVPFEGVSVRKKWDYAQPDYVTQFAVRSNIA 480 ADLDKAVESFDRYLHERGKPFVFGYFGDHQVPFEGVSVRKKWDYAQPDYVTQFAVRSNIA Sbjct: 421 ADLDKAVESFDRYLHERGKPFVFGYFGDHQVPFEGVSVRKKWDYAQPDYVTQFAVRSNIA 480 Query: 481 GGFVQRQDFLDLAFAGGVLMEAAGLEAKDGFMRANMAMRGLCGGGLEDCPNRELVGNYRN 540 GGFVQRQ+FLDLAFAGGVLMEAAGLEAKDGFMRANMAMRGLCGGGLEDCPN ELVGNYRN Sbjct: 481 GGFVQRQNFLDLAFAGGVLMEAAGLEAKDGFMRANMAMRGLCGGGLEDCPNWELVGNYRN 540 Query: 541 YLYDVLKIAR 550 YLYDVLKIAR Sbjct: 541 YLYDVLKIAR 550 A homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS117 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 118 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 235> which encodes amino acid sequence <SEQ ID 236; NGS118>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): 0.59 Possible cleavage site: 19 >>> May be a lipoprotein Amino Acid Composition of Predicted Mature Form: calculated from 22 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 8.33 threshold: 0.0 PERIPHERAL Likelihood = 8.33 modified ALOM score: −2.17 Rule: inner or outer membrane protein Rule: inner or outer membrane protein *** Reasoning Step: 2 Lipoprotein?","Inner membrane?","----- Final Results ----- bacterial outer membrane --- Certainty= 0.790(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.700(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has no homology to sequences in the databases.","The protein was expressed in E.coli as a soluble 12.98 kDa His-fusion product and then purified.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 119 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 237> which encodes amino acid sequence <SEQ ID 238; NGS119>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −4.75 Possible cleavage site: 47 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 7.69 threshold: 0.0 PERIPHERAL Likelihood = 7.69 modified ALOM score: −2.04 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.213(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_2625122 gi|2625122|gb|AAB86635.1|\\(AF031495) putative hemoglobin receptor component precursor HpuA [Neisseria gonorrhoeae] Length = 360 Score = 668 bits (1724), Expect = 0.0 Identities = 331/331 (100%), Positives = 331/331 (100%) Query: 1 VSIPTATPLPAGEVTLSSDNGNIENINTAGAGSASDAPSRSRRSLDAAPQNTSGISIRQR 60 VSIPTATPLPAGEVTLSSDNGNIENINTAGAGSASDAPSRSRRSLDAAPQNTSGISIRQR Sbjct: 30 VSIPTATPLPAGEVTLSSDNGNIENINTAGAGSASDAPSRSRRSLDAAPQNTSGISIRQR 89 Query: 61 EVEKDYFGYKSKETSFIFKTPGGAQYALSSYADPITVSYSSPKFKIPDRHAGQRLADGSR 120 EVEKDYFGYKSKETSFIFKTPGGAQYALSSYADPITVSYSSPKFKIPDRHAGQRLADGSR Sbjct: 90 EVEKDYFGYKSKETSFIFKTPGGAQYALSSYADPITVSYSSPKFKIPDRHAGQRLADGSR 149 Query: 121 IFICCSDSGATSYAEITKQDYMKFGAWIGPNGEIDLFAGGFPVGKTPPPAFSYGSSTPET 180 IFICCSDSGATSYAEITKQDYMKFGAWIGPNGEIDLFAGGFPVGKTPPPAFSYGSSTPET Sbjct: 150 IFICCSDSGATSYAEITKQDYMKFGAWIGPNGEIDLFAGGFPVGKTPPPAFSYGSSTPET 209 Query: 181 ALSKGKITYQVWGIRVRNGQFVTSSYTPPKSGSYYGTLANTPVSFITANFNSNTLAGKI 240 ALSKGKITYQVWGIRVRNGQFVTSSYTPPKSGSYYGTLANTPVSFITANFNSNTLAGKI Sbjct: 210 ALSKGKITYQVWGIRVRNGQFVTSSYTPPKSGSYYGTLANTPVSFITANFNSNTLAGKI 269 Query: 241 LGNSDYGPDVDIQNATITGPTFSGDATSGGKSGKLEGKFFGKFASTRSSEVSIGGKITFD 300 LGNSDYGPDVDIQNATITGPTFSGDATSGGKSGKLEGKFFGKFASTRSSEVSIGGKITFD Sbjct: 270 LGNSDYGPDVDIQNATITGPTFSGDATSGGKSGKLEGKFFGKFASTRSSEVSIGGKITFD 329 Query: 301 GDRSLDTVFGGVSYEKKLDDTSQDTNHLTKQ 331 GDRSLDTVFGGVSYEKKLDDTSQDTNHLTKQ Sbjct: 330 GDRSLDTVFGGVSYEKKLDDTSQDTNHLTKQ 360 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 120 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 239> which encodes amino acid sequence <SEQ ID 240; NGS120>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −7.24 Possible cleavage site: 38 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 6.42 threshold: 0.0 PERIPHERAL Likelihood = 6.42 modified ALOM score: −1.78 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.280(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gsa_AAR91313 N. gonorrhoeae glycosyltransferase LgtC |WO9610086-A1|09-JUL-1996 Length = 306 Score = 535 bits (1379), Expect = e−151 Identities = 252/253 (99%), Positives = 252/253 (99%) Query: 8 GGGNIRFIDVNPEDFAGFPLNIRHISITTYARLKLGEYIADCDKVLYLDTDVLVRDGLKP 67 GGGNIRFIDVNPEDFAGFPLNIRHISITTYARLKLGEYIADCDKVLYLDTDVLVRDGLKP Sbjct: 54 GGGNIRFIDVNPEDFAGFPLNIRHISITTYARLKLGEYIADCDKVLYLDTDVLVRDGLKP 113 Query: 68 LWDTDLGGNWVGACIDLFVERQEGYKQKIGMADGEYYFNAGVLLINLKKWRRHDIFKMSC 127 LWDTDLGGNWVGACIDLFVERQEGYKQKIGMADGEYYFNAGVLLINLKKWRRHDIFKMSC Sbjct: 114 LWDTDLGGNWVGACIDLFVERQEGYKQKIGMADGEYYFNAGVLLINLKKWRRHDIFKMSC 173 Query: 128 EWVEQYKDVMQYQDQDILNGLFKGGVCYANSRFNFMPTNYAFMANGFASRHTDPLYLDRT 187 EWVEQYKDVMQYQDQDILNGLFKGGVCYANSRFNFMPTNYAFMANGFASRHTDPLYLDRT Sbjct: 174 EWVEQYKDVMQYQDQDILNGLFKGGVCYANSRFNFMPTNYAFMANGFASRHTDPLYLDRT 233 Query: 188 NTAMPVAVSHYCGSAKPWHRDCTVWGAERFTELAGSLTTVPEEWRGKLAVPPTKRMLQRW 247 NTAMPVAVSHYCGSAKPWHRDCTVWGAERFTELAGSLTTVPEEWRGKLAVPPTKRMLQRW Sbjct: 234 NTAMPVAVSHYCGSAKPWHRDCTVWGAERFTELAGSLTTVPEEWRGKLAVPPTKRMLQRW 293 Query: 248 RKKLSARFLRKIY 260 RKKLSARFLRKIY Sbjct: 294 RKKLSARFLRKIY 306 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 121 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 241> which encodes amino acid sequence <SEQ ID 242; NGS121>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −6.22 Possible cleavage site: 37 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 3.23 threshold: 0.0 PERIPHERAL Likelihood = 3.23 modified ALOM score: −1.15 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.402(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_15281345 gi|15281345|dbj|BAB63435.1|\\(AB058945) DNA adenine methylase M.Ssu4109IB [Streptococcus suis] Length = 271 Score = 269 bits (687), Expect = 4e−71 Identities = 127/211 (60%), Positives = 158/211 (74%), Gaps = 1/211 (0%) Query: 1 MIFADPPYFLSNDGFSCQNGQMVSVNKGNWDKSKGMAADLEFYEEWLRLCYALLKPNGTI 60 MIFADPPYFLSN G S GQ+VSV+KG+WDK + EF +W+RL +LKPNGTI Sbjct: 44 MIFADPPYFLSNGGISNSGGQVVSVDKGDWDKVNSLEEKHEFNRKWIRLAKNVLKPNGTI 103 Query: 61 WVCGTFHNIYLIGYLMQTVGYHILNNITWEKPNPPPNLSCRFFTHSTETILWAKK-NKKA 119 W+ G+FHNIY +G ++ G+ ILNNITW+K NP PNLSCR+FTHSTETILWA+K +KKA Sbjct: 104 WISGSFHNIYSVGMALEQEGFKILNNITWQKTNPAPNLSCRYFTHSTETILWARKDDKKA 163 Query: 120 KHTFHYEMMKAQNNGKQMKCVWTFAPPNKTEKTFGKHPTQKPLPLLERCILSASNIGDLI 179 +H ++YE+MK N+GKQMK VW K+EK GKHPTQKP LLER IL+++ GD I Sbjct: 164 RHYYNYELMKELNDGKQMKDVWVGGLTKKSEKWAGKHPTQKPEYLLERIILASTREGDYI 223 Query: 180 FDPFMGSGTTGVAALKHGRRFCGCELEEDFL 210 DPF+GSGTTGV A + GR+F G + E D+L Sbjct: 224 LDPFVGSGTTGVVAKRLGRKFIGIDAERDYL 254 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 122 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 243> which encodes amino acid sequence <SEQ ID 244; NGS122>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −2.55 Possible cleavage site: 23 >>> May be a lipoprotein Amino Acid Composition of Predicted Mature Form: calculated from 15 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 11.46 threshold: 0.0 PERIPHERAL Likelihood = 11.46 modified ALOM score: −2.79 Rule: inner or outer membrane protein Rule: inner or outer membrane protein *** Reasoning Step: 2 Lipoprotein?","Inner membrane?","----- Final Results ----- bacterial outer membrane --- Certainty= 0.790(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.700(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has no homology to sequences in the databases: The protein was expressed in E.coli as an insoluble 14.85 kDa His-fusion product and then purified.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 123 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 245> which encodes amino acid sequence <SEQ ID 246; NGS123>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −5.65 Possible cleavage site: 20 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 4.24 threshold: 0.0 PERIPHERAL Likelihood = 4.24 modified ALOM score: −1.35 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.404(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has no homology to the sequences in the databases.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 124 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 247> which encodes amino acid sequence <SEQ ID 248; NGS124>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −5 Possible cleavage site: 18 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 1 value: −1.59 threshold: 0.0 INTEGRAL Likelihood = −1.59 Transmembrane 289-305 (289-305) PERIPHERAL Likelihood = 3.76 modified ALOM score: 0.82 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.164(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_1617515 gi|1617515|gb|AAC82509.1|\\(U365994) pilin gene inverting protein homolog PivNG [Neisseria gonorrhoeae] Length = 320 Score = 614 bits (1584), Expect = e−175 Identities = 311/320 (97%), Positivies = 316/320 (98%) Query: 1 MRNTVGLDISKLTFDATAMVGKTEHSAKFDNDSKGLDQFSDRLKSLGYQNLHICMEATGS 60 MRN VGLDISKLTF+A+AMVGKTEHSAKFDNDSKGLDQFSDRLKSLG QNLHICMEATG+ Sbjct: 1 MRNAVGLDISKLTFNASAMVGKTEHSAKFDNDSKGLDQFSDRLKSLGCQNLHICMEATGS 60 Query: 61 YYEEVADYFAQYYSVYVVNPLKISKYAESRFKRTKTDKQDAKLIAQYCRSAQESELVKRQ 120 YYEEVADYFAQYYSVYVVNPLKISKYAESRFKRTKTDKQDAKLIAQYCR A+ESELVKRQ Sbjct: 61 YYEEVADYFAQYYSVYVVNPLKISKYAESRFKRTKTDKQDAKLIAQYCRLAKESELVKRQ 120 Query: 121 KPTDEQYRLSRMTAAYAKIKSECAAMKNRHHAAKDEEAAKAYAEIIKAMNEQLEVLKEKI 180 KPTDEQYRL RMTAAYAKIKSECAAMKNRHHAAKDEEAAKAYA+IIKAMNEQLEVLKEKI Sbjct: 121 KPTDEQYRLLRMTAAYAKIKSECAAMKNRHHAAKDEEAAKAYAQIIKAMNEQLEVLKEKI 180 Query: 181 KEQTEKPNCKEGVKRLETIPAIGRMTAAVLFHHLTSSKFETSNKFAAFAGLSPQQKESGT 240 KEQTEKPNCKEGVKRLETIPAIGRMTAAVLFHHLTSSKFETSNKFAAFAGLSPQQKESGT Sbjct: 181 KEQTEKPNCKEGVKRLETIPAIGRMTAAVLFHHLTSSKFETSNKFAAFAGLSPQQKESGT 240 Query: 241 SVRGKGKLTKFGNRKLRAVLFMPAMVAVYRIRAFPDFIKRLEEKKKPKKVIIAALMRKLAV 300 SVRGKGKLTKFGNRKLRAVLFMPAMVAVYRIRAFPDFIKRLEEKKKPKKVIIAALMRKLAV Sbjct: 241 SVRGKGKLTKFGNRKLRAVLFMPAMVAVYRIRAFPDFIKRLEEKKKPKKVIIAALMRKLAV 300 Query: 301 IAYHVHKKGGDYDPSRYKSA 320 IAYHVHKKGGDYDPSRYKSA Sbjct: 301 IAYHVHKKGGDYDPSRYKSA 320 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 125 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 249> which encodes amino acid sequence <SEQ ID 250; NGS127>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −5.8 Possible cleavage site: 52 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 1.70 threshold: 0.0 PERIPHERAL Likelihood = 1.70 modified ALOM score: −0.84 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.383(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_1076012 gi|1076012|pir||B55225 stress-sensitive restriction system protein 2-Corynebacterium glutamicum (ATCC 13032) gb|AAC00044.1|(U13922) This orf may encode a typeI or typeIII restriction endonuclease which is stress-sensitive and ATP-dependent.","It contains a typical ATP binding region (Walker motif) [Corynebacterium glutamicum] Length = 632 Score = 298 bits (764), Expect = 2e−79 Identities = 199/633 (31%), Positives = 321/633 (50%), Gaps = 32/633 (5%) Query: 2 LRTYLNQLTP-PELADSVKNTVDGFMEKLSQTEPKIA-QNVLLLGNVQSGKTAQVLGVLS 59 L Y + +L + V TVD F + I+ Q VLL G+VQSGKT+ +LG++++ Sbjct: 7 LNNYITSLSDNADLREKVTATVDAFRHTVMDDFDYISDQQVLLYGDVQSGKTSHMLGIIA 66 Query: 60 ALADDGDHKVFLYLTTDSVDLQDQTVKRAKANLKNFIVLSEADDRSFMEVMKAENP--IL 117 D H + + LT+ + L QT R + +V F K+ P + Sbjct: 67 DCLDSTFHTIVI-LTSPNTRLVQQTYDRVAQAFPDTLVCDRDGYNDFRANQKSLTPRKSI 125 Query: 118 VVIKKNARVLKRWRNLFASQSSLKGYPLVIVDDEADAASLNTNSDKPKDASTINKLLND 177 VV+ K VL W +F +L G+P++I+DDEADA SLNT ++ D STIN L Sbjct: 126 VVVGKIPAVLGNWLRVFNDSGALSGHPVLIIDDEADATSLNTKVNQ--SDVSTINHQLTS 183 Query: 178 IKNSCCQSLPIQLTATPQSLLLQHEESDWQPEFIHFFEAGEKYIGGNFVFSDPPS-YIVR 236 I++ +++Q+T TPQ++LLQ ++S+W E + F GE YIGG FS+ + Y+ Sbjct: 184 IRDLATGCIYLQVTGTPQAVLLQSDDSNWAAEHVLHFAPGESYIGGQLFFSELNNPYLRL 243 Query: 237 FIDSELDDMKDESGEIAEGAKQALLSFLITCAEFALCDKANCNFALHPSYKIQDHQAFSK 296 F +++ D+ S A+ ++L+T A F L ++ C +HPS+ H+ F++ Sbjct: 244 FANTQFDEDSRFS--------DAIYTYLLTAALFLRGESLCTMLIHPSHTASSHRDFAQ 295 Query: 297 KIQAFLNDLVQAVNNGEDLAGSFKESYLDLQKTKPDIHHFDEIYEKLTALLENKQISTLV 356 + + L + + +F+ +Y L +T ++ +I L + ++ I + Sbjct: 296 EARLQLTFAFERFYEPM-IQHNFQRAYEQLAQTDSNLPPLRKILNILGGMEDDFSIH--I 352 Query: 357 VNSQTET-DFDLEKGFNIIIGGNVIGRGLTIPKLQTVYYSRTAKKPNADTFWQHRIFGY 415 VNS T + D G+NII+GGN +GRGLT LQTV+Y R +K+P ADT WQH+R+FGY Sbjct: 353 VNSDNPTVEEDWADGYNIIVGGNSLGRGLTFNNLQTVFYVRESKRPQADTLWQHARMFGY 412 Query: 416 DRDKSLLRLYIPFDVYYFFVQLNQANNLIIGQAKNSG--GNIQVIYPKNINPTRKNVLKF 473 R K +R+++P + F ++ N I Q + +I+VI + PTR NVL Sbjct: 413 KRHKDTMRVFMPATIAQTFQEVYLGNEAIKNQLDHGTHINDIRVILGDGVAPTRANVLDK 472 Query: 474 DSINQIVGGVNYFPLHPNEDNLSEINKILPSILKDEIQSDLYQIDIEDLFLVLDKLGRYV 533 + + GGVNYF P N+ ++K L + L + I + + +L+ Sbjct: 473 RKVGNLSGGVNYFAADPRIIKNVEALDKKLLAYLDKHGEDS--TIGMRAIITILNAF-TVD 529 Query: 534 PDDWNKEKFIAGVEALKAQRPSFKTYVLIKTGRKLSRATGTMLSEDDRKLGEKYPNDLFL 593 P+D + F A + + +P ++++T RK+++ TG +LS D+ L L Sbjct: 530 PNDLDLATFKAALLDFERNQPHLTARMVLRTNRKVNQGTGALLSPTDQALSRAEVAHPLL 589 Query: 594 TLYQVVGNKDKG-------WQGKDFWLPNIKLP 619 LY++ G D W W+PNIKLP Sbjct: 590 ILYRIEGVNDAAAQRGEPTWSSDPIWVPNIKLP 622 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 126 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 251> which encodes amino acid sequence <SEQ ID 252; NGS128>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −4.98 Possible cleavage site: 20 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 7.37 threshold: 0.0 PERIPHERAL Likelihood = 7.37 modified ALOM score: −1.97 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.225(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_11357195 gi|11387l95|sp|Q50976|T2F7_NEIGO TYPE II RESTRICTION ENZYME NGOFVII (ENDONUCLEASE NGOFVII) (R.NGOFVII) (R.NGOVII) pir||T10166 restriction endonuclease (EC 3.1.21.-) NgoVII-N. gonorrhoeae gb|AAA86271.1|(U43736) R.NgoVII [Neisseria gonorrhoeae] Length = 326 Score = 651 bits (1679), Expect = 0.0 Identities = 317/326 (97%), Positives = 320/326 (97%) Query: 1 MNTVFSNIANAKITEKSLNAVWMDLFKSADEVLMATGYVSNDAVVELHKILELNDHIQKI 60 MNTVFSNIANAKITEKSLNAVWMDLFKSADEVLMATGYVSNDAVVELHKILELNDHIQKI Sbjct: 1 MNTVFSNIANAKITEKSLNAVWMDLFKSADEVLMATGYVSNDAVVELHKILELNDHIQKI 60 Query: 61 DLLVGMHYLEGFSHLQYDSLCKLNDFLRHEKRGAVYVSPFVKFHGKMYSFKNYQKINGLI 120 DLLVGMHYLEGFSHLQYDSLCKLNDFLRHEKRGAVYVSPFVKFHGKMYSFKNYQKINGLI Sbjct: 61 DLLVGMHYLEGFSHLQYDSLCKLNDFLRHEKRGAVYVSPFVKFHGKMYSFKNYQKINGLI 120 Query: 121 GSANLTCFWDSTERTYETMLHLNGKPAQILQADIQSTIHKLGKNIQEVERPSKFIEHNSH 180 GSANLTCFWDSTERTYETMLHLNGKPAQILQADIQSTIHKLGKNIQEVERPSKFIEHNSH Sbjct: 121 GSANLTCFWDSTERTYETMLHLNGKPAQILQADIQSTIHKLGKNIQEVERPSKFIEHNSH 180 Query: 181 LENCLGVQKIAPEQIRQLFAQTSEYHFSIPAKTEEKSNLNVFFGEGRRDKRGFVKPRPWY 240 LENCLGVQKIAPEQIRQLFAQTSEYHFSIPAKTEEKSNLNVFFGEGRRDKRGFVKPRPWY Sbjct: 181 LENCLGVQKIAPEQIRQLFAQTSEYHFSIPAKTEEKSNLNVFFGEGRRDKRGFVKPRPWY 240 Query: 241 EVELIVSKDITSQEGYPVLKSFTVITDDGWQFQCKTSGDYSKNFRSENDLKTLGKWIKGR 300 EVELIVSKDITSQEGYPVLKSFTVITDDGWQFQCKTSGDYSK + +LKTLGKWIKGR Sbjct: 241 EVELIVSKDITSQEGYPVLKSFTVITDDGWQFQCKTSGDYSKTSTQKMNLKTLGKWIKGR 300 Query: 301 LESHGCLQNNEKITHETLREYGNDHF 326 LESHGCLQNNEKITHETLREYGN+ F Sbjct: 301 LESHGCLQNNEKITHETLREYGNESF 326 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 127 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 253> which encodes amino acid sequence <SEQ ID 254; NGS 129>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −5.5 Possible cleavage site: 48 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 10.03 threshold: 0.0 PERIPHERAL Likelihood = 10.03 modified ALOM score: −2.51 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.545(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_15804186 gi|15804186|ref|NP_290225.1|\\(NC_002655) DNA-damage- inducible protein [Escherichia coli O157:H7 EDL933] ref|NP_312547.1|(NC_002695) DNA-damage-inducible protein [Escherichia coli O157:H7] gb|AAG58789.1|AE005591_13 (AE005591) DNA-damage-inducible protein [Escherichia coli O157:H7 EDL933] dbj|BAB37943.1|(AP002566) DNA-damage-inducible protein [Escherichia coli O157:H7] Length = 278 Score = 340 bits (872), Expect = 2e−92 Identities = 161/266 (60%), Positives = 197/266 (73%) Query: 1 MTTENNAFENAKHIDETGNEYWSARTLQQILEYSEWRNFQRAIDKAITACETSGNDKNHH 60 M + FE +H G E+WSAR L +L+Y +WRNFQ+ + +A ACE S + H Sbjct: 5 MNEHQPFEEIRHYGTEGQEFWSARELAPLLDYRDWRNFQKVLAPATQACEASNQAASDH 64 Query: 61 FVETNKMIALGKGGQREVADYRLSRYACYLIVQNGDPSKSVIAAGQTYFAVQARRQELQD 120 FVET KM+LG G QRE+ D LSRYACYL+VQNGDP+K VIAAGQTYFA+Q RRQEL D Sbjct: 65 FVETTKMVVLGSGAQRELEDVHLSRYACYLVVQNGDPAKPVIAAGQTYFAIQTRRQELAD 124 Query: 121 EAAFRSLGEDKQRLLLRRQLREHNTDLAAAAKDAGVEKPVEYAVFQNHGYRGLYGGLDKQ 180 + AF+ L ED++RL LR +L+EHN L AA+ A V ++A+FQNHGY+GLYGGLD++ Sbjct: 125 DEAFKQLREDEKRLFLRNELKEHNKQLVEAAQQAAVATATDFAIFQNHGYQGLYGGLDQK 184 Query: 181 GIHSPRGLKKSQRILDHMNASEPAANLFRATQTEEKLRRKNIQGKTQANRVHFEVGQKVR 240 IH KGLKKSQ+ILDHM ++E AANLFRATQTEEKL+R + K QAN HF+VG KVR Sbjct: 185 AIEQLKGLKKSQKILDHMGSTELAANLFRATQTEEKLKRDGVNSKQQANTTHFDVGSKVR 244 Query: 241 QTIEELGGIMPENQPVPEKSIKQLEN 266 QTI+ELGG MPE P P+ SIKQLEN Sbjct: 245 QTIQELGGTMPEELPTPQVSIKQLEN 270 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 128 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 255> which encodes amino acid sequence <SEQ ID 256; NGS130>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −3.68 Possible cleavage site: 14 >>> Seems to have an uncleavable N-term signal seq Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 3 value: −3.45 threshold: 0.0 INTEGRAL Likelihood = −3.45 Transmembrane 68-84 (68-92) INTEGRAL Likelihood = −1.59 Transmembrane 10-26 (10-26) INTEGRAL Likelihood = −1.44 Transmembrane 46-62 (45-62) PERIPHERAL Likelihood = 1.48 modified ALOM score: 1.19 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.238(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_17988861 gi|17998861|ref|NP_541494.1|\\(NC_003318) hypothetical protein [Brucella melitensis] gb|AAL53758.1|(AE009687) hypothetical protein [Brucella melitensis] Length = 99 Score = 108 bits (270), Expect = 3e−23 Identities = 59/91 (64%), Positives = 69/91 (74%) Query: 11 LLFSCMLAVTCPTRLIGFFALRNRTLSRRAQTVMEAAPGCVLISVIAPYFVSDKPHELIA 70 L M +VT TR+ G+ LRNRTLS RA VMEAAPGCVLISVIAP FVSDKP LIA Sbjct: 8 LTILAMASVTYLTRIGGYVLLRNRTLSNRAMAVMEAAPGCVLISVIAPDFVSDKPANLIA 67 Query: 71 IALTAFAACRFSMLFTVLIGVGSSGISGWLM 101 +A+T FAA RFSML TVLIG+G++ I +L+ Sbjct: 6 LAVTVFAATRFSMLPTVLIGMGAASICRYLI 98 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 129 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 257> which encodes amino acid sequence <SEQ ID 258; NGS131>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −1.65 Possible cleavage site: 43 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 7.05 threshold: 0.0 PERIPHERAL Likelihood = 7.05 modified ALOM score: −1.91 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.152(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_16760390 gi|16760390|ref NP_456007.1|\\(NC_003198) hypothetical protein [Salmonella enterica subsp.","enterica serovar Typhi] emb|CAD01841.1|(AL621270) hypothetical protein [Salmonella enterica subsp.","enterica serovar Typhi] Length = 227 Score = 104 bits (259), Expect = 2e−21 Identities = 68/221 (30%), Positivies = 115/221 (51%), Gaps = 11/221 (4%) Query 2 DKEKVLDKIKKCLALGRSVNEHEAAQALRQAQALMEKYKVNAEDIALSKVSEQKAD--RK 59 D++K ++K+KK LAL S N HEAA ALR+A+ LM+ + + DIA+S + E + Sbjct: 3 DQDKHIEKLKKLLALAASGNPHEAALALRRARKLMDVHGITHSDIAMSDIDETISHYWPT 62 Query: 60 MAFKLAGWQWGVANMIADIFGCKSYQRGKT---MMFYGIGNRAETSAYAFDVVYRQISAD 116 + + + G+ N+I + FG S T + FYG RA +AY ++V+ RQ+ Sbjct: 63 GSLRPPRYMLGLMNIIREAFGVNSIIHPGTYPGVGFYGNRERAALAAYTWEVLARQLKKA 122 Query: 117 RRKFLKT-CRAGKPSHRTYLADPFCGGWIASAWETVKKFEMSDEEKAIMDGYKKKEYPDM 175 R++++ + K + RT D+F GW+ + ++ F ++D+E+ +M + + +YP Sbjct: 123 RQQYISAQNKRIKTATRTSRGDQFAEGWVLAVISEIQSFALTDDERELMQQWLEHKYPQT 182 Query: 176 AEARTRDAKSSILQGSKMEYEALTRGMESGKQVKLHYAVNG 216 R R S G Y G G+ V+LH V+G Sbjct: 183 QTTRARKPGRS-RNGDASRY----AGFREGQNVRLHRPVSG 218 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 130 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 259> which encodes amino acid sequence <SEQ ID 260; NGS132>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −4.06 Possible cleavage site: 30 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 2.49 threshold: 0.0 PERIPHERAL Likelihood = 2.49 modified ALOM score: −1.00 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.075(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has no homology to sequences in the databases.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 131 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 261> which encodes amino acid sequence <SEQ ID 262; NGS133>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): 1.64 Possible cleavage site: 53 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 3.82 threshold: 0.0 PERIPHERAL Likelihood = 3.82 modified ALOM score: −1.26 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.068(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has no homology to the following sequences in the databases: Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 132 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 263> which encodes amino acid sequence <SEQ ID 264; NGS135>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −4.67 Possible cleavage site: 39 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 5.52 threshold: 0.0 PERIPHERAL Likelihood = 5.52 modified ALOM score: −1.60 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.457(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has no homology to sequences in the databases: Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 133 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 265> which encodes amino acid sequence <SEQ ID 266; NGS136>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −3.97 Possible cleavage site: 15 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 11.35 threshold: 0.0 PERIPHERAL Likelihood = 11.35 modified ALOM score: −2.77 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.523(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has no homology to sequences in the databases: Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 134 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 267> which encodes amino acid sequence <SEQ ID 268; NGS 137>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −8.52 Possible cleavage site: 51 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 2.81 threshold: 0.0 PERIPHERAL Likelihood = 2.81 modified ALOM score: −1.06 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.374(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has no homology to sequences in the databases.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 135 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 269> which encodes amino acid sequence <SEQ ID 270; NGS138>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −7 Possible cleavage site: 36 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 10.66 threshold: 0.0 PERIPHERAL Likelihood = 10.66 modified ALOM score: −2.63 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.415(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_13559865 gi|13559865|ref|NP_112075.1|\\(NC_002730) terminase small subunit [Bacteriophage HK620] gb|AAK28890.1|AF335538_42 (AF335538) terminase small subunit [Bacteriophage HK620] Length = 140 Score = 125 bits (313), Expect = 5e−28 Identities = 56/122 (45%), Positivies = 85/122 (68%) Query: 4 TKRKLGRPTDYTKDMADKICEKIANGRSLRSICAEDGVPPMKTIYRWLEANEEFRHQYAR 63 T+ K GRP+DY ++AD IC +++G SL +C G+P T++RWL +E+FR +YA+ Sbjct: 3 TEPKAGRPSDYMPEVADDICSLLSSGESLLKVCKRPGMPDKSTVFRWLAKHEDFRDKYAK 62 Query: 64 AREKQADYFAEEIIEIADSAQAESAAVSKAKLQIDARKWAASKIAPKKYGDKSELDVKSGDG 125 A E +AD EEI EIAD+A ++A V+KA+L++D RKWA +++ P+KYGDK ++ DG Sbjct: 63 ATEARADSIFEEIFEIADNAIPDAAEVAKARLRVDTRKWALARMNPRKYGDKVTNELVGKDG 124 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 136 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 271> which encodes amino acid sequence <SEQ ID 272; NGS139>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −1.49 Possible cleavage site: 32 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 8.65 threshold: 0.0 PERIPHERAL Likelihood = 8.65 modified ALOM score: −2.23 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.301(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_16127009 gi|16127009|ref|NP_421573.1|\\(NC_002696) hypothetical protein [Caulobacter crescentus] gb|AAK24741.1|(AE005943) hypothetical protein [Caulobacter crescentus] Length = 184 Score = 59.7 bits (143), Expect = 4e−08 Identities = 50/164 (30%), Positives = 74/164 (44%), Gaps = 20/164 (12%) Query: 30 ASGREFRTAYYTYPQWRFSLSFEVLRTKASVNELEKLAGFFNARKGSFESFLYEDPAD-- 87 ASG E RT+ ++ + R+ ++ ++E+ +L FF AR+G F + DPAD Sbjct: 5 ASGHERRTSPWSQSRRRYLIA----TAPRPLDEIAELVAFFEARRGRLHGFRFRDPADFK 60 Query: 88 -------NAVTDQPVGNTVQGAR-YQLVRSMGGFIEPVSAVKERP-----AVKVGGTAL 134 A DQ +G T GV + +QL ++ G E V+ +P V V G L Sbjct: 61 SCAPSVQPAAGDQAIG-TGDGVRKKAFQLRKTYGAGGEAVARTIAKPVAGTVTVAVAGVVL 119 Query: 135 AYGRDYTVTDKGVLVFNTPQPPGRPITWTGGFYFRVRFTSDTVD 178 A G G++ NT P G +T F VRF D +D Sbjct: 120 APGAFAVDVTTGLITLNTAPPAGAAVTAGFAFDTPVRFDLDRLD 163 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 137 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 273> which encodes amino acid sequence <SEQ ID 274; NGS140>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −3.86 Possible cleavage site: 31 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 1 value: −4.94 threshold: 0.0 INTEGRAL Likelihood = −4.94 Transmembrane 34-50 (31-54) PERIPHERAL Likelihood = 8.01 modified ALOM score: 1.49 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.297(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_17987625 gi|17987625|ref|NP_540259.1|\\(NC_003317) Hypothetical Phage Protein [Brucella melitensis] gb|AAL52523.1|(AE009572) Hypothetical Phage Protein [Brucella melitensis] Length = 144 Score = 72.4 bits (176), Expect = 5e−12 Identities = 43/119 (36%), Positives = 64/119 (53%), Gaps = 7/119 (5%) Query: 10 RIVEEARSWLGTPYHHHAMVKGAGVDCAMLLVAVYGAV-GLLPEGFDPRPYPQDWHLHRD 68 R++ EA W+GTPY H A G DC L+ ++A+ G+ PE +P Y DW Sbjct: 6 RVLAEAHRWIGTPYRHGASTLGVSCDCLGVRQIWRALYGVEPE--NPGVYAPDWAEVSQ 63 Query: 69 CERYLGFVTQFC--RETESPQAGDIAV--WRFGRSFSHGGILAGGGKVIHSYIGRGVVS 123 + L ++ RE +PQ GD+ W+ G + H GI+A G+ IH+Y G GV++ Sbjct: 64 GDPMLEAAVRYMVRREEHAPQPGDLLVFRWKPGFAAKHMGIMAREGRFIHAYQGHGVLA 122 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 138 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 275> which encodes amino acid sequence <SEQ ID 276; NGS141>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): 5.35 Possible cleavage site: 28 >>> Seems to have a cleavable N-term signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 29 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 8.86 threshold: 0.0 PERIPHERAL Likelihood = 8.86 modified ALOM score: −2.27 Score for OM-PP discrimination: 1.53 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: 1.53 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Outer membrane?","Score: 0.152929 Outer membrane?","Score: 0.152929 ----- Final Results ----- bacterial outer membrane --- Certainty= 0.512(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.320(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_5915870 gi|5915870|sp|Q50940|CAH_NEIGO Carbonic anhydrase precursor (Carbonate dehydratase) emb|CAA72038.1|(Y11152) carbonic anhydrase [Neisseria gonorrhoeae] Length = 252 Scare = 523 bits (1347), Expect = e−147 Identities = 252/252 (100%), Positives = 252/252 (100%) Query: 1 MPRFPRTLPRLTAVLLLACTAFSAAAHGNHTHWGYTGHDSPESWGNLSEEFRLCSTGKNQ 60 MPRFPRTLPRLTAVLLLACTAFSAAAHGNHTHWGYTGHDSPESWGNLSEEFRLCSTGKNQ Sbjct: 1 MPRFPRTLPRLTAVLLLACTAFSAAAHGNHTHWGYTGHDSPESWGNLSEEFRLCSTGKNQ 60 Query: 61 SPVNITETVSGKLPAIKVNYKPSMVDVENNGHTIQVNYPEGGNTLTVNGTYTLKQFHFH 120 SPVNITETVSGKLPAIKVNYKPSMVDVENNGHTIQVNYPEGGNTLTVNGTYTLKQFHFH Sbjct: 61 SPVNITETVSGKLPAIKVNYKPSMVDVENNGHTIQVNYPEGGNTLTVNGTYTLKQFHFH 120 Query: 121 VPSENQIKGRTFPMEAHFVHLDENKQPLVLAVLYEAGKTNGRLSSIWNVMPMTAGKVKLN 180 VPSENQIKGRTFPMEAHFVHLDENKQPLVLAVLYEAGKTNGRLSSIWNVMPMTAGKVKLN Sbjct: 121 VPSENQIKGRTFPMEAHFVHLDENKQPLVLAVLYEAGKTNGRLSSIWNVMPMTAGKVKLN 180 Query: 181 QPFDASTLLPKRLKYYRFAGSLTTPPCTEGVSWLVLKTYDHIDQAQAEKFTRAVGSENNR 240 QPFDASTLLPKRLKYYRFAGSLTTPPCTEGVSWLVLKTYDHIDQAQAEKFTRAVGSENNR Sbjct: 181 QPFDASTLLPKRLKYYRFAGSLTTPPCTEGVSWLVLKTYDHIDQAQAEKFTRAVGSENNR 240 Query: 241 PVQPLNARVVIE 252 PVQPLNARVVIE Sbjct: 241 PVQPLNARVVIE 252 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 139 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 277> which encodes amino acid sequence <SEQ ID 278; NGS142>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −0.49 Possible cleavage site: 22 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 1.22 threshold: 0.0 PERIPHERAL Likelihood = 1.22 modified ALOM score: −0.74 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.145(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_15794480 gi|15794480|ref|NP_284302.1|\\(NC_003116) hypothetical protein [Neisseria meningitidis Z2491] pir||F81851 hypothetical protein NMA1587 [imported]-Neisseria meningitidis (group A strain Z2491) emb|CAB84814.1|(AL162756) hypothetical protein [Neisseria meningitidis Z2491] Length = 181 Score = 358 bits (919), Expect = 6e−98 Identities = 173/181 (95%), Positives = 178/181 (97%) Query: 1 LKTDTARMNNLIPEHLAAYAHSDNLQIEGGHRCFSLSCQGRDTFHIRYYGEPFDGLITDT 60 +KTDTA+MNNLIPEHLAAYAHSD+LQIEG HRCFSLSCQGRDTFHIRYYGEPFDGL+TDT Sbjct: 1 MKTDTAKMNNLIPEHLAAYAHSDSLQIEGVHRCFSLSCQGRDTFHIRYYGEPFDGLMTDT 60 Query: 61 DKAPVKIVAVEAVSGDEIVLFDGAEHGYNAMFCDKYSQNQKQNRTLTDLDEYTYRVPIHL 120 DKAPVKIVAVEAVSGDEIVLFDGAEHGYNAMFCDKYS NQKQNRTLTDLDEYTYRV IHL Sbjct: 61 DKAPVKIVAVEAVSGDEIVLFDGAEHGYNAMFCDKYSPNQKQNRTLTDLDEYTYRVLIHL 120 Query: 121 YYNIDYEDEYEDFVNSEGQVPLIDGRIISFDSLKRNGFDAISIDLIDEKHSVRELLNEELS 181 YYNIDYEDEYEDFVNSEGQVPLIDGRIISFDSLKRNGFDAIS+DLIDEKHSVRELLNEELS Sbjct: 121 YYNIDYEDEYEDFVNSEGQVPLIDGRIISFDSLKRNGFDAISVDLIDEKHSVRELLNEELS 181 A homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS142 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 140 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 279> which encodes amino acid sequence <SEQ ID 280; NGS143>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −2.51 Possible cleavage site: 57 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 4 value: −15.23 threshold: 0.0 INTEGRAL Likelihood = −15.23 Transmembrane 84-100 (79-107) INTEGRAL Likelihood = −8.12 Transmembrane 259-275 (250-281) INTEGRAL Likelihood = −4.14 Transmembrane 159-175 (153-176) INTEGRAL Likelihood = −3.88 Transmembrane 216-232 (216-235) PERIPHERAL Likelihood = 1.11 modified ALOM score: 3.55 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.709(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_5764059 gi|5764059|emb|CAB53350.1|\\(AJ010260) NosR protein [Paracoccus denitrificans] Length = 724 Score = 393 bits (1009), Expect = e−108 Identities = 191/379 (50%), Positives = 249/379 (65%), Gaps = 22/379 (5%) Query: 1 LMVQRVLSVNDKAFVTADLDYELPQAYYVDDPKAPPVEISAPVEAVPAAASDTASDGIAE 60 L+VQR + +K F T DL Y+LPQ Y AP A PAA +D Sbjct: 358 LLVQREVGPIEKVFHTFDLGYQLPQKYLRSIAPAPEA---------AAPAAQAD-------- 402 Query: 61 DASAENGVSNQLWKQIWAKQGQIVVVGIALTILLLVFLFQDWIVRYEKWYDRFRFAFLT 120 E+ QLWK+IW + +I + L +L VF FQ + RYE+ + FR A+LT Sbjct: 403 ----ESQAQAQLWKRIWLDSKPKIAGLAAMLLVLTGVFFFQSFTTRYERAFYVFRMAYLT 458 Query: 121 FTLFYIGWYAQAQLSVVNTLTLFSAILTEFHWEFFLMDPIVFILWLFTAATMLLWNRGTF 180 TL ++GWYA AQLSVVN + LF +++ F W+ FL+DP+ FILW AA +L W RG + Sbjct: 459 VTLVFLGWYANAQLSVVNLMALFGSLVNGFSWQAFLLDPLTFILWFAVAAALLFWGRGAY 518 Query: 181 CGWLCPFGSLQELTNRIAKKLGVKQITVPHMLHTRLNVIKYLILFGFLAISLYDLGTAEK 240 CGWLCPFG+LQELTN++A+KL + Q T+P LH RL +KY+I G +SL + AE Sbjct: 519 CGWLCPFGALQELTNQVARKLRIPQWTLPWGLHERLWPVKYMIFLGLFGVSLMSVEQAEH 578 Query: 241 FAEVEPFKTAIILKFMCDWWFVAPAVALLIAGLFIERFFCRYLCPLGAGIALPGRFRVFD 300 AEVEPFKTAIILKF+ W FVA+A ALLIAGLF+ERF+CRYLCPLGA +A+P R R+FD Sbjct: 579 LAEVEPFKTAIILKFIRAWPFVAYAAALLIAGLFVERFYCRYLCPLGAALAIPARMRMFD 638 Query: 301 WLRRYKMCGNPCQICTHBCPVQAIAPEGDIHPNECIQCLHCQVMYHHDTRCPQVVAENKK 360 WL+RY CGNPCQ C +CPVQ+I P G+I+PNECI CLHCQV+Y +T CP V+ KK Sbjct: 639 WLKRYHECGNPCQTCARQCPVQSIHPTGEINPNECINCLHCQVLYQSETTCPVVI---KK 695 Query: 361 KQKQAAAKSGELENVSKQP 379 +++ A +G + + + P Sbjct: 696 LKRREAVAAGSMPKLGQPP 714 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 141 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 281> which encodes amino acid sequence <SEQ ID 282; NGS144>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): 1.23 Possible cleavage site: 21 >>> May be a lipoprotein Amino Acid Composition of Predicted Mature Form: calculated from 20 ALOM: Finding transmembrane regions (Klein et al.)","count: 1 value: −0.37 threshold: 0.0 INTEGRAL Likelihood = −0.37 Transmembrane 90-106 (89-106) PERIPHERAL Likelihood = 10.82 modified ALOM score: 0.57 Rule: inner or outer membrane protein Rule: inner or outer membrane protein Rule: cytoplasmic membrane protein *** Reasoning Step: 2 Lipoprotein?","Inner membrane?","----- Final Results ----- bacterial outer membrane --- Certainty= 0.790(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.734(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology no to sequences in the databases.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 142 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 283> which encodes amino acid sequence <SEQ ID 284; NGS145>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): 1.5 Possible cleavage site: 19 >>> Seems to have a cleavable N-term signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 20 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 8.70 threshold: 0.0 PERIPHERAL Likelihood = 8.70 modified ALOM score: −2.24 Score for OM-PP discrimination: −9.24 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: −9.24 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Periplasmic space?","Score: 0.924443 Periplasmic space?","Score: 0.924443 ----- Final Results ----- bacterial periplasmic space --- Certainty= 0.931(Affirmative) < succ> bacterial outer membrane --- Certainty= 0.231(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_5051426 gi|5051426|emb|CAB45007.1|\\(AJ242839) OpcA protein [Neisseria gonorrhoeae] Length = 263 Score = 531 bits (1369), Expect = e−150 Identities = 262/263 (99%), Positives = 263/263 (99%) Query: 1 MKKALLALTIAAISGTAMAQLPDFLGKGEYTVRTDISKQTLKNADLKEKHKVQKNIGFRA 60 MKKALLALTIAAISGTAMAQLPDFLGKGEYTVRTDISKQTLKNADLKEKHKVQKNIGFRA Sbjct: 1 MKKALLALTIAAISGTAMAQLPDFLGKGEYTVRTDISKQTLKNADLKEKHKVQKNIGFRA 60 Query: 61 DMPFDDIHHGMRFEVSHSRDKKDMYVVTESTTKPFGKDVEEKRTDVYAGYTYTQPISEAT 120 DMPFDDIHHGMRFEVSHSRDKKDMYVVTESTTKPFGKDV+EKRTDVYAGYTYTQPISEAT Sbjct: 61 DMPFDDIHHGMRFEVSHSRDKKDMYVVTESTTKPFGKDVKEKRTDVYAGYTYTQPISEAT 120 Query: 121 KLRAGLGLGYEKYKDAVANEKGTVSTEREAFYTKAHADLTSDLGGGWYLNPWAEVKVDLD 180 KLRAGLGLGYEKYKDAVANEKGTVSTEREAFYTKAHADLTSDLGGGWYLNPWAEVKVDLD Sbjct: 121 KLRAGLGLGYEKYKDAVANEKGTVSTEREAFYTKAHADLTSDLGGGWYLNPWAEVKVDLD 180 Query: 181 AKLKHNATVAGVSADINAKTRGWGVGVGANIGKQITDTVGIEAGPFYKHRHFKASGSFVL 240 AKLKHNATVAGVSADINAKTRGWGVGVGANIGKQITDTVGIEAGPFYKHRHFKASGSFVL Sbjct: 181 AKLKHNATVAGVSADINAKTRGWGVGVGANIGKQITDTVGIEAGPFYKHRHFKASGSFVL 240 Query: 241 DGGNIRVDPTKINEYGVRVGVKF 263 DGGNIRVDPTKINEYGVRVGVKF Sbjct: 241 DGGNIRVDPTKINEYGVRVGVKF 263 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 143 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 285> which encodes amino acid sequence <SEQ ID 286; NGS146>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): 0.1 Possible cleavage site: 51 >>> Seems to have a cleavable N-term signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 52 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 3.50 threshold: 0.0 PERIPHERAL Likelihood = 3.50 modified ALOM score: −1.20 Score for OM-PP discrimination: −15.70 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: −15.70 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Periplasmic space?","Score: 1.56979 Periplasmic space?","Score: 1.56979 ----- Final Results ----- bacterial periplasmic space --- Certainty= 0.944(Affirmative) < succ> bacterial outer membrane --- Certainty= 0.375(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_5051429 gi|5051429|emb|CAB45013.1|\\(AJ242839) hypothetical protein [Neisseria gonorrhoeae] Length = 109 Score = 216 bits (549), Expect = 2e−55 Identities = 109/109 (100%), Positives = 109/109 (100%) Query: 1 MFKRPEEIIVLILAVLWIAGTYFLAALFGADAYVLKITALTLLWSAASFLLWQKKPQPA 60 MFKRPEEIIVLILAVLWIAGTYFLAALFGADAYVLKITALTLLWSAASFLLWQKKPQPA Sbjct: 1 MFKRPEEIIVLILAVLWIAGTYFLAALFGADAYVLKITALTLLWSAASFLLWQKKPQPA 60 Query: 61 YLAAAARLPDHLLVAVASESIGRTRFFTLACIMDVQHNLSPDSRNRRLSV 109 YLAAAARLPDHLLVAVASESIGRTRFFTLACIMDVQHNLSPDSRNRRLSV Sbjct: 61 YLAAAARLPDHLLVAVASESIGRTRFFTLACIMDVQHNLSPDSRNRRLSV 109 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 144 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 287> which encodes amino acid sequence <SEQ ID 288; NGS147>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −4.09 Possible cleavage site: 40 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 1 value: −2.28 threshold: 0.0 INTEGRAL Likelihood = −2.28 Transmembrane 36-52 (36-52) PERIPHERAL Likelihood = 5.20 modified ALOM score: 0.96 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.191(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_6606516 gi|6606516|gb|AAF19189.1|AF200716_2 \\(AF200716) trafficking protein B [Neisseria gonorrhoeae] Length = 139 Score = 274 bits (700), Expect = 7e−73 Identities = 139/139 (100%), Positivies = 139/139 (100%) Query: 2 MILLDTNVISEPLRPQPNERVVAWLDSLILEDVYLSAITVAELRLGVALLLNGKKKNVLH 61 MILLDTNVISEPLRPQPNERVVAWLDSLILEDVYLSAITVAELRLGVALLLNGKKKNVLH Sbjct: 1 MILLDTNVISEPLRPQPNERVVAWLDSLILEDVYLSAITVAELRLGVALLLNGKKKNVLH 60 Query: 62 ERLEQSILPLFAGRILPFDEPVAAIYAQIRSYAKTHGKEIAAADGYIAATAKQHSLTVAT 121 ERLEQSILPLFAGRILPFDEPVAAIYAQIRSYAKTHGKEIAAADGYIAATAKQHSLTVAT Sbjct: 61 ERLEQSILPLFAGRILPFDEPVAAIYAQIRSYAKTHGKEIAAADGYIAATAKQHSLTVAT 120 Query: 122 RDTGSFFAADVAVFNPWHD 140 RDTGSFFAADVAVFNPWHD Sbjct: 121 RDTGSFFAADVAVFNPWHD 139 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 145 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 289> which encodes amino acid sequence <SEQ ID 290; NGS148>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): 0.86 Possible cleavage site: 47 >>> Seems to have a cleavable N-term signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 48 ALOM: Finding transmembrane regions (Klein et al.)","count: 5 value: −15.44 threshold: 0.0 INTEGRAL Likelihood = −15.44 Transmembrane 157-173 (142-181) INTEGRAL Likelihood = −12.15 Transmembrane 62-78 (56-83) INTEGRAL Likelihood = −6.32 Transmembrane 194-210 (191-212) INTEGRAL Likelihood = −4.30 Transmembrane 87-103 (85-104) INTEGRAL Likelihood = −2.60 Transmembrane 121-137 (121-142) PERIPHERAL Likelihood = 2.92 modified ALOM score: 3.59 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.718(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_15777859|gi|15777859|gb|AAL05955.1|\\(AY048756) putative cadmium binding protein [Staphylococcus aureus] Length = 209 Score = 354 bits (908), Expect = 1e−96 Identities = 177/208 (85%), Positives = 194/208 (93%) Query: 14 MRCFMFSTVITAAVLYIATAVDLLVILLIFFARANTRKEYRDIYIGQYLGSVILILVSLF 73 MRC M TV+TAAVLYIATAVDLLVILLIFFARA TRKEYRDIY+GQYLGS+ILILVSLF Sbjct: 1 MRCIMIQTVVAAAVLYIATAVDLLVILLIFFARAKTRKEYRDIYVGQYLGSIILILVSLF 60 Query: 74 LAFVLNYVPEKWVLGLLGLIPIYLGIKVAIYDDCEGEKRAKKELDEKGLSKLVGIVALVT 133 LAFVLNYVPEKW+LGLLGLIPIYLGIKVAIYDDCEGEKRAKKEL+EKGLSKLVG VA+VT Sbjct: 61 LAFVLNYVPEKWILGLLGLIPIYLGIKVAIYDDCEGEKRAKKELNEKGLSKLVGTVAIVT 120 Query: 134 VASCGADNIGLFVPYFVTLDLVDLLVTLLVFLILIFVLVYTAQRLANISGVGEIVEKFSR 193 +ASCGADNIGLFVPYFVTL + +LL+TL VFLILIF LV+TAQ+LANI G+GEIVEKFSR Sbjct: 121 IASCGADNIGLFVPYFVTLSVTNLLLTLFVFLILIFFLVFTAQKLANIPGIGEIVEKFSR 180 Query: 194 WIMAVIYIGLGLFIIIENNTIRTIISII 221 WIMA+IYI LGLFIIIEN+TI+TI+ I Sbjct: 181 WIMAIIYIALGLFIIIENDTIQTILGFI 208 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 146 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 291> which encodes amino acid sequence <SEQ ID 292; NGS149>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −0.63 Possible cleavage site: 43 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 2.12 threshold: 0.0 PERIPHERAL Likelihood = 2.12 modified ALOM score: −0.92 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.122(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_15675455 gi|15675455|ref|NP_269629.1|\\(NC_002737) conserved hypothetical protein [Streptococcus pyogenes] [Streptococcus pyogenes M1 GAS] gb|AAK34350.1|(AE006588) conserved hypothetical protein [Streptococcus pyogenes M1 GAS] Length = 224 Score = 106 bits (264), Expect = 3e−22 Identities = 63/151 (41%), Positives = 85/151 (55%), Gaps = 12/151 (7%) Query: 20 LSALQHYAFCPRQCALIHNEQAWAENYLTAQGKALHERVDSDE-PETCKGVRFEWTVHVL 78 LS +QH+ FC RQ ALIH EQ W +N TA G+ LH + D+ E K + + + Sbjct: 11 LSGIQHFQFCKRQWALIHIEQQWLDNEATAHGQVLHTKADNPYIKEKRKELLVSRAMPIS 70 Query: 79 ADKLGISGILDLVE---------VDTKTGRLKP--VEYKRGKPKPDPGDEIQLCAQGLCL 127 + +LG+SGI+D+VE + K G+ P VEYKRGKPK D D +QL AQ +CL Sbjct: 71 SAELGLSGIMDVVEFYKDDQGVSLRGKRGKWLPKVVEYKRGKPKKDTRDIVQLVAQTMCL 130 Query: 128 EEMTGQTVSEGALWYMQTRHRVPVVFSDGLR 158 EE ++EG L+Y RV V + LR Sbjct: 131 EETLDCDINEGCLYYHSVNQRVIVPMTSALR 161 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 147 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 293> which encodes amino acid sequence <SEQ ID 294; NGS150>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −0.71 Possible cleavage site: 19 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 1 value: −0.85 threshold: 0.0 INTEGRAL Likelihood = −0.85 Transmembrane 79-95 (79-96) PERIPHERAL Likelihood = 6.52 modified ALOM score: 0.67 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.134(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has no homology to sequences in the databases.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 148 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 295> which encodes amino acid sequence <SEQ ID 296; NGS151>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): 3.47 Possible cleavage site: 23 >>> Seems to have a cleavable N-term signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 24 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 14.32 threshold: 0.0 PERIPHERAL Likelihood = 14.32 modified ALOM score: −3.36 Score for OM-PP discrimination: −32.29 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: −32.29 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Periplasmic space?","Score: 3.22889 Periplasmic space?","Score: 3.22889 ----- Final Results ----- bacterial periplasmic space --- Certainty= 0.933(Affirmative) < succ> bacterial outer membrane --- Certainty= 0.253(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gsa_AAY75310 Neisseria gonorrheae ORF 649 protein sequence SEQ ID N0:2094|WO9957280-A2|21-MAR-2000 Length = 103 Score = 35.4 bits (80), Expect = 0.32 Identities = 25/85 (29%), Positives = 38/85 (44%), Gaps = 5/85 (5%) Query: 7 ILTGILLATALPASAHGMHKSKPLAMDELPPIQQYFKRAETCYNKAGNKADFARN-NTK 65 + T T+ PA H H SK L P C++Y +R Y GN + N + Sbjct: 13 VSTTAAAGTSEPAHRHTKHISKA-NKQMLHPECRKYLERRAAWYRSQGNVQELRENKKAR 71 Query: 66 FLFQALPAADLGQRKQMCQIAMDSF 90 F+ LP A ++K C+ A ++F Sbjct: 72 KAFRTLPYA---EQKIQCRAAYEAF 93 The protein was expressed in E.coli as a soluble 9.35 kDa His-fusion product and then purified.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 149 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 297> which encodes amino acid sequence <SEQ ID 298; NGS152>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −5.46 Possible cleavage site: 18 >>> Seems to have an uncleavable N-term signal seq Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 1 value: −3.19 threshold: 0.0 INTEGRAL Likelihood = −3.19 Transmembrane 368-384 (367-384) PERIPHERAL Likelihood = 0.53 modified ALOM score: 1.14 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.227(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gsa_AAY81609 Streptococcus pneumoniae type 4 protein sequence #109 |WO200006737-A2|24-MAY-2000 Length = 1237 Score = 48.1 bits (113), Expect = 4e−04 Identities = 80/312 (25%), Positives = 142/312 (44%), Gaps = 59/312 (18%) Query: 57 RRQARIRVGNLITDSLEHIRVKALLPLPL----KLPVKRI---NLPRNLPALPVRLRKTI 109 RRQ R + ++ L+H RV L P+ ++PV+++ +PR A RL++ + Sbjct: 941 RRQVR-QPQQVLVHQLQHQRVHRLRRQPVHQSQQVPVRQLPHQQVPRLQQAPVRRLQQVL 999 Query: 110 SPRQIGDALPILKLQRI--RLTLHLKPLPLHPQLGLLHIKRPVRIPLRHLAVQRTLVRLN 167 +P+ P+ + Q++ RL H + PL L +P R + L QR VRLN Sbjct: 1000 APQP--QPQPVRQPQQVSQRLNRHQRVRPLQQVLA----PQPQRQQVHRL--QRQRVRLN 1051 Query: 168 RRIKPPLLQHRLTVRRILRRSRRQPFPAQFPDRRIFIMFRHNPARRIKLCRRQLTVQGPR 227 R + LQ L A P R+ +H +R++ ++ L Q R Sbjct: 1052 RHQRVRPLQQVL---------------APQPQRQQVHRLQH---QRVRPLQQVLAPQPQR 1093 Query: 228 IRRSRPLIKLPLLRRQRIRPGRHQRTLRVKITHRLAAPIHIPVKSQRRRRPSARIRRARI 287 + R L+RQR+R +HQR + + H+L +H PV+ Q + R + ++++ + Sbjct: 1094 QQVHR-------LQRQRVRLSQHQRVRQPQQAHQL-LNLHQPVR-QPQHRQAPQLQQVPV 1144 Query: 288 APREIRPGPRIGGKRLIAARKP-QTGIRTPFESTRPAQPPRPILNIVTAQIHHIPITRR 345 + R R+ + + R+P Q +R P R + P+P+ LN H P+ R+ Sbjct: 1145 RQPQRRQVRRL---QQVPVRQPQQVPVRQP--QRRQVRRPQPVHLN------ RHQPV-RQ 1192 Query: 346 PGLIIRNGTPHR 357 P ++ + H+ Sbjct: 1193 PQQVLVHQLQHQ 1204 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 150 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 299> which encodes amino acid sequence <SEQ ID 300; NGS153>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −5.48 Possible cleavage site: 13 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 6.31 threshold: 0.0 PERIPHERAL Likelihood = 6.31 modified ALOM score: −1.76 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.150(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_15832758 gi|15832758|ref|NP_311531.1|\\(NC_002695) hypothetical protein [Escherichia coli O157:H7] dbj|BAB36927.1|(AP002562) hypothetical protein [Escherichia coli O157:H7] Length = 188 Score = 73.9 bits (180), Expect = 3e−12 Identities = 54/169 (31%), Positives = 79/169 (45%), Gaps = 15/169 (8%) Query: 12 LTQEVLKELLRYDDNTGKLYWAERPRKYFNSGLHYKSWNTGFSGKEVFLYKGRLGYLKLK 71 LT + + ELL +D +TG W + + S F GY + Sbjct: 16 LTVKRIFELLSFDKSTGVFRWKVPTQ----GRIALNSVAGAFDSN------------GYSMIM 62 Query: 72 IFKKQYNAHRLIWLFVYGKH-ASSIGHINRDKTDNRISNLRDVTHAENMKNRGKFKNNTS 130 I ++Y H L++ + + A I H+N +TDNR NLR+ EN +N KN+ S Sbjct: 63 IDGRRYKTHVLVFYITHNRWPAGQIDHNGIRTDNRPENLRECLPIENSRNIRIRKNSKS 122 Query: 131 GHTGVYFKPSKKWQARIWINRKNKILGLFEHIEDAA-KAREAASKDFG 178 G GV +HK KKW R+ + K+K G F+ +E A A EA K +G Sbjct: 123 GCRGVTWMKRQKKWNVRLGFHGKSKHFGCFDDLELAVLVAEEARDKYYG 171 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 151 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 301> which encodes amino acid sequence <SEQ ID 302; NGS154>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −6.98 Possible cleavage site: 28 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 8.12 threshold: 0.0 PERIPHERAL Likelihood = 8.12 modified ALOM score: −2.12 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.423(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_15830449 gi|15830449|ref|NP_309222.1|\\(NC_002695) hypothetical protein [Escherichia coli O157:H7] dbj|BAA94132.1|(AP000422) hypothetical protein [Escherichia coli O157:H7] dbj|BAB346l8.1|(AP002554) hypothetical protein [Escherichia coli O157:H7] Length = 148 Score = 42.7 bits (99), Expect = 0.003 Identities = 27/99 (27%), Positives = 49/99 (49%), Gaps = 10/99 (10%) Query: 37 IRPRKSKRSVEQNRRLWFLYREISEKVFIDGRRFSQDVWHE-----FLKRKFIGCIEHPN 91 + ++ KRS QN R+W + ++S +V G+R + + W + +LK K + +P Sbjct: 33 VHVKEPKRSKAQNDRMWPMLNDVSRQVLWHGQRLAPEDWKDLFTALWLKTKKLEQRSVPG 92 Query: 92 GQ----LMGISTTKLSVEEMSEYQEKIISWASNEHGVLW 126 ++G+ T+K+ M+E E I+ W E V W Sbjct: 93 IDGGVVMLGVRTSKMRKASMTELIE-IMFEFGSERNVRW 130 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 152 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 303> which encodes amino acid sequence <SEQ ID 304; NGS155>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −4 Possible cleavage site: 27 >>> Seems to have an uncleavable N-term signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 4.98 threshold: 0.0 PERIPHERAL Likelihood = 4.98 modified ALOM score: −1.50 *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.046(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_15801502 gi|15801502|ref|NP_287519.1|\\(NC_002655) putative endonuclease of prophage CP-9330 [Escherichia coli O157:H7 EDL933] ref|NP_309804.1|(NC_002695) endonuclease [Escherichia coli O157:H7] gb|AAG56131.1|AE005344_7 (AE005344) putative endonuclease of prophage CP-933O [Escherichia coli O157:H7 EDL933] dbj|BAB35200.1|(AP002556) endonuclease [Escherichia coli O157:H7] Length = 119 Score = 47.4 bits (1111, Expect = 2e−04 Identities = 38/122 (31%), Positives = 54/122 (44%), Gaps = 8/122 (6%) Query: 71 LILPYPVSANRYWRIWRNRAVRSAEAAAYKETVRRIA-QGAGAMPSEGAVAVYVRLIPKA 129 L+LPYP + N YWR + S Y+ V I Q + G +A+ + P Sbjct: 5 LVLPYPPTVNTYWERRGSTYFVSKAGERYRRAVVLIVRQQRLKLSLSGRLAIKIIABP-- 62 Query: 130 NKDGGANKTVIDLDNLKVTLDALQGVAYHNDRQVRRIAAEYGGEPVTGGGLAVEVGELE 189 +K DLDN LK LDAL D + G+PV+GG L V++ ++E Sbjct: 63 -----PDKRRRDLDNILKAPLDALTHAGVLMDDEQFDEINIVRGQPVSGGRLGVKIYKIE 117 Query: 190 ME 191 E Sbjct: 118 SE 119 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 153 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 305> which encodes amino acid sequence <SEQ ID 306; NGS156>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −2.61 Possible cleavage site: 49 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 7.96 threshold: 0.0 PERIPHERAL Likelihood = 7.96 modified ALOM score: −2.09 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.307(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gsa_AAG90098 C glutamicum protein fragment SEQ ID NO: 3852 |EP1108790-A2|26-SEP-2001 Length = 148 Score = 102 bits (253), Expect = 7e-21 Identities = 60/147 (40%).","Positives = 88/147 (59%), Gaps = 18/147 (12%) Query: 3 NAYDVADFFLSPFEEEDGEQISNLKLQKLLYYAQGYALAILNRPLFAENIEHWQHGPVVP 62 +A ++A++F++ +E D E +S LKLQKLLYY+QG +A R LF++ I WQHGPV P Sbjct: 5 SAREIAEWFVAWGDELDAE-VSPLKLQKLLYYSQGEHIAATGRKLFSDKILAWQHGPVTP 63 Query: 63 CIYRTYKKYGGSPLPAAHIEPDKYADEEL---------VVLNRVRKEQGCYTAWALRNKT 113 +Y K YG +P I+PD++ E L V ++ G Y+AWALR KT Sbjct: 64 GVYSDTKSYGRNP-----IDPDEFVSDEFNWDDYSDVSDELVTVWRKYGIYSAWALREKT 118 Query: 114 HQEAPWIQT-RQGEVIGI--AIMGEYF 137 H E+PW+ QG+ I I A + ++F Sbjct: 119 HSESPWLDAWAQGQNIEITDAALKDFF 145 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 154 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 307> which encodes amino acid sequence <SEQ ID 308; NGS157>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −5.65 Possible cleavage site: 42 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 3.98 threshold: 0.0 PERIPHERAL Likelihood = 3.98 modified ALOM score: −1.30 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.291(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_6274533 gi|6274533|gb|AAF06681.1|AF163663_6 \\(AF058689) Toul [Neisseria meningitidis] Length = 272 Score = 546 bits (1408), Expect e−154 Identities = 267/272 (98%), Positives = 271/272 (99%) Query: 19 MKGMDKLRYQRDFLNIRPIFTAGEQEYLTELSDRLPLSVLTDSVRNIEEIGIDVYSPAK 78 MKGMDKLRYQ+DFLNIRPIFTAGEQEYLTELSDRLPLSVLTDSVRNIEEIGIDVYS AK Sbjct: 1 MKGMDKLRYQQDFLNIRPIFTAGEQEYLTELSDRLPLSVLTDSVRNIEEIGIDVYSSAK 60 Query: 79 LEGNTYNQYDTQALLKLGQTAGGKLYSDAVMLINLRESYRHLLSGLDSPKPFDWLDFLKT 138 LEGNTYNQYDTQALLKLGQTAGGKLYSDAVMLINLRESYRHLLSGLDSP+PFDWLDFLKT Sbjct: 61 LEGNTYNQYDTQALLKLGQTAGGKLYSDAVMLINLRESYRHLLSGLDSPEPFDWLDFLKT 120 Query: 139 THSLISENLLEKGSGGVVRRDSVTISGTDYTPLSNPQSLDTELKWLLQEAPKIENPFDRA 198 THSLISENLLEKGSGGVVRRDSVTISGTDYTPLSNPQSLDTELKWLLQEAPKIENPFDRA Sbjct: 121 THSLISENLLEKGSGGVVRRDSVTISGTDYTPLSNPQSLDTELKWLLQEAPKIENPFDRA 180 Query: 199 VYLHNNLAYLRYFKDCNKRTARNCMTLSLMRSGFFPCVFSPDSYPAYAEAVVAYYETGDY 258 VYLHNNLAYL+YFKDCNKRTARNCMTLSLMRSGFFPCVFSPDSYPAYAEAVVAYYETGDY Sbjct: 181 VYLHNNLAYLQYFKDCNKRTARNCMTLSLMRSGFFPCVFSPDSYPAYAEAVVAYYETGDY 240 Query: 259 GLFKKYFISAYENTVNKYGPQPDVDIFRNFSI 290 GLFKKYFISAYENTVNKYGPQPDVDIFRNFS+ Sbjct: 241 GLFKKYFISAYENTVNKYGPQPDVDIFRNFSL 272 A homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS157 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 155 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 309> which encodes amino acid sequence <SEQ ID 310; NGS158>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −6.98 Possible cleavage site: 18 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 0.16 threshold: 0.0 PERIPHERAL Likelihood = 0.16 modified ALOM score: −0.53 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.185(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: > {circumflex over ( )}{circumflex over ( )} **gbp_15791833 gi|15791833|ref|NP_281656.1|\\(NC_002163) amino-acid ABC transporter ATP-binding protein [Campylobacter jejuni] pir||H81391 amino-acid ABC transporter ATP-binding protein Cj0469 [imported]-Campylobacter jejuni (strain NCTC 11168) emb|CAB75107.1|(AL139075) amino-acid ABC transporter ATP-binding protein [Campylobacter jejuni] Length = 253 Score = 301 bits (772), Expect = 6e−81 Identities = 153/244 (62%), Positives = 195/244 (79%), Gaps = 2/244 (0%) Query: 1 MALLSIRKLHKQYGSVAIQSLDLDLEKGEVIVLLGPSGCGKSTLLRCVNGLEPHQGGSI 60 M++L I L K YGS A++ ++L+++ EV+V+LGPSGCGKSTLLRC+NGLE G+I Sbjct: 1 MSILKIENLQKYYGSHHALKDINLEVKAKEVVVILGPSGCGKSTLLRCINGLEEIASGNI 60 Query: 61 VMDGVGEFGKDVS-WQTARQKVGMVFQSYELFAHMTVIENILLGPVKVQNRDRAEAEAQA 119 +D + KD W RQKVGMVFQSYELF H++V ENILLGP+KVQ R + E +A Sbjct: 61 YIDNE-KIDKDFKEWPRMRQKVGMVFQSYELFEHLSVEENILLGPMKVQKRKKDEVLKEA 119 Query: 120 GKLLERVGLLDRKNAYPRELSGGQKQRIAIVRALCLNPEVILLDEITAALDPEMVREVLE 179 LE+VGLL + +AYPRELSGGQKQRIAIVR+LC+NPE++L DE+TAALDPE+VREVLE Sbjct: 120 KIWLEKVGLLHKIHAYPRELSGGQKQRIAIVRSLCMNPELMLFDEVTAALDPEIVREVLE 179 Query: 180 VVLELAREGMSMLIVTHEMGFARKVADRIVFMDKGGIVESSDPETFFSAPKSERARQFLA 239 V+L LA+EGM+MLIVTHEMGFA+ VAD+I+FMD+G I+E +DP++FF PKSERA++FL Sbjct: 180 VMLNLAKEGMTMLIVTHEMGFAKAVADKIIFMDEGKIIEENDPKSFFKNPKSERAKKFLN 239 Query: 240 GMDY 243 DY Sbjct: 240 LFDY 243 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 156 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 311> which encodes amino acid sequence <SEQ ID 312; NGS159>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −4.16 Possible cleavage site: 13 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 6.47 threshold: 0.0 PERIPHERAL Likelihood = 6.47 modified ALOM score: −1.79 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.312(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: > {circumflex over ( )}{circumflex over ( )} **gbp_15794799 gi|1579479.9|ref|NP_284621.1|\\(NC_003116) hypothetical protein [Neisseria meningitidis Z2491] pir||B81819 hypothetical protein NMA1914 [imported]- Neisseria meningitidis (group A strain Z2491) emb|CAB85135.1|(AL162757) hypothetical protein [Neisseria meningitidis Z2491] Length = 206 Score = 265 bits (678), Expect = 8e−70 Identities = 131/146 (89%), Positives = 138/146 (93%) Query: 1 MTKLYAQIAKTEAQDDGTVKVWGYASSEAVDSDGEVVAAEAMKAAIPDYMKFGAVREMHG 60 MTKLYA+IAK E QDDGTVKVWGYASSE +DSDGEV+AA AMKAAIPDYMKFGA REMHG Sbjct: 1 MTKLYAEIAKMETQDDGTVKVWGYASSEEIDSDGEIAAAAMKAAIPDYMKFGAGREMHG 60 Query: 61 SNAAGTAIEINVEDDGRTFFGAHIVDPVAVTKVKTGVYKGFSIGGSVTARNDLNKSQITG 120 SNAAGTAIEINVEDDG TFFGAHI+DPV V+KVKTGVYKGFSIGGSVTAR+DLNKSQITG Sbjct: 61 SNAAGTAIEINVEDDGITFFGAHIIDPVVVSKVKTGVYKGFSIGGSVTARDDLNKSQITG 120 Query: 121 LKLTEISLVDRPANPDAVFTCFKADK 146 LKLTEISL+DRPANPDAV TCFKADK Sbjct: 121 LKLTEISLIDRPANPDAVSTCFKADK 146 A homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS159 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 157 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 313> which encodes amino acid sequence <SEQ ID 314; NGS160>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −8.3 Possible cleavage site: 33 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 7.85 threshold: 0.0 PERIPHERAL Likelihood = 7.85 modified ALOM score: −2.07 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.407(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: > {circumflex over ( )}{circumflex over ( )} **gbp_2126352 gi|2126352|pir||JC5218 type I site-specific deoxyribonuclease (EC 3.1.21.3) Hsd chain S [validated]-Pasteurella haemolytica gb|AAC44667.1|(U46781) HSDS [Mannheimia haemolytica] Length = 442 Score = 97.1 bits (240), Expect = 3e−19 Identities = 55/149 (36%), Positives = 81/149 (53%), Gaps = 3/149 (2%) Query: 26 EVAEYSKNRICSDKLNEHNYVGVDNLLQNREGKKLSGYVPSEGKMTEYIVNDILIGNIRP 85 ++E +I L + NY+ DN+L N G L+ +P+ + DIL NIR Sbjct: 10 DIVELISEKIKIKDLKKENYISTDNMLPNFGGITLAENLPNSASCNRFAKKDILFSNIRT 69 Query: 86 YLKKIWQADCTGGTNGDVLVIRV--TDEKVNPKYLYQVLADDKFFAFNMKHAKGAKMPRG 143 Y KK+W A+ +GG + DVLV+R TD +N +YL+ ++ D F F + A GAKMPRG Sbjct: 70 YFKKVWLAEFSGGCSPDVLVMRSKNTDILLN-EYLFLLIRSDDFINFTVISANGAKMPRG 128 Query: 144 SKAAIMQYKIPIPPLPEQEKIVAILGKFD 172 K A+ + IP + Q+K +A FD Sbjct: 129 DKNAMKGFIFNIPSIEYQKKCIANYFAFD 157 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 158 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 315> which encodes amino acid sequence <SEQ ID 316; NGS161>.","Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −5.08 Possible cleavage site: 36 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 1 value: −1.59 threshold: 0.0 INTEGRAL Likelihood = −1.59 Transmembrane 302-318 (302-318) PERIPHERAL Likelihood = 3.76 modified ALOM score: 0.82 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.164(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: pir||E81921 probable DNA-invertase NMA0772 [imported]-Neisseria meningitidis (group A strain Z2491) emb|CAB84055.1|(AL162754) putative DNA-invertase [Neisseria meningitidis Z2491] Length = 321 Score = 295 bits (755), Expect = 9e−79 Identities = 151/322 (46%), Positives = 216/322 (66%), Gaps = 3/322 (0%) Query: 14 LRNAVGLDISKLTFDATAIVGNAEYSKFDNDSKGLDQFSDRLKSLGCQNLHICMEATGN 73 +RNAVGLDIS TFD I+ KF ND +G + + + +++++CMEATGN Sbjct: 1 MRNAVGLDISAKTFDVVTIINGETDYRKFSNDEQGCKNLKEWISAKREKDIYVCMEATGN 60 Query: 74 YYEEVADYFAQYYSVYVVNPLKISKYAESRFKRTKTDKQDAKLIAQYCRSAQESELVKRQ 133 YYE+ AD A+ Y V V+NPLKI YA+ RF R K DKQDAKLIA++C++A EL KR+ Sbjct: 61 YYEQAADCLAEEYHVSVINPLKIKAYAQKRFSRVKNDKQDAKLIAEFCQTALIEELPKRE 120 Query: 134 KPTDEQYRLSRMTAAYAQIKSECAAMKNRHHAAKDEEAAKAYAEIIKAMNEQLEVLKEKI 193 KPT++QY L R+ + +Q+ + + KNR AAKD K + + +K + L +K+KI Sbjct: 121 KPTEQQYSLKRLLSLQSQLLEQQTSQKNRIRAAKDSFVQKIHEKQLKELENHLNAVKKKI 180 Query: 194 KEQTEKPN--CKEGVKRLETIPAIGRMTAAVLFHHLTSSKFETSNKFAAFAGLSPQQKES 251 +QT K + KE KRLETIP++G+ TA L +L +S FE + +FA +AGL+P Q S Sbjct: 181 -DQTIKSDKKMKELTKRLETIPSVGKTTAISLMSYLINSTFENAKQFTAYAGLNPHQNIS 239 Query: 252 GTSVRGKGKLTKFGNRKLRAVLFMPAMVAYRIRAFPDFIKRLEEKKKPKKVIIAALMRKL 311 GTSV K K+TK+GNR++R LFM A+VA++ FP F RL++ KKPK +II ALMRK+ Sbjct: 240 GTSVNKKSKMTKYGNRRIRGSLFMAALVAFKNNYFPAFTNRLKKAKKPKMLIIGALMRKI 299 Query: 312 AVIAYHVHKKGGDYDPSRYKSA 333 V+A++++K D+D +RY++A Sbjct: 300 LVVAFNLYKTETDFDKTRYQTA 321 A homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS161 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.","Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 159 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 983> which encodes amino acid sequence <SEQ ID 984; NGS162>.","Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −0.86 Possible cleavage site: 33 >>> Seems to have no N-terminal signal seq.","Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)","count: 0 value: 4.08 threshold: 0.0 PERIPHERAL Likelihood = 4.08 modified ALOM score: −1.32 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.032(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: ref|NP_312507.1|(NC_002695) hypothetical protein [Escherichia coli O157:H7] gb|AAG5B749.1|AE005587_7 (AE005587) putative adhesin [Escherichia coli O157:H7 EDL933] dbj|BAB37903.1|(AP002566) hypothetical protein [Escherichia coli O157:H7] Length = 1588 Score = 120 bits (302), Expect = 4e−26 Identities = 109/359 (30%), Positives = 170/359 (46%), Gaps = 65/359 (18%) Query: 22 AVALGSSSTASGEYSYASGYNSVASGNKSYAAGYASVASAEGSVVIGDSRQVKPEADQGV 81 + A+G + A G+YS A G + A G S A G ++++ + S+ +G S + + Sbjct: 93 STAVGYDAIAEGQYSSAIGSKTHAIGGASMAFGVSAISEGDRSIALGASSYSLGQYSMAL 152 Query: 82 AVGSKATVKNKAKQRVVVGSEAKVNAERGIAIGKEAKAGGKTTNTLLDGPAYYADAIAVG 141 SKA K + +G +K IA+G KA T + +IA+G Sbjct: 153 GRYSKAL----GKLSIAMGDSSKAEGANAIALGNATKA----TEIM---------SIALG 195 Query: 142 YQAEAGKGGAIALGKQAKATKQNGMALGVESEAAGDFSTAVGNESKAKGQGG-------- 193 A A K ++ALG + A+++N +A+G E+EAA + +TA+GN +KAKG Sbjct: 196 DTANASKAYSMALGASSVASEENAIAIGAETEAA-ENATAIGNNAKAKGTNSMAMGFGSL 254 Query: 194 ------VGLGNQSKAEADFAVAV--GNKAEATKE------------NSLVIGRYARANGN 233 + LGN S+A AD A+A+ GNKA+ N++ +G + A G+ Sbjct: 255 ADKVNTIALGNGSQALADNAIAIGQGNKADGVDAIALGNGSQSRGLNTIALGTASNATGD 314 Query: 234 HSVSLGSRSEIKDGVSNSVAPGYGSVASENNVVSVAYKETPQSTELSYRKIVGVDDGV-- 291 S++LGS S +G+ NSVA G S+A +N VSV RKIV V +G Sbjct: 315 KSLALGSNSS-ANGI-NSVALGADSIADLDNTVSVGNSSLK-------RKIVNVKNGAIK 365 Query: 292 -NDFDAVNVRQLKAMQGQNMAELFSVRSEVRGVAASSAALSALTPLSYDANNPTQFMVG 349 + +DA+N QL A+ SV + G AA +T +Y+ N ++ VG Sbjct: 366 SDSYDAINGSQLYAISD-------SVAKRLGGGAAVDVDDGTVTAPTYNLKNGSKNNVG 417 Score = 86.3 bits (212), Expect = 1e−15 Identities = 68/253 (26%), Positives = 118/253 (45%), Gaps = 39/253 (15%) Query: 28 SSTASGEYSYASGYNSVASGNKSYAAGYASVASAEGSVVIGDSRQVKPEADQGVAVGSKA 87 S+ +G + G + A + Y S ++ +G V IG G+KA Sbjct: 38 SALVAGGMLSSFGALANAGNDNGQGVDYGSGSAGDGWVAIGK--------------GAKA 83 Query: 88 -TVKNKAKQRVVVGSEAKVNAERGIAIGKEAKAGGKTTNTLLDGPAYYADAIAVGYQAEA 146 T N + VG +A + AIG + A G + +A G A + Sbjct: 84 NTFMNTSGSSTAVGYDAIAEGQYSSAIGSKTHAIGGAS-------------MAFGVSAIS 130 Query: 147 GKGGAIALGKQAKATKQNGMALGVESEAAGDFSTAVGNESKAKGQGGVGLGNQSKAEADF 206 +IALG + + Q MALG S+A G S A+G+ SKA+G + LGN +KA Sbjct: 131 EGDRSIALGASSYSLGQYSMALGRYSKALGKLSIAMGDSSKAEGANAIALGNATKATEIM 190 Query: 207 AVAVGNKAEATKENSLVIGRYARANGNHSVSLGSRSEIKDGV-----------SNSVAPG 255 ++A+G+ A A+K S+ +G + A+ +++++G+ +E + +NS+A G Sbjct: 191 SIALGDTANASKAYSMALGASSVASEENAIAIGAETEAAENATAIGNNAKAKGTNSMAMG 250 Query: 256 YGSVASENNVVSV 268 +GS+A + N +++ Sbjct: 251 FGSLADKVNTIAL 263 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.","EXAMPLE 160 Further open reading frames were identified in gonococcus <SEQ IDs 317/318 to 8621/8622>.","These polypeptide and nucleotide sequences are useful for studying gonococcus, for diagnostic purposes, as antibiotic targets, and as vaccine antigens.","It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention."]],"string":"[\n [\n \"Gonococcal proteins and nucleic acids\",\n \"The invention provides proteins from gonococcus (Neisseria gonorrhoeae), including amino acid sequences, the corresponding nucleotide sequences, expression data, and serological data.\",\n \"The proteins are useful antigens for vaccines, immunogenic compositions, and/or diagnostics.\",\n \"They are also useful for distinguishing between gonococcus and meningococcus and, in particular, between gonococcus and serogroup B meningococcus.\"\n ],\n [\n \"1.A protein comprising an amino acid sequence selected from the group consisting of SEQ IDs 26, 72, 230, 984, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314 and 316: 2.A protein having 50% or greater sequence identity to a protein according to claim 1.3.A protein comprising a fragment of an amino acid sequence selected from the group consisting of 26, 72, 230, 984, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204,206,208, 210, 212,214, 216, 218,220,222, 224, 226, 228, 232,234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314 and 316.4.An antibody which specifically binds to a protein according to any one of claims 1 to 3.5.A nucleic acid molecule which encodes a protein according to any one of claims 1 to 3.6.A nucleic acid molecule according to claim 5, comprising a nucleotide sequence selected from the group consisting of SEQ IDs 25, 71, 229, 983, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313 and 315.7.A nucleic acid molecule comprising a fragment of a nucleotide sequence selected from the group consisting of SEQ IDs 25, 71, 229, 983, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313 and 315.8.A nucleic acid molecule comprising a nucleotide sequence complementary to a nucleic acid molecule according to any one of claims 5 to 7.9.A nucleic acid molecule comprising a nucleotide sequences having 50% or greater sequence identity to a nucleic acid molecule according to any one of claims 5 to 8.10.A nucleic acid molecule which can hybridise to a nucleic acid molecule according to any one of claims 5 to 9 under high stringency conditions.\",\n \"11.A composition comprising a protein, a nucleic acid molecule, or an antibody according to any preceding claim.\",\n \"12.A composition according to claim 11 being a vaccine composition or a diagnostic composition.\",\n \"13.A composition according to claim 11 or claim 12 for use as a pharmaceutical.\",\n \"14.The use of a composition according to claim 13 in the manufacture of a medicament for the treatment or prevention of infection due to streptococcus bacteria, particularly N.gonorrhoeae.\",\n \"15.A process for distinguishing N.gonorrhoeae from N.meningitidis, comprising the steps of: (a) contacting a protein, a nucleic acid molecule, or an antibody according to any one of claims 1 to 10 with a biological sample under conditions suitable for the formation of an antibody-antigen complexes; and (b) detecting said complexes.\"\n ],\n [\n \"<SOH> BACKGROUND ART <EOH>Neisseria gonorrhoeae is a bacterial pathogen.\",\n \"There is currently no effective vaccine against N.gonorrhoeae infection.\",\n \"It is an object of the invention to provide proteins and nucleic acid useful in vaccine study and/or manufacture.\",\n \"N.gonorrhoeae is related to N.meningitidis.\",\n \"Sequence data are now available for serogroup B of meningococcus [e.g.\",\n \"WO99/24578; WO99/36544; WO99/57280; WO00/22430; WO00/66791; Tettelin et al.\",\n \"(2000) Science 287:1809-1815] and also for serogroup A [Parkhill et al.\",\n \"(2000) Nature 404:502-506].\",\n \"It is a further object of the invention to provide proteins and nucleic acid useful in distinguishing between gonococcus and meningococcus and, in particular, between gonococcus and serogroup B meningococcus.\"\n ],\n [\n \"<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>There are no drawings.\",\n \"detailed-description description=\\\"Detailed Description\\\" end=\\\"lead\\\"?\"\n ],\n [\n \"TECHNICAL FIELD This invention relates to proteins from the bacterium Neisseria gonorrhoeae, and more particularly to such proteins which do not have corresponding homologs or orthologs in serogroup B N.meningitidis.\",\n \"BACKGROUND ART Neisseria gonorrhoeae is a bacterial pathogen.\",\n \"There is currently no effective vaccine against N.gonorrhoeae infection.\",\n \"It is an object of the invention to provide proteins and nucleic acid useful in vaccine study and/or manufacture.\",\n \"N.gonorrhoeae is related to N.meningitidis.\",\n \"Sequence data are now available for serogroup B of meningococcus [e.g.\",\n \"WO99/24578; WO99/36544; WO99/57280; WO00/22430; WO00/66791; Tettelin et al.\",\n \"(2000) Science 287:1809-1815] and also for serogroup A [Parkhill et al.\",\n \"(2000) Nature 404:502-506].\",\n \"It is a further object of the invention to provide proteins and nucleic acid useful in distinguishing between gonococcus and meningococcus and, in particular, between gonococcus and serogroup B meningococcus.\",\n \"DISCLOSURE OF THE INVENTION The invention provides proteins comprising the N.gonorrhoeae amino acid sequences disclosed in the examples (the even-numbered SEQ IDS 2 to 8622).\",\n \"159 of these have no homolog in serogroup B meningococcus and these have been given a name in the form “NGSn”.\",\n \"It also provides proteins comprising amino acid sequences having sequence identity to the N.gonorrhoeae amino acid sequences disclosed in the examples.\",\n \"Depending on the particular sequence, the degree of sequence identity is preferably greater than 50% (e.g.\",\n \"60%, 70%, 80%, 90%, 95%, 99% or more).\",\n \"These proteins include homologs, orthologs, allelic variants and functional mutants.\",\n \"Typically, 50% identity or more between two proteins is considered to be an indication of functional equivalence.\",\n \"Identity between proteins is preferably determined by the Smith-Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affine gap search with parameters gap open penalty=12 and gap extension penalty=1.The invention further provides proteins comprising fragmnents of the N.gonorrhoeae amino acid sequences disclosed in the examples.\",\n \"The fragments should comprise at least n consecutive amino acids from the sequences and, depending on the particular sequence, n is 7 or more (e.g.\",\n \"8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more).\",\n \"Preferably the fragments comprise one or more epitopes from the sequence.\",\n \"Other preferred fragments are (a) the N-terminal signal peptides of the proteins disclosed in the examples, and (b) the proteins disclosed in the examples, but without their N-terminal signal peptides.\",\n \"The proteins of the invention can, of course, be prepared by various means (e.g.\",\n \"recombinant expression, purification from Neisseria, chemical synthesis etc.)\",\n \"and in various forms (e.g.\",\n \"native, fusions etc.).\",\n \"They are preferably prepared in substantially pure form (i.e.\",\n \"substantially free from other N.gonorrhoeae or host cell proteins).\",\n \"The proteins of the invention are preferably Neisserial proteins, more preferably N.gonorrhoeae proteins.\",\n \"The invention provides antibodies which bind to these proteins.\",\n \"These may be polyclonal or monoclonal and may be produced by any suitable means.\",\n \"The antibodies may include a detectable label.\",\n \"The invention provides nucleic acid comprising the N.gonorrhoeae nucleotide sequences disclosed in the examples.\",\n \"In addition, the invention provides nucleic acid comprising nucleotide sequences having sequence identity to the N.gonorrhoeae nucleotide sequences disclosed in the examples.\",\n \"Furthermore, the invention provides nucleic acid which can hybridise to the N.gonorrhoeae nucleic acid disclosed in the examples, preferably under “high stringency” conditions (e.g.\",\n \"65° C. in a 0.1×SSC, 0.5% SDS solution).\",\n \"Nucleic acid comprising fragments of these sequences are also provided.\",\n \"These should comprise at least n consecutive nucleotides from the N.gonorrhoeae sequences and, depending on the particular sequence, n is 10 or more (e.g.\",\n \"12, 14, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more).\",\n \"The invention also provides nucleic acid encoding the proteins and protein fragments of the invention.\",\n \"The invention includes nucleic acid comprising sequences complementary to those described above (e.g.\",\n \"for antisense or probing purposes).\",\n \"Nucleic acid according to the invention can be prepared in many ways (e.g.\",\n \"by chemical synthesis, from genomic or cDNA libraries, from the organism itself etc.)\",\n \"and can take various forms (e.g.\",\n \"single stranded, double stranded, vectors, probes etc.).\",\n \"In addition, the term “nucleic acid” includes DNA and RNA, and also their analogues, such as those containing modified backbones, and also peptide nucleic acids (PNA) etc.\",\n \"The invention provides vectors comprising nucleotide sequences of the invention (e.g.\",\n \"cloning or expression vectors) and host cells transformed with such vectors.\",\n \"The invention provides compositions comprising protein, antibody, and/or nucleic acid according to the invention.\",\n \"These compositions may be suitable as immunogenic compositions, for instance, or as diagnostic reagents, or as vaccines.\",\n \"The invention also provides nucleic acid, protein, or antibody according to the invention for use as medicaments (e.g.\",\n \"as vaccines) or as diagnostic reagents.\",\n \"It also provides the use of nucleic acid, protein, or antibody according to the invention in the manufacture of: (i) a medicament for treating or preventing infection due to Neisseria; (ii) a diagnostic reagent for detecting the presence of Neisseria or of antibodies raised against Neisseria; and/or (iii) a reagent which can raise antibodies against Neisseria.\",\n \"Said Neisseria may be any species, but is preferably N.gonorrhoeae.\",\n \"The invention also provides a method of treating a patient, comprising administering to the patient a therapeutically effective amount of nucleic acid, protein, and/or antibody of the invention.\",\n \"According to further aspects, the invention provides various processes.\",\n \"A process for producing proteins of the invention is provided, comprising the step of culturing a host cell of to the invention under conditions which induce protein expression.\",\n \"A process for producing protein or nucleic acid of the invention is provided, wherein the protein or nucleic acid is synthesised in part or in whole using chemical means.\",\n \"A process for detecting polynucleotides of the invention is provided, comprising the steps of: (a) contacting a nucleic probe according to the invention with a biological sample under hybridising conditions to form duplexes; and (b) detecting said duplexes.\",\n \"A process for detecting proteins of the invention is provided, comprising the steps of: (a) contacting an antibody of the invention with a biological sample under conditions suitable for the formation of an antibody-antigen complexes; and (b) detecting said complexes.\",\n \"A process for distinguishing N.gonorrhoeae from N.meningitidis is provided, comprising the steps of: (a) contacting an antibody of the invention with a biological sample under conditions suitable for the formation of an antibody-antigen complexes; and (b) detecting said complexes.\",\n \"Alternatively, the steps may be (a) contacting nucleic acid of the invention with a biological sample under conditions suitable for the nucleic acid hybridisation; and (b) detecting any such hybridisation.\",\n \"Alternatively, the steps may be (a) contacting a protein of the invention with a biological sample (e.g.\",\n \"blood or serum) under conditions suitable for the formation of an antibody-antigen complexes; and (b) detecting said complexes.\",\n \"A summary of standard techniques and procedures which may be employed in order to perform the invention (e.g.\",\n \"to utilise the disclosed sequences for vaccination or diagnostic purposes) follows.\",\n \"This summary is not a limitation on the invention, but gives examples that may be used, but are not required.\",\n \"General The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art.\",\n \"Such techniques are explained fully in the literature eg.\",\n \"Sambrook Molecular Cloning; A Laboratory Manual, Second Edition (1989) or Third Edition (2000); DNA Cloning, Volumes I and II (D. N Glover ed.\",\n \"1985); Oligonucleotide Synthesis (M. J. Gait ed, 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds.\",\n \"1984); Transcription and Translation (B. D. Hames & S. J. Higgins eds.\",\n \"1984); Animal Cell Culture (R. I. Freshney ed.\",\n \"1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the Methods in Enzymology series (Academic Press, Inc.), especially volumes 154 & 155; Gene Transfer Vectors for Mammalian Cells (J. H. Miller and M. P. Calos eds.\",\n \"1987, Cold Spring Harbor Laboratory); Mayer and Walker, eds.\",\n \"(1987), Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Scopes, (1987) Protein Purification: Principles and Practice, Second Edition (Springer-Verlag, N.Y.), and Handbook of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell eds 1986).\",\n \"Standard abbreviations for nucleotides and amino acids are used in this specification.\",\n \"Definitions A composition containing X is “substantially free of” Y when at least 85% by weight of the total X+Y in the composition is X. Preferably, X comprises at least about 90% by weight of the total of X+Y in the composition, more preferably at least about 95% or even 99% by weight.\",\n \"The term “comprising” means “including” as well as “consisting” e.g.\",\n \"a composition “comprising” X may consist exclusively of X or may include something additional e.g.\",\n \"X+Y.\",\n \"The term “heterologous” refers to two biological components that are not found together in nature; The components may be host cells, genes, or regulatory regions, such as promoters.\",\n \"Although the heterologous components are not found together in nature, they can function together, as when a promoter heterologous to a gene is operably linked to the gene.\",\n \"Another example is where a Neisseria sequence is heterologous to a mouse host cell.\",\n \"A further examples would be two epitopes from the same or different proteins which have been assembled in a single protein in an arrangement not found in nature.\",\n \"An “origin of replication” is a polynucleotide sequence that initiates and regulates replication of polynucleotides, such as an expression vector.\",\n \"The origin of replication behaves as an autonomous unit of polynucleotide replication within a cell, capable of replication under its own control.\",\n \"An origin of replication may be needed for a vector to replicate in a particular host cell.\",\n \"With certain origins of replication, an expression vector can be reproduced at a high copy number in the presence of the appropriate proteins within the cell, Examples of origins are the autonomously replicating sequences, which are effective in yeast; and the viral T-antigen, effective in COS-7 cells.\",\n \"A “mutant” sequence is defined as DNA, RNA or amino acid sequence differing from but having sequence identity with the native or disclosed sequence.\",\n \"Depending on the particular sequence, the degree of sequence identity between the native or disclosed sequence and the mutant sequence is preferably greater than 50% (eg.\",\n \"60%, 70%, 80%, 90%, 95%, 99% or more, calculated using the Smith-Waterman algorithm as described above).\",\n \"As used herein, an “allelic variant” of a nucleic acid molecule, or region, for which nucleic acid sequence is provided herein is a nucleic acid molecule, or region, that occurs essentially at the same locus in the genome of another or second isolate, and that, due to natural variation caused by, for example, mutation or recombination, has a similar but not identical nucleic acid sequence.\",\n \"A coding region allelic variant typically encodes a protein having similar activity to that of the protein encoded by the gene to which it is being compared.\",\n \"An allelic variant can also comprise an alteration in the 5′ or 3′ untranslated regions of the gene, such as in regulatory control regions (eg.\",\n \"see U.S. Pat.\",\n \"No.\",\n \"5,753,235).\",\n \"Expression Systems The Neisseria nucleotide sequences can be expressed in a variety of different expression systems; for example those used with mammalian cells, baculoviruses, plants, bacteria, and yeast.\",\n \"i. Mammalian Systems Mammalian expression systems are known in the art.\",\n \"A mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3′) transcription of a coding sequence (eg.\",\n \"structural gene) into mRNA.\",\n \"A promoter will have a transcription initiating region, which is usually placed proximal to the 5′ end of the coding sequence, and a TATA box, usually located 25-30 base pairs (bp) upstream of the transcription initiation site.\",\n \"The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site.\",\n \"A mammalian promoter will also contain an upstream promoter element, usually located within 100 to 200 bp upstream of the TATA box.\",\n \"An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation [Sambrook et al.\",\n \"(1989) “Expression of Cloned Genes in Mammalian Cells.” In Molecular Cloning: A Laboratory Manual, 2nd ed.].\",\n \"Mammalian viral genes are often highly expressed and have a broad host range; therefore sequences encoding mammalian viral genes provide particularly useful promoter sequences.\",\n \"Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), and herpes simplex virus promoter.\",\n \"In addition, sequences derived from non-viral genes, such as the murine metallotheionein gene, also provide useful promoter sequences.\",\n \"Expression may be either constitutive or regulated (inducible), depending on the promoter can be induced with glucocorticoid in hormone-responsive cells.\",\n \"The presence of an enhancer element (enhancer), combined with the promoter elements described above, will usually increase expression levels.\",\n \"An enhancer is a regulatory DNA sequence that can stimulate transcription up to 1000-fold when linked to homologous or beterologous promoters, with synthesis beginning at the normal RNA start site.\",\n \"Enhancers are also active when they are placed upstream or downstream from the transcription initiation site, in either normal or flipped orientation, or at a distance of more than 1000 nucleotides from the promoter [Maniatis et al.\",\n \"(1987) Science 236:1237; Alberts et al.\",\n \"(1989) Molecular Biology of the Cell, 2nd ed.].\",\n \"Enhancer elements derived from viruses may be particularly useful, because they usually have a broader host range.\",\n \"Examples include the SV40 early gene enhancer [Dijkema et al (1985) EMBO J.\",\n \"4:7611 and the enhancer/promoters derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus [Gorman et al.\",\n \"(1982b) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"79:6777] and from human cytomegalovirus [Boshart et al.\",\n \"(1985) Cell 41:521].\",\n \"Additionally, some enhancers are regulatable and become active only in the presence of an inducer, such as a hormone or metal ion [Sassone-Corsi and Borelli (1986) Trends Genet.\",\n \"2:215; Maniatis et al.\",\n \"(1987) Science 236:1237].\",\n \"A DNA molecule may be expressed intracellularly in mammalian cells.\",\n \"A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon.\",\n \"If desired, the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.\",\n \"Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in mammalian cel Whitelaw (1988) “Termination and 3′ end processing of eukaryotic RNA, In Transcription and splicing (ed.\",\n \"B. D. Hames and D. M. Glover); Proudfoot (1989) Trends Biochem.\",\n \"Sci.\",\n \"14:105].\",\n \"These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA.\",\n \"Examples of transcription terminater/polyadenylation signals include those derived from SV40 [Sambrook et al (1989) “Expression of cloned genes in cultured mammalian cells.” In Molecular Cloning: A Laboratory Manual].\",\n \"Usually, the above described components, comprising a promoter, polyadenylation signal, and transcription termination sequence are put together into expression constructs.\",\n \"Enhancers, introns with functional splice donor and acceptor sites, and leader sequences may also be included in an expression construct, if desired.\",\n \"Expression constructs are often maintained in a replicon, such as an extrachromosomal element (eg.\",\n \"plasmids) capable of stable maintenance in a host, such as mammalian cells or bacteria.\",\n \"Mammalian replication systems include those derived from animal viruses, which require trans-acting factors to replicate.\",\n \"For example, plasmids containing the replication systems of papovaviruses, such as SV40 [Gluzman (1981) Cell 23:175] or polyomavirus, replicate to extremely high copy number in the presence of the appropriate viral T antigen.\",\n \"Additional examples of mammalian replicons include those derived from bovine papillomavirus and Epstein-Barr virus.\",\n \"Additionally, the replicon may have two replicaton systems, thus allowing it to be maintained, for example, in mammalian cells for expression and in a prokaryotic host for cloning and amplification.\",\n \"Examples of such mammalian-bacteria shuttle vectors include pMT2 [Kaufman et al.\",\n \"(1989) Mol.\",\n \"Cell.\",\n \"Biol.\",\n \"9:946] and pHEBO [Shimizu et al.\",\n \"(1986) Mol.\",\n \"Cell.\",\n \"Biol.\",\n \"6:1074].\",\n \"The transformation procedure used depends upon the host to be transformed.\",\n \"Methods for introduction of heterologous polynucleotides into mammalian cells are known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.\",\n \"Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg.\",\n \"Hep G2), and a number of other cell lines.\",\n \"ii.\",\n \"Baculovirus Systems The polynucleotide encoding the protein can also be inserted into a suitable insect expression vector, and is operably linked to the control elements within that vector.\",\n \"Vector construction employs techniques which are known in the art.\",\n \"Generally, the components of the expression system include a transfer vector, usually a bacterial plasmid, which contains both a fragment of the baculovirus genome, and a convenient restriction site for insertion of the heterologous gene or genes to be expressed; a wild type baculovirus with a sequence homologous to the baculovirus-specific fragment in the transfer vector (this allows for the homologous recombination of the heterologous gene in to the baculovirus genome); and appropriate insect host cells and growth media.\",\n \"After inserting the DNA sequence encoding the protein into the transfer vector, the vector and the wild type viral genome are transfected into an insect host cell where the vector and viral genome are allowed to recombine.\",\n \"The packaged recombinant virus is expressed and recombinant plaques are identified and purified.\",\n \"Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego Calif. (“MaxBac” kit).\",\n \"These techniques are generally known to those skilled in the art and fully described in Summers & Smith, Texas Agricultural Experiment Station Bulletin No.1555(1987) (“Summers & Smith”).\",\n \"Prior to inserting the DNA sequence encoding the protein into the baculovirus genome, the above described components, comprising a promoter, leader (if desired), coding sequence, and transcription termination sequence, are usually assembled into an intermediate transplacement construct (transfer vector).\",\n \"This may contain a single gene and operably linked regulatory elements; multiple genes, each with its owned set of operably linked regulatory elements; or multiple genes, regulated by the same set of regulatory elements.\",\n \"Intermediate transplacement constructs are often maintained in a replicon, such as an extra-chromosomal element (e.g.\",\n \"plasmids) capable of stable maintenance in a host, such as a bacterium.\",\n \"The replicon will have a replication system, thus allowing it to be maintained in a suitable host for cloning and amplification.\",\n \"Currently, the most commonly used transfer vector for introducing foreign genes into AcNPV is pAc373.Many other vectors, known to those of skill in the art, have also been designed.\",\n \"These include, for example, pVL985 (which alters the polyhedrin start codon from ATG to ATT, and which introduces a BamH1 cloning site 32 basepairs downstream from the ATT; see Luckow and Summers, Virology (1989)17:31.The plasmid usually also contains the polyhedrin polyadenylation signal (Miller et al.\",\n \"(1988) Ann.\",\n \"Rev.\",\n \"Microbiol., 42:177) and a prokaryotic ampicillin-resistance (amp) gene and origin of replication for selection and propagation in E. coli.\",\n \"Baculovirus transfer vectors usually contain a baculovirus promoter.\",\n \"A baculovirus promoter is any DNA sequence capable of binding a baculovirus RNA polymerase and initiating the downstream (5′to 3′) transcription of a coding sequence (eg.\",\n \"structural gene) into mRNA.\",\n \"A promoter will have a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence.\",\n \"This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site.\",\n \"A baculovirus transfer vector may also have a second domain called an enhancer, which, if present, is usually distal to the structural gene.\",\n \"Expression may be either regulated or constitutive.\",\n \"Structural genes, abundantly transcribed at late times in a viral infection cycle, provide particularly useful promoter sequences.\",\n \"Examples include sequences derived from the gene encoding the viral polyhedron protein, Friesen et al., (1986) “The Regulation of Baculovirus Gene Expression,” in: The Molecular Biology of Baculoviruses (ed.\",\n \"Walter Doerfler); EPO Publ.\",\n \"Nos.\",\n \"127 839 and 155 476; and the gene encoding the p10 protein, Vlak et al., (1988), J. Gen. Virol.\",\n \"69:765.DNA encoding suitable signal sequences can be derived from genes for secreted insect or baculovirus proteins, such as the baculovirus polyhedrin gene (Carbonell et al.\",\n \"(1988) Gene, 73:409).\",\n \"Alternatively, since the signals for mammalian cell posttranslational modifications (such as signal peptide cleavage, proteolytic cleavage, and phosphorylation) appear to be recognized by insect cells, and the signals required for secretion and nuclear accumulation also appear to be conserved between the invertebrate cells and vertebrate cells, leaders of non-insect origin, such as those derived from genes encoding human α-interferon, Maeda et al., (1985), Nature 315:592; human gastrin-releasing peptide, Lebacq-Verheyden et al., (1988), Molec.\",\n \"Cell.\",\n \"Biol.\",\n \"8:3129; human IL-2, Smith et al., (1985) Proc.\",\n \"Nat'l Acad.\",\n \"Sci.\",\n \"USA, 82:8404; mouse IL-3, (Miyajima et al., (1987) Gene 58:273; and human glucocerebrosidase, Martin et al.\",\n \"(1988) DNA, 7:99, can also be used to provide for secretion in insects.\",\n \"A recombinant polypeptide or polyprotein may be expressed intracellularly or, if it is expressed with the proper regulatory sequences, it can be secreted.\",\n \"Good intracellular expression of nonfused foreign proteins usually requires heterologous genes that ideally have a short leader sequence containing suitable translation initiation signals preceding an ATG start signal.\",\n \"If desired, methionine at the N-terminus may be cleaved from the mature protein by in vitro incubation with cyanogen bromide.\",\n \"Alternatively, recombinant polyproteins or proteins which are not naturally secreted can be secreted from the insect cell by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in insects.\",\n \"The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the translocation of the protein into the endoplasmic reticulum.\",\n \"After insertion of the DNA sequence and/or the gene encoding the expression product precursor of the protein, an insect cell host is co-transformed with the heterologous DNA of the transfer vector and the genomic DNA of wild type baculovirus—usually by co-transfection.\",\n \"The promoter and transcription termination sequence of the construct will usually comprise a 2-5 kb section of the baculovirus genome.\",\n \"Methods for introducing heterologous DNA into the desired site in the baculovirus virus are known in the art.\",\n \"(See Summers & Smith supra; Ju et al.\",\n \"(1987); Smith et al., Mol.\",\n \"Cell.\",\n \"Biol.\",\n \"(1983) 3:2156; and Luckow and Summers (1989)).\",\n \"For example, the insertion can be into a gene such as the polyhedrin gene, by homologous double crossover recombination; insertion can also be into a restriction enzyme site engineered into the desired baculovirus gene.\",\n \"Miller et al., (1989), Bioessays 4:91.The DNA sequence, when cloned in place of the polyhedrin gene in the expression vector, is flanked both 5′ and 3′ by polyhedrin-specific sequences and is positioned downstream of the polyhedrin promoter.\",\n \"The newly formed baculovirus expression vector is subsequently packaged into an infectious recombinant baculovirus.\",\n \"Homologous recombination occurs at low frequency (between about 1% and about 5%); thus, the majority of the virus produced after cotransfection is still wild-type virus.\",\n \"Therefore, a method is necessary to identify recombinant viruses.\",\n \"An advantage of the expression system is a visual screen allowing recombinant viruses to be distinguished.\",\n \"The polyhedrin protein, which is produced by the native virus, is produced at very high levels in the nuclei of infected cells at late times after viral infection.\",\n \"Accumulated polyhedrin protein forms occlusion bodies that also contain embedded particles.\",\n \"These occlusion bodies, up to 15 μm in size, are highly refractile, giving them a bright shiny appearance that is readily visualized under the light microscope.\",\n \"Cells infected with recombinant viruses lack occlusion bodies.\",\n \"To distinguish recombinant virus from wild-type virus, the transfection supernatant is plaqued onto a monolayer of insect cells by techniques known to those skilled in the art.\",\n \"Namely, the plaques are screened under the light microscope for the presence (indicative of wild-type virus) or absence (indicative of recombinant virus) of occlusion bodies.\",\n \"“Current Protocols in Microbiology” Vol.\",\n \"2 (Ausubel et al.\",\n \"eds) at 16.8 (Supp.\",\n \"10, 1990); Summers & Smith, supra; Miller et al.\",\n \"(1989).\",\n \"Recombinant baculovirus expression vectors have been developed for infection into several insect cells.\",\n \"For example, recombinant baculoviruses have been developed for, inter alia: Aedes aegypti , Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni (WO 89/046699; Carbonell et al., (1985) J. Virol.\",\n \"56:153; Wright (1986) Nature 321:718; Smith et al., (1983) Mol.\",\n \"Cell.\",\n \"Biol.\",\n \"3:2156; and see generally, Fraser, et al.\",\n \"(1989) In Vitro Cell.\",\n \"Dev.\",\n \"Biol.\",\n \"25:225).\",\n \"Cells and cell culture media are commercially available for both direct and fusion expression of heterologous polypeptides in a baculovirus/expression system; cell culture technology is generally known to those skilled in the art.\",\n \"See, eg.\",\n \"Summers & Smith supra.\",\n \"The modified insect cells may then be grown in an appropriate nutrient medium, which allows for stable maintenance of the plasmid(s) present in the modified insect host.\",\n \"Where the expression product gene is under inducible control, the host may be grown to high density, and expression induced.\",\n \"Alternatively, where expression is constitutive, the product will be continuously expressed into the medium and the nutrient medium must be continuously circulated, while removing the product of interest and augmenting depleted nutrients.\",\n \"The product may be purified by such techniques as chromatography, eg.\",\n \"HPLC, affinity chromatography, ion exchange chromatography, etc.\",\n \"; electrophoresis; density gradient centrifugation; solvent extraction, etc.\",\n \"As appropriate, the product may be further purified, as required, so as to remove substantially any insect proteins which are also present in the medium, so as to provide a product which is at least substantially free of host debris, eg.\",\n \"proteins, lipids and polysaccharides.\",\n \"In order to obtain protein expression, recombinant host cells derived from the transformants are incubated under conditions which allow expression of the recombinant protein encoding sequence.\",\n \"These conditions will vary, dependent upon the host cell selected.\",\n \"However, the conditions are readily ascertainable to those of ordinary skill in the art, based upon what is known in the art.\",\n \"iii.\",\n \"Plant Systems There are many plant cell culture and whole plant genetic expression systems known in the art.\",\n \"Exemplary plant cellular genetic expression systems include those described in patents, such as: U.S. Pat.\",\n \"Nos.\",\n \"5,693,506; 5,659,122; and 5,608,143.Additional examples of genetic expression in plant cell culture has been described by Zenk, Phytochemistry 30:3861-3863 (1991).\",\n \"Descriptions of plant protein signal peptides may be found in addition to the references described above in Vaulcombe et al., Mol.\",\n \"Gen. Genet.\",\n \"209:3340 (1987); Chandler et al., Plant Molecular Biology 3:407-418 (1984); Rogers, J. Biol.\",\n \"Chem.\",\n \"260:3731-3738 (1985); Rothstein et al., Gene 55:353-356 (1987); Whittier et al., Nucleic Acids Research 15:2515-2535 (1987); Wirsel et al., Molecular Microbiology 3:3-14 (1989); Yu et al., Gene 122:247-253 (1992).\",\n \"A description of the regulation of plant gene expression by the phytohormone, gibberellic acid and secreted enzymes induced by gibberellic acid can be found in R. L. Jones and J. MacMillin, Gibberellins: in: Advanced Plant Physiology,.\",\n \"Malcolm B. Wilkins, ed., 1984 Pitman Publishing Limited, London, pp.\",\n \"21-52.References that describe other metabolically-regulated genes: Sheen, Plant Cell, 2:1027-1038(1990); Maas et al., EMBO J.\",\n \"9:3447-3452 (1990); Benkel and Hickey, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"84:1337-1339 (1987).\",\n \"Typically, using techniques known in the art, a desired polynucleotide sequence is inserted into an expression cassette comprising genetic regulatory elements designed for operation in plants.\",\n \"The expression cassette is inserted into a desired expression vector with companion sequences upstream and downstream from the expression cassette suitable for expression in a plant host.\",\n \"The companion sequences will be of plasmid or viral origin and provide necessary characteristics to the vector to permit the vectors to move DNA from an original cloning host, such as bacteria, to the desired plant host.\",\n \"The basic bacterial/plant vector construct will preferably provide a broad host range prokaryote replication origin; a prokaryote selectable marker; and, for Agrobacterium transformations, T DNA sequences for Agrobacterium-mediated transfer to plant chromosomes.\",\n \"Where the heterologous gene is not readily amenable to detection, the construct will preferably also have a selectable marker gene suitable for determining if a plant cell has been transformed.\",\n \"A general review of suitable markers, for example for the members of the grass family, is found in Wilmink and Dons, 1993, Plant Mol.\",\n \"Biol.\",\n \"Reptr, 11(2):165-185.Sequences suitable for permitting integration of the heterologous sequence into the plant genome are also recommended.\",\n \"These might include transposon sequences and the like for homologous recombination as well as Ti sequences which permit random insertion of a beterologous expression cassette into a plant genome.\",\n \"Suitable prokaryote selectable markers include resistance toward antibiotics such as ampicillin or tetracycline.\",\n \"Other DNA sequences encoding additional functions may also be present in the vector, as is known in the art.\",\n \"The nucleic acid molecules of the subject invention may be included into an expression cassette for expression of the protein(s) of interest.\",\n \"Usually, there will be only one expression cassette, although two or more are feasible.\",\n \"The recombinant expression cassette will contain in addition to the heterologous protein encoding sequence the following elements, a promoter region, plant 5′ untranslated sequences, initiation codon depending upon whether or not the structural gene comes equipped with one, and a transcription and translation termination sequence.\",\n \"Unique restriction enzyme sites at the 5′ and 3′ ends of the cassette allow for easy insertion into a pre-existing vector.\",\n \"A heterologous coding sequence may be for any protein relating to the present invention.\",\n \"The sequence encoding the protein of interest will encode a signal peptide which allows processing and translocation of the protein, as appropriate, and will usually lack any sequence which might result in the binding of the desired protein of the invention to a membrane.\",\n \"Since, for the most part, the transcriptional initiation region will be for a gene which is expressed and translocated during germination, by employing the signal peptide which provides for translocation, one may also provide for translocation of the protein of interest.\",\n \"In this way, the protein(s) of interest will be translocated from the cells in which they are expressed and may be efficiently harvested.\",\n \"Typically secretion in seeds are across the aleurone or scutellar epithelium layer into the endosperm of the seed.\",\n \"While it is not required that the protein be secreted from the cells in which the protein is produced, this facilitates the isolation and purification of the recombinant protein.\",\n \"Since the ultimate expression of the desired gene product will be in a eucaryotic cell it is desirable to determine whether any portion of the cloned gene contains sequences which will be processed out as introns by the host's splicosome machinery.\",\n \"If so, site-directed mutagenesis of the “intron” region may be conducted to prevent losing a portion of the genetic message as a false intron code, Reed and Maniatis, Cell 41:95-105, 1985.The vector can be microinjected directly into plant cells by use of micropipettes to mechanically transfer the recombinant DNA.\",\n \"Crossway, Mol.\",\n \"Gen. Genet, 202:179-185, 1985.The genetic material may also be transferred into the plant cell by using polyethylene glycol, Krens, et al., Nature, 296, 72-74, 1982.Another method of introduction of nucleic acid segments is high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface, Klein, et al., Nature, 327,70-73, 1987 and Knudsen and Muller, 1991, Planta, 185:330-336 teaching particle bombardment of barley endosperm to create transgenic barley.\",\n \"Yet another method of introduction would be fusion of protoplasts with other entities, either minicells, cells, lysosomes or other fusible lipid-surfaced bodies, Fraley, et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA, 79,1859-1863, 1982.The vector may also be introduced into the plant cells by electroporation.\",\n \"(Fromm et al., Proc.\",\n \"Natl Acad.\",\n \"Sci.\",\n \"USA 82:5824, 1985).\",\n \"In this technique, plant protoplasts are electroporated in the presence of plasmids containing the gene construct.\",\n \"Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids.\",\n \"Electroporated plant protoplasts reform the cell wall, divide, and form plant callus, All plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be transformed by the present invention so that whole plants are recovered which contain the transferred gene.\",\n \"It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to all major species of sugarcane, sugar beet, cotton, fruit and other trees, legumes and vegetables.\",\n \"Some suitable plants include, for example, species from the genera Fragaria, Lotus, Medicago, Onobryclis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersion, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hererocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucum is, Browaalia, Glycine, Lolium, Zea, Triticum, Sorghum, and Datura.\",\n \"Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts containing copies of the heterologous gene is first provided.\",\n \"Callus tissue is formed and shoots may be induced from callus and subsequently rooted.\",\n \"Alternatively, embryo formation can be induced from the protoplast suspension.\",\n \"These embryos germinate as natural embryos to form plants.\",\n \"The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins.\",\n \"It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa.\",\n \"Shoots and roots normally develop simultaneously.\",\n \"Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture.\",\n \"If these three variables are controlled, then regeneration is fully reproducible and repeatable.\",\n \"In some plant cell culture systems, the desired protein of the invention may be excreted or alternatively, the protein may be extracted from the whole plant.\",\n \"Where the desired protein of the invention is secreted into the medium, it may be collected.\",\n \"Alternatively, the embryos and embryoless-half seeds or other plant tissue may be mechanically disrupted to release any secreted protein between cells and tissues.\",\n \"The mixture may be suspended in a buffer solution to retrieve soluble proteins.\",\n \"Conventional protein isolation and purification methods will be then used to purify the recombinant protein.\",\n \"Parameters of time, temperature pH, oxygen, and volumes will be adjusted through routine methods to optimize expression and recovery of heterologous protein.\",\n \"iv.\",\n \"Bacterial Systems Bacterial expression techniques are known in the art.\",\n \"A bacterial promoter is any DNA sequence capable of binding bacterial RNA polymerase and initiating the downstream (3′) transcription of a coding sequence (eg.\",\n \"structural gene) into mRNA.\",\n \"A promoter will have a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence.\",\n \"This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site.\",\n \"A bacterial promoter may also have a second domain called an operator, that may overlap an adjacent RNA polymerase binding site at which RNA synthesis begins.\",\n \"The operator permits negative regulated (inducible) transcription, as a gene repressor protein may bind the operator and thereby inhibit transcription of a specific gene.\",\n \"Constitutive expression may occur in the absence of negative regulatory elements, such as the operator.\",\n \"In addition, positive regulation may be achieved bya gene activator protein binding sequence, which, if present is usually proximal (5′) to the RNA polymerase binding sequence.\",\n \"An example of a gene activator protein is the catabolite activator protein (CAP), which helps initiate transcription of the lac operon in Escherichia coli (E. coli) [Raibaud et al.\",\n \"(1984) Annu.\",\n \"Rev.\",\n \"Genet.\",\n \"18:173].\",\n \"Regulated expression may therefore be either positive or negative, thereby either enhancing or reducing transcription.\",\n \"Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences.\",\n \"Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) [Chang et al.\",\n \"(1977) Nature 198:1056], and maltose.\",\n \"Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp) [Goeddel et al.\",\n \"(1980) Nuc.\",\n \"Acids Res.\",\n \"8:4057; Yelverton et al.\",\n \"(1981) Nucl.\",\n \"Acids Res.\",\n \"9:731; U.S. Pat.\",\n \"No.\",\n \"4,738,921; EP-A-0036776 and EP-A-0121775].\",\n \"The g-laotamase (bla) promoter system [Weissmann (1981) “The cloning of interferon and other mistakes.” In Interferon 3 (ed.\",\n \"1.Gresser)], bacteriophage lambda P L [Shimatake et al.\",\n \"(1981) Nature 292:128] and T5 [U.S. Pat.\",\n \"No.\",\n \"4,689,406] promoter systems also provide useful promoter sequences.\",\n \"In addition, synthetic promoters which do not occur in nature also function as bacterial promoters.\",\n \"For example, transcription activation sequences of one bacterial or bacteriophage promoter may be joined with the operon sequences of another bacterial or bacteriophage promoter, creating a synthetic hybrid promoter [U.S. Pat.\",\n \"No.\",\n \"4,551,433].\",\n \"For example, the tac promoter is a hybrid trp-lac promoter comprised of both trp promoter and lac operon sequences that is regulated by the lac repressor [Amann et al.\",\n \"(1983) Gene 25:167; de Boer et al.\",\n \"(1983) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"80:21].\",\n \"Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription.\",\n \"A naturally occurring promoter of non-bacterial origin can also be coupled with a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes.\",\n \"The bacteriophage T7 RNA polymeraselpromoter system is an example of a coupled promoter system [Studier et al.\",\n \"(1986) J. Mol.\",\n \"Biol.\",\n \"189:113; Tabor et al.\",\n \"(1985) Proc Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"82:1074].\",\n \"In addition, a hybrid promoter can also be comprised of a bacteriophage promoter and an E. coli operator region (EPO-A-0 267 851).\",\n \"In addition to a functioning promoter sequence, an efficient ribosome binding site is also useful for the expression of foreign genes in prokaryotes.\",\n \"In E. coli, the ribosome binding site is called the Shine-Dalgarno (SD) sequence and includes an initiation codon (ATG) and a sequence 3-9 nucleotides in length located 3-11 nucleotides upstream of the initiation codon [Shine et al.\",\n \"(1975) Nature 254:34].\",\n \"The SD sequence is thought to promote binding of mRNA to the ribosome by the pairing of bases between the SD sequence and the 3′ and of E. coli 16S rRNA [Steitz et al.\",\n \"(1979) “Genetic signals and nucleotide sequences in messenger RNA.” In Biological Regulation and Development: Gene Expression (ed.\",\n \"R. F. Goldberger)].\",\n \"To express eukaryotic genes and prokaryotic genes with weak ribosome-binding site [Sambrook et al.\",\n \"(1989) “Expression of cloned genes in Escherichia coli.” In Molecular Cloning: A Laboratory Manual].\",\n \"A DNA molecule may be expressed intracellularly.\",\n \"A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus will always be a methionine, which is encoded by the ATG start codon.\",\n \"If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide or by either in vivo on in vitro incubation with a bacterial methionine N-terminal peptidase (EPO-A-O 219 237).\",\n \"Fusion proteins provide an alternative to direct expression.\",\n \"Usually, a DNA sequence encoding the N-terminal portion of an endogenous bacterial protein, or other stable protein, is fused to the 5′ end of heterologous coding sequences.\",\n \"Upon expression, this construct will provide a fusion of the two amino acid sequences.\",\n \"For example, the bacteriophage lambda cell gene can be linked at the 5′ terminus of a foreign gene and expressed in bacteria.\",\n \"The resulting fusion protein preferably retains a site for a processing enzyme (factor Xa) to cleave the bacteriophage protein from the foreign gene [Nagai et al.\",\n \"(1984) Nature 309:810].\",\n \"Fusion proteins can also be made with sequences from the lacZ [Jia et al.\",\n \"(1987) Gene 60:197], trpE [Allen et al.\",\n \"(1987) J. Biotechnol.\",\n \"5:93; Makoff et al.\",\n \"(1989) J. Gen. Microbiol.\",\n \"135:11], and Chey [EP-A-0 324 647] genes.\",\n \"The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site.\",\n \"Another example is a ubiquitin fusion protein.\",\n \"Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (eg.\",\n \"ubiquitin specific processing-protease) to cleave the ubiquitin from the foreign protein.\",\n \"Through this method, native foreign protein can be isolated [Miller et al.\",\n \"(1989) Bio/Technology 7:698].\",\n \"Alternatively, foreign proteins can also be secreted from the cell by creating chimeric DNA molecules that encode a fusion protein comprised of a signal peptide sequence fragment that provides for secretion of the foreign protein in bacteria [U.S. Pat.\",\n \"No.\",\n \"4,336,336].\",\n \"The signal sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell.\",\n \"The protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria).\",\n \"Preferably there are processing sites, which can be cleaved either in vivo or in vitro encoded between the signal peptide fragment and the foreign gene.\",\n \"DNA encoding suitable signal sequences can be derived from genes for secreted bacterial proteins, such as the E. coli outer membrane protein gene (ompA) [Masui et al.\",\n \"(1983), in: Experimental Manipulation of Gene Expression; Ghrayeb et al.\",\n \"(1984) EMBO J.\",\n \"3:2437] and the E. coli alkaline phosphatase signal sequence (phoA) [Oka et al.\",\n \"(1985) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"82:7212].\",\n \"As an additional example, the signal sequence of the alpha-amylase gene from various Bacillus strains can be used to secrete heterologous proteins from B. subtilis [Palva et al.\",\n \"(1982) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 79:5582; EP-A-0 244 042].\",\n \"Usually, transcription termination sequences recognized by bacteria are regulatory regions located 3′ to the translation stop codon, and thus together with the promoter flank the coding sequence.\",\n \"These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA.\",\n \"Transcription termination sequences frequently include DNA sequences of about 50 nucleotides capable of forming stem loop structures that aid in terminating transcription.\",\n \"Examples include transcription termination sequences derived from genes with strong promoters, such as the trp gene in E. coli as well as other biosynthetic genes.\",\n \"Usually, the above described components, comprising a promoter, signal sequence (if desired), coding sequence of interest, and transcription termination sequence, are put together into expression constructs.\",\n \"Expression constructs are often maintained in a replicon, such as an extracbromosomal element (eg.\",\n \"plasmids) capable of stable maintenance in a host, such as bacteria.\",\n \"The replicon will have a replication system, thus allowing it to be maintained in a prokaryotic host either for expression or for cloning and amplification.\",\n \"In addition, a replicon may be either a high or low copy number plasmid.\",\n \"A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and usually about 10 to about 150.A host containing a high copy number plasmid will preferably contain at least about 10, and more preferably at least about 20 plasmids.\",\n \"Either a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host.\",\n \"Alternatively, the expression constructs can be integrated into the bacterial genome with an integrating vector.\",\n \"Integrating vectors usually contain at least one sequence homologous to the bacterial chromosome that allows the vector to integrate.\",\n \"Integrations appear to result from recombinations between homologous DNA in the vector and the bacterial chromosome.\",\n \"For example, integrating vectors constructed with DNA from various Bacillus strains integrate into the Bacillus chromosome (EP-A-0 127 328).\",\n \"Integrating vectors may also be comprised of bacteriophage or transposon sequences.\",\n \"Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of bacterial strains that have been transformed.\",\n \"Selectable markers can be expressed in the bacterial host and may include genes which render bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin), and tetracycline [Davies et al.\",\n \"(1978) Annu.\",\n \"Rev.\",\n \"Microbiol.\",\n \"32:469].\",\n \"Selectable markers may also include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways.\",\n \"Alternatively, some of the above described components can be put together in transformation vectors.\",\n \"Transformation vectors are usually comprised of a selectable market that is either maintained in a replicon or developed into an integrating vector, as described above.\",\n \"Expression and transformation vectors, either extra-chromosomal replicons or integrating vectors, have been developed for transformation into many bacteria.\",\n \"For example, expression vectors have been developed for, inter alia, the following bacteria: Bacillus subtilis [Palva et al.\",\n \"(1982) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953; WO 84/04541], Escherichia coli [Shimatake et al.\",\n \"(1981) Nature 292:128; Amann et al.\",\n \"(1985) Gene 40:183; Studier et al.\",\n \"(1986) J. Mol.\",\n \"Biol.\",\n \"189:113; EP-A-0 036 776,EP-A-0 136 829 and EP-A-0 136 907], Streptococcus cremoris [Powell et al.\",\n \"(1988) Appl.\",\n \"Environ.\",\n \"Microbiol.\",\n \"54:655]; Streptococcus lividans [Powell et al.\",\n \"(1988) Appl.\",\n \"Environ.\",\n \"Microbiol.\",\n \"54:655], Streptomyces lividans [U.S. Pat.\",\n \"No.\",\n \"4,745,056].\",\n \"Methods of introducing exogenous DNA into bacterial hosts are well-known in the art, and usually include either the transformation of bacteria treated with CaCl2 or other agents, such as divalent cations and DMSO.\",\n \"DNA can also be introduced into bacterial cells by electroporation.\",\n \"Transformation procedures usually vary with the bacterial species to be transformed.\",\n \"See eg.\",\n \"[Masson et al.\",\n \"(1989) FEMS Microbiol.\",\n \"Lett.\",\n \"60:273; Palva et al.\",\n \"(1982) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953; WO 84/04541, Bacillus], [Miller et al.\",\n \"(1988) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"85:856; Wang et al.\",\n \"(1990) J. Bacteriol.\",\n \"172:949, Campylobacter], [Cohen et al.\",\n \"(1973) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"69:2110; Dower et al.\",\n \"(1988) Nucleic Acids Res.\",\n \"16:6127; Kushner (1978) “An improved method for transformation of Escherichia coli with ColE1-derived plasmids.\",\n \"In Genetic Engineering: Proceedings of the International Symposium on Genetic Engineering (eds.\",\n \"H. W. Boyer and S. Nicosia); Mandel et al.\",\n \"(1970) J. Mol.\",\n \"Biol.\",\n \"53:159; Taketo (1988) Biochim.\",\n \"Biophys.\",\n \"Acta 949:318; Escherichia], [Chassy et al.\",\n \"(1987) FEMS Microbiol.\",\n \"Lett.\",\n \"44:173 Lactobacillus]; [Fiedler et al.\",\n \"(1988) Anal.\",\n \"Biochem 170:38, Pseudomonas]; [Augustin et al.\",\n \"(1990) FEMS Microbiol.\",\n \"Lett.\",\n \"66:203, Staphylococcus], [Barany et al.\",\n \"(1980) J. Bacterial.\",\n \"144:698; Harlander (1987) “Transformation of Streptococcus lactis by electroporation, in: Streptococcal Genetics (ed.\",\n \"J. Ferretti and R. Curtiss III); Perry et al.\",\n \"(1981) Infect.\",\n \"Immun.\",\n \"32:1295; Powell et al.\",\n \"(1988) Appl.\",\n \"Environ.\",\n \"Microbial.\",\n \"54:655; Somkuti et al.\",\n \"(1987) Proc.\",\n \"4th Evr.\",\n \"Cong.\",\n \"Biotechnology 1:412, Streptococcus].\",\n \"v. Yeast Expression Yeast expression systems are also known to one of ordinary skill in the art.\",\n \"A yeast promoter is any DNA sequence capable of binding yeast RNA polymerase and initiating the downstream (3′) transcription of a coding sequence (eg.\",\n \"structural gene) into mRNA.\",\n \"A promoter will have a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence.\",\n \"This transcription initiation region usually includes an RNA polymerase binding site (the “TATA Box”) and a transcription initiation site.\",\n \"A yeast promoter may also have a second domain called an upstream activator sequence (UAS), which, if present, is usually distal to the structural gene.\",\n \"The UAS permits regulated (inducible) expression.\",\n \"Constitutive expression occurs in the absence of a UAS.\",\n \"Regulated expression may be either positive or negative, thereby either enhancing or reducing transcription.\",\n \"Yeast is a fermenting organism with an active metabolic pathway, therefore sequences encoding enzymes in the metabolic pathway provide particularly useful promoter sequences.\",\n \"Examples include alcohol dehydrogenase (ADH) (EP-A-0 284 044), enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, and pyruvate kinase (PyK) (EPO-A-0 329 203).\",\n \"The yeast PH05 gene, encoding acid phosphatase, also provides useful promoter sequences [Myanohara et al.\",\n \"(1983) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 80:1].\",\n \"In addition, synthetic promoters which do not occur in nature also function as yeast promoters.\",\n \"For example, UAS sequences of one yeast promoter maybe joined with the transcription activation region of another yeast promoter, creating a synthetic hybrid promoter.\",\n \"Examples of such hybrid promoters include the ADH regulatory sequence linked to the GAP transcription activation region (U.S. Pat.\",\n \"Nos.\",\n \"4,876,197 and 4,880,734).\",\n \"Other examples of hybrid promoters include promoters which consist of the regulatory sequences of either the ADH2, GAL4, GAL10, OR PH05 genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK (EP-A-0 164 556).\",\n \"Furthermore, a yeast promoter can include naturally occurring promoters of non-yeast origin that have the ability to bind yeast RNA polymerase and initiate transcription.\",\n \"Examples of such promoters include, inter alia, [Cohen et al.\",\n \"(1980) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 77:1078; Henikoff et al.\",\n \"(1981) Nature 283:835; Hollenberg et al.\",\n \"(1981) Curr.\",\n \"Topics Microbiol.\",\n \"Immunol.\",\n \"96:119; Hollenberg et al.\",\n \"(1979) “The Expression of Bacterial Antibiotic Resistance Genes in the Yeast Saccharomyces cerevisiae,” in: Plasmids of Medical, Environmental and Commercial Importance (eds.\",\n \"K. N. Timmis and A. Publer); Mercerau-Puigalon et al.\",\n \"(1980) Gene 11:163; Panthier et al.\",\n \"(1980) Curr.\",\n \"Genet.\",\n \"2:109;].\",\n \"A DNA molecule may be expressed intracellularly in yeast.\",\n \"A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon.\",\n \"If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.\",\n \"Fusion proteins provide an alternative for yeast expression systems, as well as in mammalian, baculovirus, and bacterial expression systems.\",\n \"Usually, a DNA sequence encoding the N-terminal portion of an endogenous yeast protein, or other stable protein, is fused to the 5′ end of heterologous coding sequences.\",\n \"Upon expression, this construct will provide a fusion of the two amino acid sequences.\",\n \"For example, the yeast or human superoxide dismutase (SOD) gene, can be linked at the 5′ terminus of a foreign gene and expressed in yeast.\",\n \"The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site.\",\n \"See eg.\",\n \"EP-A-0 196 056.Another example is a ubiquitin fusion protein.\",\n \"Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (eg.\",\n \"ubiquitin-specific processing protease) to cleave the ubiquitin from the foreign protein.\",\n \"Through this method, therefore, native foreign protein can be isolated (eg.\",\n \"WO88/024066).\",\n \"Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provide for secretion in yeast of the foreign protein.\",\n \"Preferably, there are processing sites encoded between the leader fragment and the foreign gene that can be cleaved either in vivo or in vitro.\",\n \"The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell.\",\n \"DNA encoding suitable signal sequences can be derived from genes for secreted yeast proteins, such as the yeast invertase gene (EP-A-0 012 873; JPO.\",\n \"62,096,086) and the A-factor gene (U.S. Pat.\",\n \"No.\",\n \"4,588,684).\",\n \"Alternatively, leaders of non-yeast origin, such as an interferon leader, exist that also provide for secretion in yeast (EP-A-0 060 057).\",\n \"A preferred class of secretion leaders are those that employ a fragment of the yeast alpha-factor gene, which contains both a “pre” signal sequence, and a “pro” region.\",\n \"The types of alpha-factor fragments that can be employed include the full-length pre-pro alpha factor leader (about 83 amino acid residues) as well as truncated alpha-factor leaders (usually about 25 to about 50 amino acid residues) (U.S. Pat.\",\n \"Nos.\",\n \"4,546,083 and 4,870,008; EP-A-0 324 274).\",\n \"Additional leaders employing an alpha-factor leader fragment that provides for secretion include hybrid alpha-factor leaders made with a presequence of a first yeast, but a pro-region from a second yeast alphafactor.\",\n \"(eg.\",\n \"see WO 89/02463.)\",\n \"Usually, transcription termination sequences recognized by yeast are regulatory regions located 3′ to the translation stop codon, and thus together with the promoter flank the coding sequence.\",\n \"These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA.\",\n \"Examples of transcription terminator sequence and other yeast-recognized termination sequences, such as those coding for glycolytic enzymes.\",\n \"Usually, the above described components, comprising a promoter, leader (if desired), coding sequence of interest, and transcription termination sequence, are put together into expression constructs.\",\n \"Expression constructs are often maintained in a replicon, such as an extrachromosomal element (eg.\",\n \"plasmids) capable of stable maintenance in a host, such as yeast or bacteria.\",\n \"The replicon may have two replication systems, thus allowing it to be maintained, for example, in yeast for expression and in a prokaryotic host for cloning and amplification.\",\n \"Examples of such yeast-bacteria shuttle vectors include YEp24 [Botstein et al.\",\n \"(1979) Gene 8:17-24], pCl/1 [Brake et al.\",\n \"(1984) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci USA 81:4642-4646], and YRp17 (Stinchcomb et al.\",\n \"(1982) J. Mol.\",\n \"Biol.\",\n \"158:157].\",\n \"In addition, a replicon may be either a high or low copy number plasmid.\",\n \"A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and usually about 10 to about 150.A host containing a high copy number plasmid will preferably have at least about 10, and more preferably at least about 20.Enter a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host.\",\n \"See eg.\",\n \"Brake et al., supra.\",\n \"Alternatively, the expression constructs can be integrated into the yeast genome with an integrating vector.\",\n \"Integrating vectors usually contain at least one sequence homologous to a yeast chromosome that allows the vector to integrate, and preferably contain two homologous sequences flanking the expression construct.\",\n \"Integrations appear to result from recombinations between homologous DNA in the vector and the yeast chromosome [Orr-Weaver et al.\",\n \"(1983) Methods in Enzymol.\",\n \"101:228-2451.An integrating vector may be directed to a specific locus in yeast by selecting the appropriate homologous sequence for inclusion in the vector.\",\n \"See Orr-Weaver et al., supra.\",\n \"One or more expression construct may integrate, possibly affecting levels of recombinant protein produced [Rine et al.\",\n \"(1983) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 80:6750].\",\n \"The chromosomal sequences included in the vector can occur either as a single segment in the vector, which results in the integration of the entire vector, or two segments homologous to adjacent segments in the chromosome and flanking the expression construct in the vector, which can result in the stable integration of only the expression construct.\",\n \"Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of yeast strains that have been transformed.\",\n \"Selectable markers may include biosynthetic genes that can be expressed in the yeast host, such as ADE2, HIS4, LEU2, TRP1, and ALG7, and the G418 resistance gene, which confer resistance in yeast cells to tunicamycin and G418, respectively.\",\n \"In addition, a suitable selectable marker may also provide yeast with the ability to grow in the presence of toxic compounds, such as metal.\",\n \"For example, the presence of CUP1 allows yeast to grow in the presence of copper ions [Butt et al.\",\n \"(1987) Microbiol, Rev.\",\n \"51:351].\",\n \"Alternatively, some of the above described components can be put together into transformation vectors.\",\n \"Transformation vectors are usually comprised of a selectable marker that is either maintained in a replicon or developed into an integrating vector, as described above.\",\n \"Expression and transformation vectors, either extrachromosomal replicons or integrating vectors, have been developed for transformation into many yeasts.\",\n \"For example, expression vectors have been developed for, inter alia, the following yeasts:Candida albicans [Kurtz, et at.\",\n \"(1986) Mol.\",\n \"Cell.\",\n \"Biol.\",\n \"6:142], Candida maltosa [Kunze, et at.\",\n \"(1985) J.\",\n \"Basic Microbiol, 25:141].\",\n \"Hansenula polymorpha [Gleeson, et al.\",\n \"(1986) J. Gen. Microbiol.\",\n \"132:3459; Roggenkamp et al.\",\n \"(1986) Mol.\",\n \"Gen. Genet.\",\n \"202:302], Kluyveromyces fragilis [Das, et al.\",\n \"(1984) J. Bacteriol.\",\n \"158:1165], Kluyveromyces lactis [De Louvencourt et al.\",\n \"(1983) J. Bacteriol.\",\n \"154:737; Van den Berg et al.\",\n \"(1990) Bio/Technology 8:135], Pichia guillerimondii [Kunze et al.\",\n \"(1985) J.\",\n \"Basic Microbiol.\",\n \"25:141], Pichia pastoris [Cregg, et al.\",\n \"(1985) Mol.\",\n \"Cell.\",\n \"Biol.\",\n \"5:3376; U.S. Pat.\",\n \"Nos.\",\n \"4,837,148 and 4,929,555], Saccharomyces cerevisiae [Hinnen et al.\",\n \"(1978) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 75:1929; Ito et al.\",\n \"(1983) J. Bacteriol.\",\n \"153:163], Schizosaccharomyces pombe [Beach and Nurse (1981) Nature 300:706], and Yarrowia lipolytica [Davidow, et al.\",\n \"(1985) Curr.\",\n \"Genet.\",\n \"10:380471 Gaillardin, et al.\",\n \"(1985) Curr.\",\n \"Genet.\",\n \"10:49].\",\n \"Methods of introducing exogenous DNA into yeast hosts are well-known in the art, and usually include either the transformation of spheroplasts or of intact yeast cells treated with alkali cations.\",\n \"Transformation procedures usually vary with the yeast species to be transformed.\",\n \"See eg.\",\n \"[Kurtz et al.\",\n \"(1986) Mol.\",\n \"Cell.\",\n \"Biol.\",\n \"6:142; Kunze et al.\",\n \"(1985) J.\",\n \"Basic Microbiol.\",\n \"25:141; Candida]; [Gleeson et al.\",\n \"(1986) J. Gen. Microbiol.\",\n \"132:3459; Roggenkamp et al.\",\n \"(1986) Mol.\",\n \"Gen. Genet.\",\n \"202:302; Hansenula]; [Das et al.\",\n \"(1984) J. Bacterial.\",\n \"158:1165; De Louvencourt et al.\",\n \"(1983) J. Bacteriol.\",\n \"154:1165; Van den Berg et al.\",\n \"(1990) Bio/Technology 8:135; Kluyveromyces]; [Cregg et at.\",\n \"(1985) Mol.\",\n \"Cell.\",\n \"Biol.\",\n \"5:3376; Kunze et al.\",\n \"(1985) J.\",\n \"Basic Microbiol.\",\n \"25:141; U.S. Pat.\",\n \"Nos.\",\n \"4,837,148 and 4,929,555; Pichia]; [Hinnen et al.\",\n \"(1978) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 75;1929; Ito et al.\",\n \"(1983) J. Bacteriol.\",\n \"153:163 Saccharomyces]; [Beach and Nurse (1981) Nature 300:706; Schizosaccbaromyces]; [Davidow et al.\",\n \"(1985) Curr.\",\n \"Genet.\",\n \"10:39; Gaillardin et al.\",\n \"(1985) Curr.\",\n \"Genet.\",\n \"10:49; Yarrowia].\",\n \"Antibodies As used herein, the term “antibody” refers to a polypeptide or group of polypeptides composed of at least one antibody combining site.\",\n \"An “antibody combining site” is the three-dimensional binding space with an internal surface shape and charge distribution complementary to the features of an epitope of an antigen, which allows a binding of the antibody with the antigen.\",\n \"“Antibody” includes, for example, vertebrate antibodies, hybrid antibodies, chimeric antibodies, humanised antibodies, altered antibodies, univalent antibodies, Fab proteins, and single domain antibodies.\",\n \"Antibodies against the proteins of the invention are useful for affinity chromatography, immunoassays, and distinguishing/identifying Neisseria proteins.\",\n \"Antibodies to the proteins of the invention, both polyclonal and monoclonal, may be prepared by conventional methods.\",\n \"In general, the protein is first used to immunize a suitable animal, preferably a mouse, rat, rabbit or goat.\",\n \"Rabbits and goats are preferred for the preparation of polyclonal sera due to the volume of serum obtainable, and the availability of labeled anti-rabbit and anti-goat antibodies.\",\n \"Immunization is generally performed by mixing or emulsifying the protein in saline, preferably in an adjuvant such as Freund's complete adjuvant, and injecting the mixture or emulsion parenterally (generally subcutaneously or intramuscularly).\",\n \"A dose of 50-200 μg/injection is typically sufficient.\",\n \"Immunization is generally boosted 2-6 weeks later with one or more injections of the protein in saline, preferably using Freund's incomplete adjuvant.\",\n \"One may alternatively generate antibodies by in vitro immunization using methods known in the art, which for the purposes of this invention is considered equivalent to in vivo immunization.\",\n \"Polyclonal antisera is obtained by bleeding the immunized animal into a glass or plastic container, incubating the blood at 25° C. for one hour, followed by incubating at 4° C. for 2-18 hours.\",\n \"The serum is recovered by centrifugation (eg.\",\n \"1,000g for 10 minutes).\",\n \"About 20-50 ml per bleed may be obtained from rabbits.\",\n \"Monoclonal antibodies are prepared using the standard method of Kohler & Milstein [Nature (1975) 256:495-96], or a modification thereof.\",\n \"Typically, a mouse or rat is immunized as described above.\",\n \"However, rather than bleeding the animal to extract serum, the spleen (and optionally several large lymph nodes) is removed and dissociated into single cells.\",\n \"If desired, the spleen cells may be screened (after removal of nonspecifically adherent cells) by applying a cell suspension to a plate or well coated with the protein antigen.\",\n \"B-cells expressing membrane-bound immunoglobulin specific for the antigen bind to the plate, and are not rinsed away with the rest of the suspension.\",\n \"Resulting B-cells, or all dissociated spleen cells, are then induced to fuse with myeloma cells to form hybridomas, and are cultured in a selective medium (eg.\",\n \"hypoxanthine, aminopterin, thymidine medium, “HAT”).\",\n \"The resulting hybridomas are plated by limiting dilution, and are assayed for production of antibodies which bind specifically to the immunizing antigen (and which do not bind to unrelated antigens).\",\n \"The selected MAb-secreting hybridomas are then cultured either in vitro (eg.\",\n \"in tissue culture bottles or hollow fiber reactors), or in vivo (as ascites in mice).\",\n \"If desired, the antibodies (whether polyclonal or monoclonal) may be labeled using conventional techniques.\",\n \"Suitable labels include fluorophores, chromophores, radioactive atoms (particularly 32P and 125I), electron-dense reagents, enzymes, and ligands having specific binding partners.\",\n \"Enzymes are typically detected by their activity.\",\n \"For example, horseradish peroxidase is usually detected by its ability to convert 3,3′,5,5′-tetramethylbenzidine (TMB) to a blue pigment, quantifiable with a spectrophotometer.\",\n \"“Specific binding partner” refers to a protein capable of binding a ligand molecule with high specificity, as for example in the case of an antigen and a monoclonal antibody specific therefor.\",\n \"Other specific binding partners include biotin and avidin or streptavidin, IgG and protein A, and the numerous receptor-ligand couples known in the art.\",\n \"It should be understood that the above description is not meant to categorize the various labels into distinct classes, as the same label may serve in several different modes.\",\n \"For example, 125I may serve as a radioactive label or as an electron-dense reagent.\",\n \"HRP may serve as enzyme or as antigen for a MAb.\",\n \"Further, one may combine various labels for desired effect.\",\n \"For example, MAbs and avidin also require labels in the practice of this invention: thus, one might label a MAb with biotin, and detect its presence with avidin labeled with 125I, or with an anti-biotin MAb labeled with HRP.\",\n \"Other permutations and possibilities will be readily apparent to those of ordinary skill in the art, and are considered as equivalents within the scope of the instant invention.\",\n \"Pharmaceutical Compositions Pharmaceutical compositions can comprise either polypeptides, antibodies, or nucleic acid of the invention.\",\n \"The pharmaceutical compositions will comprise a therapeutically effective amount of either polypeptides, antibodies, or polynucleotides of the claimed invention.\",\n \"The term “therapeutically effective amount” as used herein refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect.\",\n \"The effect can be detected by, for example, chemical markers or antigen levels.\",\n \"Therapeutic effects also include reduction in physical symptoms, such as decreased body temperature.\",\n \"The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration.\",\n \"Thus, it is not useful to specify an exact effective amount in advance.\",\n \"However, the effective amount for a given situation can be determined by routine experimentation and is within the judgement of the clinician.\",\n \"For purposes of the present invention, an effective dose will be from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA constructs in the individual to which it is administered.\",\n \"A pharmaceutical composition can also contain a pharmaceutically acceptable carrier.\",\n \"The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents.\",\n \"The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.\",\n \"Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles.\",\n \"Such carriers are well known to those of ordinary skill in the art.\",\n \"Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.\",\n \"A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub.\",\n \"Co., N.J. 1991).\",\n \"Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol.\",\n \"Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.\",\n \"Typically, the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.\",\n \"Liposomes are included within the definition of a pharmaceutically acceptable carrier.\",\n \"Delivery Methods Once formulated, the compositions of the invention can be administered directly to the subject.\",\n \"The subjects to be treated can be animals; in particular, human subjects can be treated.\",\n \"Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue.\",\n \"The compositions can also be administered into a lesion.\",\n \"Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications (eg.\",\n \"see WO98/20734), needles, and gene guns or hyposprays.\",\n \"Dosage treatment may be a single dose schedule or a multiple dose schedule.\",\n \"Vaccines Vaccines according to the invention may either be prophylactic (ie.\",\n \"to prevent infection) or therapeutic (ie.\",\n \"to treat disease after infection).\",\n \"Such vaccines comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid, usually in combination with “pharmaceutically acceptable carriers,” which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition.\",\n \"Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles.\",\n \"Such carriers are well known to those of ordinary skill in the art.\",\n \"Additionally, these carriers may function as immunostimulating agents (“adjuvants”).\",\n \"Furthermore, the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, etc.\",\n \"pathogens.\",\n \"Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59™ (WO 90/14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds.\",\n \"Powell & Newman, Plenum Press 1995), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE (see below), although not required) formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton, Mass.\",\n \"), (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below) either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) Ribi™ adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.)\",\n \"containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox™); (3) saponin adjuvants, such as Stimulon™ (Cambridge Bioscience, Worcester, Mass.)\",\n \"may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes); (4) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (5) cytokines, such as interleukins (eg.\",\n \"IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.\",\n \"), interferons (eg.\",\n \"gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc; and (6) other substances that act as immunostimulating agents to enhance the effectiveness of the composition.\",\n \"Alum and MF59™ are preferred.\",\n \"As mentioned above, muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuram yl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.\",\n \"The immunogenic compositions (eg.\",\n \"the immunising antigen/immunogen/polypeptide/protein/nucleic acid, pharmaceutically acceptable carrier, and adjuvant) typically will contain diluents, such as water, saline, glycerol, ethanol, etc.\",\n \"Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.\",\n \"Typically, the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.\",\n \"The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above under pharmaceutically acceptable carriers.\",\n \"Immunogenic compositions used as vaccines comprise an immunologically effective amount of the antigenic or immunogenic polypeptides, as well as any other of the above-mentioned components, as needed.\",\n \"By “immunologically effective amount”, it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention.\",\n \"This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (eg.\",\n \"nonhuman primate, primate, etc.\",\n \"), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors.\",\n \"It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.\",\n \"The immunogenic compositions are conventionally administered parenterally, eg.\",\n \"by injection, either subcutaneously, intramuscularly, or transdermally/transcutaneously (eg.\",\n \"WO98/20734).\",\n \"Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications.\",\n \"Dosage treatment may be a single dose schedule or a multiple dose schedule.\",\n \"The vaccine may be administered in conjunction with other immunoregulatory agents.\",\n \"As an alternative to protein-based vaccines, DNA vaccination may be used [eg.\",\n \"Robinson & Torres (1997) Seminars in Immunol 9:271-283; Donnelly et al.\",\n \"(1997) Annu Rev Immunol 15:617-648; later herein].\",\n \"Gene Delivery Vehicles Gene therapy vehicles for delivery of constructs including a coding sequence of a therapeutic of the invention, to be delivered to the mammal for expression in the mammal, can be administered either locally or systemically.\",\n \"These constructs can utilize viral or non-viral vector approaches in in vivo or ex vivo modality.\",\n \"Expression of such coding sequence can be induced using endogenous mammalian or heterologous promoters.\",\n \"Expression of the coding sequence in vivo can be either constitutive or regulated.\",\n \"The invention includes gene delivery vehicles capable of expressing the contemplated nucleic acid sequences.\",\n \"The gene delivery vehicle is preferably a viral vector and, more preferably, a retroviral, adenoviral, adeno-associated viral (AAV), herpes viral, or alphavirus vector.\",\n \"The viral vector can also be an astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, or togavirus viral vector.\",\n \"See generally, Jolly (1994) Cancer Gene Therapy 1:51-64; Kimura (1994) Human Gene Therapy 5:845-852; Connelly (1995) Human Gene Therapy 6:185-193; and Kaplitt (1994) Nature Genetics 6:148-153.Retroviral vectors are well known in the art and we contemplate that any retroviral gene therapy vector is employable in the invention, including B, C and D type retroviruses, xenotropic retroviruses (for example, NZB-X1, NZB-X2 and NZB9-1 (see O'Neill (1985) J. Virol.\",\n \"53:160) polytropic retroviruses eg.\",\n \"MCF and MCF-MLV (see Kelly (1983) J. Virol.\",\n \"45:291), spumaviruses and lentiviruses.\",\n \"See RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985.Portions of the retroviral gene therapy vector may be derived from different retroviruses.\",\n \"For example, retrovector LTRs may be derived from a Murine Sarcoma Virus, a TRNA binding site from a Rous Sarcoma Virus, a packaging signal from a Murine Leukemia Virus, and an origin of second strand synthesis from an Avian Leukosis Virus.\",\n \"These recombinant retroviral vectors may be used to generate transduction competent retroviral vector particles by introducing them into appropriate packaging cell lines (see U.S. Pat.\",\n \"No.\",\n \"5,591,624).\",\n \"Retrovirus vectors can be constructed for site-specific integration into host cell DNA by incorporation of a chimeric integrase enzyme into the retroviral particle (see WO96/37626).\",\n \"It is preferable that the recombinant viral vector is a replication defective recombinant virus.\",\n \"Packaging cell lines suitable for use with the above-described retrovirus vectors are well known in the art, are readily prepared (see WO95/30763 and WO92/05266), and can be used to create producer cell lines (also termed vector cell lines or “VCLs”) for the production of recombinant vector particles.\",\n \"Preferably, the packaging cell lines are made from human parent cells (eg.\",\n \"HT1080 cells) or mink parent cell lines, which eliminates inactivation in human serum.\",\n \"Preferred retroviruses for the construction of retroviral gene therapy vectors include Avian Leukosis Virus, Bovine Leukemia, Virus, Murine Leukemia Virus, Mink-Cell Focus-Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis Virus and Rous Sarcoma Virus.\",\n \"Particularly preferred Murine Leukemia Viruses include 4070A and 1504A (Hartley and Rowe (1976) J Virol 19:19-25), Abelson (ATCC No.\",\n \"VR-999), Friend (ATCC No.\",\n \"VR-245), Graffi, Gross (ATCC Nol VR-590), Kirsten, Harvey Sarcoma Virus and Rauscher (ATCC No.\",\n \"VR-998) and Moloney Murine Leukemia Virus (ATCC No.\",\n \"VR-190).\",\n \"Such retroviruses may be obtained from depositories or collections such as the American Type Culture Collection (“ATCC”) in Rockville, Md.\",\n \"or isolated from known sources using commonly available techniques.\",\n \"Exemplary known retroviral gene therapy vectors employable in this invention include those described in patent applications GB2200651, EP0415731, EP0345242, EP0334301, WO89/02468; WO89/05349, WO89/09271, WO90/02806, WO90/07936, WO94/03622, WO93/25698, WO93/25234, WO93/11230, WO93/10218, WO91/02805, WO91/02825, WO95/07994, U.S. Pat.\",\n \"Nos.\",\n \"5,219,740, 4,405,712, 4,861,719, 4,980,289, 4,777,127, 5,591,624.See also Vile (1993) Cancer Res 53:3860-3864; Vile (1993) Cancer Res 53:962-967; Ram (1993) Cancer Res 53 (1993) 83-88; Takamiya (1992) J Neurosci Res 33:493-503; Baba (1993) J Neurosurg 79:729-735; Mann (1983) Cell 33:153; Cane (1984) Proc Natl Acad Sci 81:6349; and Miller (1990) Human Gene Therapy 1.Human adenoviral gene therapy vectors are also known in the art and employable in this invention, See, for example, Berkner (1988) Biotechniques 6:616 and Rosenfeld (1991) Science 252:431, and WO93/07283, WO93/06223, and WO93/07282.Exemplary known adenoviral gene therapy vectors employable in this invention include those described in the above referenced documents and in WO94/12649, WO93/03769, WO93/19191, WO94/28938, WO95/11984, WO95/00655, WO95/27071, WO95/29993, WO95/34671, WO96/05320, WO94/08026, WO94/11506, WO93/06223, WO94/24299, WO95/14102, WO95/24297, WO95/02697, WO94/28152, WO94/24299, WO95/09241, WO95/25807, WO95/05835, WO94/18922 and WO95/09654.Alternatively, administration of DNA linked to killed adenovirus as described in Curiel (1992) Hum.\",\n \"Gene Ther.\",\n \"3:147-154 may be employed.\",\n \"The gene delivery vehicles of the invention also include adenovirus associated virus (AAV) vectors.\",\n \"Leading and preferred examples of such vectors for use in this invention are the AAV-2 based vectors disclosed in Srivastava, WO93/09239.Most preferred AAV vectors comprise the two AAV inverted terminal repeats in which the native D-sequences are modified by substitution of nucleotides, such that at least 5 native nucleotides and up to 18 native nucleotides, preferably at least 10 native nucleotides up to 18 native nucleotides, most preferably 10 native nucleotides are retained and the remaining nucleotides of the D-sequence are deleted or replaced with non-native nucleotides.\",\n \"The native D-sequences of the AAV inverted terminal repeats are sequences of 20 consecutive nucleotides in each AAV inverted terminal repeat (ie.\",\n \"there is one sequence at each end) which are not involved in HP formation.\",\n \"The non-native replacement nucleotide may be any nucleotide other than the nucleotide found in the native D-sequence in the same position.\",\n \"Other employable exemplary AAV vectors are pWP-19, pWN-1, both of which are disclosed in Nahreini (1993) Gene 124:257-262.Another example of such an AAV vector is psub201 (see Samulski (1987) J. Virol.\",\n \"61:3096).\",\n \"Another exemplary AAV vector is the Double-D ITR vector.\",\n \"Construction of the Double-D ITR vector is disclosed in U.S. Pat.\",\n \"No.\",\n \"5,478,745.Still other vectors are those disclosed in Carter U.S. Pat.\",\n \"No.\",\n \"4,797,368 and Muzyczka U.S. Pat.\",\n \"No.\",\n \"5,139,941, Chartejee U.S. Pat.\",\n \"No.\",\n \"5,474,935, and Kotin WO94/288157.Yet a further example of an AAV vector employable in this invention is SSV9AFABTKneo, which contains the AFP enhancer and albumin promoter and directs expression predominantly in the liver.\",\n \"Its structure and construction are disclosed in Su (1996) Human Gene Therapy 7:463-470.Additional AAV gene therapy vectors are described in U.S. Pat.\",\n \"Nos.\",\n \"5,354,678, 5,173,414, 5,139,941, and 5,252,479.The gene therapy vectors of the invention also include herpes vectors.\",\n \"Leading and preferred examples are herpes simplex virus vectors containing a sequence encoding a thymidine kinase polypeptide such as those disclosed in U.S. Pat.\",\n \"No.\",\n \"5,288,641 and EP0176170 (Roizman).\",\n \"Additional exemplary herpes simplex virus vectors include HFEM/ICP6-LacZ disclosed in WO95/04139 (Wistar Institute), pHSVlac described in Geller (1988) Science 241:1667-1669 and in WO90/09441 and WO92/07945, HSV Us3::pgC-lacZ described in Fink (1992) Human Gene Therapy 3:11-19 and HSV 7134, 2 RH 105 and GAL4 described in EP 0453242 (Breakefield), and those deposited with the ATCC with accession numbers VR-977 and VR-260.Also contemplated are alpha virus gene therapy vectors that can be employed in this invention.\",\n \"Preferred alpha virus vectors are Sindbis viruses vectors.\",\n \"Togaviruses, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Middleberg virus (ATCC VR-370), Ross River virus (ATCC VR-373; ATCC VR-1246), Venezuelan equine encephalitis virus (ATCC VR923; ATCC VR-1250; ATCC VR-1249; ATCC VR-532), and those described in U.S. Pat.\",\n \"Nos.\",\n \"5,091,309, 5,217,879, and WO92/10578.More particularly, those alpha virus vectors described in U.S. Ser.\",\n \"No.\",\n \"08/405,627, filed Mar.\",\n \"15, 1995, WO94/21792, WO92/10578, WO95/07994, U.S. Pat.\",\n \"Nos.\",\n \"5,091,309 and 5,217,879 are employable.\",\n \"Such alpha viruses may be obtained from depositories or collections such as the ATCC in Rockville, Md.\",\n \"or isolated from known sources using commonly available techniques.\",\n \"Preferably, alphavirus vectors with reduced cytotoxicity are used (see U.S. Ser.\",\n \"No.\",\n \"08/679640).\",\n \"DNA vector systems such as eukaryotic layered expression systems are also useful for expressing the nucleic acids of the invention.\",\n \"See WO95/07994 for a detailed description of eukaryotic layered expression systems.\",\n \"Preferably, the eukaryotic layered expression systems of the invention are derived from alphavirus vectors and most preferably from Sindbis viral vectors.\",\n \"Other viral vectors suitable for use in the present invention include those derived from poliovirus, for example ATCC VR-58 and those described in Evans, Nature 339 (1989) 385 and Sabin (1973) J. Biol.\",\n \"Standardization 1:115; rhinovirus, for example ATCC VR-11 10 and those described in Arnold (1990) J Cell Biochem L401; pox viruses such as canary pox virus or vaccinia virus, for example ATCC VR-111 and ATCC VR-2010 and those described in Fisher-Hoch (1989) Proc Natl Acad Sci 86:317; Flexner (1989) Ann NY Acad Sci 569:86, Flexner (1990) Vaccine 8:17; in U.S. Pat.\",\n \"Nos.\",\n \"4,603,112 and 4,769,330 and WO89/01973; SV40 virus, for example ATCC VR-305 and those described in Mulligan (1979) Nature 277:108 and Madzak (1992) J Gen Virol 73:1533; influenza virus, for example ATCC VR-797 and recombinant influenza viruses made employing reverse genetics techniques as described in U.S. Pat.\",\n \"No.\",\n \"5,166,057 and in Enami (1990) Proc Natl Acad Sci 87:3802-3805; Enami & Palese (1991) J Virol 65:2711-2713 and Luytjes (1989) Cell 59:110, (see also McMichael (1983) NEI Med 309:13, and Yap (1978) Nature 273:238 and Nature (1979) 277:108); human immunodeficiency virus as described in EP-0386882 and in Buchschacher (1992) J. Virol.\",\n \"66:2731; measles virus, for example ATCC VR-67 and VR-1247 and those described in EP-0440219; Aura virus, for example ATCC VR-368; Bebaru virus, for example ATCC VR-600 and ATCC VR-1240; Cabassou virus, for example ATCC VR-922; Chikungunya virus, for example ATCC VR-64 and ATCC VR-1241; Fort Morgan Virus, for example ATCC VR-924; Getah virus, for example ATCC VR-369 and ATCC VR-1243; Kyzylagach virus, for example ATCC VR-927; Mayaro virus, for example ATCC VR-66; Mucambo virus, for example ATCC VR-580 and ATCC VR-1244; Ndumu virus, for example ATCC VR-371; Pixuna virus, for example ATCC VR-372 and ATCC VR-1245; Tonate virus, for example ATCC VR-925; Triniti virus, for example ATCC VR-469; Una virus, for example ATCC VR-374; Whataroa virus, for example ATCC VR-926; Y-62-33 virus, for example ATCC VR-375; O'Nyong virus, Eastern encephalitis virus, for example ATCC VR-65 and ATCC VR-1242; Western encephalitis virus, for example ATCC VR-70, ATCC VR-1251, ATCC VR-622 and ATCC VR-1252; and coronavirus, for example ATCC VR-740 and those described in Hamre (1966) Proc Soc Exp Biol Med 121:190.Delivery of the compositions of this invention into cells is not limited to the above mentioned viral vectors.\",\n \"Other delivery methods and media may be employed such as, for example, nucleic acid expression vectors, polycationic condensed DNA linked or unlinked to killed adenovirus alone, for example see U.S. Ser.\",\n \"No.\",\n \"08/366,787, filed Dec. 30, 1994 and Curiel (1992) Hum Gene Ther 3:147-154 ligand linked DNA, for example see Wu (1989) J Biol Chem 264:16985-16987, eucaryotic cell delivery vehicles cells, for example see U.S. Ser.\",\n \"No.\",\n \"08/240,030, filed May 9, 1994, and U.S. Ser.\",\n \"No.\",\n \"08/404,796, deposition of photopolymerized hydrogel materials, hand-held gene transfer particle gun, as described in U.S. Pat.\",\n \"No.\",\n \"5,149,655, ionizing radiation as described in U.S. Pat.\",\n \"No.\",\n \"5,206,152 and in WO92/11033, nucleic charge neutralization or fusion with cell membranes.\",\n \"Additional approaches are described in Philip (1994) Mol Cell Biol 14:2411-2418 and in Woffendin (1994) Proc Natl Acad Sci 91:1581-1585.Particle mediated gene transfer may be employed, for example see U.S. Ser.\",\n \"No.\",\n \"60/023,867.Briefly, the sequence can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, as described in Wu & Wu (1987) J. Biol.\",\n \"Chem.\",\n \"262:4429-4432, insulin as described in Hucked (1990) Biochem Pharmacol 40:253-263, galactose as described in Plank (1992) Bioconjugate Chem 3:533-539, lactose or transferrin.\",\n \"Naked DNA may also be employed.\",\n \"Exemplary naked DNA introduction methods are described in WO 90/11092 and U.S. Pat.\",\n \"No.\",\n \"5,580,859.Uptake efficiency may be improved using biodegradable latex beads.\",\n \"DNA coated latex beads are efficiently transported into cells after endocytosis initiation by the beads.\",\n \"The method may be improved further by treatment of the beads to increase hydrophobicity and thereby facilitate disruption of the endosome and release of the DNA into the cytoplasm.\",\n \"Liposomes that can act as gene delivery vehicles are described in U.S. Pat.\",\n \"No.\",\n \"5,422,120, WO95/13796, WO94/23697, WO91/14445 and EP-524,968.As described in U.S. Ser.\",\n \"No.\",\n \"60/023,867, on non-viral delivery, the nucleic acid sequences encoding a polypeptide can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then be incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, insulin, galactose, lactose, or transferrin.\",\n \"Other delivery systems include the use of liposomes to encapsulate DNA comprising the gene under the control of a variety of tissue-specific or ubiquitously-active promoters.\",\n \"Further non-viral delivery suitable for use includes mechanical delivery systems such as the approach described in Woffendin et al (1994) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 91(24):11581-11585.Moreover, the coding sequence and the product of expression of such can be delivered through deposition of photopolymerized hydrogel materials.\",\n \"Other conventional methods for gene delivery that can be used for delivery of the coding sequence include, for example, use of hand-held gene transfer particle gun, as described in U.S. Pat.\",\n \"No.\",\n \"5,149,655; use of ionizing radiation for activating transferred gene, as described in U.S. Pat.\",\n \"No.\",\n \"5,206,152 and WO92/11033.Exemplary liposome and polycationic gene delivery vehicles are those described in U.S. Pat.\",\n \"Nos.\",\n \"5,422,120 and 4,762,915; in WO 95/13796; WO94/23697; and WO91/14445; in EP-0524968; and in Stryer, Biochemistry, pages 236-240 (1975) W.H.\",\n \"Freeman, San Francisco; Szoka (1980) Biochem Biophys Acta 600:1; Bayer (1979) Biochem Biophys Acta 550:464; Rivnay (1987) Meth Enzymol 149:119; Wang (1987) Proc Natl Acad Sci 84:7851; Plant (1989) Anal Biochem 176:420.A polynucleotide composition can comprises therapeutically effective amount of a gene therapy vehicle, as the term is defined above.\",\n \"For purposes of the present invention, an effective dose will be from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA constructs in the individual to which it is administered.\",\n \"Delivery Methods Once formulated, the polynucleotide compositions of the invention can be administered (1) directly to the subject; (2) delivered ex vivo, to cells derived from the subject; or (3) in vitro for expression of recombinant proteins.\",\n \"The subjects to be treated can be mammals or birds.\",\n \"Also, human subjects can be treated.\",\n \"Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue.\",\n \"The compositions can also be administered into a lesion.\",\n \"Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications (eg.\",\n \"see WO98/20734), needles, and gene guns or hyposprays.\",\n \"Dosage treatment may be a single dose schedule or a multiple dose schedule.\",\n \"Methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and described in eg.\",\n \"WO93/14778.Examples of cells useful in ex vivo applications include, for example, stem cells, particularly hem atopoetic, lymph cells, macrophages, dendritic cells, or tumor cells.\",\n \"Generally, delivery of nucleic acids for both ex vivo and in vitro applications can be accomplished by the following procedures, for example, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei, all well known in the art.\",\n \"Polynucleotide and Polypeptide Pharmaceutical Compositions In addition to the pharmaceutically acceptable carriers and salts described above, the following additional agents can be used with polynucleotide and/or polypeptide compositions.\",\n \"A. Polypeptides One example are polypeptides which include, without limitation: asioloorosomucoid (ASOR); transferrin; asialoglycoproteins; antibodies; antibody fragments; ferritin; interleukins; interferons, granulocyte, macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), stem cell factor and erythropoietin.\",\n \"Viral antigens, such as envelope proteins, can also be used.\",\n \"Also, proteins from other invasive organisms, such as the 17 amino acid peptide from the circumsporozoite protein of plasmodium falciparum known as RII.\",\n \"B. Hormones, Vitamins, etc.\",\n \"Other groups that can be included are, for example: hormones, steroids, androgens, estrogens, thyroid hormone, or vitamins, folic acid.\",\n \"C. Polyalkylenes, Polysaccharides, etc.\",\n \"Also, polyalkylene glycol can be included with the desired polynucleotides/polypeptides.\",\n \"In a preferred embodiment, the polyalkylene glycol is polyethlylene glycol.\",\n \"In addition, mono-, di-, or polysaccharides can be included.\",\n \"In a preferred embodiment of this aspect, the polysaccharide is dextran or DEAE-dextran.\",\n \"Also, chitosan and poly(lactide-co-glycolide) D. Lipids, and Liposomes The desired polynucleotidelpolypeptide can also be encapsulated in lipids or packaged in liposomes prior to delivery to the subject or to cells derived therefrom.\",\n \"Lipid encapsulation is generally accomplished using liposomes which are able to stably bind or entrap and retain nucleic acid.\",\n \"The ratio of condensed polynucleotide to lipid preparation can vary but will generally be around 1:1 (mg DNA:micromoles lipid), or more of lipid.\",\n \"For a review of the use of liposomes as carriers for delivery of nucleic acids, see, Hug and Sleight (1991) Biochim.\",\n \"Biophys.\",\n \"Acta.\",\n \"1097:1-17; Straubinger (1983) Meth.\",\n \"Enzymol.\",\n \"101:512-527.Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations.\",\n \"Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Feigner (1987) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 84:7413-7416); mRNA (Malone (1989) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 86:6077-6081); and purified transcription factors (Debs (1990) J. Biol.\",\n \"Chem.\",\n \"265:10189-10192), in functional form.\",\n \"Cationic liposomes are readily available.\",\n \"For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Feigner supra).\",\n \"Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boerhinger).\",\n \"Other cationic liposomes can be prepared from readily available materials using techniques well known in the art.\",\n \"See, eg.\",\n \"Szoka (1978) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 75:4194-4198; WO90/11092 for a description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.\",\n \"Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials.\",\n \"Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others.\",\n \"These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios.\",\n \"Methods for making liposomes using these materials are well known in the art.\",\n \"The liposomes can comprise multilammelar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs).\",\n \"The various liposome-nucleic acid complexes are prepared using methods known in the art.\",\n \"See eg.\",\n \"Straubinger (1983) Meth.\",\n \"Immunol.\",\n \"101:512-527; Szoka (1978) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 75:4194-4198; Papahadjopoulos (1975) Biochim.\",\n \"Biophys.\",\n \"Acta 394:483; Wilson (1979) Cell 17:77); Deamer & Bangham (1976) Biochim.\",\n \"Biophys.\",\n \"Acta 443:629; Ostro (1977) Biochem.\",\n \"Biophys.\",\n \"Res.\",\n \"Commun.\",\n \"76:836; Fraley (1979) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 76:3348); Enoch & Strittmatter (1979) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 76:145; Fraley (1980) J. Biol.\",\n \"Chem.\",\n \"(1980) 255:10431; Szoka & Papahadjopoulos (1978) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 75:145; and Schaefer-Ridder (1982) Science 215:166.E.\",\n \"Lipoproteins In addition, lipoproteins can be included with the polynucleotide/polypeptide to be delivered.\",\n \"Examples of lipoproteins to be utilized include: chylomicrons, HDL, IDL, LDL, and VLDL.\",\n \"Mutants, fragments, or fusions of these proteins can also be used.\",\n \"Also, modifications of naturally occurring lipoproteins can be used, such as acetylated LDL.\",\n \"These lipoproteins can target the delivery of polynucleotides to cells expressing lipoprotein receptors.\",\n \"Preferably, if lipoproteins are including with the polynucleotide to be delivered, no other targeting ligand is included in the composition.\",\n \"Naturally occurring lipoproteins comprise a lipid and a protein portion.\",\n \"The protein portion are known as apoproteins.\",\n \"At the present, apoproteins A, B, C, D, and E have been isolated and identified.\",\n \"At least two of these contain several proteins, designated by Roman numerals, AI, AII, AIV; CI, CII, CIII.\",\n \"A lipoprotein can comprise more than one apoprotein.\",\n \"For example, naturally occurring chylomicrons comprises of A, B, C & E, over time these lipoproteins lose A and acquire C & E. VLDL comprises A, B, C & E apoproteins, LDL comprises apoprotein B; and HDL comprises apoproteins A, C, & E. The amino acid of these apoproteins are known and are described in, for example, Breslow (1985) Annu Rev.\",\n \"Biochem 54:699; Law (1986) Adv.\",\n \"Exp Med.\",\n \"Biol.\",\n \"151:162; Chen (1986) J Biol Chem 261:12918; Kane (1980) Proc Natl Acad Sci USA 77:2465; and Utermann (1984) Hum Genet 65:232.Lipoproteins contain a variety of lipids including, triglycerides, cholesterol (free and esters), and phospholipids, The composition of the lipids varies in naturally occurring lipoproteins.\",\n \"For example, chylomicrons comprise mainly triglycerides.\",\n \"A more detailed description of the lipid content of naturally occurring lipoproteins can be found, for example, in Meth.\",\n \"Enzymol.\",\n \"128 (1986).\",\n \"The composition of the lipids are chosen to aid in conformation of the apoprotein for receptor binding activity.\",\n \"The composition of lipids can also be chosen to facilitate hydrophobic interaction and association with the polynucleotide binding molecule.\",\n \"Naturally occurring lipoproteins can be isolated from serum by ultracentrifugation, for instance.\",\n \"Such methods are described in Meth.\",\n \"Enzymol.\",\n \"(supra); Pitas (1980) J. Biochem.\",\n \"255:5454-5460 and Mahey (1979) J Clin.\",\n \"Invest 64:743-750.Lipoproteins can also be produced by in vitro or recombinant methods by expression of the apoprotein genes in a desired host cell.\",\n \"See, for example, Atkinson (1986) Annu Rev Biophys Chem 15:403 and Radding (1958) Biochim Biophys Acta 30: 443.Lipoproteins can also be purchased from commercial suppliers, such as Biomedical Techniologies, Inc., Stoughton, Mass., USA.\",\n \"Further description of lipoproteins can be found in Zuckermann et al.\",\n \"PCT/US97/14465.F.\",\n \"Polycationic Agents Polycationic agents can be included, with or without lipoprotein, in a composition with the desired polynucleotide/polypeptide to be delivered.\",\n \"Polycationic agents, typically, exhibit a net positive charge at physiological relevant pH and are capable of neutralizing the electrical charge of nucleic acids to facilitate delivery to a desired location.\",\n \"These agents have both in vitro, ex vivo, and in vivo applications.\",\n \"Polycationic agents can be used to deliver nucleic acids to a living subject either intramuscularly, subcutaneously, etc.\",\n \"The following are examples of useful polypeptides as polycationic agents: polylysine, polyarginine, polyornithine, and protamine.\",\n \"Other examples include histones, protamines, human serum albumin, DNA binding proteins, non-histone chromosomal proteins, coat proteins from DNA viruses, such as (X174, transcriptional factors also contain domains that bind DNA and therefore may be useful as nucleic aid condensing agents.\",\n \"Briefly, transcriptional factors such as C/CEBP, cjun, c-fos, AP-1, AP-2, AP-3, CPF, Prot-1, Sp-1, Oct-1, Oct-2, CREP, and TFIID contain basic domains that bind DNA sequences.\",\n \"Organic polycationic agents include: spermine, spermidine, and purtrescine.\",\n \"The dimensions and of the physical properties of a polycationic agent can be extrapolated from the list above, to construct other polypeptide polycationic agents or to produce synthetic polycationic agents.\",\n \"Synthetic polycationic agents which are useful include, for example, DEAE-dextran, polybrene.\",\n \"Lipofectin™, and lipofectAMINE™ are monomers that form polycationic complexes when combined with polynucleotides/polypeptides.\",\n \"Immunodiagnostic Assays Neisseria antigens of the invention can be used in immunoassays to detect antibody levels (or, conversely, anti-Neisseria antibodies can be used to detect antigen levels).\",\n \"Immunoassays based on well defined, recombinant antigens can be developed to replace invasive diagnostics methods.\",\n \"Antibodies to Neisseria proteins within biological samples, including for example, blood or serum samples, can be detected.\",\n \"Design of the immunoassays is subject to a great deal of variation, and a variety of these are known in the art.\",\n \"Protocols for the immunoassay may be based, for example, upon competition, or direct reaction, or sandwich type assays.\",\n \"Protocols may also, for example, use solid supports, or may be by immunoprecipitation.\",\n \"Most assays involve the use of labeled antibody or polypeptide; the labels may be, for example, fluorescent, chemiluminescent, radioactive, or dye molecules.\",\n \"Assays which amplify the signals from the probe are also known; examples of which are assays which utilize biotin and avidin, and enzyme-labeled and mediated immunoassays, such as ELISA assays.\",\n \"Kits suitable for immunodiagnosis and containing the appropriate labeled reagents are constructed by packaging the appropriate materials, including the compositions of the invention, in suitable containers, along with the remaining reagents and materials (for example, suitable buffers, salt solutions, etc.)\",\n \"required for the conduct of the assay, as well as suitable set of assay instructions.\",\n \"Nucleic Acid Hybridisation “Hybridization” refers to the association of two nucleic acid sequences to one another by hydrogen bonding.\",\n \"Typically, one sequence will be fixed to a solid support and the other will be free in solution.\",\n \"Then, the two sequences will be placed in contact with one another under conditions that favor hydrogen bonding.\",\n \"Factors that affect this bonding include: the type and volume of solvent; reaction temperature; time of hybridization; agitation; agents to block the non-specific attachment of the liquid phase sequence to the solid support (Denhardt's reagent or BLOTTO); concentration of the sequences; use of compounds to increase the rate of association of sequences (dextran sulfate or polyethylene glycol); and the stringency of the washing conditions following hybridization.\",\n \"See Sambrook et al.\",\n \"[supral Volume 2, chapter 9, pages 9.47 to 9.57.“Stringency” refers to conditions in a hybridization reaction that favor association of very similar sequences over sequences that differ.\",\n \"For example, the combination of temperature and salt concentration should be chosen that is approximately 120 to 200° C. below the calculated Tm of the hybrid under study.\",\n \"The temperature and salt conditions can often be determined empirically in preliminary experiments in which samples of genomic DNA immobilized on filters are hybridized to the sequence of interest and then washed under conditions of different stringencies.\",\n \"See Sambrook et al.\",\n \"at page 9.50.Variables to consider when performing, for example, a Southern blot are (1) the complexity of the DNA being blotted and (2) the homology between the probe and the sequences being detected.\",\n \"The total amount of the fragment(s) to be studied can vary a magnitude of 10, from 0.1 to lug for a plasmid or phage digest to 10−9 to 10−8 g for a single copy gene in a highly complex eukaryotic genome.\",\n \"For lower complexity polynucleotides, substantially shorter blotting, hybridization, and exposure times, a smaller amount of starting polynucleotides, and lower specific activity of probes an be used.\",\n \"For example, a single-copy yeast gene can be detected with an exposure time of only 1 hour starting with 1 μg of yeast DNA, blotting for two hours, and hybridizing for 4-8 hours with a probe of 108 cpm/μg.\",\n \"For a single-copy mammalian gene a conservative approach would start with 10 μg of DNA, blot overnight, and hybridize overnight in the presence of 10% dextran sulfate using a probe of greater than 108 cpm/μg, resulting in an exposure time of ˜24 hours.\",\n \"Several factors can affect the melting temperature (Tm) of a DNA-DNA hybrid between the probe and the fragment of interest, and consequently, the appropriate conditions for hybridization and washing.\",\n \"In many cases the probe is not 100% homologous to the fragment.\",\n \"Other commonly encountered variables include the length and total G+C content of the hybridizing sequences and the ionic strength and form amide content of the hybridization buffer.\",\n \"The effects of all of these factors can be approximated by a single equation: Tm=81+16.6(log10Ci)+0.4[%(G+C)]0.6(% formamide)−600/n-1.5(% mismatch).\",\n \"where Ci is the salt concentration (monovalent ions) and n is the length of the hybrid in base pairs (slightly modified from Meinkoth & Wahl (1984) Anal.\",\n \"Biochem.\",\n \"138: 267-284).\",\n \"In designing a hybridization experiment, some factors affecting nucleic acid hybridization can be conveniently altered.\",\n \"The temperature of the hybridization and washes and the salt concentration during the washes are the simplest to adjust.\",\n \"As the temperature of the hybridization increases (ie.\",\n \"stringency), it becomes less likely for hybridization to occur between strands that are nonhomologous, and as a result, background decreases.\",\n \"If the radiolabeled probe is not completely homologous with the immobilized fragment (as is frequently the case in gene family and interspecies hybridization experiments), the hybridization temperature must be reduced, and background will increase.\",\n \"The temperature of the washes affects the intensity of the hybridizing band and the degree of background in a similar manner.\",\n \"The stringency of the washes is also increased with decreasing salt concentrations.\",\n \"In general, convenient hybridization temperatures in the presence of 50% formamide are 42° C. for a probe with is 95% to 100% homologous to the target fragment, 37° C for 90% to 95% homology, and 32° C. for 85% to 90% homology.\",\n \"For lower homologies, formamide content should be lowered and temperature adjusted accordingly, using the equation above.\",\n \"If the homology between the probe and the target fragment are not known, the simplest approach is to start with both hybridization and wash conditions which are nonstringent.\",\n \"If non-specific bands or high background are observed after autoradiography, the filter can be washed at high stringency and reexposed.\",\n \"If the time required for exposure makes this approach impractical, several hybridization and/or washing stringencies should be tested in parallel.\",\n \"Nucleic Acid Probe Assays Methods such as PCR, branched DNA probe assays, or blotting techniques utilizing nucleic acid probes according to the invention can determine the presence of cDNA or mRNA.\",\n \"A probe is said to “hybridize” with a sequence of the invention if it can form a duplex or double stranded complex, which is stable enough to be detected.\",\n \"The nucleic acid probes will hybridize to the Neisseria nucleotide sequences of the invention (including both sense and antisense strands).\",\n \"Though many different nucleotide sequences will encode the amino acid sequence, the native Neisseria sequence is preferred because it is the actual sequence present in cells.\",\n \"mRNA represents a coding sequence and so a probe should be complementary to the coding sequence; single-stranded cDNA is complementary to mRNA, and so a cDNA probe should be complementary to the non-coding sequence.\",\n \"The probe sequence need not be identical to the Neisseria sequence (or its complement)—some variation in the sequence and length can lead to increased assay sensitivity if the nucleic acid probe can form a duplex with target nucleotides, which can be detected.\",\n \"Also, the nucleic acid probe can include additional nucleotides to stabilize the formed duplex, Additional Neisseria sequence may also be helpful as a label to detect the formed duplex.\",\n \"For example, a non-complementary nucleotide sequence may be attached to the 5′ end of the probe, with the remainder of the probe sequence being complementary to a Neisseria sequence.\",\n \"Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the a Neisseria sequence in order to hybridize therewith and thereby form a duplex which can be detected.\",\n \"The exact length and sequence of the probe will depend on the hybridization conditions (e.g.\",\n \"temperature, salt condition etc.).\",\n \"For example, for diagnostic applications, depending on the complexity of the analyte sequence, the nucleic acid probe typically contains at least 10-20 nucleotides, preferably 15-25, and more preferably at least 30 nucleotides, although it may be shorter than this.\",\n \"Short primers generally require cooler temperatures to form sufficiently stable hybrid complexes with the template.\",\n \"Probes may be produced by synthetic procedures, such as the triester method of Matteucci et al.\",\n \"[J.\",\n \"Am.\",\n \"Chem.\",\n \"Soc.\",\n \"(1981) 103:31853, or according to Urdea et al.\",\n \"[Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA (1983) 80: 7461], or using commercially available automated oligonucleotide synthesizers.\",\n \"The chemical nature of the probe can be selected according to preference.\",\n \"For certain applications, DNA or RNA are appropriate.\",\n \"For other applications, modifications may be incorporated eg.\",\n \"backbone modifications, such as phosphorothioates or methylphosphonates, can be used to increase in vivo half-life, alter RNA affinity, increase nuclease resistance etc.\",\n \"[eg.\",\n \"see Agrawal & Jyer (1995) Curr Opin Biotechnol 6:12-19; Agrawal (1996) TIBTECH 14:376-387]; analogues such as peptide nucleic acids may also be used [eg.\",\n \"see Corey (1997) TIBTECH 15:224-229; Buchardt et al.\",\n \"(1993) TIBTECH 11:384-386].\",\n \"Alternatively, the polymerase chain reaction (PCR) is another well-known means for detecting small amounts of target nucleic acid.\",\n \"The assay is described in Mullis et al.\",\n \"[Meth.\",\n \"Enzymol.\",\n \"(1987) 155:335-350] & U.S. Pat.\",\n \"Nos.\",\n \"4,683,195 & 4,683,202.Two “primer” nucleotides hybridize with the target nucleic acids and are used to prime the reaction.\",\n \"The primers can comprise sequence that does not hybridize to the sequence of the amplification target (or its complement) to aid with duplex stability or, for example, to incorporate a convenient restriction site.\",\n \"Typically, such sequence will flank the desired Neisseria sequence.\",\n \"A thermostable polymerase creates copies of target nucleic acids from the primers using the original target nucleic acids as a template.\",\n \"After a threshold amount of target nucleic acids are generated by the polymerase, they can be detected by more traditional methods, such as Southern blots.\",\n \"When using the Southern blot method, the labelled probe will hybridize to the Neisseria sequence (or its complement).\",\n \"Also, mRNA or cDNA can be detected by traditional blotting techniques described in Sambrook et al [supra].\",\n \"mRNA, or cDNA generated from mRNA using a polymerase enzyme, can be purified and separated using gel electrophoresis.\",\n \"The nucleic acids on the gel are then blotted onto a solid support, such as nitrocellulose.\",\n \"The solid support is exposed to a labelled probe and then washed to remove any unhybridized probe.\",\n \"Next, the duplexes containing the labeled probe are detected.\",\n \"Typically, the probe is labelled with a radioactive moiety.\",\n \"BRIEF DESCRIPTION OF DRAWINGS There are no drawings.\",\n \"MODES FOR CARRYING OUT THE INVENTION The following examples describe nucleic acid sequences which have been identified in N.gonorrhoeae, along with their inferred translation products.\",\n \"The examples are generally in the following format: a nucleotide sequence which has been identified in N.gonorrhoeae.\",\n \"The strain used is FA1090 [Dempsey et al.\",\n \"(1991) J. Bacteriol.\",\n \"173:5476-5486] the inferred translation product of this sequence a computer analysis (e.g.\",\n \"PSORT output) of the translation product, indicating antigenicity homologous sequences (where relevant) results of expression and purification (where relevant) These examples typically include details of sequence homology between species and strains.\",\n \"Proteins that are similar in sequence are generally similar in both structure and function, and the homology often indicates a common evolutionary origin.\",\n \"Comparison with sequences of proteins of known function is widely used as a guide for the assignment of putative protein function to a new sequence and has proved particularly useful in whole-genome analyses.\",\n \"Open reading frames (ORFs) within nucleotide sequences were predicted using the GLIMMER program [Salzberg et al.\",\n \"(1998) Nucleic Acids Res 26:544-8).\",\n \"All predicted open-reading frames longer than 60 aa were screened against the meningococcus serotype B (‘MenB’) ORFs (accession NC002183) using the BLASTP algorithm [Altschul et al.\",\n \"(1990) J. Mol.\",\n \"Biol.\",\n \"215:403-410].\",\n \"ORFs were considered to be gonococcus-specific if they showed sequence identity to a MenB ORF lower than 60% over the whole protein length, or matching the MenB ORF over less than 30% of the length.\",\n \"Open reading frames are usually shown with a N-terminal methionine.\",\n \"Where this is not the case (e.g.\",\n \"SEQ IDs 12, 18, 20, 32, 54, 62, 66, 84, 98, 102, 104, 112, 116, 118, 126, 128, 130, 134, 136, 138, 146, 152, 162, 186, 228, 238, 240, 278, 280, 288, 290, 298, 300, 308, 314), a non-ATG start codon is present, but the N-terminus amino acid will be methionine when translated using this start codon.\",\n \"If an upstream start codon is used, however, the “native” amino acid will be translated (e.g.\",\n \"if the sequence is expressed with N-terminus fusion sequences).\",\n \"Even where the first amino acid is not shown as methionine, the invention encompasses sequences in which the first amino acid is methionine.\",\n \"Various tests can be used to assess the in vivo immunogenicity of the proteins identified in the examples.\",\n \"For example, the proteins can be expressed recombinantly and used to screen patient sera by immunoblot.\",\n \"A positive reaction between the protein and patient serum indicates that the patient has previously mounted an immune response to the protein in question ie.\",\n \"the protein is an immunogen.\",\n \"This method can also be used to identify immunodominant proteins.\",\n \"The recombinant protein can also be conveniently used to prepare antibodies e.g.\",\n \"in a mouse.\",\n \"These can be used for direct confirmation that a protein is located on the cell-surface.\",\n \"Labelled antibody (e.g.\",\n \"fluorescent labelling for FACS) can be incubated with intact bacteria and the presence of label on the bacterial surface confirms the location of the protein.\",\n \"For protein expression of 14 antigens, sequences were amplified using the following primers: Restriction Sequences site NGS5 Fwd CGCGGATCCCATATG-TGGGCAGAACAACCGGC NdeI Rev CCCGCTCGAG-GTTTTCAGCAGGGGGATTG XhoI NGS7 Fwd CGCGGATCCCATATG-GCCGGTAAAGAGCAATTTAC NdeI Rev CCCGCTCGAG-AGCCAAGAAGAACCCGTTAT XhoI NGS13 Fwd CGCGGATCCGCTAGCTGCGTTGCCGACCCCG NheI Rev CCCGCTCGAG-CATGTGCCGTGCGGCGT XhoI NGS36 Fwd CGCGGATCCGCTAGC-GACACCCCGAACAATACC NheI Rev CCCGCTCGAG-AAACCTGCCCTTGATGCC XhoI NGS37 Fwd CGCGGATCCCATATG-GTAGAAGTTAAAGGCGGGG NdeI Rev CCCGCTCGAG-TTTTTTCGCGCCGCCGAA XhoI NGS38 Fwd CGCGGATCCCATATG-GCCGACGAACGCCGCC NdeI Rev CCCGCTCGAG-AAACCGATATTTAAAACCCAACAGCC XhoI NGS39 Fwd CGCGGATCCGCTAGCAACCAAGAAGGGATTACCG NheI Rev CCCGCTCGAG-TTTTTGAGCATAATGACTTTTGCCCT XhoI NGS67 Fwd CGCGGATCCCATATG-CGTGCGCACGGACACG NdeI Rev CCCGCTCGAG-GGCGGCGAGTTTTTCGC XhoI NGS106 Fwd CGCGGATCCCATATG-GCAAACAGCGGAACGATAG NdeI Rev CCCGCTCGAG-AAAATCCTGCGGGATCGGT XhoI NGS115 Fwd CGCGGATCCCATATG-GGGGGCGGCTCCGGC NdeI Rev CCCGCTCGAG-TTCGGCCAACAATGCTTCC XhoI NGSΔG115 Fwd CGCGGATCCCATATG-GATGCCCAATCTTCACAAAG NdeI Rev CCCGCTCGAG-TTCGGCCAACAATGCTTCC XhoI NGS118 Fwd CGCGGATCCCATATG-ACCGCCCTTCCCTCTGA NdeI Rev CCCGCTCGAG-CGGCTGCCATTCGCGTT XhoI NGS122 Fwd CGCGGATCCCATATG-AACCCGAACGATGCGTTTT NdeI Rev CCCGCTCGAG-AGGGTAAAACTTATTCAAATCGGCAA XhoI NGS144 Fwd CGCGGATCCCATATGGCTTCTGAAAATTCTGTAGC NdeI Rev CCCGCTCGAG-GAACACGCTTTTCATTACACCCA XhoI NGS151 Fwd CGCGGATCCCATATGCACGGTATGCATAAGAGCA NdeI Rev CCCGCTCGAG-TTGCTGATGCGGCTTTATTCG XhoI EXAMPLE 1 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 1> which encodes amino acid sequence <SEQ ID 2; NGS1>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.47 Possible cleavage site: 36 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 4.72 threshold: 0.0 PERIPHERAL Likelihood = 4.72 modified ALOM score: −1.44 Rule: cytoplasmic protein * * * Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.326(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >sp|P45941|LYQC_BACSU HYPOTHETICAL 21.5 KD PROTEIN IN CWLA-CISA INTERGENIC REGION pir||E69949 hypothetical protein yqcF-Bacillus subtilis dbj|BAA06963.1| (D32216) ORF95 [Bacillus subtilis] dbj|BAA12427.1| (P84432) YqcF [Bacillus subtilis] emb|CAB14528.1| (Z99117) yqcF [Bacillus subtilis] Length = 192 Score = 35.5 bits (81), Expect = 0.45 Identities = 36/162 (22%), Positives 77/162 (47%), Gaps 5/162 (3%) Query: 19 DSGSQYKLNIAAIPSSPNRDLKTYITLGLSKHDLHYK---SRFEILFVCSLKYDENQIFP 75 D ++I ++ P + +Y TLGLS H +NY+ + I V +++ + Sbjct: 29 DDNKSSIDILSVSDQPQEGITSYSTLGLSDHSIEGTPLRIEIV~AAMESASDIYAN 88 Query: 76 FLRWLAETIIENKKILLRGQVVYLPRSIVNS-TKMDALYVSAPFYFDDDFQVCYGEHYNI 134 L A II + G + S+ + T M + PF +++D ++ + N+ Sbjct: 89 VLSTCAFNIINSNFTCAPGVIFKNVISMYDQETDMKHIMFVPPFLWEEDLELLEFSNKNV 148 Query: 135 VFPLLVPLYKQEAELVEKKGWNAFEQFLLDNEVGNLSDMNRK 176 + + +P+ + E ++ EK G + + Q LL+++ ++ D+ R+ Sbjct: 149 TWLMALPISEGELQVAEKHG-SDYLQDLLESKQIDIFDIKRE 189 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 2 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 3> which encodes amino acid sequence <SEQ ID 4; NGS2>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −7.2 Possible cleavage site: 18 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 5.89 threshold: 0.0 PERIPHERAL Likelihood = 5.89 modified ALOM score: −1.68 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.367(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >emb|CAC01359.1|(AL390975) hypothetical protein SCP8.21 [Streptomyces coelicolor A3(2)] Length = 198 Score = 37.2.bits (85), Expect = 0.15 Identities = 29/107 (27%), Positives = 51/107 (47%), Gaps = 3/107 (2%) Query: 73 ETPEHIETLAMLASASYPDQFQLGKNIGRPWVEQSSFRHFLISLPYPYGQELEY 130 +T + + LA+LA++ G ++++G P + F L++ P ++LE Sbjct: 88 DTDKVLRPLAVLAASPQVEGVIVAPGASLDVGEPLWPGAPFTSVLVAEPGGLVEDLELDA 147 Query: 131 -MDNRFFWLLPITQTERLFLNTHSVEELETKFDEAGIDYLDINRAS 176 +D VRF LLP+T E + H L+ ++ G D D +R S Sbjct: 148 PLDPVFLPLLPMTPNEAAWKRVHGAPALQERWLNHGTDLRDPSRRS 194 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 3 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 5> which encodes amino acid sequence <SEQ ID 6; NGS3>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −1.69 Possible cleavage site: 32 >>> Seems to have a cleavable N-term signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 33 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 5 value: −10.56 threshold: 0.0 INTEGRAL Likelihood = −10.56 Transmembrane 182-198 (171-201) INTEGRAL Likelihood = −7.86 Transmembrane 251-267 (244-273) INTEGRAL Likelihood = −7.48 Transmembrane 142-158 (136-167) INTEGRAL Likelihood = −6.32 Transmembrane 55-71 (50-82) INTEGRAL Likelihood = −2.97 Transmembrane 100-116 (99-117) PERIPHERAL Likelihood = 4.72 modified ALOM score: 2.61 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.522(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >sp|P19845|NOSY_PSEST MEMBRANE PROTEIN NOSY PRECURSOR pir||S13585 nosY protein precursor-Pseudomonas stutzeri emb|CAA37717.1|(X53676) nosY [Pseudomonas stutzeri] Length = 276 Score = 163 bits (413), Expect = 2e−39 Identities = 117/275 (42%), Positives = 174/275 (62%), Gaps = 2/275 (0%) Query: 1 MNPVWIITGKEARDSLRNRWVLAAVLLLAALALSLGFLGSSPTGSVKVDPLTVTVVSLSS 60 MN VW I KE D LRNRW+LA LL A LA+ + +LG++ +G + + T+ SL+S Sbjct: 1 MNQVWNIARKELSDGLRNRWLLAISLLFAVLAGIAWLGAAASGQLGFTSIPATIASLAS 60 Query: 61 LLSIFLIPLIAMLLSYDALIGEIERGTMALLLSYPIWRNQILAGKFVGHLIILALATTAGY 120 L+ FL+PLIA+LL+YDA++GE E GT+ LLL+YP+ R QIL GKFVGH +ILALA G+ Sbjct: 61 LATFLMPLIALLLAYDAIVGEDEGGTLMLLLTYPLGRGQILLGKFVGHGLILALAVLIGF 120 Query: 121 GLAGITLQLANGGFDIAA-WKPFALLAASVILGAAFLSMGYLISAKVKERGTAAGISIG 179 G A + + L G ++ + F + +S +LG FL+ Y++S KV E+ +AAG+++G Sbjct: 121 GCAALAIALLVEGVELGMLFWAFGRFHISSTLLGWVFLAFAYVLSGKNEKSSAAGLALG 180 Query: 180 VWLFFVVIFDMALLGILVADSKQVITAPVVETVLLFNPTDIYRLLNLTGYENTAMYAGMA 239 VW F V+ +L + S+ ++ +LL NPTDIYRL+NL+G+E + G+ Sbjct: 181 VW-FLFVLVFDLVLLALLVLSEGKFNPELLPWLLLLNPTDIYRLINLSGFEGSGSAMGVL 239 Query: 240 GLSGQIGLTVPVLLTAQVLWVIIPLVLAAGIFRKR 274 L + + VL + W+ + L+LA IFR+R Sbjct: 240 SLGADLPVPAAVLCLLAWIGVSLLLAYAIFRRR 274 A homolog (amino acids 226-276) was found in serogroup A N.meninigitidis but not in serogroup B, so NGS3 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 4 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 7> which encodes amino acid sequence <SEQ ID 8; NGS4>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 1.53 Possible cleavage site: 58 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 0.63 threshold: 0.0 PERIPHERAL Likelihood = 0.63 modified ALOM score: −0.63 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.103(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >sp|Q59746|NOSZ_RHIME NITROUS-OXIDE REDUCTASE PRECURSOR (N(2)OR) (N2O REDUCTASE) gb|AAC44023.1|(U47133) nitrous-oxide reductase [Sinorhizobium meliloti] prf||2209347B nitrous-oxide reductase [Rhizobium meliloti] Length = 639 Score = 660 bits (1704), Expect = 0.0 Identities = 344/536 (64%), Positives = 407/536 (75%), Gaps = 23/536 (4%) Query: 1 MSDEKLEQNGLSRRSFLGTAA--ASGAGIAGAGLIGLAGCSKDGEQAAANASGAAPVAKA 58 MS+E+ + L+RR LGT A A+ + G L L+G G A A+A Sbjct: 1 MSNEETKMR-LNRRQMLGTTAFMAAAGAVGAGGALTLSG-------------GTATPARA 46 Query 59 QGESKPGQLSSEVGPGELDQYYGFLSGGQSGEMRLIGLPSMRELMRIPVFNMDSATGWGR 118 Q S S EV PGELD+YY F S GQSGE+R++G PSMRE+MRIPVFN SATGWG+ Sbjct: 47 QETSGS---SYEVKPGELDEYYVFFSSGQSGEIRIVGAPSMREMMRIPVFNRCSATGWGQ 103 Query: 119 TNESLKVLNGNITEETRKFLKDSGLRCYPNGDLHHPHLSFTDQTYDGRYAYANDKANNRV 178 TNES KV+ + ET +FLKD G Y NGDLHHPH SFTD TYDGRY YANDK+N+RV Sbjct: 104 TNESRKVMTEGLLPETVEFLKDQG-GLYLNGDLHHPHPSFTDGTYDGRYLYANDKSNSRV 162 Query: 179 CRVRLDVMKADIIDIPNDSGIHGIHGLRPQRYPKTGYVFANGEHITPVSGVGK-LDDAKTWN 237 CR+RLDVMK DKII +PN +HGLR Q+YPKTGYVF NGE PV GK + D ++ Sbjct: 163 CRIRLDVMKCDKIIQLPNQHTVHGLRVQKYPKTGYVFCNGEDAVPVPNDGKTMGDKNSYQ 222 Query: 238 AVYTAIDGETMEIAWQVLVDGNLDNGDADYQGKYSFATCYNSERALTVQGASSNEQDWCV 297 A++TA+DGETME+AWQV+VDGNLDN DADYQGKY FATCYNSE T+ ++EQDW V Sbjct: 223 AIFTAVDGETMEVAWQVMVDGNLDNVDADYQGKYCFATCYNSEEGFTLADMMASEQDWVV 282 Query: 298 VFDLKAIEEGIKAGDFKEVNGVLDGRABAKSKYTRYIPVFNSPHGCNASPDGKYIMPN 357 +F+LK IEE + GD+KE+ GV +LDGR S YTRY+PVPNSPHG N +PDG +++ N Sbjct: 283 IFNLKRIEEAVAKGDYKEIGGVPVLDGR--KGSPYTRYVPVPNSPHGINTAPDGIHVVAN 340 Query: 358 GKLPPTVTVLDVSKLDDLFAGKIKERDVVAEPQLGLGPLHTAFDGRGNAYTTLFIDSQM 417 GKL PTVTV DV K DDLF KI+ RD VVAEP+LGLGPLHTA+DG+GNAYTTLFIDSQ+ Sbjct: 341 GKLSPTVTVFDVRKFDDLFDDKIQARDTVVAEPELGLGPLHTAYDGKGNAYTTLFIDSQV 400 Query: 418 VKWNIDDAIKAYGEKIDPIKQKLDVHYQPGHNHTTMGETKEADGQWLVSLNKFSKDRFL 477 KWNI+DA +AY GEK+DPI+ KLDVHYQPGHNHT+MG+TKEADG+WL+SLNKFSKDR+L Sbjct: 401 CKWNIEDAKRAYAGEKVDPIRHKLDVHYQPGHTSMGQTKEADGKWLISLNKFSKDRYL 460 Query: 478 NAGPLKPECDQLIGISGDEMRLVHDNPTFAEPHDLCLVAASKLNPGKTWDRKDPWF 533 N GPLKPE DQLI ISGDEM LVHDNPTFAEPHD +V ASK+NP W+R DP+F Sbjct: 461 NVGPLKPENDQLIDISGDEMVLVHDNPTFAEPHDATIVHASKINPVHVWNRDDPFF 516 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 5 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 9> which encodes amino acid sequence <SEQ ID 10; NGS5>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 1.09 Possible cleavage site: 19 >>> Seems to have a cleavable N-term signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 20 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 7.43 threshold: 0.0 PERIPHERAL Likelihood = 7.43 modified ALOM score: −1.99 Score for OM-PP discrimination: 4.97 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: 4.97 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Outer membrane?\",\n \"score: 0.496525 ----- Final Results ----- bacterial outer membrane --- Certainty= 0.781(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.138(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has no homology with sequences in the databases.\",\n \"The protein was expressed in E.coli as an insoluble 43.56 kDa His-fusion product and then purified.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 6 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 11> which encodes amino acid sequence <SEQ ID 12; NGS6>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.93 Possible cleavage site: 36 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 6.42 threshold: 0.0 PERIPHERAL Likelihood = 6.42 modified ALOM score: −1.78 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.447(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir|F83173 outer membrane protein OprC PA3790 [imported]-Pseudomonas aeruginosa (strain PAO1) dbj|BAA05664.1|(D28119) outer membrane protein C [Pseudomonas aeruginosa] gb|AAG07177.1|AE004797_12 (AE004797) outer membrane protein OprC [Pseudomonas aeruginosa] Length = 723 Score = 77.9 bits (191), Expect = 1e−13 Identities = 58/188 (30%), Positives = 89/188 (46%), Gaps = 13/188 (6%) Query: 49 VKDLIIFDRAHGQSGTASKGGIITRNVDAILFTAQAYARYNFNPHWAAGIKAAYNYGHN 108 +TL, V+D I+F G G++++ NVDAR+ + A Y +W AY +G N Sbjct: 546 VQDFILFSYREGMMGSSTQ-----ATNVDARIMGGELGASYQLTGNWKTDASLAYAWGKN 600 Query: 109 ETDGRPPYQIRPFEAAVQADYKNYFAHGSYNIGAATRFVAKQTRGDFDMASGLGIDKREA 168 +D R QI P EA Y+ G ++ G+ R VA Q R D + +G D ++ Sbjct: 601 SSDDRALPQIPPLEARFGLTYE----EGDWSAGSLWRVVAPQNRIARDQGNVVGKDFDKS 656 Query: 169 AKGFTVADVYAGVNIKDKYGLRLGVNNVFNKKYVEYI--SGDHVLALSPS-VVYAPGRTY 225 A GF V + + L GV+N+F+K Y E++ +GD S + V PGRT+ Sbjct: 657 A-GFGVFSLNGAYRVTRNVKLSAGVDNLFDKDYTEHLNKAGDAGFGFSANETVPEPGRTF 715 Query: 226 WLSLHAAF 233 W + +F Sbjct: 716 WTKVDFSF 723 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 7 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 13> which encodes amino acid sequence <SEQ ID 14; NGS7>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Hejine) Signal Score (−7.5): 4.94 Possible cleavage site: 26 >>> Seems to have a cleavable N-term signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 27 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 0.79 threshold: 0.0 PERIPHERAL Likelihood = 0.79 modified ALOM score: −0.66 Score for OM-PP discrimination: −18.85 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: −18.85 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Periplasmic space?‘0 score: 1.8846 ----- Final Results ----- bacterial periplasmic space --- Certainty= 0.929(Affirmative) < succ> bacterial outer membrane --- Certainty= 0.211(Affimative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir||D72405 hypothetical protein-Thermotoga maritima (strain MSB8) gb|AAD35294.1|AE001705_5 (AE001705) hypothetical protein [Thermotoga maritima] Length = 300 Score = 81.8 bits (201), Expect = 1e−14 Identities = 72/289 (24%), Positives = 124/289 (41%), Gaps = 17/289 (5%) Query: 38 PAMPSVTIAVAALQGKLAKQADVSLKIWRSPDQLRAGVASGQFKVMMSPSNVGVNLRNQG 97 +TL, P P++ V + GK+ DV ++IW++P++ A + S + + P VG NL +G Sbjct: 24 PLGPALIPVVPIMDGKIP--TDVKIEIWKNPEEAVAKIVSKEVDFAVLPVTVGANLYGKG 81 Query: 98 QKVGMNILTNGITQLVCKGSAIASP-QDLVGKKILVPF-KNDMPDIVLQALLKKLKIDA 155 ++ +V + +LV A + L G+++ P + D++++ L K + Sbjct: 82 VRIKLVGVHEWKVFYLVASDDATFDGWESLRGQEVYTPHGRGQTVDVLMRYFLSKAGLTL 141 Query: 156 HK-VSITYAATPPEAVGLFPSKGYHAVILPEPMATASLLKGKTIGINVVHGFDLVKAWGQ 214 + V I YA P E V LF S LPEP + L +GK + D K WG+ Sbjct: 142 DRDVKILYAP-PQEIVALFKSGKVKYAALPEPFVSMCLDRGKVV-------LDFQKEWGK 193 Query: 215 AFDTKPLIPMAGIIANEEYFHAHKAQFDIFHQDLKNALNWILANRQNAAKIGKNYLPAPE 274 IP+AG+ E K + + L +++ W+ N ++ L P Sbjct: 194 ELGVPGRIPIAGLFVRE---GVDKETVEKVKALIDSIRWMKENLDETVQLSSEKLGIPA 250 Query: 275 PALVHGLDGAPLTVSKGSEVKNEILKFYEILMQFNPELLGGKLPDNGFF 323 L L+ + + E+ F + L + PE K+PD GF+ Sbjct: 251 KILKSSLERIEFEYVPVEKCREEVETFLKKLNELYPEGF-EKIPDEGFY 298 The protein was expressed in E.coli as an insoluble 32.89 kDa His-fusion product and then purified.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 8 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 15> which encodes amino acid sequence <SEQ ID 16; NGS8>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 2.39 Possible cleavage site: 15 >>> Seems to have a cleavable N-term signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 16 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 4 value: −8.23 threshold: 0.0 INTEGRAL Likelihood = −8.23 Transmembrane 49-65 (41-73) INTEGRAL Likelihood = −7.38 Transmembrane 83-99 (75-106) INTEGRAL Likelihood = −7.06 Transmembrane 110-126 (100-133) INTEGRAL Likelihood = −4.41 Tranamembrane 164-180 (163-187) PERIPHERAL Likelihood = 5.89 modified ALOM score: 2.15 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.429(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >sp|P38044|NRTB_SYNP7 NITRATE TRANSPORT PERMEASE PROTEIN NRTB pir||S30892 nitrate transport protein nrtB-Synechococcus sp.\",\n \"(strain PCC 7942) emb|CAA43810.1|(X61625) nitrate transporter [Synechococcus sp.]\",\n \"prf||1908370A nitrate transporter [Synechococcus sp.]\",\n \"Length = 279 Score = 67.5 bits (164), Expect = 1e−10 Identities = 54/202 (26%), Positives = 96/202 (46%), Gaps = 7/202 (3%) Query: 4 VALWAWGSAVFGEFMLPAPVEFQKSL--DLLKHFQEN-----EIGISLWRSVVGISVAL 56 +A+W SA+ G+ LP P+ V + +++ F +N +G+ + S+ +++ Sbjct: 36 LAIWQVISAILGQDRLPGPINVVANTWMPYIVEPFFDNGGTSKGLGLQILISLQRVAIGY 95 Query: 57 IAGLAAGLVAGLVAGSFKTAMALLKPVITILLAMPPIIWVVMALFWFGFGNPSVLFTIIV 116 + G++ G V G K L PVI +L +PP+ W ++L F N S +F I + Sbjct: 96 LLAACTGILVGGVLGMSKFLGKGLDPVIQVLRTVPPLAWFPISLMVFQDANTSAIFVIFI 155 Query: 117 LVAPLTFASAAVGMASVNKQHEELFDAYKLGRLKKIRYLYIPHLTGYVISSVGVAVAMGV 176 + AVG+ + + + KL + I + IP YV + + +AV + Sbjct: 156 TAIWPIIINTAVGINQIPDDYNNVARVLKLSKKDYILNILIPSTVPYVFAGLRIAVGLAW 215 Query: 177 KAVIMAELLGASKGVGARIADA 198 A++ AE+L A G+G I DA Sbjct: 216 LAIVAAEMLKADGGIGYFIWDA 237 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 9 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 17> which encodes amino acid sequence <SEQ ID 18; NGS9>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.07 Possible cleavage site: 29 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 1 value: −1.81 threshold: 0.0 INTEGRAL Likelihood = −1.81 Transmembrane 97-113 (96-113) PERIPHERAL Likelihood = 4.24 modified ALOM score: 0.86 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.172(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >sp|P97027|SSUB_BACSU PUTATIVE ALIPHATIC SULFONATES THANSPORT ATP-BINDING PROTEIN SSUB pir||G69816 nitrate ABC transporter (binding protein) homolog ygaL- B. subtilis emb|CAB07520.1|(Z93102) hypothetical 30.6 kd protein [Bacillus subtilis] emb|CAB12711.1|(Z99108) similar to nitrate ABC transporter (binding protein) [Bacillus subtilis] Length = 274 Score = 99.5 bits (247), Expect = 3e−20 Identities = 68/181 (37%), Positives 102/181 (55%), Gaps = 9/181 (4%) Query: 4 LFGPSGCGKTTVLRLIAGLETPKSGTIRNTFH-------KTGFLFQENRLPENLTAMQNI 56 L GPSGCGK+T+L++IAGL++ G++ + GF+FQE+RL LT QNI Sbjct: 56 LIGPSGCGKSTLLKIIAGLDSEYDGSVEINGRSVTAPGIQQGFIFQEHRLFPWLTVEQNI 115 Query: 57 A--IFMDNPDEGEIVALAAKVGLTAGDLNKYPTELSGGMAKRVAFLRLLLCGCDLALLDE 114 A + + +P + V ++ G YP ELSGGM++RVA R LL ++ LLDE Sbjct: 116 AADLNLKDPKVKQKVDELIEIVRLKGSEKAYPRELSGGMSQRVAITRALLREPELLLDE 175 Query: 115 PFVGLDRDLRDILVAMLVEKIERQGMACILVTHDRFEAARLSHEIMLLSAKGMNVQNVIT 174 PF LD R L +L++ ++ ILVTHD E+ L +E+ +L AK + ++ Sbjct: 176 PFGALDAFTRKHLQDVLLDIWRKKTTNILVTHDIDESVYLGNELAILKAKPGKIHKLMP 235 Query: 175 L 175 + Sbjct: 236 I 236 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 10 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 19> which encodes amino acid sequence <SEQ ID 20; NGS10>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal score (−7.5): 2.27 Possible cleavage site: 26 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 5.14 threshold: 0.0 PERIPHERAL Likelihood = 5.14 modified ALOM score: −1.53 *** Reasoning Step: 2 imb2 HYPID: 2 CFP: 0.1 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.100(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir||A82615 surface protein XF1981 [imported]-Xylella fastidiosa (strain 9a5c) gb|AAF84783.1|AE004017_6 (AE004017) surface protein [Xylella fastidiosa] Length = 1190 Score = 50.2 bits (119), Expect = 2e−05 Identities = 59/210 (28%), Positives = 92/210 (43%), Gaps = 5/210 (2%) Query: 17 SIGTSAEANAPGAALGGSSEASKFSIAEGYLASSDGYGAIAIGSAAKI-KQLEKGTIN 75 ++G A+A GA A+G + AS K S A G A + G++A+G AK + + Sbjct: 876 AVGVGTLASAEGATAVGSGAAASGKGSTAIGRNAVASADGSVALGDGARGAESYTG 935 Query: 76 HIVGNDNKGLYVDADGNVTKITVRTESEKDILSRYGQTYGAVALGFRSSSHNLFA----S 131 G N + + G+ +K RT S L + N + Sbjct: 936 KYSGLQNNTVGTVSVGDASKGETRTVSNVADAKEATDAVNLRQLDRVAQDANRYVDNKIE 995 Query: 132 SFGAFSTATAIESLAVGDSSQSTGYRSATFGSHSRALAEESLALGYETRANYGSVALGA 191 S T + SL + + G + G + A +S+A+G + A+A +VA+G Sbjct: 996 SLSEGQTFVKVNSLNNSATPIAAGVDATAIGVGATASGADSIAMGNKASADNAVAIGN 1055 Query: 192 ESVANEENTVSVSSDTLKRKIVNVIADGTED 221 SVA+ NTVSV S +R++ NVA GT D Sbjct: 1056 HSVADRANTVSVGSAGSERQVTNVAAGTAD 1085 >sp|P10858|YADA_YERPS INVASIN PRECURSOR (OUTER MEMBRANE ADHESIN) pir||S04534 invasin precursor-Yersinia pseudotuberculosis plasmid pIBI emb|CAA32088.1|(X13883) Yop1 preprotein (AA 1-434) [Yersinia pseudotuberculosis] prf||1411295A invasin [Yersinia pseudotuberculosis[ Length = 434 Score = 42.1 bits (98), Expect = 0.006 Identities = 35/134 (26%), Positives = 68/134 (50%), Gaps = 28/134 (20%) Query: 116 AVALGFRSSSHLFASSFGFSTATAIESDSSQSTGYRSATFGSHSRA-------- 167 ++A+G + + A + G+ S AT + S+A+G S++ G + T+G+ S A Sbjct: 107 SIAIGATAEAAKPAAVAVGSGSIATGVNSVAIGPLSKALGDSAVTYGASSTAQKDGVAIG 166 Query: 168 ----LAEESLALGYETRANAYGSVALGA----------------ESVANEENTVSVSSDT 207 ++ +A+G+ ++ +A SVA+G S + EN+VS+ ++ Sbjct: 167 ASDTGVAVGFNSKDAQNSVAIGHSSHVAADHGYSIAIGDHSKTDRENSVSIGHES 226 Query: 208 LKRKIVNVADGTED 221 L R++ ++A GTED Sbjct: 227 LNRQLTHLAAGTED 240 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 11 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 21> which encodes amino acid sequence <SEQ ID 22; NGS11>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −0.16 Possible cleavage site: 60 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 4.67 threshold: 0.0 PERIPHERAL Likelihood = 4.67 modified ALOM score: −1.43 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.297(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >sp|P10858|YADA_YERPS INVASIN PRECURSOR (OUTER MEMBRANE ADHESIN) pir||S04534 invasin precursor-Yersinia pseudotuberculosis plasmid pIBI emb|CAA32088.1|(X13883) Yop1 preprotein (AA 1-434) [Yersinia pseudotuberculosis] prf||1411295A invasin [Yersinia pseudotuberculosis] Length = 434 Score = 41.3 bits (96), Expect = 0.007 Identities = 27/71 (38%), Positives = 48/71 (67%), Gaps = 4/71 (5%) Query: 16 QLNRLSKRTNRVGASAAALASL-KPAQLGKNDKFAFSLGFGSYKNAQAVAMGAVFKPAEN 74 +L++L KR ++ AS+AAL SL +P +GK + F+ G G Y+++QA+A+G+ ++ E+ Sbjct: 353 RLDKLDKRVDKGLASSAALNSLFQPYGVGKVN---FTAGVGGYRSSQALAIGSGYRVNES 409 Query: 75 VLLNVAGSFAG 85 V L ++AG Sbjct: 410 VALKAGVAYAG 420 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 12 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 23> which encodes amino acid sequence <SEQ ID 24; NGS12>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −1.29 Possible cleavage site: 61 >>> Seems to have a cleavable N-term signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 62 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 6.36 threshold: 0.0 PERIPHERAL Likelihood = 6.36 modified ALOM score: −1.77 Score for OM-PP discrimination: 6.03 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: 6.03 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Outer membrane?\",\n \"score: 0.602784 ----- Final Results ----- bacterial outer membrane --- Certainty= 0.867(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.158(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has no homology with sequences in the databases.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 13 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 25> which encodes amino acid sequence <SEQ ID 26; NGS13>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.64 Possible cleavage site: 51 >>> May be a lipoprotein Amino Acid Composition of Predicted Mature Form: calculated from 21 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 1 value: −1.01 threshold: 0.0 INTEGRAL Likelihood = −1.01 Transmembrane 36-52 (36-52) PERIPHERAL Likelihood = 5.14 modified ALOM score: 0.70 Rule: inner or outer membrane protein Rule: inner or outer membrane protein Rule: cytoplasmic membrane protein *** Reasoning Step: 2 Lipoprotein?\",\n \"Inner membrane?\",\n \"----- Final Results ----- bacterial outer membrane --- Certainty= 0.790(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.742(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gb|AAC33455.1|(AF067083) outer membrane protein homolog [Vitreoscilla sp.]\",\n \"Length = 217 Score = 236 bits (602), Expect = 2e−61 Identities = 134/217 (61%), Positives = 166/217 (75%) Query: 1 MTFFKPSTVVLTASALALSGCVADPVTGQQSPNKSAMYGLGGAAVCGWGALTHSGKGAR 60 M +K +++ T +A+ALS C DP+TGQ N + + LGGAA CGIVGALTH KGAR Sbjct: 1 WKAWKKFSLMATVAAVALSACATDPMTGQVDRNNTVLGALGGAATCGIGALTHGSKGAR 60 Query: 61 NSALACGAIGAGVGGYMDYQEQRLRQNLAGTQIEIQRQGNQIRLVMPESVTFATGSAALG 120 NSALACGAIGAGVG YMD+QE++LRQ+LA TQ+E+ R G++IRLVMPES+TFATGS L Sbjct: 61 NSALACGAIGAGVGAYMDHQERQLRQSLANTQVEVNRVGDEILVMPESITFATGSYQLN 120 Query: 121 GSAQYALNTAAQTLVQYPDTTLTINGHTDNTGSDAVNNPLSQHRAQAVAYYLQTRGVAAS 180 SA +LN+ + L QY DTT+ I GHTD+TGSDA+N PLS++RA AVA YL +R VA++ Sbjct: 121 SSASTSLNSVSSVLAQYTDTTINIVGHTDSTGSDAINEPLSRNRASAVANYLVSRNVASN 180 Query: 181 RLTVYGYGSHMPVASNATVEGRAQNRRVEILINPDQR 217 R+T G G PVASN TV GRA+NRRVEI +NP QR Sbjct: 181 RITTVGAGCRQPVASNNTVAGRAENRRVEITVNPIQR 217 >gb|AAD40344.1|U88088_22 (U88088) OmpA [Pseudomonas alcaligenes] Length = 220 Score = 130 bits (328), Expect = 1e−29 Identities = 90/219 (41%), Positives = 127/219 (57%), Gaps = 6/219 (2%) Query 7 STVVLTASALALSGCVA---DPVTGQQSPNKSAMYGLGGAAVCGIVGALTHSGKGARNSA 63 S + + L+GC + + T + + A L GA ++G + +GA A Sbjct: 3 SVIAASLVIFTLTGCASIQNEDGTTKNTALYGAGGALAGAVAGALIGK-ENRAQGALIGA 61 Query: 64 LACGAIGAGVGGYMDYQEQRLRQNLAGTQIEIQRQGNQIRLVMPESVTFATGSALLLGGSA 123 G++GAG G Y D QE LR+ + G+ ++++RQG++I +VMP ++TFATG A + + Sbjct: 62 AVAGSLGAGYGYYADKQEAELREQMKGSGVQVERQGDEIVIVMPGAITFATGKAEIQPNF 121 Query: 124 QYALNTAAQTLVQYPDTTLTINGHTDNTGSDAVNNPLSQHRAQAVAYYLQTRGVAASRLT 183 LN A + YPD+ L + GHTD+ GS N LSQ RAQ+VA +L+ GV R+ Sbjct: 122 ANTLNQLAGSFPNYPDSRLIVTGHTDSVGSYEANELLSQRRAQSVAQFLRGNGVQTDRIE 181 Query: 184 VGYGHMPVASNATEGRAQNRRVEILINPDQRAVNAA 222 V G G + PVASNAT EGRAQNRRVEI + P RAV A Sbjct: 182 VIGAGPNQPVASNATAEGRAQNRRVEIKLAP--RAVQQA 218 The protein was expressed in E.coli as a soluble 22.55 kDa His-fusion product and then purified.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 14 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 27> which encodes amino acid sequence <SEQ ID 28; NOS14>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.32 Possible cleavage site: 40 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 3.39 threshold: 0.0 PERIPHERAL Likelihood = 3.39 modified ALOM score: −1.18 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.254(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has no homology with sequences in the databases.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 15 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 29> which encodes amino acid sequence <SEQ ID 30; NGS15>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −1.75 Possible cleavage site: 45 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 5.89 threshold: 0.0 PERIPHERAL Likelihood = 5.89 modified ALOM score: −1.68 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.232(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >sp|P10487|RCI1_ECOLI SHUFFLON-SPECIFIC DNA RECOMBINASE pir||S03815 probable integrase-Escherichia coli dbj|BAA77989.1|(AB027308) shufflon-specific DNA recoinbinase [Plasmid R64] Length = 384 Score = 67.1 bits (163), Expect = 3e−10 Identities = 75/301 (24%), Positives = 125/301 (40%), Gaps = 34/301 (11%) Query: 68 KVKMMTLSEAMRKYLNETLGAGRSKKMGL---RFLMEFPIGGIGIDKLKRSDFAEHVMQR 124 +++ M+LS A+ KYL + + + PI +D++ D A + R Sbjct: 5 RIRKMSLSRALDKYLKTVSVHKKGHQQEFYRSNVIKRYPIALRNMDEITTVDIATYRDVR 64 Query: 125 RRGIPELDIAPIAASTALQELQYIRSVLKHAFYVWGLEIGWQELDFAANGLKRSNMVAKS 184 I PI +T EL + S+ A WG N ++ S Sbjct: 65 LAEINPRTGKPITGNTELELALLSSLFNIARVEWG--------TCRTNPVELVEKPKVS 116 Query: 185 AIRDRLPTTEELQTLTTYFLRQWQSRKSSIPMHLIMWLAIYTSRRQDEICRLLFDDWHKN 244 + RDR T+E + L+YF R+ ++ +++I LA+ T+ RQ EI L W Sbjct: 117 SGRDRRLTSSEBRRLSRYF------REKNLMLYVIFHLALETAMRQGEILAL---RWEHI 167 Query: 245 DCTRPVRDLKNPNGSTGNNKEFDILPMALPVIDELPEESVRKRMLANKGIADSLVPCNGK 304 D V L P G++++ + A + +P + ++ Sbjct: 168 DLRHGVAL--PETNGHSRDVPLSRRARNFLQMMP-----------VNLHGNVFDYTAS 214 Query: 305 SVSAAWTRACKTLGIKDLRFHULREEAATRMAEDG-FTIPQNQRVTLHDGWNSLQRYVSVR 364 AW A + L I+DL FHDLRHEA +R E G + ++ ++ H N L+RY +R Sbjct: 215 GFKNAWRIATQRLRIEDLHRHDLRHEAISRFFELGSLNVMEIAAISGHRSMNMLKRYTHLR 275 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 16 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 31> which encodes amino acid sequence <SEQ ID 32; NGS16>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.64 Possible cleavage site: 20 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 4.67 threshold: 0.0 PERIPHERAL Likelihood = 4.67 modified ALOM score: −1.43 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.262(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >sp|P10484|T1M1_ECOLI TYPE I RESTRICTION ENZYME ECOR124II M PROTEIN (M.ECOR124II) pir||S02166 type I site-specific deoxyribonuclease (EC 3.1.21.3) EcoR124/3 chain hsdM-Escherichia coli plasmid R124/3 emb|CAA31541.1|(X13145) hsdM protein (AA 1-520) [Escherichia coli] Length = 520 Score = 44.4 bits (104), Expect = 0.002 Identities = 65/235 (27%), Positives = 99/235 (41%), Gaps = 55/235 (23%) Query: 107 NRKKAGGYAEYITGGSLRRLVAARRYCGEHPGVFDGAAGSG--------QLEQYIEPS 158 N K+GG E+ T + +L+A ++D AAGSG Q + +I Sbjct: 191 NAGKSGG--EFFTPQHVSKLIAQLAMHGQTHVNKIYDPAAGSGSLLLQAKKQFDNHIIEE 248 Query: 159 DFRAVEIQAEACRALLQNYPAAKVYNTSLFL--------------------YTDGEPQDC 198 F EI N+ + ++FL + D +P D Sbjct: 249 GFFGQEI----------NHTTYNLARMNMFLHNINYDKRDIKLGNTLTEPHFRDEKPFDA 298 Query: 199 TVMNPPFSIKLKDLSEDEKSRIAQEYPWKKSGV------ADEIFVLKGLE--NARRFGFF 250 V NPP+S+K + D+ + I E + +GV AD FVL L +A+ Sbjct: 299 IVSNPPYSVKW--IGSDDPTLINDER-FAPAGVLAPKSKADFAFVLHALNYLSAKGRAAI 355 Query: 251 ILFPGIAYR-KSEQRFRE-IIGNRLAE--LNRIQNAFEDTPIEVLLLVIDKDKTD 301 + FPGI YR +EQ+ R+ ++ N E ++ N F T I V +LV+ K KTD Sbjct: 356 VCFPGIFYRGGAEQKIRQYLVDNNYVETVISLAPNLFFGTTIAVNILVLSKHKTD 410 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 17 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 33> which encodes amino acid sequence <SEQ ID 34; NGS17>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.29 Possible cleavage site: 16 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 2.60 threshold: 0.0 PERIPHERAL Likelihood = 2.60 modified ALOM score: −1.02 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.284(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >ref|NP_052389.1|translocator YopD [Yersinia enterocolitica] sp|P37132|YOPD_YEREN YOPD PROTEIN gb|AAD16812.1|(AF102990) translocator YopD [Yersinia enterocolitica] Length = 306 Score = 32.1 bits (72), Expect = 8.2 Identities = 29/93 (31%), Positives = 43/93 (46%), Gaps = 17/93 (18%) Query: 13 MLAAKRAAKESTRQERAVKRAGTVRNVDRNRLSARSKAQKENIARMLSGAKVSEDEALTC 72 +L R A+E Q+R ++ T+ AQKE +A M+SGAK+ A+ Sbjct: 89 LLELARKAREMGLQQRDIENKATI------------SAQKEQVAEMVSGAKLMIANAVVS 136 Query: 73 GIMMRLSLQDMRYACNQELINFAEHIVKQVQRL 105 GIM S ++ +E+ IVKQ Q L Sbjct: 137 GIMAATSTVASAFSIAKEV-----KIVKQEQIL 164 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 18 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 35> which encodes amino acid sequence <SEQ ID 36; NGS18>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −2.56 Possible cleavage site: 38 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 4.56 threshold: 0.0 PERIPHERAL Likelihood = 4.56 modified ALOM score: −1.41 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.397(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has no homology with sequences in the databases.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 19 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 37> which encodes amino acid sequence <SEQ ID 38; NGS19>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.12 Possible cleavage site: 43 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 8.49 threshold: 0.0 PERIPHERAL Likelihood = 8.49 modified ALOM score: −2.20 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.250(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >ref|NP_043483.1|orf14 [Bacteriophage HP1] sp|P51716|YO14_BPHP1 HYPOTHETICAL 14.9 KD PROTEIN IN REP-HOL INTERGENIC REGION (ORF14) pir||S69520 hypothetical protein 14-phage HP1 gb|AAB09199.1|(U24159) orf14 [Bacteriophage HP1] Length = 133 Score = 73.3 bits (179), Expect = 1e−12 Identities = 44/129 (34%), Positives = 74/129 (57%), Gaps = 6/129 (4%) Query: 1 MFIPAALHKDEHSAYGVTIPDLPGCFSCGDTVEEAVANARSAAYMHIDGMIEDGGFKNLA 60 M P + K + Y V++PD+PGCFS GDT+ EA+ NA+ A HI+GM+ED + L Sbjct: 1 NLYPICIEK-VNDGYVVSVPDVPGCFSAGDTLSEAMLNAKEAISFHIEGMLEDD--EELP 57 Query: 61 VSS-IADLSQEPDYHGATWVMIEIDPAKISRQQIRFNVSWPQYLLDRVDEY--TSANHET 117 S+ I +P+Y ++++D + + + N++ P LL R+D++ T ++ Sbjct: 58 KSNPIEQYINQPEYKDFIVTVVDVDLTHLMGKAEKINITVPALLLHRIDQFIATHPEYKN 117 Query: 118 RSGFLAKAA 126 RS FL++ A Sbjct: 118 RSNFLSQLA 126 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 20 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 39> which encodes amino acid sequence <SEQ ID 40; NGS20>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −0.1 Possible cleavage site: 19 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 7.58 threshold: 0.0 PERIPHERAL Likelihood = 7.58 modified ALOM score: −2.02 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.057(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has no homology with sequences in the databases.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 21 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 41> which encodes amino acid sequence <SEQ ID 42; NGS21>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.52 Possible cleavage site: 52 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 5.83 threshold: 0.0 PERIPHERAL Likelihood = 5.83 modified ALOM score: −1.67 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.311(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >ref|NP_040628.1|cI (repressor; 237) [bacteriophage lambda] ref|NP_061378.1|phage lambda repressor protein CI [Escherichia coil] sp|P03034|RPC1_LAMBD REPRESSOR PROTEIN CI pir||RPBPL repressor protein cI-phage lambda emb|CAA24991.1|(X00166) coding sequence cI gene [bacteriophage lambda] gb|AAA96581.1|(J02459) cI (repressor; 237) [bacteriophage lambda] emb|CAB96428.1|(AJ277653) phage lambda repressor protein CI [Escherichia coli] Length = 237 Score = 62.5 bits (151), Expect = 5e−09 Identities = 36/85 (42%), Positives = 51/85 (59%) Query: 2 KKRELNEIETAECAELKRIFNSKKEELKLTQYKLAEAVGVTQSAVNHYLNGTNALNASIA 61 KK+ L + + + LK I+ KK EL L+Q +A+ +G+ QS V NG NALNA A Sbjct: 4 KKKPLTQEQLEDARRKKAIYEKKKELGLSQESVADKMGMGQSGVGALFNGINALNAYNA 63 Query: 62 SQFAKILQIPVSDFSLRLAEEISSM 86 + AKIL++ V +FS +A EI M Sbjct: 64 ALLAKILKVSVEEFSPSIAREIYEM 88 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 22 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 43> which encodes amino acid sequence <SEQ ID 44; NGS22>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −2.6 Possible cleavage site: 43 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 7.74 threshold: 0.0 PERIPHERAL Likelihood = 7.74 modified ALOM score: −2.05 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.072(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir||S30432 hypothetical protein-Streptomyces clavuligerus plasmid pSCL Length = 307 Score = 43.6 bits (102), Expect = 0.002 Identities = 25/86 (29%), Positives = 49/86 (56%), Gaps = 2/86 (2%) Query: 6 MGMAFKT-GIPRGRFVLVLCDCANDDGLCYPSQETLAEDTGFAETAVRQHIKWLKDNN 64 MGM F G+ ++ +L+ + + G C+PS++ L +D G + + V++ +L N Sbjct: 1 MGMVFAAEGLDGSEKLLLLGYTNWTDPYGYCWPSEDRLVDDCGTSRSTVQRTKRKLVKKN 60 Query: 65 FIKSARRQRGR-ERKSDIYRINVALL 89 ++S RR+ + E S++ R+N+ LL Sbjct: 61 LLRSVRRKNSKGEPISNLSRVNLPLL 86 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 23 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 45> which encodes amino acid sequence <SEQ ID 46; NGS23>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −2.8 Possible cleavage site: 59 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 0.05 threshold: 0.0 PERIPHERAL Likelihood = 0.05 modified ALOM score: −0.51 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Result ----- bacterial cytoplasm --- Certainty= 0.195(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >sp|P07905|DNAC_ECOLI DNA REPLICATION PROTEIN DNAC pir||XMECNC DNA replication protein dnaC-Escherichia coli (strain K-12) Length = 245 Score = 110 bits (275), Expect = 2e−23 Identities = 75/224 (33%), Positives = 116/224 (51%), Gaps = 23/224 (10%) Query: 50 EAADEMAAYAETLRRGAMRDA----------LEKRIGRSGIAPRFRNCRIENYAV--SDS 97 + +E+ A+ + +GA+R A +++ RSGI P +NC ENY V Sbjct: 24 KTGEELLAWQK--EQGAIRSAALERENRAMKMQRTFNRSGIRPLHQNCSFENYRVECEGQ 81 Query: 98 IPGMARAKAAAAEYAANFADVLQTGRSMIFSGRRGTGKNHLACGIAREVIAAGKSALVIT 157 + +++A+ E+ N A S IFSG+ GTGKNHLA I E++ GKS L+IT Sbjct: 82 MNALSKARQYVEEFDGNIA-------SFIFSGKPGTGKNHLAAAICNELLLRGKSVLIIT 134 Query: 158 VGDMLRTVKDSF--GGGGEAGAVGIFVKPDLLVLDEFGAGSLSETDGRILFSVVNARYER 215 V D++ +KD+F G E + DLLV+DE G + S+ + I+ +V+ R Sbjct: 135 VADIMSAMKDTFRNSGTSEEQLLNDLSNVDLLVIDEIGVQTESKYEKVIINQIVDRRSSS 194 Query: 216 LMPMLVLTNLTAEAFRENTDARIRDRLRDGGGKLIPFDWESTRA 259 P +LTN E + R+ DR+R G + F+W+SYR+ Sbjct: 195 KRPTGHLTNSNMEEMTKLLGERVMDRMRLGNSLWVIFNWDSYRS 238 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 24 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 47> which encodes amino acid sequence <SEQ ID 48; NGS24>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.76 Possible cleavage site: 26 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 1.43 threshold: 0.0 PERIPHERAL Likelihood = 1.43 modified ALOM score: −0.79 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.112(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >ref|NP_053228.1|pXO2-73 [Bacillus anthracis] gb|AAF13678.1|AF188935_76 (AF188935) pXO2-73 [Bacillus anthracis] Length = 541 Score = 125 bits (315), Expect = 9e−28 Identities = 139/535 (25%), Positives = 254/535 (46%), Gaps = 70/535 (13%) Query: 14 PVLFIGTGMSLRYLDNSYTWDGLLSKIAIDLFGDDREYLNIKSRYCEDGRFQYEEIAEEL 73 P LFIG+G S RYL N W GL+ K + +L + EY Y + E+AE + Sbjct: 19 PFLFIGSGFSKRYL-NLEDWAGMKKFS-NLMPYEFEY------YSSTANKDWAEVAELM 70 Query: 74 QSKFDKVL--ENDPDGEFKEINDKFFENMRAGNTLSRFKIYISTLLSQLNYK----DNSN 127 F + E + KE D R + S K+ ++ L+ + YK + ++ Sbjct: 71 AKDFHPIWWKEQQFENNRKEFKD------RISSKQSPLKVEVAKYIiNSIEYKYGLDEKND 124 Query: 128 TELSELKKARKNVGSIITTNYDKLAQDIFEFNPL---IGN-DILLSNPY--GSVYKIHGC 181 E°+ LKK + IITTN+D L + IFE + IG ++L S+P +YKIHGC Sbjct: 125 KEIAALKKIV--IDGIITTNWDLLLEQIFEEQEMQVYIGQKELLFSHPLEINEIYKIHGC 182 Query: 182 VDDPSKIIITKKDYEKFKEKYELIRAQLLSLFIHNPIIFLGYNVGDENIKEILKTIFTYV 241 P +++T DY+ + EK + A+LL++FI +P+IFLGY++ D+NI++ILK I + Sbjct: 183 SSIPDSLVLTTSDYKGYNEKNAYLAAKLLTVFIEHPVIFLGYSISDDNIQQIILKAITRCL 242 Query: 242 EQNSPSANKIRRNFLLVEYEPESNNEDIVEDIDIT-GFSTIRINKIKTDNFSQIYKALA 300 +Q++ K R L+ E ED E++ +T G T+ I ++KT+++ +IY ALA Sbjct: 243 DQDNIHKLKDR----LIFVERAGQEEDSFENNSSLTIGKITVPITRVKTNDYEKIYNALA 298 Query: 301 ELTLPISAMDVRKFQSIAKEIYTGGNIKVSF---TEDMDNLNNSDKVVAIGSTKTISYNF 357 + S +R+ +S E+ + + + D+ + + V+ +G K + Sbjct: 299 QNKRKFSMKMMRQMKSQIYELVKTNDPEEKIYVVDGEYDDTQDIEFVIGLG-VKNVVEEM 357 Query: 358 QTTSEMMSN----------------YFKIIEEENS----QLLKLIDKHSIASTQYFPI-- 395 Q+ E+ ++ + +++ +E ++K+ + S QY P+ Sbjct: 358 QSNHEISADKELSEHGYGGISDIELFNELLSDEPKYDYDSIVKISLPQILRSNQYVPLFR 417 Query: 396 YGFSRICSDIHKEAVLKRQQKEKLDHFIEEINRRCKNNHSSIQSILDDENISDTYKNDAI 455 Y D ++ +K + K + F+ E ++ N S + + + + + + Sbjct: 418 YVLESSVEDELLDSKIKNKLKMRYTDFIITETQKNIKLDWQFKNLDEVLKGFPDIKV 477 Query: 456 AWG----IWNNQLSEDEVENYL---KNFVNKKN----THYKRLLCMFDYKKYADT 499 A + L+ D+++++L FV +KN T +RL ++D+ KY + Sbjct: 478 AIEQIPLLGQKNLNNCDDLDFLIKNSKFVKEKNTPERTGIRRLFRIYDWLKYGQS 532 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 25 A DNA sequence was identified in N,gonorrhoeae <SEQ ED 49> which encodes amino acid sequence <SEQ ID 50; NGS25>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 4.76837e−07 Possible cleavage site: 56 >>> May be a lipoprotein Amino Acid Composition of Predicted Mature Form: calculated from 21 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 6.68 threshold: 0.0 PERIPHERAL Likelihood = 6.68 modified ALOM score: −1.84 Rule: inner or outer membrane protein Rule: inner or outer membrane protein *** Reasoning Step: 2 Lipoprotein?\",\n \"Inner membrane?\",\n \"----- Final Results ----- bacterial outer membrane --- Certainty= 0.790(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.700(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|7433O05|pir||B70313 cytochrome-C peroxidase (EC 1.11.1.5)- Aquifex aeolicus gi|2982865|gb|AAC06485.1|(AE000675) cytochrome C peroxidase [Aquifex aeolicus] Length = 355 Score = 345 bits (885), Expect = 7e−94 Identities = 180/336 (53%), Positives = 237/336 (69%), Gaps = 12/336 (3%) Query: 59 EDQDLLKRAQGVFQPLPTVEEMQKIRPFTEEQVKLGHQLWYEPRLSKGNTVSCNSCHNLA 118 +D++LLK A+ F+PLP V E + P T E+VKLG L+Y+PRLSK +SCN+CHNLA Sbjct: 22 DDKELLKKMARQYFKPLPKVAENPQ-NPVTPEKVKLGKMLYYDPRLSKSGLISHCNTCHNLA 80 Query: 119 SAGVDNMPTSQGHKGQFGGRNSPTALNAALLGSQFWDGRAADVEEQAGGPLVNPVEMAND 178 GVDN+PTS GH+ G RN+PT NAA+ +QFWDGRA DVEEQA GP+VNP+EMAN Sbjct: 81 RYGVDNLPTSIGHRWAIGPRNAPTVYNAAIHIAQFWDGRAKDVEEQALGPIVNPIEMAN- 139 Query: 179 SQEAAAAKIAKVPEYQEFKAFP-EDGAVSFKNITTALGAFERTLLTPTKWDEYLKGNV 237 ++E A + +PEY E+FKKAFP E V ++NI A+GAFERTL+TP+++DE+LKGN Sbjct: 140 TEENAVKTLKSIPEYVELFKKAFPNEKDPVKYENIGKAIGAFERTLMTPSRFDEFLKGNT 199 Query: 238 NALSEQERKGRAFDNGCIACHNGNLGGTTFQKRGLVQGPYWK------FIEDP--KR 289 AL+EQE++G++ F++ GC+ACHNG +GG F KFG++ YWK + P K Sbjct: 200 KALTEQEKRGLKTFIEVGCVACHNGPGVGGNMFAKFGMIT-EYWKVTYPYVLVGKPAIKV 258 Query: 290 DKGEADVTKKTEDEFFFRVPGLRNVAKTYPFHNGSVWELDKAVTIMGKAQLGKDIPKED 349 D GR VTKK ED F F+VP LRN+ TYPYFH+GSVW L+ AV IM K QLGK++ + Sbjct: 259 DFGRFGVTKKEEDMFVFKVPSLRNIEHTYPYFHDGSVWSLEDAVRIMAKTQLGKELTDQQ 318 Query: 350 VDNIVVFLNALSGNVSESARTHPELPLTAPMESKPD 385 V +IV FL AL+G + + A +PELP + KP+ Sbjct: 319 VKDIVAFLKALTGKIPKHALEVPELPPSTDKTPKPE 354 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 26 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 51> which encodes amino acid sequence <SEQ ID 52; NGS26>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 0.610001 Possible cleavage site: 15 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 5.04 threshold: 0.0 PERIPHERAL Likelihood = 5.04 modified ALOM score: −1.51 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.127(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir||T13296 hypothetical protein 8-Streptococcus phage phi-O1205 gb|AAC79524.1|(U88974) ORF8 [Streptococcus thermophilus temperate bacteriophage O1205] Length = 157 Score = 62.5 bits (151), Expect = 2e−09 Identities = 53/161 (32%), Positives = 86/161 (52%), Gaps = 8/161 (4%) Query: 5 TLYRCDVQAALDYYFDSETEREDTLEAV--IGQFEVKAQSVIAYIKNQEITEKMLEGH 62 TLY + + D ET + DTLEA+ +E K + + IK+ E + + Sbjct: 3 TLYELTDQLLEIYNMDVDDET-KLDTLEAIDWTTDYENKVEGYVKVIKSLEADIEARKNE 6 Query: 63 IRQMTGKLKAAKARNQSLKDYLARNMQAAGITEIKADDGTFKASFRKSEAVVILDEAQIP 122 +++ G K+ +++ LK LA +M G T + D FK FRKSEAVV+ +E ++P Sbjct: 62 KKRLDGLNKSDQSKIDKLKTALAVSMAETGQTRV--DTTLFKVGFRKSEAVVV-NEEKLP 118 Query: 123 AEFMREAVKTEPDKTAIRXAIESGRQVAGAKIEGRKNLQIR 163 E+ K PDK +++ ++SG+ + GA +E R+NL IR Sbjct: 119 KEYQIATYK--PDKKTLKLKSGKHIEGATLEERRNLNIR 157 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 27 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 53> which encodes amino acid sequence <SEQ ID 54; NGS27>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Hejine) Signal Score (−7.5): −5.45 Possible cleavage site: 49 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 1.80 threshold: 0.0 PERIPHERAL Likelihood = 1.80 modified ALOM score: −0.86 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty = 0.559 (Affirmative) < succ> motifs: Subtilase_Asp (S, T, A, I, V)X(L, I, V, M, F)(L, I, V, M)D(D, S, T, A)G(L, I, V, M, F, C)x) (A)x(L)(I)D(D)G(I)x{2}(D) 79: DDDFL AALIDDGIVFD V A homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS27 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 28 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 55> which encodes amino acid sequence <SEQ ID 56; NGS28>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −0.19 Possible cleavage site: 61 >>> Seems to have a cleavable N-term signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 62 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 0.69 threshold: 0.0 PERIPHERAL Likelihood = 0.69 modified ALOM score: −0.64 Score for OM-PP discrimination: −24.78 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: −24.78 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Periplasmic space?\",\n \"score: 2.47798 Periplasmic space?\",\n \"score: 2.47798 ----- Final Results ----- bacterial periplasmic space --- Certainty= 0.916(Affirmative) < succ> The protein has homology with the following sequences in the databases: >prf||1306286A mobilization protein B [Escherichia coli] Length = 529 Score = 34.7 bits (78), Expect = 2.4 Identities = 24/69 (34%), Positives = 31/69 (44%), Gaps = 12/69 (17%) Query: 344 QLRARQQEIPVDYARTAVCGRIPFRRHSRPTLRSRTLGAQRRRIVPNVGQAGGIRAD--- 400 +LRA Q++P D+ +T V P R R + GA GQ G IR D Sbjct: 440 RLRAAGQDLPADFVKTTVLDNTPIRWFYRAASQESRSGA---------GQTGEIRVDVER 490 Query: 401 RTPNTQRGT 409 R P +RGT Sbjct: 491 RGPAGRRGT 499 A homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS28 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 29 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 57> which encodes amino acid sequence <SEQ ID 58; NGS29>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.61 Possible cleavage site: 31 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 4.03 threshold: 0.0 PERIPHERAL Likelihood = 4.03 modified ALOM score: −1.31 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.106(Affirmative) < succ> The protein has homology with the following sequences in the databases: emb|CAB83930.1|(AL162753) hypothetical protein NMA0640 [Neisseria meningitidis Z2491] Length = 387 Score = 653 bits (1685), Expect = 0.0 Identities = 324/388 (83%), Positives = 351/388 (89%), Gaps = 1/388 (0%) Query: 1 MNITIAAPYCSLPSEPHFNRFWYLAELLSQSHDVLLITSNFKHYDKSFRRPEDAKAASQG 60 MNITI APYCSLPSEP+FNRFWYLAE LSQSHDVLLITS F+HYDKSFRR EDA A S G Sbjct: 1 MNITIVAPYCSLPSEPYFNRFWYLAERLSQSHDVLLITSRFRHYDKSFRRHEDAAATSNG 60 Query: 61 RLKVMLLEESGYSKNVSLGRVTSHHRFVKHFEKWLENCRPGEQDVVYSAYPLIATNLLLG 120 RL+V LL+E GY KNVSL RV SH FV++ +WL + + EQD+VYSAYPL+ATNLLLG Sbjct: 61 RLRVKLLDEPGYRKNVSLARVASHRVFVRNLARWLHSPQAAEQDIVYSAYPLMATNLLLG 120 Query: 121 KHKARLGYKLIVDVQDVWPESFSSVVPFLKKIPHNLLPFASRANRAYRYADALVAVSQTY 180 KHKARLGYKLIVDVQDVWPESFSSVVPFLKK+PH LLPFASRANRAYR ADAL+AVSQTY Sbjct: 121 KHKARLGYKLIVDVQDVWPESFSSVVPFLKKVPHKLLPFASRANRAYRCADALIAVSQTY 180 Query: 181 LDRAKEANPNVPGEVVYIGADFAAIAPPPRFRSKTVRFFYLGTLSYNYDVETVCKGVRKL 240 LDRAKEANPNVPGE VYIG DFAAIA PPRFRSKTVR FYLGTLSY+YDVETVCKGVRKL Sbjct: 181 LDRAKEANPNVPGETVYIGTDFAAIA-PPRFRSKTVRLFYLGTLSYSYDVETVCKGVRKL 239 Query: 241 LDDGENVELHIMGGGPDLDRLKQYACDGIKFYGYIPYAEMMSVAKGCDIAVNAIHSYAMQ 300 LDDGENVELHIMGGGPDL++LKQY IKFYGY+PY+EMMS+AK CDIAVNAIHS+AMQ Sbjct: 240 LDDGENVELHIMGGGPDLEKLKQYENRAIKFYGYLPYSEMMSIAKACDIAVNAIHSHAMQ 299 Query: 301 SITNKLSDYMALQKPILNSQVHDEVAEVLTLLPHENYRSGDVDGFVQAAKDILKRKNDPV 360 S+TNKLSDYMALQKPILNSQ + EV ++L LLPHENYRSGDVD FVQAAK+ILKRK+DPV Sbjct: 300 SVTNKLSDYMALQKPILNSQNNAEVLDLLNLLPHENYRSGDVDSFVQAAKNILKRKDDPV 359 Query: 361 QSDEIVRRFRHDISYRKIVNLIERLANE 388 QSDEIVRRFR DISYRKIVNLIERLA+E Sbjct: 360 QSDEIVRRFRRDISYRKIVNLIERLAHE 387 >emb|CAB58324.1|(AL121855) hypothetical protein SCF62.09 [Streptomyces coelicolor A3 (2)] Length = 407 Score = 54.7 bits (130), Expect = 2e-06 Identities = 57/243 (23%), Positives = 105/243 (42%), Gaps = 24/243 (9%) Query: 99 RPGEQDVVYSAYP---LIATNLLLGKHKARLGYKLIVDVQDVWPESFSSVVPFLKKIPHN 155 R G DVV++ P L L L R G + + D D+ PE + S K + + Sbjct: 81 RVGPVDVVHACNPPDLLFLPALWL----KRRGARFVFDQHDLIPELYLSRFGRGKDLLYR 136 Query: 156 LLPFASANRAYRYADALVAVSQTYLDRAKEANPNVPGEVVYIGA-----DFAAIAPPPR 210 + R YR AD ++A +++Y D A P +V ++ F + P P Sbjct: 137 AVCALERWT--YRAADVVLATNESYKDVAIRRGGRRPDDVFVVRSAPATDRFQPVPPEPE 194 Query: 211 F-RSKTVRFFYLGTLSYNYDVETVCKGVRKLLDDGENVELH--IMGGGPDLDRLKQYA-- 265 R K YLG + V+ + + KL D+ + H +G G D + + + Sbjct: 195 LKRGKPHLLCYLGVMGPQDGVDYALRALAKLRDEVGRTDWHAVFVGSGDAFDAMVELSRS 254 Query: 266 ---CDGIKFYGYIPYAEMMSVAKGCDIAVNAIHSYAMQSIT--NKLSDYMALQKPILNSQ 320 + ++F G IP A++++ D+ ++ + ++ NK+ +YMA+ +PI++ Sbjct: 255 LGLDEQVRFTGRIPDADLVRHLSTADVCLSPDPRNPLNDVSTMNKVLEYMAMGRPIVSFD 314 Query: 321 VHD 323 + + Sbjct: 315 LRE 317 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, NGS29 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 30 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 59> which encodes amino acid sequence <SEQ ID 60; NGS30>.\",\n \"Analysis of this protein sequence reveals the following: Signal Score (−7.5): −4.8 Possible cleavage site: 46 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 1.54 threshold: 0.0 PERIPHERAL Likelihood = 1.54 modified ALOM score: −0.81 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.113(Affirmative) < succ> The protein has homology with the following sequences in the databases: fastidiosa (strain 9a5c) gb|AAF84279.1|AE003977_2 (AB003977) conserved hypothetical protein [Xylella fastidiosa] Length = 376 Score = 73.6 bits (179), Expect = 3e−12 Identities = 82/354 (23%), Positives = 143/354 (40%), Gaps = 35/354 (9%) Query: 1 MKIILTTSMSGLGGTETATVRLGRLLKRHGHDIILASSDG-PFVGEAQASGIRWQPVDFY 59 MKI+ T + +G GG E R ++ GH + L G P A+ +G+ ++ + Sbjct: 1 MKILHTEAATGCGGEEIYIYRHMLSMQAQGHHMALLCQPGAPLSTMARNAGLPVYHINMH 60 Query: 60 RGGLAGYLKSTFAYARMLRREQPDIIDCQMARVVPACALAAKIVSPKTKIICHSHGLDAA 119 G L +L+RE D+++ A AA++ +T++I S L A Sbjct: 61 --GPWRVLNGIHTVQHLLQRETFDVVNTTSHVDTLIAAAAARLT--RTRLIVRSRHLMAP 116 Query: 120 TYPKTAKLFDKLGAYIIGNCICHEREKLIRHGFPAGRIAYA---------YNTPPEFHFRK 170 K+ + L +I +H R+ LI+ G RI +T PE +++ Sbjct: 117 I--KSQLTYTYLPHRVITVSQHRDLLIKQGIQPTRIGIVPPITA-QPPWMDTDPEHAWQR 174 Query: 171 TEK-------------ECAVLGTLSRLDTRAVHLMLDILKKMVGRNIPVRLNMAGIGEE 217 ++ ++G ++ L + +LD + + N + L +AG GE Sbjct: 175 LQQTRHVVRTELGFNDNDIIVGCVAVLREAKGHRELLDAIAPLCQANPRLHLVIAGDGEP 234 Query: 218 -MDNLKAQAKRLGIEDKVTFLGGVRDLTGYFKEVDILVNTPHCVGDHGAGVGNNILEAGL 276 M +L A K L +E ++ LG D DI + G LEA Sbjct: 235 VMQHLLAHRKTLTLETQIHLLGYRHDAPRLMSGFDIFA-----LATQKEAAGTVFLEAAQ 289 Query: 277 YDTPVVTYNMAGISEMVITGQTGYCIPFGDDEAFIEAVDTLIKHPELRSQNGKA 330 P++ + G+ EM+ G + G+ A A+ TL+ + + R MG+A Sbjct: 290 AGIPIIATRVGGVPEMLQEGTNAILVTPGNQTALTNALHTLVTNNQQRHSMGRA 343 A homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS30 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 31 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 61> which encodes amino acid sequence <SEQ ID 62; NGS31>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.36 Possible cleavage site: 16 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 3.50 threshold: 0.0 PERIPHERAL Likelihood = 3.50 modified ALOM score: −1.20 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.299(Affirmative) < succ> The protein has homology with the following sequences in the databases: gb|AAB49297.1|(U84350) hypothetical hydroxylase a [Amycolatopsis orientalis] Length = 491 Score = 111 bits (278), Expect = 1e−23 Identities = 87/269 (32%), Positives = 123/269 (45%), Gaps = 15/269 (5%) Query: 1 LKNGAAFSWGSRYTEFDF----TDKFSDGPGTVYQVRRAVRDKILIEEAAKQGVEVRFGH 56 +K G F WG+R + F + K + YQV RA FD IL++ A +GV VR G Sbjct: 73 IKRGGTFRWGARPEPWTFHFGISAKMAGSTSHAYQVERAKFDDILLKNAKSKGVVVREGC 132 Query: 57 GVTAFDNSGDFARLNIETDT-GESYELTAKFVLDASGY-GRVLPRLLNLETPSHLPPRQT 114 V G+ TD G ++E++A+FV+DASG R+ ++ S Sbjct: 133 SVNDVVEDGERVTGARYTDADGNAHEVSARFVIDASGNKSRLYTKVNGRNYSEFFRSLA 192 Query: 115 HFTHIDDNITHPKFDRNKILITTHPQHRDVWIWLIPFGDNRCSVGVV---GTPKLAGES 171 F + + P+ IL W W I P D SVG V DK+ G+ Sbjct: 193 LFGYFEGGKRLPEPVSGNILSVAFDSG---WFWYIPLSDTLTSVGAVVRREDADKIQGDR 249 Query: 172 ETVLKKFVYECPHLSEILDKAVWENDFPFRSIQ---GYSANVKSLHGEHFALLGNAAEFL 228 E L + ECP++SE L A + ++ YS S L+G+AA F+ Sbjct: 250 EKALNTLIAECPLISEYLSNATRVTRYGELRVRKDYSYQQDSYWRPGMVLVGDAACFV 309 Query: 229 DPVFSSGVTIALHSAELAADLLTKQLKGE 257 DPVFSSGV +A +SA LAA + L G+ Sbjct: 310 DPVFSSGVHLATYSALLAARSINSVLAGD 338 A homolog (amino acids 280-341) was found in serogroup A N.meningitidis but not in serogroup B, so NGS31 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 32 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 63> which encodes amino acid sequence <SEQ ID 64; NGS32>.\",\n \"Analysis of this protein sequence reveals the following: Signal Score (−7.5): −3.49 Possible cleavage site: 38 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 7.80 threshold: 0.0 PERIPHERAL Likelihood = 7.80 modified ALOM score: −2.06 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.278(Affirmative) < succ> The protein has homology with the following sequences in the databases: fastidiosa (strain 9a5c) gb|AAF83310.1|AE003899_2 (AE003899) phage-related repressor protein [Xylella fastidiosa] Length = 143 Score = 87.0 bits (214), Expect = 2e−16 Identities = 40/71 (56%), Positives = 54/71 (75%) Query: 1 MFSGEQLGQAISEAIKRKNVSQKEVADHFGVKQPSVSGWIKNGRIDKKHLDKLIDYFSDV 60 M +GEQLG+AI +A++ K V+ ++A+HFGVK PSV GWIK GRI K+ L L YFSDV Sbjct: 1 MLTGEQLGRAIKQAMQLKGVTPTKMAEHFGVKAPSSVYGWIKEGRISKEKLPSLWSYFSDV 60 Query: 61 VTPSHFGIETF 71 V P+H+G+E + Sbjct: 61 VGPTHWGLEAW 71 >sp|P18680|RPC1_BPHK0 26 KD REPRESSOR PROTEIN (REGULATORY PROTEIN CI) emb|CAA34222.1|(X16093) cI gene product (AA 1-208) [Bacteriophage HK022] Length = 235 Score = 80.5 bits (197), Expect = 2e−14 Identities = 60/200 (30%), Positives = 99/200 (49%), Gaps = 15/200 (7%) Query: 22 QKEVADHFGVKQPSVSGWIKNGRIDKKHLDKLIDYFSDVVTPSHF--------GIETFRV 73 Q ++A V ++S W 1 +K DK+ S + T + + GI + Sbjct: 29 QADLARLKVTPKAISKFNGESIPRK--DESLASVLGTTAAYLHGYADDDGITVNHL 86 Query: 74 LKSNEQSSIRFPRLNAEATCGAGT-INDHYIEVVDYVTVAAAWAREKLGGNLNK-IQVIT 131 +SN+ R L+ +A+ G GT +++ +IE + + AR G + ++VIT Sbjct: 87 SRSNDY--YRVDVLDVQASAGPGTMVSNEFIEKIRAIEYTTEQARILFNGRPQESVKVIT 144 Query: 132 ARGDSMEPTIENGDVMFVDTAVEAFDGDGLYLLWYIDGLKAKRLQSTVGGGLMIISDNSS 191 RGDSME TI GD +FVD ++ FDGDG+Y+ Y + KRLQ L +ISDN++ Sbjct: 145 VRGDSMEGTINPGDEIFVDVSITCFDGDGIYVFVYGKTMHVKRLQMQ-KNRLAVISDNAA 203 Query: 192 YRTETVRGEDLNAVRIIGRI 211 Y + + + I+ ++ Sbjct: 204 YDRWYIEEGEEEQLHILAKV 223 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 33 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 65> which encodes amino acid sequence <SEQ ID 66; NGS33>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.87 Possible cleavage site: 31 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 4.88 threshold: 0.0 PERIPHERAL Likelihood = 4.88 modified ALOM score: −1.48 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.313(Affirmative) < succ> The protein has homology with the following sequences in the databases: gb|AAF31132.1|(AF069529) Gp54 [Bacteriophage HK97] Length = 273 Score = 47.4 bits (111), Expect = 3e−04 Identities = 33/123 (26%), Positives = 52/123 (41%), Gaps = 20/123 (16%) Query: 221 NGGLSGKPKNANVPRRRKTHGVPLQEIADLYNEVLGGRLPSVQVLNDTRKRAIANRWCEM 280 NGG G+ K P RRK + + + YN +G RLP +N+ RKR + + Sbjct: 160 NGGGDGQVK----PERRKAERIDYESFLNAYNTEVGDRPHAVAVNEKRKRRL-KKIIPQ 214 Query: 281 LGTAAPNGKVRFGDKETGIAFAGFFRKVA--MNPFWMGENQTGFAVGFDWIFKAGNFVK 338 L T +G F + R PF+ G+N TG+ FD++ + + Sbjct: 215 LKTPNVDG-------------FRAYVRAFVHQAKPFYFGDNDTGWTADFDYLLREDSLTG 261 Query: 339 ILE 341 + E Sbjct: 262 VRE 264 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 34 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 67> which encodes amino acid sequence <SEQ ID 68; NGS34>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.65 Possible cleavage site: 50 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 3.76 threshold: 0.0 PERIPHERAL Likelihood = 3.76 modified ALOM score: −1.25 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.310(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir||H82649 hypothetical protein XF1674 XF1569 [imported]- Xylella fastidiosa (strain 9a5c) gb|AAF84378.1|AE003986_8 (AE003986) hypothetical protein [Xylella fastidiosa] gg|AAF84483.1|AE003993_2 (AE003993) hypothetical protein [Xylella fastidiosa] Length = 316 Score = 167 bits (424), Expect = 2e−40 Identities = 108/308 (35%), Positives = 152/308 (49%), Gaps = 30/308 (9%) Query: 10 ETSVIRSLSSASLYMFTRRMFYQRRGYVWQRANHHAPICNLERVFTIGETKRLIINIPPR 69 E +VI++ A FTR F QR+ ++ HH I ++ V G K ++IN+PP Sbjct: 10 EQAVIKARCEADHLFFTRYFFKQRQQLRFRVNWHHHVIAGVVDDVIAGRRKDVVINVPPG 69 Query: 70 YSKTEIAVVNFIAWAMGRVPDCEFIHASYSAALAVNNSVQIRNLVQHEEYRAIFP-DLAL 128 SKTE+ +N +A + P F+H SYS LA+ NS R +VQ +EYRA++P ++A Sbjct: 70 SSKTELVAINVMARGLALNPYARFLHISYSDDLALLSETAREIVQSDEYRALWPLEIAD 129 Query: 129 AGESGHHWKTT-----AGGVMYXXXXXXXXXXXXXXRHREGFGGCIIIDDPHKADEARSE 183 +S W AGGV Y G+ G IIIDDP K ++A S+ Sbjct: 130 DAKSKKRWVVDGKKAGGV-YAVSLGGQVTGFRAGHAPGWQGAIIIDDPLKVEDAYSK 188 Query: 184 VRRQNVIDWFQNTVESRKNSPDTPIILIMQRLHEKDLAGWLLDGGNGEEWEHLCLPAIQE 243 R +TV+SRK SPDTPII+IMQRL + D G++ GG WE + +PA+ + Sbjct: 189 TGRSKANRKLVSTKSRASPDTPIIVIMQRLAQDDPTGFIQSGGFPGAWECIEIPALID 248 Query: 244 DG-----------------------TALWPEKHDIETLRREQAAPWFAGQYLQKPAPP 280 D + WP K + L +E YVF+GQY Q+P+P Sbjct: 249 DAYVSRLPEHVQGQVVRDAQDQDGRYSYWPYKEPLAELLALEATDRYVFSGQYQQRPSPL 308 Query: 281 DGGTFKPD 288 GG K D Sbjct: 309 GGGIIKGD 316 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 35 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 69> which encodes amino acid sequence <SEQ ID 70; NGS35>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −1.07 Possible cleavage site: 40 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 1.64 threshold: 0.0 PERIPHERAL Likelihood = 1.64 modified ALOM score: −0.83 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.020(Affirmative) < succ> The protein has homology with the following sequences in the databases: >ref|NP_047925.1|gp34 [Bacteriophage phi-C31] emb|CAA07104.1|(AJ006589) gp34 [Bacteriophage phi-C31] Length = 457 Score = 59.7 bits (143), Expect = 1e−07 Identities = 68/272 (25%), Positives = 117/272 (43%), Gaps = 49/272 (18%) Query: 226 GYSPVEQIIMTVNIALKRQVHALEYYTAGSVPDALVGVPETWSADDIRRFQEYWDLLLSG 285 G SP+ +++ AL Q + +++ G++P A+V VP T S + + R +E W SG Sbjct: 192 GCSPISYARESIGLALAAQKYGSKFFANGGAMPGAVVEVPGTMSKEGLARAREAWRAANSG 251 Query: 286 -----------ETAQRRKMRFVPGELSRNFRETRQPPLKDVYDEWLARVVCFAFSVEPTP 334 E A+ K+ P E F +T+Q + ++ AR+ F V P Sbjct: 252 VDNAHRVALLTEGAKFSKVAMSPDEAQ--FLQTRQFQVPEI-----ARI----FGVPPH- 299 Query: 335 FVAQVNRSVAETS--REQSLSDGMGSLKNWVKALIDDVLARYMDMAA--YEFVWKGEESL 390 ++ S + S EQ+++ M SL+ W++ + A + FV + + Sbjct: 300 LISDATNSTSWGSGLAEQNIAFTMFSLRPWLERIEAGFNRLLFAETADRFRFVKFNLDEI 359 Query: 391 N---PKEQAEIYAIYKNAGILTADEIRAELGKEPLP-GQG--------------QPEPDK 432 PKE+ E++++ GI + DE+RA PLP G G +PEP+ Sbjct: 360 KRGAPKERMELWSLGLQNGIYSIDEVRAAEDMTPLPDGLGEKYRVPLNLGEVGEEPEPEP 419 Query: 433 QDG----RKPEEPPNQGAEKLGKSESPMSEDE 460 P E P++ E GK + + +E Sbjct: 420 APAPPAIEPPAEEPDEEPEPEGKPDDEGATEE 451 A homolog (amino acids 641-700) was found in serogroup A N.meningitidis but not in serogroup B, so NGS35 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 36 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 71> which encodes amino acid sequence <SEQ ID 72; NGS36>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 4.3 Possible cleavage site: 26 >>> Seems to have a cleavable N-term signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 27 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 1.70 threshold: 0.0 PERIPHERAL Likelihood = 1.70 modified ALOM score: −0.84 Score for OM-PP discrimination: 0.02 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: 0.02 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Outer membrane?\",\n \"score: 0.00213559 Outer membrane?\",\n \"score: 0.00213559 ----- Final Results ----- bacterial outer membrane --- Certainty= 0.232(Affirmative) < succ> The protein has homology with the following sequences in the databases: >pir||D82437 TonB receptor-related protein VCA0625 [imported]-Vibrio cholerae (group O1 strain N16961) gb|AAF96526.1|(AE004392) TonB receptor-related protein [Vibrio cholerae] Length = 784 Score = 103 bits (256), Expect = 2e−20 Identities = 104/427 (24%), Positives = 162/427 (37%), Gaps = 100/427 (23%) Query: 31 NTEQQKELNTIVVHGKRS-ADQKGADDVYYKNVSNAYVGKEYLERYRVQSAGDVLKGLNG 89 NTEQ + T+ VHG+ DQ+ D L++ R + D+ G+ Sbjct: 57 NTEQAVD-ETVTVHGQSILTDQRTRSD---------------LDkVRGIANADIFSGITS 100 Query: 90 VYNMNTRTAGGAITPNIRGITGKGRIPVTIDGTEQTIDVWMNNYGVGDRNYLDPALFRSI 149 V + N GA+ IRG+ G+GR+P+ IDG+ Q+ GV DR Y+D L S+ Sbjct: 101 VQSNNMHNEAGALDIGIRGVQGEGRVIFIDGSLQSTHTSRGYQGVSDRTYIDTDLLSSL 160 Query: 150 AVEKSPALTRG--VKSGVGGAMSIRTIEPSDIIPEGRNWGIEVKTEFSGNTVAQKNDLRQ 207 V K + VGG ++ T+ DII + + +G+ +K Sbjct: 161 TVNKGATIESSPYASGAVGGVVNATTLGIKDIIKDDQAFGVVLK---------------- 204 Query: 208 FLGRDYRTLSPIGATADGVSGMPDVLTGYTGKPSPTALLLDEGIADTKFSGGKSHTNFKD 267 A A+ + PDV Y+ + LDE + F G Sbjct: 205 -------------ARANNHNRTPDVSGDYSEQGQ---YALDERGEHSAFKHG-------- 240 Query: 268 DRQLMLSAAFKTDITDGLAAYSHRQKGNYYAGKRGYQSYLNNPI--YGADACYDQYPDKS 325 LML ++ + + + AYS R KGN++AGK+GY+ Y P+ G + + S Sbjct: 241 --SLMLGLGYQAESFTVLAYSKRSKGNHFAGKKGYEEY-QEPVVGQGQEVVNTSFESDS 297 Query: 326 WREKDILCKSASLVPNMAVLFRPGEEIMNSHTDTKILLLKNNWYLPDNQKISLQYMDNK 385 W K S N +R H +L WY Y D K Sbjct: 298 WLFK---LASDTGTAHNADFNYR--------HHAQKAGEVLMAYWYKSSEDWEGNPYPDGK 347 Query: 386 IGFGEINPLITAWILGFAEQSLNEPVQQAPGIGTKIDSKTYKIGYEWKPQNNKWIDLQAD 445 + W LG A+ + TY Y ++P ++ W++L A+ Sbjct: 348 -------DRMPQWGLGTAKVN------------------TYSANYYYQP-DHPWLNLNAN 381 Query: 446 MWRVKTD 452 W + D Sbjct: 382 FWYTEAD 388 Score = 94.7 bits (234), Expect = 5e−18 Identities = 80/290 (27%), Positives = 126/290 (42%), Gaps = 37/290 (12%) Query: 929 SYDLDNHLFARYASRFPSLYELTAATGSGGLYGSETVAEYS----LKPEKSTNWEV 984 +Y L + +LF + +R R PSLYE T S V Y+ +KPE++ N EV Sbjct: 514 TYALTPSTQLFLKSSRTYRMPSLYETTL---------SNEVFSYNPYNPIKPEQAWNNEV 564 Query: 985 GYNFNFAPHFAKLRQGDLRLTYYSNKIKNQIDTSN--EDGGMIQ---------YDKAVSK 1033 G F + + + +L ++Y+ N IK+ I + GM + YDK Sbjct: 565 GVQFMASNSVLQDDRLNLSVSYFRNSIKDFISGGRLAKTPGMSEWQANFTFTNYDKLQLS 624 Query: 1034 GVELQSRLDSGRFFASFGGTYRLKHMVCDKGIAFKFDYYLQRVPECLEGGFGLSRFFQSL 1093 G EL + + F T + +C A C GF + Sbjct: 625 GWELGAHYQYAWILYTHPAATLYSETKICSVQQA-----QYAESDTCNSLGFAWGLTPTRI 679 Query: 1094 QPKYSLTLDVGTRFFNEKLELGMRAIHHSKAERRNYDKLIADGAGQVYARNGKPYGWHAA 1153 PK +L L+VGT+FFN+ L+ G++ +HS + N +A A Y Sbjct: 680 PPKQNLYLNVGTKFFNDTLDSGVSYHSG--KSNPSDWLAGTAANPILEIPSDY----- 732 Query: 1154 TLLDAYARYRIGKHIDLNFSVTNLANRYYLDPMSSTPVPGPGRTITFGIK 1203 +D Y++Y + + L F++ N+ +RY + P S +P PGRTIT G + Sbjct: 733 -TIDLYSQYELNANTQIFFAINNVTDRYQVRPGSVVSMPDPGRTITLGFE 781 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 37 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 73> which encodes amino acid sequence <SEQ ID 74; NGS37>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 4.47 Possible cleavage site: 21 >>> Seems to have a cleavable N-term signal seq.\",\n \"Amino Acid composition of Predicted Mature Form: calculated from 22 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 7.21 threshold: 0.0 PERIPHERAL Likelihood = 7.21 modified ALOM score: −1.94 Score for OM-PP discrimination: 16.42 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: 16.42 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Outer membrane?\",\n \"score: 1.64214 Outer membrane?\",\n \"score: 1.64214 ----- Final Results ----- bacterial outer membrane --- Certainty= 0.938(Affirmative) < succ> The protein has homology with the following sequences in the databases: >sp|Q03155|AIDA_ECOLI ADHESIN AIDA-I PRECURSOR pir||S28634 adhesin AIDA-I precursor-Escherichia coli plasmid pIB6 emb|CAA46156.1|(X65022) AIDA-I [Escherichia coli] Length = 1286 Score = 35.8 bits (81), Expect = 0.67 Identities = 34/138 (24%), Positives = 62/138 (44%), Gaps = 16/138 (11%) Query: 3 ASQLTLAVLLAAAFGSAYAVEVKGGDSSKGQLIQAAESDFLPFGSGAADIKVSTGNGLSK 62 A L + + + G+A+AV + G SS G + + E+ + G G ++ V++G ++ Sbjct: 31 AKNTLLVLAWSTIGNAPAVNISGTVSS-GGTVSSGETQIVYSGRGNSNATVNSGG-TQ 87 Query: 63 SINLEAGPAQRIRNKYGNAPINGGNQNTNVNGAANSRYLQPGDINPIA--GWFSKTRLA- 119 +N N + G+QN +GA S + G I ++ G S T L+ Sbjct: 88 IVNNGGKTTATTVN-------SSGSQNVGTSGATISTIVNSGGIQRVSSGGVASATNLSG 140 Query: 120 ---QVWYEKRANNTEVFS 134 ++ A+NT +FS Sbjct: 141 GAQNIYNLGHASNTVIFS 158 The protein was expressed in E.coli as an insoluble 32.45 kDa His-fusion product and then purified.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 38 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 75> which encodes amino acid sequence <SEQ ID 76; NGS38>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 0.34 Possible cleavage site: 24 >>> Seems to have a cleavable N-term signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 25 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 3.98 threshold: 0.0 PERIPHERAL Likelihood = 3.98 modified ALOM score: −1.30 Score for OM-PP discrimination: 2.87 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: 2.87 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Outer membrane?\",\n \"score: 0.287446 Outer membrane?\",\n \"score: 0.287446 ----- Final Results ----- bacterial outer membrane --- Certainty= 0.607(Affirmative) < succ> The protein has homology with the following sequences in the databases: >sp|Q03155|AIDA_ECOLI ADHESIN AIDA-I PRECURSOR pir||S28634 adhesin AIDA-I precursor-Escherichia coli plasmid pIB6 emb|CAA461S6.1|(X65022) AIDA-I [Escherichia coli] Length = 1286 Score = 35.8 bits (81), Expect = 0.67 Identities = 34/138 (24%), Positives = 62/138 (44%), Gaps = 16/138 (11%) Query: 3 ASQLTLAVLLAAAFGSAYAVKGGDSSKGQLIQAAESDFLPFGSGAADIKVSTGNGLSK 62 A L + + + G+A+A+V + G SS G + + E+ + G G ++ V++G ++ Sbjct: 31 AKNTLLVLAVVSTIGNAFAVNISGTVSS-GGTVSSGETQIVYSGRGNSNATVNSGG--TQ 87 Query: 63 SINLEAGPAQRIRKYGNAPINQGNQNTNVNGAANSRYLQPGDINPIA--GWFSKRLA- 119 +N N + G+QN +GA S + G I ++ G S T L+ Sbjct: 88 IVNNGGKTTATTVN-------SSGSQNVGTSGATISTIVNSGGIQRVSSGGVASATNLSG 140 Query: 120 ----QVWYEKRANNTEVFS 134 ++ A+NT +FS Sbjct: 141 GAQNIYNLGHASNTVIFS 158 >pir||G81213 conserved hypothetical protein NMB0313 [imported]-Neisseria meningitidis (group B strain MD58) gb|AAF40758.1|(AE002388) conserved hypothetical protein [Neisseria meningitidis MC58] Length = 488 Score = 84.3 bits (207), Expect = 3e−15 Identities = 111/498 (22%), Positives = 185/498 (36%), Gaps = 35/498 (7%) Query: 7 LLFLPLCTVCLAAPSNDAADERRRLLDEGSRQTQQYRESGW--LDTEQARGEVEENDGY 64 +L LPL S A+E R D SR + E+ +D E+ G+V E + Sbjct: 19 MLLLPLLA------SAAYAEETPREPDLRSRPEFRLHEAEKPIDREKPGQVREKGKVL 72 Query: 65 SIGGEIYQVGDTAEELESAIYHALNARQWHKVRQFAARYAKLPRHKPALIHLADALQKRD 124 I GE E L A+Y A+ + +R Y + + L A + + Sbjct: 73 QIDGETLL--KNPELLSRAMYSAVVSNNIAGIRVILPIYLQQAQQDKMLALYAQGILAQA 130 Query: 125 EGDFRAAGNSFQTALEAEPDNPRLLLEAGRFYAEDNQNKESAAAFEKVLKTDIPAETRPI 184 +G + A + ++ + A+PD P + + E+ QN+ +A F+++ ++P + Sbjct: 131 DGRVKEAISHYRELIAAQPDAPAVRMRLAAALFENRQNEAAADQFDRLKAENLPPQLMEQ 190 Query: 185 VENYLSELGKRRRWHGQISLGYGYNSNVNQGNGINQCVWEIAGMCLMERTLPAPTDSTFS 244 VE Y L +R W N+NQ Q + T P D T Sbjct: 191 VELYRKALRERDAWKVNGGFSVTREHNINQAPKRQQ---------YGKWTFPKQVDGTAV 241 Query: 245 SYSATAEKTVPLKGNHGVQVRGVLYGNRYTEKDKDSAAMPDYGYNGSLYAGYAYADARS 304 +Y AEK LK G + G Y K + + G +AD R Sbjct: 242 NYRLGAEKKWSLKNGWYTTAGGDVSGRVYPGNKK-------FNDMTAGVSGGIGFADRRK 294 Query: 305 SFSLLPYFEYDFRNRHTHYRAWGADADWSRTLSPHNRINSHAGAKKTGYGGQSKTYFADF 364 L + E + GA ++R +P W+ S A + G ++ +D Sbjct: 295 DAGLAVFHERRTYGNDAYSYTNGARLYFNRWQTPKWQTLSSA---EWGRLKNTRRARSDN 351 Query: 365 KQYELGAGAEFSITLKSGLLVNFDAAPKAYP-EKSSSSKEYTARLGAYRLFSGGTYLNAV 423 ++ F + + D R+ P ++ + Y R A+ GG+ L+++ Sbjct: 352 THLQISNSLVFYRNARQYMGGLDFYRERNPADRGDNFNRYGLRF-AWGQEWGGSGLSSL 410 Query: 424 LLY--RRSLYDAASFVSDNK--RRRDKQYIMMAAAGFPQWNIKGVYPELRFRRTIAHSNA 479 L + Y+ F S K RRRDK+ + + KG+ P L SN Sbjct: 411 LRLGAAKRHYEKPGFFSGFKGERRRDKELNTSLSLWHRALHFKGITPRLTLSHRETRSND 470 Query: 480 VYYRYRQNEWLLGFKYRF 497 V+ Y +N +F F Sbjct: 471 VFNEYEKNRAFVEFNKTF 488 >pir||C81790 conserved hypothetical protein NMA2174 [imported]- Neisseria meningitidis (group A strain Z2491) emb|CAB85386.1|(AL162758) conserved hypothetical protein [Neisseria meningitidis Z2491] Length = 490 Score = 84.0 bits (206), Expect = 4e−15 Identities = 111/498 (22%), Positives = 185/498 (36%), Gaps = 35/498 (7%) Query: 7 LLFLPLCTVCLAAPSNDAADERRRELLDEGSRQTQQYRESGW--LDTEQARGEVEENDGYI 64 +L LPL S A+E R D SR + E+ +D E+ G+V E + Sbjct: 23 MLLLPLLA------SAAYAEETPREPDLRSRPEFRLHEAEVKPIDREKVPGQVREKGKVL 74 Query: 65 SIGGEIYQVGDTAEELESAIYHALNARQWHKVRQFAARYAKLPRHKPALIHLADALQKRD 124 I GE E L A+Y A+ + +R Y + + L A + + Sbjct: 75 QIDGETLL--KNPELLSRAMYSAVVSNNIAGIRVILPIYLQQAQQDKMLALYAQGILAQA 132 Query: 125 EGDFRAAGNSFQTALEAEPDNPRLLLEAGRFYAEDNQNKESAAAFEKVLKTDIPAETRPI 184 +G + A + ++ + A+PD P + + E+ QN+ +A F+++ ++P + Sbjct: 133 DGRVKEAISHYRELIVAQPDAPAVRMRLAAALFENRQNEAAADQFDRLKAENLPPQLMEQ 192 Query: 185 VENYLSELGKRRWHGQISLGYGYNSNVNQGNGINQCVWEIAGMCLMERTLPAPTDSTFS 244 VE Y L +R W N+NQ Q + T P D T Sbjct: 193 VELYRRKALRERDAWKVNGGFSVTREHNINQAPKRQQ---------YGKWTFPKQVDGTAV 243 Query: 245 SYSATAEKTVPIKGNHGVQVGVLYGNRYTEKSDYGYRNGSLYAGYAYADARS 304 +Y AEK LK G + G Y K + + G +AD R Sbjct: 244 NYRLGAEKKWSLKNGWYTTAGGDVSGRVYPGNKK-------FNDMTAGVSGGIGFADRRK 296 Query: 305 SFSLLPYFEYDFRNRHTHYRAWGADADWSRTLSPHRINSHAGAKKTGYGGQSKTYFADF 364 L + E + GA ++R +P W+ S A +G ++ +D Sbjct: 297 DAGLAVFHERRTYGNDAYSYTNGARLYFNRWQTPKWQTLSSA---EWGRLKNTRRARSDN 353 Query: 365 KQYELGAGAEFSITLKSGLLVNFDAARKAYP-EKSSSSKEYTARLGAYRLFSGGTYLNAV 423 ++ F + + D R+ P ++ + Y R A+ GG+ L+++ Sbjct: 354 THLQISNSLVFYRNARQYWMGGLDFYRERNPADRGDNFNRYGLRF-AWGQEWGGSGLSSL 412 Query: 424 LLY--RRSLYDAASFVSDNK--RRRDKQYIMMAAAGFPQWNIKGVYPELRFRRTIAHSNA 479 L + Y+ F S K RRRDK+ + + KG+ P L SN Sbjct: 413 LRLGAAKRHYEKPGFFSGFKGERRRDKELNTSLSLWHRALHFKGITPRLTLSHRETRSND 472 Query: 480 VYYRYRQNEWLLGFKYRF 497 V+ Y +N + F F Sbjct: 473 VFNEYEKNRAFVEFNKTF 490 The protein was expressed in E.coli as an insoluble 52.03 kDa His-fusion product and then purified.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 39 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 77> which encodes amino acid sequence <SEQ ID 78; NGS39>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.38 Possible cleavage site: 18 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 7.16 threshold: 0.0 PERIPHERAL Likelihood = 7.16 modified ALOM score: −1.93 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.325(Affirmative) < succ> The protein has homology with the following sequences in the databases: >ref|NP_052685.1|serine protease EspP [Eseherichia coli] pir||T00317 probable serine proteinase espP, extracellular-Escherichia coli plasmid pO157 pir||T42120 probable serine proteinase espP, extracellular-Escherichia coli plasmid pO157 emb|CAA66144.1|(X97542) putative exoprotein-precursor [Escherichia coli] dbj|BAA31836.1|(AB011549) serine protease EspP [Escherichia coli] gb|AAC70088.1|(AF074613) putative exoprotein-precursor [Escherichia coli O157:H7] Length = 1300 Score = 58.9 bits (141), Expect = 2e−07 Identities = 153/687 (22%), Positives = 248/687 (35%), Gaps = 106/687 (15%) Query: 194 DLTVENKTLSDA---EFGVYALNTSMVNLSSKDNNEVKSTQVGLYSQDGGSINVDR--- 247 D +NT +DA Y N ++ +LS D E + + G + +V R Sbjct: 595 DYVAGMQNTEADAVKQNGNAYKTNNAVSDLSQPDW-ETGTFRFGTLHLENSDFSVGRNAN 653 Query: 248 --------KDNIIEGDAVALVGKGGSQNIRAS----RTLISSKSLGIHAEQAAKIAITG 295 K NI GD A + +NI R N++ S G E IT Sbjct: 654 VIGDIQASKSNITIGDTTAYIDLHAGKITGDGFGFRQNIVRGNSQG---ETLFTGGITA 710 Query: 296 ASNTIHASNAAIRSLDKSEVKIDGQITIDSNVANLARQDGSIH---LNYKDDTRITGATV 352 +TI + A ++ + TI+ N A++ Q G ++ + +TG Sbjct: 711 EDSTIVIKDKAKALFSNYVYLLNTKATIE-NGADVTTQSGMFSTSDISISGNLSMTGNPD 769 Query: 353 SDKGLVAIKPLNNTNIVADTIHYKGDVLAVNKGKVELDF----TPNILLAGRLDNFSGLT 408 D LN+ + + + ++A NK V D + +I+ + S L+ Sbjct: 770 KDNKFEPSIYLNDASYLLTPDSAR--LVMASWGPIHSTKSASIMPGHDESPLSQLS 827 Query: 409 DSKHKNLFENYVANLDSKSAGEINFNLAKDAL----WTMTGQSWLDKLEGQGTIDFNNDA 464 D K L + D G +N A + W +TG S L L+ ++ + D+ Sbjct: 828 DRTSKGLALGLLGGFDVSYRGSVNAPSASATMNNTWWQLTGDSALKTLKSTNSMVYFTDS 887 Query: 465 KTSGR--ALHIGELAGANK-FLMHLNKDGIHSDMLYVKKGTSTPQEVVVKNLSEVLDSMN 521 + + L + ELA +N + M N SD L VKK S ++ L + L Sbjct: 888 ANNKKFHTLTVDELATSNSAYAMRTNLS--ESDKLEVKKHLSGENNIL---LVDFLQKPT 942 Query: 522 YGERLRFATVTNSKNEFVNGKKYIDDTHLMEDALTVEYSAHNGXXXXXXXXXXSFNGSEM 581 ++L V+ K+ N K T D V Sbjct: 943 PEKQLNIELVSAPKTNENFKASKQTIGFSDVTPV------------------------ 978 Query: 582 TAEKAGDPYVNKTYTDNRQNVYLVKQATGNPSRNVKNINDMFDSTAHYAFT--LDTYAKR 639 + DD + T++ N K+AT N + S + AF ++ KR Sbjct: 979 ITTRETDDKI--TWSLTGYNTVANKEATRNAAA--------LFSVPYKAFLNEVNNLNKR 1028 Query: 640 EGERAFSTLDKKEGDWIRLTHTRVIQSNAFRFHNNDFEIGYDRFSLNEQEKKRKWGISLD 699 G+ ++ + G W R+ S F + ++G D+ K G+ L Sbjct: 1029 MGD--LRDINGEAGAWARIMSGTGSASGGFSDNYTHVQVGVDK-------KHELDGLDLF 1079 Query: 700 YGHGRTSLWNTFGKD----KIRKYELALYNTTQYIDKEGDETGYIDNVLKIGKLRNRVIA 755 G T ++ D K + LY + + D YID + K N A Sbjct: 1080 TGFTVTHTDSSASAFSGKTKSVGAGLYASAMF-----DSGAYIDLIGKYVHHDNEYTA 1134 Query: 756 RNHMGQLWGKGKYSNTLFSISTEYGRRKFLDDDKLWRITPQVQLQYSYLRGTGYRI-DNG 814 G G YS + E G R + +D W I PQ +L Y + G + D G Sbjct: 1135 -TFAGL--GTRDYSTHSWYAGAEAGYRYHVTEDA-W-IEPQAELVYGSVSGKQFAWKDQG 1189 Query: 815 INVNLSHA--NSLIGRLGLDVVRKFDG 839 +++++ N LIGR G+DV + F G Sbjct: 1190 MHLSMKDKDYNPLIGRTGVDVGKSFSG 1216 Score = 36.6 bits (83), Expect = 1.2 Identities = 97/412 (23%), Positives = 164/412 (39%), Gaps = 83/412 (20%) Query: 63 DNIVTMKSGDADADYVNNSKVLTETPYYKSKRGSNGIFAYGDKSLVKLIGEIVK--SE 120 D V G + ++ SK Y + +G + A+ S V + +N + +E Sbjct: 163 DKFVVETRGATEGADISLSKQQALERYGVNYKGEKKLIAFRAGSGVVSVKKNGRITPFNE 222 Query: 121 ISEKSKALNGGFRHIGIYS-W---QNAKVE----LSSKSDN--------------IVQGG 158 +S K + LNG F HI +S W N + + ++++ D+ +V G Sbjct: 223 VSYKPEMLNGSFVHIDDWSGWLILTNNQFDEFNNIASQGDSGSALFVYDNQKKKWVVAGT 282 Query: 159 IWGLYS----NNSSISLKGKNNVISNPKYNVFAYKKAKVDLTVENKNTLSDAEFGVYALN 214 +WG+Y+ N + K I N K N ++Y VD++ T+ + + + Sbjct: 283 VWGIYNYANGKNHAAYSKWNQTTIDNLK-NKYSY---NVDMSGAQVATIENGK--LTGTG 336 Query: 215 TSMVNLSSKDNNEVKSTQVGLYSQ----DGGSINVDRKDNIIEGDAVALVGKG-----GS 265 + ++ +KD + L S GG + D+K + GD G G GS Sbjct: 337 SDTTDIKNKDLIFTGGGDILLKSSFDNGAGGLVFNDKKTYRVNGDDFTFKGAGVDTRNGS 396 Query: 266 Q---NIR-ASRTNL--ISSKSLGIHAEQAAK------IAITGASNTIHASNAAIRSLDKS 313 NIR ++ NL I +L + Q + I GA T +N I S D Sbjct: 397 TVEWNIRYDNKDNLHKIGDGTLDVKTQNTNLKTGEGLVILGAEKTF--NNIYITSGD-G 453 Query: 314 EVKIDGQITIDSNVAN---LARQDGSIHLN-YKDDTRITGATSDKGLV--------AIK 361 V+++ + + N A+ G++ LN Y AT D G V +I Sbjct: 454 TVRLNAENALSGGEYNGIFFAKNGGTLDLNGYNQSFNKIAAT--DSGAVITNTSTKKSIL 511 Query: 362 PLNNTNIVADTIHYKG-----DVLAVNKGKVELDFTPNILLAGRLDNFSGLT 408 LNNT AD I++ DVL ++ K E ++L G +D + ++ Sbjct: 512 SLNNT---ADYIYHGNINGNLDVLQHHETKKE---NRRLILDGGVDTTNDIS 557 The protein was expressed in E.coli as an insoluble 95.92 kDa His-fusion product and then purified.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 40 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 79> which encodes amino acid sequence <SEQ ID 80; NGS40>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.18 Possible cleavage site: 17 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 7.05 threshold: 0.0 PERIPHERAL Likelihood = 7.05 modified ALOM score: −1.91 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.108(Affirmative) < succ> The protein has no homology with sequences in the databases.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 41 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 81> which encodes amino acid sequence <SEQ ID 82; NGS41>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) signal Score (−7.5): −2.47 Possible cleavage site: 17 >>> May be a lipoprotein Amino Acid Composition of Predicted Mature Form: calculated from 16 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 7.37 threshold: 0.0 PERIPHERAL Likelihood = 7.37 modified ALOM score: −1.97 Rule: inner or outer membrane protein Rule: inner or outer membrane protein *** Reasoning Step: 2 Lipoprotein?\",\n \"Inner membrane?\",\n \"----- Final Results ----- bacterial outer membrane --- Certainty= 0.790(Affirmative) < succ> The protein has no homology sequences in the databases.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 42 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 83> which encodes amino acid sequence <SEQ ID 84; NGS42>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.2 Possible cleavage site: 14 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 6.58 threshold: 0.0 PERIPHERAL Likelihood = 6.58 modified ALOM score: −1.82 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.514(Affirmative) < succ> The protein has no homology with sequences in the databases.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 43 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 85> which encodes amino acid sequence <SEQ ID 86; NGS43>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.34 Possible cleavage site: 39 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 2 value: −4.78 threshold: 0.0 INTEGRAL Likelihood = −4.78 Transmembrane 1881-1897 (1876-1898) INTEGRAL Likelihood = −1.01 Transmembrane 1966-1982 (1966-1982) PERIPHERAL Likelihood = 1.91 modified ALOM score: 1.46 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.291(Affirmative) < succ> The protein has no homology with sequences in the databases.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 44 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 87> which encodes amino acid sequence <SEQ ID 88; NGS44>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.49 Possible cleavage site: 58 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 1 value: −1.33 threshold: 0.0 INTEGRAL Likelihood = −1.33 Transmembrane 141-157 (140-157) PERIPHERAL Likelihood = 2.54 modified ALOM score: 0.77 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.153(Affirmative) < succ> The protein has no homology with sequences in the databases.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 45 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 89> which encodes amino acid sequence <SEQ ID 90; NGS45>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.07 Possible cleavage site: 46 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 2.07 threshold: 0.0 PERIPHERAL Likelihood = 2.07 modified ALOM score: −0.91 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.333(Affirmative) < succ> The protein has homology with the following sequences in the databases: >ref|NP_049512.1|putative portal protein [Bacteriophage 933W] ref|NP_050550.1|hypothetical protein [Bacteriophage VT2-Sa] gb|AAD25457.1|AF125520_52 (AF125520) putative portal protein [Bacteriophage 933W] dbj|BAA84334.1|(AP000363) hypothetical protein [Bacteriophage VT2-Sa] dbj|BAA94158.1|(AP000422) portal protein [Escherichia coli] O157:H7] Length = 714 Score = 314 bits (805), Expect = 2e−84 Identities = 213/658 (32%), Positives = 327/658 (49%), Gaps = 22/658 (3%) Query: 7 ETGVLPDKNGEPLTIG---EYRLFVGEMMNQPAWRAVADKEMDYADGRQLDNELLQKQR 62 ET + KN T + + ++ +QP WR A+K Y DG QL E+LQ + Sbjct: 4 ETNTMATKNDNGATPRFSQRQLQALCSDIDSQPKWRDAANKACAYYDGDQLPPEVLQVLK 63 Query: 63 ELGLPPAVENLITPTLLSVQGYEATIRTDWRVTADGETGGRD-VADALNFKLNRAERQSR 121 + G P + NLI PT+ V G EA RTD V +D + +A+A+N + A R Sbjct: 64 DRGQPMTIHNLIAPTVDGVLGMAKTRTDLVVMSDEPDDETEKLAEAINAEFADACRLGN 123 Query: 122 ADKACSDAFRGQIACGIGWVTRNPNPFEFPYECGVIHRNAIHWDMKSYKYDLSDARWL 181 +KA SDA+ QI G+ WVEV RN +PF ++ + RN + WD S + DLSD RWL Sbjct: 124 MNKARSDAYAEQIKAGLSWVEVRRNSDPFGPEFKVSTVSRNEVFWLSREADLSDCRWL 183 Query: 182 IRRWLLPERLAQFFPEYAGHFKAMGRGGSDWR-ISGELDGGGNTGLADAWGISGRNTV 240 +RRRW+ + FP G + + DWR + G +L AW Sbjct: 184 MRRRWMDTDEAKATFP---GMAQVIDYAIDDWRGFVDTTVTEOQPSPLMSAWEEYQSWDR 240 Query: 241 SEEFWFNETTRELAVAEVWYRRWVTADCLRDKKTGRTVEFDGANPNHREMAANGAV-LFA 299 + W R + + V+YR + + + GR V FD N A+G V + Sbjct: 241 QQNEWLQRERRRVLLQVVYYRTFERLPVI-ELSNQRVVAFDKNNLMQAVAVASGRVQVKV 299 Query: 300 ASVPRMRRAFVVGDLVVRDEPTPYPHQKFPYPFFGFREDNTGIPYVRNMKYAQDNLN 359 V R+R A+ VG + D P P FP VPF+G++R+D TG PYG + AQD +N Sbjct: 300 GRVSRIREAWFVGPHFIVDRPCSAPQGMFPLVPFWGYRKDKTGEPYGLISEAIPAQDEVN 359 Query: 360 STNSKLRWGLSAIRTVRTKGIVDMSDEQFRRNIARVDADIVLNKIEAAQPGAR--FDVSR 417 KL W L A R + + +SD I R D I LN + Q F V + Sbjct: 360 FRRIKLTWLLQAKRVIMDEDATQLSDNDLMEQIERPDGIIKLNPVRKNQKSVADVFRVEQ 419 Query: 418 DFELSAQHWQMLQDSRATIRQISGITPSVGNRGNATSGRQESIQVEQSNQSLGLVMDNF 477 DF++++Q +Q++Q+S I+ G+ +F+G ATSG S VEQ +L + DN+ Sbjct: 420 DFQVASQQFQVMQESEKLIQDTMGVYSAFLGQDSGATSGVAISNLVEQGAPLAEINDNY 479 Query: 478 RQSRSLVGELLLAMIIEDLGS-DEQTVVIEGDAVTQGRTVVINRPETDPVTGKAYLSNDL 536 + + VG LLLA +++DL VVI D + +T+V+N E D L+ND+ Sbjct: 480 QFACQQVGRLLLAYLLDDLKKRRNHAVVINRDDRQRRQTIVLN-AEGD----NGELTNDI 534 Query: 537 QNIRLKVALEDVPSTNSYRSQQLGAMSEAVKSLPPEYQAAVLPFMVSLMDIPFKDKVIEK 596 + +AL V T ++++Q MSE ++ LPP+ QA VL V+L+D+P K + +E+ Sbjct: 535 SRLNTHIALAPVQQTPAFKAQLAQHMSEVIQGLPPQVQAVVLDLWVNLLDVPQKQEFVER 594 Query: 597 IK-EVRVQETPEQI--EARIAQAVQDALAKSGNDIKRRELALKEQRTASEIKEIEARA 651 I+ + ++P+++ E + A Q AL + +++ RE+A + + ++ A A Sbjct: 595 IRAALGTPKSPDEMTPEEQEVAAQQQALQQQQAELQMREMAGRVAKLEADAARAHAAA 652 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 46 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 91> which encodes amino acid sequence <SEQ ID 92; NGS46>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.25 Possible cleavage site: 37 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 4.77 threshold: 0.0 PERIPHERAL Likelihood = 4.77 modified ALOM score: −1.45 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.281(Affirmative) < succ> The protein has homology with the following sequences in the databases: >sp|P44184|YE10_HAEIN HYPOTHETICAL PROTEIN HI1410 pir||E64028 hypothetical protein HI1410-Haemophilus influenzae (strain Rd KW20) gb|AAC23058.1|(U32820) H. influenzae predicted coding region HI1410 [Haemophilus influenzae Rd] Length = 394 Score = 150 bits (379), Expect = 3e−35 Identities = 75/168 (44%), Positives = 114/168 (67%), Gaps = 2/168 (1%) Query: 57 REIQKSMRDSVHRLLKDIWAQLGLGHYEITDFEIRGANGTLFVFSGLQSHTVDSIKSFE 116 REIQKS+ DSV ++L D++ L L F+++ +I G NG+ F F+GL+++ + SIKS Sbjct: 3 REIQKSISDSVIQMLADQIEMLSLQAFVQKTQIIGQNGSRFTFAGLKTN-ITSSIKSMT 61 Query: 117 GIDIVWVEEGHGVSKKSWDVLTPTIRKEGSEIWITLWPDMETDETYRRFIAMPSEDTWLC 176 GID+VWVEEG VSK+SWD+L PTIR++GS+I ++NP D+TY+RF+ P E Sbjct: 62 GIDVVWVEEGENVSESWDILIPTIREDGSQIIVSFNPKNILDDTYQRFVIHPPERCKSV 121 Query: 177 EINWRDNPWFPEALNRERLKAQRSMNKEDYGNIWEGRPRNVSEGAVYR 224 +NW+DNP+FP+L E ++ R + E Y +++EG P S+ A+ + Sbjct: 122 LVNWQDNPYFPKEL-MEDMEQMRERDYELYRHVYEGEPVADSDLAIIK 168 >ref|NP_050979.1|P18 [Bacteriophage APSE-1] gb|AAF03961.1|AF157835_18 (AF157835) P18 [Bacteriophage APSE-1] Length = 469 Score = 117 bits (294), Expect = 2e−25 Identities = 72/233 (30%), Positives = 110/233 (46%), Gaps = 13/233 (5%) Query: 17 LFKPCRYKVMYXXXXXXXXXXXXXXXXXXXXQRPLRILCAREIQKSMRDSVHRLLKDKVA 76 +FKP R KV + R LC RE S+ DS H +L+ +V Sbjct: 1 MFKPKRIKVYFGGRGGMKTVSFAKIALITASMHKRRFLCLREFMNSIEDSGHAVLQAEVE 60 Query: 77 QLGLGHFYEITDEEIRGANGTLFVFSGLQSHTVDSIKSFEGIDIVWVEEGHGVSKKSWDV 136 LGL + + I + I G N ++F + L + + SIKS D+ WVEE VS+KS D Sbjct: 61 TLGLQNRFRILNTYIEGINDSIFKYGQL-ARNIASIKSKHDFDVAWVEEAETVSEKSLDS 119 Query: 137 LTPTIRKEGSEIWITLNPDMETDETYRRFIA-----------MPSEDTWLCEINWRDNPW 185 L PTIRK GSE+W + NP E Y+RF+ +D ++ ++++ DNPW Sbjct: 120 LIPTIRKPGSELWFSFNPAEEDGAVYKRFVKPYKELIDTQGYYEDDDLYVGKVSYLDNPW 179 Query: 186 FPEALNRERLKAQRSMNKEDYGNIWEGRPMVSEGAVYRHEIQDAFHSGRVTL 238 P L + K +R N + + +++ G E A+ + E +A + L Sbjct: 180 LPAELKNDAQKMKRE-NYKKWRHVYGGECDANYEDALIQPEWVEAAIDAHIKL 231 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 47 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 93> which encodes amino acid sequence <SEQ ID 94; NGS47>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.87 Possible cleavage site: 31 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 4.88 threshold: 0.0 PERIPHERAL Likelihood = 4.88 modified ALOM score: −1.48 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.313(Affirmative) < succ> The protein has homology with the following sequences in the databases: >ref|NP_037739.1|Gp54 [Bacteriophage HK97] gb|AAF31132.1|(AF069529) Gp54 [Bacteriophage HK97] Length = 273 Score = 47.4 bits (111), Expect = 3e−04 Identities = 33/123 (26%), Positives = 52/123 (41%), Gaps = 20/123 (16%) Query: 242 NGGLSGKPKNANVPRRRKTHGVPLQEIADLYNEVLGGRLPSVQVLNDTRKRAIANRWCEM 301 NGG G+ K P RRK + + + YN +G RLP +N+ RKR + + Sbjct: 160 NGGGDGQVK----PERRKAERIDYESFLNAYNTEVGDRLPHAVAVNEKRKRRL-KKIIPQ 214 Query: 302 LGTAAPNGKVRFGDKETGLAWFAGFFEKVA--MNPFHGENQTGPAVGFDWIFKAGNFVK 359 L T +G F + R PF+ G+N TG+ FD++ + ++ Sbjct: 215 LKTPNVDG-------------FRAYVRAFHQAKPFFGDNDTGWTADFDYLLREDSLTG 261 Query: 360 ILE 362 + E Sbjct: 262 VRE 264 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 48 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 95> which encodes amino acid sequence <SEQ ID 96; NGS48>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −6.85 Possible cleavage site: 15 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 7.16 threshold: 0.0 PERIPHERAL Likelihood = 7.16 modified ALOM score: −1.93 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.379(Affirmative) < succ> The protein has homology with the following sequences in the databases: >dbj|BAA36059.1|(D90754) Outer membrane protein P.69 precursor [Escherichia coli] Length = 762 Score = 64.7 bits (156), Expect = 1e−09 Identities = 79/292 (27%), Positives = 121/292 (41%), Gaps = 55/292 (18%) Query: 3 NGARWTVTNDSMLKELDLSEDAQVEFSDNNK----FVKVSVSKLKGDGGVFKMYGDIV-- 56 N + W VT++S L L LS V+F+ + F ++V L G+ F M D+V Sbjct: 289 NNSVWNVTSNSNLDTLALSHST-VDFASHGSTAGTFATLNVENLSGNS-TFIMRADVVGE 346 Query: 57 ----KGESDKLITRKGSEGTHIIEYMDDAKAKTTGREYLKLVENKGNQEDNKASNKASYK 112 + D L S G H++ + TTG E L +V+ D AS AS + Sbjct: 347 GNGVNNKGDLLNISGSSAGNHVLAIRNQGSEATTGNEVLTVVKTT----DGAASFSASSQ 402 Query: 113 LNVRCTEQGGWCFALGESG------ASKKVNISTDGKRDF-------YLYPD-------- 151 + E GG+ + + ++G AS V T + PD Sbjct: 403 V-----ELGGYLYDVRKNGTNWELYASGTVPEPTPNPEPTPAPAQPPIVNPDPTPEPAPT 457 Query: 152 ---TLTPGASSSVLFGEALYQLNAVSDETLVQRMGEIHADGMPQEDNNVWIKRVGGKFSG 208 T T A + L Y LN V +TL+QRMG++ +D N+W++ GG Sbjct: 458 PKPTTTADAGGNYL--NVGYLLNYVENRTLMQRMGDLRNQ---SKDGNIWLRSYGGSLDS 512 Query: 209 SRSDYRVGGYGNRYWGFAGGFNRTGFGDKWIHYKGLMLRHLQSSYASEDYVG 260 S ++ G+ Y G G ++ D Y GL ++ S++AS DY G Sbjct: 513 FASG-KLSGFDMGYSGIQFGGDKR-LSDVMPLYVGL---YIGSTHASPDYSG 559 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 49 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 97> which encodes amino acid sequence <SEQ ID 98; NGS49>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −8.37 Possible cleavage site: 15 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 4.93 threshold: 0.0 PERIPHERAL Likelihood = 4.93 modified ALOM score: −1.49 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.355(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: gi|11282647|pir|H81959 patch repair protein (EC 3.1.-.-) NMA0429 [imported]-Neisseria meningitidis (group A strain Z2491) gi|7379179|emb|CAB83728.1|(AL162753) patch repair protein [Neisseria meningitidis Z2491] Length = 140 Score = 256 bits (628), Expect = 8e−68 Identities = 131/140 (93%), Positives = 132/140 (93%) Query: 1 MTDIFTPSKRSFVMSKIHSKETKPEVLVRKFLFSQGFRYRKNDKRYAGKPDIVLPKYKTV 60 MTDIFT SKRSFVM KIHSKETKPEVLVRKFLF QGFRYRKNDKRY GKPDIVL KYKTV Sbjct: 1 MTDIFTTSKRSFVMLKIHSKETKPEVLVRFLFFQGFRYRKNDKRYVGKPDIVLSKYKTV 60 Query: 61 VFIHGCFWHGHSCNKGHIPKSNMDFWLEKITKNRERDIKNETELEKIGFKVIVVWECELK 120 VFIHGCFW+GHSCNKGHIPKSN DFWLEKITKN ERDIKNETELEKIGFKVIVVWECELK Sbjct: 61 VFIHGCFWYGHSCNKGHIPKSNTDFWLEKITKNCERDIKNETELEKIGFKVIVVWECELK 120 Query: 121 NKAICRERLNRLVEEIKDAV 140 NKAICRERLNRLV EIKDAV Sbjct: 121 NKAICRERLNRLVREIKDAV 140 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 50 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 99> which encodes amino acid sequence <SEQ ID 100; NGS50>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −6.6 Possible cleavage site: 50 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 7.80 threshold: 0.0 PERIPHERAL Likelihood = 7.80 modified ALOM score: −2.06 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.398(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|11352963|pir||G81959 conserved hypothetical protein NMA0428 [imported]-Neisseria meningitidis (group A strain Z2491) gi|7379178|emb|CAB83727.1|(AL162753) conserved hypothetical protein [Neisseria meningitidis Z2491] Length = 548 Score = 371 bits (954), Expect = e−102 Identities = 189/197 (95%), Positives = 194/197 (97%) Query: 1 VKGESGVDIENWKNKLPEKEREPVEVILNRLEDSELTNKEQAEVISALHSIIPEYPYYHW 60 VKGESGVDIE+WKNKLPEKEREPVEVILNRLEDSELTNKEQAEVISALHSIIPEYPYYHW Sbjct: 350 VKGESGVDIEDWKNKLPEKEREPVEVILNRLEDSELTNKEQAEVISALHSIIPEYPYYHW 409 Query: 61 RHLHQDLHTACNDFYNEKKDYLSAAIEAVKVFEDKVQKQTGLHSIDGRELIEKAFGSKKS 120 RHLHQDLHTACNDFYNEKKDYLSAAIEAVKVFEDKVQKQTGLHSIDGRELIE+AFGSK S Sbjct: 410 RHLHQDLHTACNDFYNEKKDYLSAAIEAVKVFEDKVQKQTGLHSIDGRELIEQAFGSKKS 469 Query: 121 MLLLTNNKTQAEQNLEDGLEQLACGTWTGFRNPVQHELRANLSPSIFNDKDALDLISLVS 180 +LLLTNNKT+AEQNLEDGLEQLACGTWTGFRNPVQHELRANLSPSIFNDKDALDLISLVS Sbjct: 470 ILLLTNNKTKAEQNLEDGLEQLACGTWTGFRNPVQHELRANLSPSIFNDKDALDLISLVS 529 Query: 181 YLLRKVEQTKKRAKPTS 197 YLLRKVEQTKKR+K S Sbjct: 530 YLLRKVEQTKKRSKVVS 546 >gi|10955124|ref|NP_059780.1|ymh [Agrobacterium tumefaciens] gi|5738274|gb|AAB91582.2|(AF242881) ymh [Agrobacterium tumefaciens] Length = 266 Score = 58.7 bits (141), Expect = 5e−08 Identities = 40/127 (31%), Positives = 69/127 (53%), Gaps = 5/127 (3%) Query: 61 RHLHQDLHTACNDFYNEKKDYLSAAIEAVKVFEDKVQKQTGLHSIDGRELIEKAFGSKKS 120 R +H D+ C + +Y A +EAVK DK++++TGL + DG L+++AF Sbjct: 137 RGVHPDVLRFCREEL-LVDNYFHAVLEAVKSVADKIRQTGL-TDDGAVLVDRAFSGDAP 194 Query: 121 MLLLTNNTQAEQNEDGLEQLACGTWTGFRNPVQHELRANLSPSIFNDKDALDLISLVS 180 ML + ++++E+ + G L GT++ FRN H R + S +DA DL S+ S Sbjct: 195 MLAINELQSESEKGEQRGFSNLVKGTFSMFRNTTAHAPRIHWQMS---KEDAEDLFSMFS 251 Query: 181 YLLRKVE 187 + R+++ Sbjct: 252 LMHRRID 258 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, NGS50 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 51 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 101> which encodes amino acid sequence <SEQ ID 102; NGS51>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 0.14 Possible cleavage site: 42 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 5.67 threshold: 0.0 PERIPHERAL Likelihood = 5.67 modified ALOM score: −1.63 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.145(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir||G81959 conserved hypothetical protein NMA0428 [imported]-Neisseria meningitidis (group A strain Z2491) emb|CAB83727.1|(AL162753) conserved hypothetical protein [Neisseria meningitidis Z2491] Length = 548 Score = 532 bits (1371), Expect = e−150 Identities = 272/285 (95%), Positives = 280/285 (97%) Query: 1 MSEEKLKMSFEPTVIEHLGVKMYSHTVPAIAELIANAYDACATEVEVRLFDKPEHKIVIK 60 MSEEKLKMSFEPTVIEHLGVKMYSHTVPAIAELIANAYDACATEVEVRLFDKPEHKIVIK Sbjct: 1 MSEEKLKMSFEPTVIEHLGVKMYSHTVPAIAELIANAYDACATEVEVRLFDKPEHKIVIK 60 Query: 61 DNGIGMSFDEINDFYLRIGRNRREEKQASPCGRIPTGKKGLGKLALFRLGNKIEISTIQG 120 DNGIGMSFDEINDFYLRIGRNRREEKQASPCGRIPTGKKGLGKLALF LGNKIEISTIQG Sbjct: 61 DNGIGMSFDEINDFYLRIGRNRREEKQASPCGRIPTGKKGLGKLALFRLGNKIEISTIQG 120 Query: 121 NERVTFTLDYAEIKKSERIYQPEFQKESVKPNTENGTTITLTELTKKQGYPLDNYVGHLS 180 NERVTFTLDYAEI++S+ IYQPEF+KESV+ N E+GTTITLTELTKKQGYPLDNYV HLS Sbjct: 121 NERVTFTIDYAEIRRSKGIYQPEFRKESVESNIESGTTITLTELTKKQGYPLDNYVEHLS 180 Query: 181 RLFDFPAQDFKIKVSLNGSEPRIIDGNLKYNLVTPQFEWEYQDLATNISSLSSKFEQYEY 240 RLFDFPAQDFKIKVSLNGSEP+IIDGNLKY+LVTPQFEWEYQDLATNISSLSSKFEQYEY Sbjct: 181 RLFDFPAQDFKIKVSLNGSEPKIIDGNLKYDLVTPQFEWEYQDLATNISSLSSKFEQYEY 240 Query: 241 SGLIQGKFITTEKPLKNNMKGITLFANGRMVNMPEFFTDSESSHF 285 SGLIQGKFITTEKPLKNNMKGITLFANGRMVNMPEFFTDSESSHF Sbjct: 241 SGLIQGKFITTEKPLKNNMKGITLFANGRMVNMPEFFTDSESSHF 285 >emb|CAC22276.1|(AJ302030) putative heat shock protein [Listeria monocytogenes] Length = 181 Score = 70.2 bits (171), Expect = 2e−11 Identities = 57/173 (32%), Positives = 90/173 (51%) , Gaps = 10/173 (5%) Query: 1 MSEEKLKMSFEPTVIEHLGVKMYSHTVPAIAELIANAYDACATEVEVRLFDKPEHKIVIK 60 MSE++ + +P ++E LG +Y++ + ELIANAYDA A V V E+K++++ Sbjct: 1 MSEKEYNLDIDPRILELLGPHLYTNIYYILGELIANAYDADAKNVYVIDRIDEENKLIVE 60 Query: 61 DNGIGMSFD--EINDFYLRIGRNRREEKQASPC---GRIPTGKKGLGKLALFRLGNKIEI 115 D+G GMS++ ++ +F L + + R S R G+KG+GKLA + + I Sbjct: 61 DDGSGMSYENKDVKNF-LSVAKESRTNAINSYTKLNNRRKMGRKGVGKLASLSVSENVNI 119 Query: 116 STIQGNERVFTLDYAEI-KKSERIYQPEFQKESVKPNTENGTTITLTELTKK 167 TI+ E+ F L I KK E I + +K +GT I +T T K Sbjct: 120 KTIKDGEKSGFVLSRKVINKKLEAINEDTISFIKIK---NHGTAIEMTNPTYK 169 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, NGS51 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 52 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 103> which encodes amino acid sequence <SEQ ID 104; NGS52>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.5 Possible cleavage site: 49 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 7.64 threshold: 0.0 PERIPHERAL Likelihood = 7.64 modified ALOM score: −2.03 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.213(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: 2.1.1.73) NMA0427 [imported]-Neisseria meningitidis (group A strain Z2491) gi|7379177|emb|CAB83726.1|(AL162753) modification methylase (cytosine- specific DNA methylase) [Neisseria meningitidis Z2491] Length = 351 Score = 310 bits (794), Expect = 8e−84 Identities = 152/154 (98%), Positives = 153/154 (98%) Query: 1 LGMENGFPKIMAGHQDETDFMHSCAGLSDINLKRLALIPKNGGNRLAFAHIPELQLECFI 60 LGMENGFPKI+AGHQDETDFMHSCAGLSDINLKRLALIPKNGGNRLAFAHIPELQLECFI Sbjct: 198 LGMENGFPKIIAGHQDETDFMHSCAGLSDINLKRLALIPKNGGNRLAFAHIPELQLECFI 257 Query: 61 GKDNSFKDTFGRLWWDKPAPTITTKFFSISNGRAFAHPEEDRALSLREGATLQSFPRNYVF 120 GKDNSFKDTFGRLWWDKPAPTITTKFFSISNGRAFAHPEEDRALSLREGATLQSFPRNYVF Sbjct: 258 GKDNSFKDTFGRLWWDKPAPTITTKFFSISNGRAFAHPEEDRALSLREGATLQSFPRNYVF 317 Query: 121 KAGSRDKIARLIGNAVPPMYTEKIGRAIVDNIEC 154 KAGSRDKIARLIGNAVPPMY EKIGRAIVDNIEC Sbjct: 318 KAGSRDKIARLIGNAVPPMYAEKIGRAIVDNIEC 351 >gi|1274411|sp|P25265|MTD2_HERAU MODIFICATION METHYLASE HGIDII (CYTOSINE-SPECIFIC METHYLTRANSFERASE HGIDII) (M.HGIDII) gi|538661|pir||JT0594 site-specific DNA-methyltransferase (cytosine-specific) (EC 2.1.1.73)-Herpetosiphon aurantiacus gi|487731|emb|CAA38941.1|(X55141) methyltransferase [Herpetosiphon aurantiacus] Length = 354 Score = 95.6 bits (237), Expect = 3e−19 Identities = 62/142 (43%), Positives = 82/142 (57%), Gaps = 9/142 (6%) Query: 12 AGHQDETDFM HSCAGLSDINLKRLALIPKNGGNRLAFAHIP-ELQLECFIGKD-NSFKDT 69 +G E D MH+ + L DINL+R+ G +A P EL EC + S+ Sbjct: 200 SGGHWEGDSMHAASRLEDINLRRIQHSVPGG----TWADWPEELIAECHKKESGESYGSV 255 Query: 70 FGRLWWDKPAPTITTKFFSISNGEFAHPEEDRALSLREGATLQSFPRNYVFKAGSRDK-- 127 +GR+ WDK APTITT+ NGRF HPE+DRA+SLRE A LQ+FPR+Y F + K Sbjct: 256 YGRMEWDKVAPTITTQCNGYGNGRFGHPEQDRAISLRALLQTFPRSYQFAPEGQLKFK 315 Query: 128 -IARLIGNAVPPMYTEKIGRAI 148 ++R IGNAVP I ++I Sbjct: 316 TVSRQIGNAVPVALGRVIAKSI 337 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, NGS52 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 53 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 105> which encodes amino acid sequence <SEQ ID 106; NGS53>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −7.56 Possible cleavage site: 31 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 5.36 threshold: 0.0 PERIPHERAL Likelihood = 5.36 modified ALOM score: −1.57 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.189(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|11256915|pir||F81959 site-specific DNA-methyltransferase (cytosine- specific) (EC2.1.1.73) NMA0427 [imported]-Neisseria meningitidis (group A strain Z2491) gi|7379177|emb|CAB83726.1|(AL162753) modification methylase (cytosine- specific DNA methylase) [Neisseria meningitidis Z2491] Length = 351 Score = 247 bits (606), Expect = 5e−65 Identities = 124/149 (83%), Positives = 127/149 (85%) Query: 1 LQPETLEKELGLKKNDDDLILIGCSPCQYWSVIQTDKRKSEKSKSLLLEFQRFVEYFNPG 60 LQPETLEKELGLKKNDDDLILIGCSPCQYWSVIQTDKRKSEKSKSLLLEFQRFVEYFNPG Sbjct: 59 LQPETLEKELGLKKNDDDLILIGCSPCQYWSVIQTDKRKSEKSKSLLLEFQRFVEYFNPG 118 Query: 61 YVVVENVPGILSRMKESGLDNFIKLLEEKGFTVHFGIHNTADYGIPQSRKRFTLIANRIT 120 YVVVENVPGILSRMKESGLDNFIKLLEEKGFTVHFGIHNTADYGIPQSRKRFTLIANRIT Sbjct: 119 YVVVENVPGILSRMKESGLDNFIKLLEEKGFTVHFGIHNTADYGIPQSRKRFTLIANRIT 178 Query: 121 KKSWNQSSIRANGLRYAMFWEWKTAFPKL 149 K+ L + FPK+ Sbjct: 179 KEKLEPVKYSGKRLTVRDVLGMENGFPKI 207 >gi|127441|sp|P25265|MTD2_HERAU MODIFICATION METHYLASE HGIDII (CYTOSINE- SPECIFIC METHYLTRANSFERASE HGIDII) (M.HGIDII) gi|538661|pir||JT0594 site-specific DNA-methyltransferase (cytosine- specific) (EC 2.1.1.73)-Herpetosiphon aurantiacus gi|48773|emb|CAA38941.1|(X55141) methyltransferase [Herpetosiphon aurantiacus] Length = 354 Score = 71.9 bits (169), Expect = 4e−12 Identities = 39/105 (37%), Positives = 57/105 (54%), Gaps = 1/105 (0%) Query: 12 LKKNDDDLILIGCSPCQYWSVIQTDKRKSEKSKSLLLEFQRFVEYFNPGYVVVENVPGIL 71 L N+IL+GC+PCQ +S T K ++ LL EF R + P + +ENVP + Sbjct: 64 LYPNNQHKILVGCAPCQDFSQY-TKKSRTGTKWQLLTEFSRLIREIEPDIISMENVPEVR 122 Query: 72 SRMKESGLDNFIKLLEEKGFTHFGIHNTADYGIPQSSRKRFTLIA 116 + + +NFI+ LE+ G+ V + + DYGIPQ R R L A Sbjct: 123 TFNRGEVFNFIQSLEQLGYWSHSVVHCPDYGIPQQRDRLVLFA 167 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, NGS53 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 54 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 107> which encodes amino acid sequence <SEQ ID 108; NGS54>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −6.82 Possible cleavage site: 50 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 6.89 threshold: 0.0 PERIPHERAL Likelihood = 6.89 modified ALOM score: −1.88 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.253(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: gi|1074456|pir||D64155 hypothetical protein HI0597-Haemophilus influenzae (strain Rd KW20) gi|1573586|gb|AAC22254.1|(U32741) conserved hypothetical protein [Haemophilus influenzae Rd] Length = 272 Score = 188 bits (459), Expect = 2e−47 Identities = 95/100 (95%), Positives = 97/100 (97%) Query: 1 MNLPFRAMVSDLGGTLLTPEHLVGDLTIDTLRVLEQKGVDIILATGRNHTDMSSILGKIG 60 MNLPFRAMVSDL GTLLTPEHLVGDLTIDTLR LEQKGVDIILATGRNHTD+SSILGKIG Sbjct: 1 MNLPFRAMVSDLDGTLLTPEHLVGDLTIDTLRALEQKGVDIILATGRNHTDVSSILGKIG 60 Query: 61 AERAVMITSNGARVRDLQGNLLYSNSLPEELVLELYKTSY 100 AERAVMITSNGARVRDLQGNLLYSNSLPEELVLELYKT + Sbjct: 61 AERAVMITSNGARVRDLQGNLLYSNSLPEELVLELYKTPF 100 A homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS54 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 55 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 109> which encodes amino acid sequence <SEQ ID 110; NGS55>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.46 Possible cleavage site: 37 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 3.02 threshold: 0.0 PERIPHERAL Likelihood = 3.02 modified ALOM score: −1.10 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.311(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: aeruginosa (strain PAO1) gi|9948791|gb|AAG06104.1|AEOO4699_9 (AE004699) probable FMN oxidoreductase [Pseudomonas aeruginosa] Length = 411 Score = 279 bits (686), Expect = 2e−74 Identities = 157/375 (41%), Positives 228/375 (59%), Gaps 10/375 (2%) Query: 1 MEEQLAQNDQ-PSEKLVRLYGAWAEGGAGVLVTGNVMVAESGKGSINDVLISDDRALKLM 59 MEE +4 Q PSE4+L+RLY AWA+GGAG+L++GNVMV V++ DD LE Sbjct: 24 MEENMADAAQAPSERLMRLYQAWADGGAGLLISGNVMVDSRAMTGPGGVVLEDDAQLEKF 83 Query: 60 KKWAKARTQNDTLLIMQINKAGKQSPAVKTPLAPSAVPL--GMNGFINPPRELSADE 117 ++WA+ +QINH G+Q A + + APSAVPL GM+ P+ + Sbjct: 84 RRWARIGRSAGAGFWLGINHPGRQMQANLGQQAWAPSAVPLELGGMSRHFATPKAMDEAM 143 Query: 118 INGLIQQFVQTAKIAEQAGFSGVQIYAVHGYLISQFLSPHHNRRQDQWGGSLENRMRFLL 177 I +IQ+F ++ +AE+AGFSGV+I+A HGYL+SQFLSP NRR D WGGSLENR R LL Sbjct: 144 IAEVIQRFARSAGLAERAGFSGVEIHHGYLLSQFLSPLSNRRSDAWGGSLENRARLLL 203 Query: 178 ETYTAIRAAGKDFLVGVXLNSADFQKGGFDESESVQVVQKLSEMGIDFIEVSGGNYESP 237 E A+RA F V VKLNSADFQ+GGF ++ +VV+ L +G+D +E+SGG+YE+P Sbjct: 204 EIVRAVRAEVAPGFAVAVKLNSADFQRGGFSADDAREVVRMLDGLGVDLVELSGGSYEAP 263 Query: 238 QMLA-AKDS-TRKREAFFIDYAEKARAASQAPLIITGGFRSQTAMEDALSSGHLDLVGIA 295 M A+D T REA+F+++A RAA++ P+++TGG R + E L+SG +D+VGI Sbjct: 264 AMQGEARDGRTLAREAYFVEFARDIRAAARMPVMVTGGIRRRVAEQVLASG-VDMVGIG 322 Query: 296 RPFALVPDLANKMQNRTYQTVQADRIQTGVDKKAGAMLEMNWYMTQMDLIGQGKQSN 355 A+ P+L + Q I + +K ++ M Q+ + +G+ +N Sbject: 323 TALAIEPNLPRDWRAGKDSAPQLRPI----TWENKPLASLANMAVKFQLRKLSRGRATN 378 Query: 356 PKIVGVESIAENFAG 370 P++ ++ AG Sbjct: 379 PRVSPLCALLAQQAG 393 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 56 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 111> which encodes amino acid sequence <SEQ ID 112; NGS56>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.69 Possible cleavage site: 54 >>> Seems to have an uncleavable N-term signal seq Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 4.29 threshold: 0.0 PERIPHERAL Likelihood = 4.29 modified ALOM score: −1.36 *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.042(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|7444004|pir||D70029 transcription regulator ArsR family homolog yvbA- Bacillus subtilis gi|2635892|emb|CAB15384.1|(Z99121) similar to transcriptional regulator (ArsR family) [Bacillus subtilis] Length = 90 Score = 51.3 bits (118), Expect = 3e−06 Identities = 24/65 (36%), Positives = 42/65 (63%), Gaps = 1/65 (1%) Query: 15 IFTVLSDENRHQILHVLWKHGRMNVNELTEHLHLSRPAVSHHLKIMLQAGAVAVEQVGKE 74 +F +SD R +IL +L K G M ++ EH ++S+P++SHHL I+QA ++ + G+ Sbjct: 4 VFKAISDPTRRKILDLL-KGGDMTAGDIAEHFNISHPSISHHLNILKQAEVISDHRKGQF 62 Query: 75 RFYSI 79 +YS+ Sbjct: 63 IYYSL 67 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 57 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 113> which encodes amino acid sequence <SEQ ID 114; NGS57>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −2.57 Possible cleavage site: 55 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 5.67 threshold: 0.0 PERIPHERAL Likelihood = 5.67 modified ALOM score: −1.63 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.160(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|10444407|gb|AAG17897.1|AF297971_1 (AF297971) restriction endonuclease R.NgoMIII [Neisseria gonorrhoeae] Length = 213 Score = 319 bits (818), Expect = 1e−86 Identities = 156/156 (100%), Positives = 156/156 (100%) Query: 1 LYKQYADWNRLSYNAPIYVGKAVPKGWRQARNSDNALNQSTELFHRLKEHSRSIAAVSDL 60 LYKQYADWNRLSYNAPIYVGKAVPKGWRQARNSDNALNQSTELFHRLKEHSRSIAAVSDL Sbjct: 58 LYKQYADWNRLSYNAPIYVGKAVPKGWRQARNSDNALNQSTELFHRLKEHSRSIAAVSDL 117 Query: 61 DPSDFMCRFVIFEGAGSDMIGTIEAALIKLHKPLWNSCDGFGNHDPGKGRYEQAKSDWD 120 DPSDFMCRFVIFEGAGSDMIGTIEAALIKLHKPLWNSCDGFGNHDPGKGRYEQAKSDWD Sbjct: 118 DPSDFMCRFVIFEGAGSDMIGTIEAALIKLHKPLWNSCDGFGNHDPGKGRYEQAKSDWD 177 Query: 121 VLHSGRVWADRLNGIPNSYESILENINTHLEIIKRK 156 VLHSGRVWADRLNGIPNSYESILENINTHLEIIKRK Sbjct: 178 VLHSGRVWADRLNGIPNSYESILENINTHLEIIKRK 213 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 58 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 115> which encodes amino acid sequence <SEQ ID 116; NGS58>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −1.92 Possible cleavage site: 16 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 5.41 threshold: 0.0 PERIPHERAL Likelihood = 5.41 modified ALOM score: −1.58 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.107(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|10444408|gb|AAG17898.1|AF29797_2 (AF297971) DNA cytosine methyltransferase M.NgoMIII [Neisseria gonorrhoeae] Length = 377 Score = 759 bits (1960), Expect = 0.0 Identities = 377/377 (100%), Positives = 377/377 (100%) Query: 1 MKSLEIFSGAGGLAKGLELAGFQHASFIELNKDACNSLRSNFNPKLVYQGDVADFDLSSQ 60 MKSLEIFSGAGGLAKGLELAGFQHASFIELNKDACNSLRSNFNPKLVYQGDVADFDLSSQ Sbjct: 1 MKSLEIFSGAGGLAKGLELAGFQHASFIELNKDACNSLRSNFNPKLVYQGDVADFDLSSQ 60 Query: 61 EGIEVIAGGPPCQPFSLGGKHLAHEDRRDMFPHAVRYVEYYRPKAFIFENVKGLLRKSFA 120 EGIEVIAGGPPCQPFSLGGKHLAHEDRRDMFPHAVRYVEYYRPKAFIFENVKGLLRKSFA Sbjct: 61 EGIEVIAGGPPCQPFSLGGKHLAHEDRRDMFPHAVRYVEYYRPKAFIFENVKGLLRKSFA 120 Query: 121 DYFEYILLRLTYPNLGILQNEDWKGHLTRLKEIEFNLYKGIKYKVSYQLLNAADYGVPQK 180 DYFEYILLRLTYPNLGILQNEDWKGHLTRLKEIEFNLYKGIKYKVSYQLLNAADYGVPQK Sbjct: 121 DYFEYILLRLTYPNLGILQNEDWKGHLTRLKEIEFNLYKGIKYKVSYQLLNAADYGVPQK 180 Query: 181 RERVVIVGIRADLDIDWKFPKRTHSEDRLNWEKYVTGEYWEKHNEPKRFNKDIAEKLQKK 240 RERVVIVGIRADLDIDWKFPKRTHSEDRLNWEKYVTGEYWEKHNEPKRFNKDIAEKLQKK Sbjct: 181 RERVVIVGIRADLDIDWKFPKRTHSEDRLNWEKYVTGEYWEKHNEPKRFNKDIAEKLQKK 240 Query: 241 YGIFEPEKKPWQTVRDTLSDIPHPLGNHKITGHEYRDGARIYPGHTGSGIDEPSKTIKAG 300 YGIFEPEKKPWQTVRDTLSDIPHPLGNHKITGHEYRDGARIYPGHTGSGIDEPSKTIKAG Sbjct: 241 YGIFEPEKKPWQTVRDTLSDIPHPLGNHKITGHEYRDGARIYPGHTGSGIDEPSKTIKAG 300 Query: 301 GHGVPGGENMIRYDDGTVRYFTSYEAKLLQTFPEEFVISGAWGEAMRQIGNAVPVKLSEI 360 GHGVPGGENMIRYDDGTVRYFTSYEAKLLQTFPEEFVISGAWGEAMRQIGNAVPVKLSEI Sbjct: 301 GHGVPGGENMIRYDDGTVRYFTSYEAKLLQTFPEEFVISGAWGEAMRQIGNAVPVKLSEI 360 Query: 361 LGKHLMGVLSEKSSLHN 377 LGKHLMGVLSEKSSLHN Sbjct: 361 LGKHLMGVLSEKSSLHN 377 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 59 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 117> which encodes amino acid sequence <SEQ ID 118; NGS59>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.82 Possible cleavage site: 60 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 2.86 threshold: 0.0 PERIPHERAL Likelihood = 2.86 modified ALOM score: −1.07 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.197(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|11353338|pir||F81882 hypothetical protein NMA1155 [imported] Neisseria meningitidis (group A strain Z2491) gi|7379848|emb|CABB4417.1|(AL162755) hypothetical protein NMA1155 [Neisseria meningitidis Z2491] Length = 120 Score = 131 bits (329), Expect = 2e−30 Identities = 64/68 (94%), Positives = 67/68 (98%) Query: 1 LSDISASRAAYMDVQKQYPFETVAVCVLPNHIHAIWTLPPDDADYSLLRRLIKTKFSAYS 60 +S+ISASRAAYMDVQKQYPFETVAVCVLPNHIHAIWTLPPDDADYSLLRRLIKTKFSAYS Sbjct: 1 MSNISASRAAYMDVQKQYPFETVAVCVLPNHIHAIWTLPPDDADYSLLRRLIKTKFSAYS 60 Query: 61 PHTKNLGA 68 P+TKNL A Sbjct: 61 PYTKNLSA 68 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 60 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 119> which encodes amino acid sequence <SEQ ID 120; NGS60>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.14 Possible cleavage site: 16 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 2.76 threshold: 0.0 PERIPHERAL Likelihood = 2.76 modified ALOM score: −1.05 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.330(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|11281269|pir||D81804 hypothetical protein NMA1789 [imported]-Neisseria meningitidis (group A strain Z2491) gi|7380430|emb|CAB85016.1|(AL162757) hypothetical protein [Neisseria meningitidis Z2491] Length = 243 Score = 154 bits (389), Expect 5e−37 Identities = 82/85 (96%), Positives = 82/85 (96%) Query: 12 MNTKTELQKLLEEDISTLKETLIRVDALPPRYVRSIATPIVRRWLIDKQLNILAKEIGLT 71 MNTKTELQKLLEEDISTL ETLI DALPPRYVRSIATPIVRRWLIDKQLNILAKEIGLT Sbjct: 1 MNTKTELQKLLEEDISTLTETLICADALPPRYVRSIATPIVRRWLIDKQLNILAKEIGLT 60 Query: 72 IELPILDTSLVFEKLSTLENKVNFY 96 IELPILDTSLVFEKLSTLENKVNFY Sbjct: 61 IELPILDTSLVFEKLSTLENKVNFY 85 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, NGS60 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 61 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 121> which encodes amino acid sequence <SEQ ID 122; NGS61>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −6.88 Possible cleavage site: 32 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 5.52 threshold: 0.0 PERIPHERAL Likelihood = 5.52 modified ALOM score: −1.60 Rule: cytoplasmic protein *** Reasoning step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.300(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|11281269|pir||D81804 hypothetical protein NMA1789 [imported]-Neisseria meningitidis (group A strain Z2491) gi|7380430|emb|CAB5016.1|(AL162757) hypothetical protein [Neisseria meningitidis Z2491] Length = 243 Score = 193 bits (491), Expect = 5e−49 Identities = 96/101 (95%), Positives = 97/101 (95%) Query: 1 MAGGVYLGGKIISPIYHSSQEFSGEPIIYAETNIILCPAEKFLTLKRVFHNGNIFNMNQI 60 MAGGVYLGG+ IS IYHSSQEFSGEPIIYAETNIILCPAEKFLTLKRVFHNGNIFNMNQI Sbjct: 86 MAGGVYLGGEFISSIYHSSQEFSGEPIIYAEPNIILCPAEKFLTLKRVFHNGNIFNMNQI 145 Query: 61 ITFLSNKQGGVRFDKNYDKYKTWQVAIEKAANFLKLGNPYN 101 ITFLSNKQGGV FDKNYDKYKTWQVAIEKAANFLKLGNPYN Sbjct: 146 ITFLSNKQGGVHFDKNYDKYKTWQVAIEKAANFLKLGNPYN 186) As a homolog (amino acids 1-96) was found in serogroup A N.meningitidis but not in serogroup B, NGS61 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 62 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 123> which encodes amino acid sequence <SEQ ID 124; NGS62>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −2.43 Possible cleavage site: 44 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 4.72 threshold: 0.0 PERIPHERAL Likelihood = 4.72 modified ALOM score: −1.44 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.324(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> A homolog was found in serogroup A N.meninigitidis but not in serogroup B, so NGS62 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 63 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 125> which encodes amino acid sequence <SEQ ID 126; NGS63>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 0.74 Possible cleavage site: 24 >>> Seems to have a cleavable N-term signal seq.\",\n \"Amino Acid Composition of predicted Mature Form: calculated from 25 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 10.72 threshold: 0.0 PERIPHERAL Likelihood = 10.72 modified ALOM score: −2.64 Score for OM-PP discrimination: −22.14 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: −22.14 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Periplasmic space?\",\n \"Score: 2.21378 Periplasmic space?\",\n \"Score: 2.21378 ----- Final Results ----- bacterial periplasmic space --- Certainty= 0.931(Affirmative) < succ> bacterial outer membrane --- Certainty= 0.237(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|11284146|pir||B81142 hypothetical protein NMB0912 [imported]-Neisseria meningitidis (group B strain MD5B) gi|7226150|gb|AAF41320.1|(AE002443) hypothetical protein [Neisseria meningitidis MC58] Length = 208 Score = 51.7 bits (119), expect = 3e−06 Identities = 30/72 (41%), Positives = 40/72 (54%) Query: 5 LLKNWKPLLILSAIAFFAVSWQLDRAAQYRRGYGAAVSEVSERLKAAAVEHAEHARKSSA 64 LLK WKP+ +L I +W DRA +YR G AA +E+S RLK +E A+ AR + Sbjct: 43 LLKYWKPVGVLLLIVLIFTAWHFDRAEKYRMGREAAAAEISNRLKDGYIEQAKQARSAEQ 102 Query: 65 AYQAQKAAEEEK 76 A A R+ K Sbjct: 103 KAAAAPAERQTK 114 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 64 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 127> which encodes amino acid sequence <SEQ ID 128; NGS64>.\",\n \"Analysis of this protein sequence reveals the following: McG: Examining signal sequence (McGeoch) Length of UR: 0 Peak Value of UR: 2.99 Net Charge of CR: 4 Discriminant Score: 5.35 GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −2.53 Possible cleavage site: 33 >>> Seems to have an uncleavable N-term signal seq Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 3.23 threshold: 0.0 PERIPHERAL Likelihood = 3.23 modified ALOM score: −1.15 *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.054(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has no homology with sequences in the databases.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 65 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 129> which encodes amino acid sequence <SEQ ID 130; NGS65>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.61 Possible cleavage site: 61 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 3.34 threshold: 0.0 PERIPHERAL Likelihood = 3.34 modified ALOM score: −1.17 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.236(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|1175791|sp|P44189|YE18_HAEIN HYPOTHETICAL PROTEIN HI1418 gi|1074769|pir||A64029 hypothetica1 protein HI1418-Haemophilus influenzae (strain Rd KW20) gi|1574254|gb|AAC23068.1|(U32821) H. influenzae predicted coding region HI1418 [Haemophilus influenzae Rd] Length = 201 Score = 104 bits (251), Expect = 1e−21 Identities = 58/119 (48%), Positives = 72/119 (59%), Gaps = 4/119 (3%) Query: 51 LKMQNTISFSFKSQNRTQI-LGAEPFCLGDVAEILQIQNAR---QLPLKDQGIQKSS 106 +K Q S F+FK VR + E WFC DV IL N+R Q K G+ K Sbjct: 14 MKNQIQFSTFFKDLPVRTILDPKGEFFCGTDVCHILGYTNSRKALQDHCKQGGVTKRY 73 Query: 107 VATKKGNQELLFINEPNLYRVIFRSRKAEAVFQDWIFEEVIPQIRKTGGYQITPKTTA 165 TK +QE+ FINEPNLYR+I +SRK EA F+ W+FEEV+PQIRKTG YQ+ P+ A Sbjct: 74 TPTKSADQEMTFINEPNLYRLIIKSRKPEAEPFEAWVFEEVLPQIRKTGKYQLQPQQLA 132 >gi|11281012|pir||A81144 hypothetical protein NMB0900 [imported]-Neisseria meningitidis (group B strain MD58) gi|7226137|gb|AAF41308.1|(AE002442) hypothetical protein [Neisseria meningitidis MC58] Length = 305 Score = 104 bits (249), Expect = 2e−21 Identities = 73/137 (53%), Positives = 93/137 (67%), Gaps = 2/137 (1%) Query: 190 YSMIHQRFNVEAVEGIPADKLPEAVAYHALTLHTG-LAGEVPDREPLPAPQPALPISGN 248 +S + +F E +PA++ PE ++ + + + G L GEV DREPLPAPQPALPISGN Sbjct: 164 WSAVKSKFGCSYKE-VPAEQFPELSMGRVAVENGVLYGEVLDREPLPAPQPALPISGN 222 Query: 249 ALADIAAMVYYGTRHIELGKDVSAPLKQLGCKQAVTMWTVWHETRSILKRSVAALEVLRG 308 AL D+A V YG I++G+DVS PLKQLGCRQAVTMWTVW ETRS LK + ALE L Sbjct: 223 ALYDLAVAVRYGAWAIQMGRDVSLPLKQLGCKQAVTMWTVWAETRSRLKAAANALEALNA 282 Query: 309 YADKDASGRIAACLEGI 325 +AD + + +I L I Sbjct: 283 HADAEHAAKIRPMLPEI 299 >gi|7460273|pir||T13267 hypothetical protein-Lactococcus lactis phage BK5-T gi|928839|gb|AAA98590.1|(L44593) ORF266; putative [Lactococcus phage BK5-T] Length = 266 Score = 75.9 bits (179), Expect = 6e−13 Identities = 42/111 (37%), Positives = 63/111 (55%), Gaps = 3/111 (2%) Query: 55 NTISVFSFKSQNVRTQILGAEPWFCLGDVAEILQIQNAR---QLPLKDQGIQKSSVATKK 111 N + F+F + VRT ++ EPWF DVA + +N R + +KD+ ++S + T Sbjct: 2 NELQNFNFNNLPVRTVLINDEPWFVGKDVAIAIGYKNFRDALKSHVKDKYKRESRITTPS 61 Query: 112 GNQELLFINEPNLYRVIFRSRKAEAVKFQDWIFEBVIPQIRKTGGYQITPK 162 G Q + I+EP LY++ S+ A FQDW++EEV+P IRK G Y K Sbjct: 62 GVQSVTVISEPGLYQLAGESKLPSAEPFQDWEELPTIRKHGAYMTDAK 112 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 66 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 131> which encodes amino acid sequence <SEQ ID 132; NGS66>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.12 Possible cleavage site: 53 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 8.96 threshold: 0.0 PERIPHERAL Likelihood = 8.96 modified ALOM score: −2.29 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.402(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has no homology with sequences in the databases.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 67 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 133> which encodes amino acid sequence <SEQ ID 134; NGS67>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 5.71 Possible cleavage site: 22 >>> Seems to have a cleavable N-term signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 23 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 2.81 threshold: 0.0 PERIPHERAL Likelihood = 2.81 modified ALOM score: −1.06 Score for OM-PP discrimination: −32.34 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: −32.34 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Periplasmic space?\",\n \"Score: 3.23391 Periplasmic space?\",\n \"Score: 3.23391 ----- Final Results ----- bacterial periplasmic space --- Certainty= 0.928(Affirmative) < succ> bacterial outer membrane --- Certainty= 0.199(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|7475078|pir||H69834 hypothetical protein yhjQ-Bacillus subtilis gi|2226189|emb|CAA74479.1|(Y14081) hypothetical protein [Bacillus subtilis] gi|2633396|emb|CAB12900.1|(Z99109) yhjQ [Bacillus subtilis] Length = 108 Score = 32.9 bits (74), Expect = 2.1 Identities = 27/98 (27%), Positives = 44/98 (44%), Gaps = 4/98 (4%) Query: 54 CLDAGQVCLTHCLSLLTQGDTSMSDCAVAVRQMLALCGAVHDLAAQNSPLTRDAAKVCLE 113 C+ A C T CL Q +S C R+ +C +SP ++ +C+ Sbjct: 15 CMKACNHCFTKCLEESVQ--HHLSGCIRLDRECADICALAVKAMQTDSPFMKEICALCAD 72 Query: 114 ACKQCAKACKEHSAHHAECKACYESCLDCIKECEKLAA 151 C+ C C +H H C+AC ++C C ++C +AA Sbjct: 73 ICEACGTECGKHD--HDHCQACAKACFTCAEQCRSMAA 108 >gi|7479923|pir||T36241 hypothetical protein SCE39.31c-Streptomyces coelicolor gi|4582392|emb|CAB40339.1|(AL049573) hypothetical protein [Streptomyces coelicolor A3(2)] Length = 136 Score = 30.9 bits (69), Expect = 7.7 Identities = 27/102 (26%), Positives = 43/102 (41%), Gaps = 6/102 (5%) Query: 54 CLDAGQVCLTHCLSLLTQGDTSMSDCAVAVRQMLALCGAVHDLAAQ----NSPLTRDAAK 109 C A C CLS T D ++C +C A + ++ ++ +TR + Sbjct: 34 CAQACTACADACLSEPTVAD--LTKCIRTDMDCADVCTATAAVLSRHTGYDANVTRAVLQ 91 Query: 110 VCLEACKQCAKACKEHSAHHAECKACYESCLDCIKECEKLAA 151 C C C C H+ H C+ C E+C C + C++L A Sbjct: 92 ACATVCAACGDECARHAGMHEHCRVCAEACRSCEQACQELLA 133 The protein was expressed in E.coli as a soluble 14.19 kDa His-fusion product and then purified.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 68 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 135> which encodes amino acid sequence <SEQ ID 136; NGS68>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.05 Possible cleavage site: 38 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 5.25 threshold: 0.0 PERIPHERAL Likelihood = 5.25 modified ALOM score: −1.55 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.220(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|11353493|pir||A81795 hypothetical protein NMA2214 [imported]-Neisseria meningitidis (group A strain Z2491) gi|7380833|emb|CAB85425.1|(AL162758) hypothetical protein [Neisseria meningitidis Z2491] Length = 208 Score = 263 bits (673), Expect = 3e−69 Identities = 140/145 (96%), Positives = 143/145 (98%) Query: 1 LDWRGNKPLGAAELADLKPLYKDFMYWERGLHMYKASAVVPTGYVRVGNTAPLCGEDTQR 60 LDWRGNKPLGAAELADLKPLYKDFMYWERGLHMYKASAVVPTGYVRVGNTAPL GEDTQR Sbjct: 44 LDWQGNKPLGAAELADLKPLYKDFMYWERGLHMYKASAVVPTGYVRVGNTAPLVGEDTQR 103 Query 63 YASFWGDGYDVYRQLRWRQIPEKQRKAFKKAAKSKNTVMFAGREYGISKQNLSDVWDDFE 120 YASFWGDGYDVYRQLRW+QIPEKQRKAFKKAAKSK TVMFAGREYGISKQNLSDVWDDFE Sbjct: 104 YASFWGDGYDVYRQLRWQQIPEKQRKAFKKAAKSKKTVMFAGREYGISKQNLSDVWDDFE 163 Query: 121 DAMELAKAFPCLSSLFLTKWHKNLYE 145 DAMELAKAFPCLSSLFLTKWHKNLY+ Sbjct: 164 DAMELAKAFPCLSSLFLTKWHKNLYD 188 >gi|11280955|pir|B81219 hypothetical protein NMB0273 [imported]-Neisseria meningitidis (group B strain MD58) gi|7225497|gb|AAF40727.1|(AE002383) hypothetical protein [Neisseria meningitidis MC58] Length = 141 Score = 216 bits (550), Expect = 5e−55 Identities = 117/121 (96%), Positives = 119/121 (97%) Query: 25 MYWERGLHMYKASAVVPTGYVRVGNTAPLCGEDTQRYASFWGDGYDVYRQLRWRQIPEKQ 84 MYWERGLHMYKASAVVPTGYVRVGNTAP CGEDTQRYASFWGDGYDVYRQLRW+QIPEKQ Sbjct: 1 MYWERGLHMYKASAVVPTGYVRVGNTAPLVGEDTQRYASFWGDGYDVYRQLRWQQIPEKQ 60 Query: 85 RKAFKKAAKSKNTVMFAGREYGISKQNLSDVWDDFEDAMELKAFPCLSSLFLTKWHKNLY 144 RKAFKKAAKSK TVMFAGREYGISKQNLSDVWDDFEDAMELKAFPCLSSLFLTKWHKNLY Sbjct: 61 RKAFKKAAKSKKTVMFAGREYGISKQNLSDVWDDFEDAMELKAFPCLSSLFLTKWHKNLY 120 Query: 145 E 145 + Sbjct: 121 D 121 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 69 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 137> which encodes amino acid sequence <SEQ ID 138; NGS69>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.63 Possible cleavage site: 43 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 6.84 threshold: 0.0 PERIPHERAL Likelihood = 6.84 modified ALOM score: −1.87 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.361(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|7464552|pir||C64707 hypothetical protein HP1499-Helicobacter pylori (strain 26695) gi|2314683|gb|AAD08545.1|(AE000648) H. pylori predicted coding region HP1499 [Helicobacter pylori 26695] Length = 272 Score = 38.2 bits (88), Expect = 0.13 Identities = 34/123 (27%), Positives = 58/123 (46%), Gaps = 10/123 (8%) Query: 3 EFKPIFGQDFGLSKKEAIRKVLKWLPSHLKFTLMVAQGIQG------FHPKAVFWKNDKN 56 EF+ I G DF + ++IR +L ++ K + FHPK + N K Sbjct: 54 EFEIIVGLDFKTTDSKSIRFLLDLNKTYKKLRFYCYGDKENNKTDIVFHPKIYMFDNGK- 112 Query: 57 EYYALIGSSNLTHAAFNSNYEAN-ILTKISEQDFIKVKSWADEI--AMKSIPVSEDWLEE 113 E ++IGS+NLT +N+E N I T+ + ++ + + I A +E++L+ Sbjct: 113 EKTSIIGSTNLTKGGLENNFEVNTIFTEKKPLYYTQLNAIYNSIKYADSLFTPNEEYLQN 172 Query: 114 YQE 116 Y E Sbjct: 173 YNE 175 A homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS69 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 70 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 139> which encodes amino acid sequence <SEQ ID 140; NGS70>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −1.18 Possible cleavage site: 22 >>> Seems to have a cleavable N-term signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 23 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 4.19 threshold: 0.0 PERIPHERAL Likelihood = 4.19 modified ALOM score: −1.34 Score for OM-PP discrimination: −5.89 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: −5.89 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Periplasmic space?\",\n \"Score: 0.588927 Periplasmic space?\",\n \"Score: 0.588927 ----- Final Results ----- bacterial periplasmic space --- Certainty= 0.849(Affirmative) < succ> bacterial outer membrane --- Certainty= 0.106(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|11353344|pir||A81886 hypothetical protein NMAl183 [imported]-Neisseria meningitidis (group A strain Z2491) gi|7379875|emb|CAB84445.1|(AL162755) hypothetical protein NMA1183 [Neisseria meningitidis Z2491] Length = 74 Score = 58.7 bits (141), Expect = 2e−08 Identities = 30/43 (69%), Positives = 32/43 (73%) Query: 62 IGGFGGVGGFGGLKPALVYRNFRIIATNRPAATRARPRQTTVA 104 +G|||G+|G|GGLKP|LVY|N||IIATNRPAATRA|PR|TTVA Sbjct: 32 MGNIDGIDGSGGLKPTLVYWNHCIIATNRPAATRAHPRHTTVA 74 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 71 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 141> which encodes amino acid sequence <SEQ ID 142; NGS71>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −1.98 Possible cleavage site: 28 >>> Seems to have a cleavable N-term signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 29 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 5.04 threshold: 0.0 PERIPHERAL Likelihood = 5.04 modified ALOM score: −1.51 Score for OM-PP discrimination: −9.17 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: −9.17 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Periplasmic space?\",\n \"Score: 0.916744 Periplasmic space?\",\n \"Score: 0.916744 ----- Final Results ----- bacterial periplasmic space --- Certainty= 0.923(Affirmative) < succ> bacterial outer membrane --- Certainty= 0.146(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has no homology with sequences in the databases.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 72 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 143> which encodes amino acid sequence <SEQ ID 144; NGS72>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −3.18 Possible cleavage site: 42 >>> Seems to have an uncleavable N-term signal seq Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 4 value: −8.76 threshold: 0.0 INTEGRAL Likelihood = −8.76 Transmembrane 11-27 (8-37) INTEGRAL Likelihood = −6.90 Transmembrane 80-96 (75-102) INTEGRAL Likelihood = −2.39 Transmembrane 98-114 (98-114) INTEGRAL Likelihood = −1.12 Transmembrane 47-63 (47-64) PERIPHERAL Likelihood = 3.55 modified ALOM score: 2.25 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.450(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|11354095|pir||H81995 probable transmembrane transport protein NMA0047 [imported]-Neisseria meningitidis (group A strain Z2491) gi|7378822|emb|CAB83364.1|(AL162752) putative transmembrane transport protein [Neisseria meningitidis Z2491] Length = 405 Score = 257 bits (658), Expect = 5e−68 Identities = 152/162 (93%), Positives = 154/162 (94%) Query: 1 MTHTASKTPKLWAVIAAAAFILLITIGMRMTLGLFVQPVVNTTELNIAQFSLIITVFQLM 60 MTHTASKTPK W I AAAFILLITIGMRMTLGLFVQPVVNTTEL+IAQFSLII VFQLM Sbjct: 1 MTHTASKTPKFWLTITAAAFILLITIGMRMTLGLFVQPVVNTTELNIAQFSLIITVFQLM 60 Query: 61 WGVLQPLSGALADRFGAFRVLSGGALLLVCACLIASNIPTYWGLMIAVGLLLAFGTGSGG 120 WGV QPLSGALADRFGAFRVLSGGA+LLVCACLIA NIPTYWGLMIAVGLLLAFGTGSGG Sbjct: 61 WGVSQPLSGALADRFGAFRVLSGGAVLLVCACLIAPNIPTYWGLMIAVGLLLAFGTGSGG 120 Query: 121 FSIIMGQVAAQVPTHKRGLASGLVNAGGSAGQFLFAPLVQGL 162 FSIIMGQVAAQVP HKRGLASGLVNAGGSAGQFLFAPLVQGL Sbjct: 121 FSIIMGQVAAQVPAHKRGLASGLVNAGGSAGQFLFAPLVQGL 162 >gi|11351469|pir||F83484 probable MFS transporter PA1286 [imported]- Pseudomonas aeruginosa (strain PAO1) gi|9947221|gb|AAG04675.1|AE004558_4 (AE004558) probable MFS transporter [Pseudomonas aeruginosa] Length = 399 Score = 72.5 bits (177), Expect = 3e−12 Identities = 53/149 (35%), Positives = 81/149 (53%) Query: 14 VIAAAAFILLITIGMRMTLGLFVQPVVNTTELNIAQFSLIITVFQLMWGVLQPLSGALAD 73 ++ + A IL +++G+R GLF+ P+ F+ I + L+WG+ QP +GALAD Sbjct: 8 ILLSGALILALSLGVRHGFGLFLAPMSADFGWGREVFAFAIALQNLVWGLAQPFTGALAD 67 Query: 74 RFGAFRVLSGGALLLVCACLIASNIPTYWGIMIAVGLLLAPGTGSGGFSIIMGQVAAQVP 133 R+GA R + G LL ++ + GL ++ GLL+ G FS+I+G V VP Sbjct: 68 RYGAARAVLVGGLLYALGLVLMGLSQSASGLSLSAGLLIGLGLSGTSFSVILGAVGRAVP 127 Query: 134 THKRGLASGLVNAGGSAGQFLPAPLVQGL 162 +R +A G+ +A GS GQF P GL Sbjct: 128 AEQRSMAMGISSAAGSFGQFAMLPGTLGL 156 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, NGS72 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 73 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 145> which encodes amino acid sequence <SEQ ID 146; NGS73>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.26 Possible cleavage site: 52 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 3 value: −3.72 threshold: 0.0 INTEGRAL Likelihood = −3.72 Transmembrane 172-188 (171-190) INTEGRAL Likelihood = −2.97 Transmembrane 119-135 (114-137) INTEGRAL Likelihood = −1.86 Transmembrane 209-225 (205-225) PERIPHERAL Likelihood = 4.88 modified ALOM score: 1.24 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.249(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gi|11354095|pir||H81995 probable transmembrane transport protein NMA0047 [imported]-Neisseria meningitidis (group A strain Z2491) gi|7378822|emb|CAB83364.1|(AL162752) putative transmembrane transport protein [Neisseria meningitidis Z2491] Length = 405 Score = 222 bits (567), Expect = 3e−57 Identities = 116/123 (94%), Positives = 117/123 (94%), Gaps = 4/123 (3%) Query: 103 QGLVVLPEVGWTGTFYVWGAIALLILPVSWWLAGGNNGGNNAAHTQHTQATHGQSLGEAV 162 QGLVVLPEVGWTGTFYVWGAIALL LPVSWWLA GGNNAAHTQH QATHGQSLGEAV Sbjct: 160 QGLVVLPEVGWTGTFYVWGAIALLTLPVSWWLA----GGNNAAHTQHAQATHGQSLGEAV 215 Query: 163 KTAFKTPSYILLHLSFFACGFHIAFLVTHLPTEVALCGLPATVASTSIAIIGLANIAGCV 222 KTAFKTPSYILLHLSFFACGFHIAFLVTHLPTEVALCGLPATVASTSIAIIGLANIAGC+ Sbjct: 216 KTAFKTPSYILLHLSFFACGFHIAFLVTHLPTEVALCGLPATVASTSIAIIGLANIAGCI 275 Query: 223 FSG 225 FSG Sbjct: 276 FSG 278 >gi|11351469|pir||F83484 probable MFS transporter PA1286 [imported]-Pseudomonas aeruginosa (strain PAO1) gi|9947221|gb|AAG04675.1|AE004558_4 (AE004558) probable MFS transporter [Pseudomonas aeruginosa] Length = 399 Score = 54.4 bits (130), Expect = 1e−06 Identities = 37/115 (32%), Positives = 56/115 (48%), Gaps = 10/115 (8%) Query: 111 VGWTGTFYVWGAIALLILPVSWWLAGGNNGGNNAAHTQHTQATHGQSLGEAVKTAFKTPS 170 +GW+ G + LI+P++ + H QSLGEA++ A Sbjct: 160 LGWSSALLALGLLVALIVPLAGLM----------KDRPLPPQGHEQSLGEALREACAHSG 209 Query: 171 YILLHLSFFACGFHIAFLVTHLPTEVALCGLPATVASTSIAIIGLANIAGCVFSG 225 + LL L FF CGF + F+ HLP + LPA V +T +A++GL N+ G +G Sbjct: 210 FWLLALGFFVCGFQVVFIGVHLPAYLVDQHLPAQVGTTVLALVGLFNVFGTYIAG 264 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS73 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 74 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 147> which encodes amino acid sequence <SEQ ID 148; NGS74>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 0.41 Possible cleavage site: 30 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 2 value: −1.49 threshold: 0.0 INTEGRAL Likelihood = −1.49 Transmembrane 15-31 (15-31) INTEGRAL Likelihood = −1.28 Transmembrane 8-99 (83-99) PERIPHERAL Likelihood = 1.59 modified ALOM score: 0.80 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.160(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir||H81995 probable transmembrane transport protein NMA0047 [imported]- Neisseria meningitidis (group A strain Z2491) emb|CAB3364.1|(AL162752) putative transmembrane transport protein [Neisseria meningitidis Z2491] Length = 405 Score = 148 bits (374), Expect = 2e−35 Identities = 97/106 (91%), Positives = 103/106 (96%) Query: 1 MVLIYIFSPKTDLNFYIFAAALGFTWLATVAPTAAVTGKLFGTRYLATLFGLVMLTHQIG 60 M+LIYIFSPKTDLNFYIFAAALGFTWLATV PTA++TGKLFGTRYLATLFGL ML+HQIG Sbjct: 300 MILIYIFSPKTDLNFYIFAAALGFTWLATVTPTASITGKLFGTRYLATLFGLTMLSHQIG 359 Query: 61 GFLGSYIGGIVITQFGDYGWMWYADAVLAGTAALLVLPVREPRTAA 106 GFLGSYIGGIVITQFGDYGWMWYADA+LAGTAALL LP+REPRTAA Sbjct: 360 GFLGSYIGGIVITQFGDYGWMWYADALLAGTAALLNLPIREPRTAA 405 >pir||F83484 probable MFS transporter PA1286 [imported]-Pseudomonas aeruginosa (strain PAO1) gb|AAGO4675.1|AE004558_4 (AE004558) probable MFS transporter [Pseudomonas aeruginosa Length = 399 Score = 59.0 bits (142), Expect = 2e−08 Identities = 40/101 (39%), Positives = 61/101 (59%) Query: 1 MVLIYIFSPKTDLFYIFAAALGFTWLATVAPTAAVGKLFGTRYLATLFGLVMLTHQIG 60 +++++++ P + + Y F A+G WL+TV T LFG R L+ L G+V L HQ+G Sbjct: 286 VIVLFLWLPLSVYSAYAFGVAMGLLWLSTVPLTNGTTLFGVRNLSMLGGIVFLFHQLG 345 Query: 61 GFLGSYIGGIVITQFGDYGWMWYADAVLAGTAALLVLPVRE 101 FLG ++GG+V + G Y +W +L+ AALL PVRE Sbjct: 346 AFLGGWLGGVVYDRTGSYDLVWQLSILLSLLAALLNWPVRE 386 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, NGS74 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 75 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 149> which encodes amino acid sequence <SEQ ID 150; NGS75>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.2 Possible cleavage site: 22 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 4.45 threshold: 0.0 PERIPHERAL Likelihood = 4.45 modified ALOM score: −1.39 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.237(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: pir||S04419 type II site-specific deoxyribonuclease (EC 3.1.21.4) NgoPII- Neisseria gonorrhoeae emb|CAA368B7.1|(X52661) NgoPII restriction and modification [N. gonorrhoeae] prf||1613419A NgoPII endonuclease [Neisseria gonorrhoeae] Length = 278 Score = 240 bits (614), Expect = 4e−63 Identities = 128/129 (99%), Positives = 128/129 (99%) Query: 1 MNIIDAIINLANNPVVGVNSHSQSNNRANQAGDALEEYVKDLFSGSFNLNETQRIARHAK 60 MNIIDAIINLANNPVVGV SHSQSNNRANQAGDALEEYVKDLFSGSFNLNETQRIARHAK Sbjct: 1 MNIIDAIINLANNPVVGVESHSQSNNRANQAGDALEEYVKDLFSGSFNLNETQRIARHAK 60 Query: 61 VFSYLGNNSNPPDAMLRNGDAIEVKKIESKDSALALNSSHPKSKLSVDDSMLTKACKDAE 120 VFSYLGNNSNPPDAMLRNGDAIEVKKIESKDSALALNSSHPKSKLSVDDSMLTKACKDAE Sbjct: 61 VFSYLGNNSNPPDAMLRNGDAIEVKKIESKDSALALNSSHPKSKLSVDDSMLTKACKDAE 120 Query: 121 KWEEKDIIY 129 KWEEKDIIY Sbjct: 121 KWEEKDIIY 129 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 76 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 151> which encodes amino acid sequence <SEQ ID 152; NGS76>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −1.73 Possible cleavage site: 13 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 9.39 threshold: 0.0 PERIPHERAL Likelihood = 9.39 modified ALOM score: −2.38 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.272(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: pir||S04419 type II site-specific deoxyribonuclease (EC 3.1.21.4) NgoPII- Neisseria gonorrhoeae emb|CAA36887.1|(X52661) NgoPII restriction and modification [N. gonorrhoeae] prf||1613419A NgoPII endonuclease [Neisseria gonorrhoeae] Length = 278 Score = 268 bits (687), Expect = 2e−71 Identities = 136/136 (100%), Positives = 136/136 (100%) Query: 1 LAMVYGIDYCADAECYLKIKNQIKEGIGNIGGIQFAETKELGRVNRIDPLNITYLRVRGM 60 LAMVYGIDYCADAECYLKIKNQIKEGIGNIGGIQFAETKELGRVNRIDPLNITYLRVRGM Sbjct: 143 LAMVYGIDYCADAECYLKIKNQIKEGIGNIGGIQFAETKELGRVNRIDPLNITYLRVRGM 202 Query: 61 WGIENPWFVFNYIYQRNMEKSFNFMAIINEDKWNSFNNTDKLLAIQDSKLAISDIKIKNP 120 WGIENPWFVFNYIYQRNMEKSFNFMAIINEDKWNSFNNTDKLLAIQDSKLAISDIKIKNP Sbjct: 203 WGIENPWFVFNYIYQRNMEKSFNFMAIINEDKWNSFNNTDKLLAIQDSKLAISDIKIKNP 262 Query: 121 NNPARLRNAKLITYHL 136 NNPARLRNAKLITYHL Sbjct: 263 NNPARLRNAKLITYHL 278 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 77 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 153> which encodes amino acid sequence <SEQ ID 154; NGS77>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.51 Possible cleavage site: 58 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 3.18 threshold: 0.0 PERIPHERAL Likelihood = 3.18 modified ALOM score: −1.14 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.127(Affirmative) <succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir|||CTNHP2 site-specific DNA-methyltransferase (cytosine-specific) (EC 2.1.1.73) NgoPII-Neisseria gonorrhoeae emb|CAA30038.1|(X06965) NgoPII methylase (AA 1-341) [Neisseria gonorrhoeae] emb|CAA3688B.1|(K52661) NgoPII restriction and modification [Neisseria gonorrhoeae] gb|AAA170419.1|(L14564) cytosine methylase [Neisseria gonorrhoeae] prf||1613419B NgoPII methylase [Neisseria gonorrhoeae] Length = 341 Score = 682 bits (1761), Expect = 0.0 Identities = 341/341 (100%), Positives = 341/341 (100%) Query: 1 MQNSSPTTYNPMKIISLFSGCGGLDLGFEKAGFEIPAANEYDKTIWATFKANHPKTHLIE 60 MQNSSPTTYNPMKIISLFSGCGGLDLGFEKAGFEIPAANEYDKTIWATFKANHPKTHLIE Sbjct: 1 MQNSSPTTYNPMKIISLFSGCGGLDLGFEKAGFEIPAANEYDKTIWATFKANHPKTHLIE 60 Query: 61 GDIRKIKEEDFPEEIDGIIGGPPCQSWSEAGALRGIDDARGQLFFDYIRILKSKQPKFFL 120 GDIRKIKEEDFPEEIDGIIGGPPCQSWSEAGALRGIDDARGQLFFDYIRILKSKQPKFFL Sbjct: 61 GDIRKIKEEDFPEEIDGIIGGPPCQSWSEAGALRGIDDARGQLFFDYIRILKSKQPKFFL 120 Query: 121 AENVSGMLANRHNGAVQNLLKMFDGCGYDVTLTMANAKDYGVAQERKRVFYIGFRKDLEI 180 AENVSGMLANRHNGAVQNLLKMFDGCGYDVTLTMANAKDYGVAQERKRVFYIGFRKDLEI Sbjct: 121 AENVSGMLANRHNGAVQNLLKMFDGCGYDVTLTMANAKDYGVAQERKRVFYIGFRKDLEI 180 Query: 181 KFSFPKGSTVEDKDKITLKDVIWDLQDTAVPSAPQNKTNPDAVNNNEYFTGSFSPIFMSR 240 KFSFPKGSTVEDKDKITLKDVIWDLQDTAVPSAPQNKTNPDAVNNNEYFTGSFSPIFMSR Sbjct: 181 KFSFPKGSTVEDKDKITLKDVIWDLQDTAVPSAPQNKTNPDAVNNNEYFTGSFSPIFMSR 240 Query: 241 NRVKAWDEQGFTVQASGRQCQLHPQAPKMEKHGANDYRFAAGKETLYRRMTVREVARIQG 300 NRVKAWDEQGFTVQASGRQCQLHPQAPKMEKHGANDYRFAAGKETLYRRMTVREVARIQG Sbjct: 241 NRVKAWDEQGFTVQASGRQCQLHPQAPKMEKHGANDYRFAAGKETLYRRMTVREVARIQG 300 Query: 301 FPDNFKFIYQNVNDAYKMIGNAVPVNLAYEIAAAIKKTLER 341 FPDNFKFIYQNVNDAYKMIGNAVPVNLAYEIAAAIKKTLER Sbjct: 301 FPDNFKFIYQNVNDAYKMIGNAVPVNLAYEIAAAIKKTLER 341 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 78 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 155> which encodes amino acid sequence <SEQ ID 156; NGS78>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −8.33 Possible cleavage site: 24 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 2.17 threshold: 0.0 PERIPHERAL Likelihood = 2.17 modified ALOM score: −0.93 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.220(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir||H82000 hypothetical protein NMA0089 [imported]-Neisseria meningitidis (group A strain Z2491) emb|CAB83405.1|(AL162752) hypothetical protein NMA0089 [Neisseria meningitidis Z2491] Length = 226 Score = 422 bits (1085), Expect = e−117 Identities = 217/228 (95%), Positives = 220/228 (96%), Gaps = 2/228 (0%) Query: 1 MERYKNAVRKDKAAELYLLNLSLSRELFHVVSIFEIVLRNKIDICFRQEFKDRNRLYDSI 60 MERYKNAV KDKAAELYLLNLSLSRELFHVVSIFEIVLRNKIDIC +Q FKD N LY+SI Sbjct: 1 MERYKNAVGKDKAAELYLLNLSLSRELFHVVSIFEIVLRNKIDICLQQAFKDGNWLYNSI 60 Query: 61 QPQTNPALKYQGCFLRNGTKESAELIKVALSKIQNNSGGKFDHNQLVAGLGFGFWRYLFA 120 QPQTNPALKYQGCFLRNGTKESAELIKVALSKIQNNSGGKFDHNQLVAGLGFGFWRYLFA Sbjct: 61 QPQTNPALKYQGCFLRNGTKESAELIKVALSKIQNNSGGKFDHNQLVAGLGFGFWRYLFA 120 Query: 121 GGKDAQFDAAGKVLMKVFPKKPKSTPSVQYNQKWIFRELSNINNFRNRLAHHEPICFSFK 180 GGKDAQFDA GKVLMKVFPKKPKSTPSVQ+NQKWIFRELSNINNFRNRLAHHEPIC FK Sbjct: 121 GGKDAQFDATGKVLMKVFPKKPKSTPSVQHNQKWIFRELSNINNFRNRLAHHEPIC--FK 178 Query: 181 GAIKDTGYARNIHQSIFELLNYMDVDTASVFSHFSDQVIAVCDEIDKL 228 GAIKDTGYARNIHQSIFELLNYMDVDTASVFSHFSDQVIAVCDEIDKL Sbjct: 179 GAIKDTGYARNIHQSIFELLNYMDVDTASVFSHFSDQVIAVCDEIDKL 226 >ref|NP_053274.1|Hypothetical gene [Agrobacterium tumefaciens] dbj|BAA87659.1|(AB016260) Hypothetical gene [Agrobacterium tumefaciens] Length = 380 Score = 43.6 bits (102), Expect = 0.002 Identities = 53/215 (24%), Positives = 86/215 (39%), Gaps = 42/215 (19%) Query: 5 KNAVRKDKAAELYLLNLSLSRELFHVVSIFEIVLRNKIDICFRQEFKDRNRLYDSIQPQT 64 K ++ A LYL N +++ + +++ E+ LRN +D F Sbjct: 55 KGGNHEEYAMALYLYNARVAKAFLYPLNVAEVTLRNAVDGILVARFG------------- 101 Query: 65 NPALKYQGCFLRNGTKESAELIKVALSKIQNNSGGKFDHNQLVAGLGFGFWRYLFAGGKD 124 A +Q R+ T L L K +G +Q+VA L F FW LF Sbjct: 102 --ANWHQDATFRDQTLTGNGL--ATLDKAIQRAGAGAARDQIVATLTFDFWSNLFR---- 153 Query: 125 AQFDAAGKVLMKVPKKPKSTPSVQYNQKWIFRELSN----INNFRNRLAHHEPICFSFK 180 ++ + + + + P +Q+ + +E+ N IN FRNR+AHHEP+ Sbjct: 154 PEYGGLWRTTVNI------AFPHLQHGESR--QEIQNINKPINFRNRVAHHEPVL---- 201 Query: 181 GAIKDTGYARNIHQSIFELLNYMDVDTASVFSSFS 215 D +IH I L+ +TA+ H S Sbjct: 202 ----DLNVT-DIHAKIVRLIELRCAETATWMKHHS 231 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS78 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 79 A DNA sequence was identified in N.gonorrhoeae <SEQ D 157> which encodes amino acid sequence <SEQ ID 158; NGS79>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): 2.07 Possible cleavage site: 17 >>> Seems to have a cleavable N-term signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 18 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 9.49 threshold: 0.0 PERIPHERAL Likelihood = 9.49 modified ALOM score: −2.40 Score for OM-PP discrimination: −11.72 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: −11.72 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Periplasmic space?\",\n \"Score: 1.17242 Periplasmic space?\",\n \"Score: 1.17242 ----- Final Results ----- bacterial periplasmic space --- Certainty= 0.932(Affirmative) < succ> bacterial outer membrane --- Certainty= 0.240(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> A homolog (amino acids 23-85) was found in serogroup A N.meningitidis but not in serogroup B, so NGS79 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 80 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 159> which encodes amino acid sequence <SEQ ID 160; NGS80>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −8.49 Possible cleavage site: 57 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 4.98 threshold: 0.0 PERIPHERAL Likelihood = 4.98 modified ALOM score: −1.50 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.428(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir||E81080 conserved hypothetical protein NMB1466 [imported]-Neisseria meningitidis (group B strain MD58) gb|AAF41823.1|(AE002496) conserved hypothetical protein [Neisseria meningitidis MC58] Length = 243 Score = 148 bits (375), Expect = 7e−35 Identities = 82/189 (43%), Positives = 109/189 (57%) Query: 120 VDRMFNMAGNHFARLGISSGSGVHYWNARDFSEQAFHAEVGYRYRNSRLEWGFRPFVKQNR 179 + R N +HF GISG GVHYW+ +DFSEQ+ GY+ R+ +G PFV+QN Sbjct: 1 MSREINAGRHHFLYGGISGGGVHYWDNKDFSEQSLRLSFGYKNRSVTRSFGIVPFVEQNL 60 Query: 180 LGNNRYTANTGIVLDYSRRLNEKWRSTQSFQYGRKQYHDEYIAKRYNSKTISVSGTFSYY 239 LG +RY G D+S+RL+E+WR T + K Y ++ A RY+S T Y Sbjct: 61 LGGSRYNFVGGFNADFSQRLSERWRLTLNAGNMWKHYQEDRTAARYDSHMPLAGATLMYS 120 Query: 240 AMSAWQLYGGISGNFDNTVEKEQASBRYGVSLGTVKILDGGLGLKLGAGYTKRIFKAPAT 299 A W LYGG + T E EQAS R G+ +G VK DGGLGL+ YT+R+F AP T Sbjct: 121 APKDWLLYGGADWSHNITKEAEQASIRKGLRVGAVKTFDGGLGLRANLRYTRRMFDAPGT 180 Query: 300 LIYNFTRRD 308 ++Y F R+D Sbjct: 181 IVYRFPRKD 189 >gb|AAD11779.1|(AF118122) putative outer membrane protein OmpU [Neisseria meningitidis] Length = 488 Score = 72.1 bits (176), Expect = 7e−12 Identities = 71/300 (23%), Positives = 128/300 (42%), Gaps = 17/300 (5%) Query: 3 EAADLYRELLSERPDLVYPRFDLGVMLFEDKQYREALVQLHRAE-EVLPPDMRQLAREYI 61 EA YREL++ +PD R L LF+++Q A Q R + E LPP + + Y Sbjct: 136 EAISHYRELIAAQPDAPAVRMRLAAALFDNRQNEAAADQFDRLKAENLPPQLMEQVELYR 195 Query: 62 RQAEAVQAWHPSFNMNYEQTDNVNNASLSRDIVINGRKWIKSEDSLPKRANT--IRYELG 119 + AW + + + N+N A + KW + PK+ +G + Y LG Sbjct: 196 KALRERDAWKVNGGFSVTREHNINQAPKRQQR----GLW-----TFPKQVDGTAVNYRLG 246 Query: 120 VDRMFNMAGNHFARLGISGSGVHYWNARDFSEQAFHAEVGYRYRNSRLEWGFRPFVKQNR 179 ++ +++ + G SG Y + F++ G + + R + G F ++ Sbjct: 247 AEKKWSLKNGWYTTAGGDVSGRVYPGNKKFNDMTAGVSGGIGFADRRKDAGLAVFHERRT 306 Query: 180 LGNNRYTANTGIVLDYSRRLNEKWRSTQSFQYGRKQYHDEYLAKRYNSKTISVSGTFSYY 239 GN+ Y+ G L ++R KW++ S ++GR + R ++ + +S + +Y Sbjct: 307 YGNDAYSYTNGARLYFNRWQTPKWQTLSSAEWGRLK---NTRRARSDNTHLQISNSLVFY 363 Query: 240 AMSAWQLYGGISGMFD-NTVEKEQASRRYGVSLGTVKILDG-GLGLKLGAGYTKRIFKAP 297 + GG+ + N ++ RYG+ + G GL L G KR ++ P Sbjct: 364 RNARQYWMGGLDFYRERNPADRGDNFNRYGLRFAWGQEWGGSGLSSLLRLGAAKRHYEKP 423 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 81 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 161> which encodes amino acid sequence <SEQ ID 162; NGS81>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −6.25 Possible cleavage site: 15 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 7.05 threshold: 0.0 PERIPHERAL Likelihood = 7.05 modified ALOM score: −1.91 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.232(Affirmative) < succ> The protein has homology with the following sequences in the databases: gi|10803654|ref|NP_046052.1|putative ISH4 transposase [Halobacterium sp.\",\n \"NRC-1] gi|7484109|pir||T08324 probable transposase H1306-Halobacterium sp.\",\n \"(strain NRC-1) insertion sequence ISH4 plasmid pNRC100 gi|2822385|gb|AAC82891.1|(AF016485) putative ISH4 transposase [Halobacterium sp.\",\n \"NRC-1] gi|10580476|gb|AAG19350.1|(AE005029) Vng0918h [Halobacterium sp.\",\n \"NRC-1] Length = 294 Score = 52.1 bits (124), Expect = 4e−06 Identities = 36/139 (25%), Positives = 63/139 (44%), Gaps = 7/139 (5%) Query: 31 CPHCQSTHFVKNGKDCGNQRFLCRDCKKSFVEQTGTILYNTQKDIEVWEKYIHCMIE-KY 89 CP C++ ++ G QR+LC+DC ++F +QTGT+ ++ + W ++ I Sbjct: 28 CPSCRAESVIRYGSYRVFQRYLCKDCDRTFNDQTGTVFEHSAVALRKWFLAVYTYIRLNT 87 Query: 90 PLRKCAEICKINLATAFTWRHKILDALQNMMNEVELDGIVQADETYSTISYKGHHKNFNL 149 +R+ ++ T + + L AL L+G V+ DE Y KG ++ Sbjct: 88 SIRQLDAEIDVSYKTVYRRVQRFLRALD--APRPHLEGPVEIDEFYVKAGLKGRERD--- 142 Query: 150 PRPAHKRGTRATKRGISKE 168 +P+ RG RG E Sbjct: 143 -QPSRSRGLSTRGRGTYAE 160 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 82 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 163> which encodes amino acid sequence <SEQ ID 164; NGS82>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −4.66 Possible cleavage site: 57 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 1 value: −0.85 threshold: 0.0 INTEGRAL Likelihood = −0.85 Transmembrane 76-92 (76-92) PERIPHERAL Likelihood = 1.75 modified ALOM score: 0.67 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.134(Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi|586070|sp|Q07605|T4BA_BACCO RESTRICTION ENZYME BGCI ALPHA SUBUNIT [INCLUDES: ADENINE-SPECIFIC METHYLTRANSFERASE ACTIVITY] gi|1075788|pir||A53125 restriction enzyme BcgI alpha chain-Bacillus coagulans gi|304140|gb|AAA16626.1|(L17341) restriction endonuclease alpha subunit [Bacillus coagulans] Length = 637 Score = 91.4 bits (226), Expect = 1e−17 Identities = 78/256 (30%), Positives = 123/256 (47%), Gaps = 42/256 (16%) Query: 1 MFALAASNMILRGDGKANLHQSSCFMTDFQDLIKNPKPETGLKRPNVGFLNPPYAQSKSD 60 +F +A +NMILRGDGK+NL + +C F + I N G+ N +NPPY+Q+K+D Sbjct: 394 LFTIATTNMILRGDGKSNLIRDNCLT--FDNTIMN---GYGI---NKILMNPPYSQAKND 445 Query: 61 AELH--ELYFVKEMLKMLAEGGTGIAIIPVSCVIAPSK----AKSEIVKYHRLKAVMSMP 114 H EL F+++ L+ML GG AI+P S ++ ++ K +I+K H L+ V+++ Sbjct: 446 QTQHLSELSFIQQALEMLVVGGKLCAIVPQSTMVGKNRHDKARKKQILKQHTLETVITLN 505 Query: 115 SELFYPVGTVTCIVVFEAHKPHFQTVVIDPDTQEEISTKKACENTWFGYWRDDGFEKTKH 174 + F+ VG CIV+F+A H + ++ F + DDG KH Sbjct: 506 KDTFHGVGVNPCIVIFKAGIKHPEN-----------------KRVSFVNFEDDGHVVRKH 548 Query: 175 LGRIDLYDRWQGIKARWLEHYL-----NNEVHTGESVTAFVTDNDEWVAEAYLETDYSKI 229 +G + G + EH L + + T V + D DEW+ Y D Sbjct: 549 VGLVG-----DGTEKGKREHLLAVLAGDEDDGTDLIVKTAIKDTDEWLHSFYYFND-GIP 602 Query: 230 TRADFEQVVREFALFQ 245 + DF + V + FQ Sbjct: 603 SEDDFYKTVANYLTFQ 618 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 83 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 165> which encodes amino acid sequence <SEQ ID 166; NGS83>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −8.04 Possible cleavage site: 43 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 1 value: −1.44 threshold: 0.0 INTEGRAL Likelihood = −1.44 Transmembrane 55-71 (55-71) PERIPHERAL Likelihood = 4.03 modified ALOM score: 0.79 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.157 (Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi|586071|sp|Q07606|T4BB_BACCO RESTRICTION ENZYNE BGCI BETA SUBUNIT gi|1075789|pir||B53125 restriction enzyme BcgI beta chain-Bacillus coagulans gi|304141|gb|AAA16627.1|(L17341) restriction endonuclease beta subunit [Bacillus coagulans] Length = 341 Score = 44.0 bits (103), Expect = 0.002 Identities = 46/195 (23%), Positives = 79/195 (39%), Gaps = 23/195 (11%) Query: 4 LQEIFDVSYGSKLDLNKMSSFNPTINFVGRSGKNNGVTASVDLLKNTKPYPAGLLTVALG 63 + ++FDV G +D NK ++R NG +D K K Y L + +G Sbjct: 12 ISDLFDVVIGKTIDGNKAQRNENGTPYITRKATRNGFEFMIDGEKE-KLYSGKLPVITIG 70 Query: 64 GSVLSTFLQNKPFYTAQNVAVLNPKTEMTEQQKLFYCAAIFANAYRFSACGREANRT-LR 122 F+Q F+T V + PK ++ L Y + NA + + N T L+ Sbjct: 71 NETSKPFVQEFHFFTGTKVNICIPKLDLNRNH-LLYITTMIENATKMFSYSYTINSTRLK 129 Query: 123 QL--FVPSLDEIPSW--------------VESVNLNPSAGVTEPKLKESLDLPVVRQSKR 166 L +P E P W ++ ++ + GV++ + + L + Sbjct: 130 SLKILLPIKGEEPDWDYMNTYISKILSNMEKNFDVQQNDGVSDLRSLKDLSW----SQFK 185 Query: 167 LPEIFTIQNGIAATK 181 +DEIF+I +G+ TK Sbjct: 186 MDEIFSINSGVRLTK 200 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 84 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 167> which encodes amino acid sequence <SEQ ID 168; NGS84>.\",\n \"Analysis of this protein sequence reveals the following: Signal Score (−7.5): 3.15 Possible cleavage site: 33 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 1.22 threshold: 0.0 PERIPHERAL Likelihood = 1.22 modified ALOM score: −0.74 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.072(Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi|2495432|sp|P55409|Y4DJ_RHISN HYPOTHETICAL TRANSCRIPTIONAL REGULATOR Y4DJ gi|7465604|pir||T02773 y4dJ protein-Rhizobium sp.\",\n \"plasmid pNGR234a gi|2182353|gb|AAB91639.1|(AE000069) Y4dJ [Rhizobium sp.\",\n \"NGR234] Length = 77 Score = 44.4 bits (104), Expect = 7e−04 Identities = 25/61 (40%), Positives = 36/61 (58%) Query: 92 KAGGETFVSLRKGFTQSELATAAGLPQPYLSRIENSKQSLQDKTVQKLANALGVSPLE 151 K G F LR +KG TQ E+ +G Q YLS +E +++ T+ +LA ALGVS +E Sbjct: 5 KLVGSNFARLREEKGLTQEEEARSGFSQQYLSSLERGRRNPTVITLYELAQALGVSHVE 64 Query: 152 V 152 + Sbjct: 65 L 65 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 85 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 169> which encodes amino acid sequence <SEQ ID 170; NGS85>.\",\n \"Analysis of this protein sequence reveals the following: Signal Score (−7.5): −6.09 Possible cleavage site: 15 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 2.92 threshold: 0.0 PERIPHERAL Likelihood = 2.92 modified ALOM score: −1.08 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.480(Affirmative) < succ> The protein has no homology with sequences in the databases.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 86 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 171> which encodes amino acid sequence <SEQ ID 172; NGS86>.\",\n \"Analysis of this protein sequence reveals the following: Signal Score (−7.5): −2.92 Possible cleavage site: 21 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 1 value: −2.76 threshold: 0.0 INTEGRAL Likelihood = −2.76 Transmembrane 179-195 (179-195) PERIPHERAL Likelihood = 2.17 modified ALOM score: 1.05 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.210 (Affirmative) < succ> The protein has homology with the following sequences in the databases: >sp|Q05205|PPB_LYSEN ALKALINE PHOSPHATASE PRECURSOR (APASE) pir||A42467 alkaline phosphatase (EC 3.1.3.1) phoA precursor-Lysobacter enzymogenes emb|CAA39978.1|(X56656) alkaline phosphatase [Lysobacter enzymogenes] Length = 539 Score = 37.5 bits (86), Expect = 0.40 Identities = 28/82 (34%), Positives = 43/82 (52%), Gaps = 8/82 (9%) Query: 189 VALGLQAYWDVAGANNGATGQSPNIKTAQVPAKITRRNADGTTDTFGGGSARKSAAASVS 248 V GL A W+V+ A + AQV +++ R+ GT D + G+A A AS S Sbjct: 458 VLRGLMA-WNVSSA------AGKTLTGAQVKLQVSDRST-GTYDLYRAGAAWTEANASYS 509 Query: 249 GIEAGKKVTAVIPAVRGAVAYA 270 G+ G K+ +V+P+ GA + A Sbjct: 510 GVSLGSKIGSVVPSATGAQSIA 531 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 87 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 173> which encodes amino acid sequence <SEQ ID 174; NGS97>.\",\n \"Analysis of this protein sequence reveals the following: Signal Score (−7.5): 0.18 Possible cleavage site: 35 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 1.70 threshold: 0.0 PERIPHERAL Likelihood = 1.70 modified ALOM score: −0.84 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.138(Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi|12514207|gb|AAG55499.1|AE005289_17 (AE005289) unknown protein encoded by cryptic prophage CP-933M [Escherichia coli O157:H7] gi|12514720|gb|AAG55907.1|AE005324_10 (AE005324) unknown protein encoded by prophage CP-933N [Escherichia coli O157:H7] Length = 108 Score = 30.9 bits (69), Expect = 9.1 Identities = 21/55 (38%), Positives = 28/55 (50%), Gaps = 3/55 (5%) Query: 1 MAAPVSLEEFKQRIGVEHDRRDDFFLSVIDGVSAAAEAYIGRSLLAADYVGRYDG 55 M A ++LEE K + V+HD DD + + +A AYI S D V R DG Sbjct: 1 MTALLTLEEIKAHLRVDHDADDDMLMDKVRQATAVLLAYIQGS---RDKVIREDG 52 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 88 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 175> which encodes amino acid sequence <SEQ ID 176; NGS88>.\",\n \"Analysis of this protein sequence reveals the following: Signal Score (−7.5): −3.69 Possible cleavage site: 43 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 6.05 threshold: 0.0 PERIPHERAL Likelihood = 6.05 modified ALOM score: −1.71 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.227(Affirmative) < succ> The protein has no homology with sequences in the databases.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 89 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 177> which encodes amino acid sequence <SEQ ID 178; NGS89>.\",\n \"Analysis of this protein sequence reveals the following: Signal Score (−7.5): −4.77 Possible cleavage site: 26 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 1.38 threshold: 0.0 PERIPHERAL Likelihood = 1.38 modified ALOM score: −0.78 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.284(Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi|9634142|ref|NP_037684.1|gp24 [Enterobacteria phage HK022] gi|6863134|gb|AAF30375.1|AF069308_23 (AF069308) gp24 [Enterobacteria phage HK022] Length = 1183 Score = 44.9 bits (102), Expect = 0.006 Identities = 38/127 (29%), Positives = 64/127 (49%), Gaps = 11/127 (8%) Query: 851 NKALRDKINLIDGNGAGSVNERVEAVRSTADGNAAAVQTHARSI---NG-LEAQYTVK-- 904 NKA + +N + + ++ + +T +GN +A+ T+A++I NG L A Y +K Sbjct: 989 NKASINSLNQTFSDYQQATATQINGITATVNGNTSAITTNAQAIANVNGDLSAMYNIKVG 1048 Query: 905 VDANGK--VAGFGLATTPKNGTPESKFIVNADRFGI-GAAGKADVFPFVVDTQKNRVGIN 961 V +NG+ AG G+ +S+ I ADRF + AAG + PFV+ Q + I Sbjct: 1049 VSSNGQYYAAGMGIGVENTPSGMQSQVIFLADRFAVTTAAGNSVALPFVI--QNGQTFIR 1106 Query: 962 GELVVNG 968 + +G Sbjct: 1107 ASFIQDG 1113 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 90 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 179> which encodes amino acid sequence <SEQ ID 180; NGS90>.\",\n \"Analysis of this protein sequence reveals the following: Signal Score (−7.5): −2.82 Possible cleavage site: 24 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 4 value: −9.66 threshold: 0.0 INTEGRAL Likelihood = −9.66 Transmembrane 321-337 (317-349) INTEGRAL Likelihood = −6.48 Transmembrane 351-367 (340-371) INTEGRAL Likelihood = −5.73 Transmembrane 907-923 (903-926) INTEGRAL Likelihood = −0.00 Transmembrane 430-446 (430-446) PERIPHERAL Likelihood = 2.17 modified ALOM score: 2.43 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.486 (Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi|12514839|gb|AAG56002.1|AE005332_9 (AE005332) putative tail component of prophage CP-933X [Escherichia coli O157:H7] Length = 1026 Score = 111 bits (279), Expect = 3e−23 Identities = 78/274 (28%), Positives = 146/274 (52%), Gaps = 10/274 (3%) Query: 69 AAGNQAQQASEKVRAEVGKIGSGLSGLTKLLAGLATADFAKSVLDTADAMQSINSQVRQV 128 AA + ++A ++ +++ +I + G+T AG A ++ AD S+N++++Q Sbjct: 45 AAAREQRRALAELHSQLTEIRASAVGMTGAFAG---AFATGHLISLADEWSSVNARLKQA 101 Query: 129 TSSETEYLAVQQQLLDTANRTRASLESTANLYVSTSRALKDYGYTQQEILKFTEAANNAM 188 + S E+ + Q+ L+D + RT + A L+ ++ ++++YGY+ ++LK TEA + + Sbjct: 102 SQSSDEFASSQKVLMDISQRTGTAFSDNAALFARSAASMREYGYSADDVLKVTEAISTGL 161 Query: 189 TIGGVGAQQQAAALMQLSQALGSGVLQGDEFKSISEAAPILLDTIAEYMGKSRDEIKKLG 248 I G + + + Q SQAL GVL+G+EF S++E+ ++ +A MG +R ++K + Sbjct: 162 KISGASTAEAGSVITQFSQALAQGVLRGEEFNSVNESGDRIVRALAAGMGVARKDLKAMA 221 Query: 249 SEGKLTADVIFKAISGASEKFGEQAAKMPVTMGQALTVFSNNWQSMVSKLLNDSGTMSGI 308 +GKLTAD + A+ ++ A MP T+ ++T N + + V G + Sbjct: 222 DDGKLTADKVVPALISQLGILRDEYAAMPETVSSSITKVENAFMAWV-------GGANEA 274 Query: 309 AAVIKLIADNLNLVVPIVAGFAVAVAAAVAPTLA 342 + V K ++ LN V + A AV A VA +A Sbjct: 275 SGVTKTLSGMLNGVAGQIDNVATAVGALVAVGVA 308 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 91 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 181> which encodes amino acid sequence <SEQ ID 182; NGS91>.\",\n \"Analysis of this protein sequence reveals the following: Signal Score (−7.5): −0.63 Possible cleavage site: 36 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 0.16 threshold: 0.0 PERIPHERAL Likelihood = 0.16 modified ALOM score: −0.53 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.250(Affirmative) < succ> The protein has homology with the following sequences in the databases: (AF237934) putative integrase/recombinase [Pasteurella multocida] Length = 329 Score = 449 (206.9 bits), Expect = 4.4e−91, Sum P(2) = 4.4e−91 Identities = 93/196 (47%), Positives = 129/196 (65%) Query: 56 IFADLIRRYLSEVTPSKRGAREESYRIGRALKTPLAKVRLADLRPQDFADWRDQRLQEVS 115 IF D+I RY +EV+ +K+GAR E R+ R L+ ++ + + DLR +DF +W RL EVS Sbjct: 55 IFRDVIERYQNEVSITKKGARNEIIRLNRFLRYDISNLYIRDLRKEDFEEWIRIRLTEVS 114 Query: 116 PTSVGRELTTLSAVCEHAMKEWGLLRENPVRKISKPKKSRARTRRPTEQEIADICAALLY 175 SV REL T+S+V A+ +WG + +P+ I KPK S R R +EQ+I I Y Sbjct: 115 DASVRRELVTISSVLTTAINKWGYISRHPMTGIEKPKNSAERKERYSEQDIKTILETARY 174 Query: 176 RPNEKPKMAVQRVAVAVLFAIETAMRAGEICGLKWADVNMRRRIAHLPITKNGDSRDVPL 235 ++ P QRVA+A+LFAIETAMRAGEI +KW +V + +RI HLP TKNG SRDVPL Sbjct: 175 CEDKLPITLKQRVAIAMLFAIETAMRAGEIASIKWDNVFLEKRIVHLPTTKNGHSRDVPL 234 Query: 236 SLRAAELIEQLRGIDD 251 S RA LI +++ +++ Sbjct: 235 SQRAVALILKMKEVEN 250 Score = 248 (114.3 bits), Expect = 4.4e−91, Sum P(2) = 4.4e−91 Identities = 48/76 (63%), Positives = 57/76 (75%) Query: 254 VFSLDAKSLDVLFRRARDNCGIQGLHFHDTRREALTRLSKKVPVEVLAKISGHRDLRILL 313 VF +SL FR + CG++ LHFHDTRREALTRLSKKV V LAKISGHRDLRIL Sbjct: 254 VFQTTPESLSTTFRVLKKECGLEHLHFHDTRREALTRLSKKVDVMTLAKISGHRDLRILQ 313 Query: 314 NVYYRPDMADIAKMLD 329 N YY P+M+++A +LD Sbjct: 314 NTYYAPNMSEVANLLD 329 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 92 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 183> which encodes amino acid sequence <SEQ ID 184; NGS92>.\",\n \"Analysis of this protein sequence reveals the following: Signal Score (−7.5): −7.85 Possible cleavage site: 25 >>> Seems to have an uncleavable N-term signal seq Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 1 value: −8.33 threshold: 0.0 INTEGRAL Likelihood = −8.33 Transmembrane 6-22 (1-25) PERIPHERAL Likelihood = 5.99 modified ALOM score: 2.17 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.433 (Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi|963205|ref|NP_048840.1|A484L [Paramecium bursaria Chlorella virus 1] gi|7461623|pir|T17986 hypothetical protein A484L-Chlorella virus PBCV-1 gi|1620155|gb|AAC96851.1|(U42580) A484L [Paramecium bursaria Chlorella virus 1] Length = 155 Score = 31.6 bits (70), Expect = 3.5 Identities = 20/72 (27%), Positives = 36/72 (49%) Frame = +1 Query: 52 LQINLKMLEKRIDFLVENIDKYYQQYGSYPNNFDFISTKTDFTTESYCDFWDKNIAGYGN 231 + +NLKM I F +DKY +QY +Y N F T+ + + ++ + +I N Sbjct: 23 IAVNLKMNGVSIPF----VDKYSKQYPTYTKNALFHVTRFNNAYQKTFEYKNISIDTINN 78 Query: 232 CYFVKNDKDYTI 267 + +++D Y I Sbjct: 79 LFSIRDDVLYNI 90 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 93 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 185> which encodes amino acid sequence <SEQ ID 186; NGS93>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.08 Possible cleavage site: 14 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 0.79 threshold: 0.0 PERIPHERAL Likelihood = 0.79 modified ALOM score: −0.66 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.320(Affirmative) < succ> The protein has homology with the following sequences in the databases: >gb|AAG22017.1|AF288038_2 (AF288038) putative HsdR ]Streptococcus thermophilus] Length = 740 Score = 674 bits (1738), Expect = 0.0 Identities = 364/746 (48%), Positives = 489/746 (64%), Gaps = 40/746 (5%) Query: 14 NENSRVKIPAVLHLMRLGYDYLSLKNANW---DRQTNIFPEIFVDSLCRINPDLPPDDAR 70 +E +RV+IPA HLMRLGY YL D +TNI IF + + N D Sbjct: 8 SELTRVQIPAAFHLMRLGYTYLPHNGKEIMGRDPETNILISIFREQFLKFNNYATDLDVE 67 Query: 71 RLLADIRLELDNEDLGQKFYERLTNQSGGKKLIDFQNFDNNSFHVVTELPCINGDEAFRP 130 R L +I++ELD DLG+ FY R+ + SG +D++N +NN+FH+ E+ C NG + FRP Sbjct: 68 RELNNIKIELDQNDLGRAFYNRIVSDSG-PTYVDWENPENNTFHLALEVTCQNGGDEFRP 126 Query: 131 DIALLVNGMPLVFIEVKKPN----NKGGIGEERERMGKRAKNPKFRRFINITQFMIFSNN 186 DI + +NG+PL +IEVK+PN K I E+ R R +N +FRRF NITQ + FS+N Sbjct: 127 DIVIFINGLPLSYIEVKQPNAIRDGKTAIQSEQSRTAVRFENRRFRRFNNITQLISFSDN 186 Query: 187 MEYDDGATEPAQGAFYASSACGKPVFNYFREEHKXXXXXXXXXXXXXXXXXVLQDNNLPV 246 + Y G + QG+FY S+A K FN F+EE + VL+D N Sbjct: 187 LPYISGQGQQKQGSFYCSNAFSKTKFNAFKEEREEELIYSIRSLGEEEIDAVLKDVNRFA 246 Query: 247 IKHSPEFISNKSPDTPTNRILTSLLCRERLSFLLQHGLTYVK--ASQGLVQ--KHIMRYP 302 +K PEF +N+ P TP N ++SL ++RL FLL++GL YV+ + G +Q KH+MRYP Sbjct: 247 LKSQPEFKTNQDPSTPCNTFISSLYQKKRLLFLLRYGLAYVEEHSKDGTIQLQKHVMRYP 306 Query: 303 QLFATLAIEKHLANGGKKGVIWHTQGSGKTALAYYNTRYLTHYYAKQGIVPKFYFIVDRL 362 Q FAT AIE + G +KGVIWHTQGSGKTAL+Y+N RYLT+Y++KQGIVP+FYF+VDRL Sbjct: 307 QFFATKAIEDAIGKGVRKGVIWHTQGSGKTALSYFNIRYLTNYFSKQGIVPQFYFVVDRL 366 Query: 363 DLLKQAQREFTARDLVVHTIDSREAFAADIKSAQTLHNHAGKAEITVVNIQKFQDDPDVV 422 DL QA REFT R L V I+S Q L+ ++ VVNIQKF+D+ D+ Sbjct: 367 DLADQATREFTKRGLKVKRINS----------PQELNEKHDAYQVAVVNIQKFKDNSDLT 416 Query: 423 ARNDYDLAIQRVYFLDEVHRSYNPKGSFLANLNQSDVNAVKIGLTGTPLI-----GVTA- 476 + YDL Q +YF+DE HRSYN KGS+L NL +D NA+KI LTGTPLI G T Sbjct: 417 DHSGYDLNRQNIYFIDEAHRSYNEKGSYLPNLYNADKNAIKIALTGTPLITYKKDGKTKE 476 Query: 477 GNVNTRELFGDYIHKYYYNASIADGYTLRLIREEIGSRYKAQLQEALAQLEIEKGSFDRK 536 + TR++FGDYIHKYYYN SI DG+TLRL+RE+I + YK LQ EI +G + Sbjct: 477 SHATTRDIFGDYIHKYYYNQSIDDGFTLRLMREDIETSYKETLQTI--NEEILRGDLSKD 534 Query: 537 EIYAHPHFVHPMLDYILDDFAKFRKTN-QDESLGAMVVCDSAEQARQL---FEHFQTASD 592 +I+AHP +V PMLG+IL+DF + R D+S+G M+VCDS++QAR++ E ++ + Sbjct: 535 DIFAHPRYVSPMLDFILEDFNRARDVVFDDDSIGGMIVCDSSKQAREIEKQLEERRSRGE 594 Query: 593 HNFTAALILHDVGTKEERDQWVKDFKAGKIDILFVYNMLLTGFDAPRLKKLYLGRLIKAH 652 N T+ALILHD G KE + V+ ++ GKID++ VY+MLLTGFDAPRLK+LYLGR IKAH Sbjct: 595 TNITSALILHDEGDKEYKKDRVESYREGKIDLVIVYSMLLTGFDAPRLKRLYLGRKIKAH 654 Query: 653 NLLQTLTRVNRTYKSYRYGYVVDFADIEREFDKTNRAYWDELSEN-----LGDEIGS-YS 706 NLLQTLTRVNR YK Y++GYV+DFADI +EFDKTNRAY +EL+ E G+++ + + Sbjct: 655 NLLQTLTRVNRPYKDYQFGYVIDFADISKEFDKTNRAYLEELNQEYDPKNTGEDVENVFG 714 Query: 707 QLFKTAEEIEQEIADIKNALFDFDTE 732 LF +A+EI +++ + L ++ TE Sbjct: 715 SLFVSADEISKQLEKSETILMNPTE 740 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 94 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 187> which encodes amino acid sequence <SEQ ID 188; NGS94>.\",\n \"Analysis of this protein sequence reveals the following: Signal Score (−7.5): −3.19 Possible cleavage site: 35 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 5.73 threshold: 0.0 PERIPHERAL Likelihood = 5.73 modified ALOM score: −1.65 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.302(Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi|10717100|gb|AAG22014.1|AF288037_3 (AF288037) putative HsdS [Streptococcus thermophilus] Length = 402 Score = 154 bits (389), Expect = 2e−36 Identities = 123/348 (35%), Positives = 168/348 (47%), Gaps = 48/348 (13%) Query: 73 GKTAFVDILDDGEVAFGSTEFIVLRAKNET--NPEFLYYFAISPDFRKRAIECMEGTSGR 130 GKT ++ DGE ++ IV +E+ +FLYYF + F G++ + Sbjct: 74 GKT----VIFDGEDSYFQDSNIVWIENDESKVTNQFLYYFLQTNPFIT-----TNGSTIK 124 Query: 131 QRVNENALKTLELPIPEPQIQQSIAAVLSALDKKIALNKQINARLEEMAKTLYDYWFVQF 190 + N+N T +P Q Q I +L LDKKI +N QIN LE MAKTLYDYWFVQF Sbjct: 125 RLYNDNLRDTKIPNVPSIQQQNQITDILGTLDKKIQINNQINQELEAMAKTLYDYWFVQF 184 Query: 191 DFPDANGKPYKSSGGDMVFDETLKREIPKGWGSIELQSCL---AKIPNTTKILNKDIKDF 247 DFPD NGKPYKSSGG MV++ LKREIP+GWG+ +L S L + N K N++ K + Sbjct: 185 DFPDQNGKPYKSSGGKMVYNPELKREIPEGWGAEKLSSLLKIGKETTNPKKFPNEEFKYY 244 Query: 248 --------GKYPVVD----QSQDFICGFTNKEKSILNPQDAHIIFGDHTRIVKLVNFQYA 295 G Y + +S F + S LNP +I+ + F Sbjct: 245 SIPEFDTTGTYSLERGESIKSNKFKFKVEKNDLLVSKLNPWFNRVIYNLEENAIASTEF--- 301 Query: 296 RGADGTQVILSNNERMPNYLFYQIINQIDLSSY------GYARHFK-----FLKEFKIIL 344 ++ R YQ+ + Y G + K + F+I Sbjct: 302 -------IVWKTFNRFEKNFLYQVATGKEFIEYCTRFATGTSNSHKRVSPDIMVGFQIPF 354 Query: 345 PSKDISQKYNEIANTFFVKVRNNLKQNHHLTQLRDFLLPMLMNGQVSV 392 I QK+ EI ++ +V N +QN LTQLRD++LPMLMNGQV V Sbjct: 355 EKTHI-QKFGEIIDSIRTQVLQNNEQNQELTQLRDWILPMLMNGQVKV 401 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 95 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 189> which encodes amino acid sequence <SEQ ID 190; NGS95>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −6.12 Possible cleavage site: 19 >>> Seems to have an uncleavable N-term signal seq Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 3 value: −10.51 threshold: 0.0 INTEGRAL Likelihood = −10.51 Transmembrane 112-128 (109-132) INTEGRAL Likelihood = −4.46 Transmembrane 50-66 (46-70) INTEGRAL Likelihood = −2.23 Transmembrane 7-23 (7-23) PERIPHERAL Likelihood = 4.19 modified ALOM score: 2.60 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.520 (Affirmative) < succ> The protein has homology with the following sequences in the databases: >pir||G69096 hypothetical protein MTH1717-Methanobacterium thermoautotrophicum (strain Delta H) gb|AAB86189.1|(AE000928) unknown [Methanothermobacter thermautotrophicus] Length = 557 Score = 35.4 bits (80), Expect = 0.50 Identities = 25/80 (31%), Positives = 47/80 (58%), Gaps = 5/80 (6%) Query: 52 LLFYFLIPFIATATVLWLSKYLGKDEFKQGEVKELEYVNDNFLPSYLGYFFVALSIPDNN 111 L+F+F+ P + TATVL + K + ++ F++ EV L + +PS++ ++ IP++ Sbjct: 92 LVFFFISPLLGTATVLVIYK-VARETFEREEVALLSAFLFSMVPSFVAR--TSVFIPESM 148 Query: 112 LFLLFVMYGIIFLLVSCSKS 131 LL GI+++LV K+ Sbjct: 149 GLLL--TSGILYMLVKYLKT 166 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 96 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 191> which encodes amino acid sequence <SEQ ID 192; NGS96>.\",\n \"Analysis of this protein sequence reveals the following: GyH: Examining signal sequence (von Heijne) Signal Score (−7.5): −7.76 Possible cleavage site: 28 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 6.15 threshold: 0.0 PERIPHERAL Likelihood = 6.15 modified ALOM score: −1.73 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.362(Affirmative) < succ> The protein has no homology with sequences in the databases.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 97 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 193> which encodes amino acid sequence <SEQ ID 194; NGS97>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −0.48 Possible cleavage site: 13 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 8.86 threshold: 0.0 PERIPHERAL Likelihood = 8.86 modified ALOM score: −2.27 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.127(Affirmative) < succ> The protein has homology with the following sequences in the databases: >ref|NP_052265.1|P2 J homolog; baseplate or base of tail fibre [Enterobacteria phage 186] gb|AAC34162.1|(U32222) P2 J homolog; baseplate or base of tail fibre [Enterobacteria phage 186] Length = 302 Score = 112 bits (280), Expect = 3e−24 Identities = 65/151 (43%), Positives = 85/151 (56%), Gaps = 1/151 (0%) Query: 1 MGNSRLSQLPAPAAIEETDFEGIFARKKAALTALCPESIRETVAQTLELESEPLTIDLQQ 60 M LS LP P +EE DFE I A + A L +L PE +E VA+TL LESEP+ LQ+ Sbjct: 1 MATVDLSLLPVPDVVEELDFETILAERIATLISLYPEDQQEAVARTLALESEPIVKLLQE 60 Query: 61 QAYQELLVRNRINEAVKANLLAYAQGSDLDHIAAQYGLSRKTIRXXXXXXXXXXXXEYET 120 AY+E++ R R+NEA +A +LAYA+ SDLD++ A + + R +R E E Sbjct: 61 NAYREVIWRQRVNEAARAGMLAYARDSDLDNLGANFNVERLVVRPADDTTIPPTPAEMEL 120 Query: 121 DDAFRARV-QAHPEKYAAGPRTAYEAHAIDA 150 D FR R+ QA AG AYE H A Sbjct: 121 DADFRLRIQQAFEGMSVAGSTGAYEFHGRSA 151 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 98 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 195> which encodes amino acid sequence <SEQ ID 196; NGS98>.\",\n \"Analysis of this protein sequence reveals the following: Signal Score (−7.5): −3.68 Possible cleavage site: 33 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 4.61 threshold: 0.0 PERIPHERAL Likelihood = 4.61 modified ALOM score: −1.42 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.182(Affirmative) < succ> The protein has no homology with sequences in the databases.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 99 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 197> which encodes amino acid sequence <SEQ ID 198; NGS99>.\",\n \"Analysis of this protein sequence reveals the following: Signal Score (−7.5): −4.87 Possible cleavage site: 19 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 4.93 threshold: 0.0 PERIPHERAL Likelihood = 4.93 modified ALOM score: −1.49 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.189(Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi|10172952|dbj|BAB04058.1|(AP001508) BH0339-unknown conserved protein in others [Bacillus halodurans] Length = 283 Score = 83.7 bits (206), Expect = 1e−15 Identities = 59/156 (37%), Positives = 87/156 (54%), Gaps = 8/156 (5%) Query: 10 VRGPVQLAFAQSIDPIVPPEVSITRMAVTNEKDLEKERTMGRKYIVPYVVYRVHGFISAN 69 VRGPV + A SIDPI IT+ + D TMG K+ V + VY G I+ Sbjct: 129 VRGPVSIHTATSIDPIDIVSTQITKSVNSVTGDKRSSDTMGMKHRVDFGVYVFKGSINTQ 188 Query: 70 LAAKTGFSDDDLAKLWQALTLMFEHDRSAAR--GEMARKLVFKHDSALGSQPAHKLFD 127 LA KTGF+++D K+ +AL +FE+D S+AR G M K+ ++H S LG + K+ Sbjct: 189 LAEKTGFTNEDAEKIKRALITLFENDSSSARPDGSMEVHKVYWWEHSSKLGQYSSAKVHR 248 Query: 128 AVKVERVNGESGTPASGFGDYKISVVSDGLNGVSVE 163 ++K+E ++ TP S F DY + + L+G+ VE Sbjct: 249 SLKIE---SKTDTPKS-FDDYAVELYE--LDGLGVE 278 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 100 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 199> which encodes amino acid sequence <SEQ ID 200; NGS100>.\",\n \"Analysis of this protein sequence reveals the following: Signal Score (−7.5): −3.03 Possible cleavage site: 18 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 6.63 threshold: 0.0 PERIPHERAL Likelihood = 6.63 modified ALOM score: −1.83 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.185(Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi|1175791|sp|P44189|YE18_HAEIN HYPOTHETICAL PROTEIN HI1418 gi|1074769|pir||A64029 hypothetical protein HI1418-Haemophilus influenzae (strain Rd KW20) gi|1574254|gb|AAC23068.1|(U32821) H. influenzae predicted coding region HI1418 [Haemophilus influenzae Rd] Length = 201 Score = 144 bits (364), Expect = 1e−33 Identities = 71/109 (65%), Positives = 79/109 (72%) Query: 8 NFQQNSVRTVADNKGELWFLANDVCEILGYTNPRRTVDLHCKSRGVTKRYTPTTSGEQEM 67 NF+ VR + D KGE WF DVC ILGYTN R+ + HCK GVTKRYTPT S +QEM Sbjct: 24 NFKDLPVRVILDPKGEFWFCGTDVCHILGYTNSRKALQDHCKQGGVTKRYTPTKSADQEM 83 Query: 68 TYINEPNLYRLIIKSRKPAAEAFEEWVMETVLPAIRKTGGCQVGPKTTA 116 T+INEPNLYRLIIKSRKP AE FE WV E VLP IRKTG Q+ P+ A Sbjct: 84 TFINEPNLYRLIIKSEKPEAEPFEAWVFEEVLPQIRKTGKYQLQPQQLA 132 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 101 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 201> which encodes amino acid sequence <SEQ ID 202; NGS101>.\",\n \"Analysis of this protein sequence reveals the following: Signal Score (−7.5): −1.23 Possible cleavage site: 47 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 3.55 threshold: 0.0 PERIPHERAL Likelihood = 3.55 modified ALOM score: −1.21 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.126(Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi|9632520|ref|NP_049514.1|hypothetical protein [Bacteriophage 933W] gi|9633449|ref|NP_050552.1|hypothetical protein [Bacteriophage VT2-Sa] gi|4585431|gb|AAD25459.1|AF125520_54 (AF125520) hypothetical protein [Bacteriophage 933W] gi|5881645|dbj|BAA84336.1|(AP000363) hypothetical protein [Bacteriophage VT2-Sa] gi|7649882|dbj|BAA94l60.1|(AP000422) hypothetical protein [Escherichia coli O157:H7] Length = 404 Score = 177 bits (449), Expect = 3e−43 Identities = 130/425 (30%), Positives = 204/425 (47%), Gaps = 27/425 (6%) Query: 7 TAYGDPQAMMKQAAGLFAMHMQRNSTLNRLAGKMPAGTA-GAEATLRKQTTQHMPVVRCQ 65 T QA LF + S +N L + A A + KQT+ PVVR Sbjct: 2 TTVTSAQANKLYQVALFTAANRNRSMVNILTEQQEAPKAVSPDKKSTKQTSAGAPVVRIT 61 Query: 66 DLTRGMGDEIRFNLVNPVSALPIMGDNTAEGRGVGMSLSEAGLRVNQARFPVDGGGTMTN 125 DL + GDE+ F++++ +S P MGD EGRG +S ++ L++NQ R VD GG M+ Sbjct: 62 DLNKQAGDEVTFSIMHKLSKRPTMGDERVEGRGEDLSHADFSLKINQGRHLVDAGGRMSQ 121 Query: 126 QRSPADYRALIRPAAQSLMDRYADQTLLVHMAGARGFHDNIEWGVPLAGDPKFNDYAVNP 185 QR+ + + R + + DQ +VH+AGARG + +P A P+F +N Sbjct: 122 QRTKFNLASSARTLLGTYFNDLQDQCAIVHLAGARGDFVADDTILPTAEHPEFKKIMIND 181 Query: 186 VKAPSKNRHFTASGDAVTGVGDNGGELKIASTDLFTMDTVDSMRTVLDQIPLPPPIVKFE 245 V P+ +RHF GD +I + D+F++ VD++ +D++ P V+ Sbjct: 182 VLPPTHDRHFFG--------GDATSFEQIEAADIFSIGLVDNLSLFIDEMAHPLQPVRLS 233 Query: 246 GDKAAGDSPLRVWLLSPAQYNRF---AADPKFRQLQASAIARASQANQNPLFLGDAGLWN 302 GD+ G+ P V ++P Q+N + + + Q+ A+ RA N +PLF G+ +W Sbjct: 234 GDELHGEDPYYVLYVTPRQWNDWYTSTSGKDWNQMMVRAVNRAKGFN-HPLFKGECAMWR 292 Query: 303 GFILVKMP-RPIRFYAGDEMKYCADKFSEAESGLKIPASFADKFAVDRSVILGGQAVLEA 361 ++ K PIRFY G ++ + + A +DR++++LG QA+ A Sbjct: 293 NILVRKYAGMPIRFYQGSKVLVSENNLTATTK------EVAAATNIDRAMLLGAQALANA 346 Query: 362 FANTGKHGGMPFFWSEKELDHGNRVETLVGTIRGVAKTRFAVDVGGGAKEITDYGVTVVD 421 + G+ G F EK+ D NR E + I G+ K RF G ++ D+GV VD Sbjct: 347 Y---GQKAGGHFNMVEKKTDMDNRTEIAISWINGLKKIRFPEKSG----KMQDHGVIAVD 399 Query: 422 TVVPL 426 T V L Sbjct: 400 TAVKL 404 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 102 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 203> which encodes amino acid sequence <SEQ ID 204; NGS 102>.\",\n \"Analysis of this protein sequence reveals the following: Signal Score (−7.5): −6.09 Possible cleavage site: 15 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 2.92 threshold: 0.0 PERIPHERAL Likelihood = 2.92 modified ALOM score: −1.08 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.480(Affirmative) < succ The protein has no homology with sequences in the databases.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 103 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 205> which encodes amino acid sequence <SEQ ID 206; NGS 103>.\",\n \"Analysis of this protein sequence reveals the following: Signal Score (−7.5): −1.29 Possible cleavage site: 34 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 1 value: −0.00 threshold: 0.0 INTEGRAL Likelihood = −0.00 Transmembrane 22-38 (22-38) PERIPHERAL Likelihood = 4.88 modified ALOM score: 0.50 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.100(Affirmative) < succ> The protein has homology with the following sequences in the databases: >gi|11277848|pir||E81145 replicative DNA helicase NMB0885 [imported]-Neisseria meningitidis (group B strain MD58) gi|7226124|gb|AAF41296.1|(AE002441) replicative DNA helicase [Neisseria meningitidis MC58] Length = 468 Score = 233 bits (594), Expect = 5e−60 Identities = 158/456 (34%), Positives = 245/456 (53%), Gaps = 34/456 (7%) Query: 15 SVGAEQNILGGILIEPTAIARCA-ILTPEKFYQAQHRIIFRALLDMAAANEPIDIITLND 73 S+ AEQ++LGG+++E A R A +++ E FY+ +HR+IFR++ + + P D+IT+ + Sbjct: 23 SMEAEQSVLGGLMLENPAWDRIADVVSGEDFYRHEHRLIFRSIAKLINESRPADVITVQE 82 Query: 74 KLEARGEAENAGGLAYLIDLNQNTPSAKNISRYVGIVNDRFVERGLLKASAAIEKIAVSK 133 L+ E E AGG YLI L QNTPSA NI RY IV +R + R L + I + A + Sbjct: 83 DLQRNEELEAAGGFEYLITLAWNTPSAANIRRYAEIVRERSIMRQLAEVGTEIARSAYNP 142 Query: 134 DGGTVAEKLSKAADELAAVGKDAVKRETKTFGQTVEDLIGGLDKRLDGVR--------FG 185 G + L +A +++ + + K +K + DL+ + +R+D + G Sbjct: 143 QGRDAGQLLDEAENKVFQIAESTAK--SKQGFLEMPDLLKEVVQRIDMLYSRDNPDEVTG 200 Query: 186 LPTGLMKLDGMTGGLPDGNLIVIAARPSMGKTVLAENIARFALKQGK-AVHFQSYEMSAV 244 +PTG + LD T GL G+LI++A RPSMGKT + NIA +G+ V S EM Sbjct: 201 VPTGFIDLDKKTSGLQPGDLIIVAGRPSMGKTAFSINIAEHVAVEGRLPVAVFSMEMGGA 260 Query: 245 ELARRGMAAECNIPMQNLKTGNLTQSDYANM----------PIYVSQAKEWKFDVNCDLL 294 +L R + + + LKTG L + + P+Y+ + Sbjct: 261 QLVMRMLGSVGRLDQSVLKTGRLEDEHWGRLNEAVVKLSDAPVYIDETPGLTALELRARA 320 Query: 295 NVDELCFLAKEKKLTTGLDLLVVDHLHIMPRAGRDE--VAELGNISRRLKNLAAELNTPV 352 F K L L+V+D+L +M +GR + +ELG ISR LK LA EL P+ Sbjct: 321 RRLARQFNNK-------LGLIVIDYLQLMAGSGRSDNRASELGEISRSLKALAKELQVPI 373 Query: 353 VLVAQLNRGNTKQADKRPNMADIRGSGAIEQDANIIIMPHRESYYDGNENP--SIAELII 410 + ++QL+R + DKRP M+D+R SGAIEQDA++I+ +R+ YY+ ++P +AE II Sbjct: 374 IALSQLSRTVESRTDKRPMMSDLRESGAIEQDADLIMFLMYRDEYYN-QDSPMKGLAECII 432 Query: 411 AKNRDGEMGTVVCGWKGQFMKFEEEPDLAWQAPKHD 446 K+R+G +G + W GQF KF+ + +A D Sbjct: 433 GKHRNGPVGKIFLTWTGQFTKFDNAAYIPEEAKIED 468 >gi|11277846|pir||E81876 probable replicative DNA helicase (EC 3.6.1.-) NMA1105 [imported]- Neisseria meningitidis (group A strain Z2491) gi|7379799|emb|CAB84367.1|(AL162755) putative replicative DNA helicase [Neisseria meningitidis Z2491] Length = 468 Score = 230 bits (588), Expect = 2e−59 Identities = 158/456 (34%), Positives = 244/456 (52%), Gaps = 34/456 (7%) Query: 15 SVGAEQNILGGILIEPTAIARCA-ILTPEKFYQAQHRIIFRALLDMAAANEPIDIITLND 73 S+ AEQ++LGG+++E A R A +++ E FY+ +HR+IFR++ + + P D+IT+ + Sbjct: 23 SMEAEQSVLGGLMLENPAWDRIADVVSGEDFYRHEHRLIFRSIAKLINESRPADVITVQE 82 Query: 74 KLEARGEAENAGGLAYLIDLNQNTPSAKNISRYVGIVNDRFVERGLLKASAAIEKIAVSK 133 L+ E E AGG YLI L QNTPSA NI RY IV +R + R L + I + A + Sbjct: 83 DLQRNEELEAAGGFEYLITLAQNTPSAANIRRYAEIVRERSIMQLAEVGTEIARSAYNP 142 Query: 134 DGGTVAEKLSKAADELAAVGKDAVKRETKTFGQTVEDLIGGLDKRLDGVR--------FG 185 G + L +A +++ + + K +K + DL+ + +R+D + G Sbjct: 143 QGRDAGQLLDEAENKVFQIAESTAK--SKQGFLEMPDLLKEVVQRIDMLYSRDNPDEVTG 200 Query: 186 LPTGLMKLDGMTGGLPDGNLIVIAARPSMGKTVLAENIARFALKQGK-AVHFQSYEMSAV 244 + TG + LD T GL G+LI++A RPSMGKT + NIA +GK V S EM Sbjct: 201 VSTGFIDLDKKTSGLQPGDLIIVAGRPSMGKTAFSINIAEHVAVEGKLPVAVFSMEMGGA 260 Query: 245 ELARRGMAAECNIPMQNLKTGNLTQSDYANM----------PIYVSQAKEWKFDVNCDLL 294 +L R + + + LKTG L + + P+Y+ + Sbjct: 261 QLVMRMLGSVGRLDQSVLKTGRLEDEHWGRLNEAVVKLSDAPVYIDETPGLTALELRARA 320 Query: 295 NVDELCFLAKEKKLTTGLDLLVVDHLHIMPRAGRDE--VAELGNISRRLKNLAAELNTPV 352 F K L L+V+D+L +M +GR + +ELG ISR LK LA EL P+ Sbjct: 321 RRLARQFNNK-------LGLIVIDYLQLMAGSGRSDNRASELGEISRSLKALAKELQVPI 373 Query: 353 VLVAQLNRGNTKQADKRPNMADIRGSGAIEQDANIIIMPHRESYYDGNENP--SIAELII 410 + ++QL+R + DKRP M+D+R SGAIEQDA++I+ +R+ YY+ ++P +AE II Sbjct: 374 IALSQLSRTVESRTDKRPMMSDLRESGAIEQDADLIMFMYRDEYYN-QDSPMKGLAECII 432 Query: 411 AKNRDGEMGTVVCGWKGQFMKFEEEPDLAWQAPKHD 446 K+R+G +G + W GQF KF+ + +A D Sbjct: 433 GKHRNGPVGKIFLTWTGQFTKFDNAAYIPEEAKIED 468 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 104 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 207> which encodes amino acid sequence <SEQ ID 208; NGS104>.\",\n \"Analysis of this protein sequence reveals the following: Signal Score (−7.5): −2.11 Possible cleavage site: 15 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 5.04 threshold: 0.0 PERIPHERAL Likelihood = 5.04 modified ALOM score: −1.51 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.220(Affirmative) < succ The protein has homology with the following sequences in the databases: >gi|7515458|pir||T13296 hypothetical protein 8-Streptococcus phage phi- O1205 gi|2444088|gb|AAC79524.1|(U88974) ORF8 [Streptococcus thermophilus temperate bacteriophage O1205] Length = 157 Score = 62.1 bits (150), Expect = 3e−09 Identities = 53/161 (32%), Positives = 86/161 (52%), Gaps = 8/161 (4%) Query: 5 TLYRCAADVQAGLDYYFDSETEREDTLEAV--IGQFEVKAQSVIAYIKNQEITEKMLEGH 62 TLY + + D ET + DTLEA+ +E K + + IK+ E + + Sbjct: 3 TLYELTDQLLEIYNMDVDDET-KLDTLEAIDWTTDYENKVEGYVKVIKSLEADIEARKNE 61 Query: 63 IRQMTGKLKAAKARNQSLKDYLARNMQAAGITEIKADDGTFKASFRKSEAVVILDEAQIP 122 +++ G K+ +++ LK LA +M G T + D FK FRKSEAVV+ +E ++P Sbjct: 62 KKRLDGLNKSDQSKIDKLKTALAVSMAETGQTRV--DTTLFKVGFRKSEAVVV-NEEEKLP 118 Query: 123 AEFMREAVKTEPDKTAIRKAIESGRQVAGAKIEGRKNLQIR 163 E+ K PDK +++ ++SG+ + GA +E R+NL IR Sbjct: 119 KEYQIATYK--PDKKTLKBLLKSGKHIEGATLEERRNLNIR 157 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 105 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 209> which encodes amino acid sequence <SEQ ID 210; NGS 105>.\",\n \"Analysis of this protein sequence reveals the following: Signal Score (−7.5): −5.52 Possible cleavage site: 31 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 2.60 threshold: 0.0 PERIPHERAL Likelihood = 2.60 modified ALOM score: −1.02 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.135(Affirmative) < succ> The protein has no homology with sequences in the databases.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 106 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 211> which encodes amino acid sequence <SEQ ID 212; NGS 106>.\",\n \"Analysis of this protein sequence reveals the following: Signal Score (−7.5): 4.8 Possible cleavage site: 26 >>> Seems to have a cleavable N-term signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 27 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 7.80 threshold: 0.0 PERIPHERAL Likelihood = 7.80 modified ALOM score: −2.06 Score for OM-PP discrimination: 4.38 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: 4.38 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Outer membrane?\",\n \"Score: 0.437687 Outer membrane?\",\n \"Score: 0.437687 ----- Final Results ----- bacterial outer membrane --- Certainty= 0.768(Affirmative) < succ> The protein has no homology with sequences in the databases, although it is similar to HMW1 from Haemophilus influenzae.\",\n \"The protein was expressed in E.coli as an insoluble 43.56 kDa His-fusion product and then purified.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 107 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 213> which encodes amino acid sequence <SEQ ID 214; NGS 107>.\",\n \"Analysis of this protein sequence reveals the following: Signal Score (−7.5): −3.83 Possible cleavage site: 51 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 4.61 threshold: 0.0 PERIPHERAL Likelihood = 4.61 modified ALOM score: −1.42 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.146(Affirmative) < succ> The protein has no homology with sequences in the databases.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 108 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 215> which encodes amino acid sequence <SEQ ID 216; NGS108>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −6.14 Possible cleavage site: 19 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 8.43 threshold: 0.0 PERIPHERAL Likelihood = 8.43 modified ALOM score: −2.19 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.574(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir||G81977 probable lipoprotein NMA0586 [imported]-Neisseria meningitidis (group A strain Z2491) emb|CAB83877.1|(AL162753) putative lipoprotein [Neisseria meningitidis Z2491] Length = 280 Score = 52.9 bits (126), Expect = 5e−06 Identities = 43/134 (32%), Positives = 63/134 (46%), Gaps = 23/134 (17%) Query: 174 LGDIRGVATDEDKLPKAGSFQYEGRAFGGNGVLSKESLDNHNGVFRYTIDFDRRKGSGSI 233 +GDI G T DKLP+ G Y G AFG D+ +G YTIDF ++G G I Sbjct: 156 IGDIAGEHTSFDKLPEGGRATYRGTAFGS---------DDASGKLTYTIDFAAKQGHGKI 206 Query: 234 EGMEQYGKIKLEEAAIERIPYRESGSSLGLKDRVSYFGVNEGVAMLEKDNEIKKYHLGIF 293 E ++ ++ ++ AA + P ++ + + ++L E Y LGIF Sbjct: 207 EHLKS-PELNVDLAASDIKPDKKRHAVI-------------SGSVLYNQAEKGSYSLGIF 252 Query: 294 GEAANEVAGAVSQE 307 G A EVAG+ E Sbjct: 253 GGQAQEVAGSAEVE 266 >pir||D81032 hypothetical protein NMB1870 [imported]-Neisseria meningitidis (group B strain MD58) gb|AAF42204.1|(AE002537) hypothetical protein [Neisseria meningitidis MC58] Length = 320 Score = 50.6 bits (120), Expect = 3e−05 Identities = 50/168 (29%), Positives 76/168 (44%), Gaps = 28/168 (16%) Query: 136 VYEQPYSVVRGYFGYSRKDGNPIEGDGQNPEEIPFDLYLGDIRGVATDEDKLPKAGSFQY 195 VY+Q +S + + +D E G+ + F +GDI G T DKLP+ G Y Sbjct: 163 VYKQSHSALTAFQTEQIQDS---EHSGKMVAKRQFR--IGDIAGEHTSFDKLPEGGRATY 217 Query: 196 EGRAFGGNGVLSKESLDNHNGVFRYTIDFDRRKGSGSIEGMEQYGKIKLEEAAIERIPYR 255 G AFG D+ G YTIDF ++G+G IE ++ ++ ++ AA + P Sbjct: 218 RGTAFGS---------DDAGGKLTYTIDFAAKQGNGKIEHLKS-PELNVDLAAADIKPDG 267 Query: 256 ESGSSLGLKDRVSYFGVNEGVAMLEKDNEIKKYHLGIFGEAANEVAGA 303 + + + ++L E Y LGIFG A EVAG+ Sbjct: 268 KRHAVI-------------SGSVLYNQAEKGSYSLGIFGGKAQEVAGS 302 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 109 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 217> which encodes amino acid sequence <SEQ ID 218; NGS109>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.39 Possible cleavage site: 25 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 7.00 threshold: 0.0 PERIPHERAL Likelihood = 7.00 modified ALOM score: −1.90 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.353(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >pir||A82012 hypothetical protein NMA0179 [imported]-Neisseria meningitidis (group A strain Z2491) emb|CAB83494.1|(AL162752) hypothetical protein NMA0179 [Neisseria meningitidis Z2491] Length = 97 Score = 183 bits (464), Expect = 1e−45 Identities = 92/97 (94%), Positives = 95/97 (97%) Query: 44 MKANDKLNRQIDVLQKQSAAIHNEAYIEMNTLLYRHREVVSIHNRKADYAEKGKERIALF 103 MK NDKLNRQIDVLQKQSAAIHNEAYIEMNTLLYRHREVVS+HNRKADYAEKGKE+IALF Sbjct: 1 MKTNDKLNRQIDVLQKQSAAIHNEAYIEMNTLLYRHREVVSVHNRKADYAEKGKEQIALF 60 Query: 104 PRGLNGITKLPAAVLLPERPYHFDMKEVLYIFSRIPR 140 PRGLNGITKLPAAVLLPERPYHFDMKEVL+IFS IPR Sbjct: 61 PRGLNGITKLPAAVLLPERPYHFDMKEVLHIFSWIPR 97 As a homolog was found in serogroup A N.meningitidis but not in serogroup B, NGS109 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 110 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 219> which encodes amino acid sequence <SEQ ID 220; NGS110>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −2.76 Possible cleavage site: 41 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 1 value: −0.00 threshold: 0.0 INTEGRAL Likelihood = −0.00 Transmembrane 88-104 (88-104) PERIPHERAL Likelihood = 7.69 modified ALOM score: 0.50 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.100(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has no homology with sequences in the databases.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 111 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 221> which encodes amino acid sequence <SEQ ID 222; NGS111>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −5.89 Possible cleavage site: 21 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 2.44 threshold: 0.0 PERIPHERAL Likelihood = 2.44 modified ALOM score: −0.99 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.293(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology with the following sequences in the databases: >gb|AAC45840.1|(AF001598) restriction endonuclease [Neisseria gonorrhoeae] Length = 374 Score = 539 bits (1390), Expect = e−152 Identities = 285/285 (100%), Positives = 285/285 (100%) Query: 1 MGFIEPFLSSYTPLSRDYVQARTNRKRQTLLSKIVYTHSGFQRSVTENSNIRQINFLIKT 60 MGFIEPFLSSYTPLSRDYVQARTNRKRQTLLSKIVYTHSGFQRSVTENSNIRQINFLIKT Sbjct: 90 MGFIEPFLSSYTPLSRDYVQARTNRKRQTLLSKIVYTHSGFQRSVTENSNIRQINFLIKT 149 Query: 61 LVEHPQGKLNKKEIAAMMLVDLKTFQQDYLTETELNDYFQQGIESGFIERKYNQISYLWN 120 LVEHPQGKLNKKEIAAMMLVDLKTFQQDYLTETELNDYFQQGIESGFIERKYNQISYLWN Sbjct: 150 LVEHPQGKLNKKEIAAMMLVDLKTFQQDYLTETELNDYFQQGIESGFIERKYNQISYLWN 209 Query: 121 LLDKLDDLKRVGDDLYFAEDAQRIFGNLDEITVRKRDPYLHRLYKNQLQEESEEHYGNVK 180 LLDKLDDLKRVGDDLYFAEDAQRIFGNLDEITVRKRDPYLHRLYKNQLQEESEEHYGNVK Sbjct: 210 LLDKLDDLKRVGDDLYFAEDAQRIFGNLDEITVRKRDPYLHRLYKNQLQEESEEHYGNVK 269 Query: 181 CMLEKLAYPVLIASHIKPFILSDDTEAYDPNNGLLLSRTLDSLFDLKYISFDDEGNMVKS 240 CMLEKLAYPVLIASHIKPFILSDDTEAYDPNNGLLLSRTLDSLFDLKYISFDDEGNMVKS Sbjct: 270 CMLEKLAYPVLIASHIKPFILSDDTEAYDPNNGLLLSRTLDSLFDLKYISFDDEGNMVKS 329 Query: 241 KRLSDDVWRRWCDVKLDNNLLNDKRKSYLAYHRELMLQEDQEFHI 285 KRLSDDVWRRWCDVKLDNNLLNDKRKSYLAYHRELMLQEDQEFHI Sbjct: 330 KRLSDDVWRRWCDVKLDNNLLNDKRKSYLAYHRELMLQEDQEFHI 374 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 112 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 223> which encodes amino acid sequence <SEQ ID 224; NGS112>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −9.08 Possible cleavage site: 54 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 1 value: −1.22 threshold: 0.0 INTEGRAL Likelihood = −1.22 Transmembrane 160-176 (160-177) PERIPHERAL Likelihood = 0.58 modified ALOM score: 0.74 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.149(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_12644572 gi|12644572|sp|Q50973|T2B1_NEIGO TYPE II RESTRICTION ENZYME NGOBI (ENDONUCLEASE NGOBI) (R.NGOBI) (R.NGOI) gb|AAB03207.2|(U42459) NgoI restriction endonuclease R.NgoI [N. gonorrhoeae] Length = 350 Score = 694 bits (1791), Expect = 0.0 Identities = 349/350 (99%), Positives = 349/350 (99%) Query: 1 MTLEEQQAKEALDGIIKKSRVHLYKPIQIAEILYHDRCIKQLDFLNLDTYRNQSKRWRDE 60 MTLEEQQAKEALDGIIKKSRVHLYKPIQIAEILYHDRCIKQLDFLNLDTYRNQSKRWRDE Sbjct: 1 MTLEEQQAKEALDGIIKKSRVHLYKPIQIAEILYHDRCIKQLDFLNLDTYRNQSKRWRDE 60 Query: 61 ICRRFLGISTSSAKFQDNLFEKNAIPPEKLAVLGTLNRQSDGGVESTITKQFFNRFSQM 120 ICRRFLGISTSSAKFQDNLFEKNAIPPEKLAVLGTLNRQSDGGVESTITKQFFNRFSQM Sbjct: 61 ICRRFLGISTSSAKFQDNLFEKNAIPPEKLAVLGTLNRQSDGGVESTITKQFFNRFSQM 120 Query: 121 SEALAYVGNTDRYSFQLSEFLNLFWLEPGLKRSIDKIYEIVVYALFDALVSELGITVSID 180 SEALAYVGNTDRYSFQLSEFLNLFWLEPGLKRSIDKIYEIVVYALFDALVSELGITVSID Sbjct: 121 SEALAYVGNTDRYSFQLSEFLNLFWLEPGLKRSIDKIYEIVVYALFDALVSELGITVSID 180 Query: 181 FPKENLFLWEEYQDFAEKIITMPKNEHLKLPAKIHRVGVTNAADRGLDMWSNFGLAIQVK 240 FPKENLFLWEEYQDFAEKIITMPKNEHLKLPAKIHRVGVTNAADRGLDMWSNFGLAIQVK Sbjct: 181 FPKENLFLWEEYQDFAEKIITMPKNEHLKLPAKIHRVGVTNAADRGLDMWSNFGLAIQVK 240 Query: 241 HLSLDEELAEDIVSSISADRIVIVCKKAEQSVIVSLLTQIGWKSRIQNIVTEDDLISWYE 300 HLSLDEELAEDIVSSISADRIVIVCKKAEQSVIVSLLTQIGWKSRIQNIVTEDDLISWYE Sbjct: 241 HLSLDEELAEDIVSSISADRIVIVCKKAEQSVIVSLLTQIGWKSRIQNIVTEDDLISWYE 300 Query: 301 KALRQQYPIAEALLENIKTEIMREFPAVNEANEFLDFAQNRGYDITVTHF 350 KALRQQYPIAEALLENIKTEIMREFPAVNEANEFLDFAQNRGYDITVTHF Sbjct: 301 KALRQQYPIAEALLENIKTEIMREFPAVNEANEFLDFAQNRGYDITVTHF 350 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 113 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 225> which encodes amino acid sequence <SEQ ID 226; NGS113>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −1.7 Possible cleavage site: 43 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 4 value: −9.77 threshold: 0.0 INTEGRAL Likelihood = −9.77 Transmembrane 187-203 (183-208) INTEGRAL Likelihood = −7.22 Transmembrane 25-41 (19-46) INTEGRAL Likelihood = −4.14 Transmembrane 139-155 (138-155) INTEGRAL Likelihood = −2.87 Transmembrane 86-102 (85-102) PERIPHERAL Likelihood = 1.27 modified ALOM score: 2.45 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.491(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_15902668 gi|15902668|ref|NP_358218.1|\\\\(NC_003098) ABC transporter membrane-spanning permease-glutamine transport [Streptococcus pneumoniae R6] gb|AAK99428.1|(AE008440) ABC transporter membrane-spanning permease- glutamine transport [Streptococcus pneumoniae R6] Length = 226 Score = 218 bits (556), Expect = 7e−56 Identities = 113/218 (51%), Positives = 155/218 (70%) Query: 1 MNWPYLIDAVPKFADAAKLTLELSVYGVVLSLLFLPVAVVTAYRIRPFYALARAYIELS 60 M+W + +P + A LTL ++V+G++ S L GL V+++ YRI +A AYIEILS Sbjct: 1 MNWPYLIDAVPKFADAAKLTLELSVYGVVLSLLFGLPVAVVTAYRIRPFYALARAYIELS 60 RNTPLLIQLFFLY+GLP++GI C + LVFLG SYMAE+ R+G+ A+ + Q G Query: 61 RNTPLLIQLFFLYYGLPKMGIKWDGFTCGVIALVFLGASYMAEAVRAGILAVPKGQVGAG 120 RNTPLLIQLFFLY+GLP++GI C +LVFLG SYMAE+ R+G+ A+ + G G Sbjct: 61 RNTPLLIQLFFLYFGLPRIGIVLSSEVCATLGLVFLGGSYMAESFRSGLEAISQTQQEIG 120 Query: 121 KAIGLSRFQVFRYVELPQVWAVAVPAIGANILFLMKETSVVSTVGIAELLFVTKDVIGMD 180 AIGL+ QVFRYV LPQ AVA+P+ AN++FL+KETSV S V +A+L++V KD+IG+ Sbjct: 121 LAIGLTPLQVFRYVVLPQATAVALPSFSANVIFLIKETSVFSAVALADLMYVAKDLIGLY 180 Query: 181 YKTNEALFLLFAAYLIILLPVSLLARRIENRVRSAKYG 218 Y+T+ AL +L AYLI+LLP+SL+ IE R+R A +G Sbjct: 181 YETDIALAMLVVAYLIMLLPISLVFSWIERRIRHAGFG 218 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 114 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 227> which encodes amino acid sequence <SEQ ID 228; NGS 114>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −0.46 Possible cleavage site: 17 >>> Seems to have a cleavable N-term signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 18 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 3 value: −5.36 threshold: 0.0 INTEGRAL Likelihood = −5.36 Transmembrane 50-66 (47-67) INTEGRAL Likelihood = −4.83 Transmembrane 183-199 (176-200) INTEGRAL Likelihood = −1.81 Transmembrane 72-88 (72-88) PERIPHERAL Likelihood = 0.26 modified ALOM score: 1.57 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.314(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_15902667 gi|15902667|ref|NP_358217.1|\\\\(NC_003098) ABC transporter membrane-spanning permease-glutamine transport [Streptococcus pneumoniae R6] gb|AAK99427.1|(AE008440) ABC transporter membrane-spanning permease- glutamine transport [Streptococcus pneumoniae R6] Length = 225 Score = 218 bits (555), Expect = 9e−56 Identities = 111/206 (53%), Positives = 151/206 (72%) Query: 3 EGLLLTAQISLISVAASCVLGTLFGLVLRSRNRLVRFVGRFYLETIRIVPILVWLFGLYF 62 +GL +T IS++SV S + GT+ G+++ S +R++RF+ R YLE IRI+P LV LF +YF Sbjct: 20 QGLGVTIGISILSVLLSMMFGTVMGIIMTSHSRIIRFLTRLYLEFIRIMPQLVLLFIVYF 79 Query: 63 GLSVWTGIHIGGFWVCVWVFSLWGVAEMGKLVRGALESIEKHQVESGLAPGLSRGQVFRC 122 GL+ I+I G + VF+LWG AEMGDLVRGA+ S+ KHQ ESG A GL+ Q++ Sbjct: 80 GLARNFNINISGETSAIIVFTLWGTAEMGDLVRGAITSLPKHQFESGQALGLTNVQLYYH 139 Query: 123 IELPQSIRRVLPGAVNLFTRMIKTSSLAWLIGVIEVVKVGQQIIENSLLTQPNASFWVYG 182 I +PQ +RR+LP A+NL TRMIKT+SL LIGV+EV KVGQQII+++ LT P ASFW+YG Sbjct: 140 IIIPQVLRRLLPQAINLVTRMIKTTSLVVLIGVVEVTKVGQQIIDSNRLTIPTASFWIYG 199 Query: 183 LIFMLYFFCCWPLSLLAAKLEQKWEH 208 I +LYF C+P+S L+ LE+ W + Sbjct: 200 TILVLYFAVCYPISKLSTHLEKHWRN 225 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 115 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 229> which encodes amino acid sequence <SEQ ID 230; NGS 115>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −0.639999 Possible cleavage site: 38 >>> May be a lipoprotein Amino Acid Composition of Predicted Mature Form: calculated from 23 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 5.25 threshold: 0.0 PERIPHERAL Likelihood = 5.25 modified ALOM score: −1.55 Rule: inner or outer membrane protein Rule: inner or outer membrane protein *** Reasoning Step: 2 Lipoprotein?\",\n \"Inner membrane?\",\n \"----- Final Results ----- bacterial outer membrane --- Certainty= 0.790(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.700(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_4588485 gi|4588485|gb|AAD26123.1|\\\\(AF109148) antigenic protein [Actinobacillus pleuropneumoniae] Length = 278 Score = 407 bits (1045), Expect = e−112 Identities = 212/282 (75%), Positives = 242/282 (85%), Gaps = 7/282 (2%) Query: 1 MKLNAKLKALLASAAIAVGLTACGGGSGDAQSSQSSGAA-TVAAIKEKGVIRIGVFGDKP 59 MKL+ LK LLA+A A LTAC +A ++QSS A +VA IKEKGVIRIGVFGDKP Sbjct: 1 MKLSTTLKTLLATAITAFALTACD----NANNAQSSTAKDSVAQIKEKGVIRIGVFGDKP 56 Query: 60 PFGYVDANGKNQGFDVEIAKDLAKDLLGSPDKVEFVLTEAANRVEYVRSGKVDLILANFT 119 PFGYVDANGK+QGFDVEIAK++A DLLGS DKVEFVLTEAANRVEY++S KVDLILANFT Sbjct: 57 PFGYVDANGKSQGFDVEIAKEIANDLLGSSDKVEFVLTEAANRVEYLKSNKVDLILANFT 116 Query: 120 QTPERAEAVDFADPYMKVALGVVSPKNKPITDMAQLKDQTLLVNKGTTADAFFTKSHPEV 179 +TPERAE VDFA PYM VALGVVSPK + I+D+ QL+ +TLLVNKGTTADA+FTK+HPE+ Sbjct: 117 KTPERAEVVDFAAPYMNVALGVVSPKVRLISDLKQLEGKTLLVNKGTTADAYFTKNHPEI 176 Query: 180 KLLKFDQNTETFDALKDGRGVALAHDNALLWAWAKENPNFEVAIGNLGPAEFIAPAVQKG 239 LLKFDQNTETFDALKDGRGVALAHDNAL+WAWAKENP F+VAIG++GPAE IAPVQKG Sbjct: 177 NLLKFDQNTETFDALKDGRGVALAHDNALVWAWAKENTFDVAIGSVGPAEQIAPAVQKG 236 Query: 240 NADLLNWVNGEIAAMKKDGRLKAAYEKTLLPVYGEKVKPEAL 281 N LL+ +N EIA K +G+LKAAYEKTL+PVYG+ KPE L Sbjct: 237 NQALLDVINKEIAEFKTNGKLKAAYERKTLVPVYGD--KPELL 276 The protein was expressed in E.coli as a soluble 28.16 kDa His-fusion product, lacking its leader peptide and its poly-glycine sequence (GGGSG), and then purified.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 116 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 231> which encodes amino acid sequence <SEQ ID 232; NGS116>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −7.13 Possible cleavage site: 61 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 1 value: −1.86 threshold: 0.0 INTEGRAL Likelihood = −1.86 Transmembrane 51-67 (51-67) PERIPHERAL Likelihood = 1.54 modified ALOM score: 0.87 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.174(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has no homology to sequences in the databases.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 117 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 233> which encodes amino acid sequence <SEQ ID 234; NGS117>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): 0.25 Possible cleavage site: 40 >>> Seems to have a cleavable N-term signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 41 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 2 value: −4.57 threshold: 0.0 INTEGRAL Likelihood = −4.57 Transmembrane 100-116 (99-118) INTEGRAL Likelihood = −1.59 Transmembrane 54-70 (54-70) PERIPHERAL Likelihood = 0.53 modified ALOM score: 1.41 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.283(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_15793413 gi|15793413|ref|NP_283235.1|\\\\(NC_003116) putative integral membrane protein [Neisseria meningitidis Z2491] pir||C81957 probable integral membrane protein NMA0408 [imported]- Neisseria meningitidis (group A strain Z2491) emb|CAB83707.1|(AL162753) putative integral membrane protein [Neisseria meningitidis Z2491] Length = 550 Score = 1115 bits (2885), Expect = 0.0 Identities = 539/550 (98%), Positives = 545/550 (99%) Query: 1 MVAYAFLFLFVTAAVLLIVRSHYRWTYFFASALFVFLAGGMLMLTAQWQRALNFASVWFV 60 MVAY FLFLFVTAA++LI+RSHYRWTYFFASALFVFLAGGMLMLTAQWQRALNFASVWFV Sbjct: 1 MVAYVFLFLFVTAALVLIIRSHYRWTYFFASALFVFLAGGMLMLTAQWQRALNFASVWFV 60 Query: 61 VLILFHRLKIHYYKQPLLISDFLLIADWRNWETLFHYKEAVIGMAGLLALAGYAVFGWSG 120 VLILFHRLKIHYYKQPLLISDFLLIADWRNWETLFHYKEAVIGMAGLLALA YAVFGWSG Sbjct: 61 VLILFHRLKIHYYKQPLLISDFLLIADWRNWETLFHYKEAVIGMAGLLALAAYAVFGWSG 120 Query: 121 ADSLGMPWRWAGAVLFAAAFVSVRHFSKHPGAVKTWLDSLPDDGRDVFLNLPMSCRAVFF 180 ADSL +PWRWAGAVLFAAAFVS+RHFSKHPGAVKTWLDSLPDDGRDVFLNLPMSCRAVFF Sbjct: 121 ADSLDVPWRWAGAVLFAAAFVSMRHFSKHPGAVKTWLDSLPDDGRDVFLNLPMSCRAVFF 180 Query: 181 QVPVFEGDGEAFARQMPSETRPYGMSDEKPDIVVTLMESTLDPHCFDFAAAKIPDLKMFG 240 QVPVFEGDGEAFARQMPSETRP GMSDEKPDIVVTLMESTLDPHCFDFAAAKIPDLKMFG Sbjct: 181 QVPVFEGDGEAFARQMPSETRPCGMSDEKPDIVVTLMESTLDPHCFDFAAAKIPDLKMFG 240 Query: 241 RQEDTVFSSPLRVHTFGGATWKSEFAFLAGVPSTDFGALASGVFYSVVPHLQTGFVRNLR 300 RQEDTVFSSPLRVHTFGGATWKSEFAFLAGVPSTDFGALASGVFYSVVPHLQTGFVRNLR Sbjct: 241 RQEDTVFSSPLRVHTFGGATWKSEFAFLAGVPSTDFGALASGVFYSVVPHLQTGFVRNLR 300 Query: 301 EHGYFCVALSPFTKGNYNAKAAYDHFGFNLMFQPQDLGYPAPMGKNLWHISSEEMMQYAR 360 EHGYFCVALSPFTKGNYNAKAAYDHFGFNLMFQPQDLGYPAPMGKNLWHISSEEMMQYAR Sbjct: 301 EHGYFCVALSPFTKGNYNAKAAYDHFGFNLMFQPQDLGYPAPMGKNLWHISSEEMMQYAR 360 Query: 361 MILEKRHPDLENVRQPMFVYVLTMKEHGPYRTDTDNVFDLDAPDLNAKTVSALNDYIGRI 420 MILEKRHPDLENVRQPMFVYVLTMKEHGPYRTDTDNVFDLDAPDLNAKTVSALNDYIGRI Sbjct: 361 MILEKRHPDLENVRQPMFVYVLTMKEHGPYRTDTDNVFDLDAPDLNAKTVSALNDYIGRI 420 Query: 421 ADLDKAVESFDRYLHERGKPFVFGYFGDHQVPFEGVSVRKKWDYAQPDYVTQFAVRSNIA 480 ADLDKAVESFDRYLHERGKPFVFGYFGDHQVPFEGVSVRKKWDYAQPDYVTQFAVRSNIA Sbjct: 421 ADLDKAVESFDRYLHERGKPFVFGYFGDHQVPFEGVSVRKKWDYAQPDYVTQFAVRSNIA 480 Query: 481 GGFVQRQDFLDLAFAGGVLMEAAGLEAKDGFMRANMAMRGLCGGGLEDCPNRELVGNYRN 540 GGFVQRQ+FLDLAFAGGVLMEAAGLEAKDGFMRANMAMRGLCGGGLEDCPN ELVGNYRN Sbjct: 481 GGFVQRQNFLDLAFAGGVLMEAAGLEAKDGFMRANMAMRGLCGGGLEDCPNWELVGNYRN 540 Query: 541 YLYDVLKIAR 550 YLYDVLKIAR Sbjct: 541 YLYDVLKIAR 550 A homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS117 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 118 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 235> which encodes amino acid sequence <SEQ ID 236; NGS118>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): 0.59 Possible cleavage site: 19 >>> May be a lipoprotein Amino Acid Composition of Predicted Mature Form: calculated from 22 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 8.33 threshold: 0.0 PERIPHERAL Likelihood = 8.33 modified ALOM score: −2.17 Rule: inner or outer membrane protein Rule: inner or outer membrane protein *** Reasoning Step: 2 Lipoprotein?\",\n \"Inner membrane?\",\n \"----- Final Results ----- bacterial outer membrane --- Certainty= 0.790(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.700(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has no homology to sequences in the databases.\",\n \"The protein was expressed in E.coli as a soluble 12.98 kDa His-fusion product and then purified.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 119 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 237> which encodes amino acid sequence <SEQ ID 238; NGS119>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −4.75 Possible cleavage site: 47 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 7.69 threshold: 0.0 PERIPHERAL Likelihood = 7.69 modified ALOM score: −2.04 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.213(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_2625122 gi|2625122|gb|AAB86635.1|\\\\(AF031495) putative hemoglobin receptor component precursor HpuA [Neisseria gonorrhoeae] Length = 360 Score = 668 bits (1724), Expect = 0.0 Identities = 331/331 (100%), Positives = 331/331 (100%) Query: 1 VSIPTATPLPAGEVTLSSDNGNIENINTAGAGSASDAPSRSRRSLDAAPQNTSGISIRQR 60 VSIPTATPLPAGEVTLSSDNGNIENINTAGAGSASDAPSRSRRSLDAAPQNTSGISIRQR Sbjct: 30 VSIPTATPLPAGEVTLSSDNGNIENINTAGAGSASDAPSRSRRSLDAAPQNTSGISIRQR 89 Query: 61 EVEKDYFGYKSKETSFIFKTPGGAQYALSSYADPITVSYSSPKFKIPDRHAGQRLADGSR 120 EVEKDYFGYKSKETSFIFKTPGGAQYALSSYADPITVSYSSPKFKIPDRHAGQRLADGSR Sbjct: 90 EVEKDYFGYKSKETSFIFKTPGGAQYALSSYADPITVSYSSPKFKIPDRHAGQRLADGSR 149 Query: 121 IFICCSDSGATSYAEITKQDYMKFGAWIGPNGEIDLFAGGFPVGKTPPPAFSYGSSTPET 180 IFICCSDSGATSYAEITKQDYMKFGAWIGPNGEIDLFAGGFPVGKTPPPAFSYGSSTPET Sbjct: 150 IFICCSDSGATSYAEITKQDYMKFGAWIGPNGEIDLFAGGFPVGKTPPPAFSYGSSTPET 209 Query: 181 ALSKGKITYQVWGIRVRNGQFVTSSYTPPKSGSYYGTLANTPVSFITANFNSNTLAGKI 240 ALSKGKITYQVWGIRVRNGQFVTSSYTPPKSGSYYGTLANTPVSFITANFNSNTLAGKI Sbjct: 210 ALSKGKITYQVWGIRVRNGQFVTSSYTPPKSGSYYGTLANTPVSFITANFNSNTLAGKI 269 Query: 241 LGNSDYGPDVDIQNATITGPTFSGDATSGGKSGKLEGKFFGKFASTRSSEVSIGGKITFD 300 LGNSDYGPDVDIQNATITGPTFSGDATSGGKSGKLEGKFFGKFASTRSSEVSIGGKITFD Sbjct: 270 LGNSDYGPDVDIQNATITGPTFSGDATSGGKSGKLEGKFFGKFASTRSSEVSIGGKITFD 329 Query: 301 GDRSLDTVFGGVSYEKKLDDTSQDTNHLTKQ 331 GDRSLDTVFGGVSYEKKLDDTSQDTNHLTKQ Sbjct: 330 GDRSLDTVFGGVSYEKKLDDTSQDTNHLTKQ 360 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 120 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 239> which encodes amino acid sequence <SEQ ID 240; NGS120>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −7.24 Possible cleavage site: 38 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 6.42 threshold: 0.0 PERIPHERAL Likelihood = 6.42 modified ALOM score: −1.78 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.280(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gsa_AAR91313 N. gonorrhoeae glycosyltransferase LgtC |WO9610086-A1|09-JUL-1996 Length = 306 Score = 535 bits (1379), Expect = e−151 Identities = 252/253 (99%), Positives = 252/253 (99%) Query: 8 GGGNIRFIDVNPEDFAGFPLNIRHISITTYARLKLGEYIADCDKVLYLDTDVLVRDGLKP 67 GGGNIRFIDVNPEDFAGFPLNIRHISITTYARLKLGEYIADCDKVLYLDTDVLVRDGLKP Sbjct: 54 GGGNIRFIDVNPEDFAGFPLNIRHISITTYARLKLGEYIADCDKVLYLDTDVLVRDGLKP 113 Query: 68 LWDTDLGGNWVGACIDLFVERQEGYKQKIGMADGEYYFNAGVLLINLKKWRRHDIFKMSC 127 LWDTDLGGNWVGACIDLFVERQEGYKQKIGMADGEYYFNAGVLLINLKKWRRHDIFKMSC Sbjct: 114 LWDTDLGGNWVGACIDLFVERQEGYKQKIGMADGEYYFNAGVLLINLKKWRRHDIFKMSC 173 Query: 128 EWVEQYKDVMQYQDQDILNGLFKGGVCYANSRFNFMPTNYAFMANGFASRHTDPLYLDRT 187 EWVEQYKDVMQYQDQDILNGLFKGGVCYANSRFNFMPTNYAFMANGFASRHTDPLYLDRT Sbjct: 174 EWVEQYKDVMQYQDQDILNGLFKGGVCYANSRFNFMPTNYAFMANGFASRHTDPLYLDRT 233 Query: 188 NTAMPVAVSHYCGSAKPWHRDCTVWGAERFTELAGSLTTVPEEWRGKLAVPPTKRMLQRW 247 NTAMPVAVSHYCGSAKPWHRDCTVWGAERFTELAGSLTTVPEEWRGKLAVPPTKRMLQRW Sbjct: 234 NTAMPVAVSHYCGSAKPWHRDCTVWGAERFTELAGSLTTVPEEWRGKLAVPPTKRMLQRW 293 Query: 248 RKKLSARFLRKIY 260 RKKLSARFLRKIY Sbjct: 294 RKKLSARFLRKIY 306 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 121 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 241> which encodes amino acid sequence <SEQ ID 242; NGS121>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −6.22 Possible cleavage site: 37 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 3.23 threshold: 0.0 PERIPHERAL Likelihood = 3.23 modified ALOM score: −1.15 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.402(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_15281345 gi|15281345|dbj|BAB63435.1|\\\\(AB058945) DNA adenine methylase M.Ssu4109IB [Streptococcus suis] Length = 271 Score = 269 bits (687), Expect = 4e−71 Identities = 127/211 (60%), Positives = 158/211 (74%), Gaps = 1/211 (0%) Query: 1 MIFADPPYFLSNDGFSCQNGQMVSVNKGNWDKSKGMAADLEFYEEWLRLCYALLKPNGTI 60 MIFADPPYFLSN G S GQ+VSV+KG+WDK + EF +W+RL +LKPNGTI Sbjct: 44 MIFADPPYFLSNGGISNSGGQVVSVDKGDWDKVNSLEEKHEFNRKWIRLAKNVLKPNGTI 103 Query: 61 WVCGTFHNIYLIGYLMQTVGYHILNNITWEKPNPPPNLSCRFFTHSTETILWAKK-NKKA 119 W+ G+FHNIY +G ++ G+ ILNNITW+K NP PNLSCR+FTHSTETILWA+K +KKA Sbjct: 104 WISGSFHNIYSVGMALEQEGFKILNNITWQKTNPAPNLSCRYFTHSTETILWARKDDKKA 163 Query: 120 KHTFHYEMMKAQNNGKQMKCVWTFAPPNKTEKTFGKHPTQKPLPLLERCILSASNIGDLI 179 +H ++YE+MK N+GKQMK VW K+EK GKHPTQKP LLER IL+++ GD I Sbjct: 164 RHYYNYELMKELNDGKQMKDVWVGGLTKKSEKWAGKHPTQKPEYLLERIILASTREGDYI 223 Query: 180 FDPFMGSGTTGVAALKHGRRFCGCELEEDFL 210 DPF+GSGTTGV A + GR+F G + E D+L Sbjct: 224 LDPFVGSGTTGVVAKRLGRKFIGIDAERDYL 254 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 122 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 243> which encodes amino acid sequence <SEQ ID 244; NGS122>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −2.55 Possible cleavage site: 23 >>> May be a lipoprotein Amino Acid Composition of Predicted Mature Form: calculated from 15 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 11.46 threshold: 0.0 PERIPHERAL Likelihood = 11.46 modified ALOM score: −2.79 Rule: inner or outer membrane protein Rule: inner or outer membrane protein *** Reasoning Step: 2 Lipoprotein?\",\n \"Inner membrane?\",\n \"----- Final Results ----- bacterial outer membrane --- Certainty= 0.790(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.700(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has no homology to sequences in the databases: The protein was expressed in E.coli as an insoluble 14.85 kDa His-fusion product and then purified.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 123 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 245> which encodes amino acid sequence <SEQ ID 246; NGS123>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −5.65 Possible cleavage site: 20 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 4.24 threshold: 0.0 PERIPHERAL Likelihood = 4.24 modified ALOM score: −1.35 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.404(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has no homology to the sequences in the databases.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 124 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 247> which encodes amino acid sequence <SEQ ID 248; NGS124>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −5 Possible cleavage site: 18 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 1 value: −1.59 threshold: 0.0 INTEGRAL Likelihood = −1.59 Transmembrane 289-305 (289-305) PERIPHERAL Likelihood = 3.76 modified ALOM score: 0.82 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.164(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_1617515 gi|1617515|gb|AAC82509.1|\\\\(U365994) pilin gene inverting protein homolog PivNG [Neisseria gonorrhoeae] Length = 320 Score = 614 bits (1584), Expect = e−175 Identities = 311/320 (97%), Positivies = 316/320 (98%) Query: 1 MRNTVGLDISKLTFDATAMVGKTEHSAKFDNDSKGLDQFSDRLKSLGYQNLHICMEATGS 60 MRN VGLDISKLTF+A+AMVGKTEHSAKFDNDSKGLDQFSDRLKSLG QNLHICMEATG+ Sbjct: 1 MRNAVGLDISKLTFNASAMVGKTEHSAKFDNDSKGLDQFSDRLKSLGCQNLHICMEATGS 60 Query: 61 YYEEVADYFAQYYSVYVVNPLKISKYAESRFKRTKTDKQDAKLIAQYCRSAQESELVKRQ 120 YYEEVADYFAQYYSVYVVNPLKISKYAESRFKRTKTDKQDAKLIAQYCR A+ESELVKRQ Sbjct: 61 YYEEVADYFAQYYSVYVVNPLKISKYAESRFKRTKTDKQDAKLIAQYCRLAKESELVKRQ 120 Query: 121 KPTDEQYRLSRMTAAYAKIKSECAAMKNRHHAAKDEEAAKAYAEIIKAMNEQLEVLKEKI 180 KPTDEQYRL RMTAAYAKIKSECAAMKNRHHAAKDEEAAKAYA+IIKAMNEQLEVLKEKI Sbjct: 121 KPTDEQYRLLRMTAAYAKIKSECAAMKNRHHAAKDEEAAKAYAQIIKAMNEQLEVLKEKI 180 Query: 181 KEQTEKPNCKEGVKRLETIPAIGRMTAAVLFHHLTSSKFETSNKFAAFAGLSPQQKESGT 240 KEQTEKPNCKEGVKRLETIPAIGRMTAAVLFHHLTSSKFETSNKFAAFAGLSPQQKESGT Sbjct: 181 KEQTEKPNCKEGVKRLETIPAIGRMTAAVLFHHLTSSKFETSNKFAAFAGLSPQQKESGT 240 Query: 241 SVRGKGKLTKFGNRKLRAVLFMPAMVAVYRIRAFPDFIKRLEEKKKPKKVIIAALMRKLAV 300 SVRGKGKLTKFGNRKLRAVLFMPAMVAVYRIRAFPDFIKRLEEKKKPKKVIIAALMRKLAV Sbjct: 241 SVRGKGKLTKFGNRKLRAVLFMPAMVAVYRIRAFPDFIKRLEEKKKPKKVIIAALMRKLAV 300 Query: 301 IAYHVHKKGGDYDPSRYKSA 320 IAYHVHKKGGDYDPSRYKSA Sbjct: 301 IAYHVHKKGGDYDPSRYKSA 320 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 125 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 249> which encodes amino acid sequence <SEQ ID 250; NGS127>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −5.8 Possible cleavage site: 52 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 1.70 threshold: 0.0 PERIPHERAL Likelihood = 1.70 modified ALOM score: −0.84 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.383(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_1076012 gi|1076012|pir||B55225 stress-sensitive restriction system protein 2-Corynebacterium glutamicum (ATCC 13032) gb|AAC00044.1|(U13922) This orf may encode a typeI or typeIII restriction endonuclease which is stress-sensitive and ATP-dependent.\",\n \"It contains a typical ATP binding region (Walker motif) [Corynebacterium glutamicum] Length = 632 Score = 298 bits (764), Expect = 2e−79 Identities = 199/633 (31%), Positives = 321/633 (50%), Gaps = 32/633 (5%) Query: 2 LRTYLNQLTP-PELADSVKNTVDGFMEKLSQTEPKIA-QNVLLLGNVQSGKTAQVLGVLS 59 L Y + +L + V TVD F + I+ Q VLL G+VQSGKT+ +LG++++ Sbjct: 7 LNNYITSLSDNADLREKVTATVDAFRHTVMDDFDYISDQQVLLYGDVQSGKTSHMLGIIA 66 Query: 60 ALADDGDHKVFLYLTTDSVDLQDQTVKRAKANLKNFIVLSEADDRSFMEVMKAENP--IL 117 D H + + LT+ + L QT R + +V F K+ P + Sbjct: 67 DCLDSTFHTIVI-LTSPNTRLVQQTYDRVAQAFPDTLVCDRDGYNDFRANQKSLTPRKSI 125 Query: 118 VVIKKNARVLKRWRNLFASQSSLKGYPLVIVDDEADAASLNTNSDKPKDASTINKLLND 177 VV+ K VL W +F +L G+P++I+DDEADA SLNT ++ D STIN L Sbjct: 126 VVVGKIPAVLGNWLRVFNDSGALSGHPVLIIDDEADATSLNTKVNQ--SDVSTINHQLTS 183 Query: 178 IKNSCCQSLPIQLTATPQSLLLQHEESDWQPEFIHFFEAGEKYIGGNFVFSDPPS-YIVR 236 I++ +++Q+T TPQ++LLQ ++S+W E + F GE YIGG FS+ + Y+ Sbjct: 184 IRDLATGCIYLQVTGTPQAVLLQSDDSNWAAEHVLHFAPGESYIGGQLFFSELNNPYLRL 243 Query: 237 FIDSELDDMKDESGEIAEGAKQALLSFLITCAEFALCDKANCNFALHPSYKIQDHQAFSK 296 F +++ D+ S A+ ++L+T A F L ++ C +HPS+ H+ F++ Sbjct: 244 FANTQFDEDSRFS--------DAIYTYLLTAALFLRGESLCTMLIHPSHTASSHRDFAQ 295 Query: 297 KIQAFLNDLVQAVNNGEDLAGSFKESYLDLQKTKPDIHHFDEIYEKLTALLENKQISTLV 356 + + L + + +F+ +Y L +T ++ +I L + ++ I + Sbjct: 296 EARLQLTFAFERFYEPM-IQHNFQRAYEQLAQTDSNLPPLRKILNILGGMEDDFSIH--I 352 Query: 357 VNSQTET-DFDLEKGFNIIIGGNVIGRGLTIPKLQTVYYSRTAKKPNADTFWQHRIFGY 415 VNS T + D G+NII+GGN +GRGLT LQTV+Y R +K+P ADT WQH+R+FGY Sbjct: 353 VNSDNPTVEEDWADGYNIIVGGNSLGRGLTFNNLQTVFYVRESKRPQADTLWQHARMFGY 412 Query: 416 DRDKSLLRLYIPFDVYYFFVQLNQANNLIIGQAKNSG--GNIQVIYPKNINPTRKNVLKF 473 R K +R+++P + F ++ N I Q + +I+VI + PTR NVL Sbjct: 413 KRHKDTMRVFMPATIAQTFQEVYLGNEAIKNQLDHGTHINDIRVILGDGVAPTRANVLDK 472 Query: 474 DSINQIVGGVNYFPLHPNEDNLSEINKILPSILKDEIQSDLYQIDIEDLFLVLDKLGRYV 533 + + GGVNYF P N+ ++K L + L + I + + +L+ Sbjct: 473 RKVGNLSGGVNYFAADPRIIKNVEALDKKLLAYLDKHGEDS--TIGMRAIITILNAF-TVD 529 Query: 534 PDDWNKEKFIAGVEALKAQRPSFKTYVLIKTGRKLSRATGTMLSEDDRKLGEKYPNDLFL 593 P+D + F A + + +P ++++T RK+++ TG +LS D+ L L Sbjct: 530 PNDLDLATFKAALLDFERNQPHLTARMVLRTNRKVNQGTGALLSPTDQALSRAEVAHPLL 589 Query: 594 TLYQVVGNKDKG-------WQGKDFWLPNIKLP 619 LY++ G D W W+PNIKLP Sbjct: 590 ILYRIEGVNDAAAQRGEPTWSSDPIWVPNIKLP 622 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 126 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 251> which encodes amino acid sequence <SEQ ID 252; NGS128>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −4.98 Possible cleavage site: 20 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 7.37 threshold: 0.0 PERIPHERAL Likelihood = 7.37 modified ALOM score: −1.97 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.225(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_11357195 gi|11387l95|sp|Q50976|T2F7_NEIGO TYPE II RESTRICTION ENZYME NGOFVII (ENDONUCLEASE NGOFVII) (R.NGOFVII) (R.NGOVII) pir||T10166 restriction endonuclease (EC 3.1.21.-) NgoVII-N. gonorrhoeae gb|AAA86271.1|(U43736) R.NgoVII [Neisseria gonorrhoeae] Length = 326 Score = 651 bits (1679), Expect = 0.0 Identities = 317/326 (97%), Positives = 320/326 (97%) Query: 1 MNTVFSNIANAKITEKSLNAVWMDLFKSADEVLMATGYVSNDAVVELHKILELNDHIQKI 60 MNTVFSNIANAKITEKSLNAVWMDLFKSADEVLMATGYVSNDAVVELHKILELNDHIQKI Sbjct: 1 MNTVFSNIANAKITEKSLNAVWMDLFKSADEVLMATGYVSNDAVVELHKILELNDHIQKI 60 Query: 61 DLLVGMHYLEGFSHLQYDSLCKLNDFLRHEKRGAVYVSPFVKFHGKMYSFKNYQKINGLI 120 DLLVGMHYLEGFSHLQYDSLCKLNDFLRHEKRGAVYVSPFVKFHGKMYSFKNYQKINGLI Sbjct: 61 DLLVGMHYLEGFSHLQYDSLCKLNDFLRHEKRGAVYVSPFVKFHGKMYSFKNYQKINGLI 120 Query: 121 GSANLTCFWDSTERTYETMLHLNGKPAQILQADIQSTIHKLGKNIQEVERPSKFIEHNSH 180 GSANLTCFWDSTERTYETMLHLNGKPAQILQADIQSTIHKLGKNIQEVERPSKFIEHNSH Sbjct: 121 GSANLTCFWDSTERTYETMLHLNGKPAQILQADIQSTIHKLGKNIQEVERPSKFIEHNSH 180 Query: 181 LENCLGVQKIAPEQIRQLFAQTSEYHFSIPAKTEEKSNLNVFFGEGRRDKRGFVKPRPWY 240 LENCLGVQKIAPEQIRQLFAQTSEYHFSIPAKTEEKSNLNVFFGEGRRDKRGFVKPRPWY Sbjct: 181 LENCLGVQKIAPEQIRQLFAQTSEYHFSIPAKTEEKSNLNVFFGEGRRDKRGFVKPRPWY 240 Query: 241 EVELIVSKDITSQEGYPVLKSFTVITDDGWQFQCKTSGDYSKNFRSENDLKTLGKWIKGR 300 EVELIVSKDITSQEGYPVLKSFTVITDDGWQFQCKTSGDYSK + +LKTLGKWIKGR Sbjct: 241 EVELIVSKDITSQEGYPVLKSFTVITDDGWQFQCKTSGDYSKTSTQKMNLKTLGKWIKGR 300 Query: 301 LESHGCLQNNEKITHETLREYGNDHF 326 LESHGCLQNNEKITHETLREYGN+ F Sbjct: 301 LESHGCLQNNEKITHETLREYGNESF 326 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 127 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 253> which encodes amino acid sequence <SEQ ID 254; NGS 129>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −5.5 Possible cleavage site: 48 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 10.03 threshold: 0.0 PERIPHERAL Likelihood = 10.03 modified ALOM score: −2.51 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.545(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_15804186 gi|15804186|ref|NP_290225.1|\\\\(NC_002655) DNA-damage- inducible protein [Escherichia coli O157:H7 EDL933] ref|NP_312547.1|(NC_002695) DNA-damage-inducible protein [Escherichia coli O157:H7] gb|AAG58789.1|AE005591_13 (AE005591) DNA-damage-inducible protein [Escherichia coli O157:H7 EDL933] dbj|BAB37943.1|(AP002566) DNA-damage-inducible protein [Escherichia coli O157:H7] Length = 278 Score = 340 bits (872), Expect = 2e−92 Identities = 161/266 (60%), Positives = 197/266 (73%) Query: 1 MTTENNAFENAKHIDETGNEYWSARTLQQILEYSEWRNFQRAIDKAITACETSGNDKNHH 60 M + FE +H G E+WSAR L +L+Y +WRNFQ+ + +A ACE S + H Sbjct: 5 MNEHQPFEEIRHYGTEGQEFWSARELAPLLDYRDWRNFQKVLAPATQACEASNQAASDH 64 Query: 61 FVETNKMIALGKGGQREVADYRLSRYACYLIVQNGDPSKSVIAAGQTYFAVQARRQELQD 120 FVET KM+LG G QRE+ D LSRYACYL+VQNGDP+K VIAAGQTYFA+Q RRQEL D Sbjct: 65 FVETTKMVVLGSGAQRELEDVHLSRYACYLVVQNGDPAKPVIAAGQTYFAIQTRRQELAD 124 Query: 121 EAAFRSLGEDKQRLLLRRQLREHNTDLAAAAKDAGVEKPVEYAVFQNHGYRGLYGGLDKQ 180 + AF+ L ED++RL LR +L+EHN L AA+ A V ++A+FQNHGY+GLYGGLD++ Sbjct: 125 DEAFKQLREDEKRLFLRNELKEHNKQLVEAAQQAAVATATDFAIFQNHGYQGLYGGLDQK 184 Query: 181 GIHSPRGLKKSQRILDHMNASEPAANLFRATQTEEKLRRKNIQGKTQANRVHFEVGQKVR 240 IH KGLKKSQ+ILDHM ++E AANLFRATQTEEKL+R + K QAN HF+VG KVR Sbjct: 185 AIEQLKGLKKSQKILDHMGSTELAANLFRATQTEEKLKRDGVNSKQQANTTHFDVGSKVR 244 Query: 241 QTIEELGGIMPENQPVPEKSIKQLEN 266 QTI+ELGG MPE P P+ SIKQLEN Sbjct: 245 QTIQELGGTMPEELPTPQVSIKQLEN 270 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 128 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 255> which encodes amino acid sequence <SEQ ID 256; NGS130>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −3.68 Possible cleavage site: 14 >>> Seems to have an uncleavable N-term signal seq Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 3 value: −3.45 threshold: 0.0 INTEGRAL Likelihood = −3.45 Transmembrane 68-84 (68-92) INTEGRAL Likelihood = −1.59 Transmembrane 10-26 (10-26) INTEGRAL Likelihood = −1.44 Transmembrane 46-62 (45-62) PERIPHERAL Likelihood = 1.48 modified ALOM score: 1.19 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.238(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_17988861 gi|17998861|ref|NP_541494.1|\\\\(NC_003318) hypothetical protein [Brucella melitensis] gb|AAL53758.1|(AE009687) hypothetical protein [Brucella melitensis] Length = 99 Score = 108 bits (270), Expect = 3e−23 Identities = 59/91 (64%), Positives = 69/91 (74%) Query: 11 LLFSCMLAVTCPTRLIGFFALRNRTLSRRAQTVMEAAPGCVLISVIAPYFVSDKPHELIA 70 L M +VT TR+ G+ LRNRTLS RA VMEAAPGCVLISVIAP FVSDKP LIA Sbjct: 8 LTILAMASVTYLTRIGGYVLLRNRTLSNRAMAVMEAAPGCVLISVIAPDFVSDKPANLIA 67 Query: 71 IALTAFAACRFSMLFTVLIGVGSSGISGWLM 101 +A+T FAA RFSML TVLIG+G++ I +L+ Sbjct: 6 LAVTVFAATRFSMLPTVLIGMGAASICRYLI 98 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 129 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 257> which encodes amino acid sequence <SEQ ID 258; NGS131>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −1.65 Possible cleavage site: 43 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 7.05 threshold: 0.0 PERIPHERAL Likelihood = 7.05 modified ALOM score: −1.91 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.152(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_16760390 gi|16760390|ref NP_456007.1|\\\\(NC_003198) hypothetical protein [Salmonella enterica subsp.\",\n \"enterica serovar Typhi] emb|CAD01841.1|(AL621270) hypothetical protein [Salmonella enterica subsp.\",\n \"enterica serovar Typhi] Length = 227 Score = 104 bits (259), Expect = 2e−21 Identities = 68/221 (30%), Positivies = 115/221 (51%), Gaps = 11/221 (4%) Query 2 DKEKVLDKIKKCLALGRSVNEHEAAQALRQAQALMEKYKVNAEDIALSKVSEQKAD--RK 59 D++K ++K+KK LAL S N HEAA ALR+A+ LM+ + + DIA+S + E + Sbjct: 3 DQDKHIEKLKKLLALAASGNPHEAALALRRARKLMDVHGITHSDIAMSDIDETISHYWPT 62 Query: 60 MAFKLAGWQWGVANMIADIFGCKSYQRGKT---MMFYGIGNRAETSAYAFDVVYRQISAD 116 + + + G+ N+I + FG S T + FYG RA +AY ++V+ RQ+ Sbjct: 63 GSLRPPRYMLGLMNIIREAFGVNSIIHPGTYPGVGFYGNRERAALAAYTWEVLARQLKKA 122 Query: 117 RRKFLKT-CRAGKPSHRTYLADPFCGGWIASAWETVKKFEMSDEEKAIMDGYKKKEYPDM 175 R++++ + K + RT D+F GW+ + ++ F ++D+E+ +M + + +YP Sbjct: 123 RQQYISAQNKRIKTATRTSRGDQFAEGWVLAVISEIQSFALTDDERELMQQWLEHKYPQT 182 Query: 176 AEARTRDAKSSILQGSKMEYEALTRGMESGKQVKLHYAVNG 216 R R S G Y G G+ V+LH V+G Sbjct: 183 QTTRARKPGRS-RNGDASRY----AGFREGQNVRLHRPVSG 218 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 130 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 259> which encodes amino acid sequence <SEQ ID 260; NGS132>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −4.06 Possible cleavage site: 30 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 2.49 threshold: 0.0 PERIPHERAL Likelihood = 2.49 modified ALOM score: −1.00 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.075(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has no homology to sequences in the databases.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 131 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 261> which encodes amino acid sequence <SEQ ID 262; NGS133>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): 1.64 Possible cleavage site: 53 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 3.82 threshold: 0.0 PERIPHERAL Likelihood = 3.82 modified ALOM score: −1.26 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.068(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has no homology to the following sequences in the databases: Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 132 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 263> which encodes amino acid sequence <SEQ ID 264; NGS135>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −4.67 Possible cleavage site: 39 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 5.52 threshold: 0.0 PERIPHERAL Likelihood = 5.52 modified ALOM score: −1.60 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.457(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has no homology to sequences in the databases: Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 133 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 265> which encodes amino acid sequence <SEQ ID 266; NGS136>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −3.97 Possible cleavage site: 15 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 11.35 threshold: 0.0 PERIPHERAL Likelihood = 11.35 modified ALOM score: −2.77 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.523(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has no homology to sequences in the databases: Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 134 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 267> which encodes amino acid sequence <SEQ ID 268; NGS 137>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −8.52 Possible cleavage site: 51 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 2.81 threshold: 0.0 PERIPHERAL Likelihood = 2.81 modified ALOM score: −1.06 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.374(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has no homology to sequences in the databases.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 135 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 269> which encodes amino acid sequence <SEQ ID 270; NGS138>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −7 Possible cleavage site: 36 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 10.66 threshold: 0.0 PERIPHERAL Likelihood = 10.66 modified ALOM score: −2.63 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.415(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_13559865 gi|13559865|ref|NP_112075.1|\\\\(NC_002730) terminase small subunit [Bacteriophage HK620] gb|AAK28890.1|AF335538_42 (AF335538) terminase small subunit [Bacteriophage HK620] Length = 140 Score = 125 bits (313), Expect = 5e−28 Identities = 56/122 (45%), Positivies = 85/122 (68%) Query: 4 TKRKLGRPTDYTKDMADKICEKIANGRSLRSICAEDGVPPMKTIYRWLEANEEFRHQYAR 63 T+ K GRP+DY ++AD IC +++G SL +C G+P T++RWL +E+FR +YA+ Sbjct: 3 TEPKAGRPSDYMPEVADDICSLLSSGESLLKVCKRPGMPDKSTVFRWLAKHEDFRDKYAK 62 Query: 64 AREKQADYFAEEIIEIADSAQAESAAVSKAKLQIDARKWAASKIAPKKYGDKSELDVKSGDG 125 A E +AD EEI EIAD+A ++A V+KA+L++D RKWA +++ P+KYGDK ++ DG Sbjct: 63 ATEARADSIFEEIFEIADNAIPDAAEVAKARLRVDTRKWALARMNPRKYGDKVTNELVGKDG 124 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 136 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 271> which encodes amino acid sequence <SEQ ID 272; NGS139>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −1.49 Possible cleavage site: 32 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 8.65 threshold: 0.0 PERIPHERAL Likelihood = 8.65 modified ALOM score: −2.23 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.301(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_16127009 gi|16127009|ref|NP_421573.1|\\\\(NC_002696) hypothetical protein [Caulobacter crescentus] gb|AAK24741.1|(AE005943) hypothetical protein [Caulobacter crescentus] Length = 184 Score = 59.7 bits (143), Expect = 4e−08 Identities = 50/164 (30%), Positives = 74/164 (44%), Gaps = 20/164 (12%) Query: 30 ASGREFRTAYYTYPQWRFSLSFEVLRTKASVNELEKLAGFFNARKGSFESFLYEDPAD-- 87 ASG E RT+ ++ + R+ ++ ++E+ +L FF AR+G F + DPAD Sbjct: 5 ASGHERRTSPWSQSRRRYLIA----TAPRPLDEIAELVAFFEARRGRLHGFRFRDPADFK 60 Query: 88 -------NAVTDQPVGNTVQGAR-YQLVRSMGGFIEPVSAVKERP-----AVKVGGTAL 134 A DQ +G T GV + +QL ++ G E V+ +P V V G L Sbjct: 61 SCAPSVQPAAGDQAIG-TGDGVRKKAFQLRKTYGAGGEAVARTIAKPVAGTVTVAVAGVVL 119 Query: 135 AYGRDYTVTDKGVLVFNTPQPPGRPITWTGGFYFRVRFTSDTVD 178 A G G++ NT P G +T F VRF D +D Sbjct: 120 APGAFAVDVTTGLITLNTAPPAGAAVTAGFAFDTPVRFDLDRLD 163 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 137 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 273> which encodes amino acid sequence <SEQ ID 274; NGS140>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −3.86 Possible cleavage site: 31 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 1 value: −4.94 threshold: 0.0 INTEGRAL Likelihood = −4.94 Transmembrane 34-50 (31-54) PERIPHERAL Likelihood = 8.01 modified ALOM score: 1.49 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.297(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_17987625 gi|17987625|ref|NP_540259.1|\\\\(NC_003317) Hypothetical Phage Protein [Brucella melitensis] gb|AAL52523.1|(AE009572) Hypothetical Phage Protein [Brucella melitensis] Length = 144 Score = 72.4 bits (176), Expect = 5e−12 Identities = 43/119 (36%), Positives = 64/119 (53%), Gaps = 7/119 (5%) Query: 10 RIVEEARSWLGTPYHHHAMVKGAGVDCAMLLVAVYGAV-GLLPEGFDPRPYPQDWHLHRD 68 R++ EA W+GTPY H A G DC L+ ++A+ G+ PE +P Y DW Sbjct: 6 RVLAEAHRWIGTPYRHGASTLGVSCDCLGVRQIWRALYGVEPE--NPGVYAPDWAEVSQ 63 Query: 69 CERYLGFVTQFC--RETESPQAGDIAV--WRFGRSFSHGGILAGGGKVIHSYIGRGVVS 123 + L ++ RE +PQ GD+ W+ G + H GI+A G+ IH+Y G GV++ Sbjct: 64 GDPMLEAAVRYMVRREEHAPQPGDLLVFRWKPGFAAKHMGIMAREGRFIHAYQGHGVLA 122 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 138 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 275> which encodes amino acid sequence <SEQ ID 276; NGS141>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): 5.35 Possible cleavage site: 28 >>> Seems to have a cleavable N-term signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 29 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 8.86 threshold: 0.0 PERIPHERAL Likelihood = 8.86 modified ALOM score: −2.27 Score for OM-PP discrimination: 1.53 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: 1.53 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Outer membrane?\",\n \"Score: 0.152929 Outer membrane?\",\n \"Score: 0.152929 ----- Final Results ----- bacterial outer membrane --- Certainty= 0.512(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.320(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_5915870 gi|5915870|sp|Q50940|CAH_NEIGO Carbonic anhydrase precursor (Carbonate dehydratase) emb|CAA72038.1|(Y11152) carbonic anhydrase [Neisseria gonorrhoeae] Length = 252 Scare = 523 bits (1347), Expect = e−147 Identities = 252/252 (100%), Positives = 252/252 (100%) Query: 1 MPRFPRTLPRLTAVLLLACTAFSAAAHGNHTHWGYTGHDSPESWGNLSEEFRLCSTGKNQ 60 MPRFPRTLPRLTAVLLLACTAFSAAAHGNHTHWGYTGHDSPESWGNLSEEFRLCSTGKNQ Sbjct: 1 MPRFPRTLPRLTAVLLLACTAFSAAAHGNHTHWGYTGHDSPESWGNLSEEFRLCSTGKNQ 60 Query: 61 SPVNITETVSGKLPAIKVNYKPSMVDVENNGHTIQVNYPEGGNTLTVNGTYTLKQFHFH 120 SPVNITETVSGKLPAIKVNYKPSMVDVENNGHTIQVNYPEGGNTLTVNGTYTLKQFHFH Sbjct: 61 SPVNITETVSGKLPAIKVNYKPSMVDVENNGHTIQVNYPEGGNTLTVNGTYTLKQFHFH 120 Query: 121 VPSENQIKGRTFPMEAHFVHLDENKQPLVLAVLYEAGKTNGRLSSIWNVMPMTAGKVKLN 180 VPSENQIKGRTFPMEAHFVHLDENKQPLVLAVLYEAGKTNGRLSSIWNVMPMTAGKVKLN Sbjct: 121 VPSENQIKGRTFPMEAHFVHLDENKQPLVLAVLYEAGKTNGRLSSIWNVMPMTAGKVKLN 180 Query: 181 QPFDASTLLPKRLKYYRFAGSLTTPPCTEGVSWLVLKTYDHIDQAQAEKFTRAVGSENNR 240 QPFDASTLLPKRLKYYRFAGSLTTPPCTEGVSWLVLKTYDHIDQAQAEKFTRAVGSENNR Sbjct: 181 QPFDASTLLPKRLKYYRFAGSLTTPPCTEGVSWLVLKTYDHIDQAQAEKFTRAVGSENNR 240 Query: 241 PVQPLNARVVIE 252 PVQPLNARVVIE Sbjct: 241 PVQPLNARVVIE 252 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 139 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 277> which encodes amino acid sequence <SEQ ID 278; NGS142>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −0.49 Possible cleavage site: 22 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 1.22 threshold: 0.0 PERIPHERAL Likelihood = 1.22 modified ALOM score: −0.74 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.145(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_15794480 gi|15794480|ref|NP_284302.1|\\\\(NC_003116) hypothetical protein [Neisseria meningitidis Z2491] pir||F81851 hypothetical protein NMA1587 [imported]-Neisseria meningitidis (group A strain Z2491) emb|CAB84814.1|(AL162756) hypothetical protein [Neisseria meningitidis Z2491] Length = 181 Score = 358 bits (919), Expect = 6e−98 Identities = 173/181 (95%), Positives = 178/181 (97%) Query: 1 LKTDTARMNNLIPEHLAAYAHSDNLQIEGGHRCFSLSCQGRDTFHIRYYGEPFDGLITDT 60 +KTDTA+MNNLIPEHLAAYAHSD+LQIEG HRCFSLSCQGRDTFHIRYYGEPFDGL+TDT Sbjct: 1 MKTDTAKMNNLIPEHLAAYAHSDSLQIEGVHRCFSLSCQGRDTFHIRYYGEPFDGLMTDT 60 Query: 61 DKAPVKIVAVEAVSGDEIVLFDGAEHGYNAMFCDKYSQNQKQNRTLTDLDEYTYRVPIHL 120 DKAPVKIVAVEAVSGDEIVLFDGAEHGYNAMFCDKYS NQKQNRTLTDLDEYTYRV IHL Sbjct: 61 DKAPVKIVAVEAVSGDEIVLFDGAEHGYNAMFCDKYSPNQKQNRTLTDLDEYTYRVLIHL 120 Query: 121 YYNIDYEDEYEDFVNSEGQVPLIDGRIISFDSLKRNGFDAISIDLIDEKHSVRELLNEELS 181 YYNIDYEDEYEDFVNSEGQVPLIDGRIISFDSLKRNGFDAIS+DLIDEKHSVRELLNEELS Sbjct: 121 YYNIDYEDEYEDFVNSEGQVPLIDGRIISFDSLKRNGFDAISVDLIDEKHSVRELLNEELS 181 A homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS142 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 140 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 279> which encodes amino acid sequence <SEQ ID 280; NGS143>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −2.51 Possible cleavage site: 57 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 4 value: −15.23 threshold: 0.0 INTEGRAL Likelihood = −15.23 Transmembrane 84-100 (79-107) INTEGRAL Likelihood = −8.12 Transmembrane 259-275 (250-281) INTEGRAL Likelihood = −4.14 Transmembrane 159-175 (153-176) INTEGRAL Likelihood = −3.88 Transmembrane 216-232 (216-235) PERIPHERAL Likelihood = 1.11 modified ALOM score: 3.55 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.709(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_5764059 gi|5764059|emb|CAB53350.1|\\\\(AJ010260) NosR protein [Paracoccus denitrificans] Length = 724 Score = 393 bits (1009), Expect = e−108 Identities = 191/379 (50%), Positives = 249/379 (65%), Gaps = 22/379 (5%) Query: 1 LMVQRVLSVNDKAFVTADLDYELPQAYYVDDPKAPPVEISAPVEAVPAAASDTASDGIAE 60 L+VQR + +K F T DL Y+LPQ Y AP A PAA +D Sbjct: 358 LLVQREVGPIEKVFHTFDLGYQLPQKYLRSIAPAPEA---------AAPAAQAD-------- 402 Query: 61 DASAENGVSNQLWKQIWAKQGQIVVVGIALTILLLVFLFQDWIVRYEKWYDRFRFAFLT 120 E+ QLWK+IW + +I + L +L VF FQ + RYE+ + FR A+LT Sbjct: 403 ----ESQAQAQLWKRIWLDSKPKIAGLAAMLLVLTGVFFFQSFTTRYERAFYVFRMAYLT 458 Query: 121 FTLFYIGWYAQAQLSVVNTLTLFSAILTEFHWEFFLMDPIVFILWLFTAATMLLWNRGTF 180 TL ++GWYA AQLSVVN + LF +++ F W+ FL+DP+ FILW AA +L W RG + Sbjct: 459 VTLVFLGWYANAQLSVVNLMALFGSLVNGFSWQAFLLDPLTFILWFAVAAALLFWGRGAY 518 Query: 181 CGWLCPFGSLQELTNRIAKKLGVKQITVPHMLHTRLNVIKYLILFGFLAISLYDLGTAEK 240 CGWLCPFG+LQELTN++A+KL + Q T+P LH RL +KY+I G +SL + AE Sbjct: 519 CGWLCPFGALQELTNQVARKLRIPQWTLPWGLHERLWPVKYMIFLGLFGVSLMSVEQAEH 578 Query: 241 FAEVEPFKTAIILKFMCDWWFVAPAVALLIAGLFIERFFCRYLCPLGAGIALPGRFRVFD 300 AEVEPFKTAIILKF+ W FVA+A ALLIAGLF+ERF+CRYLCPLGA +A+P R R+FD Sbjct: 579 LAEVEPFKTAIILKFIRAWPFVAYAAALLIAGLFVERFYCRYLCPLGAALAIPARMRMFD 638 Query: 301 WLRRYKMCGNPCQICTHBCPVQAIAPEGDIHPNECIQCLHCQVMYHHDTRCPQVVAENKK 360 WL+RY CGNPCQ C +CPVQ+I P G+I+PNECI CLHCQV+Y +T CP V+ KK Sbjct: 639 WLKRYHECGNPCQTCARQCPVQSIHPTGEINPNECINCLHCQVLYQSETTCPVVI---KK 695 Query: 361 KQKQAAAKSGELENVSKQP 379 +++ A +G + + + P Sbjct: 696 LKRREAVAAGSMPKLGQPP 714 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 141 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 281> which encodes amino acid sequence <SEQ ID 282; NGS144>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): 1.23 Possible cleavage site: 21 >>> May be a lipoprotein Amino Acid Composition of Predicted Mature Form: calculated from 20 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 1 value: −0.37 threshold: 0.0 INTEGRAL Likelihood = −0.37 Transmembrane 90-106 (89-106) PERIPHERAL Likelihood = 10.82 modified ALOM score: 0.57 Rule: inner or outer membrane protein Rule: inner or outer membrane protein Rule: cytoplasmic membrane protein *** Reasoning Step: 2 Lipoprotein?\",\n \"Inner membrane?\",\n \"----- Final Results ----- bacterial outer membrane --- Certainty= 0.790(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.734(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology no to sequences in the databases.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 142 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 283> which encodes amino acid sequence <SEQ ID 284; NGS145>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): 1.5 Possible cleavage site: 19 >>> Seems to have a cleavable N-term signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 20 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 8.70 threshold: 0.0 PERIPHERAL Likelihood = 8.70 modified ALOM score: −2.24 Score for OM-PP discrimination: −9.24 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: −9.24 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Periplasmic space?\",\n \"Score: 0.924443 Periplasmic space?\",\n \"Score: 0.924443 ----- Final Results ----- bacterial periplasmic space --- Certainty= 0.931(Affirmative) < succ> bacterial outer membrane --- Certainty= 0.231(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_5051426 gi|5051426|emb|CAB45007.1|\\\\(AJ242839) OpcA protein [Neisseria gonorrhoeae] Length = 263 Score = 531 bits (1369), Expect = e−150 Identities = 262/263 (99%), Positives = 263/263 (99%) Query: 1 MKKALLALTIAAISGTAMAQLPDFLGKGEYTVRTDISKQTLKNADLKEKHKVQKNIGFRA 60 MKKALLALTIAAISGTAMAQLPDFLGKGEYTVRTDISKQTLKNADLKEKHKVQKNIGFRA Sbjct: 1 MKKALLALTIAAISGTAMAQLPDFLGKGEYTVRTDISKQTLKNADLKEKHKVQKNIGFRA 60 Query: 61 DMPFDDIHHGMRFEVSHSRDKKDMYVVTESTTKPFGKDVEEKRTDVYAGYTYTQPISEAT 120 DMPFDDIHHGMRFEVSHSRDKKDMYVVTESTTKPFGKDV+EKRTDVYAGYTYTQPISEAT Sbjct: 61 DMPFDDIHHGMRFEVSHSRDKKDMYVVTESTTKPFGKDVKEKRTDVYAGYTYTQPISEAT 120 Query: 121 KLRAGLGLGYEKYKDAVANEKGTVSTEREAFYTKAHADLTSDLGGGWYLNPWAEVKVDLD 180 KLRAGLGLGYEKYKDAVANEKGTVSTEREAFYTKAHADLTSDLGGGWYLNPWAEVKVDLD Sbjct: 121 KLRAGLGLGYEKYKDAVANEKGTVSTEREAFYTKAHADLTSDLGGGWYLNPWAEVKVDLD 180 Query: 181 AKLKHNATVAGVSADINAKTRGWGVGVGANIGKQITDTVGIEAGPFYKHRHFKASGSFVL 240 AKLKHNATVAGVSADINAKTRGWGVGVGANIGKQITDTVGIEAGPFYKHRHFKASGSFVL Sbjct: 181 AKLKHNATVAGVSADINAKTRGWGVGVGANIGKQITDTVGIEAGPFYKHRHFKASGSFVL 240 Query: 241 DGGNIRVDPTKINEYGVRVGVKF 263 DGGNIRVDPTKINEYGVRVGVKF Sbjct: 241 DGGNIRVDPTKINEYGVRVGVKF 263 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 143 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 285> which encodes amino acid sequence <SEQ ID 286; NGS146>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): 0.1 Possible cleavage site: 51 >>> Seems to have a cleavable N-term signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 52 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 3.50 threshold: 0.0 PERIPHERAL Likelihood = 3.50 modified ALOM score: −1.20 Score for OM-PP discrimination: −15.70 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: −15.70 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Periplasmic space?\",\n \"Score: 1.56979 Periplasmic space?\",\n \"Score: 1.56979 ----- Final Results ----- bacterial periplasmic space --- Certainty= 0.944(Affirmative) < succ> bacterial outer membrane --- Certainty= 0.375(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_5051429 gi|5051429|emb|CAB45013.1|\\\\(AJ242839) hypothetical protein [Neisseria gonorrhoeae] Length = 109 Score = 216 bits (549), Expect = 2e−55 Identities = 109/109 (100%), Positives = 109/109 (100%) Query: 1 MFKRPEEIIVLILAVLWIAGTYFLAALFGADAYVLKITALTLLWSAASFLLWQKKPQPA 60 MFKRPEEIIVLILAVLWIAGTYFLAALFGADAYVLKITALTLLWSAASFLLWQKKPQPA Sbjct: 1 MFKRPEEIIVLILAVLWIAGTYFLAALFGADAYVLKITALTLLWSAASFLLWQKKPQPA 60 Query: 61 YLAAAARLPDHLLVAVASESIGRTRFFTLACIMDVQHNLSPDSRNRRLSV 109 YLAAAARLPDHLLVAVASESIGRTRFFTLACIMDVQHNLSPDSRNRRLSV Sbjct: 61 YLAAAARLPDHLLVAVASESIGRTRFFTLACIMDVQHNLSPDSRNRRLSV 109 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 144 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 287> which encodes amino acid sequence <SEQ ID 288; NGS147>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −4.09 Possible cleavage site: 40 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 1 value: −2.28 threshold: 0.0 INTEGRAL Likelihood = −2.28 Transmembrane 36-52 (36-52) PERIPHERAL Likelihood = 5.20 modified ALOM score: 0.96 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.191(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_6606516 gi|6606516|gb|AAF19189.1|AF200716_2 \\\\(AF200716) trafficking protein B [Neisseria gonorrhoeae] Length = 139 Score = 274 bits (700), Expect = 7e−73 Identities = 139/139 (100%), Positivies = 139/139 (100%) Query: 2 MILLDTNVISEPLRPQPNERVVAWLDSLILEDVYLSAITVAELRLGVALLLNGKKKNVLH 61 MILLDTNVISEPLRPQPNERVVAWLDSLILEDVYLSAITVAELRLGVALLLNGKKKNVLH Sbjct: 1 MILLDTNVISEPLRPQPNERVVAWLDSLILEDVYLSAITVAELRLGVALLLNGKKKNVLH 60 Query: 62 ERLEQSILPLFAGRILPFDEPVAAIYAQIRSYAKTHGKEIAAADGYIAATAKQHSLTVAT 121 ERLEQSILPLFAGRILPFDEPVAAIYAQIRSYAKTHGKEIAAADGYIAATAKQHSLTVAT Sbjct: 61 ERLEQSILPLFAGRILPFDEPVAAIYAQIRSYAKTHGKEIAAADGYIAATAKQHSLTVAT 120 Query: 122 RDTGSFFAADVAVFNPWHD 140 RDTGSFFAADVAVFNPWHD Sbjct: 121 RDTGSFFAADVAVFNPWHD 139 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 145 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 289> which encodes amino acid sequence <SEQ ID 290; NGS148>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): 0.86 Possible cleavage site: 47 >>> Seems to have a cleavable N-term signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 48 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 5 value: −15.44 threshold: 0.0 INTEGRAL Likelihood = −15.44 Transmembrane 157-173 (142-181) INTEGRAL Likelihood = −12.15 Transmembrane 62-78 (56-83) INTEGRAL Likelihood = −6.32 Transmembrane 194-210 (191-212) INTEGRAL Likelihood = −4.30 Transmembrane 87-103 (85-104) INTEGRAL Likelihood = −2.60 Transmembrane 121-137 (121-142) PERIPHERAL Likelihood = 2.92 modified ALOM score: 3.59 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.718(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_15777859|gi|15777859|gb|AAL05955.1|\\\\(AY048756) putative cadmium binding protein [Staphylococcus aureus] Length = 209 Score = 354 bits (908), Expect = 1e−96 Identities = 177/208 (85%), Positives = 194/208 (93%) Query: 14 MRCFMFSTVITAAVLYIATAVDLLVILLIFFARANTRKEYRDIYIGQYLGSVILILVSLF 73 MRC M TV+TAAVLYIATAVDLLVILLIFFARA TRKEYRDIY+GQYLGS+ILILVSLF Sbjct: 1 MRCIMIQTVVAAAVLYIATAVDLLVILLIFFARAKTRKEYRDIYVGQYLGSIILILVSLF 60 Query: 74 LAFVLNYVPEKWVLGLLGLIPIYLGIKVAIYDDCEGEKRAKKELDEKGLSKLVGIVALVT 133 LAFVLNYVPEKW+LGLLGLIPIYLGIKVAIYDDCEGEKRAKKEL+EKGLSKLVG VA+VT Sbjct: 61 LAFVLNYVPEKWILGLLGLIPIYLGIKVAIYDDCEGEKRAKKELNEKGLSKLVGTVAIVT 120 Query: 134 VASCGADNIGLFVPYFVTLDLVDLLVTLLVFLILIFVLVYTAQRLANISGVGEIVEKFSR 193 +ASCGADNIGLFVPYFVTL + +LL+TL VFLILIF LV+TAQ+LANI G+GEIVEKFSR Sbjct: 121 IASCGADNIGLFVPYFVTLSVTNLLLTLFVFLILIFFLVFTAQKLANIPGIGEIVEKFSR 180 Query: 194 WIMAVIYIGLGLFIIIENNTIRTIISII 221 WIMA+IYI LGLFIIIEN+TI+TI+ I Sbjct: 181 WIMAIIYIALGLFIIIENDTIQTILGFI 208 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 146 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 291> which encodes amino acid sequence <SEQ ID 292; NGS149>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −0.63 Possible cleavage site: 43 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 2.12 threshold: 0.0 PERIPHERAL Likelihood = 2.12 modified ALOM score: −0.92 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.122(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_15675455 gi|15675455|ref|NP_269629.1|\\\\(NC_002737) conserved hypothetical protein [Streptococcus pyogenes] [Streptococcus pyogenes M1 GAS] gb|AAK34350.1|(AE006588) conserved hypothetical protein [Streptococcus pyogenes M1 GAS] Length = 224 Score = 106 bits (264), Expect = 3e−22 Identities = 63/151 (41%), Positives = 85/151 (55%), Gaps = 12/151 (7%) Query: 20 LSALQHYAFCPRQCALIHNEQAWAENYLTAQGKALHERVDSDE-PETCKGVRFEWTVHVL 78 LS +QH+ FC RQ ALIH EQ W +N TA G+ LH + D+ E K + + + Sbjct: 11 LSGIQHFQFCKRQWALIHIEQQWLDNEATAHGQVLHTKADNPYIKEKRKELLVSRAMPIS 70 Query: 79 ADKLGISGILDLVE---------VDTKTGRLKP--VEYKRGKPKPDPGDEIQLCAQGLCL 127 + +LG+SGI+D+VE + K G+ P VEYKRGKPK D D +QL AQ +CL Sbjct: 71 SAELGLSGIMDVVEFYKDDQGVSLRGKRGKWLPKVVEYKRGKPKKDTRDIVQLVAQTMCL 130 Query: 128 EEMTGQTVSEGALWYMQTRHRVPVVFSDGLR 158 EE ++EG L+Y RV V + LR Sbjct: 131 EETLDCDINEGCLYYHSVNQRVIVPMTSALR 161 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 147 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 293> which encodes amino acid sequence <SEQ ID 294; NGS150>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −0.71 Possible cleavage site: 19 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 1 value: −0.85 threshold: 0.0 INTEGRAL Likelihood = −0.85 Transmembrane 79-95 (79-96) PERIPHERAL Likelihood = 6.52 modified ALOM score: 0.67 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.134(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has no homology to sequences in the databases.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 148 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 295> which encodes amino acid sequence <SEQ ID 296; NGS151>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): 3.47 Possible cleavage site: 23 >>> Seems to have a cleavable N-term signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 24 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 14.32 threshold: 0.0 PERIPHERAL Likelihood = 14.32 modified ALOM score: −3.36 Score for OM-PP discrimination: −32.29 Rule: outer membrane or periplasmic protein Score for OM-PP discrimination: −32.29 Rule: outer membrane or periplasmic protein *** Reasoning Step: 2 Periplasmic space?\",\n \"Score: 3.22889 Periplasmic space?\",\n \"Score: 3.22889 ----- Final Results ----- bacterial periplasmic space --- Certainty= 0.933(Affirmative) < succ> bacterial outer membrane --- Certainty= 0.253(Affirmative) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gsa_AAY75310 Neisseria gonorrheae ORF 649 protein sequence SEQ ID N0:2094|WO9957280-A2|21-MAR-2000 Length = 103 Score = 35.4 bits (80), Expect = 0.32 Identities = 25/85 (29%), Positives = 38/85 (44%), Gaps = 5/85 (5%) Query: 7 ILTGILLATALPASAHGMHKSKPLAMDELPPIQQYFKRAETCYNKAGNKADFARN-NTK 65 + T T+ PA H H SK L P C++Y +R Y GN + N + Sbjct: 13 VSTTAAAGTSEPAHRHTKHISKA-NKQMLHPECRKYLERRAAWYRSQGNVQELRENKKAR 71 Query: 66 FLFQALPAADLGQRKQMCQIAMDSF 90 F+ LP A ++K C+ A ++F Sbjct: 72 KAFRTLPYA---EQKIQCRAAYEAF 93 The protein was expressed in E.coli as a soluble 9.35 kDa His-fusion product and then purified.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 149 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 297> which encodes amino acid sequence <SEQ ID 298; NGS152>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −5.46 Possible cleavage site: 18 >>> Seems to have an uncleavable N-term signal seq Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 1 value: −3.19 threshold: 0.0 INTEGRAL Likelihood = −3.19 Transmembrane 368-384 (367-384) PERIPHERAL Likelihood = 0.53 modified ALOM score: 1.14 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.227(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gsa_AAY81609 Streptococcus pneumoniae type 4 protein sequence #109 |WO200006737-A2|24-MAY-2000 Length = 1237 Score = 48.1 bits (113), Expect = 4e−04 Identities = 80/312 (25%), Positives = 142/312 (44%), Gaps = 59/312 (18%) Query: 57 RRQARIRVGNLITDSLEHIRVKALLPLPL----KLPVKRI---NLPRNLPALPVRLRKTI 109 RRQ R + ++ L+H RV L P+ ++PV+++ +PR A RL++ + Sbjct: 941 RRQVR-QPQQVLVHQLQHQRVHRLRRQPVHQSQQVPVRQLPHQQVPRLQQAPVRRLQQVL 999 Query: 110 SPRQIGDALPILKLQRI--RLTLHLKPLPLHPQLGLLHIKRPVRIPLRHLAVQRTLVRLN 167 +P+ P+ + Q++ RL H + PL L +P R + L QR VRLN Sbjct: 1000 APQP--QPQPVRQPQQVSQRLNRHQRVRPLQQVLA----PQPQRQQVHRL--QRQRVRLN 1051 Query: 168 RRIKPPLLQHRLTVRRILRRSRRQPFPAQFPDRRIFIMFRHNPARRIKLCRRQLTVQGPR 227 R + LQ L A P R+ +H +R++ ++ L Q R Sbjct: 1052 RHQRVRPLQQVL---------------APQPQRQQVHRLQH---QRVRPLQQVLAPQPQR 1093 Query: 228 IRRSRPLIKLPLLRRQRIRPGRHQRTLRVKITHRLAAPIHIPVKSQRRRRPSARIRRARI 287 + R L+RQR+R +HQR + + H+L +H PV+ Q + R + ++++ + Sbjct: 1094 QQVHR-------LQRQRVRLSQHQRVRQPQQAHQL-LNLHQPVR-QPQHRQAPQLQQVPV 1144 Query: 288 APREIRPGPRIGGKRLIAARKP-QTGIRTPFESTRPAQPPRPILNIVTAQIHHIPITRR 345 + R R+ + + R+P Q +R P R + P+P+ LN H P+ R+ Sbjct: 1145 RQPQRRQVRRL---QQVPVRQPQQVPVRQP--QRRQVRRPQPVHLN------ RHQPV-RQ 1192 Query: 346 PGLIIRNGTPHR 357 P ++ + H+ Sbjct: 1193 PQQVLVHQLQHQ 1204 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 150 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 299> which encodes amino acid sequence <SEQ ID 300; NGS153>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −5.48 Possible cleavage site: 13 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 6.31 threshold: 0.0 PERIPHERAL Likelihood = 6.31 modified ALOM score: −1.76 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.150(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_15832758 gi|15832758|ref|NP_311531.1|\\\\(NC_002695) hypothetical protein [Escherichia coli O157:H7] dbj|BAB36927.1|(AP002562) hypothetical protein [Escherichia coli O157:H7] Length = 188 Score = 73.9 bits (180), Expect = 3e−12 Identities = 54/169 (31%), Positives = 79/169 (45%), Gaps = 15/169 (8%) Query: 12 LTQEVLKELLRYDDNTGKLYWAERPRKYFNSGLHYKSWNTGFSGKEVFLYKGRLGYLKLK 71 LT + + ELL +D +TG W + + S F GY + Sbjct: 16 LTVKRIFELLSFDKSTGVFRWKVPTQ----GRIALNSVAGAFDSN------------GYSMIM 62 Query: 72 IFKKQYNAHRLIWLFVYGKH-ASSIGHINRDKTDNRISNLRDVTHAENMKNRGKFKNNTS 130 I ++Y H L++ + + A I H+N +TDNR NLR+ EN +N KN+ S Sbjct: 63 IDGRRYKTHVLVFYITHNRWPAGQIDHNGIRTDNRPENLRECLPIENSRNIRIRKNSKS 122 Query: 131 GHTGVYFKPSKKWQARIWINRKNKILGLFEHIEDAA-KAREAASKDFG 178 G GV +HK KKW R+ + K+K G F+ +E A A EA K +G Sbjct: 123 GCRGVTWMKRQKKWNVRLGFHGKSKHFGCFDDLELAVLVAEEARDKYYG 171 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 151 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 301> which encodes amino acid sequence <SEQ ID 302; NGS154>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −6.98 Possible cleavage site: 28 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 8.12 threshold: 0.0 PERIPHERAL Likelihood = 8.12 modified ALOM score: −2.12 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.423(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_15830449 gi|15830449|ref|NP_309222.1|\\\\(NC_002695) hypothetical protein [Escherichia coli O157:H7] dbj|BAA94132.1|(AP000422) hypothetical protein [Escherichia coli O157:H7] dbj|BAB346l8.1|(AP002554) hypothetical protein [Escherichia coli O157:H7] Length = 148 Score = 42.7 bits (99), Expect = 0.003 Identities = 27/99 (27%), Positives = 49/99 (49%), Gaps = 10/99 (10%) Query: 37 IRPRKSKRSVEQNRRLWFLYREISEKVFIDGRRFSQDVWHE-----FLKRKFIGCIEHPN 91 + ++ KRS QN R+W + ++S +V G+R + + W + +LK K + +P Sbjct: 33 VHVKEPKRSKAQNDRMWPMLNDVSRQVLWHGQRLAPEDWKDLFTALWLKTKKLEQRSVPG 92 Query: 92 GQ----LMGISTTKLSVEEMSEYQEKIISWASNEHGVLW 126 ++G+ T+K+ M+E E I+ W E V W Sbjct: 93 IDGGVVMLGVRTSKMRKASMTELIE-IMFEFGSERNVRW 130 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 152 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 303> which encodes amino acid sequence <SEQ ID 304; NGS155>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −4 Possible cleavage site: 27 >>> Seems to have an uncleavable N-term signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 4.98 threshold: 0.0 PERIPHERAL Likelihood = 4.98 modified ALOM score: −1.50 *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.046(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_15801502 gi|15801502|ref|NP_287519.1|\\\\(NC_002655) putative endonuclease of prophage CP-9330 [Escherichia coli O157:H7 EDL933] ref|NP_309804.1|(NC_002695) endonuclease [Escherichia coli O157:H7] gb|AAG56131.1|AE005344_7 (AE005344) putative endonuclease of prophage CP-933O [Escherichia coli O157:H7 EDL933] dbj|BAB35200.1|(AP002556) endonuclease [Escherichia coli O157:H7] Length = 119 Score = 47.4 bits (1111, Expect = 2e−04 Identities = 38/122 (31%), Positives = 54/122 (44%), Gaps = 8/122 (6%) Query: 71 LILPYPVSANRYWRIWRNRAVRSAEAAAYKETVRRIA-QGAGAMPSEGAVAVYVRLIPKA 129 L+LPYP + N YWR + S Y+ V I Q + G +A+ + P Sbjct: 5 LVLPYPPTVNTYWERRGSTYFVSKAGERYRRAVVLIVRQQRLKLSLSGRLAIKIIABP-- 62 Query: 130 NKDGGANKTVIDLDNLKVTLDALQGVAYHNDRQVRRIAAEYGGEPVTGGGLAVEVGELE 189 +K DLDN LK LDAL D + G+PV+GG L V++ ++E Sbjct: 63 -----PDKRRRDLDNILKAPLDALTHAGVLMDDEQFDEINIVRGQPVSGGRLGVKIYKIE 117 Query: 190 ME 191 E Sbjct: 118 SE 119 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 153 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 305> which encodes amino acid sequence <SEQ ID 306; NGS156>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −2.61 Possible cleavage site: 49 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 7.96 threshold: 0.0 PERIPHERAL Likelihood = 7.96 modified ALOM score: −2.09 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.307(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gsa_AAG90098 C glutamicum protein fragment SEQ ID NO: 3852 |EP1108790-A2|26-SEP-2001 Length = 148 Score = 102 bits (253), Expect = 7e-21 Identities = 60/147 (40%).\",\n \"Positives = 88/147 (59%), Gaps = 18/147 (12%) Query: 3 NAYDVADFFLSPFEEEDGEQISNLKLQKLLYYAQGYALAILNRPLFAENIEHWQHGPVVP 62 +A ++A++F++ +E D E +S LKLQKLLYY+QG +A R LF++ I WQHGPV P Sbjct: 5 SAREIAEWFVAWGDELDAE-VSPLKLQKLLYYSQGEHIAATGRKLFSDKILAWQHGPVTP 63 Query: 63 CIYRTYKKYGGSPLPAAHIEPDKYADEEL---------VVLNRVRKEQGCYTAWALRNKT 113 +Y K YG +P I+PD++ E L V ++ G Y+AWALR KT Sbjct: 64 GVYSDTKSYGRNP-----IDPDEFVSDEFNWDDYSDVSDELVTVWRKYGIYSAWALREKT 118 Query: 114 HQEAPWIQT-RQGEVIGI--AIMGEYF 137 H E+PW+ QG+ I I A + ++F Sbjct: 119 HSESPWLDAWAQGQNIEITDAALKDFF 145 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 154 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 307> which encodes amino acid sequence <SEQ ID 308; NGS157>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −5.65 Possible cleavage site: 42 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 3.98 threshold: 0.0 PERIPHERAL Likelihood = 3.98 modified ALOM score: −1.30 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.291(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: {circumflex over ( )} **gbp_6274533 gi|6274533|gb|AAF06681.1|AF163663_6 \\\\(AF058689) Toul [Neisseria meningitidis] Length = 272 Score = 546 bits (1408), Expect e−154 Identities = 267/272 (98%), Positives = 271/272 (99%) Query: 19 MKGMDKLRYQRDFLNIRPIFTAGEQEYLTELSDRLPLSVLTDSVRNIEEIGIDVYSPAK 78 MKGMDKLRYQ+DFLNIRPIFTAGEQEYLTELSDRLPLSVLTDSVRNIEEIGIDVYS AK Sbjct: 1 MKGMDKLRYQQDFLNIRPIFTAGEQEYLTELSDRLPLSVLTDSVRNIEEIGIDVYSSAK 60 Query: 79 LEGNTYNQYDTQALLKLGQTAGGKLYSDAVMLINLRESYRHLLSGLDSPKPFDWLDFLKT 138 LEGNTYNQYDTQALLKLGQTAGGKLYSDAVMLINLRESYRHLLSGLDSP+PFDWLDFLKT Sbjct: 61 LEGNTYNQYDTQALLKLGQTAGGKLYSDAVMLINLRESYRHLLSGLDSPEPFDWLDFLKT 120 Query: 139 THSLISENLLEKGSGGVVRRDSVTISGTDYTPLSNPQSLDTELKWLLQEAPKIENPFDRA 198 THSLISENLLEKGSGGVVRRDSVTISGTDYTPLSNPQSLDTELKWLLQEAPKIENPFDRA Sbjct: 121 THSLISENLLEKGSGGVVRRDSVTISGTDYTPLSNPQSLDTELKWLLQEAPKIENPFDRA 180 Query: 199 VYLHNNLAYLRYFKDCNKRTARNCMTLSLMRSGFFPCVFSPDSYPAYAEAVVAYYETGDY 258 VYLHNNLAYL+YFKDCNKRTARNCMTLSLMRSGFFPCVFSPDSYPAYAEAVVAYYETGDY Sbjct: 181 VYLHNNLAYLQYFKDCNKRTARNCMTLSLMRSGFFPCVFSPDSYPAYAEAVVAYYETGDY 240 Query: 259 GLFKKYFISAYENTVNKYGPQPDVDIFRNFSI 290 GLFKKYFISAYENTVNKYGPQPDVDIFRNFS+ Sbjct: 241 GLFKKYFISAYENTVNKYGPQPDVDIFRNFSL 272 A homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS157 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 155 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 309> which encodes amino acid sequence <SEQ ID 310; NGS158>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −6.98 Possible cleavage site: 18 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 0.16 threshold: 0.0 PERIPHERAL Likelihood = 0.16 modified ALOM score: −0.53 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.185(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: > {circumflex over ( )}{circumflex over ( )} **gbp_15791833 gi|15791833|ref|NP_281656.1|\\\\(NC_002163) amino-acid ABC transporter ATP-binding protein [Campylobacter jejuni] pir||H81391 amino-acid ABC transporter ATP-binding protein Cj0469 [imported]-Campylobacter jejuni (strain NCTC 11168) emb|CAB75107.1|(AL139075) amino-acid ABC transporter ATP-binding protein [Campylobacter jejuni] Length = 253 Score = 301 bits (772), Expect = 6e−81 Identities = 153/244 (62%), Positives = 195/244 (79%), Gaps = 2/244 (0%) Query: 1 MALLSIRKLHKQYGSVAIQSLDLDLEKGEVIVLLGPSGCGKSTLLRCVNGLEPHQGGSI 60 M++L I L K YGS A++ ++L+++ EV+V+LGPSGCGKSTLLRC+NGLE G+I Sbjct: 1 MSILKIENLQKYYGSHHALKDINLEVKAKEVVVILGPSGCGKSTLLRCINGLEEIASGNI 60 Query: 61 VMDGVGEFGKDVS-WQTARQKVGMVFQSYELFAHMTVIENILLGPVKVQNRDRAEAEAQA 119 +D + KD W RQKVGMVFQSYELF H++V ENILLGP+KVQ R + E +A Sbjct: 61 YIDNE-KIDKDFKEWPRMRQKVGMVFQSYELFEHLSVEENILLGPMKVQKRKKDEVLKEA 119 Query: 120 GKLLERVGLLDRKNAYPRELSGGQKQRIAIVRALCLNPEVILLDEITAALDPEMVREVLE 179 LE+VGLL + +AYPRELSGGQKQRIAIVR+LC+NPE++L DE+TAALDPE+VREVLE Sbjct: 120 KIWLEKVGLLHKIHAYPRELSGGQKQRIAIVRSLCMNPELMLFDEVTAALDPEIVREVLE 179 Query: 180 VVLELAREGMSMLIVTHEMGFARKVADRIVFMDKGGIVESSDPETFFSAPKSERARQFLA 239 V+L LA+EGM+MLIVTHEMGFA+ VAD+I+FMD+G I+E +DP++FF PKSERA++FL Sbjct: 180 VMLNLAKEGMTMLIVTHEMGFAKAVADKIIFMDEGKIIEENDPKSFFKNPKSERAKKFLN 239 Query: 240 GMDY 243 DY Sbjct: 240 LFDY 243 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 156 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 311> which encodes amino acid sequence <SEQ ID 312; NGS159>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −4.16 Possible cleavage site: 13 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 6.47 threshold: 0.0 PERIPHERAL Likelihood = 6.47 modified ALOM score: −1.79 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.312(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: > {circumflex over ( )}{circumflex over ( )} **gbp_15794799 gi|1579479.9|ref|NP_284621.1|\\\\(NC_003116) hypothetical protein [Neisseria meningitidis Z2491] pir||B81819 hypothetical protein NMA1914 [imported]- Neisseria meningitidis (group A strain Z2491) emb|CAB85135.1|(AL162757) hypothetical protein [Neisseria meningitidis Z2491] Length = 206 Score = 265 bits (678), Expect = 8e−70 Identities = 131/146 (89%), Positives = 138/146 (93%) Query: 1 MTKLYAQIAKTEAQDDGTVKVWGYASSEAVDSDGEVVAAEAMKAAIPDYMKFGAVREMHG 60 MTKLYA+IAK E QDDGTVKVWGYASSE +DSDGEV+AA AMKAAIPDYMKFGA REMHG Sbjct: 1 MTKLYAEIAKMETQDDGTVKVWGYASSEEIDSDGEIAAAAMKAAIPDYMKFGAGREMHG 60 Query: 61 SNAAGTAIEINVEDDGRTFFGAHIVDPVAVTKVKTGVYKGFSIGGSVTARNDLNKSQITG 120 SNAAGTAIEINVEDDG TFFGAHI+DPV V+KVKTGVYKGFSIGGSVTAR+DLNKSQITG Sbjct: 61 SNAAGTAIEINVEDDGITFFGAHIIDPVVVSKVKTGVYKGFSIGGSVTARDDLNKSQITG 120 Query: 121 LKLTEISLVDRPANPDAVFTCFKADK 146 LKLTEISL+DRPANPDAV TCFKADK Sbjct: 121 LKLTEISLIDRPANPDAVSTCFKADK 146 A homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS159 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 157 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 313> which encodes amino acid sequence <SEQ ID 314; NGS160>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −8.3 Possible cleavage site: 33 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 7.85 threshold: 0.0 PERIPHERAL Likelihood = 7.85 modified ALOM score: −2.07 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.407(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: > {circumflex over ( )}{circumflex over ( )} **gbp_2126352 gi|2126352|pir||JC5218 type I site-specific deoxyribonuclease (EC 3.1.21.3) Hsd chain S [validated]-Pasteurella haemolytica gb|AAC44667.1|(U46781) HSDS [Mannheimia haemolytica] Length = 442 Score = 97.1 bits (240), Expect = 3e−19 Identities = 55/149 (36%), Positives = 81/149 (53%), Gaps = 3/149 (2%) Query: 26 EVAEYSKNRICSDKLNEHNYVGVDNLLQNREGKKLSGYVPSEGKMTEYIVNDILIGNIRP 85 ++E +I L + NY+ DN+L N G L+ +P+ + DIL NIR Sbjct: 10 DIVELISEKIKIKDLKKENYISTDNMLPNFGGITLAENLPNSASCNRFAKKDILFSNIRT 69 Query: 86 YLKKIWQADCTGGTNGDVLVIRV--TDEKVNPKYLYQVLADDKFFAFNMKHAKGAKMPRG 143 Y KK+W A+ +GG + DVLV+R TD +N +YL+ ++ D F F + A GAKMPRG Sbjct: 70 YFKKVWLAEFSGGCSPDVLVMRSKNTDILLN-EYLFLLIRSDDFINFTVISANGAKMPRG 128 Query: 144 SKAAIMQYKIPIPPLPEQEKIVAILGKFD 172 K A+ + IP + Q+K +A FD Sbjct: 129 DKNAMKGFIFNIPSIEYQKKCIANYFAFD 157 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 158 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 315> which encodes amino acid sequence <SEQ ID 316; NGS161>.\",\n \"Analysis of this protein sequence reveals the following: GvH Examining signal sequence (von Heijne) Signal Score (−7.5): −5.08 Possible cleavage site: 36 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 1 value: −1.59 threshold: 0.0 INTEGRAL Likelihood = −1.59 Transmembrane 302-318 (302-318) PERIPHERAL Likelihood = 3.76 modified ALOM score: 0.82 Rule: cytoplasmic membrane protein *** Reasoning Step: 2 ----- Final Results ----- bacterial inner membrane --- Certainty= 0.164(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial cytoplasm --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: pir||E81921 probable DNA-invertase NMA0772 [imported]-Neisseria meningitidis (group A strain Z2491) emb|CAB84055.1|(AL162754) putative DNA-invertase [Neisseria meningitidis Z2491] Length = 321 Score = 295 bits (755), Expect = 9e−79 Identities = 151/322 (46%), Positives = 216/322 (66%), Gaps = 3/322 (0%) Query: 14 LRNAVGLDISKLTFDATAIVGNAEYSKFDNDSKGLDQFSDRLKSLGCQNLHICMEATGN 73 +RNAVGLDIS TFD I+ KF ND +G + + + +++++CMEATGN Sbjct: 1 MRNAVGLDISAKTFDVVTIINGETDYRKFSNDEQGCKNLKEWISAKREKDIYVCMEATGN 60 Query: 74 YYEEVADYFAQYYSVYVVNPLKISKYAESRFKRTKTDKQDAKLIAQYCRSAQESELVKRQ 133 YYE+ AD A+ Y V V+NPLKI YA+ RF R K DKQDAKLIA++C++A EL KR+ Sbjct: 61 YYEQAADCLAEEYHVSVINPLKIKAYAQKRFSRVKNDKQDAKLIAEFCQTALIEELPKRE 120 Query: 134 KPTDEQYRLSRMTAAYAQIKSECAAMKNRHHAAKDEEAAKAYAEIIKAMNEQLEVLKEKI 193 KPT++QY L R+ + +Q+ + + KNR AAKD K + + +K + L +K+KI Sbjct: 121 KPTEQQYSLKRLLSLQSQLLEQQTSQKNRIRAAKDSFVQKIHEKQLKELENHLNAVKKKI 180 Query: 194 KEQTEKPN--CKEGVKRLETIPAIGRMTAAVLFHHLTSSKFETSNKFAAFAGLSPQQKES 251 +QT K + KE KRLETIP++G+ TA L +L +S FE + +FA +AGL+P Q S Sbjct: 181 -DQTIKSDKKMKELTKRLETIPSVGKTTAISLMSYLINSTFENAKQFTAYAGLNPHQNIS 239 Query: 252 GTSVRGKGKLTKFGNRKLRAVLFMPAMVAYRIRAFPDFIKRLEEKKKPKKVIIAALMRKL 311 GTSV K K+TK+GNR++R LFM A+VA++ FP F RL++ KKPK +II ALMRK+ Sbjct: 240 GTSVNKKSKMTKYGNRRIRGSLFMAALVAFKNNYFPAFTNRLKKAKKPKMLIIGALMRKI 299 Query: 312 AVIAYHVHKKGGDYDPSRYKSA 333 V+A++++K D+D +RY++A Sbjct: 300 LVVAFNLYKTETDFDKTRYQTA 321 A homolog was found in serogroup A N.meningitidis but not in serogroup B, so NGS161 protein and nucleic acid are useful for distinguishing between gonococcus and serogroup B N.meningitidis.\",\n \"Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 159 A DNA sequence was identified in N.gonorrhoeae <SEQ ID 983> which encodes amino acid sequence <SEQ ID 984; NGS162>.\",\n \"Analysis of this protein sequence reveals the following: GvH: Examining signal sequence (von Heijne) Signal Score (−7.5): −0.86 Possible cleavage site: 33 >>> Seems to have no N-terminal signal seq.\",\n \"Amino Acid Composition of Predicted Mature Form: calculated from 1 ALOM: Finding transmembrane regions (Klein et al.)\",\n \"count: 0 value: 4.08 threshold: 0.0 PERIPHERAL Likelihood = 4.08 modified ALOM score: −1.32 Rule: cytoplasmic protein *** Reasoning Step: 2 ----- Final Results ----- bacterial cytoplasm --- Certainty= 0.032(Affirmative) < succ> bacterial periplasmic space --- Certainty= 0.000(Not Clear) < succ> bacterial outer membrane --- Certainty= 0.000(Not Clear) < succ> bacterial inner membrane --- Certainty= 0.000(Not Clear) < succ> The protein has homology to the following sequences in the databases: ref|NP_312507.1|(NC_002695) hypothetical protein [Escherichia coli O157:H7] gb|AAG5B749.1|AE005587_7 (AE005587) putative adhesin [Escherichia coli O157:H7 EDL933] dbj|BAB37903.1|(AP002566) hypothetical protein [Escherichia coli O157:H7] Length = 1588 Score = 120 bits (302), Expect = 4e−26 Identities = 109/359 (30%), Positives = 170/359 (46%), Gaps = 65/359 (18%) Query: 22 AVALGSSSTASGEYSYASGYNSVASGNKSYAAGYASVASAEGSVVIGDSRQVKPEADQGV 81 + A+G + A G+YS A G + A G S A G ++++ + S+ +G S + + Sbjct: 93 STAVGYDAIAEGQYSSAIGSKTHAIGGASMAFGVSAISEGDRSIALGASSYSLGQYSMAL 152 Query: 82 AVGSKATVKNKAKQRVVVGSEAKVNAERGIAIGKEAKAGGKTTNTLLDGPAYYADAIAVG 141 SKA K + +G +K IA+G KA T + +IA+G Sbjct: 153 GRYSKAL----GKLSIAMGDSSKAEGANAIALGNATKA----TEIM---------SIALG 195 Query: 142 YQAEAGKGGAIALGKQAKATKQNGMALGVESEAAGDFSTAVGNESKAKGQGG-------- 193 A A K ++ALG + A+++N +A+G E+EAA + +TA+GN +KAKG Sbjct: 196 DTANASKAYSMALGASSVASEENAIAIGAETEAA-ENATAIGNNAKAKGTNSMAMGFGSL 254 Query: 194 ------VGLGNQSKAEADFAVAV--GNKAEATKE------------NSLVIGRYARANGN 233 + LGN S+A AD A+A+ GNKA+ N++ +G + A G+ Sbjct: 255 ADKVNTIALGNGSQALADNAIAIGQGNKADGVDAIALGNGSQSRGLNTIALGTASNATGD 314 Query: 234 HSVSLGSRSEIKDGVSNSVAPGYGSVASENNVVSVAYKETPQSTELSYRKIVGVDDGV-- 291 S++LGS S +G+ NSVA G S+A +N VSV RKIV V +G Sbjct: 315 KSLALGSNSS-ANGI-NSVALGADSIADLDNTVSVGNSSLK-------RKIVNVKNGAIK 365 Query: 292 -NDFDAVNVRQLKAMQGQNMAELFSVRSEVRGVAASSAALSALTPLSYDANNPTQFMVG 349 + +DA+N QL A+ SV + G AA +T +Y+ N ++ VG Sbjct: 366 SDSYDAINGSQLYAISD-------SVAKRLGGGAAVDVDDGTVTAPTYNLKNGSKNNVG 417 Score = 86.3 bits (212), Expect = 1e−15 Identities = 68/253 (26%), Positives = 118/253 (45%), Gaps = 39/253 (15%) Query: 28 SSTASGEYSYASGYNSVASGNKSYAAGYASVASAEGSVVIGDSRQVKPEADQGVAVGSKA 87 S+ +G + G + A + Y S ++ +G V IG G+KA Sbjct: 38 SALVAGGMLSSFGALANAGNDNGQGVDYGSGSAGDGWVAIGK--------------GAKA 83 Query: 88 -TVKNKAKQRVVVGSEAKVNAERGIAIGKEAKAGGKTTNTLLDGPAYYADAIAVGYQAEA 146 T N + VG +A + AIG + A G + +A G A + Sbjct: 84 NTFMNTSGSSTAVGYDAIAEGQYSSAIGSKTHAIGGAS-------------MAFGVSAIS 130 Query: 147 GKGGAIALGKQAKATKQNGMALGVESEAAGDFSTAVGNESKAKGQGGVGLGNQSKAEADF 206 +IALG + + Q MALG S+A G S A+G+ SKA+G + LGN +KA Sbjct: 131 EGDRSIALGASSYSLGQYSMALGRYSKALGKLSIAMGDSSKAEGANAIALGNATKATEIM 190 Query: 207 AVAVGNKAEATKENSLVIGRYARANGNHSVSLGSRSEIKDGV-----------SNSVAPG 255 ++A+G+ A A+K S+ +G + A+ +++++G+ +E + +NS+A G Sbjct: 191 SIALGDTANASKAYSMALGASSVASEENAIAIGAETEAAENATAIGNNAKAKGTNSMAMG 250 Query: 256 YGSVASENNVVSV 268 +GS+A + N +++ Sbjct: 251 FGSLADKVNTIAL 263 Based on this analysis, it was predicted that this protein from N.gonorrhoeae, and its epitopes, could be useful antigens for vaccines or diagnostics.\",\n \"EXAMPLE 160 Further open reading frames were identified in gonococcus <SEQ IDs 317/318 to 8621/8622>.\",\n \"These polypeptide and nucleotide sequences are useful for studying gonococcus, for diagnostic purposes, as antibiotic targets, and as vaccine antigens.\",\n \"It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10467657"}}},{"rowIdx":380,"cells":{"text":{"kind":"list like","value":[["Dry shaver for variable heights of cut","A dry shaver (11) has a single cutter blade (15) and a single cooperating shear blade (17).","The shear blade (17) is an elongate flexible foil, the thickness of which is different at different regions (21, 22) along the elongate extent of the foil (17).","The foil (17) is mounted (27) for displacement in its elongate direction so that a foil region (21, 22) of a particular thickness may be selectively aligned with the cutter blade (15).","The spacing between a skin engaging surface of the shear blade (17) and the opposite face of the shear blade (17) which cooperates with the cutter blade (15) is thus selectively variable.","In a further embodiment, the dry shaver is of the rotary type and has a plurality of interchangeable shear blades of different thicknesses.","In a still further embodiment, a rotary dry shaver has a mechanism for varying the spacing between the outer surface of a cover member overlying a shear blade and a cooperating rotary cutter blade."],["1.A dry shaver unit comprising: at least one cutter blade; and a cooperating shear blade; wherein a spacing between a skin-engaging surface of the shaver unit and regions of the shear blade which cooperate with the cutter blade can be varied.","2.A dry shaver unit according to claim 1, wherein different spacings between the skin-engaging surface of the shaver unit and the regions of a shear blade which cooperate with a cutter blade are selectively available.","3.A dry shaver according to claim 2, wherein the shear blade is an elongate flexible foil, the thickness of which is different at different regions along the elongate extent of the elongate flexible foil; and wherein the elongate flexible foil is mounted for displacement in its elongate direction so that a foil region of a particular thickness may be selectively aligned with the cutter blades whereby a spacing between a skin engaging surface of the shear blade and the opposite face of the shear blade which cooperates with the cutter blade is selectively variable.","4.A dry shaver according to claim 1, wherein said at least one cutter blade is a rotary cutter blade, wherein the cooperating shear blade is located over the rotary cutter blade, and wherein the spacing between the skin engaging surface of the shear blade and the opposite face thereof for cooperation with the cutter blade can be varied.","5.A dry shaver according to claim 4, wherein the shaver is provided with a plurality of shear blades and the spacing between the skin engaging surface of one of the shear blades and the opposite face thereof for cooperation with an associated cutter blade is different from the spacing between the corresponding surface and opposite face of at least one other of the plurality of shear blades.","6.A dry shaver according to claim 5, comprising at least two rotary cutter blades, wherein each rotary cutter blade has a general axis of symmetry and the axis of symmetry of at least one rotary cutter blade is oriented differently from the axis of symmetry of at least one other rotary cutter blade with respect to a generally longitudinal axis of the dry shaver as whole, so that only one of the rotary cutter blades can be used for shaving purposes at one time.","7.A dry shaver according to claim 5 further comprising: at least two cutting head assemblies, each head assembly having at least one cutter blade and a respective overlying cooperating shear blade, wherein a spacing between a skin engaging surface and an opposite face of the shear blade in at least one of the cutting head assemblies is different from a corresponding spacing in other head assemblies, and the head assemblies are interchangeably mountable on a body portion of the dry shaver.","8.A dry shaver according to claim 4 further comprising a plurality of blade assemblies, each blade assembly comprising a cutter blade and an overlying cooperating shear blade, wherein the blade assemblies are interchangeably mountable on the dry shaver.","9.","(canceled) 10.A dry shaver according to claim 1, wherein the cutter blade comprises at least one rotary cutter blade, and wherein the cooperating shear blade is located over the rotary cutter blade and comprises a shear blade and cutter blade assembly, and further comprising a cover member overlying the shear blade, and a displacement mechanism for effecting controlled movement of the shear blade and cutter blade assembly towards and away from the cover member, so that the spacing between a skin engaging surface of the cover member and the face of the shear blade which cooperates with the cutter blade is variable.","11.A dry shaver according to claim 2, wherein said at least one cutter blade is a rotary cutter blade, wherein the cooperating shear blade is located over the rotary cutter blade, and wherein the spacing between the skin engaging surface of the shear blade and the opposite face thereof for cooperation with the cutter blade can be varied.","12.A dry shaver according to claim 6 further comprising: at least two cutting head assemblies, each head assembly having at least one cutter blade and a respective overlying cooperating shear blade, wherein a spacing between a skin engaging surface and an opposite face of the shear blade in at least one of the cutting head assemblies is different from a corresponding spacing in other head assemblies, and the head assemblies are interchangeably mountable on a body portion of the dry shaver.","13.A dry shaver according to claim 5 further comprising a plurality of blade assemblies, each blade assembly comprising a cutter blade and an overlying cooperating shear blade, wherein the blade assemblies are interchangeably mountable on the dry shaver.","14.A dry shaver according to claim 6 further comprising a plurality of blade assemblies, each blade assembly comprising a cutter blade and an overlying cooperating shear blade, wherein the blade assemblies are interchangeably mountable on the dry shaver."],["<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention This invention relates to dry shavers having a cutter blade and a cooperating shear blade arrangement.","The invention relates at least both to electric dry shavers of the type having a reciprocating cutter and a shear blade arrangement in the form of a perforated foil with a plurality of perforations, and also to dry shavers of the type having a rotary cutter member or blade underlying an external shear member provided with apertures for entry of the hairs to be cut.","The history of shaving to date has been the search for the ever closer shave.","In effectuating a close shave, certain skin and hair types encounter the problem of an inflammation due to re-entry into the skin of too short cut (tangentially exiting) hairs.","It is an aim of the present invention to prevent or protect against this by purposefully cutting the hair long at a sufficient distance from the skin surface so as to prevent any possible re-entry into the skin of the close cut hairs.","The invention is thus directed at cutting the hairs long enough away from the skin surface to prevent their re-entry.","In shaving close, certain skin and hair types thus experience or develop inflammation due to re-entry into the skin of the closely shaved hairs.","It is therefore a purpose of the present invention both to allow for a normal close shave and to allow also for a cut of hair at some distance from the skin surface, the distance from the skin surface being sufficient so as to prevent re-entry of the cut hairs into the skin.","2.Description of the Prior Art GB-A-1,569,096 describes a dry shaver having a flexible perforated shear foil supported in a frame and a reciprocatable cutter which cooperates with the shear foil.","The shear foil is supported in an arched position between two side members of the frame and a cutter blade or shear head is pressed against the interior side of the shear foil by means of a spring.","Similar such units are described in U.S. Pat.","No.","3,729,821 and in a diversity of other patent documents.","The cutter blade may be mounted for reciprocation in a direction substantially parallel to the axis of curvature of the shear blade foil, or alternatively, the cutter blade may be driven in oscillatory motion about this axis.","In a particular example of the shear foil type dry shaver, there is described in U.S. Pat.","No.","3,694,916 an arrangement in which a reciprocatable cutter cooperates with a shearing blade having at least two shearing fields of different perforations.","The shearing blade or foil is movable to bring a selected shearing field into the cooperating shearing disposition with the cutter blade.","The arrangement enables a field of perforations which is most suitable for a particular type of skin to be selected but makes no reference to cutting hair at different lengths.","The selected field of perforations is changed, but not the depth or height of cut.","A diversity of rotary cutter shavers are also known, and reference may be directed inter alia to U.S. Pat.","No.","5,007,168, EP-A1-0,241,082, and EP-A1-0,484,795, all of which embody the rotary principle already indicated above.","In WO-A-97/45235, there is described a rotary shaving apparatus in which radially directed hair entry apertures are of either broad or narrow dimension in the circumferential direction.","The thickness of the shear blade in which the hair entry apertures are defined varies, depending on the dimension of the hair entry apertures.","The shear blade is of greater thickness in a region thereof containing broad hair entry apertures than it is in a region containing narrow hair entry apertures.","Thus the blade has a wavy or undulating configuration in the circumferential direction, with the depth or thickness of the blade increasing or reducing as between broad hair aperture entry regions and narrow hair entry aperture regions.","The purpose of the arrangement is said to be to enable both short and long hairs to be caught and severed effectively with the end result of a close shave, while avoiding, as far as possible, irritation of the skin."],["<SOH> BRIEF SUMMARY OF THE INVENTION <EOH>It is a general object of the invention to provide a dry shaver unit directed to overcoming the inflammation problems discussed hereinabove.","According to the invention in a first and most general aspect, there is provided a dry shaver unit comprising at least one cutter blade and a cooperating shear blade arrangement in which provision is made for the spacing between a skin-engaging surface of the shaver unit and regions of the shear blade arrangement which cooperate with the cutter blade to be increased as compared with the spacing required to achieve a “close shave”.","It is a particular object of the present invention to provide a dry shaver in which the depth of cut is variable, so that hair to be cut may be left at a variable length over certain areas of the skin as required, in order inter alia to avoid the development of a shaving rash due to re-entry into the skin of too-short tangentially cut hairs exiting the skin at an acute angle.","According to the invention in a first specific aspect, there is therefore provided a dry shaver unit wherein different spacings between a skin-engaging surface of the shaver unit and regions of a shear blade arrangement which cooperate with a cutter blade are selectively available.","In a particular embodiment of dry shaver according to the invention, a single cutter blade cooperates with a single shear blade and the spacing between a skin engaging surface of the shear blade and the opposite face of the shear blade which cooperates with the cutter blade (i.e.","the cutter blade surface or side of the shear blade) is selectively variable.","In a favoured construction, the shear blade is an elongate flexible foil, the thickness of which is different at different regions along the elongate extent of the foil, and the foil is mounted for displacement in its elongate direction so that a foil region of a particular thickness may be selectively aligned with the cutter blade.","The foil may vary in thickness in a substantially continuous manner over its elongate extent from a minimum thickness portion to a maximum thickness portion, or alternatively the foil may comprise a sequence of foil portions, each of which has a different thickness, the foil portions being flexibly interconnected with one another.","A control member is suitably attached to the foil, the control member being displaceable in selective manner in the elongate direction of the foil.","In a preferred arrangement, the control member comprises a control stud extending through a slot defined in a wall portion of the shaver, an external region of the control stud being manually engageable to effect displacement of the foil in its elongate direction.","The foil may be retained in a particular selected position such as by frictional interaction between control stud and slot.","In an alternative arrangement, opposite elongate ends of the foil may be engaged at opposite ends of a rocking mechanism, displacement of the foil in its elongate direction being effected by rocking displacement of the rocking mechanism.","In a further aspect of the invention, there is provided a dry shaver in which said at least one cutter blade is a rotary cutter blade, the or each cutter blade having an overlying cooperating shear blade, wherein the spacing between the skin engaging surface of the shear blade and the opposite face thereof for cooperation with the cutter blade is increased as compared with the spacing required to achieve a “close shave”.","In a particular rotary construction of dry shaver according to the invention, the shaver is provided with a plurality of shear blades and the spacing between the skin engaging surface of one of the shear blades and the opposite face thereof for cooperation with an associated cutter blade (i.e.","the cutter blade surface or side of the shear blade) is different from the spacing between the corresponding surface and opposite face of at least one other of the plurality of shear blades.","Thus, for example, one of the shear blades may provide for a close shave and another shear blade for a less close shave For a shaver having a head with two or three blades used simultaneously, the invention may provide two or more sets of blades for use on the head, one of the sets of blades providing for a close shave and another of the sets of blades enabling a less close shave.","It will be appreciated that more than two such sets may be provided, i, a third set for example facilitating a shave intermediate between a close shave and a long shave.","In a variant of this aspect of the invention, a dry shaver may comprise at least two rotary cutter blades, wherein each rotary cutter blade has a general axis of symmetry and the axis of symmetry of at least one rotary cutter blade is oriented differently from the axis of symmetry of at least one other rotary cutter blade with respect to a generally longitudinal axis of the dry shaver as whole, so that only one of at said least one rotary cutter blade and said at least one other rotary cutter blade can be used for shaving purposes at one time.","Thus in this variant of the invention, the shaver is provided with two or more shaving head regions, each of which faces away from the general longitudinal axis of the unit in a different direction, so that only one of the head regions can be used at a time.","Each head region may have two or more blades and as previously indicated, the shaver unit may be provided with multiple sets of blades so that one head region may be set up for a close shave and another head region set up for a less close shave.","Because this embodiment of the invention has two or more head regions facing in different directions, the unit may be set up to enable the user to choose a close shave or a less close shave simply by applying the appropriate head region to the skin.","In yet another variant of this aspect of the invention, the shaver unit may have three head regions, each of which faces in a different direction away from the general longitudinal axis of the unit, the three head regions or head assemblies being spaced apart by substantially one hundred and twenty degrees about this longitudinal axis.","In any variant of dry shaver according to the rotary aspect of the invention, the shaver unit may be provided in combination with at least two cutting head assemblies, each head assembly having at least one cutter blade and the or each cutter blade having a respective overlying cooperating shear blade, wherein the or each shear blade of one of the head assemblies has a spacing between its skin engaging surface and its opposite face which is the same for the or all of the shear blade(s) of said one of the head assemblies but is different from the corresponding spacing for the or all of the shear blade(s) of the other of the head assemblies, and the head assemblies are interchangeably mountable on a body portion of the dry shaver.","In addition to being provided with two or more interchangeable head assemblies, multiple sets of blades providing for different depths of cut may also be included in the shaver kit.","Thus one head may be set up with blades suitable for a close shave, and another of the head assemblies set up for the less close shave.","The user wishing to change from one class of shave to the other simply exchanges the heads appropriately.","Thus the invention extends to a shaver kit including a shaver body unit and two or more head assemblies interchangeably associatable with the body unit which contains the drive features of the shaver, and the kit may optionally include multiple blade assemblies for individual assocation as required with any of the head assemblies.","Extensive flexibility of use is thus available, either by interchanging a head assembly or set of blades as required, or, for the arrangement provided with multiple head regions or head assemblies disposed in different angular orientations, merely by applying the appropriate head region to the skin, with optional advance mounting of blades of an appropriate depth of cut on the selected head region before use, as may be required.","The dry shaver of the invention, in any of the rotary embodiments, may be provided in combination with a plurality of blade assemblies, each blade assembly comprising a cutter blade and an overlying cooperating shear blade, wherein the blade assemblies are interchangeably mountable on the dry shaver.","Thus, as already indicated, in a dry shaver kit according to this aspect of the invention and comprising any of the foregoing features of the invention, the kit may include multiple sets of blade assemblies, interchangeably mountable on the dry shaver, so that the user may select an appropriate depth of cut by mounting a blade or blades suitable for that depth of cut on a head assembly of the dry shaver unit or kit, as required.","According to yet another aspect of the invention in its rotary configuration, there is provided a dry shaver comprising at least one rotary cutter blade, wherein the spacing between a skin-engaging surface of the dry shaver and the cutter blade is selectively variable and a value may be selected for said spacing which is increased as compared with the spacing required to achieve a “close shave” Thus in this variant of the invention, depth of cut is available in an infinitely variable manner similar to that afforded by the foil embodiment of the invention, but without necessitating either individual blade assemblies to be exchanged or the head assembly as a whole to be swapped for an alternative head assembly.","In this aspect, the invention thus offers the immediately selectively variable depth of cut of the foil embodiment combined with features of the rotary construction.","In a particular construction of the invention in this additional aspect, the shaver comprises at least one rotary cutter blade, the or each of which has an overlying cooperating shear blade and a cover member overlying the shear blade, the spacing between a skin engaging surface of the cover member and the face of the shear blade which cooperates with the cutter blade being variable.","In this arrangement, the shaver may include a displacement mechanism for effecting controlled movement of a shear blade and cutter blade assembly towards and away from the cover member.","Thus in these foregoing embodiments, the advantages of immediate or direct, controlled substantially infinitely variable adjustment of the depth of cut within predetermined limits is achieved for a rotary shaving unit also.","The invention also extends to a blade assembly for a dry shaver comprising a rotary cutter blade and a cooperating shear blade, wherein the spacing between a skin engaging surface of the shear blade and the opposite face thereof for cooperation with the cutter blade is increased as compared with the spacing required to achieve a “close shave”.","The invention likewise additionally encompasses a head assembly for a dry shaver, comprising at least one rotary cutter blade, the or each cutter blade having an overlying cooperating shear blade, wherein the spacing between the skin engaging surface of the shear blade and the opposite face thereof for cooperation with the cutter blade is increased as compared with the spacing required to achieve a “close shave”.","Thus the invention embraces in addition blade assemblies and head assemblies incorporating the features of the invention as independent units, whether in combination with a dry shaver or as entirely separate assemblies.","The invention likewise extends to any kit of parts comprising a dry shaver drive unit, one or more head assemblies, and/or one or more blade assemblies, provided in combination or in any commercial association, in so far as such units incorporate the features of the invention or together cooperate in defining equipment lying within the scope of the invention.","The invention also extends to a dry shaver substantially as described herein with reference to and as shown in any one or more of the accompanying drawings."],["BACKGROUND OF THE INVENTION 1.Field of the Invention This invention relates to dry shavers having a cutter blade and a cooperating shear blade arrangement.","The invention relates at least both to electric dry shavers of the type having a reciprocating cutter and a shear blade arrangement in the form of a perforated foil with a plurality of perforations, and also to dry shavers of the type having a rotary cutter member or blade underlying an external shear member provided with apertures for entry of the hairs to be cut.","The history of shaving to date has been the search for the ever closer shave.","In effectuating a close shave, certain skin and hair types encounter the problem of an inflammation due to re-entry into the skin of too short cut (tangentially exiting) hairs.","It is an aim of the present invention to prevent or protect against this by purposefully cutting the hair long at a sufficient distance from the skin surface so as to prevent any possible re-entry into the skin of the close cut hairs.","The invention is thus directed at cutting the hairs long enough away from the skin surface to prevent their re-entry.","In shaving close, certain skin and hair types thus experience or develop inflammation due to re-entry into the skin of the closely shaved hairs.","It is therefore a purpose of the present invention both to allow for a normal close shave and to allow also for a cut of hair at some distance from the skin surface, the distance from the skin surface being sufficient so as to prevent re-entry of the cut hairs into the skin.","2.Description of the Prior Art GB-A-1,569,096 describes a dry shaver having a flexible perforated shear foil supported in a frame and a reciprocatable cutter which cooperates with the shear foil.","The shear foil is supported in an arched position between two side members of the frame and a cutter blade or shear head is pressed against the interior side of the shear foil by means of a spring.","Similar such units are described in U.S. Pat.","No.","3,729,821 and in a diversity of other patent documents.","The cutter blade may be mounted for reciprocation in a direction substantially parallel to the axis of curvature of the shear blade foil, or alternatively, the cutter blade may be driven in oscillatory motion about this axis.","In a particular example of the shear foil type dry shaver, there is described in U.S. Pat.","No.","3,694,916 an arrangement in which a reciprocatable cutter cooperates with a shearing blade having at least two shearing fields of different perforations.","The shearing blade or foil is movable to bring a selected shearing field into the cooperating shearing disposition with the cutter blade.","The arrangement enables a field of perforations which is most suitable for a particular type of skin to be selected but makes no reference to cutting hair at different lengths.","The selected field of perforations is changed, but not the depth or height of cut.","A diversity of rotary cutter shavers are also known, and reference may be directed inter alia to U.S. Pat.","No.","5,007,168, EP-A1-0,241,082, and EP-A1-0,484,795, all of which embody the rotary principle already indicated above.","In WO-A-97/45235, there is described a rotary shaving apparatus in which radially directed hair entry apertures are of either broad or narrow dimension in the circumferential direction.","The thickness of the shear blade in which the hair entry apertures are defined varies, depending on the dimension of the hair entry apertures.","The shear blade is of greater thickness in a region thereof containing broad hair entry apertures than it is in a region containing narrow hair entry apertures.","Thus the blade has a wavy or undulating configuration in the circumferential direction, with the depth or thickness of the blade increasing or reducing as between broad hair aperture entry regions and narrow hair entry aperture regions.","The purpose of the arrangement is said to be to enable both short and long hairs to be caught and severed effectively with the end result of a close shave, while avoiding, as far as possible, irritation of the skin.","BRIEF SUMMARY OF THE INVENTION It is a general object of the invention to provide a dry shaver unit directed to overcoming the inflammation problems discussed hereinabove.","According to the invention in a first and most general aspect, there is provided a dry shaver unit comprising at least one cutter blade and a cooperating shear blade arrangement in which provision is made for the spacing between a skin-engaging surface of the shaver unit and regions of the shear blade arrangement which cooperate with the cutter blade to be increased as compared with the spacing required to achieve a “close shave”.","It is a particular object of the present invention to provide a dry shaver in which the depth of cut is variable, so that hair to be cut may be left at a variable length over certain areas of the skin as required, in order inter alia to avoid the development of a shaving rash due to re-entry into the skin of too-short tangentially cut hairs exiting the skin at an acute angle.","According to the invention in a first specific aspect, there is therefore provided a dry shaver unit wherein different spacings between a skin-engaging surface of the shaver unit and regions of a shear blade arrangement which cooperate with a cutter blade are selectively available.","In a particular embodiment of dry shaver according to the invention, a single cutter blade cooperates with a single shear blade and the spacing between a skin engaging surface of the shear blade and the opposite face of the shear blade which cooperates with the cutter blade (i.e.","the cutter blade surface or side of the shear blade) is selectively variable.","In a favoured construction, the shear blade is an elongate flexible foil, the thickness of which is different at different regions along the elongate extent of the foil, and the foil is mounted for displacement in its elongate direction so that a foil region of a particular thickness may be selectively aligned with the cutter blade.","The foil may vary in thickness in a substantially continuous manner over its elongate extent from a minimum thickness portion to a maximum thickness portion, or alternatively the foil may comprise a sequence of foil portions, each of which has a different thickness, the foil portions being flexibly interconnected with one another.","A control member is suitably attached to the foil, the control member being displaceable in selective manner in the elongate direction of the foil.","In a preferred arrangement, the control member comprises a control stud extending through a slot defined in a wall portion of the shaver, an external region of the control stud being manually engageable to effect displacement of the foil in its elongate direction.","The foil may be retained in a particular selected position such as by frictional interaction between control stud and slot.","In an alternative arrangement, opposite elongate ends of the foil may be engaged at opposite ends of a rocking mechanism, displacement of the foil in its elongate direction being effected by rocking displacement of the rocking mechanism.","In a further aspect of the invention, there is provided a dry shaver in which said at least one cutter blade is a rotary cutter blade, the or each cutter blade having an overlying cooperating shear blade, wherein the spacing between the skin engaging surface of the shear blade and the opposite face thereof for cooperation with the cutter blade is increased as compared with the spacing required to achieve a “close shave”.","In a particular rotary construction of dry shaver according to the invention, the shaver is provided with a plurality of shear blades and the spacing between the skin engaging surface of one of the shear blades and the opposite face thereof for cooperation with an associated cutter blade (i.e.","the cutter blade surface or side of the shear blade) is different from the spacing between the corresponding surface and opposite face of at least one other of the plurality of shear blades.","Thus, for example, one of the shear blades may provide for a close shave and another shear blade for a less close shave For a shaver having a head with two or three blades used simultaneously, the invention may provide two or more sets of blades for use on the head, one of the sets of blades providing for a close shave and another of the sets of blades enabling a less close shave.","It will be appreciated that more than two such sets may be provided, i, a third set for example facilitating a shave intermediate between a close shave and a long shave.","In a variant of this aspect of the invention, a dry shaver may comprise at least two rotary cutter blades, wherein each rotary cutter blade has a general axis of symmetry and the axis of symmetry of at least one rotary cutter blade is oriented differently from the axis of symmetry of at least one other rotary cutter blade with respect to a generally longitudinal axis of the dry shaver as whole, so that only one of at said least one rotary cutter blade and said at least one other rotary cutter blade can be used for shaving purposes at one time.","Thus in this variant of the invention, the shaver is provided with two or more shaving head regions, each of which faces away from the general longitudinal axis of the unit in a different direction, so that only one of the head regions can be used at a time.","Each head region may have two or more blades and as previously indicated, the shaver unit may be provided with multiple sets of blades so that one head region may be set up for a close shave and another head region set up for a less close shave.","Because this embodiment of the invention has two or more head regions facing in different directions, the unit may be set up to enable the user to choose a close shave or a less close shave simply by applying the appropriate head region to the skin.","In yet another variant of this aspect of the invention, the shaver unit may have three head regions, each of which faces in a different direction away from the general longitudinal axis of the unit, the three head regions or head assemblies being spaced apart by substantially one hundred and twenty degrees about this longitudinal axis.","In any variant of dry shaver according to the rotary aspect of the invention, the shaver unit may be provided in combination with at least two cutting head assemblies, each head assembly having at least one cutter blade and the or each cutter blade having a respective overlying cooperating shear blade, wherein the or each shear blade of one of the head assemblies has a spacing between its skin engaging surface and its opposite face which is the same for the or all of the shear blade(s) of said one of the head assemblies but is different from the corresponding spacing for the or all of the shear blade(s) of the other of the head assemblies, and the head assemblies are interchangeably mountable on a body portion of the dry shaver.","In addition to being provided with two or more interchangeable head assemblies, multiple sets of blades providing for different depths of cut may also be included in the shaver kit.","Thus one head may be set up with blades suitable for a close shave, and another of the head assemblies set up for the less close shave.","The user wishing to change from one class of shave to the other simply exchanges the heads appropriately.","Thus the invention extends to a shaver kit including a shaver body unit and two or more head assemblies interchangeably associatable with the body unit which contains the drive features of the shaver, and the kit may optionally include multiple blade assemblies for individual assocation as required with any of the head assemblies.","Extensive flexibility of use is thus available, either by interchanging a head assembly or set of blades as required, or, for the arrangement provided with multiple head regions or head assemblies disposed in different angular orientations, merely by applying the appropriate head region to the skin, with optional advance mounting of blades of an appropriate depth of cut on the selected head region before use, as may be required.","The dry shaver of the invention, in any of the rotary embodiments, may be provided in combination with a plurality of blade assemblies, each blade assembly comprising a cutter blade and an overlying cooperating shear blade, wherein the blade assemblies are interchangeably mountable on the dry shaver.","Thus, as already indicated, in a dry shaver kit according to this aspect of the invention and comprising any of the foregoing features of the invention, the kit may include multiple sets of blade assemblies, interchangeably mountable on the dry shaver, so that the user may select an appropriate depth of cut by mounting a blade or blades suitable for that depth of cut on a head assembly of the dry shaver unit or kit, as required.","According to yet another aspect of the invention in its rotary configuration, there is provided a dry shaver comprising at least one rotary cutter blade, wherein the spacing between a skin-engaging surface of the dry shaver and the cutter blade is selectively variable and a value may be selected for said spacing which is increased as compared with the spacing required to achieve a “close shave” Thus in this variant of the invention, depth of cut is available in an infinitely variable manner similar to that afforded by the foil embodiment of the invention, but without necessitating either individual blade assemblies to be exchanged or the head assembly as a whole to be swapped for an alternative head assembly.","In this aspect, the invention thus offers the immediately selectively variable depth of cut of the foil embodiment combined with features of the rotary construction.","In a particular construction of the invention in this additional aspect, the shaver comprises at least one rotary cutter blade, the or each of which has an overlying cooperating shear blade and a cover member overlying the shear blade, the spacing between a skin engaging surface of the cover member and the face of the shear blade which cooperates with the cutter blade being variable.","In this arrangement, the shaver may include a displacement mechanism for effecting controlled movement of a shear blade and cutter blade assembly towards and away from the cover member.","Thus in these foregoing embodiments, the advantages of immediate or direct, controlled substantially infinitely variable adjustment of the depth of cut within predetermined limits is achieved for a rotary shaving unit also.","The invention also extends to a blade assembly for a dry shaver comprising a rotary cutter blade and a cooperating shear blade, wherein the spacing between a skin engaging surface of the shear blade and the opposite face thereof for cooperation with the cutter blade is increased as compared with the spacing required to achieve a “close shave”.","The invention likewise additionally encompasses a head assembly for a dry shaver, comprising at least one rotary cutter blade, the or each cutter blade having an overlying cooperating shear blade, wherein the spacing between the skin engaging surface of the shear blade and the opposite face thereof for cooperation with the cutter blade is increased as compared with the spacing required to achieve a “close shave”.","Thus the invention embraces in addition blade assemblies and head assemblies incorporating the features of the invention as independent units, whether in combination with a dry shaver or as entirely separate assemblies.","The invention likewise extends to any kit of parts comprising a dry shaver drive unit, one or more head assemblies, and/or one or more blade assemblies, provided in combination or in any commercial association, in so far as such units incorporate the features of the invention or together cooperate in defining equipment lying within the scope of the invention.","The invention also extends to a dry shaver substantially as described herein with reference to and as shown in any one or more of the accompanying drawings.","BRIEF DESCRIPTION OF THE DRAWINGS A number of embodiments of the invention will now be described having regard to the accompanying drawings in which: FIG.","1 is a diagrammatic representation of a portion of human skin, in the region of the surface of the skin, in section, showing inner and outer skin layers and hair growth, and representing also a particular condition in which an individual's body hair is subject to growth in a manner more horizontal than vertical, FIG.","2 is a side sectional view of an adjustable foil blade in a dry shaver in accordance with the present invention, FIG.","3 is a pictorial representation of an embodiment of foil blade suitable for a dry shaver according to the invention, FIG.","4 is a side sectional representation of an alternative manner of adjustable foil blade mounting for a dry shaver in accordance with the present invention, FIG.","5 is a diagrammatic view in section through a shear blade of the kind illustrated in FIGS.","2 to 4, showing a known variant in which a sharp cutting edge is defined by a downturn around the periphery of the hair entry openings in the foil, FIG.","6 is a pictorial view of a rotary blade razor of known construction, to which the present invention may be applied in an alternative embodiment, FIG.","7 is a simplified pictorial view of an alternative rotary blade razor, having two blades as compared with the three-bladed configuration of FIG.","6, to which again the present invention may be applied in an alternative embodiment, FIG.","8 is a side sectional view of a shear blade of conventional construction for use in the shaver of FIG.","6 or that of FIG.","7, FIG.","9 is a side sectional view of a modified shear blade for rotary embodiment of dry shaver in accordance with the present invention, FIG.","10 is a pictorial representation from beneath of a shaver head construction providing for adjustability of the spacing between the cutting blades and the skin surface in a twin-bladed embodiment of dry shaver of a kind similar to that shown in FIG.","6, and FIG.","11 is a longitudinal sectional view on the longitudinal centre line of the arrangement of FIG.","10.DETAILED DESCRIPTION OF THE DRAWINGS FIG.","1 is a representation in sectional view of a portion of the surface of the skin of an individual.","As shown in FIG.","1, the outer layer 1 of the skin is underlaid by an inner layer 2.Hairs 3 and 3a grow outwardly through the skin from respective root regions 4 and 4a and emerge through the outer layer 1 of the skin.","In the normal way, as indicated by hair 3, a hair should emerge from the skin outer layer I and extend outwardly above the skin surface substantially at right angles to the skin surface.","Depending on the region of the skin surface in question, certain hairs such as that designated by reference 3a in FIG.","1 do not grow upwardly substantially perpendicularly or vertically with respect to the skin surface but run tangentially close to but underneath the skin surface.","Eventually, such a hair emerges from the skin surface, as indicated at reference 3b, but in a highly tangential manner.","If an individual suffering from such tangential hair growth wishes to effect a close shave and cuts the hair relatively close to the skin outer surface, a condition develops in which the sharp edge of the cut hair burrows back beneath the surface of the skin, as indicated at reference 3c, rather than emerging from the skin and growing externally in the normal manner.","Such ingrowth occurs if the sharp, cut edge of the hair becomes engaged or caught beneath the surface of the outer skin layer 1.If growth then continues with the hair 3 running into and under the skin, the inevitable result is some kind of inflammation.","It is therefore preferable to prevent the development of any such inflammation and the present invention addresses this problem.","The concept underlying the avoidance of such skin conditions is to cut the hair slightly above the surface of the skin in areas of the body surface where the possibility of such ingrowing conditions arising is likely to be greatest.","The advantages of its being possible to cut hair in accordance with the principles of the present invention are not however limited only to skin conditions such as that illustrated with reference to FIG.","1, but the invention is applicable to all skin circumstances where shaving bumps or other skin eruptions or imperfections militate against an excessively close shave.","The invention provides the user of a dry shaver with the ability to selectively cut body hair either tightly or to a greater length, depending on the region of the skin in question, i.e.","the area currently being shaved.","An embodiment of the invention applicable to a foil type dry shaver is shown in FIG.","2.A removable shaver head portion 11 is mounted on a shaver body portion 12.The head 11 is seated on the body portion 12 to enclose the upper or drive portion 13 of the shaver body portion 12 in conventional manner.","A generally conventional drive arrangement is applied such as by drive member 14 to an outwardly curved or convex cutter blade 15 to provide either reciprocating movement in the axial direction of the curved cutter blade 15, or alternatively oscillatory or circumferential motion about this axis.","A spring arrangement such as a helical spring 16 urges the curved displaceable cutter blade 15 against the inner surface of a cooperating perforated foil blade 17, which serves as a substantially stationary shear member for the movable blade cutter 15 under shaving conditions, i.e.","foil blade 17 remains substantially static with respect to cutter blade 15 during an actual shaving operation.","The perforated foil blade 17 is received in a bent-over configuration within an open top region of the shaver head 11.The foil blade 17 is received and accommodated in curved guide grooves extending along opposite inner walls of the shaver head 11.One such guide groove is indicated by reference 27.It is a particular feature of the invention that this perforated foil which serves as the shearing blade 17 has a thickness which varies along the length of the foil, as shown in FIGS.","2 and 3.It is also possible for the size, i.e.","the width or breadth of the hair-entry apertures, also their configuration, to vary with the thickness of the foil, e.g.","the apertures being narrow where the foil is thin and wider where the foil is thick.","Wide and narrow apertures may also be intermingled.","Referring now also to FIG.","3 in particular, there is shown a favoured embodiment of the perforated foil blade or shear cutter 17, formed as an integral unit, in which the perforated central region of the blade 17, designated by cross-hatching, is surrounded by side 18 and end 19 members or blade regions, which are unperforated and define therefore lateral members and end or cross-members of the blade structure.","The blade 17 has a thinner region 21 towards one axial end in its generally longitudinal direction and a thicker region 22 towards its other axial end.","In the region of the thicker end 22, an aperture 23 is provided through which a fastener 24, FIG.","2, enables the blade 17 according to the invention to be coupled to an operating or control button 25, which is displaceable on the exterior of the shaver head 11 in the longitudinal direction of the blade 17 and is guided by a guide slot 26 provided in the housing of the shaver head 11.In an alternative arrangement (not illustrated), the flexible foil blade may be supported in a separate frame structure having end and side members.","The end edges of the foil blade are fastened to the end bars of the frame structure and guiding grooves provided in opposite inner walls of the shearing or shaver head housing again serve to guide the lateral or side members of the frame, which are flexible, in similar manner to the flexible side edges of the integral blade structure of FIG.","3.The perforated foil blade again varies in thickness along its longitudinal extent from one end edge to the other.","In either arrangement, integral or frame-mounted, a foil portion of the desired thickness is selectively brought into a cutting disposition with respect to the cutter blade 15 by controlled sliding movement of the foil blade 17 in its longitudinal direction This is facilitated by the external button arrangement 25 which runs in the guide slot 26 along the surface of the shaver unit head 11.In this manner, a required depth of cut may be readily chosen and speedily varied in controlled manner as required by the user.","Retention of a particular selected position of the foil 17 may be achieved by cooperating frictional interaction between button 25 and slot 26, e.g.","by button 25 and connection or fastener portion 24 defining a “nip” or light gripping engagement between inner and outer face regions of head 11 bordering slot 26.In order to provide guidance for the side edges 1a of the variable thickness shear blade 17, the guide grooves 27 in the shaver head have a dimension between the opposite side surfaces of the guiding groove 27 which may vary but is always appropriate for the thickness dimension of the blade 17 in the relevant region.","In order to ensure that the thinner regions of the foil 17 are appropriately guided, the guide grooves 27 then may be provided, if necessary, with appropriate resilient means, such as leaf springs or the like, to urge the foil 17 towards an appropriate side face of the groove 27, and thereby take up as it were any possible intervening redundant space between a thinner portion of the foil 17 and the opposite side wall of the guiding groove 27.Alternatively, in either embodiment of the foil, the side edges 18 or the corresponding side frame members may be arranged to have a uniform thickness throughout the longitudinal extent of the shear blade or foil 17, so that the provision of resilient means in the guide grooves is not then required.","The installed foil blade 17 is stretched as it were over the curved surface of cutter blade 15 at the cutting end of the shaver head unit 11.The foil blade 17 is tensioned in the outward direction by the spring-loading (16), underlying cutter blade 15, which also serves for holding it in place.","In the embodiment as shown in FIGS.","2 and 3 of a foil 17 suitable for use with the invention, variation in thickness is substantially continuous from one end of the foil 17 to the other, from a minimum thickness portion 21 at a first end, to a maximum thickness region 22 at the other end.","In this way, a substantially infinite variation of thickness can be selected between the two extremes.","The side edges of the foil 18 may however be of constant or uniform thickness over the entire longitudinal extent of the blade 17, depending on the properties of the material selected for the foil 17, as already discussed above, or the side frame members, in a framed embodiment, may be likewise of uniform thickness.","In an alternative embodiment (not illustrated), the foil may have a number of sections, each of which is of a different thickness and configuration, and each section being connected to an adjacent section by for example a hinge portion or by any other suitable manner of interconnection.","The sections are therefore joined for flexure, i.e.","to be flexible when the continuous foil structure is formed from the individual sections, and define a flexible continuous foil.","The sections may for example be joined by being bonded to one another in some suitable manner.","In such an embodiment, the foil has therefore a number of discrete portions or sections, each of which has its own individual thickness, so that a corresponding depth of cut applies.","Again, an appropriate depth of cut is readily selected in controlled manner by sliding movement of the foil to the requisite location.","Variable aperture size and configuration and/or intermingled sizes and configurations of aperture in different regions of the foil may also be provided in this constructional variant.","In an alternative mounting arrangement shown in FIG.","4, the structure and arrangement of the shaver head housing 11′ and shaver body 12′, as well as the cutter 15′ structure, correspond to the arrangements of FIG.","2, features similar to those of FIG.","2 being designated by use of the same reference numerals with the addition of an apostrophe.","However, in order to secure longitudinal displacement of the perforated foil blade 17′, the sliding button-controlled arrangement of FIG.","2 is replaced by a pivotal structure, in which end edges of the foil blade 17′ are connected 34, 35 to respective ends of a pivotally mounted rocker mechanism 36 arranged 37 for rocking movement within the shaver head housing 11′.","Any suitable means may be provided for connecting the foil blade 17′ to cross-bar portions of the rocker lever structure 36, such as fasteners 34, 35.Likewise, any suitable arrangement may be provided to secure controlled pivoting 37 of the rocking blade mounting frame or structure 36, for example a lever arm or knob at the end of a pivot axis mounting 37, or alternatively, displacement may be effected merely by sliding the perforated foil blade 17′ over the surface of the cutter blade 15′, using the fingers.","In either arrangement, the rocker member 36 is suitably pivotally mounted by way of pins or a shaft 37, bearingly accommodated on the interior of the side walls of the head housing 11′.","Retention of the foil blade 17′ in a particular longitudinal disposition with respect to the blade 15′ may again be achieved such as by suitable frictional cooperation or interaction between pivot mounting 37 and the bearing features by which pivot axis or shaft 37 is accommodated in the walls of the head housing 11′, or by any other suitable equivalent arrangement, such as for example a locking screw or the like associated with a pivot position adjusting knob.","A multiplicity of possible arrangements will be apparent for achieving substantial locking of the foil 17′ in a particular selected shaving disposition by securing the rocker arm or lever 36 in a particular pivoted disposition following selective controlled displacement of the foil to align a designated region of the foil with the cutter blade.","In any variant of the variable thickness foil embodiment of the invention, the regions of different foil thickness may be differentiated from one another such as by for example colour coding.","FIG.","5 shows detail of a feature by which an especially effective cutting or shearing action may be achieved for a foil shear blade.","In the arrangement shown, in the formation of the hair entry openings 28 in the foil structure shear blade 17, the periphery of each entry opening 28 is turned slightly downwards, so that the peripheral region 29 surrounding the opening 28 protrudes below the general level of the remainder of the underside of the foil shear blade 17 to define, as it were, a lip.","An especially effective shearing type action is then achieved when the cutter blade 15 interacts with this peripheral region 29 of the foil shear blade 17 surrounding the hair entry opening 28.Referring now to FIG.","6, an embodiment of the invention applicable to a rotary type dry shaver will now be described.","In a typical construction, a rotary dry shaver 41 may have a number of cutter units 42, typically three as shown in the drawing, or alternatively two units 42, in each case located at the cutting end face 43 of the shaver body 44.The cutting drive and mounting arrangements of this variant of the invention are generally in accordance with substantially conventional arrangements well-known in the industry and only the particular deviations from such constructions required by the present invention are now described in detail.","In such constructions, each cutter unit has an external or shear blade, which has hair entry apertures or slits and is underlaid, in the assembled condition, by a rotating cutter blade, which severs hairs extending into the cutting region of the cutter blade through the slits or perforations (apertures) of the shear blade.","According to the invention, each of the rotary blade assemblies 42 is equipped with a shearing blade unit having a particular thickness or depth dimension of the hair entry slits or perforations, so as to provide for a corresponding depth of cut.","Preferably, each blade assembly provides the same depth of cut.","In a favoured arrangement according to the invention, two or more interchangeable head assemblies may be provided, each having blades providing a particular depth of cut, e.g.","“close” shave or “long” cut, so that a user can choose a head assembly providing for a tight cut or a head assembly providing for a less close shave.","Thus the invention provides a shaving kit incorporating a dry shaver body unit which includes drive features, one or more head assemblies which are interchangeable with the drive portion or body of the shaver and two or more sets of blades which can be interchangeably associated with any of the head assemblies.","In this manner, a user may set up one head assembly with each blade providing for a close shave and a second head assembly on which each blade allows a more generous cut.","For regions of the skin where the close shave is desired, the user applies the close shave blades by mounting the appropriate head assembly on the drive portion of the shaver unit.","Where it is necessary or desirable to apply a less close shave, the user then exchanges the close shave head assembly for that enabling the less close shave.","Referring now to FIG.","7, there is shown, in pictorial view, a rotary shaver having two cutter heads, each of which is angled away from the longitudinal axis of the cutter unit, so that only one head can be used at any given time.","Each cutter head is located on an angled end face region of the shaver, so that each cutter can be engaged individually against the skin, without engagement of the other cutter, located on the other angled face.","In a variant, each angled face could carry two blades, in a two-bladed cutter head assembly.","The shaver would then have two twin-blade head assemblies, one on each angled cutter face.","In a shaver of this kind in accordance with the invention, all cutter blades and each head assembly are interchangeable with other blade units or head assemblies respectively.","As already indicated, the shaver unit in this embodiment also may be provided as a kit, with for example two or more detachable and interchangeable head assemblies and two or more sets of blades providing for different depths of cut.","The user sets up one of the head assemblies with blades suitable for a close shave and the other head assembly for a less close shave.","The two head assemblies can be mounted on the base portion of the shaver unit for use in the manner already described, namely close shave where feasible and less close shave where necessary.","Additional head assemblies may also be included in the kit of parts as also may additional sets of blades providing for still different depths of cut.","In a further variant of the arrangement provided in FIG.","7, the base unit may accommodate three heads, each angled outwardly from the generally longitudinal axis of the base portion which also defines the gripping or handle portion of the unit, so that the three head regions or head assembly mounting portions are spaced equidistantly around this axis substantially at 120° from each other.","The unit may then be set up to provide for three different depths of cut and the user applies the appropriate depth of cut by rotating the unit so that the selected head region can be applied to the skin.","In this manner, particular ease of use is facilitated with maximum interchangeability and flexibility as regards depths of cut, by the diverse options available for interchange or exchange of head assemblies and blade assemblies.","Referring now to FIG.","8, which shows an external cutting member or shear blade 51 of generally conventional configuration, a head 42 equipped with this type of shear blade 51 may be regarded as providing a minimum depth of cut, for a tight shave.","As shown in this drawing, the cutting member 51, which is in the general shape of a top-hat, or an inverted cup, has a series of generally radially-oriented, optionally skewed, hair entry slits or apertures 52, in an outwardly-directed generally disc-shaped region 55 of the blade 51, which is upwardly-oriented when mounted on the cutting unit 42.The outer surface of this disc-shaped region 55 of cutting member 51 comes into contact with the skin during a shaving operation.","From the side of this disc-shaped region 55 opposite to the skin-engaging surface, at the periphery of the disc, a generally annular peripheral wall portion 56 extends (downwardly in the mounted condition on cutting unit 42) to terminate in an externally-directed holding flange 53, by which the shear blade is secured in position on the shaver cutting unit 42.Centrally of the generally disc-shaped region 55, radially inward of the slits 52, there is a provided a central dished portion 54, which is set back from the skin-engaging surface defined by the outer face of region 55 at the slots 52.In order to provide for a more generous shave, in other words a less tight or close shave leaving a longer beard length, at least one other cutter 61 is provided, in the form of a shear blade of greater thickness, as shown in FIG.","9, so that the depth of cut is greater when that particular cutter is applied to the face.","The general construction of cutter or shear blade 61 is similar to blade 51, so that blades 51 and 61 are interchangeable on the dry shaver of FIG.","6 or that of FIG.","7.In the shaver of FIG.","6, preferably all of the blades provide for a close shave or all of the blades provide for a less close shave.","However, in the two head shaver of FIG.","7 or in a three head variant of this design, the blade or blades of one of the heads may provide for a close shave and the blades of the other or another of the heads provide for a less close shave, so that different regions of the face may be shaved according to need, either to provide a smooth close shave or a less tight cut.","Thus in the FIG.","7 variant, a tight shave or a less close shave may be achieved without requiring the user to change either a blade assembly or a head assembly, although this option may also be present within the scope of the invention.","Again as already discussed, the invention in this aspect offers extensive flexibility of use, not only by selecting the appropriate head region to be applied to the skin in the two-headed arrangement of FIG.","7 or the alternative three-headed variant discussed previously, but further flexibility and interchangeability is facilitated by the inclusion of additional head assemblies and/or additional blade assemblies in a shaver kit provided in accordance with the principles of the is invention.","The only dimensional difference between the blades 51 and 61 is in the thickness of the blade in the outer skin-engaging portion.","Slits 62, flange 63 and central dished region 64 correspond to portions 52, 53 and 54 of the standard cutter 51 of FIG.","8, but the slits 62 are substantially greater in depth than the slits 52 of blade 51.Accordingly, when this shear blade 61 is applied to the face, the hairs are cut at a longer length than in the case of the blade 51.In use of this embodiment of the invention, the second cutter 61 (FIG.","9) is to be applied to skin areas where the cut is not to be too tight, so as to avoid the dermatological and other skin problems previously identified, while the first cutter 51 (FIG.","8) will be used to achieve a tight shave and good appearance where this does not create any skin hazard or potential damage.","Yet another cutter unit may provide another thickness of shear blade, so that a still further depth of cut may be effected, as required.","Thus, at least two possible depths of cut are selectively available to the user during operation of the dry shaver.","In the three-blade shaver of FIG.","6, completely separate head units 45 may be provided for, first of all, a standard cut, and secondly and alternatively, for situations where a less tight cut is required, this second alternative head having all of the shear blades dimensioned to provide greater spacing from the skin surface according to the arrangement of FIG.","9, while the first head has shear blades in accordance with FIG.","8.Referring again to FIG.","6, head unit 45 is separable from the body 44 of the shaver unit, so that the entire head structure may thus be removed and exchanged for one providing for such a different depth of cut by virtue of the shear blades of all of the cutting units of the alternative head portion 45 being provided with shear blades of different slit or aperture depth from the depth applicable to a standard cut.","Again in this variant, at least two possible depths of cut are then available to the user, simply by head interchange.","Thus each head, standard and alternative, has shear blades of uniform cut capability for all blades of that head, but the depth of cut achievable varies as between the heads.","It is known in the rotary shaver art to provide head and blade assemblies that can be readily dismantled for cleaning and maintenance.","Any known construction of head or blade assembly of this kind may be applied to the improved blade arrangements and techniques provided by the present invention.","In particular, such known constructions may be applied to the interchangeable head assemblies, and/or individual interchangeable blade units, as provided the present invention.","According to the present invention, the dismantling capability of head and blade assemblies is further developed to provide extensive interchangeability on the drive portion of the shaver as between head assemblies on the drive base and blade assemblies on the head assemblies.","Thus a rotary shaver in accordance with the invention having a base portion suitable for mounting a single head assembly may be provided as a kit including two head assemblies, each of which has blades enabling a different depth of cut.","One head assembly may provide for a close shave and the other head assembly for a less close shave.","In addition, further sets of blades or blade assemblies may be provided, so that still different depths of cut may also be enabled by mounting a further alternative set of blades on one of the heads.","In a variant of the invention, the base drive unit may accommodate more than one head assembly at the same time.","Both head assemblies can be driven simultaneously or selectively by the base drive unit.","One of the head assemblies is provided with blades suitable for a close shave and the other with blades suitable for a less close shave.","One or other of the head assemblies can then be applied to the skin, depending on the depth of cut required.","Yet a further variant of this aspect of the invention provides for the single drive unit to have no less than three head assemblies, all capable of use without requiring demounting of a head assembly and its replacement by an alternative.","In any of the variants of the invention, the system may be provided as a kit of parts, with a single base unit or drive portion and two or three or more head assemblies.","Each head assembly may be pre-provided with a set of blades of a particular depth of cut.","In addition or alternatively, further blade assemblies may be included in the kit of parts, for interchangeable placement or mounting on any of the head assemblies as may be required.","Great flexibility and adaptability in use is thus available in accordance with the principles of the invention.","All head assemblies are readily removable from the base drive portion and all blade assemblies are readily removable from the respective head assembly.","All sub-assemblies of the system are fully interchangeable, so that any head assembly may be exchanged for any other head assembly and any head assembly may receive any blade assembly.","Thus any head portion of the unit may receive mounted on it, blade units of any depth of cut.","Thus in a kit of parts according to the invention, the shaver unit is interchangeably associatable with any one of a plurality or multiplicity of heads.","Each head may receive a plurality or multiplicity of blades and any blade may be demountably associated with any head.","Where angled heads are provided for selective optional use without necessarily exchanging a head for an alternative head unit, the principles of full interchangeability and exchangeability continue to prevail, for ease of advance selection of the particular depth of cut required on any of the angled head regions.","In FIGS.","10 and 11, there is shown an alternative arrangement in which the actual depth of cut for a pair of rotary cutters may be selectively varied during use.","The arrangement illustrated is for a two head cutter rotary dry shaver.","As shown, there is an inner shear blade structure 81, against the underside of which rotary cutters 85 exercise the cutting action.","An outer “dummy” shear blade or external cutter cover arrangement 91 is fixedly mounted in the cutter head 71 for cooperation with the inner shear blade structure 81 to provide for variable spacing between shear blade 81 and outer “dummy” blade or cover 91.The rotary blades 85 are rotatably mounted in known manner and are driven in rotary motion by likewise known drive arrangements (not illustrated).","According to the present invention, the outer “dummy” shear blade or external cutter cover arrangement is a fixed outer perforated cover 91 and is associated with the inner shear blade structure 81 for variable depth of shave or cut, the shear blade structure 81 being mounted to be displaceable towards and away from the outer cover 91.The outer surface 92 of this cover or “dummy” shear blade 91 engages the skin.","The cover or spacer head 91 is suitably provided with a series of apertures 93 which may be of relatively large size compared with the slits 82 of the inner shear blade 81, through which apertures 93 the facial hair to be cut passes to be gripped by the perforations or slits 82 of the shear blade 81 and cut in the usual way by the rotary blades 85.The adjustable arrangement enables the shear blade structure 81 to be moved towards and away from the outer cover 91, so that the depth of cut may be selectively varied in controlled manner by the user, depending on the portion of the skin to be shaved.","Any one of a diversity of cover-to-blade space-adjusting constructions may be provided to enable the requisite adjustment.","A particularly favoured arrangement for securing this spacing adjustment is shown in FIGS.","10 and 11.In accordance with this arrangement, the dummy external cutter or cover 91 provided with the apertured region 93, which generally overlies the slit region of the shear blade 81, also has a substantially solid central region 94.The general configuration of this additional cover or dummy cutter 91 is therefore broadly similar to that of the actual shear blade 81.Variants are however also possible, to the extent that the cover 91 may be of more open work type construction, with the apertures 93 being defined between a series of radial and circumferential bars, wire type members, or perforated plates, and the central bridging region of “dummy” shear blade 91 is also not an essential structural feature.","In order to secure the inward and outward displaceability of shear blade structure 81 towards and away from the fixed cover 91, FIGS.","10 and 11 provide a control member 95 which is in the form of a peripheral endless band slideable in an external slot 96 in the external side wall 72 of the shaver unit head region 71.At four locations on its endless path, the peripheral band 95 has camming members 97, 98 which cooperate with mating camming members 87, 88 provided on a support structure 89 which either comprises or carries the shear blade 87.Movement of the endless band 95 in the direction indicated by arrow “a” in FIG.","8 causes the shear blade structure 81 to be pulled away from the fixed cover 91 and therefore increases the depth of shave or cut, while movement of the band 95 in the direction indicated by arrow “b” in FIG.","8 produces the converse effect, namely moves the shear blade 81 closer to the fixed blade 91.A structure of this type is known in itself and is applied in an existing dry shaver arrangement in which the shear blades are mounted in a quasi-floating manner.","The structure is such that the rotor blades remain at all times in fixed proximity to the shear blade and travel inwards and outwards with the shear blade, being flexibly coupled to the drive arrangements so that rotation of the cutter blades may be effected at all locations of the shear blade.","In this manner therefore the principles of the invention may be embodied in a dry shaver of the rotary cutter blade type also.","The invention in this embodiment therefore provides substantially infinitely variable selective choice of a preferred depth of cut for any region of the body to be shaved, by varying the displacement between the skin-engaging outer or upper surface 92 of the cover or dummy shear blade 91 and the inner face of the cooperating shear blade 81, where the rotary action of the underlying cutter blades 85 effects actual severance of the hair.","Thus according to the present variant of the invention, there is again achieved a dry shaver unit in which a variable depth of cut is selectively available at the user's choice.","The control arrangement 95 shown in FIG.","10 is exemplary only.","The arrangements described and proposals indicated herein for shear blade 81 positional control relative to the dummy blade or outer cover 91 are also exemplary only and in no way limit the scope of the invention as defined by the appended claims.","In particular, alternative control or drive arrangements for displacing the cover portion relative to the cutter blade may be devised and provided, including, but not limited to, mechanical inversions of the arrangements described and illustrated.","The words “comprises/comprising” and the word “having” when used herein with reference to the present invention are used to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof."]],"string":"[\n [\n \"Dry shaver for variable heights of cut\",\n \"A dry shaver (11) has a single cutter blade (15) and a single cooperating shear blade (17).\",\n \"The shear blade (17) is an elongate flexible foil, the thickness of which is different at different regions (21, 22) along the elongate extent of the foil (17).\",\n \"The foil (17) is mounted (27) for displacement in its elongate direction so that a foil region (21, 22) of a particular thickness may be selectively aligned with the cutter blade (15).\",\n \"The spacing between a skin engaging surface of the shear blade (17) and the opposite face of the shear blade (17) which cooperates with the cutter blade (15) is thus selectively variable.\",\n \"In a further embodiment, the dry shaver is of the rotary type and has a plurality of interchangeable shear blades of different thicknesses.\",\n \"In a still further embodiment, a rotary dry shaver has a mechanism for varying the spacing between the outer surface of a cover member overlying a shear blade and a cooperating rotary cutter blade.\"\n ],\n [\n \"1.A dry shaver unit comprising: at least one cutter blade; and a cooperating shear blade; wherein a spacing between a skin-engaging surface of the shaver unit and regions of the shear blade which cooperate with the cutter blade can be varied.\",\n \"2.A dry shaver unit according to claim 1, wherein different spacings between the skin-engaging surface of the shaver unit and the regions of a shear blade which cooperate with a cutter blade are selectively available.\",\n \"3.A dry shaver according to claim 2, wherein the shear blade is an elongate flexible foil, the thickness of which is different at different regions along the elongate extent of the elongate flexible foil; and wherein the elongate flexible foil is mounted for displacement in its elongate direction so that a foil region of a particular thickness may be selectively aligned with the cutter blades whereby a spacing between a skin engaging surface of the shear blade and the opposite face of the shear blade which cooperates with the cutter blade is selectively variable.\",\n \"4.A dry shaver according to claim 1, wherein said at least one cutter blade is a rotary cutter blade, wherein the cooperating shear blade is located over the rotary cutter blade, and wherein the spacing between the skin engaging surface of the shear blade and the opposite face thereof for cooperation with the cutter blade can be varied.\",\n \"5.A dry shaver according to claim 4, wherein the shaver is provided with a plurality of shear blades and the spacing between the skin engaging surface of one of the shear blades and the opposite face thereof for cooperation with an associated cutter blade is different from the spacing between the corresponding surface and opposite face of at least one other of the plurality of shear blades.\",\n \"6.A dry shaver according to claim 5, comprising at least two rotary cutter blades, wherein each rotary cutter blade has a general axis of symmetry and the axis of symmetry of at least one rotary cutter blade is oriented differently from the axis of symmetry of at least one other rotary cutter blade with respect to a generally longitudinal axis of the dry shaver as whole, so that only one of the rotary cutter blades can be used for shaving purposes at one time.\",\n \"7.A dry shaver according to claim 5 further comprising: at least two cutting head assemblies, each head assembly having at least one cutter blade and a respective overlying cooperating shear blade, wherein a spacing between a skin engaging surface and an opposite face of the shear blade in at least one of the cutting head assemblies is different from a corresponding spacing in other head assemblies, and the head assemblies are interchangeably mountable on a body portion of the dry shaver.\",\n \"8.A dry shaver according to claim 4 further comprising a plurality of blade assemblies, each blade assembly comprising a cutter blade and an overlying cooperating shear blade, wherein the blade assemblies are interchangeably mountable on the dry shaver.\",\n \"9.\",\n \"(canceled) 10.A dry shaver according to claim 1, wherein the cutter blade comprises at least one rotary cutter blade, and wherein the cooperating shear blade is located over the rotary cutter blade and comprises a shear blade and cutter blade assembly, and further comprising a cover member overlying the shear blade, and a displacement mechanism for effecting controlled movement of the shear blade and cutter blade assembly towards and away from the cover member, so that the spacing between a skin engaging surface of the cover member and the face of the shear blade which cooperates with the cutter blade is variable.\",\n \"11.A dry shaver according to claim 2, wherein said at least one cutter blade is a rotary cutter blade, wherein the cooperating shear blade is located over the rotary cutter blade, and wherein the spacing between the skin engaging surface of the shear blade and the opposite face thereof for cooperation with the cutter blade can be varied.\",\n \"12.A dry shaver according to claim 6 further comprising: at least two cutting head assemblies, each head assembly having at least one cutter blade and a respective overlying cooperating shear blade, wherein a spacing between a skin engaging surface and an opposite face of the shear blade in at least one of the cutting head assemblies is different from a corresponding spacing in other head assemblies, and the head assemblies are interchangeably mountable on a body portion of the dry shaver.\",\n \"13.A dry shaver according to claim 5 further comprising a plurality of blade assemblies, each blade assembly comprising a cutter blade and an overlying cooperating shear blade, wherein the blade assemblies are interchangeably mountable on the dry shaver.\",\n \"14.A dry shaver according to claim 6 further comprising a plurality of blade assemblies, each blade assembly comprising a cutter blade and an overlying cooperating shear blade, wherein the blade assemblies are interchangeably mountable on the dry shaver.\"\n ],\n [\n \"<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention This invention relates to dry shavers having a cutter blade and a cooperating shear blade arrangement.\",\n \"The invention relates at least both to electric dry shavers of the type having a reciprocating cutter and a shear blade arrangement in the form of a perforated foil with a plurality of perforations, and also to dry shavers of the type having a rotary cutter member or blade underlying an external shear member provided with apertures for entry of the hairs to be cut.\",\n \"The history of shaving to date has been the search for the ever closer shave.\",\n \"In effectuating a close shave, certain skin and hair types encounter the problem of an inflammation due to re-entry into the skin of too short cut (tangentially exiting) hairs.\",\n \"It is an aim of the present invention to prevent or protect against this by purposefully cutting the hair long at a sufficient distance from the skin surface so as to prevent any possible re-entry into the skin of the close cut hairs.\",\n \"The invention is thus directed at cutting the hairs long enough away from the skin surface to prevent their re-entry.\",\n \"In shaving close, certain skin and hair types thus experience or develop inflammation due to re-entry into the skin of the closely shaved hairs.\",\n \"It is therefore a purpose of the present invention both to allow for a normal close shave and to allow also for a cut of hair at some distance from the skin surface, the distance from the skin surface being sufficient so as to prevent re-entry of the cut hairs into the skin.\",\n \"2.Description of the Prior Art GB-A-1,569,096 describes a dry shaver having a flexible perforated shear foil supported in a frame and a reciprocatable cutter which cooperates with the shear foil.\",\n \"The shear foil is supported in an arched position between two side members of the frame and a cutter blade or shear head is pressed against the interior side of the shear foil by means of a spring.\",\n \"Similar such units are described in U.S. Pat.\",\n \"No.\",\n \"3,729,821 and in a diversity of other patent documents.\",\n \"The cutter blade may be mounted for reciprocation in a direction substantially parallel to the axis of curvature of the shear blade foil, or alternatively, the cutter blade may be driven in oscillatory motion about this axis.\",\n \"In a particular example of the shear foil type dry shaver, there is described in U.S. Pat.\",\n \"No.\",\n \"3,694,916 an arrangement in which a reciprocatable cutter cooperates with a shearing blade having at least two shearing fields of different perforations.\",\n \"The shearing blade or foil is movable to bring a selected shearing field into the cooperating shearing disposition with the cutter blade.\",\n \"The arrangement enables a field of perforations which is most suitable for a particular type of skin to be selected but makes no reference to cutting hair at different lengths.\",\n \"The selected field of perforations is changed, but not the depth or height of cut.\",\n \"A diversity of rotary cutter shavers are also known, and reference may be directed inter alia to U.S. Pat.\",\n \"No.\",\n \"5,007,168, EP-A1-0,241,082, and EP-A1-0,484,795, all of which embody the rotary principle already indicated above.\",\n \"In WO-A-97/45235, there is described a rotary shaving apparatus in which radially directed hair entry apertures are of either broad or narrow dimension in the circumferential direction.\",\n \"The thickness of the shear blade in which the hair entry apertures are defined varies, depending on the dimension of the hair entry apertures.\",\n \"The shear blade is of greater thickness in a region thereof containing broad hair entry apertures than it is in a region containing narrow hair entry apertures.\",\n \"Thus the blade has a wavy or undulating configuration in the circumferential direction, with the depth or thickness of the blade increasing or reducing as between broad hair aperture entry regions and narrow hair entry aperture regions.\",\n \"The purpose of the arrangement is said to be to enable both short and long hairs to be caught and severed effectively with the end result of a close shave, while avoiding, as far as possible, irritation of the skin.\"\n ],\n [\n \"<SOH> BRIEF SUMMARY OF THE INVENTION <EOH>It is a general object of the invention to provide a dry shaver unit directed to overcoming the inflammation problems discussed hereinabove.\",\n \"According to the invention in a first and most general aspect, there is provided a dry shaver unit comprising at least one cutter blade and a cooperating shear blade arrangement in which provision is made for the spacing between a skin-engaging surface of the shaver unit and regions of the shear blade arrangement which cooperate with the cutter blade to be increased as compared with the spacing required to achieve a “close shave”.\",\n \"It is a particular object of the present invention to provide a dry shaver in which the depth of cut is variable, so that hair to be cut may be left at a variable length over certain areas of the skin as required, in order inter alia to avoid the development of a shaving rash due to re-entry into the skin of too-short tangentially cut hairs exiting the skin at an acute angle.\",\n \"According to the invention in a first specific aspect, there is therefore provided a dry shaver unit wherein different spacings between a skin-engaging surface of the shaver unit and regions of a shear blade arrangement which cooperate with a cutter blade are selectively available.\",\n \"In a particular embodiment of dry shaver according to the invention, a single cutter blade cooperates with a single shear blade and the spacing between a skin engaging surface of the shear blade and the opposite face of the shear blade which cooperates with the cutter blade (i.e.\",\n \"the cutter blade surface or side of the shear blade) is selectively variable.\",\n \"In a favoured construction, the shear blade is an elongate flexible foil, the thickness of which is different at different regions along the elongate extent of the foil, and the foil is mounted for displacement in its elongate direction so that a foil region of a particular thickness may be selectively aligned with the cutter blade.\",\n \"The foil may vary in thickness in a substantially continuous manner over its elongate extent from a minimum thickness portion to a maximum thickness portion, or alternatively the foil may comprise a sequence of foil portions, each of which has a different thickness, the foil portions being flexibly interconnected with one another.\",\n \"A control member is suitably attached to the foil, the control member being displaceable in selective manner in the elongate direction of the foil.\",\n \"In a preferred arrangement, the control member comprises a control stud extending through a slot defined in a wall portion of the shaver, an external region of the control stud being manually engageable to effect displacement of the foil in its elongate direction.\",\n \"The foil may be retained in a particular selected position such as by frictional interaction between control stud and slot.\",\n \"In an alternative arrangement, opposite elongate ends of the foil may be engaged at opposite ends of a rocking mechanism, displacement of the foil in its elongate direction being effected by rocking displacement of the rocking mechanism.\",\n \"In a further aspect of the invention, there is provided a dry shaver in which said at least one cutter blade is a rotary cutter blade, the or each cutter blade having an overlying cooperating shear blade, wherein the spacing between the skin engaging surface of the shear blade and the opposite face thereof for cooperation with the cutter blade is increased as compared with the spacing required to achieve a “close shave”.\",\n \"In a particular rotary construction of dry shaver according to the invention, the shaver is provided with a plurality of shear blades and the spacing between the skin engaging surface of one of the shear blades and the opposite face thereof for cooperation with an associated cutter blade (i.e.\",\n \"the cutter blade surface or side of the shear blade) is different from the spacing between the corresponding surface and opposite face of at least one other of the plurality of shear blades.\",\n \"Thus, for example, one of the shear blades may provide for a close shave and another shear blade for a less close shave For a shaver having a head with two or three blades used simultaneously, the invention may provide two or more sets of blades for use on the head, one of the sets of blades providing for a close shave and another of the sets of blades enabling a less close shave.\",\n \"It will be appreciated that more than two such sets may be provided, i, a third set for example facilitating a shave intermediate between a close shave and a long shave.\",\n \"In a variant of this aspect of the invention, a dry shaver may comprise at least two rotary cutter blades, wherein each rotary cutter blade has a general axis of symmetry and the axis of symmetry of at least one rotary cutter blade is oriented differently from the axis of symmetry of at least one other rotary cutter blade with respect to a generally longitudinal axis of the dry shaver as whole, so that only one of at said least one rotary cutter blade and said at least one other rotary cutter blade can be used for shaving purposes at one time.\",\n \"Thus in this variant of the invention, the shaver is provided with two or more shaving head regions, each of which faces away from the general longitudinal axis of the unit in a different direction, so that only one of the head regions can be used at a time.\",\n \"Each head region may have two or more blades and as previously indicated, the shaver unit may be provided with multiple sets of blades so that one head region may be set up for a close shave and another head region set up for a less close shave.\",\n \"Because this embodiment of the invention has two or more head regions facing in different directions, the unit may be set up to enable the user to choose a close shave or a less close shave simply by applying the appropriate head region to the skin.\",\n \"In yet another variant of this aspect of the invention, the shaver unit may have three head regions, each of which faces in a different direction away from the general longitudinal axis of the unit, the three head regions or head assemblies being spaced apart by substantially one hundred and twenty degrees about this longitudinal axis.\",\n \"In any variant of dry shaver according to the rotary aspect of the invention, the shaver unit may be provided in combination with at least two cutting head assemblies, each head assembly having at least one cutter blade and the or each cutter blade having a respective overlying cooperating shear blade, wherein the or each shear blade of one of the head assemblies has a spacing between its skin engaging surface and its opposite face which is the same for the or all of the shear blade(s) of said one of the head assemblies but is different from the corresponding spacing for the or all of the shear blade(s) of the other of the head assemblies, and the head assemblies are interchangeably mountable on a body portion of the dry shaver.\",\n \"In addition to being provided with two or more interchangeable head assemblies, multiple sets of blades providing for different depths of cut may also be included in the shaver kit.\",\n \"Thus one head may be set up with blades suitable for a close shave, and another of the head assemblies set up for the less close shave.\",\n \"The user wishing to change from one class of shave to the other simply exchanges the heads appropriately.\",\n \"Thus the invention extends to a shaver kit including a shaver body unit and two or more head assemblies interchangeably associatable with the body unit which contains the drive features of the shaver, and the kit may optionally include multiple blade assemblies for individual assocation as required with any of the head assemblies.\",\n \"Extensive flexibility of use is thus available, either by interchanging a head assembly or set of blades as required, or, for the arrangement provided with multiple head regions or head assemblies disposed in different angular orientations, merely by applying the appropriate head region to the skin, with optional advance mounting of blades of an appropriate depth of cut on the selected head region before use, as may be required.\",\n \"The dry shaver of the invention, in any of the rotary embodiments, may be provided in combination with a plurality of blade assemblies, each blade assembly comprising a cutter blade and an overlying cooperating shear blade, wherein the blade assemblies are interchangeably mountable on the dry shaver.\",\n \"Thus, as already indicated, in a dry shaver kit according to this aspect of the invention and comprising any of the foregoing features of the invention, the kit may include multiple sets of blade assemblies, interchangeably mountable on the dry shaver, so that the user may select an appropriate depth of cut by mounting a blade or blades suitable for that depth of cut on a head assembly of the dry shaver unit or kit, as required.\",\n \"According to yet another aspect of the invention in its rotary configuration, there is provided a dry shaver comprising at least one rotary cutter blade, wherein the spacing between a skin-engaging surface of the dry shaver and the cutter blade is selectively variable and a value may be selected for said spacing which is increased as compared with the spacing required to achieve a “close shave” Thus in this variant of the invention, depth of cut is available in an infinitely variable manner similar to that afforded by the foil embodiment of the invention, but without necessitating either individual blade assemblies to be exchanged or the head assembly as a whole to be swapped for an alternative head assembly.\",\n \"In this aspect, the invention thus offers the immediately selectively variable depth of cut of the foil embodiment combined with features of the rotary construction.\",\n \"In a particular construction of the invention in this additional aspect, the shaver comprises at least one rotary cutter blade, the or each of which has an overlying cooperating shear blade and a cover member overlying the shear blade, the spacing between a skin engaging surface of the cover member and the face of the shear blade which cooperates with the cutter blade being variable.\",\n \"In this arrangement, the shaver may include a displacement mechanism for effecting controlled movement of a shear blade and cutter blade assembly towards and away from the cover member.\",\n \"Thus in these foregoing embodiments, the advantages of immediate or direct, controlled substantially infinitely variable adjustment of the depth of cut within predetermined limits is achieved for a rotary shaving unit also.\",\n \"The invention also extends to a blade assembly for a dry shaver comprising a rotary cutter blade and a cooperating shear blade, wherein the spacing between a skin engaging surface of the shear blade and the opposite face thereof for cooperation with the cutter blade is increased as compared with the spacing required to achieve a “close shave”.\",\n \"The invention likewise additionally encompasses a head assembly for a dry shaver, comprising at least one rotary cutter blade, the or each cutter blade having an overlying cooperating shear blade, wherein the spacing between the skin engaging surface of the shear blade and the opposite face thereof for cooperation with the cutter blade is increased as compared with the spacing required to achieve a “close shave”.\",\n \"Thus the invention embraces in addition blade assemblies and head assemblies incorporating the features of the invention as independent units, whether in combination with a dry shaver or as entirely separate assemblies.\",\n \"The invention likewise extends to any kit of parts comprising a dry shaver drive unit, one or more head assemblies, and/or one or more blade assemblies, provided in combination or in any commercial association, in so far as such units incorporate the features of the invention or together cooperate in defining equipment lying within the scope of the invention.\",\n \"The invention also extends to a dry shaver substantially as described herein with reference to and as shown in any one or more of the accompanying drawings.\"\n ],\n [\n \"BACKGROUND OF THE INVENTION 1.Field of the Invention This invention relates to dry shavers having a cutter blade and a cooperating shear blade arrangement.\",\n \"The invention relates at least both to electric dry shavers of the type having a reciprocating cutter and a shear blade arrangement in the form of a perforated foil with a plurality of perforations, and also to dry shavers of the type having a rotary cutter member or blade underlying an external shear member provided with apertures for entry of the hairs to be cut.\",\n \"The history of shaving to date has been the search for the ever closer shave.\",\n \"In effectuating a close shave, certain skin and hair types encounter the problem of an inflammation due to re-entry into the skin of too short cut (tangentially exiting) hairs.\",\n \"It is an aim of the present invention to prevent or protect against this by purposefully cutting the hair long at a sufficient distance from the skin surface so as to prevent any possible re-entry into the skin of the close cut hairs.\",\n \"The invention is thus directed at cutting the hairs long enough away from the skin surface to prevent their re-entry.\",\n \"In shaving close, certain skin and hair types thus experience or develop inflammation due to re-entry into the skin of the closely shaved hairs.\",\n \"It is therefore a purpose of the present invention both to allow for a normal close shave and to allow also for a cut of hair at some distance from the skin surface, the distance from the skin surface being sufficient so as to prevent re-entry of the cut hairs into the skin.\",\n \"2.Description of the Prior Art GB-A-1,569,096 describes a dry shaver having a flexible perforated shear foil supported in a frame and a reciprocatable cutter which cooperates with the shear foil.\",\n \"The shear foil is supported in an arched position between two side members of the frame and a cutter blade or shear head is pressed against the interior side of the shear foil by means of a spring.\",\n \"Similar such units are described in U.S. Pat.\",\n \"No.\",\n \"3,729,821 and in a diversity of other patent documents.\",\n \"The cutter blade may be mounted for reciprocation in a direction substantially parallel to the axis of curvature of the shear blade foil, or alternatively, the cutter blade may be driven in oscillatory motion about this axis.\",\n \"In a particular example of the shear foil type dry shaver, there is described in U.S. Pat.\",\n \"No.\",\n \"3,694,916 an arrangement in which a reciprocatable cutter cooperates with a shearing blade having at least two shearing fields of different perforations.\",\n \"The shearing blade or foil is movable to bring a selected shearing field into the cooperating shearing disposition with the cutter blade.\",\n \"The arrangement enables a field of perforations which is most suitable for a particular type of skin to be selected but makes no reference to cutting hair at different lengths.\",\n \"The selected field of perforations is changed, but not the depth or height of cut.\",\n \"A diversity of rotary cutter shavers are also known, and reference may be directed inter alia to U.S. Pat.\",\n \"No.\",\n \"5,007,168, EP-A1-0,241,082, and EP-A1-0,484,795, all of which embody the rotary principle already indicated above.\",\n \"In WO-A-97/45235, there is described a rotary shaving apparatus in which radially directed hair entry apertures are of either broad or narrow dimension in the circumferential direction.\",\n \"The thickness of the shear blade in which the hair entry apertures are defined varies, depending on the dimension of the hair entry apertures.\",\n \"The shear blade is of greater thickness in a region thereof containing broad hair entry apertures than it is in a region containing narrow hair entry apertures.\",\n \"Thus the blade has a wavy or undulating configuration in the circumferential direction, with the depth or thickness of the blade increasing or reducing as between broad hair aperture entry regions and narrow hair entry aperture regions.\",\n \"The purpose of the arrangement is said to be to enable both short and long hairs to be caught and severed effectively with the end result of a close shave, while avoiding, as far as possible, irritation of the skin.\",\n \"BRIEF SUMMARY OF THE INVENTION It is a general object of the invention to provide a dry shaver unit directed to overcoming the inflammation problems discussed hereinabove.\",\n \"According to the invention in a first and most general aspect, there is provided a dry shaver unit comprising at least one cutter blade and a cooperating shear blade arrangement in which provision is made for the spacing between a skin-engaging surface of the shaver unit and regions of the shear blade arrangement which cooperate with the cutter blade to be increased as compared with the spacing required to achieve a “close shave”.\",\n \"It is a particular object of the present invention to provide a dry shaver in which the depth of cut is variable, so that hair to be cut may be left at a variable length over certain areas of the skin as required, in order inter alia to avoid the development of a shaving rash due to re-entry into the skin of too-short tangentially cut hairs exiting the skin at an acute angle.\",\n \"According to the invention in a first specific aspect, there is therefore provided a dry shaver unit wherein different spacings between a skin-engaging surface of the shaver unit and regions of a shear blade arrangement which cooperate with a cutter blade are selectively available.\",\n \"In a particular embodiment of dry shaver according to the invention, a single cutter blade cooperates with a single shear blade and the spacing between a skin engaging surface of the shear blade and the opposite face of the shear blade which cooperates with the cutter blade (i.e.\",\n \"the cutter blade surface or side of the shear blade) is selectively variable.\",\n \"In a favoured construction, the shear blade is an elongate flexible foil, the thickness of which is different at different regions along the elongate extent of the foil, and the foil is mounted for displacement in its elongate direction so that a foil region of a particular thickness may be selectively aligned with the cutter blade.\",\n \"The foil may vary in thickness in a substantially continuous manner over its elongate extent from a minimum thickness portion to a maximum thickness portion, or alternatively the foil may comprise a sequence of foil portions, each of which has a different thickness, the foil portions being flexibly interconnected with one another.\",\n \"A control member is suitably attached to the foil, the control member being displaceable in selective manner in the elongate direction of the foil.\",\n \"In a preferred arrangement, the control member comprises a control stud extending through a slot defined in a wall portion of the shaver, an external region of the control stud being manually engageable to effect displacement of the foil in its elongate direction.\",\n \"The foil may be retained in a particular selected position such as by frictional interaction between control stud and slot.\",\n \"In an alternative arrangement, opposite elongate ends of the foil may be engaged at opposite ends of a rocking mechanism, displacement of the foil in its elongate direction being effected by rocking displacement of the rocking mechanism.\",\n \"In a further aspect of the invention, there is provided a dry shaver in which said at least one cutter blade is a rotary cutter blade, the or each cutter blade having an overlying cooperating shear blade, wherein the spacing between the skin engaging surface of the shear blade and the opposite face thereof for cooperation with the cutter blade is increased as compared with the spacing required to achieve a “close shave”.\",\n \"In a particular rotary construction of dry shaver according to the invention, the shaver is provided with a plurality of shear blades and the spacing between the skin engaging surface of one of the shear blades and the opposite face thereof for cooperation with an associated cutter blade (i.e.\",\n \"the cutter blade surface or side of the shear blade) is different from the spacing between the corresponding surface and opposite face of at least one other of the plurality of shear blades.\",\n \"Thus, for example, one of the shear blades may provide for a close shave and another shear blade for a less close shave For a shaver having a head with two or three blades used simultaneously, the invention may provide two or more sets of blades for use on the head, one of the sets of blades providing for a close shave and another of the sets of blades enabling a less close shave.\",\n \"It will be appreciated that more than two such sets may be provided, i, a third set for example facilitating a shave intermediate between a close shave and a long shave.\",\n \"In a variant of this aspect of the invention, a dry shaver may comprise at least two rotary cutter blades, wherein each rotary cutter blade has a general axis of symmetry and the axis of symmetry of at least one rotary cutter blade is oriented differently from the axis of symmetry of at least one other rotary cutter blade with respect to a generally longitudinal axis of the dry shaver as whole, so that only one of at said least one rotary cutter blade and said at least one other rotary cutter blade can be used for shaving purposes at one time.\",\n \"Thus in this variant of the invention, the shaver is provided with two or more shaving head regions, each of which faces away from the general longitudinal axis of the unit in a different direction, so that only one of the head regions can be used at a time.\",\n \"Each head region may have two or more blades and as previously indicated, the shaver unit may be provided with multiple sets of blades so that one head region may be set up for a close shave and another head region set up for a less close shave.\",\n \"Because this embodiment of the invention has two or more head regions facing in different directions, the unit may be set up to enable the user to choose a close shave or a less close shave simply by applying the appropriate head region to the skin.\",\n \"In yet another variant of this aspect of the invention, the shaver unit may have three head regions, each of which faces in a different direction away from the general longitudinal axis of the unit, the three head regions or head assemblies being spaced apart by substantially one hundred and twenty degrees about this longitudinal axis.\",\n \"In any variant of dry shaver according to the rotary aspect of the invention, the shaver unit may be provided in combination with at least two cutting head assemblies, each head assembly having at least one cutter blade and the or each cutter blade having a respective overlying cooperating shear blade, wherein the or each shear blade of one of the head assemblies has a spacing between its skin engaging surface and its opposite face which is the same for the or all of the shear blade(s) of said one of the head assemblies but is different from the corresponding spacing for the or all of the shear blade(s) of the other of the head assemblies, and the head assemblies are interchangeably mountable on a body portion of the dry shaver.\",\n \"In addition to being provided with two or more interchangeable head assemblies, multiple sets of blades providing for different depths of cut may also be included in the shaver kit.\",\n \"Thus one head may be set up with blades suitable for a close shave, and another of the head assemblies set up for the less close shave.\",\n \"The user wishing to change from one class of shave to the other simply exchanges the heads appropriately.\",\n \"Thus the invention extends to a shaver kit including a shaver body unit and two or more head assemblies interchangeably associatable with the body unit which contains the drive features of the shaver, and the kit may optionally include multiple blade assemblies for individual assocation as required with any of the head assemblies.\",\n \"Extensive flexibility of use is thus available, either by interchanging a head assembly or set of blades as required, or, for the arrangement provided with multiple head regions or head assemblies disposed in different angular orientations, merely by applying the appropriate head region to the skin, with optional advance mounting of blades of an appropriate depth of cut on the selected head region before use, as may be required.\",\n \"The dry shaver of the invention, in any of the rotary embodiments, may be provided in combination with a plurality of blade assemblies, each blade assembly comprising a cutter blade and an overlying cooperating shear blade, wherein the blade assemblies are interchangeably mountable on the dry shaver.\",\n \"Thus, as already indicated, in a dry shaver kit according to this aspect of the invention and comprising any of the foregoing features of the invention, the kit may include multiple sets of blade assemblies, interchangeably mountable on the dry shaver, so that the user may select an appropriate depth of cut by mounting a blade or blades suitable for that depth of cut on a head assembly of the dry shaver unit or kit, as required.\",\n \"According to yet another aspect of the invention in its rotary configuration, there is provided a dry shaver comprising at least one rotary cutter blade, wherein the spacing between a skin-engaging surface of the dry shaver and the cutter blade is selectively variable and a value may be selected for said spacing which is increased as compared with the spacing required to achieve a “close shave” Thus in this variant of the invention, depth of cut is available in an infinitely variable manner similar to that afforded by the foil embodiment of the invention, but without necessitating either individual blade assemblies to be exchanged or the head assembly as a whole to be swapped for an alternative head assembly.\",\n \"In this aspect, the invention thus offers the immediately selectively variable depth of cut of the foil embodiment combined with features of the rotary construction.\",\n \"In a particular construction of the invention in this additional aspect, the shaver comprises at least one rotary cutter blade, the or each of which has an overlying cooperating shear blade and a cover member overlying the shear blade, the spacing between a skin engaging surface of the cover member and the face of the shear blade which cooperates with the cutter blade being variable.\",\n \"In this arrangement, the shaver may include a displacement mechanism for effecting controlled movement of a shear blade and cutter blade assembly towards and away from the cover member.\",\n \"Thus in these foregoing embodiments, the advantages of immediate or direct, controlled substantially infinitely variable adjustment of the depth of cut within predetermined limits is achieved for a rotary shaving unit also.\",\n \"The invention also extends to a blade assembly for a dry shaver comprising a rotary cutter blade and a cooperating shear blade, wherein the spacing between a skin engaging surface of the shear blade and the opposite face thereof for cooperation with the cutter blade is increased as compared with the spacing required to achieve a “close shave”.\",\n \"The invention likewise additionally encompasses a head assembly for a dry shaver, comprising at least one rotary cutter blade, the or each cutter blade having an overlying cooperating shear blade, wherein the spacing between the skin engaging surface of the shear blade and the opposite face thereof for cooperation with the cutter blade is increased as compared with the spacing required to achieve a “close shave”.\",\n \"Thus the invention embraces in addition blade assemblies and head assemblies incorporating the features of the invention as independent units, whether in combination with a dry shaver or as entirely separate assemblies.\",\n \"The invention likewise extends to any kit of parts comprising a dry shaver drive unit, one or more head assemblies, and/or one or more blade assemblies, provided in combination or in any commercial association, in so far as such units incorporate the features of the invention or together cooperate in defining equipment lying within the scope of the invention.\",\n \"The invention also extends to a dry shaver substantially as described herein with reference to and as shown in any one or more of the accompanying drawings.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS A number of embodiments of the invention will now be described having regard to the accompanying drawings in which: FIG.\",\n \"1 is a diagrammatic representation of a portion of human skin, in the region of the surface of the skin, in section, showing inner and outer skin layers and hair growth, and representing also a particular condition in which an individual's body hair is subject to growth in a manner more horizontal than vertical, FIG.\",\n \"2 is a side sectional view of an adjustable foil blade in a dry shaver in accordance with the present invention, FIG.\",\n \"3 is a pictorial representation of an embodiment of foil blade suitable for a dry shaver according to the invention, FIG.\",\n \"4 is a side sectional representation of an alternative manner of adjustable foil blade mounting for a dry shaver in accordance with the present invention, FIG.\",\n \"5 is a diagrammatic view in section through a shear blade of the kind illustrated in FIGS.\",\n \"2 to 4, showing a known variant in which a sharp cutting edge is defined by a downturn around the periphery of the hair entry openings in the foil, FIG.\",\n \"6 is a pictorial view of a rotary blade razor of known construction, to which the present invention may be applied in an alternative embodiment, FIG.\",\n \"7 is a simplified pictorial view of an alternative rotary blade razor, having two blades as compared with the three-bladed configuration of FIG.\",\n \"6, to which again the present invention may be applied in an alternative embodiment, FIG.\",\n \"8 is a side sectional view of a shear blade of conventional construction for use in the shaver of FIG.\",\n \"6 or that of FIG.\",\n \"7, FIG.\",\n \"9 is a side sectional view of a modified shear blade for rotary embodiment of dry shaver in accordance with the present invention, FIG.\",\n \"10 is a pictorial representation from beneath of a shaver head construction providing for adjustability of the spacing between the cutting blades and the skin surface in a twin-bladed embodiment of dry shaver of a kind similar to that shown in FIG.\",\n \"6, and FIG.\",\n \"11 is a longitudinal sectional view on the longitudinal centre line of the arrangement of FIG.\",\n \"10.DETAILED DESCRIPTION OF THE DRAWINGS FIG.\",\n \"1 is a representation in sectional view of a portion of the surface of the skin of an individual.\",\n \"As shown in FIG.\",\n \"1, the outer layer 1 of the skin is underlaid by an inner layer 2.Hairs 3 and 3a grow outwardly through the skin from respective root regions 4 and 4a and emerge through the outer layer 1 of the skin.\",\n \"In the normal way, as indicated by hair 3, a hair should emerge from the skin outer layer I and extend outwardly above the skin surface substantially at right angles to the skin surface.\",\n \"Depending on the region of the skin surface in question, certain hairs such as that designated by reference 3a in FIG.\",\n \"1 do not grow upwardly substantially perpendicularly or vertically with respect to the skin surface but run tangentially close to but underneath the skin surface.\",\n \"Eventually, such a hair emerges from the skin surface, as indicated at reference 3b, but in a highly tangential manner.\",\n \"If an individual suffering from such tangential hair growth wishes to effect a close shave and cuts the hair relatively close to the skin outer surface, a condition develops in which the sharp edge of the cut hair burrows back beneath the surface of the skin, as indicated at reference 3c, rather than emerging from the skin and growing externally in the normal manner.\",\n \"Such ingrowth occurs if the sharp, cut edge of the hair becomes engaged or caught beneath the surface of the outer skin layer 1.If growth then continues with the hair 3 running into and under the skin, the inevitable result is some kind of inflammation.\",\n \"It is therefore preferable to prevent the development of any such inflammation and the present invention addresses this problem.\",\n \"The concept underlying the avoidance of such skin conditions is to cut the hair slightly above the surface of the skin in areas of the body surface where the possibility of such ingrowing conditions arising is likely to be greatest.\",\n \"The advantages of its being possible to cut hair in accordance with the principles of the present invention are not however limited only to skin conditions such as that illustrated with reference to FIG.\",\n \"1, but the invention is applicable to all skin circumstances where shaving bumps or other skin eruptions or imperfections militate against an excessively close shave.\",\n \"The invention provides the user of a dry shaver with the ability to selectively cut body hair either tightly or to a greater length, depending on the region of the skin in question, i.e.\",\n \"the area currently being shaved.\",\n \"An embodiment of the invention applicable to a foil type dry shaver is shown in FIG.\",\n \"2.A removable shaver head portion 11 is mounted on a shaver body portion 12.The head 11 is seated on the body portion 12 to enclose the upper or drive portion 13 of the shaver body portion 12 in conventional manner.\",\n \"A generally conventional drive arrangement is applied such as by drive member 14 to an outwardly curved or convex cutter blade 15 to provide either reciprocating movement in the axial direction of the curved cutter blade 15, or alternatively oscillatory or circumferential motion about this axis.\",\n \"A spring arrangement such as a helical spring 16 urges the curved displaceable cutter blade 15 against the inner surface of a cooperating perforated foil blade 17, which serves as a substantially stationary shear member for the movable blade cutter 15 under shaving conditions, i.e.\",\n \"foil blade 17 remains substantially static with respect to cutter blade 15 during an actual shaving operation.\",\n \"The perforated foil blade 17 is received in a bent-over configuration within an open top region of the shaver head 11.The foil blade 17 is received and accommodated in curved guide grooves extending along opposite inner walls of the shaver head 11.One such guide groove is indicated by reference 27.It is a particular feature of the invention that this perforated foil which serves as the shearing blade 17 has a thickness which varies along the length of the foil, as shown in FIGS.\",\n \"2 and 3.It is also possible for the size, i.e.\",\n \"the width or breadth of the hair-entry apertures, also their configuration, to vary with the thickness of the foil, e.g.\",\n \"the apertures being narrow where the foil is thin and wider where the foil is thick.\",\n \"Wide and narrow apertures may also be intermingled.\",\n \"Referring now also to FIG.\",\n \"3 in particular, there is shown a favoured embodiment of the perforated foil blade or shear cutter 17, formed as an integral unit, in which the perforated central region of the blade 17, designated by cross-hatching, is surrounded by side 18 and end 19 members or blade regions, which are unperforated and define therefore lateral members and end or cross-members of the blade structure.\",\n \"The blade 17 has a thinner region 21 towards one axial end in its generally longitudinal direction and a thicker region 22 towards its other axial end.\",\n \"In the region of the thicker end 22, an aperture 23 is provided through which a fastener 24, FIG.\",\n \"2, enables the blade 17 according to the invention to be coupled to an operating or control button 25, which is displaceable on the exterior of the shaver head 11 in the longitudinal direction of the blade 17 and is guided by a guide slot 26 provided in the housing of the shaver head 11.In an alternative arrangement (not illustrated), the flexible foil blade may be supported in a separate frame structure having end and side members.\",\n \"The end edges of the foil blade are fastened to the end bars of the frame structure and guiding grooves provided in opposite inner walls of the shearing or shaver head housing again serve to guide the lateral or side members of the frame, which are flexible, in similar manner to the flexible side edges of the integral blade structure of FIG.\",\n \"3.The perforated foil blade again varies in thickness along its longitudinal extent from one end edge to the other.\",\n \"In either arrangement, integral or frame-mounted, a foil portion of the desired thickness is selectively brought into a cutting disposition with respect to the cutter blade 15 by controlled sliding movement of the foil blade 17 in its longitudinal direction This is facilitated by the external button arrangement 25 which runs in the guide slot 26 along the surface of the shaver unit head 11.In this manner, a required depth of cut may be readily chosen and speedily varied in controlled manner as required by the user.\",\n \"Retention of a particular selected position of the foil 17 may be achieved by cooperating frictional interaction between button 25 and slot 26, e.g.\",\n \"by button 25 and connection or fastener portion 24 defining a “nip” or light gripping engagement between inner and outer face regions of head 11 bordering slot 26.In order to provide guidance for the side edges 1a of the variable thickness shear blade 17, the guide grooves 27 in the shaver head have a dimension between the opposite side surfaces of the guiding groove 27 which may vary but is always appropriate for the thickness dimension of the blade 17 in the relevant region.\",\n \"In order to ensure that the thinner regions of the foil 17 are appropriately guided, the guide grooves 27 then may be provided, if necessary, with appropriate resilient means, such as leaf springs or the like, to urge the foil 17 towards an appropriate side face of the groove 27, and thereby take up as it were any possible intervening redundant space between a thinner portion of the foil 17 and the opposite side wall of the guiding groove 27.Alternatively, in either embodiment of the foil, the side edges 18 or the corresponding side frame members may be arranged to have a uniform thickness throughout the longitudinal extent of the shear blade or foil 17, so that the provision of resilient means in the guide grooves is not then required.\",\n \"The installed foil blade 17 is stretched as it were over the curved surface of cutter blade 15 at the cutting end of the shaver head unit 11.The foil blade 17 is tensioned in the outward direction by the spring-loading (16), underlying cutter blade 15, which also serves for holding it in place.\",\n \"In the embodiment as shown in FIGS.\",\n \"2 and 3 of a foil 17 suitable for use with the invention, variation in thickness is substantially continuous from one end of the foil 17 to the other, from a minimum thickness portion 21 at a first end, to a maximum thickness region 22 at the other end.\",\n \"In this way, a substantially infinite variation of thickness can be selected between the two extremes.\",\n \"The side edges of the foil 18 may however be of constant or uniform thickness over the entire longitudinal extent of the blade 17, depending on the properties of the material selected for the foil 17, as already discussed above, or the side frame members, in a framed embodiment, may be likewise of uniform thickness.\",\n \"In an alternative embodiment (not illustrated), the foil may have a number of sections, each of which is of a different thickness and configuration, and each section being connected to an adjacent section by for example a hinge portion or by any other suitable manner of interconnection.\",\n \"The sections are therefore joined for flexure, i.e.\",\n \"to be flexible when the continuous foil structure is formed from the individual sections, and define a flexible continuous foil.\",\n \"The sections may for example be joined by being bonded to one another in some suitable manner.\",\n \"In such an embodiment, the foil has therefore a number of discrete portions or sections, each of which has its own individual thickness, so that a corresponding depth of cut applies.\",\n \"Again, an appropriate depth of cut is readily selected in controlled manner by sliding movement of the foil to the requisite location.\",\n \"Variable aperture size and configuration and/or intermingled sizes and configurations of aperture in different regions of the foil may also be provided in this constructional variant.\",\n \"In an alternative mounting arrangement shown in FIG.\",\n \"4, the structure and arrangement of the shaver head housing 11′ and shaver body 12′, as well as the cutter 15′ structure, correspond to the arrangements of FIG.\",\n \"2, features similar to those of FIG.\",\n \"2 being designated by use of the same reference numerals with the addition of an apostrophe.\",\n \"However, in order to secure longitudinal displacement of the perforated foil blade 17′, the sliding button-controlled arrangement of FIG.\",\n \"2 is replaced by a pivotal structure, in which end edges of the foil blade 17′ are connected 34, 35 to respective ends of a pivotally mounted rocker mechanism 36 arranged 37 for rocking movement within the shaver head housing 11′.\",\n \"Any suitable means may be provided for connecting the foil blade 17′ to cross-bar portions of the rocker lever structure 36, such as fasteners 34, 35.Likewise, any suitable arrangement may be provided to secure controlled pivoting 37 of the rocking blade mounting frame or structure 36, for example a lever arm or knob at the end of a pivot axis mounting 37, or alternatively, displacement may be effected merely by sliding the perforated foil blade 17′ over the surface of the cutter blade 15′, using the fingers.\",\n \"In either arrangement, the rocker member 36 is suitably pivotally mounted by way of pins or a shaft 37, bearingly accommodated on the interior of the side walls of the head housing 11′.\",\n \"Retention of the foil blade 17′ in a particular longitudinal disposition with respect to the blade 15′ may again be achieved such as by suitable frictional cooperation or interaction between pivot mounting 37 and the bearing features by which pivot axis or shaft 37 is accommodated in the walls of the head housing 11′, or by any other suitable equivalent arrangement, such as for example a locking screw or the like associated with a pivot position adjusting knob.\",\n \"A multiplicity of possible arrangements will be apparent for achieving substantial locking of the foil 17′ in a particular selected shaving disposition by securing the rocker arm or lever 36 in a particular pivoted disposition following selective controlled displacement of the foil to align a designated region of the foil with the cutter blade.\",\n \"In any variant of the variable thickness foil embodiment of the invention, the regions of different foil thickness may be differentiated from one another such as by for example colour coding.\",\n \"FIG.\",\n \"5 shows detail of a feature by which an especially effective cutting or shearing action may be achieved for a foil shear blade.\",\n \"In the arrangement shown, in the formation of the hair entry openings 28 in the foil structure shear blade 17, the periphery of each entry opening 28 is turned slightly downwards, so that the peripheral region 29 surrounding the opening 28 protrudes below the general level of the remainder of the underside of the foil shear blade 17 to define, as it were, a lip.\",\n \"An especially effective shearing type action is then achieved when the cutter blade 15 interacts with this peripheral region 29 of the foil shear blade 17 surrounding the hair entry opening 28.Referring now to FIG.\",\n \"6, an embodiment of the invention applicable to a rotary type dry shaver will now be described.\",\n \"In a typical construction, a rotary dry shaver 41 may have a number of cutter units 42, typically three as shown in the drawing, or alternatively two units 42, in each case located at the cutting end face 43 of the shaver body 44.The cutting drive and mounting arrangements of this variant of the invention are generally in accordance with substantially conventional arrangements well-known in the industry and only the particular deviations from such constructions required by the present invention are now described in detail.\",\n \"In such constructions, each cutter unit has an external or shear blade, which has hair entry apertures or slits and is underlaid, in the assembled condition, by a rotating cutter blade, which severs hairs extending into the cutting region of the cutter blade through the slits or perforations (apertures) of the shear blade.\",\n \"According to the invention, each of the rotary blade assemblies 42 is equipped with a shearing blade unit having a particular thickness or depth dimension of the hair entry slits or perforations, so as to provide for a corresponding depth of cut.\",\n \"Preferably, each blade assembly provides the same depth of cut.\",\n \"In a favoured arrangement according to the invention, two or more interchangeable head assemblies may be provided, each having blades providing a particular depth of cut, e.g.\",\n \"“close” shave or “long” cut, so that a user can choose a head assembly providing for a tight cut or a head assembly providing for a less close shave.\",\n \"Thus the invention provides a shaving kit incorporating a dry shaver body unit which includes drive features, one or more head assemblies which are interchangeable with the drive portion or body of the shaver and two or more sets of blades which can be interchangeably associated with any of the head assemblies.\",\n \"In this manner, a user may set up one head assembly with each blade providing for a close shave and a second head assembly on which each blade allows a more generous cut.\",\n \"For regions of the skin where the close shave is desired, the user applies the close shave blades by mounting the appropriate head assembly on the drive portion of the shaver unit.\",\n \"Where it is necessary or desirable to apply a less close shave, the user then exchanges the close shave head assembly for that enabling the less close shave.\",\n \"Referring now to FIG.\",\n \"7, there is shown, in pictorial view, a rotary shaver having two cutter heads, each of which is angled away from the longitudinal axis of the cutter unit, so that only one head can be used at any given time.\",\n \"Each cutter head is located on an angled end face region of the shaver, so that each cutter can be engaged individually against the skin, without engagement of the other cutter, located on the other angled face.\",\n \"In a variant, each angled face could carry two blades, in a two-bladed cutter head assembly.\",\n \"The shaver would then have two twin-blade head assemblies, one on each angled cutter face.\",\n \"In a shaver of this kind in accordance with the invention, all cutter blades and each head assembly are interchangeable with other blade units or head assemblies respectively.\",\n \"As already indicated, the shaver unit in this embodiment also may be provided as a kit, with for example two or more detachable and interchangeable head assemblies and two or more sets of blades providing for different depths of cut.\",\n \"The user sets up one of the head assemblies with blades suitable for a close shave and the other head assembly for a less close shave.\",\n \"The two head assemblies can be mounted on the base portion of the shaver unit for use in the manner already described, namely close shave where feasible and less close shave where necessary.\",\n \"Additional head assemblies may also be included in the kit of parts as also may additional sets of blades providing for still different depths of cut.\",\n \"In a further variant of the arrangement provided in FIG.\",\n \"7, the base unit may accommodate three heads, each angled outwardly from the generally longitudinal axis of the base portion which also defines the gripping or handle portion of the unit, so that the three head regions or head assembly mounting portions are spaced equidistantly around this axis substantially at 120° from each other.\",\n \"The unit may then be set up to provide for three different depths of cut and the user applies the appropriate depth of cut by rotating the unit so that the selected head region can be applied to the skin.\",\n \"In this manner, particular ease of use is facilitated with maximum interchangeability and flexibility as regards depths of cut, by the diverse options available for interchange or exchange of head assemblies and blade assemblies.\",\n \"Referring now to FIG.\",\n \"8, which shows an external cutting member or shear blade 51 of generally conventional configuration, a head 42 equipped with this type of shear blade 51 may be regarded as providing a minimum depth of cut, for a tight shave.\",\n \"As shown in this drawing, the cutting member 51, which is in the general shape of a top-hat, or an inverted cup, has a series of generally radially-oriented, optionally skewed, hair entry slits or apertures 52, in an outwardly-directed generally disc-shaped region 55 of the blade 51, which is upwardly-oriented when mounted on the cutting unit 42.The outer surface of this disc-shaped region 55 of cutting member 51 comes into contact with the skin during a shaving operation.\",\n \"From the side of this disc-shaped region 55 opposite to the skin-engaging surface, at the periphery of the disc, a generally annular peripheral wall portion 56 extends (downwardly in the mounted condition on cutting unit 42) to terminate in an externally-directed holding flange 53, by which the shear blade is secured in position on the shaver cutting unit 42.Centrally of the generally disc-shaped region 55, radially inward of the slits 52, there is a provided a central dished portion 54, which is set back from the skin-engaging surface defined by the outer face of region 55 at the slots 52.In order to provide for a more generous shave, in other words a less tight or close shave leaving a longer beard length, at least one other cutter 61 is provided, in the form of a shear blade of greater thickness, as shown in FIG.\",\n \"9, so that the depth of cut is greater when that particular cutter is applied to the face.\",\n \"The general construction of cutter or shear blade 61 is similar to blade 51, so that blades 51 and 61 are interchangeable on the dry shaver of FIG.\",\n \"6 or that of FIG.\",\n \"7.In the shaver of FIG.\",\n \"6, preferably all of the blades provide for a close shave or all of the blades provide for a less close shave.\",\n \"However, in the two head shaver of FIG.\",\n \"7 or in a three head variant of this design, the blade or blades of one of the heads may provide for a close shave and the blades of the other or another of the heads provide for a less close shave, so that different regions of the face may be shaved according to need, either to provide a smooth close shave or a less tight cut.\",\n \"Thus in the FIG.\",\n \"7 variant, a tight shave or a less close shave may be achieved without requiring the user to change either a blade assembly or a head assembly, although this option may also be present within the scope of the invention.\",\n \"Again as already discussed, the invention in this aspect offers extensive flexibility of use, not only by selecting the appropriate head region to be applied to the skin in the two-headed arrangement of FIG.\",\n \"7 or the alternative three-headed variant discussed previously, but further flexibility and interchangeability is facilitated by the inclusion of additional head assemblies and/or additional blade assemblies in a shaver kit provided in accordance with the principles of the is invention.\",\n \"The only dimensional difference between the blades 51 and 61 is in the thickness of the blade in the outer skin-engaging portion.\",\n \"Slits 62, flange 63 and central dished region 64 correspond to portions 52, 53 and 54 of the standard cutter 51 of FIG.\",\n \"8, but the slits 62 are substantially greater in depth than the slits 52 of blade 51.Accordingly, when this shear blade 61 is applied to the face, the hairs are cut at a longer length than in the case of the blade 51.In use of this embodiment of the invention, the second cutter 61 (FIG.\",\n \"9) is to be applied to skin areas where the cut is not to be too tight, so as to avoid the dermatological and other skin problems previously identified, while the first cutter 51 (FIG.\",\n \"8) will be used to achieve a tight shave and good appearance where this does not create any skin hazard or potential damage.\",\n \"Yet another cutter unit may provide another thickness of shear blade, so that a still further depth of cut may be effected, as required.\",\n \"Thus, at least two possible depths of cut are selectively available to the user during operation of the dry shaver.\",\n \"In the three-blade shaver of FIG.\",\n \"6, completely separate head units 45 may be provided for, first of all, a standard cut, and secondly and alternatively, for situations where a less tight cut is required, this second alternative head having all of the shear blades dimensioned to provide greater spacing from the skin surface according to the arrangement of FIG.\",\n \"9, while the first head has shear blades in accordance with FIG.\",\n \"8.Referring again to FIG.\",\n \"6, head unit 45 is separable from the body 44 of the shaver unit, so that the entire head structure may thus be removed and exchanged for one providing for such a different depth of cut by virtue of the shear blades of all of the cutting units of the alternative head portion 45 being provided with shear blades of different slit or aperture depth from the depth applicable to a standard cut.\",\n \"Again in this variant, at least two possible depths of cut are then available to the user, simply by head interchange.\",\n \"Thus each head, standard and alternative, has shear blades of uniform cut capability for all blades of that head, but the depth of cut achievable varies as between the heads.\",\n \"It is known in the rotary shaver art to provide head and blade assemblies that can be readily dismantled for cleaning and maintenance.\",\n \"Any known construction of head or blade assembly of this kind may be applied to the improved blade arrangements and techniques provided by the present invention.\",\n \"In particular, such known constructions may be applied to the interchangeable head assemblies, and/or individual interchangeable blade units, as provided the present invention.\",\n \"According to the present invention, the dismantling capability of head and blade assemblies is further developed to provide extensive interchangeability on the drive portion of the shaver as between head assemblies on the drive base and blade assemblies on the head assemblies.\",\n \"Thus a rotary shaver in accordance with the invention having a base portion suitable for mounting a single head assembly may be provided as a kit including two head assemblies, each of which has blades enabling a different depth of cut.\",\n \"One head assembly may provide for a close shave and the other head assembly for a less close shave.\",\n \"In addition, further sets of blades or blade assemblies may be provided, so that still different depths of cut may also be enabled by mounting a further alternative set of blades on one of the heads.\",\n \"In a variant of the invention, the base drive unit may accommodate more than one head assembly at the same time.\",\n \"Both head assemblies can be driven simultaneously or selectively by the base drive unit.\",\n \"One of the head assemblies is provided with blades suitable for a close shave and the other with blades suitable for a less close shave.\",\n \"One or other of the head assemblies can then be applied to the skin, depending on the depth of cut required.\",\n \"Yet a further variant of this aspect of the invention provides for the single drive unit to have no less than three head assemblies, all capable of use without requiring demounting of a head assembly and its replacement by an alternative.\",\n \"In any of the variants of the invention, the system may be provided as a kit of parts, with a single base unit or drive portion and two or three or more head assemblies.\",\n \"Each head assembly may be pre-provided with a set of blades of a particular depth of cut.\",\n \"In addition or alternatively, further blade assemblies may be included in the kit of parts, for interchangeable placement or mounting on any of the head assemblies as may be required.\",\n \"Great flexibility and adaptability in use is thus available in accordance with the principles of the invention.\",\n \"All head assemblies are readily removable from the base drive portion and all blade assemblies are readily removable from the respective head assembly.\",\n \"All sub-assemblies of the system are fully interchangeable, so that any head assembly may be exchanged for any other head assembly and any head assembly may receive any blade assembly.\",\n \"Thus any head portion of the unit may receive mounted on it, blade units of any depth of cut.\",\n \"Thus in a kit of parts according to the invention, the shaver unit is interchangeably associatable with any one of a plurality or multiplicity of heads.\",\n \"Each head may receive a plurality or multiplicity of blades and any blade may be demountably associated with any head.\",\n \"Where angled heads are provided for selective optional use without necessarily exchanging a head for an alternative head unit, the principles of full interchangeability and exchangeability continue to prevail, for ease of advance selection of the particular depth of cut required on any of the angled head regions.\",\n \"In FIGS.\",\n \"10 and 11, there is shown an alternative arrangement in which the actual depth of cut for a pair of rotary cutters may be selectively varied during use.\",\n \"The arrangement illustrated is for a two head cutter rotary dry shaver.\",\n \"As shown, there is an inner shear blade structure 81, against the underside of which rotary cutters 85 exercise the cutting action.\",\n \"An outer “dummy” shear blade or external cutter cover arrangement 91 is fixedly mounted in the cutter head 71 for cooperation with the inner shear blade structure 81 to provide for variable spacing between shear blade 81 and outer “dummy” blade or cover 91.The rotary blades 85 are rotatably mounted in known manner and are driven in rotary motion by likewise known drive arrangements (not illustrated).\",\n \"According to the present invention, the outer “dummy” shear blade or external cutter cover arrangement is a fixed outer perforated cover 91 and is associated with the inner shear blade structure 81 for variable depth of shave or cut, the shear blade structure 81 being mounted to be displaceable towards and away from the outer cover 91.The outer surface 92 of this cover or “dummy” shear blade 91 engages the skin.\",\n \"The cover or spacer head 91 is suitably provided with a series of apertures 93 which may be of relatively large size compared with the slits 82 of the inner shear blade 81, through which apertures 93 the facial hair to be cut passes to be gripped by the perforations or slits 82 of the shear blade 81 and cut in the usual way by the rotary blades 85.The adjustable arrangement enables the shear blade structure 81 to be moved towards and away from the outer cover 91, so that the depth of cut may be selectively varied in controlled manner by the user, depending on the portion of the skin to be shaved.\",\n \"Any one of a diversity of cover-to-blade space-adjusting constructions may be provided to enable the requisite adjustment.\",\n \"A particularly favoured arrangement for securing this spacing adjustment is shown in FIGS.\",\n \"10 and 11.In accordance with this arrangement, the dummy external cutter or cover 91 provided with the apertured region 93, which generally overlies the slit region of the shear blade 81, also has a substantially solid central region 94.The general configuration of this additional cover or dummy cutter 91 is therefore broadly similar to that of the actual shear blade 81.Variants are however also possible, to the extent that the cover 91 may be of more open work type construction, with the apertures 93 being defined between a series of radial and circumferential bars, wire type members, or perforated plates, and the central bridging region of “dummy” shear blade 91 is also not an essential structural feature.\",\n \"In order to secure the inward and outward displaceability of shear blade structure 81 towards and away from the fixed cover 91, FIGS.\",\n \"10 and 11 provide a control member 95 which is in the form of a peripheral endless band slideable in an external slot 96 in the external side wall 72 of the shaver unit head region 71.At four locations on its endless path, the peripheral band 95 has camming members 97, 98 which cooperate with mating camming members 87, 88 provided on a support structure 89 which either comprises or carries the shear blade 87.Movement of the endless band 95 in the direction indicated by arrow “a” in FIG.\",\n \"8 causes the shear blade structure 81 to be pulled away from the fixed cover 91 and therefore increases the depth of shave or cut, while movement of the band 95 in the direction indicated by arrow “b” in FIG.\",\n \"8 produces the converse effect, namely moves the shear blade 81 closer to the fixed blade 91.A structure of this type is known in itself and is applied in an existing dry shaver arrangement in which the shear blades are mounted in a quasi-floating manner.\",\n \"The structure is such that the rotor blades remain at all times in fixed proximity to the shear blade and travel inwards and outwards with the shear blade, being flexibly coupled to the drive arrangements so that rotation of the cutter blades may be effected at all locations of the shear blade.\",\n \"In this manner therefore the principles of the invention may be embodied in a dry shaver of the rotary cutter blade type also.\",\n \"The invention in this embodiment therefore provides substantially infinitely variable selective choice of a preferred depth of cut for any region of the body to be shaved, by varying the displacement between the skin-engaging outer or upper surface 92 of the cover or dummy shear blade 91 and the inner face of the cooperating shear blade 81, where the rotary action of the underlying cutter blades 85 effects actual severance of the hair.\",\n \"Thus according to the present variant of the invention, there is again achieved a dry shaver unit in which a variable depth of cut is selectively available at the user's choice.\",\n \"The control arrangement 95 shown in FIG.\",\n \"10 is exemplary only.\",\n \"The arrangements described and proposals indicated herein for shear blade 81 positional control relative to the dummy blade or outer cover 91 are also exemplary only and in no way limit the scope of the invention as defined by the appended claims.\",\n \"In particular, alternative control or drive arrangements for displacing the cover portion relative to the cutter blade may be devised and provided, including, but not limited to, mechanical inversions of the arrangements described and illustrated.\",\n \"The words “comprises/comprising” and the word “having” when used herein with reference to the present invention are used to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10467689"}}},{"rowIdx":381,"cells":{"text":{"kind":"list like","value":[["Extracts derived from chenopodium plants and uses thereof","The present invention relates to pesticides.","More particularly, the present invention relates to botanical pesticides.","In particular, the present invention relates to compositions and methods for controlling plant-infesting pests with plant extracts and notably with compositions comprising oil extracts derived from Chenopodium sp.","plant material.","The invention further relates to compositions comprising such extracts as pesticidal compositions and providing the advantages of minimal development of resistance thereto, minimal toxicity to mammals, minimal residual activity and environmental compatibility.","The pesticidal compositions of the present invention comprises α-terpinene, ρ-cymene, limonene, carvacrol, carveol, nerol, thymol, and carvone."],["1.An essential oil extract derived from a Chenopodium species, comprising: from at least 30% to at least 65% α-terpinene, from at least 8% to at least 26% ρ-cymene, from at least 5% to at least 24% limonene, from at least 0.04% to at least 1% carvacrol, from at least 0.1% to at least 2% carveol, from at least 0.1% to at least 2.0% nerol, from at least 0.04% to at least 2% thymol, and from at least 0.04% to at least 1% carvone, and wherein said extract is active against one or more pests chosen from insects, acari, and fungi.","2.","(Canceled) 3.","(Canceled) 4.","(Canceled) 5.The essential oil extract according to claim 1, wherein said species is Chenopodium ambrosioides.","6.A pesticidal composition, comprising an effective amount of the essential oil extract of claim 1 and a suitable emulsifier, spreader or sticking agent, and carrier.","7.The pesticidal composition according to claim 6, comprising between 0.125% to 10% (by volume) essential oil extract.","8.The pesticidal composition according to claim 7, comprising between 0.25% to 5% (by volume) of said essential oil extract.","9.The pesticidal composition according to claim 6, wherein said composition comprises between 5% to 50% (by volume) of said essential oil extract.","10.The pesticidal composition according to claim 9, comprising between 10% to 25% (by volume) of said essential oil extract.","11.The pesticidal composition according to claim 10, comprising between 1% to 15% (by volume) of a suitable emulsifier, and between 50% to 70% (by volume) of a suitable carrier or solvent.","12.The pesticidal composition according to claim 11, comprising between 2% to 20% (by volume) of a suitable spreader or sticking agent.","13.A pesticidal composition, comprising between 30% to 50% (by volume) of the essential oil extract of claim 1, between 0.5% to 25% (by volume) of a suitable emulsifier, and between 10% to 50% (by volume) water.","14.A method for controlling one or more pests chosen from phytophagous acari, phytophagous insects, and phytophagous fungi, which comprises applying to a locus where control is desired a pesticidally-effective amount of the pesticidal composition of claim 6.15.","(Canceled) 16.","(Canceled) 17.The method according to claim 14, wherein said locus is soil.","18.The method according to claim 14, wherein said locus is a plant.","19.The essential oil extract according to claim 1, further comprising ascaridole at a proportion of 9.86% or less.","20.The essential oil extract according to claim 1, further comprising ascaridole at a proportion of 1% or less.","21.The essential oil extract according to claim 20, wherein said extract has trace or undetectable amounts of ascaridole.","22.A formulation for indoor or outdoor fumigation, comprising: between 1% to 15% (by volume) of the essential oil extract of claim 1.23.A formulation for topical application, comprising: between 1% to 10% (by volume) of the essential oil extract of claim 1.24.The formulation of claim 23, wherein the formulation further comprises between 2% to 5% (by volume) of an emollient, between 0.1% to 1% (by volume) of a stabilizer and between 0.1% to 0.5% (by volume) of a preservative.","25.The formulation of claim 23, wherein the formulation further comprises between 1% to 10% (by volume) of an emulsifier, between 2% to 5% (by volume) of a coat or skin conditioner, between 0.1% to 1% (by volume) of a stabilizer, between 0.1% to 1% (by volume) of a preservative and between 0.1% to 0.5% (by volume) of an antioxidant.","26.A microemulsion formulation, comprising: between 30% to 50% (by volume) of the essential oil extract of claim 1, between 0.5% to 25% (by volume) of a suitable emulsifier, and between 10% to 50% (by volume) water.","27.The formulation of claim 26, wherein said formulation is for soil delivery.","28.A method for controlling phytophagous pests, which comprises applying to a locus where control is desired a pesticidally-effective amount of the formulation according to any one of claims 22, 23, or 26."],["<SOH> BACKGROUND OF THE INVENTION <EOH>Plant feeding mites are among the most voracious phytophagous pests of crops.","To combat these pests, synthetic pesticides have been developed.","These synthetic chemical pesticides, however, often have detrimental environmental effects that are harmful to humans and other animals and therefore do not meet the guidelines developed by most Integrated Pest Management programs.","Moreover, resistance to these products has been found to develop with many of the new products put on the market (Georghiou, 1990; Nauen et al., 2001).","Although resistance follows a highly complex genetic and biochemical process, it can generally develop rapidly with synthetic products because their active ingredients rely on one or more molecules of the same class.","The organism can therefore respond to the toxin by developing physiological, behavioral or morphological defense mechanisms to neutralize the effect of the molecule (Roush and MacKenzie, 1987).","Acari, such as spider mites, in particular, are extremely difficult to control with pesticides.","Tetranychus urticae (the two-spotted spider mite), for example, has accumulated a considerable number of genes conferring resistance to all major classes of acaricides.","Resistance to many registered acaricides have been reported, for example, resistance has been reported to hexythiazox, abamectin, and clofentezine.","Furthermore, many of these pesticides have been found to exacerbate pest infestation by destroying the natural predators of mites (U.S. Pat.","No.","5,839,224).","Additionally, many synthetic insecticides have been found to stimulate mite reproduction.","For example, it was found that mites reproduce many times faster when exposed to carbaryl, methyl parathion, or dimethoate in the laboratory than untreated populations (Flint, 1990).","As a result, there are very few pesticides remaining that are effective against spider mites (Georghiou, 1990).","In the Farm Chemical Handbook (Meister, 1999), for example, only 48 products out of a total of 2,050 listed acaricides and insecticides (or 2.4%), were identified as acaricides and only 69 of these products (or 3.4%) were identified as both acaricides and insecticides.","The control of insect infestation has also proven to be difficult.","For example, European chafer Rhizotrogus majalis Razoumowsky (Coleoptera: Scarabaeidae) insects are an exotic species to Canada and the United States and are commonly known as Scarab beetles.","Throughout the northeast, where the beetle has become established, R. majalis causes major damage to turfgrass, consuming the roots of home lawns as well as urban recreational areas and golf courses.","The larvae of this species are extremely difficult and expensive to control using conventional insecticides.","In several areas of the U.S. insecticide resistance has already developed within populations.","The control of plant fungi is another source for growing concern.","Phytopathogenic fungi are of great economic importance since fungal growth on plants or on parts of plants inhibits production of root, stem, foliage, fruit or seed, and the overall quality of a cultivated crop.","About 25 percent of all fungal diseases in agriculture and horticulture are caused by powdery mildew phytopathogens (U.S. Pat.","Nos.","5,882,689 and 5,496,568).","In cucumber crops, for example, powdery mildew caused by the plant pathogens, Erysiphe cichoracearum and Sphaerotheca fuliginea , is one of the most problematic disease.","The fungus responsible for Gray Mold, Botrytis cinerea, can attack more than 200 species of cultivated plants and especially those growing in a greenhouse environment.","It is a saprophyte that attacks dead or senescent plant tissue.","B. cinerea is especially damaging to stems (causing stem rot), when it enters scars left by pruning of lower leaves.","Following attack, the plant dies, causing heavy economic losses to the grower.","Control of stem rot is attempted by treating leaf scars after leaf pruning.","Although benomyl is presently used, it will likely be removed from the market shortly.","Moreover, iprodione (another pesticide), is less effective because of developing fungal resistance.","In spite of their strong fungicidal effect, agricultural fungicides currently in use have been a source of problems, because the required amount is ever increasing as tolerance of target plant pathogens increases.","Moreover, many fungicides are synthetic agents, which when used, pose possible drawbacks for humans, animals and the environment.","Thus, effective commercial products (to combat fungal attacks on crops), which reduce the dose of synthetic chemicals spread into the environment, are in need; keeping in mind a regard for environmental and human safety issues.","As an alternative to synthetic agents, botanical pesticides offer the advantage of being naturally derived compounds that are safe to both humans and the environment.","Specifically, botanical pesticides offer such advantages as being inherently less toxic than conventional pesticides, generally affecting only the target pest and closely related organisms, and are often effective in very small quantities.","In addition, botanical pesticides often decompose quickly and, therefore, are ideal for use as a component of Integrated Pest Management (IPM) programs.","There are few published reports of the acaricidal properties of botanical pesticides.","For example, U.S. Pat.","No.","4,933,371 describes the use of saponins extracted from various plants (i.e., yucca, quillaja, agave, tobacco and licorice) as acaricides.","This patent also describes the use of linalool extracted from the oil of various plants such as Ceylon's cinnamon, sassafras, orange flower, bergamot, Artemisia balchanorum, ylang ylang, rosewood and other oil extracts as acaricides.","These methods, however, require the extraction of one active substance from the plant which often does not meet desired levels of toxicity towards acari.","Plant essential oils are a complex mixture of compounds of which many can be biologically active against insect and mite pests, the compounds acting individually or in synergy with each other, to either repel or kill the pests by contact.","These components are plant secondary metabolites or allelochemicals produced by plants as a defense mechanism against plant feeding pests (Ceske and Kaufman, 1999).","Because of the complexity of the mixture, it has been observed that pests do not easily develop resistance to these products as they can to synthetic pesticides or botanical pesticides comprising a single active compound.","In this respect, Feng and Isman (1995) demonstrated that repeated treatments of pure azadirachtin, a major active constituent of neem oil, against the green peach aphid led to a 9-fold resistance after 40 generations.","However, repeated exposure during 40 generations to crude neem extracts did not lead to resistance.","There remains a need to provide new and effective pesticidal products which overcome the shortcomings of products known in the art.","For example, there remains a need for broad spectrum compositions which are less likely to enable pests to develop resistance thereto.","There also remains a need to provide a method to combat pests at any given locus, using a composition which is not toxic to animals, especially to mammals, nor to any beneficial predator/parasitoid insects."],["<SOH> SUMMARY OF THE INVENTION <EOH>The invention provides essential oil extracts derived from Chenopodium sp.","comprising, α-terpinene, ρ-cymene, limonene, carvacrol, carveol, nerol, thymol, and carvone, wherein these extracts have one or more activities selected from the group of acaricidal, pesticidal, insecticidal, and fungicidal activities.","One or more of these extracts can be formulated into compositions that enable their application into different environments such as plants, soil, animals and buildings.","If accordance with another aspect of the invention there is provided composition comprising one or more Chenopodium sp.","-derived essential oil extracts, wherein said extract comprises α-terpinene, ρ-cymene, limonene, carvacrol, carveol, nerol, thymol, and carvone, in combination with an emulsifier, carrier, spreader and/or sticking agent, to enable application of the composition to a specific environment, wherein the composition has one or more activities selected from the group of acaricidal, pesticidal, insecticidal, and fungicidal activities."],["FIELD OF INVENTION The present invention relates to the field of pesticides for controlling plant-infesting pests.","BACKGROUND OF THE INVENTION Plant feeding mites are among the most voracious phytophagous pests of crops.","To combat these pests, synthetic pesticides have been developed.","These synthetic chemical pesticides, however, often have detrimental environmental effects that are harmful to humans and other animals and therefore do not meet the guidelines developed by most Integrated Pest Management programs.","Moreover, resistance to these products has been found to develop with many of the new products put on the market (Georghiou, 1990; Nauen et al., 2001).","Although resistance follows a highly complex genetic and biochemical process, it can generally develop rapidly with synthetic products because their active ingredients rely on one or more molecules of the same class.","The organism can therefore respond to the toxin by developing physiological, behavioral or morphological defense mechanisms to neutralize the effect of the molecule (Roush and MacKenzie, 1987).","Acari, such as spider mites, in particular, are extremely difficult to control with pesticides.","Tetranychus urticae (the two-spotted spider mite), for example, has accumulated a considerable number of genes conferring resistance to all major classes of acaricides.","Resistance to many registered acaricides have been reported, for example, resistance has been reported to hexythiazox, abamectin, and clofentezine.","Furthermore, many of these pesticides have been found to exacerbate pest infestation by destroying the natural predators of mites (U.S. Pat.","No.","5,839,224).","Additionally, many synthetic insecticides have been found to stimulate mite reproduction.","For example, it was found that mites reproduce many times faster when exposed to carbaryl, methyl parathion, or dimethoate in the laboratory than untreated populations (Flint, 1990).","As a result, there are very few pesticides remaining that are effective against spider mites (Georghiou, 1990).","In the Farm Chemical Handbook (Meister, 1999), for example, only 48 products out of a total of 2,050 listed acaricides and insecticides (or 2.4%), were identified as acaricides and only 69 of these products (or 3.4%) were identified as both acaricides and insecticides.","The control of insect infestation has also proven to be difficult.","For example, European chafer Rhizotrogus majalis Razoumowsky (Coleoptera: Scarabaeidae) insects are an exotic species to Canada and the United States and are commonly known as Scarab beetles.","Throughout the northeast, where the beetle has become established, R. majalis causes major damage to turfgrass, consuming the roots of home lawns as well as urban recreational areas and golf courses.","The larvae of this species are extremely difficult and expensive to control using conventional insecticides.","In several areas of the U.S. insecticide resistance has already developed within populations.","The control of plant fungi is another source for growing concern.","Phytopathogenic fungi are of great economic importance since fungal growth on plants or on parts of plants inhibits production of root, stem, foliage, fruit or seed, and the overall quality of a cultivated crop.","About 25 percent of all fungal diseases in agriculture and horticulture are caused by powdery mildew phytopathogens (U.S. Pat.","Nos.","5,882,689 and 5,496,568).","In cucumber crops, for example, powdery mildew caused by the plant pathogens, Erysiphe cichoracearum and Sphaerotheca fuliginea, is one of the most problematic disease.","The fungus responsible for Gray Mold, Botrytis cinerea, can attack more than 200 species of cultivated plants and especially those growing in a greenhouse environment.","It is a saprophyte that attacks dead or senescent plant tissue.","B. cinerea is especially damaging to stems (causing stem rot), when it enters scars left by pruning of lower leaves.","Following attack, the plant dies, causing heavy economic losses to the grower.","Control of stem rot is attempted by treating leaf scars after leaf pruning.","Although benomyl is presently used, it will likely be removed from the market shortly.","Moreover, iprodione (another pesticide), is less effective because of developing fungal resistance.","In spite of their strong fungicidal effect, agricultural fungicides currently in use have been a source of problems, because the required amount is ever increasing as tolerance of target plant pathogens increases.","Moreover, many fungicides are synthetic agents, which when used, pose possible drawbacks for humans, animals and the environment.","Thus, effective commercial products (to combat fungal attacks on crops), which reduce the dose of synthetic chemicals spread into the environment, are in need; keeping in mind a regard for environmental and human safety issues.","As an alternative to synthetic agents, botanical pesticides offer the advantage of being naturally derived compounds that are safe to both humans and the environment.","Specifically, botanical pesticides offer such advantages as being inherently less toxic than conventional pesticides, generally affecting only the target pest and closely related organisms, and are often effective in very small quantities.","In addition, botanical pesticides often decompose quickly and, therefore, are ideal for use as a component of Integrated Pest Management (IPM) programs.","There are few published reports of the acaricidal properties of botanical pesticides.","For example, U.S. Pat.","No.","4,933,371 describes the use of saponins extracted from various plants (i.e., yucca, quillaja, agave, tobacco and licorice) as acaricides.","This patent also describes the use of linalool extracted from the oil of various plants such as Ceylon's cinnamon, sassafras, orange flower, bergamot, Artemisia balchanorum, ylang ylang, rosewood and other oil extracts as acaricides.","These methods, however, require the extraction of one active substance from the plant which often does not meet desired levels of toxicity towards acari.","Plant essential oils are a complex mixture of compounds of which many can be biologically active against insect and mite pests, the compounds acting individually or in synergy with each other, to either repel or kill the pests by contact.","These components are plant secondary metabolites or allelochemicals produced by plants as a defense mechanism against plant feeding pests (Ceske and Kaufman, 1999).","Because of the complexity of the mixture, it has been observed that pests do not easily develop resistance to these products as they can to synthetic pesticides or botanical pesticides comprising a single active compound.","In this respect, Feng and Isman (1995) demonstrated that repeated treatments of pure azadirachtin, a major active constituent of neem oil, against the green peach aphid led to a 9-fold resistance after 40 generations.","However, repeated exposure during 40 generations to crude neem extracts did not lead to resistance.","There remains a need to provide new and effective pesticidal products which overcome the shortcomings of products known in the art.","For example, there remains a need for broad spectrum compositions which are less likely to enable pests to develop resistance thereto.","There also remains a need to provide a method to combat pests at any given locus, using a composition which is not toxic to animals, especially to mammals, nor to any beneficial predator/parasitoid insects.","SUMMARY OF THE INVENTION The invention provides essential oil extracts derived from Chenopodium sp.","comprising, α-terpinene, ρ-cymene, limonene, carvacrol, carveol, nerol, thymol, and carvone, wherein these extracts have one or more activities selected from the group of acaricidal, pesticidal, insecticidal, and fungicidal activities.","One or more of these extracts can be formulated into compositions that enable their application into different environments such as plants, soil, animals and buildings.","If accordance with another aspect of the invention there is provided composition comprising one or more Chenopodium sp.","-derived essential oil extracts, wherein said extract comprises α-terpinene, ρ-cymene, limonene, carvacrol, carveol, nerol, thymol, and carvone, in combination with an emulsifier, carrier, spreader and/or sticking agent, to enable application of the composition to a specific environment, wherein the composition has one or more activities selected from the group of acaricidal, pesticidal, insecticidal, and fungicidal activities.","BRIEF DESCRIPTION OF THE DRAWINGS FIG.","1 shows the chemical content of three lots or pools of oil samples extracted from whole plant parts above root (00MC-21P, 00MC-24P and 00M-29P).","FIG.","2 shows the average mortality (%) of the two-spotted spider mite (TSSM: Tetranychus urticae) when tested with solutions of individual compounds present in the essential oil of Chenopodium ambrosioides.","Results adjusted for control mortality with Abbott's formula.","FIG.","3 shows the average mortality (%) of the greenhouse whitefly (GWF; Trialeurodes vaporaiorum) when tested with solutions of individual compounds present in the essential oil of Chenopodium ambrosioides.","Results adjusted for control mortality with Abbott's formula.","FIG.","4 shows adult spider mite (Tetranychus urticae) mortality obtained with bioassays using the RTU formulation of Chenopodium ambrosioides and commercial preparations of natural and synthetic insecticides.","FIG.","5 shows spider mite egg (Tetranychus urticae) mortality, using the RTU formulation of Chenopodium ambrosioides oil.","FIG.","6 shows spider mite nymph (Tetranychus urticae) mortality, using the RTU Chenopodium extract formulation and commercial preparations of synthetic and natural products.","FIG.","7 shows the mortality of adult spider mites 48 h following introduction on faba bean leaves treated one hour previously with the RTU formulation and selected natural acaricides.","FIG.","8 shows red mite, Panonychus ulmi mortality, using the RTU formulation.","FIG.","9 shows insect mortality (%) obtained with bioassays using the RTU formulation of Chenopodium ambrosioides.","FIG.","10 shows mortality of adult female twospotted spider mites 48 hours following applications.","FIG.","11 shows mortality of adult female European red mite 24 hours following applications.","FIG.","12 shows egg hatch (%) of the twospotted spider mite, 10 days following applications.","FIG.","13 shows egg hatch (%) of European red mite 10 days following applications.","FIG.","14 shows mortality of adult female two-spotted spider mites 48 hours following introduction on leaf discs treated with EC25% and Dicofol one hour previously.","FIG.","15 shows mortality of green peach aphids (Myzus persicae (Sulz.))","48 hours following application of 0.125, 0.25, 0.5, 1.0 and 2.0% concentrations of formulation EC25% and the commercially available bioinsecticides Neem Rose Defense® and Safer's Trounce® FIG.","16 shows lethal concentrations (LC50 and LC90) in % of EC25% for the green peach aphid (Myzus persicae (Sulz.))","calculated with 48 hour mortality data.","FIG.","17 shows average number of green peach aphids (Myzus persicae (Sulz.))","per cm2 of treated Verbena speciosa shoot following application of 0.25, 0.50 and 1.0% concentrations of EC25% and the commercially available bioinsecticides Neem Rose Defense® and Safer's Trounce® FIG.","18 shows mortality of Western flower thrips (Frankliniella occidentalis (Perg.))","24 hours following application of six concentrations (0.05, 0.125, 0.18, 0.25, 0.5 and 1.0%) of formulation EC25% and the commercially available bioinsecticides Neem Rose Defense® and Safer's Trounce® FIG.","19 shows lethal concentrations (LC50 and LC90) in mg/cm2 of EC25% for the Western flower thrips (Frankliniella occidentalis (Perg.))","calculated with 24 hour mortality data.","FIG.","20 shows average number of Western flower thrips/cm2 (WFT: Frankliniella occidentalis (Perg.))","per treatment as a percentage of thrips present on leaves treated with the control during a greenhouse bioassay using two concentrations (0.25 and 1.0%) of EC25% and two commercially available bioinsecticides Neem Rose Defense® and Safer's Trounce® FIG.","21 shows mortality of greenhouse whiteflies (Trialeurodes vaporariorum (Westw.))","20 hours following application of five concentrations (0.0625, 0.125, 0.25, 0.5 and 1%) of formulation EC25% and the commercially available insecticides Neem Rose Defense®, Safer's Trounce® and Thiodan® FIG.","22 shows lethal concentrations (LC50 and LC90) in mg/cm2 of EC25% for the greenhouse whitefly (Trialeurodes vaporariorum (Westw.))","calculated with 20 hour mortality data.","FIG.","23 shows mortality of Encarsia formosa 24 hours following application of four concentrations (0.0625, 0.125, 0.25, 0.5 and 1.0%) of formulation EC25% and the commercially available bioinsecticides, Neem Rose Defense® and Safer's Trounce® FIG.","24 shows the effect of three concentrations of EC25% with the tomato canker caused by the fungus Botrytis cinerea.","FIG.","25 shows the percentage control of the tomato canker following treatment with a selection of products 1 and 8 days following inoculation with Botrytis cinerea.","FIG.","26 shows the percentage (%) infection among blocks with powdery mildew.","FIG.","27 shows the effect of EC25% and reference products on Powdery Mildew.","FIG.","28 shows daily mean and total emergence of adult thrips from soil treated with different volumes of EC25% at 0.5% concentration.","FIG.","29 shows the percent survival of R. majalis 7 days after treatment with EC25%.","FIG.","30 shows EC25% median lethal concentrations (LC50), standard error of log LC50, 95 percent confidence interval and slope/intercept equation for two 7 day trials with R. majalis.","FIG.","31 shows average soil percent organic matter and pH±standard error determined for each tray in both R. majalis 7 day trial.","FIG.","32 shows treatments tested with the European chafer, Rhizotrogus majalis.","FIG.","33 shows counts for live European chafers on turfgrass plots.","FIG.","34 shows treatments evaluated with the HCB on turfgrass plots.","FIG.","35 shows counts for live HCB on turfgrass plots.","FIG.","36 shows mean mortality (%) of Amblyseius fallacis adult females following the direct application of several concentrations of EC25% and commercially available insecticides.","FIG.","37 shows contact toxicity of EC25% oil formulation on adult females of Amblyseius fallacis.","Probit analysis.","FIG.","38 shows mean percent mortality of Phytoseiulus persimilis adult females to different insecticide treatments.","FIG.","39 shows overall percent mean mortality of adult wasps Aphidius colemani following direct application with EC25% and commercially available insecticides.","FIG.","40 shows male and female mean mortality (%) of Aphidius colemani adult wasps following direct application with EC25% and commercially available insecticides.","FIG.","41 shows contact toxicity of EC25% oil formulation on adult wasps Aphidius colemani.","Probit analysis.","FIG.","42 shows mortality of adult wasps Aphidius colemani following exposure to EC25% and commercially available insecticide residues.","FIG.","43 shows probit analysis of adult wasps Aphidius colemani 24 H and 48 H following exposure to EC25% residues.","FIG.","44 shows the effect of treatment on Aphidius colemani emergence from treated mummies.","FIG.","45 shows fecundity assessment of female Aphidius colemani following contact with EC25% residues.","FIG.","46 shows mean mortality of Orius insidiosus second instar nymphs following application with EC25% and commercially available insecticides.","FIG.","47 shows mean mortality of Orius insidiosus adults following EC25% and other insecticide treatments.","FIG.","48 shows fecundity of Orius insidiosus females surviving insecticide treatments.","FIG.","49 shows probit analysis of Orius insidosus second instar nymphs following application with EC25%.","FIG.","50 shows probit analysis of Orius insidiosus adults following application with EC25%.","DETAILED DESCRIPTION OF THE INVENTION Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.","“Locus” means a site which is infested or could be infested with acari, insects, fungi, or other pests and may include, but is not restricted to, domestic, urban, agricultural, horticultural, and forest environments.","“Essential Oil Extract” means the volatile, aromatic oils obtained by steam or hydro-distillation of plant material and may include, but are not restricted to, being primarily composed of terpenes and their oxygenated derivatives.","Essential oils can be obtained from, for example, plant parts including, for example, flowers, leaves, seeds, roots, stems, bark, wood, etc.","“Active Constituents” means the constituents of the essential oil extract to which the pesticidal, acaricidal, insecticidal, and/or fungicidal activity is attributed.","The essential oil extract of the present invention generally comprises the active constituents including: α-terpinene, ρ-cymene, limonene, carvacrol, carveol, nerol, thymol, and carvone.","The term “partially purified”, when used in reference to an essential oil extract means that the extract is in a form that is relatively free of proteins, nucleic acids, lipids, carbohydrates or other materials with which it is naturally associated in a plant.","As disclosed herein, an essential oil extract of the invention is considered to be partially purified.","In addition, the individual components of the essential oil extract can be further purified using routine and well known methods as provided herein.","Other chemistry terms herein are used according to conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (ed.","Parker, S., 1985), McGraw-Hill, San Francisco, incorporated herein by reference.","The present invention provides essential oil extracts derived from Chenopodium sp.","comprising, α-terpinene, ρ-cymene, limonene, carvacrol, carveol, nerol, thymol, and carvone.","These extracts have one or more activities selected from the group of acaricidal, pesticidal, insecticidal, and fungicidal activities.","The present invention also provides for the use of one or more of these essential oil extracts to prepare compositions that enable their application into different environments such as plants, soil, animals and buildings.","1.Essential Oil Extract Plant Material Plant material that may be used in the present invention includes plant material derived from the genus Chenopodium sp.","taken individually or in a group and may include, but is not restricted to, the leaf, flowers, roots, seeds, and stems.","As is known by persons skilled in the art, the chemical composition and efficacy of an essential oil extract varies with the phenological age of the plant (Jackson et al., 1994), percent humidity of the harvested material (Chialva et al., 1983), the plant parts chosen for extraction (Jackson et al., 1994; and Chialva et al., 1983), and the method of extraction (Perez-Souto, 1992).","Methods well-known in the art can be adapted by a person of ordinary skill in the art to achieve the desired yield and quality of the essential oil extract of the present invention.","In one embodiment, the plant material is derived from Chenopodium ambrosioides.","Pre-Treatment of Plant Material In addition to such parameters as the phenological age of the plant, the percent humidity of the harvested material, the plant parts chosen for extraction, and the method of extraction, the chemical composition and efficacy of an essential oil extract may be affected by pre-treatment of the plant material.","For example, when a plant is stressed, several biochemical processes are activated and many compounds, in addition to those constitutively expressed, are synthesized as a response.","In addition to pests, fungi, and other pathogenic attacks, stressors include drought, heat, water and mechanical wounding.","Moreover, persons of skill in the art will also recognize that combinations of stressors may be used.","For example, the effects of mechanical wounding can be increased by the addition of compounds that are naturally synthesized by plants when stressed.","Such compounds include jasmonic acid (JA).","In addition, analogs of oral secretions of insects can also be used in this way, to enhance the reaction of plants to stressors.","In one embodiment, the essential oil extracts of the present invention are derived from plant material which has been pre-treated, for example by stressing the plant by chemical or mechanical wounding, drought, heat, or cold, or a combination thereof, before plant material collection and extraction.","Harvesting the Plant Material for Extraction and Optional Storage Treatment The plant material may be used immediately after harvesting.","In one embodiment the fresh plant material having a humidity level of >75% is used.","Otherwise, it may be desirable to store the plant material for a period of time, prior to performing the extraction procedure(s).","In another embodiment wilted plant material having a humidity level of 40 to 60% is used.","In another embodiment dry plant material having a humidity level of <20% is used.","In a further embodiment, the plant material is treated prior to storage.","In such cases, the treatment may include drying, freezing, lyophilisizing, or some combination thereof.","Extraction of the Essential Oil Extract and Validation of Constituents Essential oil extracts can be extracted from plant material by standard techniques known in the art.","A variety of strategies are available for extracting essential oils from plant material, the choice of which depends on the ability of the method to extract the constituents in the extract of the present invention.","Examples of suitable methods for extracting essential oil extracts include, but are not limited to, hydro-distillation, direct steam distillation (Durbeck 1997), solvent extraction, and Microwave Assisted Process (MAP™) (Belanger et al., 1991).","In one embodiment, plant material is treated by boiling the plant material in water to release the volatile constituents into the water which can be recovered after distillation and cooling.","In another embodiment, plant material is treated with steam to cause the essential oils within the cell membranes to diffuse out and form mixtures with the water vapor.","The steam and volatiles can then be condensed and the oil collected.","In another embodiment, organic solvents are used to extract organically soluble compounds found in essential oils.","Non-limiting examples of such organic solvents include methanol, ethanol, hexane, and methylene chloride.","In a further embodiment, microwaves are used to excite water molecules in the plant tissue which causes cells to rupture and release the essential oils trapped in the extracellular tissues of the plant material.","To confirm the presence of the constituents of the present invention in the essential oil extract, a variety of analytical techniques well known to those of skill in the art may be employed.","Such techniques include, for example, chromatographic separation of organic molecules (e.g., gas chromatography) or by other analytical techniques (e.g., mass spectroscopy) useful to identify molecules falling within the scope of the invention.","The invention provides an essential oil extract derived from Chenopodium sp.","comprising α-terpinene, ρ-cymene, limonene, carvacrol, carveol, nerol, thymol, and carvone.","In one embodiment, the essential oil extract comprises at least 30% α-terpinene, 8% ρ-cymene, 5% limonene, trace carvacrol, 0.1% carveol, 0.1% nerol, trace thymol, and trace carvone.","In one embodiment, the essential oil extract comprises at least one of each compound selected from the range for each compounds of at least 30%, 32%, 34%, 38%, 40%, 44%, 48%, 50%, 55%, 60 %, 65% α-terpinene; 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26% ρ-cymene, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%,22%,24% limonene, trace, 0.04%, 0.08%, 0.10%, 0.12%, 0.14%, 0.2%, 0.5%, 0.8%, 1.0% carvacrol, 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 1.4%, 1.6%, 1.8%, 2.0% carveol, 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 1.4%, 1.6%, 1.8%, 2.0% nerol, trace, 0.04%, 0.08%, 0.10%,0.12%, 0.14%, 0.2%, 0.5%, 0.8%, 1.0%, 1.4%, 1.6%, 1.8%, 2.0% thymol, and trace, 0.04%, 0.08%, 0.10%, 0.12%, 0.14%, 0.2%, 0.5%, 0.8%, 1.0% carvone.","2.Activity of the Essential Oil Extract Following extraction of an essential oil extract of the invention, it may be desirable to test the efficacy of the extracts for acaricidal, pesticidal, insecticidal, and fungicidal activities.","Any number of tests familiar to a worker skilled in the art may be used to test the activity of the extracts, compositions, and formulations of the invention.","Deternination of Acaricidal Activity of the Essential Oil Extract Acaricidal activity of an essential oil extract may be evaluated by using a variety of bioassays known in the art (Ebeling and Pence, 1953; Ascher and Cwilich, 1960; Dittrich, 1962; Lippold, 1963; Foot and Boyce, 1966; and Busvine, 1980).","a) Contact Efficacy with the Adult Stage One exemplary method that may be used tests the contact efficacy of the essential oil extract, or formulations thereof, with the adult stage of a mite species.","For example, adult mites may be placed on their dorsum with a camel hair brush on a double-sided sticking tape glued to a 9 cm Petri dish.","Essential oil extracts and/or formulations may then be applied to the test subjects by spraying with the spray nozzle of a Potter Spray Tower mounted on a stand and connected to a pressure gauge set at 3 psi.","Mites that fail to respond to probing with a fine camel hair brush with movements of the legs, proboscis or abdomen are considered dead.","In one embodiment, the contact efficacy of an essential oil extract, or formulations thereof, is determined using the two-spotted spider mite (Tetranychus urticae), at the adult stage, as a model test subject.","A person skilled in the art, however, will readily understand that other species of acari can be used.","b) Ovicidal Activity The ovicidal effect can be determined by treating mite eggs with concentrations of essential oil extracts, or formulations thereof.","For example, adult female T. urticae may be transferred to 2 cm diameter leaf disks cut out of lima bean leaves and left for four hours for oviposition.","When at least 20 eggs/disk are laid, adult mites may then be removed.","Essential oil extracts and/or formulations may then be applied by spraying the test subjects.","Egg hatch is assessed daily and for 10 days following treatment by counting the number of eggs remaining on the leaf disks and the number of live and dead nymphs present.","Percent egg hatch is determined with live nymphs only.","The nymphs are considered dead if no movement is observed after repeated gentle probing with a single-hair brush.","In one embodiment the ovicidal activity of an essential oil extract, or formulations thereof, is determined with mite eggs of the two-spotted spider mite (Tetranychus urticae), as a model test subject.","A person skilled in the art, however, will readily understand that other species of acari can be used.","Determination of Insecticidal Activity of the Essential Oil Extract Similar bioassays can be conducted to evaluate the insecticidal activity of an essential oil extract, or formulations thereof, by utilizing an insect model.","In one embodiment, the greenhouse whitefly (Trialeurodes vaporariorum (Westw.))","is used as a model test subject in an insecticide bioassay.","For example, Whitefly adults may be glued to a black 5 cm×7,5 cm plastic card sprayed with Tangle-Trap® (Gempler's Co.) to obtain at least 20 active adults per card.","Each card is sprayed with the essential oil extract, composition, or formulation and allowed to dry.","The cards are then placed sideways on a Styrofoam rack in a closed clear plastic container of 5 L with moistened foam on the bottom to keep humidity high (>90% R.H.).","The plastic container is stored in a growth chamber at 24° C. and 16 L:8 D photoperiod.","Mortality is evaluated 20 hours following treatment by gently probing the whitefly with a single-hair brush under the binocular microscope.","Absence of movement (antennae, leg, wing) following probing is recorded as dead.","A person skilled in the art, however, will readily understand that other insect species can be used.","Determination of Fungicidal Activity of the Essential Oil Extract The fungicidal activity of an essential oil extract, or formulations thereof, can also be evaluated by utilizing a fungal model evaluated by using a variety of bioassays known in the art.","Examples of such known bioassays include the following.","Laboratory Tests The fungicidal efficacy of an essential oil can be done in the laboratory using several methods.","One method incorporates the test samples in an agar overlay in a Petri dish.","A second method would use a filter disk saturated with the test samples and placed on top of untreated agar.","Both systems are challenged with fungal plugs cut from lawns of indicator organisms at the same stage of growth.","The plates will be incubated at 30° C. for 5-10 days with visual observations and the zone of inhibition measured and recorded.","A positive control, i.e.","a commercially available fungicide and a negative control, i.e.","water are tested in the same way.","Greenhouse Tests Greenhouse tests may also be employed to evaluate fungicidal efficacy.","For example, the effect of the essential oil extracts, or formulations thereof, may be tested on host plants infected by a disease organism such as, for example, Botrytis cinerea, Erysiphe cichoracearum or Sphaerotheca fuliginea, Rhizoctonia solani, and Phytophthora infestans, by observing the percent damage or presence of lesions on the host plant after treatment and against controls.","Botrytis cinerea.","Tomato plants are seeded and grown following current commercial practices for greenhouse tomato production.","About 2 months following seeding, lesions are made on the leaves and the stem (5 lesions/plant) and inoculated with a suspension of 3×106 spores of B. cinerea, 2 ml per lesion.","Treatments are then applied to the plants.","A positive control, i.e.","a commercially available fungicide and a negative control, i.e.","water are also tested and all treatments are done in a randomized block design.","The length of lesions are measured every two weeks over a period of 3 months, then the number of fruit, the total weight of fruit and the average weight of fruit are calculated during the entire production period of the plant.","The experiment is repeated and the effect of treatments is subjected to an analysis of variance (ANOVA) and means are compared with a LSD test.","Erysiphe cichoracearum or Sphaerotheca fuliginea.","These disease organisms are obligatory parasites that do not have the capacity to survive in absence of its host.","Therefore to provide the inoculum for the test, cucumber leaves are taken from an infested greenhouse.","The conidia present on these leaves will transfer onto cucumber plants grown for the experiment one or two months previously.","New plants are periodically infested in this manner in order to increase the inoculum.","Treatments are then applied to the plants before or after inoculation depending on the type of fungicide used.","A positive control, i.e.","a commercially available fungicide and a negative control, i.e.","water are also tested and all treatments are done in a randomized block design.","The effect of the disease is evaluated on individual leaves of all plants using a index of infestation from 0 to 5 (0=absence of blemish and 5=80-100% of the leaf surface with blemishes).","The degree of the infestation is evaluated 3, 7, and 14 days following inoculation and reported in averages per plant.","The experiment is repeated and the effect of treatments is subjected to an analysis of variance (ANOVA) and means are compared with a LSD test.","Rhizoctonia solani.","An isolate of Rhizoctonia solani is produced on a culture media (PDA) 3 days before inoculation and a plug of the disease is then transferred to Erlenmeyer flasks filled with a YMG broth for 5 days.","The mycelium is filtered, suspended in distilled water and blended.","Seeds of tomato are used and sterilized on the surface using successive ethanol 70%, bleach and distilled water solutions.","A suitable sterile potting soil mix is used in which 60 mg blended mycelium is inoculated per 100 g of potting soil.","Tests are done in bedding boxes of 72 cells/box and 3 boxes are used per treatment.","The boxes are spread out in a randomized arrangement in a controlled atmosphere growth chamber the following conditions: 20° C. during the day and 16° C at night, 16 hours of light, 162 umol of light intensity and 60% humidity.","The boxes are incubated in the growing chambers during 3 weeks.","Treatments are then applied to the young plants before or after inoculation depending on the type of fungicide used.","A positive control, i.e.","a commercially available fungicide and a negative control, i.e.","water are also tested and all treatments are done in a randomized block design.","Plants are examined each week and the incidence of the disease is measured as well as the degree of infestation on a scale of 0 to 5 (0=absence of infestation and 5=80-100% of the leaf surface attacked).","The experiment is repeated and the effect of treatments is subjected to an analysis of variance (ANOVA) and means are compared with a LSD test.","Phytophthora infestans.","On tomato plants.","Tomato plants are seeded and grown following current commercial practices for greenhouse tomato production.","About 2 months following seeding, leaves and stems are inoculated with a suspension of 1×104 spores of P. Infestans until the plant surfaces are completely covered.","Treatments are then applied.","A positive control, i.e.","a commercially available fungicide and a negative control, i.e.","water are also tested and all treatments are done in a randomized block design.","Percent damage or presence of lesions is evaluated every 3-4 days for a period of 2 weeks on leaves that had been identified previously (15-30 leaves per plant).","The experiment is repeated and the effect of treatments is subjected to an analysis of variance (ANOVA) and means are compared with a LSD test.","On potato plants.","Potato tubers are sown and grown in pots of 6-8 inches.","About 1,5 months after seeding, the leaves and stems of the plants are inoculated with a suspension of 1×104 spores of P. Infestans until the plant surfaces are completely covered.","Treatments are then applied.","A positive control, i.e.","a commercially available fungicide and a negative control, i.e.","water are also tested and all treatments are done in a randomized block design.","Percent damage or presence of lesions is evaluated every 3-4 days for a period of 2 weeks on leaves that had been identified previously (15-30 leaves per plant).","The experiment is repeated and the effect of treatments is subjected to an analysis of variance (ANOVA) and means are compared with a LSD test.","3.Formulations of the Essential Oil Extract Formulations containing the essential oil extracts of the present invention can be prepared by known techniques to form emulsions, aerosols, sprays, or other liquid preparations, dusts, powders or solid preparations.","These types of formulations can be prepared, for example, by combining with pesticide dispersible liquid carriers and/or dispersible solid carriers known in the art and optionally with carrier vehicle assistants, e.g., conventional pesticide surface-active agents, including emulsifying agents and/or dispersing agents.","The choice of dispersing and emulsifying agents and the amount combined is determined by the nature of the formulation, the intended form of application of the formulation to a specific environment (e.g., plant, animal, soil, building), and the ability of the agent to facilitate the dispersion of the essential oil extract of the present invention while not significantly diminishing the pesticidal, acaricidal, insecticidal, and/or fungicidal activity of the essential oil extract.","Non-limiting examples of conventional carriers include liquid carriers, including aerosol propellants which are gaseous at normal temperatures and pressures, such as Freon; inert dispersible liquid diluent carriers, including inert organic solvents, such as aromatic hydrocarbons (e.g., benzene, toluene, xylene, alkyl naphthalenes), halogenated especially chlorinated, aromatic hydrocarbons (e.g., chloro-benzenes), cycloalkanes (e.g., cyclohexane), paraffins (e.g., petroleum or mineral oil fractions), chlorinated aliphatic hydrocarbons (e.g., methylene chloride, chloroethylenes), alcohols (e.g., methanol, ethanol, propanol, butanol, glycol), as well as ethers and esters thereof (e.g., glycol monomethyl ether), amines (e.g., ethanolamine), amides (e.g., dimethyl sormamide), sulfoxides (e.g., dimethyl sulfoxide), acetonitrile, ketones (e.g., acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone), and/or water; as well as inert dispersible finely divided solid carriers such as ground natural minerals (e.g., kaolins, clays, vermiculite, alumina, silica, chalk, i.e., calcium carbonate, talc, attapulgite, montmorillonite, kieselguhr), and ground synthetic minerals (e.g., highly dispersed silicic acid, silicates).","Surface-active agents, i.e., conventional carrier vehicle assistants, that can be employed with the present invention include, without limitation, emulsifying agents, such as non-ionic and/or anionic emulsifying agents (e.g., polyethylene oxide esters of fatty acids, polyethylene oxide ethers of fatty alcohols, alkyl sulfates, alkyl sulfonates, aryl sulfonates, albumin hydrolyzates, and especially alkyl arylpolyglycol ethers, magnesium stearate, sodium oleate); and/or dispersing agents such as lignin, sulfite waste liquors, methyl cellulose.","Emulsifiers that can be used to solubilize the essential oil extracts of the present invention in water include blends of anionic and non-ionic emulsifiers.","Examples of commercial anionic emulsifiers that can be used include, but are not limited to: Rhodacal™ DS-10, Cafax™ DB-45, Stepanol™ DEA, Aerosol™ OT-75, Rhodacal™ A246L, Rhodafac™ RE-610, and Rhodapex™ CO-436, Rhodacal™ CA, Stepanol™ WAC.","Examples of commercial non-ionic emulsifiers that can be used include, but are not limited to: Igepal™ CO-887, Macol™ NP-9.5, Igepal™ CO-430, Rhodasurf™ ON-870, Alkamuls™ EL-719, Alkamuls™ EL-620, Alkamide™ L9DE, Span™ 80, Tween™ 80, Alkamuls™ PSMO-5, Atlas™ G1086, and Tween™ 20, Igepal™ CA-630, Toximul™ R, Toximul™ S, Polystep™ A7 and Polystep™ B1.If desired, colourants such as inorganic pigments, for example, iron oxide, titanium oxide, and Prussian Blue, and organic dyestuffs, such as alizarin dyestuffs, azo dyestuffs or metal phthalocyanine dyestuffs, and trace elements, such as salts of iron, manganeses, boron, copper, cobalt, molybdenum and zinc may be used.","Spreader and sticking agents, such as carboxymethyl cellulose, natural and synthetic polymers (e.g., gum arabic, polyvinyl alcohol, and polyvinyl acetate), can also be used in the formulations.","Examples of commercial spreaders and sticking agents which can be used in the formulations include, but are not limited to, Schercoat™ P110, Pemulen™ TR2, and Carbose™ 514 H, Umbrella™, Toximul™858 and Latron™CS-7.Time-release formulations are also contemplated by the present invention.","For example, formulations which have been encapsulated and/or pelletized.","In one embodiment, the formulation is a sprayable ready-to-use (RTU) formulation suitable, for example, for delivery by fogging, aerosol spraying, and pump spraying methods for application in a variety of environments.","In a further embodiment, the formulation can also be prepared as an emulsifiable concentrate (EC) which can be diluted before use.","In one embodiment, the emulsifiable concentrate comprises between 5% to 50% (by volume) Chenopodium-derived essential oil extract in combination with a suitable emulsifier, carrier, and spreader and/or sticking agent to enable application of the formulation to a specific environment.","In another embodiment, the emulsifiable concentrate comprises between 10% to 25% (by volume) Chenopodium-derived essential oil extract in combination with a suitable emulsifier, carrier, and spreader and/or sticking agent to enable application of the formulation to a specific environment.","In a further embodiment, the emulsifiable concentrate comprises between 10% to 25% (by volume) Chenopodium-derived essential oil extract in combination with between 1% to 15% (by volume) of a suitable emulsifier, and between 50% to 70% (by volume) of a suitable carrier or solvent to enable application of the formulation to a specific environment.","In another embodiment, the emulsifiable concentrate may also comprise 2% to 20% (by volume) of a suitable spreader and/or sticking agent.","The person skilled in the art, however, will understand that these concentrations can be modified in accordance with particular needs so that the formulation is acaricidal, insecticidal, and/or fungicidal, but not phytotoxic.","Non-limiting examples of suitable emulsifiers that can be used in preparing emulsifiable concentrates of the present invention include: Rhodapex™ CO-436, Rhodapex™ CO-433, Igepal™ CO-430, Igepal™ CA-630, Igepal™ CO-887, Isopropanol, canola oil, Alkamuls™ EL-719, Rhodacal™ DS-10, Macol™ NP-9.5, Tergitol™ TMN-3, Tergitol™ TMN-6, Tergitol™ TMN-10, Morwet™ D425, and Tween™ 80.Suitable carriers or solvents that may be used include, but are not limited to, Isopar™ M, THFA™, ethyl lactate, butyl lactate, Soygold™ 1000, M-Pyrol, Propylene glycol, Agsolex™ 12, Agsolex™ BLO, Light mineral oil, Polysolve™ TPM, and Finsolv™ TN.","Examples of suitable spreaders and/or sticking agents include, but are not limited to, Latex emulsion, Umbrella™, Adsee™ 775, Witconol™ 14, Toximul™ 858, Latron™ B-1956, Latron™ CS-7, Latron™ AG-44M, T-Mulz™ AO-2, T-Mulz™ 1204, Silwet™ L-774.Formulations containing the essential oil extracts of the present invention can be prepared by known techniques to enable application to specific environments.","Plant Formulations The formulation can be prepared for application to plants and plant environments, for example, household/domestic plants, greenhouse plants, agricultural plants, and horticultural plants.","In one embodiment, the formulation contains a final concentration of between 0.125% to 10% (by volume) of the Chenopodium-derived essential oil extract in combination with a suitable emulsifier, carrier, and spreader and/or sticking agent to enable sprayable application to a plant.","In another embodiment, the formulation contains between 0.25% to 5% (by volume) of the Chenopodium-derived essential oil extract in combination with a suitable carrier, emulsifier, and spreader and/or sticking agent, to enable sprayable application to a plant.","Fumigant Formulations for Closed and/or Open Environments The formulation can also be prepared for application as a fumigant for both outdoor as well as indoor application, for example in closed environments, such as greenhouses, animal barns or sheds, human domiciles, and other buildings.","Persons of skill in the art will appreciate the various methods for preparing such fumigants, for example, as fogging concentrates and smoke generators.","A fogging concentrate is generally a liquid formulation for application through a fogging machine to create a fine mist that can be distributed throughout a closed and/or open environment.","Such fogging concentrates can be prepared using known techniques to enable application through a fogging machine.","For example, the formulation may have the following general composition: Ingredient % Chenopodium extract (active ingredient) 1-15 Spreader/sticker 1-5 Organic solvent To 100% Smoke generators, which are generally a powder formulation which is burned to create a smoke fumigant.","Such smoke generators can also be prepared using known techniques.","For example, the formulation may have the following general composition: Ingredient % Chenopodium extract (active ingredient) 1-10 Absorbent silica 3-5 Pyrotechnic ingredients 3-10 Free flow aid 1-5 Filler To 100% Animal Formulations The formulation can be prepared in a form suitable for application to animals and animal environments, such as barns or sheds.","In one embodiment, the formulation is prepared in a form suitable for topical application on an animal for controlling insects, acari, pests and fungi.","Persons of skill in the art will appreciate the various methods for preparing such topical formulations, for example as powders or sprayable formulations.","In one embodiment, the topical formulation is an aerosol and may have the following general composition: Ingredient % Chenopodium extract (active ingredient) 1-5 Emollient (coat conditioner) 2-5 Stabilizer 0.1-1 Preservative 0.1-0.5 Organic solvent To 100% In another embodiment, the formulation is a water-based pump spray formulation having the following general composition: Ingredient % Chenopodium extract (active ingredient) 1-10 Emulsifier(s) 2-10 Coat conditioner 2-5 Stabilizer 0.1-1 Preservative 0.1-1 Antioxidant 0.1-0.5 Water To 100% In a further embodiment, the formulation is an aerosol formulation for application to an animal environment, such as a barn or shed, having the following general composition: Ingredient % Chenopodium extract (active ingredient) 3-10 Stabilizer 0.1-1 Preservative 0.1-0.5 Organic solvent To 100% Human Formulations The formulation can be prepared in a form suitable for topical application on humans, for example as a repellent.","Persons of skill in the art will appreciate the various methods for preparing such topical formulations, for example as lotions or sprayable formulations.","In one embodiment, the formulation is a solvent-based sprayable formulation having the following general composition: Ingredient % Chenopodium extract (active ingredient) 1-5 Emollient (skin conditioner) 2-5 Stabilizer 0.1-1 Organic solvent To 100% In another embodiment, the formulation is a water-based sprayable formulation having the following general composition: Ingredient % Chenopodium extract (active ingredient) 1-5 Emulsifier(s) 1-5 Skin conditioner 2-5 Stabilizer 0.1-1 Preservative 0.1-1 Antioxidant 0.1-0.5 Water To 100% In a further embodiment, the formulation is a topical lotion formulation having the following general composition: Ingredient % Chenopodium extract (active ingredient) 1-10 Emulsifier(s) 1-10 Skin conditioner 2-10 Thickener(s) 0.2-5 Stabilizer 0.1-1 Preservative 0.1-1 Antioxidant 0.1-0.5 Water To 100% Soil Formulations In a further embodiment, the formulation of the present invention can be prepared as a microemulsion.","Microemulsions are low-viscosity, optically transparent dispersions of two immiscible liquids which are stabilized by at least one ionic or nonionic surfactant.","In the case of microemulsions, the particle diameters are in the range from about 5-100 nm suspended in a continuous phase.","The interfacial tension between the two phases is extremely low.","The viscosity of many microemulsions of the oil and water type (O/W) is comparable with that of water.","In contrast to microemulsions, “macroemulsions” have high viscosities and their particle diameter is in the range from about 10 to 100 micrometers.","Macroemulsions are milky white in color and, upon heating, tend toward phase separation or toward sedimentation of the dispersed substances.","It is commonly believed that pesticidal microemulsions can provide superior efficacy relative to macroemulsion formulas having the same levels of active ingredients.","It is believed that the small size of the emulsion droplets may allow for better transport of the pesticide through cell membranes (plant and insect) thereby resulting in enhanced efficacy.","Microemulsions are considered to be infinitely stable, thereby providing improved stability over traditional macroemulsion systems.","Accordingly, microemulsion formulations of the present invention may be particularly suitable for certain applications, for example soil delivery.","Microemulsion formulations of the present invention can be prepared using various known methods known in the art.","In one embodiment, the essential oil extract of the present invention can be combined with a combination of emulsifiers to create a microemulsion.","For example, a microemulsion can be prepared in a three phase process.","Phase 1: Selection of Emulsifiers A selection of potential primary emulsifiers is tested for miscibility with the active ingredient.","Solubility is evaluated by visually assessing for a clear stable solution.","Each pesticide/emulsifier mixture is mixed with the required volume of water and the quality of emulsion/microemulsion produced is recorded.","Emulsifiers producing the best emulsions are taken through into phase 2.Emulsion/microemulsion quality is determined by visual assessment.","Transparent microemulsions are the best, white emulsions with a bluish hue are second, plain white emulsions are next and any emulsion showing phase separation is last.","Phase 2: Selection of Secondary and Tertiary Emulsifiers Upon completion of phase 1, the most promising primary emulsifier systems are blended with other emulsifiers in various ratios until an emulsifier blend is identified which provides the desired physical properties.","The optimized system generally consists of one main emulsifier and two or more co-emulsifiers/co-solvents.","These co-emulsifiers/co-solvents tend to broaden the thermal phase stability range of the microemulsion system.","Ternary phase diagrams are used in this phase of development to assist in the selection of emulsifier ratios.","The best formulation at this stage is often one containing one anionic emulsifier and two non-ionics of different types.","The criteria for assessing the microemulsion properties are again physical appearance as a function of time and temperature.","Phase 3: Optimization of Emulsifier Levels Having established the best emulsifier combination, the final phase is optimizing the levels and ratios of each emulsifier component in the system.","In one embodiment, the microemulsion comprises between 30% to 50% (by volume) Chenopodium-derived essential oil extract, between 0.5% to 25% (by volume) of a suitable emulsifier, and between 10% to 50% water.","Non-limiting examples of emulsifiers that can be used in preparing microemulsions of the present invention include: Igepal™ CA-630, Rhodasurf™ ON-870, Alkamuls™ EL-719, Tween™ 80, Alkamuls™ PSMO-5, Rhodapex™ CO-436, Rhodafac™ RE-610, Rhodacal™ CA, Ammonyx™ CO, Aeros™ OT-S, Ammonyx™ LO, Stepanol™ WAC, Soprophor™ BSU, and Rhodaca™1 IPAM.","It is contemplated that the formulations of the essential oil extract can be combined with a controlled release delivery system in order to time-release the bioactive agents.","Such controlled release delivery systems include methods, known in the art, of encapsulation, dissolution, or incorporation of the active ingredient.","It is further contemplated that the formulations of the present invention can be prepared in combination with nutrients (fertilizers) or herbicides.","Thus the skilled artisan will appreciate that the instant invention may be further formulated to provide various dissolution rates and/or be prepared in combination with nutrients (fertilizers) or herbicides.","4.Use of Essential Oil Extract Formulations The essential oil extracts of the present invention can be used for controlling pests by applying a pesticidally effective amount of the essential oil extract and/or formulation of the present invention to the locus to be protected.","The essential oil extract formulations can be applied in a suitable manner known in the art such as, for example, spraying, atomizing, vaporizing, scattering, dusting, watering, squirting, sprinkling, pouring, fumigating, and the like.","Loci, for the application of essential oil extracts or formulations thereof include, but are not limited to, agricultural, horticultural, forest, plantation, orchard, nursery, organically grown crops, turfgrass and urban environments.","Specifically, the essential oil extracts or formulations thereof may be applied to soil or plants/trees at one or several sites including the leaves, petioles, stems, seeds, roots, flower, cones, bark, wood or tubers.","It is further contemplated that the essential oil extract may be formulated for seed treatment either as a pre-treatment for storage or sowing.","For example, the seed may form part of a pelleted composition or, alternatively, may be soaked, sprayed, dusted or fumigated with a formulation of the present invention.","The essential oil extract, and formulations thereof, can be applied to the surface of the soil to control soil surface or soil-inhabiting pests.","The essential oil extract of the present invention can also be used as part of an Organic Production system and Integrated Pest Management program.","For example, in conjunction with augmentation of beneficial insects and mites.","It is further contemplated that the essential oil extract, and formulations thereof, can be applied to ‘fertigation’ i.e.","fertilization via the irrigation system of plants in greenhouses.","For example, an essential oil microemulsion can be added to the water in small concentrations (0.1-0.5% AI) and as water is being irrigated to individual plants, the latter would be treated for soil-inhibating pests (insects and disease pathogens).","5.Effect of the Essential Oil Extract or Formulations on Beneficial Insects and Mites Natural enemies of phytophagous pests include both predators and parasitoid.","Predators are generally as large, or larger than the prey they feed on.","They are quite capable of moving around to search out their food, and they usually consume many pests during their lifetime.","Parasitoids, or parasitic insects, are smaller than their prey.","One or more parasitoids grow and develop in or on a single host.","The host is slowly destroyed as the parasitic larva(e) feed and mature.","Such beneficial insects and mites can help prevent or delay the development of pesticide resistance by reducing the number of pesticides required to control a pest.","They will also feed on the resistant pests that survive a pesticide application.","Integrated pest management (IPM) programs take advantage of the biological pest control provided by beneficial insects and mites by conserving or augmenting natural enemies.","When chemical controls are necessary in an IPM program, pesticides recommended are those that have minimal impact on naturally occurring beneficials.","Essential oil extracts of the present invention, and formulations thereof, may be tested for their effect on beneficial insects and mites, i.e., predators and parasitoids, by means of standardized IOBC (International Organization for Biologicial Control) testing methods as illustrated in Example XIV for integration into IPM programs.","The invention now being generally described, it will be more readily understood by references to the following examples, which are included for purposes of illustration only and are not intended to limit the invention unless so stated.","EXAMPLES Example I Phytochemical Profile of an Essential Oil Extract Derived from Chenopodium ambrosioides Whole plants of C. ambrosioides were harvested.","Plant material used for extraction purposes comprised the whole plant above root.","Essential oil extracts were extracted from the plant material by steam distillation, i.e., distillation in water (DW) and/or direct steam distillation (DSD).","Distillation in water was carried out in a 380 L distillator with a capacity for processing ca.","20 kg of plant material.","During the process of DW, plant material was completely immersed in an appropriate volume of water which was then brought to a boil by the application of heat with a steam coil located at the base of the still body.","In DSD, the plant material was supported within the still body and packed uniformly and loosely to provide for the smooth passage of steam through it.","Steam was produced by an external generator and allowed to diffuse through the plant material from the bottom of the tank.","The rate of entry of the steam was set at (300 ml/min).","With both methods, the oil constituents are released from the plant material and with the water vapor are allowed to cool in a condenser to separate into two components, oil and water.","The essential oil extracts were analyzed by capillary gas chromatography (GC) equipped with a flame ionization detector (FID).","GC was carried out using a Varian 6000 series Vista and peak areas were computed by a Varian DS 654 integrator.","SPB-1 (30 m×0.25 mm φ, 0.25 μm) and Supelcowax (30 m×0.25 mm φ, 0.25 μm) fused silca columns were used.","Compounds in the sample come off the column at different times in minutes (Rt's or Retention Times) and these are compared to known standards and the compounds can thus be identified.","When GC-FID gave ambiguous identification of certain compounds, Mass Spectrometry (MS) was used to compare the mass spectra of the compounds with a database of known spectra.","The relative amount of each component of the essential oil extracts was determined for different lots of a variety of C. ambroisiodes.","Each lot represents pooled extractions taken from a crop within one harvest date.","FIG.","1 shows the phytochemical profile of the essential oil extract taken from three different lots.","Lot No.","00MC-21P indicates an ascaridole content of 9.86%; Lot No.","00MC-24P has an ascaridole content of 6.39% and 00MC-29P has an ascaridole content of 3.6%.","The activity of the extract is not apparently affected by the variability in relative amount of ascaridole as results from bioassays with these lots suggest.","Example II Determination of the Active Constituents of the Essential Oil Extract Extensive testing was done in order to determine the active constituents of the essential oil extract.","All compounds present in the oil were tested except for trans-ρ-mentha-2,8dien-1-ol and cis-ρ-mentha-2,8-dien-1-ol because they were unavailable.","All compounds tested were obtained commercially (Sigma-Aldrich) except for ascaridole and iso-ascaridole that were isolated from a sample of our extract by Laboratoires LaSeve, Chicoutimi Qc.","Acaricidal Activity Tests with the Two-Spotted Spider Mite (TSSM: Tetranychus urticae) To test acaricidal activity, thirty adult female mites were placed on their dorsum with a camel hair brush on a double-sided sticking tape glued to a 9 cm Petri dish.","Three dishes were prepared for each concentration of each compound tested and the control (e.g., water) for a total of 90 mites per treatment per treatment day.","One (1) ml of each preparation and of microfiltered water as control was added with a Gilson Pipetman™ P-1000 to the reservoir of the spray nozzle of a Potter Spray Tower mounted on a stand and connected to a pressure gauge set at 3 P.S.I.","Petri dishes were weighed before and immediately after each application to calculate the amount of oil deposited (mg/cm2) with each sample tested.","The entire procedure was followed three times to give a total number of 270 mites tested with each treatment.","Mite mortality was assessed 24 and 48 h after treatment.","Mites that failed to respond to probing with a fine camel hair brush with movements of the legs, proboscis or abdomen were considered dead.","Individual compounds were tested at 0.125, 0.50, 1.0 and 2.0% concentrations with the two-spotted spider mite (TSSM: Tetranychus urticae).","Results are illustrated in FIG.","2.Comparisons were made with mortality data obtained with the 1% concentration of each compound and it was observed that carvacrol is the most active compound (90% mortality of TSSM) followed by carveol (82% mortality), nerol (82% mortality), thymol (78% mortality), carvone (78% mortality) and α-terpineol (71% mortality).","Other compounds gave less than 40% mortality.","No mortality was recorded for ascaridole at 1% .","Although 3% mortality was obtained with a solution of 0.125% ascaridole, we believe that this is an erroneous udependable result because too few individuals were tested (n=125) and the standard deviation is high (13), compared to the higher number of individuals tested at the higher concentrations of this compound (n=300 each at 0.5% and 1.0%) where no mortality was recorded.","The results obtained with individual compounds, do not indicate that the compounds present in large quantities in the oil, i.e α-terpinene, ρ-cymene, limonene, ascaridole, iso-ascaridole, have a great impact on the biological activity of the extract.","Mortality obtained with each of these compounds tested at 1% concentration was 17% or less.","Ascaridole and iso-ascaridole at 1% concentration had no effect on the spider mite (0% mortality).","Carvacrol, carveol, nerol, thymol and carvone on the other hand may have a much greater impact on the activity of the oil (>70% of TSSM at a 1% concentration) even though each of these compounds are present in relatively small quantities (<1%) Insecticidal Activity Tests with the Greenhouse Whitefly (GWF: Trialeurodes vaporariorum) Tests were also done using compounds that had demonstrated the higher degree of activity, i.e.","carvacrol, nerol and thymol with the greenhouse whitefly (Trialeurodes vaporariorum) our model bioassay for insecticidal effect.","Whitefly adults were glued to a black 5 cm×7,5 cm plastic card sprayed with Tangle-Trap® (Gempler's Co.) by placing cards directly in the greenhouse colony cage until at least 20 adults have alighted on each card.","Cards were observed before spraying under the binocular scope to remove all dead and immobile whiteflies.","Only active whiteflies were kept for the experiment.","Four cards were used per treatment.","Each card was sprayed at 6 psi with 300 μl of emulsion using a BADGER 100-F® (Omer DeSerres Co., Montréeal, Canada) paintbrush sprayer mounted on a frame at a distance of 14.5 cm from the spray nozzle in an exhaust chamber.","Cards were weighed immediately before and after spraying to calculate the amount of active ingredient deposited in mg/cm2.Cards were allowed to dry under the exhaust chamber and then placed sideways on a Styrofoam rack in a closed clear plastic container of 5 L with moistened foam on the bottom to keep humidity high (>90% R.H.).","The plastic container was stored in a growth chamber at 24° C. and 16 L:8 D photoperiod.","This procedure was repeated three times.","Mortality was evaluated 20 hours following treatment by gently probing the whitefly with a single-hair brush under the binocular microscope.","Absence of movement (antennae, leg, wing) following probing was recorded as dead.","Relative efficacy of the compounds were compared by transforming mortality data to arcsin{square root}p and then subjecting to an ANOVA analysis using SAS® software (SAS Institute 1988).","Results with the GWF, shown in FIG.","3, confirm the important biological activity of these three compounds.","Example III Ready-To-Use Acaricidal Formulations A ready-to-use (RTU) sprayable insecticidal formulation having as the active ingredient an extract of Chenopodium was prepared.","In one embodiment, this formulation contains between 0.125% and 10% (by volume) of the essential oil extract, an emulsifier, a spreader and sticking agent, and a carrier.","Examples of basic RTU formulations are as follows.","Ingredient Amount (%) Amount (%) Amount (%) Essential oil 1.00 1.00 1.00 extract Rodacal IPAM 0.50 0.83 0.83 Igepal CA-630 — 0.50 — Macol NP 9.5 — — 0.50 Water 98.5 97.67 97.67 Examples of formulations with added polymers for added residual effect are as follows.","Ingredient Amount (%) Amount (%) Amount (%) Essential oil extract 1.00 1.00 1.00 Rhodacal IPAM 0.83 0.83 0.83 Igepal CA-630 0.50 0.50 0.50 Carboset 514H 2.00 — — Pemulen TR2 — 0.05 — Schercoat P110 — — 5.00 Propylene glycol — 2.00 — Water 95.67 95.62 92.67 Example IV Acaricidal Efficacy of the Essential Oil Extract (RTU Formulation) Efficacy trials were conducted using a Ready-to-use (RTU) formulation of the present invention comprising: 1.00% Essential oil extract; 0.83% Rhodacal IPAM; 0.50% Igepal CA-630; 0.05% Pemulen TR2; 2.00% Propylene glycol; and 95.62% Water.","Thirty adult female mites were placed on their dorsum with a camel hair brush on a double-sided adhesive tape glued to a 9 cm Petri dish.","Three dishes were prepared for each concentration of each formulations or products tested and the control, (e.g.","water), for a total of 90 mites per treatment per treatment day.","One (1) ml of each preparation and of microfiltered water as control was added with a Gilson Pipetman™P-1000 to the reservoir of the spray nozzle of a Potter Spray Tower mounted on a stand and connected to a pressure gauge set at 3 P.S.I.","Petri dishes were weighed before and immediately after each application to calculate the amount of oil deposited (mg/cm2) with each sample tested.","The ready-to-use formulation was tested according to the method mentioned above to identify the minimum concentration needed for the desired mortality (>95%) at different concentrations (00.125, 0.25, 0.5, 0.75, and 1%) in order to compare the relative efficacy of this RTU formulation and other acaricidal products (synthetic and natural) presently on the market.","The entire procedure was followed three times to give a total number of 270 mites tested with each treatment.","Mite mortality was assessed 24 and 48 h after treatment.","Mites that failed to respond to probing with a fine camel hair brush with movements of the legs, proboscis or abdomen were considered dead.","In order to obtain LC50 values (Lethal Concentration in mg/cm2 is the amount of product needed to kill 50% of the test organism; therefore the lower the LC50 value the more toxic the product) results of the 48 h counts were subjected to Probit analysis using POLO computer program (LeOra Software, 1987).","Mortalities were entered with corresponding weighed dose (mg/cm2) to take into consideration variability in the application rate.","The results obtained with these bioassays are shown in FIG.","4.Although the toxicity tests presented herein were performed with female mites, it will be clear to a person skilled in the art that those results show that the mortality that would have been observed for male mites would have been the same if not higher knowing that male mites are smaller than females.","Example V Effect on the Egg and Nymphal Stages of the Spider Mite (RTU Formulation) The RTU formulation (comprising: 1.00% Essential oil extract; 0.83% Rhodacal IPAM; 0.50% Igepal CA-630; 0.05% Pemulen TR2; 2.00% Propylene glycol; and 95.62% Water) was also tested on the egg and the nymphal stages of the spider mite.","The ovicidal effect was determined with eggs of the twospotted spider mite following treatment with concentrations of the RTU formulation.","Adult female T. urticae are transferred to 2 cm diameter leaf disks cut out of lima bean leaves and left for four hours for oviposition.","When at least 20 eggs/disk are laid, adult mites are then removed.","Leaf disks are moist and then sprayed and Petri dishes are weighed before and after treatment and stored after treatment.","Egg hatch is assessed daily and for 10 days following treatment by counting the number of eggs remaining on the leaf disks and the number of live and dead nymphs present.","Percent egg hatch is determined with live nymphs only.","The nymphs are considered dead if no movement is observed after repeated gentle probing with a single-hair brush.","Results of the test on the egg stage (FIG.","5) indicate that the RTU formulation has some effect on the eggs with 30% mortality using a 0.5% solution of the oil.","It is expected that a higher concentration of the oil should show greater efficacy on eggs.","Similarly to the effect of the RTU formulation on the nymphal stage, even at the 0.5% concentration, the RTU gave higher results (95.8%) than the existing commercial preparations of either Avid (80.1%) or Safer Soap (61.7%) (FIG.","6).","Example VI Residual Effect of the RTU Formulations of the Present Invention and Comparison Thereof with Commercially Available Acaricidal Products The residual effect of the RTU formulation (comprising: 1.00% Essential oil extract; 0.83% Rhodacal IPAM; 0.50% Igepal CA-630; 0.05% Pemulen TR2; 2.00% Propylene glycol; and 95.62% Water) was also tested with the spider mite and compared to natural and synthetic products already on the market, (i.e.","Kelthane™, Avid™, Safer's™ Soap and Wilson's dormant oil).","The procedure for this test involved the preparation of vials containing a nutrient solution in which individual faba bean leaves were placed.","Eighteen leaves were prepared for each concentration tested and each were sprayed with the indicated concentration until run-off and allowed to dry.","Ten spider mites were placed on nine of the leaves one hour after spraying and ten were placed on the other nine leaves one day following treatment.","Mortality was observed 24 and 48 hr following mite introduction on the leaves.","The entire procedure was repeated three times.","The results of the residual effect of the different products when the mite is introduced on the plant one hour following treatment are shown in FIG.","7.These results indicate that there is a residual effect of the RTU and that this effect is greater than in the Safer product.","However, it is inferior to the residual effect of synthetic products such as Kelthane and Avid.","These results show the RTU formulation's very low persistence in the environment (about 23 mortality of spider mites when the pest is introduced on the plant one hour after treatment with the product).","The RTU formulation is therefore compatible with the recommendations of the Integrated Pest Management program which supports control methods that do not harm natural enemy populations and permit rapid re-entry of workers to the tested area and uninterrupted periods of harvest while assuring safety to workers and consumers.","Example VII Acaricidal Activity of the Extracts on Other Acari (RTU Formulation) To confirm the efficacy of the formulations of the present invention on plant infesting acari in general, certain bioassays were performed on another plant infesting mite, the European red mite, Panonychus ulmi, a mite which shows a close taxonomical relationship with T. Urticae.","The RTU formulation (comprising: 1.00% Essential oil extract; 0.83% Rhodacal IPAM; 0.50% Igepal CA-630; 0.05% Pemulen TR2; 2.00% Propylene glycol; and 95.62% Water) was thus tested on the red mite Panonychus ulmi, a pest of apple orchards, following the same protocol described for contact efficacy on adult spider mites in order to confirm its broad effect as an acaricide.","The results confirm the effectiveness of the essential oil extract as a contact acaricide (FIG.","8) which is not exclusively active on T. Urticae.","Example VIII Insecticidal Efficacy of the Essential Oil Extract (RTU Formulation) Similar efficacy tests were also performed on several insect species that are serious pests of cultivated plants.","The species tested were the greenhouse whitefly, Trialeurodes vaporariorum; the Western flower thrips, Frankliniella occidentalis; the green peach aphid, Myzus persicae; and the silverleaf whitefly, Bermisia argentifolii following the same protocol described in Example XI (C) below.","Results presented in FIG.","9 indicate that the RTU product (comprising: 1.00% Essential oil extract; 0.83% Rhodacal IPAM; 0.50% Igepal CA-630; 0.05% Pemulen TR2; 2.00% Propylene glycol; and 95.62% Water) is toxic to all organisms tested.","LC50 could be calculated for the greenhouse whitefly and the green peach aphid and results (LC50 of 0.00131 mg/ cm2 and 0.0009 mg/cm respectively) show that the product is as or more effective to these insects as the spider mite.","Example IX Emulsifiable Concentrate Formulation An emulsifiable concentrate formulation with an extract of Chenopodium ambrosioides was also prepared.","The concentrate contains between 10 to 25% essential oil extract, emulsifiers, a spreader/sticker, and a carrier.","Examples of emulsifiable concentrate formulations are as follows.","Amount Amount Amount Amount Amount Amount Ingredient (%) (%) (%) (%) (%) (%) Essential 25 25 25 25 25 25 oil extract Rhodopex 5 2.5 — — 1.25 — CO-436 Rhodopex — — — — — — CO-433 Igepal CO- — 2.5 — — 1.25 2.5 430 Igepal CA- — — 5 2.5 — — 630 Igepal CO- — — — 2.5 — — 887 Isopropanol — — 10 — — — Isopar M — — 60 70 — — Macol NP — — — — — 2.5 95 THFA 70 70 — — 72.5 70 Example X Acaricidal Efficacy of the Essential Oil Extract (Emulsifiable Concentrate Formulation) Contact and residual bioassays were conducted in the laboratory to test the efficacy of the essential oil extract of the present invention.","A 25% emulsifiable concentrate (EC25%) formulation of oil was tested against the adult and eggs of the twospotted spider mite and the European red mite.","The EC25% formulation tested comprised: 25% Essential oil extract; 2.5% Rhodopex CO-436; 2.5% Igepal CO-430; and 70% THFA.","The twospotted spider mite was reared on Lima bean plants (Phaseolus sp.)","and the European red mite on apple leaves cv McIntosh (Malus domestica Borkhausen).","Contact Efficacy with the Adult Stage The methodology used for adults was the same for both species.","Twospotted spider mite adults were treated with four concentrations of oil of a North American herbaceous plant (0.125, 0.25, 0.5 and 1.0% active ingredient (AI) EC25%; Urgel Delisle et Associes, Saint-Charles-sur-Richelieu, QC, Canada), neem oil (Neem Rose Defense® EC 90%; Green Light, San Antonio Tex, USA) at 0.7% AI, insecticidal soap (Safer's Trounce® EC 20% potassium salts of fatty acids with 0.2% pyrethrins; Safer Ltd. Scaborough, ON, Canada) at 1% AI and a water control.","European red mite.","adults were treated with five concentrations (0.0312, 0.0625, 0.125, 0.25 and 0.5%) of EC25%, abamectin (Avid® EC1.9%; Novartis, Greensboro, N.C., USA) at 0.006% AI and a water control.","Twenty-five mature female mites were deposited dorsally on a 1 cm2 piece of double-coated tape glued on a glass microscope slide.","For each treatment period, four slides were prepared for each treatment or acaricide application as defined above.","Solutions for each treatment were prepared on the treatment day and each slide was sprayed at a pressure of 0.42 kg/cm2 under an exhaust chamber with 250 μl of solution using a Badger 100-F® paint brush sprayer (Badger Air-Brush Co., Franklin Park, Ill., USA) mounted on a frame at a distance of 15 cm from the slide.","The slides were weighed immediately before and after spraying to calculate the amount of active ingredient deposited per surface area (mg/cm2); this quantity varied less than 15% between slides.","After spraying, the slides were placed on a styrofoam rack in a closed clear plastic container with a wet foam at the bottom to keep moisture high (90% R.H.).","The container was stored in a growth chamber at 24° C. and 16 L:8 D photoperiod.","This experimental procedure was repeated on three consecutive days in a complete block design where treatment period was considered a block.","Mortality was assessed under a binocular microscope 48 (twospotted spider mite) and 24 hours (European red mite) following treatment.","Because European red mite mortality in the control group at 48 hours was high, it was judged to be inadequate for statistical evaluation.","Mites were considered dead if movement was imperceptible after repeated gentle probing with a single-hair brush.","Data were transformed by arcsin{square root}p and subjected to an ANOVA statistical analysis using SAS® software (SAS Institute, 1988).","The LC50 and LC90 (in mg/cm2 of AI) of EC25% were calculated with PROBIT analysis using POLO-PC® software (LeOra Software, 1987).","EC25% at 1% concentration and insecticial soap at 1% were most effective at controlling the adult twospotted spider mites causing 99.2 and 100% mortality respectively (FIG.","10).","At 0.5, 0.25 and 0.125% EC25% resulted in 94.7, 76.8 and 68% mortality respectively.","The least effective treatment was neem oil, which at the recommended dose caused only 22.1% mortality.","The LC50 and LC90 of EC25% for the twospotted spider mite were 0.009 mg/cm2 (99% confidence interval 0.0082-0.0099 mg/cm2) and 0.0292 mg/cm2 (99% confidence interval 0.0268-0.0321 mg/cm2) respectively (significant at P=0.01).","In comparison, the LC50 of insecticial soap had been determined by the manufacturer to be 0.016 mg/cm2.At 0.5% concentration, EC25% was significantly more toxic (97.1% mortality) to P. ulmi adults than abamectin (82.4%) (FIG.","11).","Treatments with EC25% at concentrations ranging from 0.0625 to 0.25% gave statistically the same control level as abamectin.","The LC50 and LC90 of EC25% for the red spider mite were 0.0029 mg/cm2 (99% confidence interval 0.0019-0.0038 mg/cm2) and 0.014 mg/cm2 (99% confidence interval 0.0108-0.0203 mg/cm2).","EC25% gave <80% control of the adult stage of the two mites species at low doses.","Ovicidal Activity The ovicidal effect of the following products was determined with eggs of the twospotted spider mite and the European red mite: six concentrations of EC25% (0.0625, 0.125, 0.25, 0.5, 1 and 2%), neem oil at 0.7% AI, insecticidal soap at 1% AI and abamectin at 0.006% and a water control.","Twenty adult female T. urticae were transferred to 2 cm diameter leaf disks cut out of lima bean leaves and left for four hours for oviposition.","Female P. ulmi were left for 24 hours to lay their eggs on 2 cm diameter leaf disks of apple leaves.","When at least 20 eggs/disk were laid, adult mites were then removed with a soft brush Leaf disks were kept on moist soft cotton swabs placed in small (4 cm diameter) plastic Petri dishes.","Three leaf disks were prepared for each treatment or acaricide application.","Leaf disks were sprayed and Petri dishes were weighed before treatment and stored after treatment as for the slides used in the bioassay with adults.","This experimental procedure was repeated on three consecutive days in a complete block design where treatment period was considered a block.","Egg hatch was assessed daily and for 10 days following treatment by counting the number of eggs remaining on the leaf disks and the number of live and dead nymphs present.","Percent egg hatch was determined with live nymphs only.","The nymphs were considered dead if no movement was observed after repeated gentle probing with a single-hair brush.","All nymphs (alive and dead) were removed daily from the leaf disks.","Percent egg hatch (number of nymphs/total number of eggs on leaf disk×100) were transformed with arcsin {square root} and subjected to an ANOVA statistical analysis using SAS® software (SAS Institute, 1988).","Egg hatch for the twospotted spider mite was significantly reduced by abamectin (8.0% egg hatch) and neem oil (2.1%) (FIG.","12).","Egg hatch was reduced to 67 and 40% with 1.0 and 2.0% concentrations of EC25% respectively and to 61.3% with insecticial soap.","Egg hatch for the European Red mite was significantly reduced compared to the control treatment with the recommended doses of insecticial soap (27.2% egg hatch), abamectin (11.0%) and neem oil (14.2%) (FIG.","13).","Residual Bioassay with Adult Twospotted Spider Mites Leaf discs measuring 2 cm in diameter of bean leaves were sprayed on both sides with a VEGA 2000 sprayer (Thayer & Chandler Co., Lake Bluff, Ill., USA) at 0.42 kg/cm2 to runoff with 6.25 ml of each the following solutions: 2, 4, 8, and 16% of 99B-245, the recommended dose of dicofol (Kelthane® 35WP, Rohm and Haas Co., Philadelphia, Pa., USA) at 0.037% AI and a water control.","Each treatment consisted of eight discs.","One hour after treatment, 10 spider mites were transferred to each disc.","Mortality was evaluated 48 hours following transfer of mites to the leaf discs.","The procedure was repeated three times on three subsequent days.","EC25% at 2, 4, 8 and 16% concentrations caused 23.0, 18.3, 13.9 and 32.5% mortality respectively to the adult spider mites when mites were introduced on bean leaves, 1 hr after treatment (FIG.","14).","Dicofol's residual activity was significantly higher (99.5% mortality) than any of the EC25% concentrations.","EC25% was as effective as the insecticidal soap and synthetic acaricide abamectin to control adult twospotted spider mite and the European red mite.","EC25% decreased egg hatch, but not as effectively as abamectin or neem oil.","It may be important however to continue these investigations to determine the viability of emerged nymphs treated with the essential oil product because some botanicals, such as neem mixtures have shown growth-inhibiting properties to various pests (Rembald, 1989) and pulegone decreased larval growth of southern armyworm, Spodoptera eridania (Grunderson et al., 1985).","Furthermore we demonstrated that when adult mites are introduced one hour after treatment, the mortality rate was statistically comparable to that of the control (FIG.","14).","A botanical such as EC25% may be an alternative to the more toxic or incompatible products.","A contact acaricide with low residual activity can be used for treatments of localized infestations, before scheduled introductions of natural enemy populations or in absence of the natural enemy, i.e.","treating at night in absence of diurnal parasitoids or predators.","Plant essential oils may be phytotoxic (Isman, 1999).","The oil used for EC25% was evaluated on several edible and ornamental plants for its phytotoxic effects and results indicate that at the recommended dose, i.e.","0.5%, there were no observable effects on the leaves and flowers of tested plants (H. Chiasson, unpublished results).","Example XI Insecticidal Efficacy of the Essential Oil Extract (Emulsifiable Concentrate Formulation) Efficacy trials were conducted (laboratory and small-scale greenhouse trials) using the emulsifiable concentrate formulation of the present invention (EC at 25% of chenopodium oil comprising: 25% Essential oil extract; 2.5% Rhodopex CO-436; 2.5% Igepal CO-430; and 70% THFA) with the following organisms: the green peach aphid (Myzus persicae), the Western flower thrips (Frankliniella occidentalis), the greenhouse whitefly (Trialeurodes vaporariorium) as well as the parasitoid Encarsia formosa.","All bioassays were conducted in the laboratory of the Horticultural Research and Development Center (HRDC) of Agriculture and Agri-food Canada in Saint-Jean-sur-Richelieu, Quebec, Canada.","A.","Contact bioassays in the laboratory and greenhouse using EC25% and commercially available bioinsecticides with the green peach aphid (Myzus persicae (Sulz.))","Laboratory Bioassay Five concentrations (0.125, 0.25, 0.5, 1 and 2%) of formulation EC25% were compared to commercial preparations of Neem Rose Defense® at 0.5% (EC 90% hydrophobic Neem oil), Safer's Trounce® at 1% (EC 20% with 0.2% pyrethrin) and a water control.","Each treatment was repeated 12 times and each replicate consisted of a 2 month old shoot (10-15 cm) of Verbena speciosa ‘Imagination’ placed in a plastic Aqua-Pick® (tube used by florist to keep stems of cut flowers wet) filled with 10 ml of water.","Aqua Picks were secured on a block of Styrofoam placed on the bottom of a 1 l transparent plastic container modified with screened sides and top to permit aeration.","Green peach aphids (Myzus persicae (Sulz.))","were collected in plastic containers from a rearing cage maintained in a greenhouse colony.","Ten adults were transferred to each Verbena shoot.","The shoot was sprayed at 8 psi under an exhaust chamber for about 15 seconds (long enough to cover the whole shoot) with a VEGA 2000® paintbrush sprayer equipped with a 20 ml reservoir (Thayer & Chandler Co., Lake Bluff, Ill., USA).","Each shoot and plastic container was then stored in a growth chamber at 24° C., 65% R.H. and 16 L:8 N photoperiod.","The entire procedure was repeated four times.","Mortality was evaluated 48 hours following treatment by probing the aphid for movement with a small brush; absence of movement was recorded as dead.","To evaluate the relative efficacy of EC25%, Neem Rose Defense® and Safer's Trounce®, percentage mortality data were transformed to arcsin{square root}p and subjected to ANOVA analysis using SAS® software (SAS Institute 1988).","LC50 and LC90 were calculated using mortality results by PROBIT analysis using POLO-PC® software (LaOra Software 1994).","Product concentrations (%) were used because data on quantity of active material deposited were not available.","Results show that EC25% at 2.0% concentration was more effective (92.3% mortality) at controlling the green peach aphid than EC25% at 1% concentration (71.7%) and Safer's Trounce® (55.2%) though not significantly (FIG.","15 ).","This lack of distinction between treatments may be due to the low number (n) of aphids tested.","Treatments with EC25% at concentrations of 0.5% and less and with Neem Rose Defense® resulted in <50% mortality of the aphids and results were not significantly different to those obtained with the water control.","The LC50 and LC90 of EC25% for the green peach aphid was 0.63 (in % concentration) (Confidence Interval 0.47%-0.79%) and 1.84% (Confidence Interval of 1.39%-2.95%) respectively (FIG.","16).","Greenhouse Bioassay Three concentrations (0.25, 0.5 and 1%) of formulation EC25%, Neem Rose Defense® at 0.5% (EC 90% hydrophobic Neem oil), Safer's Trounce® at 1% (EC 20% with 0.2% pyrethrin) and a water control were tested with the green peach aphid (Myzus persicae (Sulz.)).","Fifteen plants (replicates) of two month old Verbena speciosa ‘Imagination’ (10-15 cm) grown in small plastic insertions cells (used for potting plants) filled with Pro-Mix BX® were used for each treatment.","Each insertion cell was glued to the bottom of a 1 l transparent plastic container with screened sides and top, to permit aeration.","Green peach aphids were collected in plastic containers from a rearing cage maintained in a HRDC greenhouse and ten adults were transferred to each plant.","The whole plant was sprayed for 15 seconds on average, at 8 psi under an exhaust chamber with a VEGA 2000® paintbrush sprayer equipped with a 20 ml reservoir (Thayer & Chandler Co., Lake Bluff, Ill., USA).","Spraying was done three times over the course of the experiment, i.e.","on days 0, 7 and 14.Containers with the sprayed plants were kept in a greenhouse under shade for the duration of the experiment.","Counts were done on days 7, 14 (prior to spraying) and on day 21 by dismantling five of the fifteen replicates in each treatment.","Aphids were individually counted when numbers were small (<50).","For larger numbers, plants were shaken over a clear 250 ml container filled with soapy water over a black and white grid to evaluate the number of aphids present.","Plant leaf surface (cm2) was measured with an area meter LI-3100® (LI-COR Inc., Lincoln, Nebr., USA) and counts were averaged to number of aphids/cm2 for each treatment and transformed to square root (x+0.5) for ANOVA analysis with SAS® software to evaluate the efficacy of the different treatments.","Counts within treatments did not differ significantly (P=0.3647) from one sampling day to the other, so results within treatments were pooled and averaged for the whole experiment.","All concentrations of EC25% and Safer's Trounce® were more effective in controlling the aphids than the water control (FIG.","17 ).","EC25% at 0.5% and 1.0% and Safer's Trounce were significantly more effective in reducing the number of aphids/cm2 than Neem Rose Defense® and EC25% at 0.25%.","Both 0.5% and 1.0% EC25% concentrations were more effective (0.5 aphids/cm2 and 0.0 aphids/cm2 respectively) than Safer's Trounce® (0.9 aphids/cm2) though not significantly.","B.","Contact bioassays in the laboratory and greenhouse with the western flower thrips (Frankliniella occidentalis (Perg.))","using EC25% formulation and two commercially available bioinsecticides.","Laboratory bioassay Six concentrations (0.05, 0.18, 0.125, 0.25, 0.5 and 1%) of formulation EC25%, Neem Rose Defensee at 0.7% (EC 90% hydrophobic Neem oil), Safer's Trounce® at 1% (EC 20% with 0.2% pyrethrin) and a water control were tested with the Western flower thrips (WFT: Frankliniella occidentalis (Perg.)).","WFT were collected in plastic containers by tapping infested Lima bean leaves over white paper.","Ten WFT (either adults or 3rd or 4th instar nymphs) were transferred to a closed 250 ml transparent plastic container.","Wet dental cotton was inserted through the lid for use as a water source.","Four replicates were prepared for each treatment.","Containers were sprayed at 6 psi under an exhaust chamber for 15 seconds with a VEGA 2000® paintbrush sprayer equipped with a 20 ml reservoir (Thayer & Chandler Co., Lake Bluff, Ill., USA).","Containers were weighed just before and after spraying to calculate the amount of active ingredient deposited in mg/cm2.Containers were then stored in a growth chamber at 24° C., 65% R.H. and 16 L:8 D photoperiod.","The entire procedure was repeated four times.","Mortality was evaluated 24 hours following treatment under a binocular scope by probing WFT with a small brush.","Absence of movement was recorded as dead.","The efficacy of EC25% was compared to Neem Rose Defense® and Safer's Trounce® and data were transformed by arcsin{square root}p and subjected to ANOVA analysis using SAS® software (SAS Institute 1988).","The LC50 and LC90 (in mg/cm2 of active ingredients) were calculated mortality results by PROBIT analysis using POLO-PC® software (LaOra Software 1987).","Formulation EC25% at 0.5% and 1.0% were significantly more effective (98.8% and 95.8% mortality respectively) in controlling the WFT than all other treatments except for Safer's Trounce® (82.7% mortality) (FIG.","18).","EC25% at 0.25% caused significantly more mortality (63.7%) than the control (10.8%) but all remaining treatments did not.","The LC50 and LC90 of EC25% for thrips was determined as 0.0034 mg/cm2 (Confidence Interval: 0.0027-0.0039 mg/cm2) and 0.0079 mg/cm2 (Confidence Interval: 0.0067-0.0099 mg/cm2) respectively (FIG.","19).","Greenhouse Bioassay Two concentrations (0.25% and 1%) of formulation EC25%, Neem Rose Defense® at 0.7% (EC 90% hydrophobic Neem oil), Safer's Trounce® at 1% (EC 20% with 0.2% pyrethrin) and a water control were used to evaluate their relative efficacy in controlling the Western Flower thrips (WFT: Frankliniella occidentalis (Perg.))","in a greenhouse setting.","Ten 10 day-old Lima bean plants (Phaseolus sp.)","were prepared for each treatment.","One leaf and the cotyledons of each plant were removed to keep only one leaf per plant grown in Pro-Mix BX® in a plastic insertion cell (used for potting plants) glued to the bottom of a clear plastic container (1 l) with screened sides and top.","WFT were collected in small plastic containers by tapping infested bean leaves over white paper and lifted with a small brush.","Ten adult thrips (or 3rd or 4th instar larvae) were transferred on each single leaf of each plant/insertion cell which were sprayed to drip point at 6 psi under an exhaust chamber with a VEGA 2000® paintbrush sprayer equipped with a 20 ml reservoir (Thayer & Chandler Co., Lake Bluff, Ill., USA).","Spraying was done on days 0, 8 and 14.Each replicate/plastic container was then kept in a greenhouse under shade for the duration of the experiment.","Counts were made on days 8 and 14 (prior to spraying) and on days 21 and 28.All live stages present on the whole plant were counted under a binocular scope and the leaf surface was measured by comparing it to a series of pre-measured hand-made leaf-size patterns.","On the last day of the experiment (day 28), the leaf was cut and its surface was measured with an area meter LI-3100® (LI-COR Inc., Lincoln, Nebr., USA).","Counts were calculated as average number of thrips/cm2 per treatment.","In order to compare treatments, average counts were then calculated as a percentage of thrips present on the control plants: (n/cm2 on treated plants/n/cm2 on control plants)×100 The control treatment therefore had a value of zero and other treatments had positive or negative values indicating that more or less thrips were present respectively in relation to the control treatment.","At the end of the experiment on day 28, leaves treated with EC25% at a concentration of 1.0% had 69.3% less WFT than leaves treated with the control while leaves treated with Safer's Trounce® had 101.1% more WFT (FIG.","20).","Leaves treated with Neem Rose Defense® had slightly more thrips (19.3%) than the control on day 28.Leaves treated with EC25% at 0.25% concentration had 52.3% more thrips than the control on day 28.C.","Contact bioassay in the laboratory with the greenhouse whitefly (Trialeurodes vaporariorium (Westw.))","using EC25% and commercially available insecticides Laboratory Bioassay Five concentrations (0.0625, 0.125, 0.25, 0.5 and 1%) of formulation EC25%, Neem Rose Defense® at 0.7% (EC 90% hydrophobic Neem oil), Safer's Trounce® at 1.0% (EC 20% with 0.2% pyrethrin), Thiodan® at 0.044% (50 WP) and a water control were used to evaluate their relative efficacy in controlling the greenhouse whitefly (Trialeurodes vaporariorium (Westw.)).","Whitefly adults were collected with an insect aspirator from HRDC greenhouses and glued to a black 5 cm×7,5 cm plastic card sprayed with Tangle-Trap® (Gempler's Co.) by emptying the aspirator over the card to obtain at least 20 adults per card.","Cards were observed before spraying under the binocular scope to remove all dead and immobile whiteflies.","Only active whiteflies were kept for the experiment.","Four cards were used per treatment.","Each card was sprayed at 6 psi with 300 μl of emulsion using a BADGER 100-F® (Omer DeSerres Co., Montreal, Canada) paintbrush sprayer mounted on a frame at a distance of 14.5 cm from the spray nozzle in an exhaust chamber.","Cards were weighed immediately before and after spraying to calculate the amount of active ingredient deposited in mg/cm2.Cards were allowed to dry under the exhaust chamber and then placed sideways on a Styrofoam rack in a closed clear plastic container of 5 L with moistened foam on the bottom to keep humidity high (>90% R.H.).","The plastic container was stored in a growth chamber at 24° C. and 16 L:8 D photoperiod.","This procedure was repeated three times.","Mortality was evaluated 20 hours following treatment by gently probing the whitefly with a single-hair brush under the binocular microscope.","Absence of movement (antennae, leg, wing) following probing was recorded as dead.","Relative efficacy of EC25% and the two commercially available bioinsecticides, Neem Rose Defense® and Safer's Trounce®, and the synthetic insecticide Thiodan®, were compared by transforming mortality data to arcsin{square root}p and then subjecting to an ANOVA analysis using SAS® software (SAS Institute 1988).","LC50 and LC90 (in mg/cm2 of active ingredients) were calculated by PROBIT analysis using POLO-PC® software (LaOra Software 1987).","Formulation EC25% at concentrations 0.5% and 1.0% were significantly more effective (98.9% and 100.0% mortality respectively) at controlling the greenhouse whitefly than all other treatments except for Safer's Trounce® (98.0% mortality) (FIG.","21).","Formulation EC25% at 0.125% concentration and Neem Rose Defense® were significantly more effective than the control treatment but significantly less effective than EC25% at 0.25, 0.5 and 1.0% concentrations and Safer's Trounce®.","Thiodan and EC25% at 0.0625% concentration were as effective as the control treatment.","LC50 and LC90 were 0.0066 mg/cm2 (conf.","int: 0.0054-0.0076 mg/cm2) and 0.0141 mg/cm2 (conf.","int: 0.0121-0.0172mg/cm2) respectively (FIG.","22).","D. Contact bioassay in the laboratory with the parasitoïd (Encarsia formosa) using EC25% and commercially available bioinsecticides Laboratory Bioassay Four concentrations (0.0625, 0.125, 0.25 and 0.5%) of formulation EC25%, Neem Rose Defense® at 0.7% (EC-90%), Safer's Trounce® at 1.0% (EC 20.2%) and a water control were tested with the parasitoïd Encarsia formosa (EF) (obtained from Koppert Co. Ltd).","EF were kept in a growth chamber at 24° C., 16 L:8 N photoperiod and 65% R.H. until emergence.","Sixty newly emerged adult EF were transferred with a mouth aspirator into plastic Solo® cups of 20 ml.","Cups were sprayed at 6 psi under an exhaust chamber with 250 ml of solution with a Badger 100-F® paintbrush sprayer (Omer de Serre Co., Montreal, Canada) mounted on a frame at a fixed distance of 14.5 cm.","Solo® cups were weighed just before and after spraying to calculate the amount of active ingredient deposited in mg/cm2.Once sprayed, the EF were gently transferred with a small brush from the Solo cups to small clear plastic Petri dishes (10 EF/Petri) lined with a filter paper wetted with a 5% sugar solution as a food source.","Four replicates were prepared for each treatment.","The Petri dishes were then placed in a tray and stored in a growth chamber at 24° C., 65% R.H. and 16 L:8 D photoperiod.","The entire procedure was repeated three times.","Mortality was evaluated 24 hours following treatment under a binocular scope by observing the EF.","Absence of movement was recorded as dead.","The effect of EC25% was compared to Neem Rose Defense® and Safer's Trounce® using mortality data transformed by arcsin{square root}p and subjected to ANOVA analysis using SAS® software (SAS Institute 1988).","All EC25% formulations at concentrations ranging from 0.0625 to 0.5% were significantly less effective than Safer's Trounce® at 1% (71.9%) (FIG.","23).","Results from all concentrations of EC25% and Neem Rose Defense® formulations were not significantly different than the control.","These results indicate that the recommended dose (0.5%) of EC25% can be safely used with the biological control agent, Encarsia formosa.","Example XII Fungicidal Efficacy of the Essential Oil Extract (Emulsifiable Concentrate Formulation) A.","Effect of EC on Gray Mold Caused by the Plant Pathogen, Botrytis cinerea on Greenhouse Tomatoes Tomatoes (cv Trust) were sown and grown according to practices generally used for the production of greenhouse tomatoes.","About two months after sowing, lesions are made on the stem (5 lesions/plant) and inocuated with a suspension of 3×106 spores of B. cinerea, 1 ml per lesions.","The following treatments were evaluated: 1) control (−) with water only; 2) control (+) with B. cinerea; 3) Nova® (0.3 g/l); 4) Iprodione® (Rovral 1.0 g/l), 5) EC25% (comprising: 25% Essential oil extract; 2.5% Rhodopex CO-436; 2.5% Igepal CO-430; and 70% THFA) at a concentration of 0.125%; 6) EC25% at a concentration of 0.50%; 7) EC25% at a concentration of 0.75%; 8) M. ochracea (P130A) (5×108 spores/ml); 9) Rootshield® (0.6 g/l).","The experimental design was a randomized block design with 8 blocks.","Treatments were applied 1 and 8 days following inoculation of lesions with B. cinerea.","Disease symptoms were evaluated according to the following severity index: 1=absence of symptoms, 2=necrosis (browning of tissue) around the lesion, 3=advanced necrosis and presence of spores on necrotic zones, 4=advanced necrosis, presence of spores on necrotic zones and the start of canker formation, 5=well developed canker and death of plant.","Severity of symptoms was estimated 3 and 5 weeks after plant inoculation.","The experiment was repeated and the effect of treatment was analyzed with an analysis of variance (ANOVA) and means were compared with a Least Significant Difference test (LSD).","Statistical analyses demonstrated that there was a significant difference in efficacy between treatments (FIG.","24).","Control plants demonstrated average severity of disease symptoms on 26 of the 35 control plants.","Plants from all other treatments presented a significantly lower level of severity than the control plants one day after inoculation or the first day of treatment.","The two microbial fungicides Rootshield and P1304 did not significantly control the disease with an average severity between 25.33 and 21.67 8 days after treatment (FIG.","24).","Plants treated with the botanical biopesticide EC25% was less effective in controlling the disease symptoms than the commercial products, Nova and Rovral.","However, all three concentrations of EC25% (0.125, 0.25 and 0.5% AI) decreased disease symptoms with severity indexes being 60, 44 and 37% lower respectively on plants treated with EC25% than the control plants.","Severity indexes were reduced by 38 and 32% with Nova and Rovral respectively (FIG.","25).","B.","Effect of EC25% on Powdery Mildew Caused by the Plant Pathogens, Erysiphe cichoracearum and Sphaerotheca fuliginea on Greenhouse Cucumbers The fungus responsible for powdery mildew are obligatory parasites incapable of survival in the absence of the host.","In order to produce inoculum necessary for inoculation, infested cucumber leaves are collected from a grower in July of 2002.Conidia present on these leaves were transferred on leaves of cucumber plants grown in an experimental greenhouse, one of to two months after sowing the cucumber plants (cv Straight 8).","The following treatments were evaluated: 1) negative control treated with water; 2) positive control treated with the disease organisms, E. cichoracearum and S. fliginea; 3) Nova® (0.3 g/l); 4) Benlate®+Manzate® (1.2 g/l and 4.g/l); 5) EC25% (comprising: 25% Essential oil extract; 2.5% Rhodopex CO-436; 2.5% Igepal CO-430; and 70% THFA) at 0.125% AI; 6) EC25% at 0.50% AI; 7) EC25% at 0.750% AI; 8) Mineral oil.","The experimental design was a complete randomized block design with 7 blocks.","Since it is almost impossile to inoculate an obligatory fungal parasite in an known or measurable quantity of inoculum, there is always variability in the level of infection of the plants.","A first evaluation of powdery mildew was made on all plants inoculated in the greenhouse.","The percentage of infection was analyzed in order to group 8 plants presenting the same level of infection (SE <1.5, see FIG.","26).","Severity of powdery mildew was also evaluated on each individual leaf with the use of a severity index 7 days after treatment applications and reported as average severity for each plant.","The entire experiment was repeated a few days later.","The effect of treatment was analyzed with an analysis of variance (ANOVA) and means were compared with a least significant difference test (LSD).","Statistical analysis demonstrate that there is a difference of effect between treatments.","(FIG.","27).","In fact, an increase of 45 and 39% of disease incidence was observed on control plants (water and disease).","All plants treated with the various products presented a significantly lower increase in percentage of disease severity compared to the control plants.","The botanical biopesticide EC25% was as effective as the commercial fungicide Nova in controlling the disease.","At concentrations of 0.50 and 0.75%, EC25% demonstrated better control than Nova, permitting an increase of disease symptoms of only 2 and 2.5% respectively for both concentrations compared to 4.5% for Nova.","However phytotoxicity was observed on plants treated with EC25% at those two concentrations Example XIII Effect of Essential Oil Extract for Controlling Soil Inhabiting Pests A.","Effect of Soil Treatments with EC25% at 0.5% on Soil Emergence of Adult Western Flower Thrips Lettuce plants were transplanted in plastic pots of 12 cm of diameter and grown for six weeks in a greenhouse.","The plants were heavily infested with all stages of thrips Frankliniella occidentalis Pergande and were watered and fertilized as required by good management practices.","Then, the lettuce plants were cut at the soil level and the pots were sprayed with a preparation of EC25% (comprising: 25% Essential oil extract; 2.5% Rhodopex CO-436; 2.5% Igepal CO-430; and 70% THFA) at 0.5 concentration of active ingredients.","Five volumes of EC25% were applied, 25 ml, 16.7 ml, 13.3 ml, 10 ml, and 6.67 ml per pot representing volumes of 1500 l/ha, 1000 l/ha, 800 l/ha, 600 l/ha and 400 l/ha respectively.","A total of 10 pots were sprayed per volume and control pots were sprayed with 25 ml of tap water.","The application was made using a badger sprayer connected to an air outlet to provide the required air pressure for pesticide application.","Immediately after treatments, the pots were covered with a plastic petri dish cover of 14.5 cm diameter.","The inner surface of the cover was smeared with a thin layer of tanglefoot insect glue and the joints were completely sealed with parafilm to prevent the escape of emerging adults from the soil pot or the introduction of foreign into the dish.","Treatments were applied on 7 July 2002 and counts of emerging adults thrips were made 3, 5, 7, 10 and 12 days after treatments.","At each observation time, emerging adults were removed from the glue and the cover was replaced on the top of the plastic pot and sealed with parafilm.","Counts of emerging adults over a period of 2 weeks after treatments were analysed using an ANOVA and means for each treatment were separated using a Fischer LSD test.","Most of the adult thrips emerged during the first week following treatment and this corresponds with the duration of pupal life that varies between 2 and 8 days according to the ambient temperature (FIG.","28).","Effect of treatment on the total number of adult thrips emergence from the soil was statistically highly significant ((F=5.71; dl=5; P<0.0001).).","All treatments significantly reduced thrips emergence compared to the control.","At 3 days after treatment, an important decrease in the number of emerging adult thrips was observed in the 1500 l/ha treatment compared with all other treatments.","At all other dates, the 1500 l/ha treatment was the best treatment and caused the greatest reduction in adult thrips emergence.","B.","Using EC25% and Microemulsion Formulations for Controlling Soil-Inhabiting Pests of Turf i) Laboratory Tests using EC25% with the European Chafer, Rhizotrogus maialis The formulation EC25% (comprising: 25% Essential oil extract; 2.5% Rhodopex CO-436; 2.5% Igepal CO-430; and 70% THFA) was tested with third instar R. majalis.","The insects were collected from Arnprior ON, and were kept in containers with soil at 4° C. until trials were initiated.","Grass was grown in plastic seedling trays divided into 6 compartments.","Soil within each compartment consisted of 1 part potting soil: 1 part sand to a depth of approximately 5 cm.","The grass seedling mix consisted of Kentucky Blue 65%, Annual Rye Grass 20% and Fescue 15% and was distributed at 1.5 g per compartment 2-3 weeks prior to the addition of the insects.","Ten third instar R. majalis were placed into each compartment so for a total of 60 larvae per tray.","Three replicate trays were used for two separate experiments.","One to two days after the larvae were added to the trays, treatment was initiated.","Each of the EC25% treatment concentrations; 0.0625, 0.125, 0.25, 0.5 and 1%, were prepared in 100 mL of water by the addition of 0.25, 0.5, 1, 2 and 4 mL of stock EC respectively.","The control was an EC blank, 0.375 mL of formulation excluding essential oil.","The trays were watered once per day for the seven day period after treatment.","After one week each compartment was sampled to determine the survival of the larvae.","Samples of the soil were taken from each compartment after both trials and analysed for both pH (ASTM, 1989) and organic matter content (ASTM, 1987).","Probit analysis was used with the data from the two European chafer trials in order to determine the LC50 values for EC25%.","Comparison of the LC50 values within and between each trial was conducted using a z-test.","Ninety percent (90%) R. majalis larvae survived at the 0.0625% concentrations of EC25% and 10% survived at 1% EC25% (FIG.","29).","Percent mortality from both trials were used to calculate LC50 values (FIG.","30).","Two different values were calculated for each trial since not all of the larvae were accounted for at the end of the exposure period.","The first LC50 for each trial is based on the mortality within the actual number of larvae found in each treatment replicate at the end of the test (FIG.","30).","The second LC50 is calculated based on the assumption that there were 10 larvae present at the beginning and end of the experiment within each treatment replicate but due to possible deterioration of the organism it was not identified or located.","A comparison of the LC50 using a z-test indicated that within each trial there was no significant difference between the calculated LC50s (z<0.885, z critical=1.96) Between trials it was determined that there was no significant difference (z=1.587, z critical=1.96) when comparing the first LC50 (based on the actual number of larvae found).","When the LC50s based upon 30 larvae per treatment were compared between trials a significant difference (z=2.233, critical z=1.96) was detected.","A mean LC50 and standard error for the two trials was calculated as 0.466±0.122% EC25% by averaging the two LC50s from trial one and two and these were not significantly different.","Soil Organic Matter Content and pH.","The soil organic matter content was determined to range from 8.9 to 11 with an average of 9.1 in the first trial and 10.5 in the second (FIG.","31).","The pH of the soil ranged from 4.9 to 5.0 with a mean of 4.94 in the first and 5.02 in the second trial.","These values were consistent with those measured in previous R. majalis trials.","ii) Field Tests using Microemulsion Formulations with the European Chafer, Rhizotrogus majalis The objective of this study was to measure the effect of broadcast applications of two microemulsion formulations (MEF), MEF1 and MEF2 of an essential oil extract from Chenopodium ambrosioides, on the presence of the European chafer, Rhizotrogus majalis.","The microemulsion formulations tested comprised the following: Ingredient MEF 1 MEF 2 Chenopodium extract 50 50 Rhodafac RE-610 10 10 Ammonyx LO 4 4 Stepanol WAC 1 — Water 35 35 The experiment was conducted on lawn at the Guelph Turfgrass Institute (GTI), Ontario on turfgrass that was mowed once a week and received irrigation.","Experimental design was a randomized complete block design with 4 replications and 8 treatments.","Plots were 3 m×3 m and treatments tested are listed in FIG.","32.Treatments were applied on Aug. 27, 2002, immediately after pre-treatment assessment of the European chafer, with a sprayer in water at 0.25 L/m2.The European chafers were at the second instar when treatments were applied and at the third instar by the end of the experiment.","The live European chafers were counted using a golf course hole-cutter (15.2 cm, ‘turf mender’).","Five plugs were taken from each plot.","Once removed, plugs were broken up and the number of chafers was recorded.","The last chafer count was done using a sod-cutter: three turf strips (0.3 m×3m) across each plot.","The turfgrass sod was removed and chafers near the soil surface were counted.","Counts were made at 5, 15 and 25 days after treatment on 2, 15, and 23 Sep. 2002, respectively.","Analysis of variance (ANOVA) on the number of total European chafes alive was parformed by the General Linear Model Procedure of SAS statistical software (SAS Institute 1990).","Fisher's Least Significant Difference test (LSD) was used for comparison of treatment means when the F-test was significant (P=0.05).","Number of European chafers was not significantly different between plots prior to treatment application.","On September 23 (25 days after the application), all insecticide treatments significantly reduced the number of chafers compared to the untreated control except treatments MEF1 (10.0 ml/m2) and MEF2 (20.0 ml/m2) (F=4.25; P<0.00046) (FIG.","33).","European chafer counts for both treatments were significantly higher than counts on plots treated with SEVIN XLR PLUS.","No significant difference in the number of chafers in turfgrass plots was observed on September 2 (5 days after application) and on September 15 (15 days after application).","For both dates, all treatments reduced chafers compared to the untreated control lower, the statistical model was not significant mainly because of the fact that European chafers were counted on a smaller area at these two dates.","iii) Field Tests with the Microemulsion Formulations MEF1 and MEF2 and the Hairy Chinch Bug, Blissus leucopterus The experiment was conducted on a commercial lawn in Guelph, Ontario on low maintenance turfgrass that was mowed once a week and received no irrigation.","Experimental design was a randomized complete block design with 4 replications and 8 treatments.","Plots were 2 m×3 m and treatments tested are listed in FIG.","34.Treatments were applied on Jul.","30, 2003, immediately after pre-treatment assessment of the HCB, with a sprayer in water at 1 L/m2.Most of the HCB population was at the third instar.","HCB were counted using the flotation method.","A cylinder (346 cm2) was inserted into the turf to a depth of about 3 or 4 inches and was flooded to the brim with water.","After 5 minutes, live HCB floating to the surface were counted.","Three counts per plot were made (by the same person) and the total HCB per plot was used for statistical analysis.","HCB were counted 5, 15 and 25 days after treatment on August 4, 12 and 24, respectively.","Analysis of variance (ANOVA) on the number of total live HCB was performed using the General Linear Model Procedure of SAS statistical software (SAS Institute, 1990).","Fisher's Least Significant Difference test (LSD) was used for comparison of treatment means when the F-test was significant (P=0.05).","Number of HCB was not significantly different between plots prior to treatment application.","All insecticide treatments significantly reduced the number of HCB compared to the untreated control on August 12, 15 days after the application (F=6.13; P<0.0005) (FIG.","35).","There were no differences between results with the Diazinon treatment and results with the two formulations.","At this date, treatment MEF2 (20.0 ml/m2) had significantly more insects than treatment MEF2 (10.0 ml/m2).","No significant difference in the number of HCB in turfgrass plots was observed on August 4 (5 days after application) and on August 24 (25 days after application).","For both dates, all treatments reduced HCB compared to the untreated control.","Example XIV Effect of Essential Oil Extract on Beneficial Pests A.","Direct Toxicity of the Essential Oil Extract on Predatory Mites Amblyseius fallacis and Phytoseiulus persimilis The purpose of this study was to evaluate the direct toxicity of the EC25%, a botanical biopesticide with two predaceous mites Amblyseius fallacis, a natural regulator of mites in integrated control orchards and Phytoseiulus persimilis, a known mite predator for the control of the twospotted mite in vegetable crops grown under glasshouses in Quebec and elsewhere.","The suitability of EC25% as a primary tool in IPM of greenhouse crops would therefore be determined.","Rearing of Tetranychus urticae and Amblyseius fallacis The phytophagous mite, Tetranychus urticae has been reared on common bean plants (Phaseolus vulgare) for several years at the Horticultural Research and Development Center, St. Jean-sur-Richelieu, Quebec.","The beans were sown at high densities of 40 to 50 plants per tray (39 cm×30 cm).","Colonies of T. urticae were kept in a growth chamber set at 25° C., 75% HR and 16 L photoperiod.","The predaceous mite Amblyseius fallacis was maintained on Tetranychus urticae and kept in a greenhouse set at 25° C., 75 HR and 16 L photoperiod.","A fan placed in front of the cage containing both Amblyseius fallacis and the twospotted spider mite provided continuous air flow to the colonies.","Trays containing bean plants infested with the twospotted spider mites were added regularly to provide sufficient food to the predator colonies.","Rearing of Phytoseiulus persimilis Colonies of Phytoseiulus persimilis were bought from Koppert Canada and reared in the laboratory in the same conditions as for A. fallacis.","The colonies originating from the shipment were maintained and acclimatized in a growth chamber set at 25 C, 70-85% RH and 16:8 (light/darkness) for two weeks.","Contact Toxicity Assay The bioassays were carried out in Petri dishes using a leaf disc method.","A wet sponge was placed in a plastic Petri dish (14 cm diameter and 1.5 high) and rings of apple leaf (cv.","McIntosh; 3.5 cm of diameter) were cut and placed upside down on the surface of a water-saturated sponge.","Sufficient numbers of all stages of the twospotted spider mite Tetranychus urticae Koch were then brushed onto each leaf disc.","A total of five leaf discs were put in a Petri dish and each Petri dish represented one replicate.","Ten replicates per treatment were prepared over a period of three weeks.","Gravid females of Amblyseius fallacies (5) or Phytoseiulus persimilis (9), were picked up at random under a stereomicroscope from leaves taken from plants used to rear the predator colonies.","They were transferred individually with a fine camel brush to a small Petri dish (5.5 cm of diameter) containing a leaf piece of the common bean, Phaseolus vulgare.","They were treated topically with 0.3 ml of pesticide solution at different dosages using a paintbrush sprayer (Vega 2000, Thayer & Chandler, Lake Bluff, Ill., USA) at 6 psi set at 14.5 cm above the treated area.","The pesticide solutions were prepared on the day of application.","Treated females were then transferred carefully and individually to each apple leaf disc.","To avoid contamination, a new camel brush was used for each concentration to transfer the treated females to leaf discs.","Petri dishes were put in a black tray and covered with transparent plastic covers and a strip of brown paper was placed on top to reduce glare and to keep the mites within the leaf disc area.","Water was added to the tray to maintain high relative humidity.","The trays were incubated in a growth chamber set at 25° C., 75% HR and 16 L Photoperiod.","Mortality was recorded 24 h and 48 h after treatment.","One and 2 replicates were set up per day respectively for A. fallacis and P. persimilis and only 11 treatments were evaluated for P. persimilis.","Treatments EC25% is an EC formulation with 25% essential oil as an active ingredient.","Seven concentrations of EC25% were prepared as follows.","The 1% concentration was prepared by mixing 0.4 ml of the formulation and 9.6 ml of tap water and successive dilutions were made from the stock solution.","The following commercially available insecticides were used at their recommended rates: Trounce® (20.2% of fatty acids and 0.2% pyrethrin) at the recommended concentration of 1%; the insect growth regulator Enstar® (s-kinoprene) at the concentration of 0.065%; and Avid® (abamectin 1.9% EC), at the concentrations of 0.0057% and 0.000855%.","A water treatment was used as a control for a total of twelve treatments with A. fallacies and 11 with P. persimilis where the Enstar treatment was dropped.","The test product EC25% was sprayed first starting from the lower to the higher concentrations.","Then the control treatment was applied followed the reference products Avid, Trounce and Enstar.","The spray apparatus was rinsed three times between treatments using successively ethanol 95%, acetone, hexane, distilled water.","Statistical Analysis Mortality percentages were transformed to logit or probit to determine which analysis gave a better fit as recommended by Robertson and Preisler (1992).","The analysis which presents the highest number of small individual Chi square (χ2) is chosen.","Probit mortality were regressed on 1+log10 (dose) for EC25%.","Concentration mortality regression lines were determined to estimate the lethal concentration to kill 50% of the predator population using the POLO-PC program (LeOra, 1987).","Toxicity values of LC50, LC90 and LC99 are given as percent (%) of active ingredient.","Data were transformed to arcsine before analysis of variance.","Comparison between treatments were analysed using GLM procedure and means were separated by the Fisher test at 5% probability (SAS, 1996).","Results Amblyseius fallacis A total of 667 adult females of Amblyseius fallacis was tested and only 12 females (1.79%) walked out of the leaf disc area; number of missing was subtracted from initial total.","Mortality in the control was 5.56% at 24 h and remained unchanged at 48 h following treatment (FIG.","36).","There was a highly significant difference between treatments at 24 h (F=30.32, df=11, P<0.001) and at 48 h (F=31.64, df=11, P<0.001).","There was no mortality after 48 h was with EC25% at the concentration of 0.125% and 3,1% 7% and 23% mortality with EC25% at 0.25%, Enstar and EC25% at 0.5% and these results were not significantly different from the control.","Note that at the concentration of 0.5% the EC25% suggested commercial rate, mortality was 23.11% which is less than the 50% limit of the IOBC for harmless pesticides.","Amongst the commercially available products, Trounce caused the highest mortality (85.11%) after 48 H. This was followed by the Avid treatments at concentrations of 0.0057% (94.8% mortality) and 0.000855% (81.5% mortality) and results did not differ significantly, demonstrating that both products are equally toxic to Amblyseius fallacis.","LC50, LC90 and LC99 values at 48 h (FIG.","37) are well above (1.01%, 3.91% and 4.12% respectively) the 0.5% effective dose used to control the spider mite pest, Tetranychus urticae)(Chiasson, unpublished results).","These results indicate that EC25% might have low or no residual toxicity to Ambiyseius fallacis and most adult females that remained alive 24 hours after the EC25% treatments continued to reproduce and were observed laying eggs.","Phytoseiulus persimilis A cohort of 555 adult females was used to evaluate the toxicity of EC25% and the commercially available Trounce and Avid with the mite predator, Phytoseiulus persimilis.","In this bioassay, 7.35% and 13.17% of the total number of gravid females escaped from the leaf disc 24 h and 48 h respectively after treatments.","They contributed to 13.06% and 18.35% of the total mortality recorded at 24 h and 48 h respectively.","The highest number of predator escapees were observed in the control treatment and in the EC25% treatments at concentrations lower than 2%.","We will discuss only mortality calculated over total number treated minus missing individuals (3rd column of FIG.","38).","Highest mortality were caused by Trounce (99,71%) followed by Avid at the concentration of 0.0057% (93.69%).The lowest mortality was observed in the treatment with EC25% at the 0.125% concentration (13.43%).","Mortality with EC25% at 0.125%, 0.25 and 0.5% were not significantly different from the control treatment.","When missing females were deducted from the initial number of adults tested, the LC50 of P. persimilis was 1.2% and 0.8% at 24 h and 48 h after treatments respectively (FIG.","37).","B.","Direct Toxicity of the Essential Oil Extract on Aphid Endoparasitoids Aphidius colemani (Hymenoptera: Brachonidae, Aphidiinae) In the present study, adult Aphidius colemani wasps were exposed to a direct spray application of EC25% and remained in permanent contact with the biopesticide residues, which is considered worse case conditions, to test the potential side effects this biopesticide may have on beneficial hymenoptera such as Aphidius colemani Rearing of Aphidius colemani Aphidius colemani wasps were purchased from.Plant Product Quebec in lots of 250 mixed mummies and adults.","The emerged wasps and the remaining mummies were directly transferred to a 5 litre plastic bag filled with air and the wasps were provided with a 10% solution of sucrose and honey (w/w) as food source and water.","Direct Contact Bioassay Six to 14 adult parasitoids less than 48 h old were transferred into a large solo cup (500 ml ca.)","using a mouth aspirator.","The solo cup was lined with a filter paper (Rothmans #1) and had two large openings drilled on the side and one on the cover to provide ventilation and these openings were covered with a fine screen to prevent escape of adult wasps and condensation of the pesticide vapour.","The filter paper was humidified with a 10% solution of sucrose and honey.","The solo cup containing the wasps was weighed and the wasps were dragged down to the bottom of the solo cup by means of successive beats on the cover with a 15 cm long stick.","They were treated with 0.3 ml of the insecticide solution using a paintbrush sprayer (Vega 2000, Thayer & Chandler, Lake Bluff, Ill., USA) at 6 psi and set at 14.5 cm above the treated area.","The solo cup was then covered and re-weighed to determine weight of pesticide used.","The treated wasps were then incubated in a growth chamber set at 18° C.-22° C. and 60-65% HR.","Assessment of treatment effects were made at 24 h and 48 h following treatment.","Residual Bioassay Ten to 20 adult wasps including at least 5 females were picked up and put in a glass Petri dish and covered.","The cover had an opening covered with a screen to enable ventilation and to prevent condensation of the pesticide vapour.","The Petri dishes were previously treated with a pesticide solution exactly in the same manner as for direct toxicity bioassay but dishes were left to dry for an hour before covering and exposing the wasps to the pesticide residues.","On the cover, two small circular holes were drilled and used to provide the wasps with water and a solution of honey and sucrose.","Mortality was recorded at 24 h and 48 h. Treatments The test product EC25%, a 25% essential oil EC formulation obtained from Codena Inc.","Seven concentrations were prepared as follows: EC25% at 8% was prepared by mixing 3.2 ml of EC25% and 6.4 ml of tap water and successive dilutions of 4%, 2%, 1%, 0.5% and 0.125% were made from the stock solution.","Commercially available insecticides were used at their respective recommended doses as positive controls: Trounce® (20.2% of fatty acids, Safer Ltd, Scarborough, Ont.)","at the recommended concentration of 1%, the insect growth regulator Enstare (s-kinoprene) at the concentration of 0.065%; Avid® (abamectin 1.9% EC) at the concentrations of 0.0057% and 0.000855%, and Thiodan® (endosulfan 50 WP) at the concentration of 5%.","The test product EC25% was used first, starting from the lowest to the highest concentration and followed by the water control and finally by Avid, Trounce, Enstar and Thiodan.","The spray apparatus was rinsed three times between treatments using successively ethanol 95%, acetone, hexane, distilled water.","Statistical Analysis Concentration was analysed as main effect and the weight of pesticide applied was tested as a covariate to correct for difference in quantity of applied pesticide.","This covariate was deleted from the model when found not significant.","Mortality regression lines were determined to estimate the lethal concentration to kill 10%, 50% and 90% of the parasitoid population using the POLO-PC program (LeOra, 1987).","Toxicity values of LC50 are given as percent of active ingredient.","Data were transformed to arcsine before analysis of variance but actual means were presented.","Comparison between treatments were analysed using GLM procedure and means were separated by Fisher test at 5% probability (SAS, 1996).","Effect of Treatments on Aphidius colemani Emergence from Mummies Myzus persicae mummies parasitized by Aphidius colemani females on leaves of cabbage (cv.","Lennox) were used in this test.","Portions of leaves bearing mummies were cut and placed in a Petri dish.","The Petri dish was weighted and treated with a pesticide solution and immediately re-weighted to determine the amount of pesticide used.","The treated Petri dish was then covered and sealed with parafilm.","The cover of the Petri had a screened opening to enable ventilation and to prevent escape of emerging Aphidius adults.","The incubation period lasted 7 days and all mummies that did not emergence as adult wasps were considered dead.","Fecundity Assessment Females that survived the pesticide residual treatments were assessed for fecundity on wheat plants infested with aphids.","Myzus persicae aphids reared on cabbage plants (c.v. Lennox) were brushed onto a pot containing 25 to 30 plants of wheat 6 days old.","Soon after, the brushed aphids climbed the wheat plants and a density of at least 100 aphids per pot was required.","Female wasps that survived the 48 h residual treatments were removed individually from the test arena by means of an aspirator and confined over pots of aphid-infested plants using ventilated transparent plastic cylinders for a period of 24 h. The females were then removed and the plant bearing parasitized aphids were incubated for a period of 10 days at 18° C. to 22° C. At the end of the incubation period, the wheat plant was cut and put in a Petri dish.","The number of parasitized aphids were counted.","Results Direct Contact Bioassay A total of 1174 adult wasps including 657 or 55.9% female parasitoids were tested.in the bioassay.","The mean quantity of pesticide solutions applied was 4.58±1.36 mg/cm which was more than double the amount of 2.0±0.2 mg/cm recommended for the typical bioassay (Mead-Briggs et al., 2000).","Mortality with EC25% at concentrations up to 1% was not significantly different than for the water control after 24 h. though at 48 h, results with EC25% treatments at the 0.5% and 1% concentrations significantly different from the control (FIG.","39).","At the 0.5% concentration of EC25%, recommended for field application, mortality varied from 18.6% to 35.2% at 24 h and 48 H after treatments respectively.","Highest mortality was observed with the Avid treatments at concentrations of 0.0057% and 0.000855% and with the EC25% treatment at concentrations of 4% and 8%.","Results in FIG.","40 show that female wasps were relatively less sensitive to treatments than adult males.","LC50 values for EC25% on A. colemani females (FIG.","41) was equal to 1.28% which is more than twice the recommended concentration of 0.5% for field application.","The LC50 for A. colemani males was lower at 0.77% but still above the 0.5% field recommended concentration of EC25%.","However, the 95% confidence limits (CL 95%) of LD50% for both males and females were overlapping and therefore their LD50% were not differently significant (Robertson and Presisler, 1992).","Residual Assay Results shown in FIGS.","42 and 43.Effect of Treatments on Aphidius colemani Emergence from Treated Mummies FIG.","44 showed that the effects of treatments on emergence of Aphidius colemani adults from treated mummies were significant (F=6.94, dl=16, P<0.0001).","The emergence rate of A. colemani decreased steadily when EC25% concentration increased and there was no emergence at the concentration of 8%.","At the recommended concentration for field application, i.e.","0.5%, emergence was 86.4% and this result was not statistically different from that observed in the control.","In the reference products tested, the highest emergence was observed in the Avid treatment with 96.1% and the lowest was Enstar at 35% emergence.","Fecundity Assessment The results of FIG.","45 indicated that females that survived the treatment were able to parasitize Myzus persicae hosts and that their reproductive functions did not seem to be affected.","There was no enough surviving female to test for the EC25% concentration of 4% and 8%.","The lowest fecundity rate was observed in the treatment of Avid with 9.1 mummies per plants compared to 23.9 mummies per plant recorded in the control treatment.","The number of mummies produced from females treated with EC25% treatments at concentrations varying from 0.125 to 2% were not significantly different from the control.","C. Direct Toxicity of the Essential Oil Extract on Predatory Minute Bug Orius insidiosus Say Various Orius species including Orius insidiosus Say (Heteroptera: Anthocoridae) are effective biological control agents of western flower thrips (WFT) Frankliniella occidentallis Pergrande (Thysanoptera: Thripidae) in sweet pepper, cucumber and other vegetable and ornamental crops (Veire de van et al., 1996).","The present study was initiated to evaluate the side effects of EC25% on the predatory bug Orius insidiosus under laboratory conditions.","Culture of Orius insidiosus Orius insidiosus stock culture was initiated with individuals obtained from a commercial supplier (Plant Prod Quebec, 3370 Le Corbusier, Laval, Quebec) and maintained in a laboratory growth chamber.","Eggs of Ephestia spp were served as a food source and snaps beans of Phaseolus vulgaris as an oviposition substrate.","The beans containing eggs were then incubated in folded brown paper until emergence.","The folded paper was used to reduce cannibalism.","Emerging nymphs were then transferred into one litre jars containing bean pods and fed with Ephestia eggs until the adult stage.","The stock culture was renewed regularly.","Direct Contact Bioassay The bioassays were carried out in small Petri dishes (5.5 cm in dia.)","using a leaf disc method.","A thin layer of agar 2% (2-3 mm) was poured into each Petri dish and a ring of apple leaf (cv.","McIntosh, 3.5 cm in dia.)","was cut and placed upside down on the surface of the agar.","At least 10 Orius insidiosus 2nd nymph instar or adults were transferred carefully using an aspirator on the surface of the apple leaf disc.","The Petri dish containing the nymphs or the adults bugs were dragged down to the bottom of the Petri dish by means of successive beats on the cover with a 15 cm long stick.","The Petri dishes were weighted and immediately, they were treated immediately with 0.3 ml of pesticide solution at different concentrations using a paintbrush sprayer (Vega 2000, Thayer & chandler, Lake Bluff, Ill., USA) at 6 psi and set at 14.5 cm above the treated area.","The Petri dishes were then re-weighted to determine the quantity of pesticide applied.","The pesticide solutions were prepared on the day of treatment.","The treated nymphs or adults were then transferred carefully to the surface of the apple leaf disc containing eggs of Ephestia spp as a source of food.","To avoid contamination, a new camel brush is used for each concentration to transfer the treated nymphs or adults to the leaf discs.","The Petri dishes were put in a tray and incubated in a growth chamber set at 25° C., 65% HR and 16 L Photoperiod.","A fan was placed in front of the tray to provide continuous air flow.","Mortality of nymphs was recorded at 1, 2, 5, 7 and 9 days after treatment when more than 80% of the nymphs became adults.","Mortality of adult predators was recorded at 24 H and 48 H following treatment.","Ten replicates were prepared per treatment and 12 treatments were evaluated on second instar nymphs and adults.","Treatments The test product is EC25%, a 25% EC essential oil formulation obtained from Codena Inc.","Seven concentrations were prepared as follow: EC25% at 8% was prepared by mixing 3.2 ml of EC25% and 6.4 ml of tap water and successive dilutions of 4%, 2%, 1%, 0.5% and 0.125% were made from the stock solution.","EC25% was compared to the recommended doses of the following commercially available insecticides: Trounce® (20.2% potassium salts of fatty acids and 0.2% pyrethrins) at the recommended concentration of 1%; the insect growth regulator Enstar® (S-kinoprene), at the recommended concentration of 0.065% and Avide® (abamectin 1.9% EC) at the concentration of 0.000855%, Thiodane® (endosulfan 50 WP) at the concentration of 5% and Cygon® (dimethoate) at the concentration of 4%.","Water was used as a negative control.","The test product EC25% was sprayed first, starting from the lowest to the highest concentration followed by the water control treatment and finally by the reference products Avid, Cygon, Enstar, Thiodan and Trounce.","The sprayer was rinsed three times between treatments using successively ethanol 95%, acetone, hexane and distilled water.","Fecundity Assessment The potential sublethal effects of EC25% on Orius insidiosus female fecundity was monitored.","Fecundity assessment was carried out on females that survived 48 h after the direct contact pesticide treatments.","Surviving females were separated from males and put individually in a Petri dish filled with a 2 mm layer of agar used as a support and an apple ring (5.5 cm) placed upside down on the agar surface along with a 3 cm long pod of faba bean (Phaseolus vulgare).","The apple leaf disc and the bean pod were used as oviposition substrates.","The Petri dish was covered with the correspondent cover and sealed with parafilm.","The Petri cover had an opening covered with fine muslin tissue for ventilation and air exchange.","Females were left undisturbed for 48 H for oviposion and then were fed with sufficient numbers of Ephestia spp eggs.","After the 48 h period, females were then transferred to another Petri dish for a second 48 H oviposition test.","During both periods, the eggs laid were counted and left to hatch for 5 days.","The eggs that do not hatch after 5 days were considered dead and not viable.","Statistical Analysis LC50 values of EC25% were determined using probit analysis with POLO software (LeOra, 1987).","Concentrations were analysed as main effects and the weight of pesticide applied was tested as a covariance to correct for difference in quantity of the applied pesticide.","This covariance was deleted from the model when found not significant.","Mortalities were analysed using General Linear model (GLM) procedure within SAS (SAS, 1996) and the number of individuals initially introduced were tested as a covariant.","Means were adjusted for covariance when appropriate and separated using the Fisher test for means comparison.","However, actual means were presented in the results section.","Results Results show (FIG.","46) that nine days following treatment application, with Orius nymphs, the most toxic treatments were in decreasing order, Trounce (99,5% mortality), Cygon (98% mortality), EC25% at 8% concentration (87.6% mortality), Avid (82.5% mortality) and EC25% at 4% concentration (79.6% mortality).","All results were significantly different from that of the control treatment (3.6% mortality).","Less than 50% mortality was obtained with the other treatments though only Thiodan (45.7%) and EC25% (35.1%) results were significantly different from the control.","Results with EC25% at the recommended concentration for field application of 0.5% were not significantly different from results obtained with the control.","Results show (FIG.","47) that the effects of the 12 treatments expressed as percent mortality of adults was significantly different at 24 H after treatment (F=55.9, df=11, p<0.0001) and at 48 H after treatment (F=63.2, df=11, p<0.0001).","The least toxic treatments of EC25% at concentration of 0.125% and 0.25% were not statistically different from the control treatment.","The treatment of EC25% at the recommended field concentration of 0.5% was the least toxic of the remaining treatments causing a mortality of 28%.","The most toxic group included Cygon (100% mortality), Trounce (98.9% mortality), EC25% at concentrations of 4 and 8% (94% and 94% respectively) and Avid (87.8%).","Fecundity Assessment The results from the fedundity assessment assay (FIG.","48) showed that almost all females tested had laid eggs.","There were few surviving females to test for fecundity following treatments with Avid, Cygon, Trounce and EC25% at concentrations of 2, 4 and 8%.","The mean number of eggs laid per female per day in the control treatment was 7.6 which was almost 4 times the minimum number of 2 eggs per female per day set by the IOBC standards for fecundity for Orius leavigatus, a closely related species of O. insidiosus.","The lowest rate was 2.8 eggs per female per day obtained in the treatment with Thiodan followed by EC25% at 0.5% concentration with 3.6 eggs per female per day and both were significantly different from the rate obtained with the water control (7.6 eggs per female per day).","The date rate of eggs laid in the EC25% treatments at concentrations of 0.25% (5 eggs) and 0.5% (5.4 eggs) were not significantly different from the number of eggs laid in the control.","The eclosion rate varied from 28.5% in the Thiodan treatment to 53% in the control.","There was 33.9% egg eclosion in the EC25% at 0.5% concentration treatment.","LC50 values for Orius nymphs were 2.65%, 9 days following treatment with EC25% (FIG.","49) and for adults were 1.14%, 2 days following treatment with EC25% (FIG.","50).","References: Ascher, K. R. S. and Cwilich, R. (1960) Laboratory Evaluation of Acaricides Against Tetranychus Telarius L. on Sugar Beet and on Beans.","Israel J Agric.","Res.","10:159-163.ASTM.","1987.Standard test methods for moisture, ash, and organic matter of peat and other organic soils.","American Standards for Testing and Materials (ASTM) D 2974-87.ASTM.","1989.Standard test method for pH of soils.","American Standards for Testing and Materials (ASTM) D 4972-89.Bélanger et al., (1991) Extraction et Détermination de Composés Volatils de L'ail (Allium sativum), Riv.","Ital.","EPPOS 2:455-461.Busvine, J. R. (1980) Recommended Methods for Measurement of Pest Resistance to Pesticides.","FAO Plant Production and Protection Paper.","pp.","49-53.Ceske, L. J. and P. B. Kaufman, (1999) Regulation of Metabolite Synthesis in Plants.","In: Natural Products from Plants.","Kaufman et al., eds.","CRC Press.","Boca Raton.","pp.","91-121.Chialva et al., (1983) Chemotaxonomy of Wormwood (Artemisia Absinthum L.).","Z. Lebensm Unters Forsch 176:363-366.Dittrich, V. (1962) A Comparative Study of Toxicological Test Methods on a Population of the Two-spotted Spider Mite (Tetranychus telarius).","J. Econ.","Entomol.","55:644-648.Duerbeck, K., et al., (1997) The Distillation of Essential Oils.","Manufacturing and Plant Constuction Handbook.","Protrade: Dept.","of Foodstuffs and Agricultural Products.","Eschborn, Germany.","pp.","21-25.Ebeling, W. and R. J. Pence.","(1953) Pesticide Formulation: Influence of Formulation on Effectiveness.","J. Agr.","Food Chem.","1:386-397.Feng, R. and M. B. Isman.","(1995) Selection for resistance to azadirachtin in the green peach aphid, Myzus persicae, Experientia 51:831-833.Flint, M. L. (1990) Pests of the Garden and Small Farm.","A Grower's Guide to Using Less Pesticide.","Statewide IPM Project.","U. of California.","Publication 3332.pp.","116-123.Foott, W. H. and H. R. Boyce.","(1966) A Modification of the Leaf-Disc Technique for Acaricide Tests.","Proc.","Entomol.","Soc.","Ont.","96:117-119.Georghiou, G. P. (1990) Overview of Insecticide Resistance.","In: Managing Resistance to Agrochemicals.","M. E. Green, H. M. Le Baron and W. K. Moberg, eds., ACS Symposium Series.","Chapter 2.Vol.","421:18-41.Gundersin et al., (1985).","Effects of the Mint Monoterpene Pulegone on Spodoptera Eridania (Lepidoptera: Noctuidae) Environ.","Entomol.","14:859-863.Isman, M. (1999) Pesticides Based on Plant Essential Oils.","Pesticide Outlook.","pp.","68-72.Jackson, S. A. L. and R. K. M. Hay.","(1994) Characteristics of varieties of thyme (Thymus vulgaris L.) for use in the UK: Oil content, composition and related characters.","J. Hort.","Sci.","69:275-281.LeOra Software.","POLO-PC, (1994) Probit or Logit Analysis.","LeOra Software, Berkeley Calif. pp.","1-28 Lippold, P. (1963) Acaricidal Testing Techniques in the Two-spotted Spider Mite.","Adv.","Acarol.","1:174-180.Mead-Briggs, et al., (2000) A laboratory test for evaluating the effects of plant protection products on the parasitic wasp, Aphidius rhopalosiphi (Destephani-Perez)(Hymenoptera: Braconidae).","Pages 13-25.In.","Guidelines to evaluate side effects of plants protection products to non target arthropods, IOBC, BART and EPPO Joint Initiative.","Candolfi, M. P., Blumel, S., Forster, R. Bakker, F. M., Grimm, C., Hassan, S. A., Heimbach, U., Mead-Briggs, M. A., Reber, B, Schmuck, R., Vogt, H.","(Edts.","), Reinham, Germany.","Meister, R. T. ed.","(1999) Farm Chemicals Handbook.","Vol.","85.Meister Publishing Company, Willoughby, Ohio.","Perez- Souto, N. et al., (1992) Use of high-performance liquid chromatographic peak deconvolution and peak labeling to identify antiparasitic components in plant extracts.","J. of Chrom.","593:209-215.Rembald, H. (1989) Azadirachtins.","Their Structure and Mode of Action.","In: Insecticides of Plant Origin.","ACS Symposium Series No.","387.pp.","150-163.Roush, R. T. and J.","A. McKenzie.","(1987) Ecological Genetics of Insecticide and Acaricide Resistance.","Ann.","Rev.","Entomol.","32:361-380 The McGraw-Hill Dictionary of Chemical Terms (ed.","Parker, S., 1985), McGraw-Hill, Sam Francisco.","U.S. Pat.","No.","4,933,371 U.S. Pat.","No.","5,839,224"]],"string":"[\n [\n \"Extracts derived from chenopodium plants and uses thereof\",\n \"The present invention relates to pesticides.\",\n \"More particularly, the present invention relates to botanical pesticides.\",\n \"In particular, the present invention relates to compositions and methods for controlling plant-infesting pests with plant extracts and notably with compositions comprising oil extracts derived from Chenopodium sp.\",\n \"plant material.\",\n \"The invention further relates to compositions comprising such extracts as pesticidal compositions and providing the advantages of minimal development of resistance thereto, minimal toxicity to mammals, minimal residual activity and environmental compatibility.\",\n \"The pesticidal compositions of the present invention comprises α-terpinene, ρ-cymene, limonene, carvacrol, carveol, nerol, thymol, and carvone.\"\n ],\n [\n \"1.An essential oil extract derived from a Chenopodium species, comprising: from at least 30% to at least 65% α-terpinene, from at least 8% to at least 26% ρ-cymene, from at least 5% to at least 24% limonene, from at least 0.04% to at least 1% carvacrol, from at least 0.1% to at least 2% carveol, from at least 0.1% to at least 2.0% nerol, from at least 0.04% to at least 2% thymol, and from at least 0.04% to at least 1% carvone, and wherein said extract is active against one or more pests chosen from insects, acari, and fungi.\",\n \"2.\",\n \"(Canceled) 3.\",\n \"(Canceled) 4.\",\n \"(Canceled) 5.The essential oil extract according to claim 1, wherein said species is Chenopodium ambrosioides.\",\n \"6.A pesticidal composition, comprising an effective amount of the essential oil extract of claim 1 and a suitable emulsifier, spreader or sticking agent, and carrier.\",\n \"7.The pesticidal composition according to claim 6, comprising between 0.125% to 10% (by volume) essential oil extract.\",\n \"8.The pesticidal composition according to claim 7, comprising between 0.25% to 5% (by volume) of said essential oil extract.\",\n \"9.The pesticidal composition according to claim 6, wherein said composition comprises between 5% to 50% (by volume) of said essential oil extract.\",\n \"10.The pesticidal composition according to claim 9, comprising between 10% to 25% (by volume) of said essential oil extract.\",\n \"11.The pesticidal composition according to claim 10, comprising between 1% to 15% (by volume) of a suitable emulsifier, and between 50% to 70% (by volume) of a suitable carrier or solvent.\",\n \"12.The pesticidal composition according to claim 11, comprising between 2% to 20% (by volume) of a suitable spreader or sticking agent.\",\n \"13.A pesticidal composition, comprising between 30% to 50% (by volume) of the essential oil extract of claim 1, between 0.5% to 25% (by volume) of a suitable emulsifier, and between 10% to 50% (by volume) water.\",\n \"14.A method for controlling one or more pests chosen from phytophagous acari, phytophagous insects, and phytophagous fungi, which comprises applying to a locus where control is desired a pesticidally-effective amount of the pesticidal composition of claim 6.15.\",\n \"(Canceled) 16.\",\n \"(Canceled) 17.The method according to claim 14, wherein said locus is soil.\",\n \"18.The method according to claim 14, wherein said locus is a plant.\",\n \"19.The essential oil extract according to claim 1, further comprising ascaridole at a proportion of 9.86% or less.\",\n \"20.The essential oil extract according to claim 1, further comprising ascaridole at a proportion of 1% or less.\",\n \"21.The essential oil extract according to claim 20, wherein said extract has trace or undetectable amounts of ascaridole.\",\n \"22.A formulation for indoor or outdoor fumigation, comprising: between 1% to 15% (by volume) of the essential oil extract of claim 1.23.A formulation for topical application, comprising: between 1% to 10% (by volume) of the essential oil extract of claim 1.24.The formulation of claim 23, wherein the formulation further comprises between 2% to 5% (by volume) of an emollient, between 0.1% to 1% (by volume) of a stabilizer and between 0.1% to 0.5% (by volume) of a preservative.\",\n \"25.The formulation of claim 23, wherein the formulation further comprises between 1% to 10% (by volume) of an emulsifier, between 2% to 5% (by volume) of a coat or skin conditioner, between 0.1% to 1% (by volume) of a stabilizer, between 0.1% to 1% (by volume) of a preservative and between 0.1% to 0.5% (by volume) of an antioxidant.\",\n \"26.A microemulsion formulation, comprising: between 30% to 50% (by volume) of the essential oil extract of claim 1, between 0.5% to 25% (by volume) of a suitable emulsifier, and between 10% to 50% (by volume) water.\",\n \"27.The formulation of claim 26, wherein said formulation is for soil delivery.\",\n \"28.A method for controlling phytophagous pests, which comprises applying to a locus where control is desired a pesticidally-effective amount of the formulation according to any one of claims 22, 23, or 26.\"\n ],\n [\n \"<SOH> BACKGROUND OF THE INVENTION <EOH>Plant feeding mites are among the most voracious phytophagous pests of crops.\",\n \"To combat these pests, synthetic pesticides have been developed.\",\n \"These synthetic chemical pesticides, however, often have detrimental environmental effects that are harmful to humans and other animals and therefore do not meet the guidelines developed by most Integrated Pest Management programs.\",\n \"Moreover, resistance to these products has been found to develop with many of the new products put on the market (Georghiou, 1990; Nauen et al., 2001).\",\n \"Although resistance follows a highly complex genetic and biochemical process, it can generally develop rapidly with synthetic products because their active ingredients rely on one or more molecules of the same class.\",\n \"The organism can therefore respond to the toxin by developing physiological, behavioral or morphological defense mechanisms to neutralize the effect of the molecule (Roush and MacKenzie, 1987).\",\n \"Acari, such as spider mites, in particular, are extremely difficult to control with pesticides.\",\n \"Tetranychus urticae (the two-spotted spider mite), for example, has accumulated a considerable number of genes conferring resistance to all major classes of acaricides.\",\n \"Resistance to many registered acaricides have been reported, for example, resistance has been reported to hexythiazox, abamectin, and clofentezine.\",\n \"Furthermore, many of these pesticides have been found to exacerbate pest infestation by destroying the natural predators of mites (U.S. Pat.\",\n \"No.\",\n \"5,839,224).\",\n \"Additionally, many synthetic insecticides have been found to stimulate mite reproduction.\",\n \"For example, it was found that mites reproduce many times faster when exposed to carbaryl, methyl parathion, or dimethoate in the laboratory than untreated populations (Flint, 1990).\",\n \"As a result, there are very few pesticides remaining that are effective against spider mites (Georghiou, 1990).\",\n \"In the Farm Chemical Handbook (Meister, 1999), for example, only 48 products out of a total of 2,050 listed acaricides and insecticides (or 2.4%), were identified as acaricides and only 69 of these products (or 3.4%) were identified as both acaricides and insecticides.\",\n \"The control of insect infestation has also proven to be difficult.\",\n \"For example, European chafer Rhizotrogus majalis Razoumowsky (Coleoptera: Scarabaeidae) insects are an exotic species to Canada and the United States and are commonly known as Scarab beetles.\",\n \"Throughout the northeast, where the beetle has become established, R. majalis causes major damage to turfgrass, consuming the roots of home lawns as well as urban recreational areas and golf courses.\",\n \"The larvae of this species are extremely difficult and expensive to control using conventional insecticides.\",\n \"In several areas of the U.S. insecticide resistance has already developed within populations.\",\n \"The control of plant fungi is another source for growing concern.\",\n \"Phytopathogenic fungi are of great economic importance since fungal growth on plants or on parts of plants inhibits production of root, stem, foliage, fruit or seed, and the overall quality of a cultivated crop.\",\n \"About 25 percent of all fungal diseases in agriculture and horticulture are caused by powdery mildew phytopathogens (U.S. Pat.\",\n \"Nos.\",\n \"5,882,689 and 5,496,568).\",\n \"In cucumber crops, for example, powdery mildew caused by the plant pathogens, Erysiphe cichoracearum and Sphaerotheca fuliginea , is one of the most problematic disease.\",\n \"The fungus responsible for Gray Mold, Botrytis cinerea, can attack more than 200 species of cultivated plants and especially those growing in a greenhouse environment.\",\n \"It is a saprophyte that attacks dead or senescent plant tissue.\",\n \"B. cinerea is especially damaging to stems (causing stem rot), when it enters scars left by pruning of lower leaves.\",\n \"Following attack, the plant dies, causing heavy economic losses to the grower.\",\n \"Control of stem rot is attempted by treating leaf scars after leaf pruning.\",\n \"Although benomyl is presently used, it will likely be removed from the market shortly.\",\n \"Moreover, iprodione (another pesticide), is less effective because of developing fungal resistance.\",\n \"In spite of their strong fungicidal effect, agricultural fungicides currently in use have been a source of problems, because the required amount is ever increasing as tolerance of target plant pathogens increases.\",\n \"Moreover, many fungicides are synthetic agents, which when used, pose possible drawbacks for humans, animals and the environment.\",\n \"Thus, effective commercial products (to combat fungal attacks on crops), which reduce the dose of synthetic chemicals spread into the environment, are in need; keeping in mind a regard for environmental and human safety issues.\",\n \"As an alternative to synthetic agents, botanical pesticides offer the advantage of being naturally derived compounds that are safe to both humans and the environment.\",\n \"Specifically, botanical pesticides offer such advantages as being inherently less toxic than conventional pesticides, generally affecting only the target pest and closely related organisms, and are often effective in very small quantities.\",\n \"In addition, botanical pesticides often decompose quickly and, therefore, are ideal for use as a component of Integrated Pest Management (IPM) programs.\",\n \"There are few published reports of the acaricidal properties of botanical pesticides.\",\n \"For example, U.S. Pat.\",\n \"No.\",\n \"4,933,371 describes the use of saponins extracted from various plants (i.e., yucca, quillaja, agave, tobacco and licorice) as acaricides.\",\n \"This patent also describes the use of linalool extracted from the oil of various plants such as Ceylon's cinnamon, sassafras, orange flower, bergamot, Artemisia balchanorum, ylang ylang, rosewood and other oil extracts as acaricides.\",\n \"These methods, however, require the extraction of one active substance from the plant which often does not meet desired levels of toxicity towards acari.\",\n \"Plant essential oils are a complex mixture of compounds of which many can be biologically active against insect and mite pests, the compounds acting individually or in synergy with each other, to either repel or kill the pests by contact.\",\n \"These components are plant secondary metabolites or allelochemicals produced by plants as a defense mechanism against plant feeding pests (Ceske and Kaufman, 1999).\",\n \"Because of the complexity of the mixture, it has been observed that pests do not easily develop resistance to these products as they can to synthetic pesticides or botanical pesticides comprising a single active compound.\",\n \"In this respect, Feng and Isman (1995) demonstrated that repeated treatments of pure azadirachtin, a major active constituent of neem oil, against the green peach aphid led to a 9-fold resistance after 40 generations.\",\n \"However, repeated exposure during 40 generations to crude neem extracts did not lead to resistance.\",\n \"There remains a need to provide new and effective pesticidal products which overcome the shortcomings of products known in the art.\",\n \"For example, there remains a need for broad spectrum compositions which are less likely to enable pests to develop resistance thereto.\",\n \"There also remains a need to provide a method to combat pests at any given locus, using a composition which is not toxic to animals, especially to mammals, nor to any beneficial predator/parasitoid insects.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>The invention provides essential oil extracts derived from Chenopodium sp.\",\n \"comprising, α-terpinene, ρ-cymene, limonene, carvacrol, carveol, nerol, thymol, and carvone, wherein these extracts have one or more activities selected from the group of acaricidal, pesticidal, insecticidal, and fungicidal activities.\",\n \"One or more of these extracts can be formulated into compositions that enable their application into different environments such as plants, soil, animals and buildings.\",\n \"If accordance with another aspect of the invention there is provided composition comprising one or more Chenopodium sp.\",\n \"-derived essential oil extracts, wherein said extract comprises α-terpinene, ρ-cymene, limonene, carvacrol, carveol, nerol, thymol, and carvone, in combination with an emulsifier, carrier, spreader and/or sticking agent, to enable application of the composition to a specific environment, wherein the composition has one or more activities selected from the group of acaricidal, pesticidal, insecticidal, and fungicidal activities.\"\n ],\n [\n \"FIELD OF INVENTION The present invention relates to the field of pesticides for controlling plant-infesting pests.\",\n \"BACKGROUND OF THE INVENTION Plant feeding mites are among the most voracious phytophagous pests of crops.\",\n \"To combat these pests, synthetic pesticides have been developed.\",\n \"These synthetic chemical pesticides, however, often have detrimental environmental effects that are harmful to humans and other animals and therefore do not meet the guidelines developed by most Integrated Pest Management programs.\",\n \"Moreover, resistance to these products has been found to develop with many of the new products put on the market (Georghiou, 1990; Nauen et al., 2001).\",\n \"Although resistance follows a highly complex genetic and biochemical process, it can generally develop rapidly with synthetic products because their active ingredients rely on one or more molecules of the same class.\",\n \"The organism can therefore respond to the toxin by developing physiological, behavioral or morphological defense mechanisms to neutralize the effect of the molecule (Roush and MacKenzie, 1987).\",\n \"Acari, such as spider mites, in particular, are extremely difficult to control with pesticides.\",\n \"Tetranychus urticae (the two-spotted spider mite), for example, has accumulated a considerable number of genes conferring resistance to all major classes of acaricides.\",\n \"Resistance to many registered acaricides have been reported, for example, resistance has been reported to hexythiazox, abamectin, and clofentezine.\",\n \"Furthermore, many of these pesticides have been found to exacerbate pest infestation by destroying the natural predators of mites (U.S. Pat.\",\n \"No.\",\n \"5,839,224).\",\n \"Additionally, many synthetic insecticides have been found to stimulate mite reproduction.\",\n \"For example, it was found that mites reproduce many times faster when exposed to carbaryl, methyl parathion, or dimethoate in the laboratory than untreated populations (Flint, 1990).\",\n \"As a result, there are very few pesticides remaining that are effective against spider mites (Georghiou, 1990).\",\n \"In the Farm Chemical Handbook (Meister, 1999), for example, only 48 products out of a total of 2,050 listed acaricides and insecticides (or 2.4%), were identified as acaricides and only 69 of these products (or 3.4%) were identified as both acaricides and insecticides.\",\n \"The control of insect infestation has also proven to be difficult.\",\n \"For example, European chafer Rhizotrogus majalis Razoumowsky (Coleoptera: Scarabaeidae) insects are an exotic species to Canada and the United States and are commonly known as Scarab beetles.\",\n \"Throughout the northeast, where the beetle has become established, R. majalis causes major damage to turfgrass, consuming the roots of home lawns as well as urban recreational areas and golf courses.\",\n \"The larvae of this species are extremely difficult and expensive to control using conventional insecticides.\",\n \"In several areas of the U.S. insecticide resistance has already developed within populations.\",\n \"The control of plant fungi is another source for growing concern.\",\n \"Phytopathogenic fungi are of great economic importance since fungal growth on plants or on parts of plants inhibits production of root, stem, foliage, fruit or seed, and the overall quality of a cultivated crop.\",\n \"About 25 percent of all fungal diseases in agriculture and horticulture are caused by powdery mildew phytopathogens (U.S. Pat.\",\n \"Nos.\",\n \"5,882,689 and 5,496,568).\",\n \"In cucumber crops, for example, powdery mildew caused by the plant pathogens, Erysiphe cichoracearum and Sphaerotheca fuliginea, is one of the most problematic disease.\",\n \"The fungus responsible for Gray Mold, Botrytis cinerea, can attack more than 200 species of cultivated plants and especially those growing in a greenhouse environment.\",\n \"It is a saprophyte that attacks dead or senescent plant tissue.\",\n \"B. cinerea is especially damaging to stems (causing stem rot), when it enters scars left by pruning of lower leaves.\",\n \"Following attack, the plant dies, causing heavy economic losses to the grower.\",\n \"Control of stem rot is attempted by treating leaf scars after leaf pruning.\",\n \"Although benomyl is presently used, it will likely be removed from the market shortly.\",\n \"Moreover, iprodione (another pesticide), is less effective because of developing fungal resistance.\",\n \"In spite of their strong fungicidal effect, agricultural fungicides currently in use have been a source of problems, because the required amount is ever increasing as tolerance of target plant pathogens increases.\",\n \"Moreover, many fungicides are synthetic agents, which when used, pose possible drawbacks for humans, animals and the environment.\",\n \"Thus, effective commercial products (to combat fungal attacks on crops), which reduce the dose of synthetic chemicals spread into the environment, are in need; keeping in mind a regard for environmental and human safety issues.\",\n \"As an alternative to synthetic agents, botanical pesticides offer the advantage of being naturally derived compounds that are safe to both humans and the environment.\",\n \"Specifically, botanical pesticides offer such advantages as being inherently less toxic than conventional pesticides, generally affecting only the target pest and closely related organisms, and are often effective in very small quantities.\",\n \"In addition, botanical pesticides often decompose quickly and, therefore, are ideal for use as a component of Integrated Pest Management (IPM) programs.\",\n \"There are few published reports of the acaricidal properties of botanical pesticides.\",\n \"For example, U.S. Pat.\",\n \"No.\",\n \"4,933,371 describes the use of saponins extracted from various plants (i.e., yucca, quillaja, agave, tobacco and licorice) as acaricides.\",\n \"This patent also describes the use of linalool extracted from the oil of various plants such as Ceylon's cinnamon, sassafras, orange flower, bergamot, Artemisia balchanorum, ylang ylang, rosewood and other oil extracts as acaricides.\",\n \"These methods, however, require the extraction of one active substance from the plant which often does not meet desired levels of toxicity towards acari.\",\n \"Plant essential oils are a complex mixture of compounds of which many can be biologically active against insect and mite pests, the compounds acting individually or in synergy with each other, to either repel or kill the pests by contact.\",\n \"These components are plant secondary metabolites or allelochemicals produced by plants as a defense mechanism against plant feeding pests (Ceske and Kaufman, 1999).\",\n \"Because of the complexity of the mixture, it has been observed that pests do not easily develop resistance to these products as they can to synthetic pesticides or botanical pesticides comprising a single active compound.\",\n \"In this respect, Feng and Isman (1995) demonstrated that repeated treatments of pure azadirachtin, a major active constituent of neem oil, against the green peach aphid led to a 9-fold resistance after 40 generations.\",\n \"However, repeated exposure during 40 generations to crude neem extracts did not lead to resistance.\",\n \"There remains a need to provide new and effective pesticidal products which overcome the shortcomings of products known in the art.\",\n \"For example, there remains a need for broad spectrum compositions which are less likely to enable pests to develop resistance thereto.\",\n \"There also remains a need to provide a method to combat pests at any given locus, using a composition which is not toxic to animals, especially to mammals, nor to any beneficial predator/parasitoid insects.\",\n \"SUMMARY OF THE INVENTION The invention provides essential oil extracts derived from Chenopodium sp.\",\n \"comprising, α-terpinene, ρ-cymene, limonene, carvacrol, carveol, nerol, thymol, and carvone, wherein these extracts have one or more activities selected from the group of acaricidal, pesticidal, insecticidal, and fungicidal activities.\",\n \"One or more of these extracts can be formulated into compositions that enable their application into different environments such as plants, soil, animals and buildings.\",\n \"If accordance with another aspect of the invention there is provided composition comprising one or more Chenopodium sp.\",\n \"-derived essential oil extracts, wherein said extract comprises α-terpinene, ρ-cymene, limonene, carvacrol, carveol, nerol, thymol, and carvone, in combination with an emulsifier, carrier, spreader and/or sticking agent, to enable application of the composition to a specific environment, wherein the composition has one or more activities selected from the group of acaricidal, pesticidal, insecticidal, and fungicidal activities.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS FIG.\",\n \"1 shows the chemical content of three lots or pools of oil samples extracted from whole plant parts above root (00MC-21P, 00MC-24P and 00M-29P).\",\n \"FIG.\",\n \"2 shows the average mortality (%) of the two-spotted spider mite (TSSM: Tetranychus urticae) when tested with solutions of individual compounds present in the essential oil of Chenopodium ambrosioides.\",\n \"Results adjusted for control mortality with Abbott's formula.\",\n \"FIG.\",\n \"3 shows the average mortality (%) of the greenhouse whitefly (GWF; Trialeurodes vaporaiorum) when tested with solutions of individual compounds present in the essential oil of Chenopodium ambrosioides.\",\n \"Results adjusted for control mortality with Abbott's formula.\",\n \"FIG.\",\n \"4 shows adult spider mite (Tetranychus urticae) mortality obtained with bioassays using the RTU formulation of Chenopodium ambrosioides and commercial preparations of natural and synthetic insecticides.\",\n \"FIG.\",\n \"5 shows spider mite egg (Tetranychus urticae) mortality, using the RTU formulation of Chenopodium ambrosioides oil.\",\n \"FIG.\",\n \"6 shows spider mite nymph (Tetranychus urticae) mortality, using the RTU Chenopodium extract formulation and commercial preparations of synthetic and natural products.\",\n \"FIG.\",\n \"7 shows the mortality of adult spider mites 48 h following introduction on faba bean leaves treated one hour previously with the RTU formulation and selected natural acaricides.\",\n \"FIG.\",\n \"8 shows red mite, Panonychus ulmi mortality, using the RTU formulation.\",\n \"FIG.\",\n \"9 shows insect mortality (%) obtained with bioassays using the RTU formulation of Chenopodium ambrosioides.\",\n \"FIG.\",\n \"10 shows mortality of adult female twospotted spider mites 48 hours following applications.\",\n \"FIG.\",\n \"11 shows mortality of adult female European red mite 24 hours following applications.\",\n \"FIG.\",\n \"12 shows egg hatch (%) of the twospotted spider mite, 10 days following applications.\",\n \"FIG.\",\n \"13 shows egg hatch (%) of European red mite 10 days following applications.\",\n \"FIG.\",\n \"14 shows mortality of adult female two-spotted spider mites 48 hours following introduction on leaf discs treated with EC25% and Dicofol one hour previously.\",\n \"FIG.\",\n \"15 shows mortality of green peach aphids (Myzus persicae (Sulz.))\",\n \"48 hours following application of 0.125, 0.25, 0.5, 1.0 and 2.0% concentrations of formulation EC25% and the commercially available bioinsecticides Neem Rose Defense® and Safer's Trounce® FIG.\",\n \"16 shows lethal concentrations (LC50 and LC90) in % of EC25% for the green peach aphid (Myzus persicae (Sulz.))\",\n \"calculated with 48 hour mortality data.\",\n \"FIG.\",\n \"17 shows average number of green peach aphids (Myzus persicae (Sulz.))\",\n \"per cm2 of treated Verbena speciosa shoot following application of 0.25, 0.50 and 1.0% concentrations of EC25% and the commercially available bioinsecticides Neem Rose Defense® and Safer's Trounce® FIG.\",\n \"18 shows mortality of Western flower thrips (Frankliniella occidentalis (Perg.))\",\n \"24 hours following application of six concentrations (0.05, 0.125, 0.18, 0.25, 0.5 and 1.0%) of formulation EC25% and the commercially available bioinsecticides Neem Rose Defense® and Safer's Trounce® FIG.\",\n \"19 shows lethal concentrations (LC50 and LC90) in mg/cm2 of EC25% for the Western flower thrips (Frankliniella occidentalis (Perg.))\",\n \"calculated with 24 hour mortality data.\",\n \"FIG.\",\n \"20 shows average number of Western flower thrips/cm2 (WFT: Frankliniella occidentalis (Perg.))\",\n \"per treatment as a percentage of thrips present on leaves treated with the control during a greenhouse bioassay using two concentrations (0.25 and 1.0%) of EC25% and two commercially available bioinsecticides Neem Rose Defense® and Safer's Trounce® FIG.\",\n \"21 shows mortality of greenhouse whiteflies (Trialeurodes vaporariorum (Westw.))\",\n \"20 hours following application of five concentrations (0.0625, 0.125, 0.25, 0.5 and 1%) of formulation EC25% and the commercially available insecticides Neem Rose Defense®, Safer's Trounce® and Thiodan® FIG.\",\n \"22 shows lethal concentrations (LC50 and LC90) in mg/cm2 of EC25% for the greenhouse whitefly (Trialeurodes vaporariorum (Westw.))\",\n \"calculated with 20 hour mortality data.\",\n \"FIG.\",\n \"23 shows mortality of Encarsia formosa 24 hours following application of four concentrations (0.0625, 0.125, 0.25, 0.5 and 1.0%) of formulation EC25% and the commercially available bioinsecticides, Neem Rose Defense® and Safer's Trounce® FIG.\",\n \"24 shows the effect of three concentrations of EC25% with the tomato canker caused by the fungus Botrytis cinerea.\",\n \"FIG.\",\n \"25 shows the percentage control of the tomato canker following treatment with a selection of products 1 and 8 days following inoculation with Botrytis cinerea.\",\n \"FIG.\",\n \"26 shows the percentage (%) infection among blocks with powdery mildew.\",\n \"FIG.\",\n \"27 shows the effect of EC25% and reference products on Powdery Mildew.\",\n \"FIG.\",\n \"28 shows daily mean and total emergence of adult thrips from soil treated with different volumes of EC25% at 0.5% concentration.\",\n \"FIG.\",\n \"29 shows the percent survival of R. majalis 7 days after treatment with EC25%.\",\n \"FIG.\",\n \"30 shows EC25% median lethal concentrations (LC50), standard error of log LC50, 95 percent confidence interval and slope/intercept equation for two 7 day trials with R. majalis.\",\n \"FIG.\",\n \"31 shows average soil percent organic matter and pH±standard error determined for each tray in both R. majalis 7 day trial.\",\n \"FIG.\",\n \"32 shows treatments tested with the European chafer, Rhizotrogus majalis.\",\n \"FIG.\",\n \"33 shows counts for live European chafers on turfgrass plots.\",\n \"FIG.\",\n \"34 shows treatments evaluated with the HCB on turfgrass plots.\",\n \"FIG.\",\n \"35 shows counts for live HCB on turfgrass plots.\",\n \"FIG.\",\n \"36 shows mean mortality (%) of Amblyseius fallacis adult females following the direct application of several concentrations of EC25% and commercially available insecticides.\",\n \"FIG.\",\n \"37 shows contact toxicity of EC25% oil formulation on adult females of Amblyseius fallacis.\",\n \"Probit analysis.\",\n \"FIG.\",\n \"38 shows mean percent mortality of Phytoseiulus persimilis adult females to different insecticide treatments.\",\n \"FIG.\",\n \"39 shows overall percent mean mortality of adult wasps Aphidius colemani following direct application with EC25% and commercially available insecticides.\",\n \"FIG.\",\n \"40 shows male and female mean mortality (%) of Aphidius colemani adult wasps following direct application with EC25% and commercially available insecticides.\",\n \"FIG.\",\n \"41 shows contact toxicity of EC25% oil formulation on adult wasps Aphidius colemani.\",\n \"Probit analysis.\",\n \"FIG.\",\n \"42 shows mortality of adult wasps Aphidius colemani following exposure to EC25% and commercially available insecticide residues.\",\n \"FIG.\",\n \"43 shows probit analysis of adult wasps Aphidius colemani 24 H and 48 H following exposure to EC25% residues.\",\n \"FIG.\",\n \"44 shows the effect of treatment on Aphidius colemani emergence from treated mummies.\",\n \"FIG.\",\n \"45 shows fecundity assessment of female Aphidius colemani following contact with EC25% residues.\",\n \"FIG.\",\n \"46 shows mean mortality of Orius insidiosus second instar nymphs following application with EC25% and commercially available insecticides.\",\n \"FIG.\",\n \"47 shows mean mortality of Orius insidiosus adults following EC25% and other insecticide treatments.\",\n \"FIG.\",\n \"48 shows fecundity of Orius insidiosus females surviving insecticide treatments.\",\n \"FIG.\",\n \"49 shows probit analysis of Orius insidosus second instar nymphs following application with EC25%.\",\n \"FIG.\",\n \"50 shows probit analysis of Orius insidiosus adults following application with EC25%.\",\n \"DETAILED DESCRIPTION OF THE INVENTION Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.\",\n \"“Locus” means a site which is infested or could be infested with acari, insects, fungi, or other pests and may include, but is not restricted to, domestic, urban, agricultural, horticultural, and forest environments.\",\n \"“Essential Oil Extract” means the volatile, aromatic oils obtained by steam or hydro-distillation of plant material and may include, but are not restricted to, being primarily composed of terpenes and their oxygenated derivatives.\",\n \"Essential oils can be obtained from, for example, plant parts including, for example, flowers, leaves, seeds, roots, stems, bark, wood, etc.\",\n \"“Active Constituents” means the constituents of the essential oil extract to which the pesticidal, acaricidal, insecticidal, and/or fungicidal activity is attributed.\",\n \"The essential oil extract of the present invention generally comprises the active constituents including: α-terpinene, ρ-cymene, limonene, carvacrol, carveol, nerol, thymol, and carvone.\",\n \"The term “partially purified”, when used in reference to an essential oil extract means that the extract is in a form that is relatively free of proteins, nucleic acids, lipids, carbohydrates or other materials with which it is naturally associated in a plant.\",\n \"As disclosed herein, an essential oil extract of the invention is considered to be partially purified.\",\n \"In addition, the individual components of the essential oil extract can be further purified using routine and well known methods as provided herein.\",\n \"Other chemistry terms herein are used according to conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (ed.\",\n \"Parker, S., 1985), McGraw-Hill, San Francisco, incorporated herein by reference.\",\n \"The present invention provides essential oil extracts derived from Chenopodium sp.\",\n \"comprising, α-terpinene, ρ-cymene, limonene, carvacrol, carveol, nerol, thymol, and carvone.\",\n \"These extracts have one or more activities selected from the group of acaricidal, pesticidal, insecticidal, and fungicidal activities.\",\n \"The present invention also provides for the use of one or more of these essential oil extracts to prepare compositions that enable their application into different environments such as plants, soil, animals and buildings.\",\n \"1.Essential Oil Extract Plant Material Plant material that may be used in the present invention includes plant material derived from the genus Chenopodium sp.\",\n \"taken individually or in a group and may include, but is not restricted to, the leaf, flowers, roots, seeds, and stems.\",\n \"As is known by persons skilled in the art, the chemical composition and efficacy of an essential oil extract varies with the phenological age of the plant (Jackson et al., 1994), percent humidity of the harvested material (Chialva et al., 1983), the plant parts chosen for extraction (Jackson et al., 1994; and Chialva et al., 1983), and the method of extraction (Perez-Souto, 1992).\",\n \"Methods well-known in the art can be adapted by a person of ordinary skill in the art to achieve the desired yield and quality of the essential oil extract of the present invention.\",\n \"In one embodiment, the plant material is derived from Chenopodium ambrosioides.\",\n \"Pre-Treatment of Plant Material In addition to such parameters as the phenological age of the plant, the percent humidity of the harvested material, the plant parts chosen for extraction, and the method of extraction, the chemical composition and efficacy of an essential oil extract may be affected by pre-treatment of the plant material.\",\n \"For example, when a plant is stressed, several biochemical processes are activated and many compounds, in addition to those constitutively expressed, are synthesized as a response.\",\n \"In addition to pests, fungi, and other pathogenic attacks, stressors include drought, heat, water and mechanical wounding.\",\n \"Moreover, persons of skill in the art will also recognize that combinations of stressors may be used.\",\n \"For example, the effects of mechanical wounding can be increased by the addition of compounds that are naturally synthesized by plants when stressed.\",\n \"Such compounds include jasmonic acid (JA).\",\n \"In addition, analogs of oral secretions of insects can also be used in this way, to enhance the reaction of plants to stressors.\",\n \"In one embodiment, the essential oil extracts of the present invention are derived from plant material which has been pre-treated, for example by stressing the plant by chemical or mechanical wounding, drought, heat, or cold, or a combination thereof, before plant material collection and extraction.\",\n \"Harvesting the Plant Material for Extraction and Optional Storage Treatment The plant material may be used immediately after harvesting.\",\n \"In one embodiment the fresh plant material having a humidity level of >75% is used.\",\n \"Otherwise, it may be desirable to store the plant material for a period of time, prior to performing the extraction procedure(s).\",\n \"In another embodiment wilted plant material having a humidity level of 40 to 60% is used.\",\n \"In another embodiment dry plant material having a humidity level of <20% is used.\",\n \"In a further embodiment, the plant material is treated prior to storage.\",\n \"In such cases, the treatment may include drying, freezing, lyophilisizing, or some combination thereof.\",\n \"Extraction of the Essential Oil Extract and Validation of Constituents Essential oil extracts can be extracted from plant material by standard techniques known in the art.\",\n \"A variety of strategies are available for extracting essential oils from plant material, the choice of which depends on the ability of the method to extract the constituents in the extract of the present invention.\",\n \"Examples of suitable methods for extracting essential oil extracts include, but are not limited to, hydro-distillation, direct steam distillation (Durbeck 1997), solvent extraction, and Microwave Assisted Process (MAP™) (Belanger et al., 1991).\",\n \"In one embodiment, plant material is treated by boiling the plant material in water to release the volatile constituents into the water which can be recovered after distillation and cooling.\",\n \"In another embodiment, plant material is treated with steam to cause the essential oils within the cell membranes to diffuse out and form mixtures with the water vapor.\",\n \"The steam and volatiles can then be condensed and the oil collected.\",\n \"In another embodiment, organic solvents are used to extract organically soluble compounds found in essential oils.\",\n \"Non-limiting examples of such organic solvents include methanol, ethanol, hexane, and methylene chloride.\",\n \"In a further embodiment, microwaves are used to excite water molecules in the plant tissue which causes cells to rupture and release the essential oils trapped in the extracellular tissues of the plant material.\",\n \"To confirm the presence of the constituents of the present invention in the essential oil extract, a variety of analytical techniques well known to those of skill in the art may be employed.\",\n \"Such techniques include, for example, chromatographic separation of organic molecules (e.g., gas chromatography) or by other analytical techniques (e.g., mass spectroscopy) useful to identify molecules falling within the scope of the invention.\",\n \"The invention provides an essential oil extract derived from Chenopodium sp.\",\n \"comprising α-terpinene, ρ-cymene, limonene, carvacrol, carveol, nerol, thymol, and carvone.\",\n \"In one embodiment, the essential oil extract comprises at least 30% α-terpinene, 8% ρ-cymene, 5% limonene, trace carvacrol, 0.1% carveol, 0.1% nerol, trace thymol, and trace carvone.\",\n \"In one embodiment, the essential oil extract comprises at least one of each compound selected from the range for each compounds of at least 30%, 32%, 34%, 38%, 40%, 44%, 48%, 50%, 55%, 60 %, 65% α-terpinene; 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26% ρ-cymene, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%,22%,24% limonene, trace, 0.04%, 0.08%, 0.10%, 0.12%, 0.14%, 0.2%, 0.5%, 0.8%, 1.0% carvacrol, 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 1.4%, 1.6%, 1.8%, 2.0% carveol, 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 1.4%, 1.6%, 1.8%, 2.0% nerol, trace, 0.04%, 0.08%, 0.10%,0.12%, 0.14%, 0.2%, 0.5%, 0.8%, 1.0%, 1.4%, 1.6%, 1.8%, 2.0% thymol, and trace, 0.04%, 0.08%, 0.10%, 0.12%, 0.14%, 0.2%, 0.5%, 0.8%, 1.0% carvone.\",\n \"2.Activity of the Essential Oil Extract Following extraction of an essential oil extract of the invention, it may be desirable to test the efficacy of the extracts for acaricidal, pesticidal, insecticidal, and fungicidal activities.\",\n \"Any number of tests familiar to a worker skilled in the art may be used to test the activity of the extracts, compositions, and formulations of the invention.\",\n \"Deternination of Acaricidal Activity of the Essential Oil Extract Acaricidal activity of an essential oil extract may be evaluated by using a variety of bioassays known in the art (Ebeling and Pence, 1953; Ascher and Cwilich, 1960; Dittrich, 1962; Lippold, 1963; Foot and Boyce, 1966; and Busvine, 1980).\",\n \"a) Contact Efficacy with the Adult Stage One exemplary method that may be used tests the contact efficacy of the essential oil extract, or formulations thereof, with the adult stage of a mite species.\",\n \"For example, adult mites may be placed on their dorsum with a camel hair brush on a double-sided sticking tape glued to a 9 cm Petri dish.\",\n \"Essential oil extracts and/or formulations may then be applied to the test subjects by spraying with the spray nozzle of a Potter Spray Tower mounted on a stand and connected to a pressure gauge set at 3 psi.\",\n \"Mites that fail to respond to probing with a fine camel hair brush with movements of the legs, proboscis or abdomen are considered dead.\",\n \"In one embodiment, the contact efficacy of an essential oil extract, or formulations thereof, is determined using the two-spotted spider mite (Tetranychus urticae), at the adult stage, as a model test subject.\",\n \"A person skilled in the art, however, will readily understand that other species of acari can be used.\",\n \"b) Ovicidal Activity The ovicidal effect can be determined by treating mite eggs with concentrations of essential oil extracts, or formulations thereof.\",\n \"For example, adult female T. urticae may be transferred to 2 cm diameter leaf disks cut out of lima bean leaves and left for four hours for oviposition.\",\n \"When at least 20 eggs/disk are laid, adult mites may then be removed.\",\n \"Essential oil extracts and/or formulations may then be applied by spraying the test subjects.\",\n \"Egg hatch is assessed daily and for 10 days following treatment by counting the number of eggs remaining on the leaf disks and the number of live and dead nymphs present.\",\n \"Percent egg hatch is determined with live nymphs only.\",\n \"The nymphs are considered dead if no movement is observed after repeated gentle probing with a single-hair brush.\",\n \"In one embodiment the ovicidal activity of an essential oil extract, or formulations thereof, is determined with mite eggs of the two-spotted spider mite (Tetranychus urticae), as a model test subject.\",\n \"A person skilled in the art, however, will readily understand that other species of acari can be used.\",\n \"Determination of Insecticidal Activity of the Essential Oil Extract Similar bioassays can be conducted to evaluate the insecticidal activity of an essential oil extract, or formulations thereof, by utilizing an insect model.\",\n \"In one embodiment, the greenhouse whitefly (Trialeurodes vaporariorum (Westw.))\",\n \"is used as a model test subject in an insecticide bioassay.\",\n \"For example, Whitefly adults may be glued to a black 5 cm×7,5 cm plastic card sprayed with Tangle-Trap® (Gempler's Co.) to obtain at least 20 active adults per card.\",\n \"Each card is sprayed with the essential oil extract, composition, or formulation and allowed to dry.\",\n \"The cards are then placed sideways on a Styrofoam rack in a closed clear plastic container of 5 L with moistened foam on the bottom to keep humidity high (>90% R.H.).\",\n \"The plastic container is stored in a growth chamber at 24° C. and 16 L:8 D photoperiod.\",\n \"Mortality is evaluated 20 hours following treatment by gently probing the whitefly with a single-hair brush under the binocular microscope.\",\n \"Absence of movement (antennae, leg, wing) following probing is recorded as dead.\",\n \"A person skilled in the art, however, will readily understand that other insect species can be used.\",\n \"Determination of Fungicidal Activity of the Essential Oil Extract The fungicidal activity of an essential oil extract, or formulations thereof, can also be evaluated by utilizing a fungal model evaluated by using a variety of bioassays known in the art.\",\n \"Examples of such known bioassays include the following.\",\n \"Laboratory Tests The fungicidal efficacy of an essential oil can be done in the laboratory using several methods.\",\n \"One method incorporates the test samples in an agar overlay in a Petri dish.\",\n \"A second method would use a filter disk saturated with the test samples and placed on top of untreated agar.\",\n \"Both systems are challenged with fungal plugs cut from lawns of indicator organisms at the same stage of growth.\",\n \"The plates will be incubated at 30° C. for 5-10 days with visual observations and the zone of inhibition measured and recorded.\",\n \"A positive control, i.e.\",\n \"a commercially available fungicide and a negative control, i.e.\",\n \"water are tested in the same way.\",\n \"Greenhouse Tests Greenhouse tests may also be employed to evaluate fungicidal efficacy.\",\n \"For example, the effect of the essential oil extracts, or formulations thereof, may be tested on host plants infected by a disease organism such as, for example, Botrytis cinerea, Erysiphe cichoracearum or Sphaerotheca fuliginea, Rhizoctonia solani, and Phytophthora infestans, by observing the percent damage or presence of lesions on the host plant after treatment and against controls.\",\n \"Botrytis cinerea.\",\n \"Tomato plants are seeded and grown following current commercial practices for greenhouse tomato production.\",\n \"About 2 months following seeding, lesions are made on the leaves and the stem (5 lesions/plant) and inoculated with a suspension of 3×106 spores of B. cinerea, 2 ml per lesion.\",\n \"Treatments are then applied to the plants.\",\n \"A positive control, i.e.\",\n \"a commercially available fungicide and a negative control, i.e.\",\n \"water are also tested and all treatments are done in a randomized block design.\",\n \"The length of lesions are measured every two weeks over a period of 3 months, then the number of fruit, the total weight of fruit and the average weight of fruit are calculated during the entire production period of the plant.\",\n \"The experiment is repeated and the effect of treatments is subjected to an analysis of variance (ANOVA) and means are compared with a LSD test.\",\n \"Erysiphe cichoracearum or Sphaerotheca fuliginea.\",\n \"These disease organisms are obligatory parasites that do not have the capacity to survive in absence of its host.\",\n \"Therefore to provide the inoculum for the test, cucumber leaves are taken from an infested greenhouse.\",\n \"The conidia present on these leaves will transfer onto cucumber plants grown for the experiment one or two months previously.\",\n \"New plants are periodically infested in this manner in order to increase the inoculum.\",\n \"Treatments are then applied to the plants before or after inoculation depending on the type of fungicide used.\",\n \"A positive control, i.e.\",\n \"a commercially available fungicide and a negative control, i.e.\",\n \"water are also tested and all treatments are done in a randomized block design.\",\n \"The effect of the disease is evaluated on individual leaves of all plants using a index of infestation from 0 to 5 (0=absence of blemish and 5=80-100% of the leaf surface with blemishes).\",\n \"The degree of the infestation is evaluated 3, 7, and 14 days following inoculation and reported in averages per plant.\",\n \"The experiment is repeated and the effect of treatments is subjected to an analysis of variance (ANOVA) and means are compared with a LSD test.\",\n \"Rhizoctonia solani.\",\n \"An isolate of Rhizoctonia solani is produced on a culture media (PDA) 3 days before inoculation and a plug of the disease is then transferred to Erlenmeyer flasks filled with a YMG broth for 5 days.\",\n \"The mycelium is filtered, suspended in distilled water and blended.\",\n \"Seeds of tomato are used and sterilized on the surface using successive ethanol 70%, bleach and distilled water solutions.\",\n \"A suitable sterile potting soil mix is used in which 60 mg blended mycelium is inoculated per 100 g of potting soil.\",\n \"Tests are done in bedding boxes of 72 cells/box and 3 boxes are used per treatment.\",\n \"The boxes are spread out in a randomized arrangement in a controlled atmosphere growth chamber the following conditions: 20° C. during the day and 16° C at night, 16 hours of light, 162 umol of light intensity and 60% humidity.\",\n \"The boxes are incubated in the growing chambers during 3 weeks.\",\n \"Treatments are then applied to the young plants before or after inoculation depending on the type of fungicide used.\",\n \"A positive control, i.e.\",\n \"a commercially available fungicide and a negative control, i.e.\",\n \"water are also tested and all treatments are done in a randomized block design.\",\n \"Plants are examined each week and the incidence of the disease is measured as well as the degree of infestation on a scale of 0 to 5 (0=absence of infestation and 5=80-100% of the leaf surface attacked).\",\n \"The experiment is repeated and the effect of treatments is subjected to an analysis of variance (ANOVA) and means are compared with a LSD test.\",\n \"Phytophthora infestans.\",\n \"On tomato plants.\",\n \"Tomato plants are seeded and grown following current commercial practices for greenhouse tomato production.\",\n \"About 2 months following seeding, leaves and stems are inoculated with a suspension of 1×104 spores of P. Infestans until the plant surfaces are completely covered.\",\n \"Treatments are then applied.\",\n \"A positive control, i.e.\",\n \"a commercially available fungicide and a negative control, i.e.\",\n \"water are also tested and all treatments are done in a randomized block design.\",\n \"Percent damage or presence of lesions is evaluated every 3-4 days for a period of 2 weeks on leaves that had been identified previously (15-30 leaves per plant).\",\n \"The experiment is repeated and the effect of treatments is subjected to an analysis of variance (ANOVA) and means are compared with a LSD test.\",\n \"On potato plants.\",\n \"Potato tubers are sown and grown in pots of 6-8 inches.\",\n \"About 1,5 months after seeding, the leaves and stems of the plants are inoculated with a suspension of 1×104 spores of P. Infestans until the plant surfaces are completely covered.\",\n \"Treatments are then applied.\",\n \"A positive control, i.e.\",\n \"a commercially available fungicide and a negative control, i.e.\",\n \"water are also tested and all treatments are done in a randomized block design.\",\n \"Percent damage or presence of lesions is evaluated every 3-4 days for a period of 2 weeks on leaves that had been identified previously (15-30 leaves per plant).\",\n \"The experiment is repeated and the effect of treatments is subjected to an analysis of variance (ANOVA) and means are compared with a LSD test.\",\n \"3.Formulations of the Essential Oil Extract Formulations containing the essential oil extracts of the present invention can be prepared by known techniques to form emulsions, aerosols, sprays, or other liquid preparations, dusts, powders or solid preparations.\",\n \"These types of formulations can be prepared, for example, by combining with pesticide dispersible liquid carriers and/or dispersible solid carriers known in the art and optionally with carrier vehicle assistants, e.g., conventional pesticide surface-active agents, including emulsifying agents and/or dispersing agents.\",\n \"The choice of dispersing and emulsifying agents and the amount combined is determined by the nature of the formulation, the intended form of application of the formulation to a specific environment (e.g., plant, animal, soil, building), and the ability of the agent to facilitate the dispersion of the essential oil extract of the present invention while not significantly diminishing the pesticidal, acaricidal, insecticidal, and/or fungicidal activity of the essential oil extract.\",\n \"Non-limiting examples of conventional carriers include liquid carriers, including aerosol propellants which are gaseous at normal temperatures and pressures, such as Freon; inert dispersible liquid diluent carriers, including inert organic solvents, such as aromatic hydrocarbons (e.g., benzene, toluene, xylene, alkyl naphthalenes), halogenated especially chlorinated, aromatic hydrocarbons (e.g., chloro-benzenes), cycloalkanes (e.g., cyclohexane), paraffins (e.g., petroleum or mineral oil fractions), chlorinated aliphatic hydrocarbons (e.g., methylene chloride, chloroethylenes), alcohols (e.g., methanol, ethanol, propanol, butanol, glycol), as well as ethers and esters thereof (e.g., glycol monomethyl ether), amines (e.g., ethanolamine), amides (e.g., dimethyl sormamide), sulfoxides (e.g., dimethyl sulfoxide), acetonitrile, ketones (e.g., acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone), and/or water; as well as inert dispersible finely divided solid carriers such as ground natural minerals (e.g., kaolins, clays, vermiculite, alumina, silica, chalk, i.e., calcium carbonate, talc, attapulgite, montmorillonite, kieselguhr), and ground synthetic minerals (e.g., highly dispersed silicic acid, silicates).\",\n \"Surface-active agents, i.e., conventional carrier vehicle assistants, that can be employed with the present invention include, without limitation, emulsifying agents, such as non-ionic and/or anionic emulsifying agents (e.g., polyethylene oxide esters of fatty acids, polyethylene oxide ethers of fatty alcohols, alkyl sulfates, alkyl sulfonates, aryl sulfonates, albumin hydrolyzates, and especially alkyl arylpolyglycol ethers, magnesium stearate, sodium oleate); and/or dispersing agents such as lignin, sulfite waste liquors, methyl cellulose.\",\n \"Emulsifiers that can be used to solubilize the essential oil extracts of the present invention in water include blends of anionic and non-ionic emulsifiers.\",\n \"Examples of commercial anionic emulsifiers that can be used include, but are not limited to: Rhodacal™ DS-10, Cafax™ DB-45, Stepanol™ DEA, Aerosol™ OT-75, Rhodacal™ A246L, Rhodafac™ RE-610, and Rhodapex™ CO-436, Rhodacal™ CA, Stepanol™ WAC.\",\n \"Examples of commercial non-ionic emulsifiers that can be used include, but are not limited to: Igepal™ CO-887, Macol™ NP-9.5, Igepal™ CO-430, Rhodasurf™ ON-870, Alkamuls™ EL-719, Alkamuls™ EL-620, Alkamide™ L9DE, Span™ 80, Tween™ 80, Alkamuls™ PSMO-5, Atlas™ G1086, and Tween™ 20, Igepal™ CA-630, Toximul™ R, Toximul™ S, Polystep™ A7 and Polystep™ B1.If desired, colourants such as inorganic pigments, for example, iron oxide, titanium oxide, and Prussian Blue, and organic dyestuffs, such as alizarin dyestuffs, azo dyestuffs or metal phthalocyanine dyestuffs, and trace elements, such as salts of iron, manganeses, boron, copper, cobalt, molybdenum and zinc may be used.\",\n \"Spreader and sticking agents, such as carboxymethyl cellulose, natural and synthetic polymers (e.g., gum arabic, polyvinyl alcohol, and polyvinyl acetate), can also be used in the formulations.\",\n \"Examples of commercial spreaders and sticking agents which can be used in the formulations include, but are not limited to, Schercoat™ P110, Pemulen™ TR2, and Carbose™ 514 H, Umbrella™, Toximul™858 and Latron™CS-7.Time-release formulations are also contemplated by the present invention.\",\n \"For example, formulations which have been encapsulated and/or pelletized.\",\n \"In one embodiment, the formulation is a sprayable ready-to-use (RTU) formulation suitable, for example, for delivery by fogging, aerosol spraying, and pump spraying methods for application in a variety of environments.\",\n \"In a further embodiment, the formulation can also be prepared as an emulsifiable concentrate (EC) which can be diluted before use.\",\n \"In one embodiment, the emulsifiable concentrate comprises between 5% to 50% (by volume) Chenopodium-derived essential oil extract in combination with a suitable emulsifier, carrier, and spreader and/or sticking agent to enable application of the formulation to a specific environment.\",\n \"In another embodiment, the emulsifiable concentrate comprises between 10% to 25% (by volume) Chenopodium-derived essential oil extract in combination with a suitable emulsifier, carrier, and spreader and/or sticking agent to enable application of the formulation to a specific environment.\",\n \"In a further embodiment, the emulsifiable concentrate comprises between 10% to 25% (by volume) Chenopodium-derived essential oil extract in combination with between 1% to 15% (by volume) of a suitable emulsifier, and between 50% to 70% (by volume) of a suitable carrier or solvent to enable application of the formulation to a specific environment.\",\n \"In another embodiment, the emulsifiable concentrate may also comprise 2% to 20% (by volume) of a suitable spreader and/or sticking agent.\",\n \"The person skilled in the art, however, will understand that these concentrations can be modified in accordance with particular needs so that the formulation is acaricidal, insecticidal, and/or fungicidal, but not phytotoxic.\",\n \"Non-limiting examples of suitable emulsifiers that can be used in preparing emulsifiable concentrates of the present invention include: Rhodapex™ CO-436, Rhodapex™ CO-433, Igepal™ CO-430, Igepal™ CA-630, Igepal™ CO-887, Isopropanol, canola oil, Alkamuls™ EL-719, Rhodacal™ DS-10, Macol™ NP-9.5, Tergitol™ TMN-3, Tergitol™ TMN-6, Tergitol™ TMN-10, Morwet™ D425, and Tween™ 80.Suitable carriers or solvents that may be used include, but are not limited to, Isopar™ M, THFA™, ethyl lactate, butyl lactate, Soygold™ 1000, M-Pyrol, Propylene glycol, Agsolex™ 12, Agsolex™ BLO, Light mineral oil, Polysolve™ TPM, and Finsolv™ TN.\",\n \"Examples of suitable spreaders and/or sticking agents include, but are not limited to, Latex emulsion, Umbrella™, Adsee™ 775, Witconol™ 14, Toximul™ 858, Latron™ B-1956, Latron™ CS-7, Latron™ AG-44M, T-Mulz™ AO-2, T-Mulz™ 1204, Silwet™ L-774.Formulations containing the essential oil extracts of the present invention can be prepared by known techniques to enable application to specific environments.\",\n \"Plant Formulations The formulation can be prepared for application to plants and plant environments, for example, household/domestic plants, greenhouse plants, agricultural plants, and horticultural plants.\",\n \"In one embodiment, the formulation contains a final concentration of between 0.125% to 10% (by volume) of the Chenopodium-derived essential oil extract in combination with a suitable emulsifier, carrier, and spreader and/or sticking agent to enable sprayable application to a plant.\",\n \"In another embodiment, the formulation contains between 0.25% to 5% (by volume) of the Chenopodium-derived essential oil extract in combination with a suitable carrier, emulsifier, and spreader and/or sticking agent, to enable sprayable application to a plant.\",\n \"Fumigant Formulations for Closed and/or Open Environments The formulation can also be prepared for application as a fumigant for both outdoor as well as indoor application, for example in closed environments, such as greenhouses, animal barns or sheds, human domiciles, and other buildings.\",\n \"Persons of skill in the art will appreciate the various methods for preparing such fumigants, for example, as fogging concentrates and smoke generators.\",\n \"A fogging concentrate is generally a liquid formulation for application through a fogging machine to create a fine mist that can be distributed throughout a closed and/or open environment.\",\n \"Such fogging concentrates can be prepared using known techniques to enable application through a fogging machine.\",\n \"For example, the formulation may have the following general composition: Ingredient % Chenopodium extract (active ingredient) 1-15 Spreader/sticker 1-5 Organic solvent To 100% Smoke generators, which are generally a powder formulation which is burned to create a smoke fumigant.\",\n \"Such smoke generators can also be prepared using known techniques.\",\n \"For example, the formulation may have the following general composition: Ingredient % Chenopodium extract (active ingredient) 1-10 Absorbent silica 3-5 Pyrotechnic ingredients 3-10 Free flow aid 1-5 Filler To 100% Animal Formulations The formulation can be prepared in a form suitable for application to animals and animal environments, such as barns or sheds.\",\n \"In one embodiment, the formulation is prepared in a form suitable for topical application on an animal for controlling insects, acari, pests and fungi.\",\n \"Persons of skill in the art will appreciate the various methods for preparing such topical formulations, for example as powders or sprayable formulations.\",\n \"In one embodiment, the topical formulation is an aerosol and may have the following general composition: Ingredient % Chenopodium extract (active ingredient) 1-5 Emollient (coat conditioner) 2-5 Stabilizer 0.1-1 Preservative 0.1-0.5 Organic solvent To 100% In another embodiment, the formulation is a water-based pump spray formulation having the following general composition: Ingredient % Chenopodium extract (active ingredient) 1-10 Emulsifier(s) 2-10 Coat conditioner 2-5 Stabilizer 0.1-1 Preservative 0.1-1 Antioxidant 0.1-0.5 Water To 100% In a further embodiment, the formulation is an aerosol formulation for application to an animal environment, such as a barn or shed, having the following general composition: Ingredient % Chenopodium extract (active ingredient) 3-10 Stabilizer 0.1-1 Preservative 0.1-0.5 Organic solvent To 100% Human Formulations The formulation can be prepared in a form suitable for topical application on humans, for example as a repellent.\",\n \"Persons of skill in the art will appreciate the various methods for preparing such topical formulations, for example as lotions or sprayable formulations.\",\n \"In one embodiment, the formulation is a solvent-based sprayable formulation having the following general composition: Ingredient % Chenopodium extract (active ingredient) 1-5 Emollient (skin conditioner) 2-5 Stabilizer 0.1-1 Organic solvent To 100% In another embodiment, the formulation is a water-based sprayable formulation having the following general composition: Ingredient % Chenopodium extract (active ingredient) 1-5 Emulsifier(s) 1-5 Skin conditioner 2-5 Stabilizer 0.1-1 Preservative 0.1-1 Antioxidant 0.1-0.5 Water To 100% In a further embodiment, the formulation is a topical lotion formulation having the following general composition: Ingredient % Chenopodium extract (active ingredient) 1-10 Emulsifier(s) 1-10 Skin conditioner 2-10 Thickener(s) 0.2-5 Stabilizer 0.1-1 Preservative 0.1-1 Antioxidant 0.1-0.5 Water To 100% Soil Formulations In a further embodiment, the formulation of the present invention can be prepared as a microemulsion.\",\n \"Microemulsions are low-viscosity, optically transparent dispersions of two immiscible liquids which are stabilized by at least one ionic or nonionic surfactant.\",\n \"In the case of microemulsions, the particle diameters are in the range from about 5-100 nm suspended in a continuous phase.\",\n \"The interfacial tension between the two phases is extremely low.\",\n \"The viscosity of many microemulsions of the oil and water type (O/W) is comparable with that of water.\",\n \"In contrast to microemulsions, “macroemulsions” have high viscosities and their particle diameter is in the range from about 10 to 100 micrometers.\",\n \"Macroemulsions are milky white in color and, upon heating, tend toward phase separation or toward sedimentation of the dispersed substances.\",\n \"It is commonly believed that pesticidal microemulsions can provide superior efficacy relative to macroemulsion formulas having the same levels of active ingredients.\",\n \"It is believed that the small size of the emulsion droplets may allow for better transport of the pesticide through cell membranes (plant and insect) thereby resulting in enhanced efficacy.\",\n \"Microemulsions are considered to be infinitely stable, thereby providing improved stability over traditional macroemulsion systems.\",\n \"Accordingly, microemulsion formulations of the present invention may be particularly suitable for certain applications, for example soil delivery.\",\n \"Microemulsion formulations of the present invention can be prepared using various known methods known in the art.\",\n \"In one embodiment, the essential oil extract of the present invention can be combined with a combination of emulsifiers to create a microemulsion.\",\n \"For example, a microemulsion can be prepared in a three phase process.\",\n \"Phase 1: Selection of Emulsifiers A selection of potential primary emulsifiers is tested for miscibility with the active ingredient.\",\n \"Solubility is evaluated by visually assessing for a clear stable solution.\",\n \"Each pesticide/emulsifier mixture is mixed with the required volume of water and the quality of emulsion/microemulsion produced is recorded.\",\n \"Emulsifiers producing the best emulsions are taken through into phase 2.Emulsion/microemulsion quality is determined by visual assessment.\",\n \"Transparent microemulsions are the best, white emulsions with a bluish hue are second, plain white emulsions are next and any emulsion showing phase separation is last.\",\n \"Phase 2: Selection of Secondary and Tertiary Emulsifiers Upon completion of phase 1, the most promising primary emulsifier systems are blended with other emulsifiers in various ratios until an emulsifier blend is identified which provides the desired physical properties.\",\n \"The optimized system generally consists of one main emulsifier and two or more co-emulsifiers/co-solvents.\",\n \"These co-emulsifiers/co-solvents tend to broaden the thermal phase stability range of the microemulsion system.\",\n \"Ternary phase diagrams are used in this phase of development to assist in the selection of emulsifier ratios.\",\n \"The best formulation at this stage is often one containing one anionic emulsifier and two non-ionics of different types.\",\n \"The criteria for assessing the microemulsion properties are again physical appearance as a function of time and temperature.\",\n \"Phase 3: Optimization of Emulsifier Levels Having established the best emulsifier combination, the final phase is optimizing the levels and ratios of each emulsifier component in the system.\",\n \"In one embodiment, the microemulsion comprises between 30% to 50% (by volume) Chenopodium-derived essential oil extract, between 0.5% to 25% (by volume) of a suitable emulsifier, and between 10% to 50% water.\",\n \"Non-limiting examples of emulsifiers that can be used in preparing microemulsions of the present invention include: Igepal™ CA-630, Rhodasurf™ ON-870, Alkamuls™ EL-719, Tween™ 80, Alkamuls™ PSMO-5, Rhodapex™ CO-436, Rhodafac™ RE-610, Rhodacal™ CA, Ammonyx™ CO, Aeros™ OT-S, Ammonyx™ LO, Stepanol™ WAC, Soprophor™ BSU, and Rhodaca™1 IPAM.\",\n \"It is contemplated that the formulations of the essential oil extract can be combined with a controlled release delivery system in order to time-release the bioactive agents.\",\n \"Such controlled release delivery systems include methods, known in the art, of encapsulation, dissolution, or incorporation of the active ingredient.\",\n \"It is further contemplated that the formulations of the present invention can be prepared in combination with nutrients (fertilizers) or herbicides.\",\n \"Thus the skilled artisan will appreciate that the instant invention may be further formulated to provide various dissolution rates and/or be prepared in combination with nutrients (fertilizers) or herbicides.\",\n \"4.Use of Essential Oil Extract Formulations The essential oil extracts of the present invention can be used for controlling pests by applying a pesticidally effective amount of the essential oil extract and/or formulation of the present invention to the locus to be protected.\",\n \"The essential oil extract formulations can be applied in a suitable manner known in the art such as, for example, spraying, atomizing, vaporizing, scattering, dusting, watering, squirting, sprinkling, pouring, fumigating, and the like.\",\n \"Loci, for the application of essential oil extracts or formulations thereof include, but are not limited to, agricultural, horticultural, forest, plantation, orchard, nursery, organically grown crops, turfgrass and urban environments.\",\n \"Specifically, the essential oil extracts or formulations thereof may be applied to soil or plants/trees at one or several sites including the leaves, petioles, stems, seeds, roots, flower, cones, bark, wood or tubers.\",\n \"It is further contemplated that the essential oil extract may be formulated for seed treatment either as a pre-treatment for storage or sowing.\",\n \"For example, the seed may form part of a pelleted composition or, alternatively, may be soaked, sprayed, dusted or fumigated with a formulation of the present invention.\",\n \"The essential oil extract, and formulations thereof, can be applied to the surface of the soil to control soil surface or soil-inhabiting pests.\",\n \"The essential oil extract of the present invention can also be used as part of an Organic Production system and Integrated Pest Management program.\",\n \"For example, in conjunction with augmentation of beneficial insects and mites.\",\n \"It is further contemplated that the essential oil extract, and formulations thereof, can be applied to ‘fertigation’ i.e.\",\n \"fertilization via the irrigation system of plants in greenhouses.\",\n \"For example, an essential oil microemulsion can be added to the water in small concentrations (0.1-0.5% AI) and as water is being irrigated to individual plants, the latter would be treated for soil-inhibating pests (insects and disease pathogens).\",\n \"5.Effect of the Essential Oil Extract or Formulations on Beneficial Insects and Mites Natural enemies of phytophagous pests include both predators and parasitoid.\",\n \"Predators are generally as large, or larger than the prey they feed on.\",\n \"They are quite capable of moving around to search out their food, and they usually consume many pests during their lifetime.\",\n \"Parasitoids, or parasitic insects, are smaller than their prey.\",\n \"One or more parasitoids grow and develop in or on a single host.\",\n \"The host is slowly destroyed as the parasitic larva(e) feed and mature.\",\n \"Such beneficial insects and mites can help prevent or delay the development of pesticide resistance by reducing the number of pesticides required to control a pest.\",\n \"They will also feed on the resistant pests that survive a pesticide application.\",\n \"Integrated pest management (IPM) programs take advantage of the biological pest control provided by beneficial insects and mites by conserving or augmenting natural enemies.\",\n \"When chemical controls are necessary in an IPM program, pesticides recommended are those that have minimal impact on naturally occurring beneficials.\",\n \"Essential oil extracts of the present invention, and formulations thereof, may be tested for their effect on beneficial insects and mites, i.e., predators and parasitoids, by means of standardized IOBC (International Organization for Biologicial Control) testing methods as illustrated in Example XIV for integration into IPM programs.\",\n \"The invention now being generally described, it will be more readily understood by references to the following examples, which are included for purposes of illustration only and are not intended to limit the invention unless so stated.\",\n \"EXAMPLES Example I Phytochemical Profile of an Essential Oil Extract Derived from Chenopodium ambrosioides Whole plants of C. ambrosioides were harvested.\",\n \"Plant material used for extraction purposes comprised the whole plant above root.\",\n \"Essential oil extracts were extracted from the plant material by steam distillation, i.e., distillation in water (DW) and/or direct steam distillation (DSD).\",\n \"Distillation in water was carried out in a 380 L distillator with a capacity for processing ca.\",\n \"20 kg of plant material.\",\n \"During the process of DW, plant material was completely immersed in an appropriate volume of water which was then brought to a boil by the application of heat with a steam coil located at the base of the still body.\",\n \"In DSD, the plant material was supported within the still body and packed uniformly and loosely to provide for the smooth passage of steam through it.\",\n \"Steam was produced by an external generator and allowed to diffuse through the plant material from the bottom of the tank.\",\n \"The rate of entry of the steam was set at (300 ml/min).\",\n \"With both methods, the oil constituents are released from the plant material and with the water vapor are allowed to cool in a condenser to separate into two components, oil and water.\",\n \"The essential oil extracts were analyzed by capillary gas chromatography (GC) equipped with a flame ionization detector (FID).\",\n \"GC was carried out using a Varian 6000 series Vista and peak areas were computed by a Varian DS 654 integrator.\",\n \"SPB-1 (30 m×0.25 mm φ, 0.25 μm) and Supelcowax (30 m×0.25 mm φ, 0.25 μm) fused silca columns were used.\",\n \"Compounds in the sample come off the column at different times in minutes (Rt's or Retention Times) and these are compared to known standards and the compounds can thus be identified.\",\n \"When GC-FID gave ambiguous identification of certain compounds, Mass Spectrometry (MS) was used to compare the mass spectra of the compounds with a database of known spectra.\",\n \"The relative amount of each component of the essential oil extracts was determined for different lots of a variety of C. ambroisiodes.\",\n \"Each lot represents pooled extractions taken from a crop within one harvest date.\",\n \"FIG.\",\n \"1 shows the phytochemical profile of the essential oil extract taken from three different lots.\",\n \"Lot No.\",\n \"00MC-21P indicates an ascaridole content of 9.86%; Lot No.\",\n \"00MC-24P has an ascaridole content of 6.39% and 00MC-29P has an ascaridole content of 3.6%.\",\n \"The activity of the extract is not apparently affected by the variability in relative amount of ascaridole as results from bioassays with these lots suggest.\",\n \"Example II Determination of the Active Constituents of the Essential Oil Extract Extensive testing was done in order to determine the active constituents of the essential oil extract.\",\n \"All compounds present in the oil were tested except for trans-ρ-mentha-2,8dien-1-ol and cis-ρ-mentha-2,8-dien-1-ol because they were unavailable.\",\n \"All compounds tested were obtained commercially (Sigma-Aldrich) except for ascaridole and iso-ascaridole that were isolated from a sample of our extract by Laboratoires LaSeve, Chicoutimi Qc.\",\n \"Acaricidal Activity Tests with the Two-Spotted Spider Mite (TSSM: Tetranychus urticae) To test acaricidal activity, thirty adult female mites were placed on their dorsum with a camel hair brush on a double-sided sticking tape glued to a 9 cm Petri dish.\",\n \"Three dishes were prepared for each concentration of each compound tested and the control (e.g., water) for a total of 90 mites per treatment per treatment day.\",\n \"One (1) ml of each preparation and of microfiltered water as control was added with a Gilson Pipetman™ P-1000 to the reservoir of the spray nozzle of a Potter Spray Tower mounted on a stand and connected to a pressure gauge set at 3 P.S.I.\",\n \"Petri dishes were weighed before and immediately after each application to calculate the amount of oil deposited (mg/cm2) with each sample tested.\",\n \"The entire procedure was followed three times to give a total number of 270 mites tested with each treatment.\",\n \"Mite mortality was assessed 24 and 48 h after treatment.\",\n \"Mites that failed to respond to probing with a fine camel hair brush with movements of the legs, proboscis or abdomen were considered dead.\",\n \"Individual compounds were tested at 0.125, 0.50, 1.0 and 2.0% concentrations with the two-spotted spider mite (TSSM: Tetranychus urticae).\",\n \"Results are illustrated in FIG.\",\n \"2.Comparisons were made with mortality data obtained with the 1% concentration of each compound and it was observed that carvacrol is the most active compound (90% mortality of TSSM) followed by carveol (82% mortality), nerol (82% mortality), thymol (78% mortality), carvone (78% mortality) and α-terpineol (71% mortality).\",\n \"Other compounds gave less than 40% mortality.\",\n \"No mortality was recorded for ascaridole at 1% .\",\n \"Although 3% mortality was obtained with a solution of 0.125% ascaridole, we believe that this is an erroneous udependable result because too few individuals were tested (n=125) and the standard deviation is high (13), compared to the higher number of individuals tested at the higher concentrations of this compound (n=300 each at 0.5% and 1.0%) where no mortality was recorded.\",\n \"The results obtained with individual compounds, do not indicate that the compounds present in large quantities in the oil, i.e α-terpinene, ρ-cymene, limonene, ascaridole, iso-ascaridole, have a great impact on the biological activity of the extract.\",\n \"Mortality obtained with each of these compounds tested at 1% concentration was 17% or less.\",\n \"Ascaridole and iso-ascaridole at 1% concentration had no effect on the spider mite (0% mortality).\",\n \"Carvacrol, carveol, nerol, thymol and carvone on the other hand may have a much greater impact on the activity of the oil (>70% of TSSM at a 1% concentration) even though each of these compounds are present in relatively small quantities (<1%) Insecticidal Activity Tests with the Greenhouse Whitefly (GWF: Trialeurodes vaporariorum) Tests were also done using compounds that had demonstrated the higher degree of activity, i.e.\",\n \"carvacrol, nerol and thymol with the greenhouse whitefly (Trialeurodes vaporariorum) our model bioassay for insecticidal effect.\",\n \"Whitefly adults were glued to a black 5 cm×7,5 cm plastic card sprayed with Tangle-Trap® (Gempler's Co.) by placing cards directly in the greenhouse colony cage until at least 20 adults have alighted on each card.\",\n \"Cards were observed before spraying under the binocular scope to remove all dead and immobile whiteflies.\",\n \"Only active whiteflies were kept for the experiment.\",\n \"Four cards were used per treatment.\",\n \"Each card was sprayed at 6 psi with 300 μl of emulsion using a BADGER 100-F® (Omer DeSerres Co., Montréeal, Canada) paintbrush sprayer mounted on a frame at a distance of 14.5 cm from the spray nozzle in an exhaust chamber.\",\n \"Cards were weighed immediately before and after spraying to calculate the amount of active ingredient deposited in mg/cm2.Cards were allowed to dry under the exhaust chamber and then placed sideways on a Styrofoam rack in a closed clear plastic container of 5 L with moistened foam on the bottom to keep humidity high (>90% R.H.).\",\n \"The plastic container was stored in a growth chamber at 24° C. and 16 L:8 D photoperiod.\",\n \"This procedure was repeated three times.\",\n \"Mortality was evaluated 20 hours following treatment by gently probing the whitefly with a single-hair brush under the binocular microscope.\",\n \"Absence of movement (antennae, leg, wing) following probing was recorded as dead.\",\n \"Relative efficacy of the compounds were compared by transforming mortality data to arcsin{square root}p and then subjecting to an ANOVA analysis using SAS® software (SAS Institute 1988).\",\n \"Results with the GWF, shown in FIG.\",\n \"3, confirm the important biological activity of these three compounds.\",\n \"Example III Ready-To-Use Acaricidal Formulations A ready-to-use (RTU) sprayable insecticidal formulation having as the active ingredient an extract of Chenopodium was prepared.\",\n \"In one embodiment, this formulation contains between 0.125% and 10% (by volume) of the essential oil extract, an emulsifier, a spreader and sticking agent, and a carrier.\",\n \"Examples of basic RTU formulations are as follows.\",\n \"Ingredient Amount (%) Amount (%) Amount (%) Essential oil 1.00 1.00 1.00 extract Rodacal IPAM 0.50 0.83 0.83 Igepal CA-630 — 0.50 — Macol NP 9.5 — — 0.50 Water 98.5 97.67 97.67 Examples of formulations with added polymers for added residual effect are as follows.\",\n \"Ingredient Amount (%) Amount (%) Amount (%) Essential oil extract 1.00 1.00 1.00 Rhodacal IPAM 0.83 0.83 0.83 Igepal CA-630 0.50 0.50 0.50 Carboset 514H 2.00 — — Pemulen TR2 — 0.05 — Schercoat P110 — — 5.00 Propylene glycol — 2.00 — Water 95.67 95.62 92.67 Example IV Acaricidal Efficacy of the Essential Oil Extract (RTU Formulation) Efficacy trials were conducted using a Ready-to-use (RTU) formulation of the present invention comprising: 1.00% Essential oil extract; 0.83% Rhodacal IPAM; 0.50% Igepal CA-630; 0.05% Pemulen TR2; 2.00% Propylene glycol; and 95.62% Water.\",\n \"Thirty adult female mites were placed on their dorsum with a camel hair brush on a double-sided adhesive tape glued to a 9 cm Petri dish.\",\n \"Three dishes were prepared for each concentration of each formulations or products tested and the control, (e.g.\",\n \"water), for a total of 90 mites per treatment per treatment day.\",\n \"One (1) ml of each preparation and of microfiltered water as control was added with a Gilson Pipetman™P-1000 to the reservoir of the spray nozzle of a Potter Spray Tower mounted on a stand and connected to a pressure gauge set at 3 P.S.I.\",\n \"Petri dishes were weighed before and immediately after each application to calculate the amount of oil deposited (mg/cm2) with each sample tested.\",\n \"The ready-to-use formulation was tested according to the method mentioned above to identify the minimum concentration needed for the desired mortality (>95%) at different concentrations (00.125, 0.25, 0.5, 0.75, and 1%) in order to compare the relative efficacy of this RTU formulation and other acaricidal products (synthetic and natural) presently on the market.\",\n \"The entire procedure was followed three times to give a total number of 270 mites tested with each treatment.\",\n \"Mite mortality was assessed 24 and 48 h after treatment.\",\n \"Mites that failed to respond to probing with a fine camel hair brush with movements of the legs, proboscis or abdomen were considered dead.\",\n \"In order to obtain LC50 values (Lethal Concentration in mg/cm2 is the amount of product needed to kill 50% of the test organism; therefore the lower the LC50 value the more toxic the product) results of the 48 h counts were subjected to Probit analysis using POLO computer program (LeOra Software, 1987).\",\n \"Mortalities were entered with corresponding weighed dose (mg/cm2) to take into consideration variability in the application rate.\",\n \"The results obtained with these bioassays are shown in FIG.\",\n \"4.Although the toxicity tests presented herein were performed with female mites, it will be clear to a person skilled in the art that those results show that the mortality that would have been observed for male mites would have been the same if not higher knowing that male mites are smaller than females.\",\n \"Example V Effect on the Egg and Nymphal Stages of the Spider Mite (RTU Formulation) The RTU formulation (comprising: 1.00% Essential oil extract; 0.83% Rhodacal IPAM; 0.50% Igepal CA-630; 0.05% Pemulen TR2; 2.00% Propylene glycol; and 95.62% Water) was also tested on the egg and the nymphal stages of the spider mite.\",\n \"The ovicidal effect was determined with eggs of the twospotted spider mite following treatment with concentrations of the RTU formulation.\",\n \"Adult female T. urticae are transferred to 2 cm diameter leaf disks cut out of lima bean leaves and left for four hours for oviposition.\",\n \"When at least 20 eggs/disk are laid, adult mites are then removed.\",\n \"Leaf disks are moist and then sprayed and Petri dishes are weighed before and after treatment and stored after treatment.\",\n \"Egg hatch is assessed daily and for 10 days following treatment by counting the number of eggs remaining on the leaf disks and the number of live and dead nymphs present.\",\n \"Percent egg hatch is determined with live nymphs only.\",\n \"The nymphs are considered dead if no movement is observed after repeated gentle probing with a single-hair brush.\",\n \"Results of the test on the egg stage (FIG.\",\n \"5) indicate that the RTU formulation has some effect on the eggs with 30% mortality using a 0.5% solution of the oil.\",\n \"It is expected that a higher concentration of the oil should show greater efficacy on eggs.\",\n \"Similarly to the effect of the RTU formulation on the nymphal stage, even at the 0.5% concentration, the RTU gave higher results (95.8%) than the existing commercial preparations of either Avid (80.1%) or Safer Soap (61.7%) (FIG.\",\n \"6).\",\n \"Example VI Residual Effect of the RTU Formulations of the Present Invention and Comparison Thereof with Commercially Available Acaricidal Products The residual effect of the RTU formulation (comprising: 1.00% Essential oil extract; 0.83% Rhodacal IPAM; 0.50% Igepal CA-630; 0.05% Pemulen TR2; 2.00% Propylene glycol; and 95.62% Water) was also tested with the spider mite and compared to natural and synthetic products already on the market, (i.e.\",\n \"Kelthane™, Avid™, Safer's™ Soap and Wilson's dormant oil).\",\n \"The procedure for this test involved the preparation of vials containing a nutrient solution in which individual faba bean leaves were placed.\",\n \"Eighteen leaves were prepared for each concentration tested and each were sprayed with the indicated concentration until run-off and allowed to dry.\",\n \"Ten spider mites were placed on nine of the leaves one hour after spraying and ten were placed on the other nine leaves one day following treatment.\",\n \"Mortality was observed 24 and 48 hr following mite introduction on the leaves.\",\n \"The entire procedure was repeated three times.\",\n \"The results of the residual effect of the different products when the mite is introduced on the plant one hour following treatment are shown in FIG.\",\n \"7.These results indicate that there is a residual effect of the RTU and that this effect is greater than in the Safer product.\",\n \"However, it is inferior to the residual effect of synthetic products such as Kelthane and Avid.\",\n \"These results show the RTU formulation's very low persistence in the environment (about 23 mortality of spider mites when the pest is introduced on the plant one hour after treatment with the product).\",\n \"The RTU formulation is therefore compatible with the recommendations of the Integrated Pest Management program which supports control methods that do not harm natural enemy populations and permit rapid re-entry of workers to the tested area and uninterrupted periods of harvest while assuring safety to workers and consumers.\",\n \"Example VII Acaricidal Activity of the Extracts on Other Acari (RTU Formulation) To confirm the efficacy of the formulations of the present invention on plant infesting acari in general, certain bioassays were performed on another plant infesting mite, the European red mite, Panonychus ulmi, a mite which shows a close taxonomical relationship with T. Urticae.\",\n \"The RTU formulation (comprising: 1.00% Essential oil extract; 0.83% Rhodacal IPAM; 0.50% Igepal CA-630; 0.05% Pemulen TR2; 2.00% Propylene glycol; and 95.62% Water) was thus tested on the red mite Panonychus ulmi, a pest of apple orchards, following the same protocol described for contact efficacy on adult spider mites in order to confirm its broad effect as an acaricide.\",\n \"The results confirm the effectiveness of the essential oil extract as a contact acaricide (FIG.\",\n \"8) which is not exclusively active on T. Urticae.\",\n \"Example VIII Insecticidal Efficacy of the Essential Oil Extract (RTU Formulation) Similar efficacy tests were also performed on several insect species that are serious pests of cultivated plants.\",\n \"The species tested were the greenhouse whitefly, Trialeurodes vaporariorum; the Western flower thrips, Frankliniella occidentalis; the green peach aphid, Myzus persicae; and the silverleaf whitefly, Bermisia argentifolii following the same protocol described in Example XI (C) below.\",\n \"Results presented in FIG.\",\n \"9 indicate that the RTU product (comprising: 1.00% Essential oil extract; 0.83% Rhodacal IPAM; 0.50% Igepal CA-630; 0.05% Pemulen TR2; 2.00% Propylene glycol; and 95.62% Water) is toxic to all organisms tested.\",\n \"LC50 could be calculated for the greenhouse whitefly and the green peach aphid and results (LC50 of 0.00131 mg/ cm2 and 0.0009 mg/cm respectively) show that the product is as or more effective to these insects as the spider mite.\",\n \"Example IX Emulsifiable Concentrate Formulation An emulsifiable concentrate formulation with an extract of Chenopodium ambrosioides was also prepared.\",\n \"The concentrate contains between 10 to 25% essential oil extract, emulsifiers, a spreader/sticker, and a carrier.\",\n \"Examples of emulsifiable concentrate formulations are as follows.\",\n \"Amount Amount Amount Amount Amount Amount Ingredient (%) (%) (%) (%) (%) (%) Essential 25 25 25 25 25 25 oil extract Rhodopex 5 2.5 — — 1.25 — CO-436 Rhodopex — — — — — — CO-433 Igepal CO- — 2.5 — — 1.25 2.5 430 Igepal CA- — — 5 2.5 — — 630 Igepal CO- — — — 2.5 — — 887 Isopropanol — — 10 — — — Isopar M — — 60 70 — — Macol NP — — — — — 2.5 95 THFA 70 70 — — 72.5 70 Example X Acaricidal Efficacy of the Essential Oil Extract (Emulsifiable Concentrate Formulation) Contact and residual bioassays were conducted in the laboratory to test the efficacy of the essential oil extract of the present invention.\",\n \"A 25% emulsifiable concentrate (EC25%) formulation of oil was tested against the adult and eggs of the twospotted spider mite and the European red mite.\",\n \"The EC25% formulation tested comprised: 25% Essential oil extract; 2.5% Rhodopex CO-436; 2.5% Igepal CO-430; and 70% THFA.\",\n \"The twospotted spider mite was reared on Lima bean plants (Phaseolus sp.)\",\n \"and the European red mite on apple leaves cv McIntosh (Malus domestica Borkhausen).\",\n \"Contact Efficacy with the Adult Stage The methodology used for adults was the same for both species.\",\n \"Twospotted spider mite adults were treated with four concentrations of oil of a North American herbaceous plant (0.125, 0.25, 0.5 and 1.0% active ingredient (AI) EC25%; Urgel Delisle et Associes, Saint-Charles-sur-Richelieu, QC, Canada), neem oil (Neem Rose Defense® EC 90%; Green Light, San Antonio Tex, USA) at 0.7% AI, insecticidal soap (Safer's Trounce® EC 20% potassium salts of fatty acids with 0.2% pyrethrins; Safer Ltd. Scaborough, ON, Canada) at 1% AI and a water control.\",\n \"European red mite.\",\n \"adults were treated with five concentrations (0.0312, 0.0625, 0.125, 0.25 and 0.5%) of EC25%, abamectin (Avid® EC1.9%; Novartis, Greensboro, N.C., USA) at 0.006% AI and a water control.\",\n \"Twenty-five mature female mites were deposited dorsally on a 1 cm2 piece of double-coated tape glued on a glass microscope slide.\",\n \"For each treatment period, four slides were prepared for each treatment or acaricide application as defined above.\",\n \"Solutions for each treatment were prepared on the treatment day and each slide was sprayed at a pressure of 0.42 kg/cm2 under an exhaust chamber with 250 μl of solution using a Badger 100-F® paint brush sprayer (Badger Air-Brush Co., Franklin Park, Ill., USA) mounted on a frame at a distance of 15 cm from the slide.\",\n \"The slides were weighed immediately before and after spraying to calculate the amount of active ingredient deposited per surface area (mg/cm2); this quantity varied less than 15% between slides.\",\n \"After spraying, the slides were placed on a styrofoam rack in a closed clear plastic container with a wet foam at the bottom to keep moisture high (90% R.H.).\",\n \"The container was stored in a growth chamber at 24° C. and 16 L:8 D photoperiod.\",\n \"This experimental procedure was repeated on three consecutive days in a complete block design where treatment period was considered a block.\",\n \"Mortality was assessed under a binocular microscope 48 (twospotted spider mite) and 24 hours (European red mite) following treatment.\",\n \"Because European red mite mortality in the control group at 48 hours was high, it was judged to be inadequate for statistical evaluation.\",\n \"Mites were considered dead if movement was imperceptible after repeated gentle probing with a single-hair brush.\",\n \"Data were transformed by arcsin{square root}p and subjected to an ANOVA statistical analysis using SAS® software (SAS Institute, 1988).\",\n \"The LC50 and LC90 (in mg/cm2 of AI) of EC25% were calculated with PROBIT analysis using POLO-PC® software (LeOra Software, 1987).\",\n \"EC25% at 1% concentration and insecticial soap at 1% were most effective at controlling the adult twospotted spider mites causing 99.2 and 100% mortality respectively (FIG.\",\n \"10).\",\n \"At 0.5, 0.25 and 0.125% EC25% resulted in 94.7, 76.8 and 68% mortality respectively.\",\n \"The least effective treatment was neem oil, which at the recommended dose caused only 22.1% mortality.\",\n \"The LC50 and LC90 of EC25% for the twospotted spider mite were 0.009 mg/cm2 (99% confidence interval 0.0082-0.0099 mg/cm2) and 0.0292 mg/cm2 (99% confidence interval 0.0268-0.0321 mg/cm2) respectively (significant at P=0.01).\",\n \"In comparison, the LC50 of insecticial soap had been determined by the manufacturer to be 0.016 mg/cm2.At 0.5% concentration, EC25% was significantly more toxic (97.1% mortality) to P. ulmi adults than abamectin (82.4%) (FIG.\",\n \"11).\",\n \"Treatments with EC25% at concentrations ranging from 0.0625 to 0.25% gave statistically the same control level as abamectin.\",\n \"The LC50 and LC90 of EC25% for the red spider mite were 0.0029 mg/cm2 (99% confidence interval 0.0019-0.0038 mg/cm2) and 0.014 mg/cm2 (99% confidence interval 0.0108-0.0203 mg/cm2).\",\n \"EC25% gave <80% control of the adult stage of the two mites species at low doses.\",\n \"Ovicidal Activity The ovicidal effect of the following products was determined with eggs of the twospotted spider mite and the European red mite: six concentrations of EC25% (0.0625, 0.125, 0.25, 0.5, 1 and 2%), neem oil at 0.7% AI, insecticidal soap at 1% AI and abamectin at 0.006% and a water control.\",\n \"Twenty adult female T. urticae were transferred to 2 cm diameter leaf disks cut out of lima bean leaves and left for four hours for oviposition.\",\n \"Female P. ulmi were left for 24 hours to lay their eggs on 2 cm diameter leaf disks of apple leaves.\",\n \"When at least 20 eggs/disk were laid, adult mites were then removed with a soft brush Leaf disks were kept on moist soft cotton swabs placed in small (4 cm diameter) plastic Petri dishes.\",\n \"Three leaf disks were prepared for each treatment or acaricide application.\",\n \"Leaf disks were sprayed and Petri dishes were weighed before treatment and stored after treatment as for the slides used in the bioassay with adults.\",\n \"This experimental procedure was repeated on three consecutive days in a complete block design where treatment period was considered a block.\",\n \"Egg hatch was assessed daily and for 10 days following treatment by counting the number of eggs remaining on the leaf disks and the number of live and dead nymphs present.\",\n \"Percent egg hatch was determined with live nymphs only.\",\n \"The nymphs were considered dead if no movement was observed after repeated gentle probing with a single-hair brush.\",\n \"All nymphs (alive and dead) were removed daily from the leaf disks.\",\n \"Percent egg hatch (number of nymphs/total number of eggs on leaf disk×100) were transformed with arcsin {square root} and subjected to an ANOVA statistical analysis using SAS® software (SAS Institute, 1988).\",\n \"Egg hatch for the twospotted spider mite was significantly reduced by abamectin (8.0% egg hatch) and neem oil (2.1%) (FIG.\",\n \"12).\",\n \"Egg hatch was reduced to 67 and 40% with 1.0 and 2.0% concentrations of EC25% respectively and to 61.3% with insecticial soap.\",\n \"Egg hatch for the European Red mite was significantly reduced compared to the control treatment with the recommended doses of insecticial soap (27.2% egg hatch), abamectin (11.0%) and neem oil (14.2%) (FIG.\",\n \"13).\",\n \"Residual Bioassay with Adult Twospotted Spider Mites Leaf discs measuring 2 cm in diameter of bean leaves were sprayed on both sides with a VEGA 2000 sprayer (Thayer & Chandler Co., Lake Bluff, Ill., USA) at 0.42 kg/cm2 to runoff with 6.25 ml of each the following solutions: 2, 4, 8, and 16% of 99B-245, the recommended dose of dicofol (Kelthane® 35WP, Rohm and Haas Co., Philadelphia, Pa., USA) at 0.037% AI and a water control.\",\n \"Each treatment consisted of eight discs.\",\n \"One hour after treatment, 10 spider mites were transferred to each disc.\",\n \"Mortality was evaluated 48 hours following transfer of mites to the leaf discs.\",\n \"The procedure was repeated three times on three subsequent days.\",\n \"EC25% at 2, 4, 8 and 16% concentrations caused 23.0, 18.3, 13.9 and 32.5% mortality respectively to the adult spider mites when mites were introduced on bean leaves, 1 hr after treatment (FIG.\",\n \"14).\",\n \"Dicofol's residual activity was significantly higher (99.5% mortality) than any of the EC25% concentrations.\",\n \"EC25% was as effective as the insecticidal soap and synthetic acaricide abamectin to control adult twospotted spider mite and the European red mite.\",\n \"EC25% decreased egg hatch, but not as effectively as abamectin or neem oil.\",\n \"It may be important however to continue these investigations to determine the viability of emerged nymphs treated with the essential oil product because some botanicals, such as neem mixtures have shown growth-inhibiting properties to various pests (Rembald, 1989) and pulegone decreased larval growth of southern armyworm, Spodoptera eridania (Grunderson et al., 1985).\",\n \"Furthermore we demonstrated that when adult mites are introduced one hour after treatment, the mortality rate was statistically comparable to that of the control (FIG.\",\n \"14).\",\n \"A botanical such as EC25% may be an alternative to the more toxic or incompatible products.\",\n \"A contact acaricide with low residual activity can be used for treatments of localized infestations, before scheduled introductions of natural enemy populations or in absence of the natural enemy, i.e.\",\n \"treating at night in absence of diurnal parasitoids or predators.\",\n \"Plant essential oils may be phytotoxic (Isman, 1999).\",\n \"The oil used for EC25% was evaluated on several edible and ornamental plants for its phytotoxic effects and results indicate that at the recommended dose, i.e.\",\n \"0.5%, there were no observable effects on the leaves and flowers of tested plants (H. Chiasson, unpublished results).\",\n \"Example XI Insecticidal Efficacy of the Essential Oil Extract (Emulsifiable Concentrate Formulation) Efficacy trials were conducted (laboratory and small-scale greenhouse trials) using the emulsifiable concentrate formulation of the present invention (EC at 25% of chenopodium oil comprising: 25% Essential oil extract; 2.5% Rhodopex CO-436; 2.5% Igepal CO-430; and 70% THFA) with the following organisms: the green peach aphid (Myzus persicae), the Western flower thrips (Frankliniella occidentalis), the greenhouse whitefly (Trialeurodes vaporariorium) as well as the parasitoid Encarsia formosa.\",\n \"All bioassays were conducted in the laboratory of the Horticultural Research and Development Center (HRDC) of Agriculture and Agri-food Canada in Saint-Jean-sur-Richelieu, Quebec, Canada.\",\n \"A.\",\n \"Contact bioassays in the laboratory and greenhouse using EC25% and commercially available bioinsecticides with the green peach aphid (Myzus persicae (Sulz.))\",\n \"Laboratory Bioassay Five concentrations (0.125, 0.25, 0.5, 1 and 2%) of formulation EC25% were compared to commercial preparations of Neem Rose Defense® at 0.5% (EC 90% hydrophobic Neem oil), Safer's Trounce® at 1% (EC 20% with 0.2% pyrethrin) and a water control.\",\n \"Each treatment was repeated 12 times and each replicate consisted of a 2 month old shoot (10-15 cm) of Verbena speciosa ‘Imagination’ placed in a plastic Aqua-Pick® (tube used by florist to keep stems of cut flowers wet) filled with 10 ml of water.\",\n \"Aqua Picks were secured on a block of Styrofoam placed on the bottom of a 1 l transparent plastic container modified with screened sides and top to permit aeration.\",\n \"Green peach aphids (Myzus persicae (Sulz.))\",\n \"were collected in plastic containers from a rearing cage maintained in a greenhouse colony.\",\n \"Ten adults were transferred to each Verbena shoot.\",\n \"The shoot was sprayed at 8 psi under an exhaust chamber for about 15 seconds (long enough to cover the whole shoot) with a VEGA 2000® paintbrush sprayer equipped with a 20 ml reservoir (Thayer & Chandler Co., Lake Bluff, Ill., USA).\",\n \"Each shoot and plastic container was then stored in a growth chamber at 24° C., 65% R.H. and 16 L:8 N photoperiod.\",\n \"The entire procedure was repeated four times.\",\n \"Mortality was evaluated 48 hours following treatment by probing the aphid for movement with a small brush; absence of movement was recorded as dead.\",\n \"To evaluate the relative efficacy of EC25%, Neem Rose Defense® and Safer's Trounce®, percentage mortality data were transformed to arcsin{square root}p and subjected to ANOVA analysis using SAS® software (SAS Institute 1988).\",\n \"LC50 and LC90 were calculated using mortality results by PROBIT analysis using POLO-PC® software (LaOra Software 1994).\",\n \"Product concentrations (%) were used because data on quantity of active material deposited were not available.\",\n \"Results show that EC25% at 2.0% concentration was more effective (92.3% mortality) at controlling the green peach aphid than EC25% at 1% concentration (71.7%) and Safer's Trounce® (55.2%) though not significantly (FIG.\",\n \"15 ).\",\n \"This lack of distinction between treatments may be due to the low number (n) of aphids tested.\",\n \"Treatments with EC25% at concentrations of 0.5% and less and with Neem Rose Defense® resulted in <50% mortality of the aphids and results were not significantly different to those obtained with the water control.\",\n \"The LC50 and LC90 of EC25% for the green peach aphid was 0.63 (in % concentration) (Confidence Interval 0.47%-0.79%) and 1.84% (Confidence Interval of 1.39%-2.95%) respectively (FIG.\",\n \"16).\",\n \"Greenhouse Bioassay Three concentrations (0.25, 0.5 and 1%) of formulation EC25%, Neem Rose Defense® at 0.5% (EC 90% hydrophobic Neem oil), Safer's Trounce® at 1% (EC 20% with 0.2% pyrethrin) and a water control were tested with the green peach aphid (Myzus persicae (Sulz.)).\",\n \"Fifteen plants (replicates) of two month old Verbena speciosa ‘Imagination’ (10-15 cm) grown in small plastic insertions cells (used for potting plants) filled with Pro-Mix BX® were used for each treatment.\",\n \"Each insertion cell was glued to the bottom of a 1 l transparent plastic container with screened sides and top, to permit aeration.\",\n \"Green peach aphids were collected in plastic containers from a rearing cage maintained in a HRDC greenhouse and ten adults were transferred to each plant.\",\n \"The whole plant was sprayed for 15 seconds on average, at 8 psi under an exhaust chamber with a VEGA 2000® paintbrush sprayer equipped with a 20 ml reservoir (Thayer & Chandler Co., Lake Bluff, Ill., USA).\",\n \"Spraying was done three times over the course of the experiment, i.e.\",\n \"on days 0, 7 and 14.Containers with the sprayed plants were kept in a greenhouse under shade for the duration of the experiment.\",\n \"Counts were done on days 7, 14 (prior to spraying) and on day 21 by dismantling five of the fifteen replicates in each treatment.\",\n \"Aphids were individually counted when numbers were small (<50).\",\n \"For larger numbers, plants were shaken over a clear 250 ml container filled with soapy water over a black and white grid to evaluate the number of aphids present.\",\n \"Plant leaf surface (cm2) was measured with an area meter LI-3100® (LI-COR Inc., Lincoln, Nebr., USA) and counts were averaged to number of aphids/cm2 for each treatment and transformed to square root (x+0.5) for ANOVA analysis with SAS® software to evaluate the efficacy of the different treatments.\",\n \"Counts within treatments did not differ significantly (P=0.3647) from one sampling day to the other, so results within treatments were pooled and averaged for the whole experiment.\",\n \"All concentrations of EC25% and Safer's Trounce® were more effective in controlling the aphids than the water control (FIG.\",\n \"17 ).\",\n \"EC25% at 0.5% and 1.0% and Safer's Trounce were significantly more effective in reducing the number of aphids/cm2 than Neem Rose Defense® and EC25% at 0.25%.\",\n \"Both 0.5% and 1.0% EC25% concentrations were more effective (0.5 aphids/cm2 and 0.0 aphids/cm2 respectively) than Safer's Trounce® (0.9 aphids/cm2) though not significantly.\",\n \"B.\",\n \"Contact bioassays in the laboratory and greenhouse with the western flower thrips (Frankliniella occidentalis (Perg.))\",\n \"using EC25% formulation and two commercially available bioinsecticides.\",\n \"Laboratory bioassay Six concentrations (0.05, 0.18, 0.125, 0.25, 0.5 and 1%) of formulation EC25%, Neem Rose Defensee at 0.7% (EC 90% hydrophobic Neem oil), Safer's Trounce® at 1% (EC 20% with 0.2% pyrethrin) and a water control were tested with the Western flower thrips (WFT: Frankliniella occidentalis (Perg.)).\",\n \"WFT were collected in plastic containers by tapping infested Lima bean leaves over white paper.\",\n \"Ten WFT (either adults or 3rd or 4th instar nymphs) were transferred to a closed 250 ml transparent plastic container.\",\n \"Wet dental cotton was inserted through the lid for use as a water source.\",\n \"Four replicates were prepared for each treatment.\",\n \"Containers were sprayed at 6 psi under an exhaust chamber for 15 seconds with a VEGA 2000® paintbrush sprayer equipped with a 20 ml reservoir (Thayer & Chandler Co., Lake Bluff, Ill., USA).\",\n \"Containers were weighed just before and after spraying to calculate the amount of active ingredient deposited in mg/cm2.Containers were then stored in a growth chamber at 24° C., 65% R.H. and 16 L:8 D photoperiod.\",\n \"The entire procedure was repeated four times.\",\n \"Mortality was evaluated 24 hours following treatment under a binocular scope by probing WFT with a small brush.\",\n \"Absence of movement was recorded as dead.\",\n \"The efficacy of EC25% was compared to Neem Rose Defense® and Safer's Trounce® and data were transformed by arcsin{square root}p and subjected to ANOVA analysis using SAS® software (SAS Institute 1988).\",\n \"The LC50 and LC90 (in mg/cm2 of active ingredients) were calculated mortality results by PROBIT analysis using POLO-PC® software (LaOra Software 1987).\",\n \"Formulation EC25% at 0.5% and 1.0% were significantly more effective (98.8% and 95.8% mortality respectively) in controlling the WFT than all other treatments except for Safer's Trounce® (82.7% mortality) (FIG.\",\n \"18).\",\n \"EC25% at 0.25% caused significantly more mortality (63.7%) than the control (10.8%) but all remaining treatments did not.\",\n \"The LC50 and LC90 of EC25% for thrips was determined as 0.0034 mg/cm2 (Confidence Interval: 0.0027-0.0039 mg/cm2) and 0.0079 mg/cm2 (Confidence Interval: 0.0067-0.0099 mg/cm2) respectively (FIG.\",\n \"19).\",\n \"Greenhouse Bioassay Two concentrations (0.25% and 1%) of formulation EC25%, Neem Rose Defense® at 0.7% (EC 90% hydrophobic Neem oil), Safer's Trounce® at 1% (EC 20% with 0.2% pyrethrin) and a water control were used to evaluate their relative efficacy in controlling the Western Flower thrips (WFT: Frankliniella occidentalis (Perg.))\",\n \"in a greenhouse setting.\",\n \"Ten 10 day-old Lima bean plants (Phaseolus sp.)\",\n \"were prepared for each treatment.\",\n \"One leaf and the cotyledons of each plant were removed to keep only one leaf per plant grown in Pro-Mix BX® in a plastic insertion cell (used for potting plants) glued to the bottom of a clear plastic container (1 l) with screened sides and top.\",\n \"WFT were collected in small plastic containers by tapping infested bean leaves over white paper and lifted with a small brush.\",\n \"Ten adult thrips (or 3rd or 4th instar larvae) were transferred on each single leaf of each plant/insertion cell which were sprayed to drip point at 6 psi under an exhaust chamber with a VEGA 2000® paintbrush sprayer equipped with a 20 ml reservoir (Thayer & Chandler Co., Lake Bluff, Ill., USA).\",\n \"Spraying was done on days 0, 8 and 14.Each replicate/plastic container was then kept in a greenhouse under shade for the duration of the experiment.\",\n \"Counts were made on days 8 and 14 (prior to spraying) and on days 21 and 28.All live stages present on the whole plant were counted under a binocular scope and the leaf surface was measured by comparing it to a series of pre-measured hand-made leaf-size patterns.\",\n \"On the last day of the experiment (day 28), the leaf was cut and its surface was measured with an area meter LI-3100® (LI-COR Inc., Lincoln, Nebr., USA).\",\n \"Counts were calculated as average number of thrips/cm2 per treatment.\",\n \"In order to compare treatments, average counts were then calculated as a percentage of thrips present on the control plants: (n/cm2 on treated plants/n/cm2 on control plants)×100 The control treatment therefore had a value of zero and other treatments had positive or negative values indicating that more or less thrips were present respectively in relation to the control treatment.\",\n \"At the end of the experiment on day 28, leaves treated with EC25% at a concentration of 1.0% had 69.3% less WFT than leaves treated with the control while leaves treated with Safer's Trounce® had 101.1% more WFT (FIG.\",\n \"20).\",\n \"Leaves treated with Neem Rose Defense® had slightly more thrips (19.3%) than the control on day 28.Leaves treated with EC25% at 0.25% concentration had 52.3% more thrips than the control on day 28.C.\",\n \"Contact bioassay in the laboratory with the greenhouse whitefly (Trialeurodes vaporariorium (Westw.))\",\n \"using EC25% and commercially available insecticides Laboratory Bioassay Five concentrations (0.0625, 0.125, 0.25, 0.5 and 1%) of formulation EC25%, Neem Rose Defense® at 0.7% (EC 90% hydrophobic Neem oil), Safer's Trounce® at 1.0% (EC 20% with 0.2% pyrethrin), Thiodan® at 0.044% (50 WP) and a water control were used to evaluate their relative efficacy in controlling the greenhouse whitefly (Trialeurodes vaporariorium (Westw.)).\",\n \"Whitefly adults were collected with an insect aspirator from HRDC greenhouses and glued to a black 5 cm×7,5 cm plastic card sprayed with Tangle-Trap® (Gempler's Co.) by emptying the aspirator over the card to obtain at least 20 adults per card.\",\n \"Cards were observed before spraying under the binocular scope to remove all dead and immobile whiteflies.\",\n \"Only active whiteflies were kept for the experiment.\",\n \"Four cards were used per treatment.\",\n \"Each card was sprayed at 6 psi with 300 μl of emulsion using a BADGER 100-F® (Omer DeSerres Co., Montreal, Canada) paintbrush sprayer mounted on a frame at a distance of 14.5 cm from the spray nozzle in an exhaust chamber.\",\n \"Cards were weighed immediately before and after spraying to calculate the amount of active ingredient deposited in mg/cm2.Cards were allowed to dry under the exhaust chamber and then placed sideways on a Styrofoam rack in a closed clear plastic container of 5 L with moistened foam on the bottom to keep humidity high (>90% R.H.).\",\n \"The plastic container was stored in a growth chamber at 24° C. and 16 L:8 D photoperiod.\",\n \"This procedure was repeated three times.\",\n \"Mortality was evaluated 20 hours following treatment by gently probing the whitefly with a single-hair brush under the binocular microscope.\",\n \"Absence of movement (antennae, leg, wing) following probing was recorded as dead.\",\n \"Relative efficacy of EC25% and the two commercially available bioinsecticides, Neem Rose Defense® and Safer's Trounce®, and the synthetic insecticide Thiodan®, were compared by transforming mortality data to arcsin{square root}p and then subjecting to an ANOVA analysis using SAS® software (SAS Institute 1988).\",\n \"LC50 and LC90 (in mg/cm2 of active ingredients) were calculated by PROBIT analysis using POLO-PC® software (LaOra Software 1987).\",\n \"Formulation EC25% at concentrations 0.5% and 1.0% were significantly more effective (98.9% and 100.0% mortality respectively) at controlling the greenhouse whitefly than all other treatments except for Safer's Trounce® (98.0% mortality) (FIG.\",\n \"21).\",\n \"Formulation EC25% at 0.125% concentration and Neem Rose Defense® were significantly more effective than the control treatment but significantly less effective than EC25% at 0.25, 0.5 and 1.0% concentrations and Safer's Trounce®.\",\n \"Thiodan and EC25% at 0.0625% concentration were as effective as the control treatment.\",\n \"LC50 and LC90 were 0.0066 mg/cm2 (conf.\",\n \"int: 0.0054-0.0076 mg/cm2) and 0.0141 mg/cm2 (conf.\",\n \"int: 0.0121-0.0172mg/cm2) respectively (FIG.\",\n \"22).\",\n \"D. Contact bioassay in the laboratory with the parasitoïd (Encarsia formosa) using EC25% and commercially available bioinsecticides Laboratory Bioassay Four concentrations (0.0625, 0.125, 0.25 and 0.5%) of formulation EC25%, Neem Rose Defense® at 0.7% (EC-90%), Safer's Trounce® at 1.0% (EC 20.2%) and a water control were tested with the parasitoïd Encarsia formosa (EF) (obtained from Koppert Co. Ltd).\",\n \"EF were kept in a growth chamber at 24° C., 16 L:8 N photoperiod and 65% R.H. until emergence.\",\n \"Sixty newly emerged adult EF were transferred with a mouth aspirator into plastic Solo® cups of 20 ml.\",\n \"Cups were sprayed at 6 psi under an exhaust chamber with 250 ml of solution with a Badger 100-F® paintbrush sprayer (Omer de Serre Co., Montreal, Canada) mounted on a frame at a fixed distance of 14.5 cm.\",\n \"Solo® cups were weighed just before and after spraying to calculate the amount of active ingredient deposited in mg/cm2.Once sprayed, the EF were gently transferred with a small brush from the Solo cups to small clear plastic Petri dishes (10 EF/Petri) lined with a filter paper wetted with a 5% sugar solution as a food source.\",\n \"Four replicates were prepared for each treatment.\",\n \"The Petri dishes were then placed in a tray and stored in a growth chamber at 24° C., 65% R.H. and 16 L:8 D photoperiod.\",\n \"The entire procedure was repeated three times.\",\n \"Mortality was evaluated 24 hours following treatment under a binocular scope by observing the EF.\",\n \"Absence of movement was recorded as dead.\",\n \"The effect of EC25% was compared to Neem Rose Defense® and Safer's Trounce® using mortality data transformed by arcsin{square root}p and subjected to ANOVA analysis using SAS® software (SAS Institute 1988).\",\n \"All EC25% formulations at concentrations ranging from 0.0625 to 0.5% were significantly less effective than Safer's Trounce® at 1% (71.9%) (FIG.\",\n \"23).\",\n \"Results from all concentrations of EC25% and Neem Rose Defense® formulations were not significantly different than the control.\",\n \"These results indicate that the recommended dose (0.5%) of EC25% can be safely used with the biological control agent, Encarsia formosa.\",\n \"Example XII Fungicidal Efficacy of the Essential Oil Extract (Emulsifiable Concentrate Formulation) A.\",\n \"Effect of EC on Gray Mold Caused by the Plant Pathogen, Botrytis cinerea on Greenhouse Tomatoes Tomatoes (cv Trust) were sown and grown according to practices generally used for the production of greenhouse tomatoes.\",\n \"About two months after sowing, lesions are made on the stem (5 lesions/plant) and inocuated with a suspension of 3×106 spores of B. cinerea, 1 ml per lesions.\",\n \"The following treatments were evaluated: 1) control (−) with water only; 2) control (+) with B. cinerea; 3) Nova® (0.3 g/l); 4) Iprodione® (Rovral 1.0 g/l), 5) EC25% (comprising: 25% Essential oil extract; 2.5% Rhodopex CO-436; 2.5% Igepal CO-430; and 70% THFA) at a concentration of 0.125%; 6) EC25% at a concentration of 0.50%; 7) EC25% at a concentration of 0.75%; 8) M. ochracea (P130A) (5×108 spores/ml); 9) Rootshield® (0.6 g/l).\",\n \"The experimental design was a randomized block design with 8 blocks.\",\n \"Treatments were applied 1 and 8 days following inoculation of lesions with B. cinerea.\",\n \"Disease symptoms were evaluated according to the following severity index: 1=absence of symptoms, 2=necrosis (browning of tissue) around the lesion, 3=advanced necrosis and presence of spores on necrotic zones, 4=advanced necrosis, presence of spores on necrotic zones and the start of canker formation, 5=well developed canker and death of plant.\",\n \"Severity of symptoms was estimated 3 and 5 weeks after plant inoculation.\",\n \"The experiment was repeated and the effect of treatment was analyzed with an analysis of variance (ANOVA) and means were compared with a Least Significant Difference test (LSD).\",\n \"Statistical analyses demonstrated that there was a significant difference in efficacy between treatments (FIG.\",\n \"24).\",\n \"Control plants demonstrated average severity of disease symptoms on 26 of the 35 control plants.\",\n \"Plants from all other treatments presented a significantly lower level of severity than the control plants one day after inoculation or the first day of treatment.\",\n \"The two microbial fungicides Rootshield and P1304 did not significantly control the disease with an average severity between 25.33 and 21.67 8 days after treatment (FIG.\",\n \"24).\",\n \"Plants treated with the botanical biopesticide EC25% was less effective in controlling the disease symptoms than the commercial products, Nova and Rovral.\",\n \"However, all three concentrations of EC25% (0.125, 0.25 and 0.5% AI) decreased disease symptoms with severity indexes being 60, 44 and 37% lower respectively on plants treated with EC25% than the control plants.\",\n \"Severity indexes were reduced by 38 and 32% with Nova and Rovral respectively (FIG.\",\n \"25).\",\n \"B.\",\n \"Effect of EC25% on Powdery Mildew Caused by the Plant Pathogens, Erysiphe cichoracearum and Sphaerotheca fuliginea on Greenhouse Cucumbers The fungus responsible for powdery mildew are obligatory parasites incapable of survival in the absence of the host.\",\n \"In order to produce inoculum necessary for inoculation, infested cucumber leaves are collected from a grower in July of 2002.Conidia present on these leaves were transferred on leaves of cucumber plants grown in an experimental greenhouse, one of to two months after sowing the cucumber plants (cv Straight 8).\",\n \"The following treatments were evaluated: 1) negative control treated with water; 2) positive control treated with the disease organisms, E. cichoracearum and S. fliginea; 3) Nova® (0.3 g/l); 4) Benlate®+Manzate® (1.2 g/l and 4.g/l); 5) EC25% (comprising: 25% Essential oil extract; 2.5% Rhodopex CO-436; 2.5% Igepal CO-430; and 70% THFA) at 0.125% AI; 6) EC25% at 0.50% AI; 7) EC25% at 0.750% AI; 8) Mineral oil.\",\n \"The experimental design was a complete randomized block design with 7 blocks.\",\n \"Since it is almost impossile to inoculate an obligatory fungal parasite in an known or measurable quantity of inoculum, there is always variability in the level of infection of the plants.\",\n \"A first evaluation of powdery mildew was made on all plants inoculated in the greenhouse.\",\n \"The percentage of infection was analyzed in order to group 8 plants presenting the same level of infection (SE <1.5, see FIG.\",\n \"26).\",\n \"Severity of powdery mildew was also evaluated on each individual leaf with the use of a severity index 7 days after treatment applications and reported as average severity for each plant.\",\n \"The entire experiment was repeated a few days later.\",\n \"The effect of treatment was analyzed with an analysis of variance (ANOVA) and means were compared with a least significant difference test (LSD).\",\n \"Statistical analysis demonstrate that there is a difference of effect between treatments.\",\n \"(FIG.\",\n \"27).\",\n \"In fact, an increase of 45 and 39% of disease incidence was observed on control plants (water and disease).\",\n \"All plants treated with the various products presented a significantly lower increase in percentage of disease severity compared to the control plants.\",\n \"The botanical biopesticide EC25% was as effective as the commercial fungicide Nova in controlling the disease.\",\n \"At concentrations of 0.50 and 0.75%, EC25% demonstrated better control than Nova, permitting an increase of disease symptoms of only 2 and 2.5% respectively for both concentrations compared to 4.5% for Nova.\",\n \"However phytotoxicity was observed on plants treated with EC25% at those two concentrations Example XIII Effect of Essential Oil Extract for Controlling Soil Inhabiting Pests A.\",\n \"Effect of Soil Treatments with EC25% at 0.5% on Soil Emergence of Adult Western Flower Thrips Lettuce plants were transplanted in plastic pots of 12 cm of diameter and grown for six weeks in a greenhouse.\",\n \"The plants were heavily infested with all stages of thrips Frankliniella occidentalis Pergande and were watered and fertilized as required by good management practices.\",\n \"Then, the lettuce plants were cut at the soil level and the pots were sprayed with a preparation of EC25% (comprising: 25% Essential oil extract; 2.5% Rhodopex CO-436; 2.5% Igepal CO-430; and 70% THFA) at 0.5 concentration of active ingredients.\",\n \"Five volumes of EC25% were applied, 25 ml, 16.7 ml, 13.3 ml, 10 ml, and 6.67 ml per pot representing volumes of 1500 l/ha, 1000 l/ha, 800 l/ha, 600 l/ha and 400 l/ha respectively.\",\n \"A total of 10 pots were sprayed per volume and control pots were sprayed with 25 ml of tap water.\",\n \"The application was made using a badger sprayer connected to an air outlet to provide the required air pressure for pesticide application.\",\n \"Immediately after treatments, the pots were covered with a plastic petri dish cover of 14.5 cm diameter.\",\n \"The inner surface of the cover was smeared with a thin layer of tanglefoot insect glue and the joints were completely sealed with parafilm to prevent the escape of emerging adults from the soil pot or the introduction of foreign into the dish.\",\n \"Treatments were applied on 7 July 2002 and counts of emerging adults thrips were made 3, 5, 7, 10 and 12 days after treatments.\",\n \"At each observation time, emerging adults were removed from the glue and the cover was replaced on the top of the plastic pot and sealed with parafilm.\",\n \"Counts of emerging adults over a period of 2 weeks after treatments were analysed using an ANOVA and means for each treatment were separated using a Fischer LSD test.\",\n \"Most of the adult thrips emerged during the first week following treatment and this corresponds with the duration of pupal life that varies between 2 and 8 days according to the ambient temperature (FIG.\",\n \"28).\",\n \"Effect of treatment on the total number of adult thrips emergence from the soil was statistically highly significant ((F=5.71; dl=5; P<0.0001).).\",\n \"All treatments significantly reduced thrips emergence compared to the control.\",\n \"At 3 days after treatment, an important decrease in the number of emerging adult thrips was observed in the 1500 l/ha treatment compared with all other treatments.\",\n \"At all other dates, the 1500 l/ha treatment was the best treatment and caused the greatest reduction in adult thrips emergence.\",\n \"B.\",\n \"Using EC25% and Microemulsion Formulations for Controlling Soil-Inhabiting Pests of Turf i) Laboratory Tests using EC25% with the European Chafer, Rhizotrogus maialis The formulation EC25% (comprising: 25% Essential oil extract; 2.5% Rhodopex CO-436; 2.5% Igepal CO-430; and 70% THFA) was tested with third instar R. majalis.\",\n \"The insects were collected from Arnprior ON, and were kept in containers with soil at 4° C. until trials were initiated.\",\n \"Grass was grown in plastic seedling trays divided into 6 compartments.\",\n \"Soil within each compartment consisted of 1 part potting soil: 1 part sand to a depth of approximately 5 cm.\",\n \"The grass seedling mix consisted of Kentucky Blue 65%, Annual Rye Grass 20% and Fescue 15% and was distributed at 1.5 g per compartment 2-3 weeks prior to the addition of the insects.\",\n \"Ten third instar R. majalis were placed into each compartment so for a total of 60 larvae per tray.\",\n \"Three replicate trays were used for two separate experiments.\",\n \"One to two days after the larvae were added to the trays, treatment was initiated.\",\n \"Each of the EC25% treatment concentrations; 0.0625, 0.125, 0.25, 0.5 and 1%, were prepared in 100 mL of water by the addition of 0.25, 0.5, 1, 2 and 4 mL of stock EC respectively.\",\n \"The control was an EC blank, 0.375 mL of formulation excluding essential oil.\",\n \"The trays were watered once per day for the seven day period after treatment.\",\n \"After one week each compartment was sampled to determine the survival of the larvae.\",\n \"Samples of the soil were taken from each compartment after both trials and analysed for both pH (ASTM, 1989) and organic matter content (ASTM, 1987).\",\n \"Probit analysis was used with the data from the two European chafer trials in order to determine the LC50 values for EC25%.\",\n \"Comparison of the LC50 values within and between each trial was conducted using a z-test.\",\n \"Ninety percent (90%) R. majalis larvae survived at the 0.0625% concentrations of EC25% and 10% survived at 1% EC25% (FIG.\",\n \"29).\",\n \"Percent mortality from both trials were used to calculate LC50 values (FIG.\",\n \"30).\",\n \"Two different values were calculated for each trial since not all of the larvae were accounted for at the end of the exposure period.\",\n \"The first LC50 for each trial is based on the mortality within the actual number of larvae found in each treatment replicate at the end of the test (FIG.\",\n \"30).\",\n \"The second LC50 is calculated based on the assumption that there were 10 larvae present at the beginning and end of the experiment within each treatment replicate but due to possible deterioration of the organism it was not identified or located.\",\n \"A comparison of the LC50 using a z-test indicated that within each trial there was no significant difference between the calculated LC50s (z<0.885, z critical=1.96) Between trials it was determined that there was no significant difference (z=1.587, z critical=1.96) when comparing the first LC50 (based on the actual number of larvae found).\",\n \"When the LC50s based upon 30 larvae per treatment were compared between trials a significant difference (z=2.233, critical z=1.96) was detected.\",\n \"A mean LC50 and standard error for the two trials was calculated as 0.466±0.122% EC25% by averaging the two LC50s from trial one and two and these were not significantly different.\",\n \"Soil Organic Matter Content and pH.\",\n \"The soil organic matter content was determined to range from 8.9 to 11 with an average of 9.1 in the first trial and 10.5 in the second (FIG.\",\n \"31).\",\n \"The pH of the soil ranged from 4.9 to 5.0 with a mean of 4.94 in the first and 5.02 in the second trial.\",\n \"These values were consistent with those measured in previous R. majalis trials.\",\n \"ii) Field Tests using Microemulsion Formulations with the European Chafer, Rhizotrogus majalis The objective of this study was to measure the effect of broadcast applications of two microemulsion formulations (MEF), MEF1 and MEF2 of an essential oil extract from Chenopodium ambrosioides, on the presence of the European chafer, Rhizotrogus majalis.\",\n \"The microemulsion formulations tested comprised the following: Ingredient MEF 1 MEF 2 Chenopodium extract 50 50 Rhodafac RE-610 10 10 Ammonyx LO 4 4 Stepanol WAC 1 — Water 35 35 The experiment was conducted on lawn at the Guelph Turfgrass Institute (GTI), Ontario on turfgrass that was mowed once a week and received irrigation.\",\n \"Experimental design was a randomized complete block design with 4 replications and 8 treatments.\",\n \"Plots were 3 m×3 m and treatments tested are listed in FIG.\",\n \"32.Treatments were applied on Aug. 27, 2002, immediately after pre-treatment assessment of the European chafer, with a sprayer in water at 0.25 L/m2.The European chafers were at the second instar when treatments were applied and at the third instar by the end of the experiment.\",\n \"The live European chafers were counted using a golf course hole-cutter (15.2 cm, ‘turf mender’).\",\n \"Five plugs were taken from each plot.\",\n \"Once removed, plugs were broken up and the number of chafers was recorded.\",\n \"The last chafer count was done using a sod-cutter: three turf strips (0.3 m×3m) across each plot.\",\n \"The turfgrass sod was removed and chafers near the soil surface were counted.\",\n \"Counts were made at 5, 15 and 25 days after treatment on 2, 15, and 23 Sep. 2002, respectively.\",\n \"Analysis of variance (ANOVA) on the number of total European chafes alive was parformed by the General Linear Model Procedure of SAS statistical software (SAS Institute 1990).\",\n \"Fisher's Least Significant Difference test (LSD) was used for comparison of treatment means when the F-test was significant (P=0.05).\",\n \"Number of European chafers was not significantly different between plots prior to treatment application.\",\n \"On September 23 (25 days after the application), all insecticide treatments significantly reduced the number of chafers compared to the untreated control except treatments MEF1 (10.0 ml/m2) and MEF2 (20.0 ml/m2) (F=4.25; P<0.00046) (FIG.\",\n \"33).\",\n \"European chafer counts for both treatments were significantly higher than counts on plots treated with SEVIN XLR PLUS.\",\n \"No significant difference in the number of chafers in turfgrass plots was observed on September 2 (5 days after application) and on September 15 (15 days after application).\",\n \"For both dates, all treatments reduced chafers compared to the untreated control lower, the statistical model was not significant mainly because of the fact that European chafers were counted on a smaller area at these two dates.\",\n \"iii) Field Tests with the Microemulsion Formulations MEF1 and MEF2 and the Hairy Chinch Bug, Blissus leucopterus The experiment was conducted on a commercial lawn in Guelph, Ontario on low maintenance turfgrass that was mowed once a week and received no irrigation.\",\n \"Experimental design was a randomized complete block design with 4 replications and 8 treatments.\",\n \"Plots were 2 m×3 m and treatments tested are listed in FIG.\",\n \"34.Treatments were applied on Jul.\",\n \"30, 2003, immediately after pre-treatment assessment of the HCB, with a sprayer in water at 1 L/m2.Most of the HCB population was at the third instar.\",\n \"HCB were counted using the flotation method.\",\n \"A cylinder (346 cm2) was inserted into the turf to a depth of about 3 or 4 inches and was flooded to the brim with water.\",\n \"After 5 minutes, live HCB floating to the surface were counted.\",\n \"Three counts per plot were made (by the same person) and the total HCB per plot was used for statistical analysis.\",\n \"HCB were counted 5, 15 and 25 days after treatment on August 4, 12 and 24, respectively.\",\n \"Analysis of variance (ANOVA) on the number of total live HCB was performed using the General Linear Model Procedure of SAS statistical software (SAS Institute, 1990).\",\n \"Fisher's Least Significant Difference test (LSD) was used for comparison of treatment means when the F-test was significant (P=0.05).\",\n \"Number of HCB was not significantly different between plots prior to treatment application.\",\n \"All insecticide treatments significantly reduced the number of HCB compared to the untreated control on August 12, 15 days after the application (F=6.13; P<0.0005) (FIG.\",\n \"35).\",\n \"There were no differences between results with the Diazinon treatment and results with the two formulations.\",\n \"At this date, treatment MEF2 (20.0 ml/m2) had significantly more insects than treatment MEF2 (10.0 ml/m2).\",\n \"No significant difference in the number of HCB in turfgrass plots was observed on August 4 (5 days after application) and on August 24 (25 days after application).\",\n \"For both dates, all treatments reduced HCB compared to the untreated control.\",\n \"Example XIV Effect of Essential Oil Extract on Beneficial Pests A.\",\n \"Direct Toxicity of the Essential Oil Extract on Predatory Mites Amblyseius fallacis and Phytoseiulus persimilis The purpose of this study was to evaluate the direct toxicity of the EC25%, a botanical biopesticide with two predaceous mites Amblyseius fallacis, a natural regulator of mites in integrated control orchards and Phytoseiulus persimilis, a known mite predator for the control of the twospotted mite in vegetable crops grown under glasshouses in Quebec and elsewhere.\",\n \"The suitability of EC25% as a primary tool in IPM of greenhouse crops would therefore be determined.\",\n \"Rearing of Tetranychus urticae and Amblyseius fallacis The phytophagous mite, Tetranychus urticae has been reared on common bean plants (Phaseolus vulgare) for several years at the Horticultural Research and Development Center, St. Jean-sur-Richelieu, Quebec.\",\n \"The beans were sown at high densities of 40 to 50 plants per tray (39 cm×30 cm).\",\n \"Colonies of T. urticae were kept in a growth chamber set at 25° C., 75% HR and 16 L photoperiod.\",\n \"The predaceous mite Amblyseius fallacis was maintained on Tetranychus urticae and kept in a greenhouse set at 25° C., 75 HR and 16 L photoperiod.\",\n \"A fan placed in front of the cage containing both Amblyseius fallacis and the twospotted spider mite provided continuous air flow to the colonies.\",\n \"Trays containing bean plants infested with the twospotted spider mites were added regularly to provide sufficient food to the predator colonies.\",\n \"Rearing of Phytoseiulus persimilis Colonies of Phytoseiulus persimilis were bought from Koppert Canada and reared in the laboratory in the same conditions as for A. fallacis.\",\n \"The colonies originating from the shipment were maintained and acclimatized in a growth chamber set at 25 C, 70-85% RH and 16:8 (light/darkness) for two weeks.\",\n \"Contact Toxicity Assay The bioassays were carried out in Petri dishes using a leaf disc method.\",\n \"A wet sponge was placed in a plastic Petri dish (14 cm diameter and 1.5 high) and rings of apple leaf (cv.\",\n \"McIntosh; 3.5 cm of diameter) were cut and placed upside down on the surface of a water-saturated sponge.\",\n \"Sufficient numbers of all stages of the twospotted spider mite Tetranychus urticae Koch were then brushed onto each leaf disc.\",\n \"A total of five leaf discs were put in a Petri dish and each Petri dish represented one replicate.\",\n \"Ten replicates per treatment were prepared over a period of three weeks.\",\n \"Gravid females of Amblyseius fallacies (5) or Phytoseiulus persimilis (9), were picked up at random under a stereomicroscope from leaves taken from plants used to rear the predator colonies.\",\n \"They were transferred individually with a fine camel brush to a small Petri dish (5.5 cm of diameter) containing a leaf piece of the common bean, Phaseolus vulgare.\",\n \"They were treated topically with 0.3 ml of pesticide solution at different dosages using a paintbrush sprayer (Vega 2000, Thayer & Chandler, Lake Bluff, Ill., USA) at 6 psi set at 14.5 cm above the treated area.\",\n \"The pesticide solutions were prepared on the day of application.\",\n \"Treated females were then transferred carefully and individually to each apple leaf disc.\",\n \"To avoid contamination, a new camel brush was used for each concentration to transfer the treated females to leaf discs.\",\n \"Petri dishes were put in a black tray and covered with transparent plastic covers and a strip of brown paper was placed on top to reduce glare and to keep the mites within the leaf disc area.\",\n \"Water was added to the tray to maintain high relative humidity.\",\n \"The trays were incubated in a growth chamber set at 25° C., 75% HR and 16 L Photoperiod.\",\n \"Mortality was recorded 24 h and 48 h after treatment.\",\n \"One and 2 replicates were set up per day respectively for A. fallacis and P. persimilis and only 11 treatments were evaluated for P. persimilis.\",\n \"Treatments EC25% is an EC formulation with 25% essential oil as an active ingredient.\",\n \"Seven concentrations of EC25% were prepared as follows.\",\n \"The 1% concentration was prepared by mixing 0.4 ml of the formulation and 9.6 ml of tap water and successive dilutions were made from the stock solution.\",\n \"The following commercially available insecticides were used at their recommended rates: Trounce® (20.2% of fatty acids and 0.2% pyrethrin) at the recommended concentration of 1%; the insect growth regulator Enstar® (s-kinoprene) at the concentration of 0.065%; and Avid® (abamectin 1.9% EC), at the concentrations of 0.0057% and 0.000855%.\",\n \"A water treatment was used as a control for a total of twelve treatments with A. fallacies and 11 with P. persimilis where the Enstar treatment was dropped.\",\n \"The test product EC25% was sprayed first starting from the lower to the higher concentrations.\",\n \"Then the control treatment was applied followed the reference products Avid, Trounce and Enstar.\",\n \"The spray apparatus was rinsed three times between treatments using successively ethanol 95%, acetone, hexane, distilled water.\",\n \"Statistical Analysis Mortality percentages were transformed to logit or probit to determine which analysis gave a better fit as recommended by Robertson and Preisler (1992).\",\n \"The analysis which presents the highest number of small individual Chi square (χ2) is chosen.\",\n \"Probit mortality were regressed on 1+log10 (dose) for EC25%.\",\n \"Concentration mortality regression lines were determined to estimate the lethal concentration to kill 50% of the predator population using the POLO-PC program (LeOra, 1987).\",\n \"Toxicity values of LC50, LC90 and LC99 are given as percent (%) of active ingredient.\",\n \"Data were transformed to arcsine before analysis of variance.\",\n \"Comparison between treatments were analysed using GLM procedure and means were separated by the Fisher test at 5% probability (SAS, 1996).\",\n \"Results Amblyseius fallacis A total of 667 adult females of Amblyseius fallacis was tested and only 12 females (1.79%) walked out of the leaf disc area; number of missing was subtracted from initial total.\",\n \"Mortality in the control was 5.56% at 24 h and remained unchanged at 48 h following treatment (FIG.\",\n \"36).\",\n \"There was a highly significant difference between treatments at 24 h (F=30.32, df=11, P<0.001) and at 48 h (F=31.64, df=11, P<0.001).\",\n \"There was no mortality after 48 h was with EC25% at the concentration of 0.125% and 3,1% 7% and 23% mortality with EC25% at 0.25%, Enstar and EC25% at 0.5% and these results were not significantly different from the control.\",\n \"Note that at the concentration of 0.5% the EC25% suggested commercial rate, mortality was 23.11% which is less than the 50% limit of the IOBC for harmless pesticides.\",\n \"Amongst the commercially available products, Trounce caused the highest mortality (85.11%) after 48 H. This was followed by the Avid treatments at concentrations of 0.0057% (94.8% mortality) and 0.000855% (81.5% mortality) and results did not differ significantly, demonstrating that both products are equally toxic to Amblyseius fallacis.\",\n \"LC50, LC90 and LC99 values at 48 h (FIG.\",\n \"37) are well above (1.01%, 3.91% and 4.12% respectively) the 0.5% effective dose used to control the spider mite pest, Tetranychus urticae)(Chiasson, unpublished results).\",\n \"These results indicate that EC25% might have low or no residual toxicity to Ambiyseius fallacis and most adult females that remained alive 24 hours after the EC25% treatments continued to reproduce and were observed laying eggs.\",\n \"Phytoseiulus persimilis A cohort of 555 adult females was used to evaluate the toxicity of EC25% and the commercially available Trounce and Avid with the mite predator, Phytoseiulus persimilis.\",\n \"In this bioassay, 7.35% and 13.17% of the total number of gravid females escaped from the leaf disc 24 h and 48 h respectively after treatments.\",\n \"They contributed to 13.06% and 18.35% of the total mortality recorded at 24 h and 48 h respectively.\",\n \"The highest number of predator escapees were observed in the control treatment and in the EC25% treatments at concentrations lower than 2%.\",\n \"We will discuss only mortality calculated over total number treated minus missing individuals (3rd column of FIG.\",\n \"38).\",\n \"Highest mortality were caused by Trounce (99,71%) followed by Avid at the concentration of 0.0057% (93.69%).The lowest mortality was observed in the treatment with EC25% at the 0.125% concentration (13.43%).\",\n \"Mortality with EC25% at 0.125%, 0.25 and 0.5% were not significantly different from the control treatment.\",\n \"When missing females were deducted from the initial number of adults tested, the LC50 of P. persimilis was 1.2% and 0.8% at 24 h and 48 h after treatments respectively (FIG.\",\n \"37).\",\n \"B.\",\n \"Direct Toxicity of the Essential Oil Extract on Aphid Endoparasitoids Aphidius colemani (Hymenoptera: Brachonidae, Aphidiinae) In the present study, adult Aphidius colemani wasps were exposed to a direct spray application of EC25% and remained in permanent contact with the biopesticide residues, which is considered worse case conditions, to test the potential side effects this biopesticide may have on beneficial hymenoptera such as Aphidius colemani Rearing of Aphidius colemani Aphidius colemani wasps were purchased from.Plant Product Quebec in lots of 250 mixed mummies and adults.\",\n \"The emerged wasps and the remaining mummies were directly transferred to a 5 litre plastic bag filled with air and the wasps were provided with a 10% solution of sucrose and honey (w/w) as food source and water.\",\n \"Direct Contact Bioassay Six to 14 adult parasitoids less than 48 h old were transferred into a large solo cup (500 ml ca.)\",\n \"using a mouth aspirator.\",\n \"The solo cup was lined with a filter paper (Rothmans #1) and had two large openings drilled on the side and one on the cover to provide ventilation and these openings were covered with a fine screen to prevent escape of adult wasps and condensation of the pesticide vapour.\",\n \"The filter paper was humidified with a 10% solution of sucrose and honey.\",\n \"The solo cup containing the wasps was weighed and the wasps were dragged down to the bottom of the solo cup by means of successive beats on the cover with a 15 cm long stick.\",\n \"They were treated with 0.3 ml of the insecticide solution using a paintbrush sprayer (Vega 2000, Thayer & Chandler, Lake Bluff, Ill., USA) at 6 psi and set at 14.5 cm above the treated area.\",\n \"The solo cup was then covered and re-weighed to determine weight of pesticide used.\",\n \"The treated wasps were then incubated in a growth chamber set at 18° C.-22° C. and 60-65% HR.\",\n \"Assessment of treatment effects were made at 24 h and 48 h following treatment.\",\n \"Residual Bioassay Ten to 20 adult wasps including at least 5 females were picked up and put in a glass Petri dish and covered.\",\n \"The cover had an opening covered with a screen to enable ventilation and to prevent condensation of the pesticide vapour.\",\n \"The Petri dishes were previously treated with a pesticide solution exactly in the same manner as for direct toxicity bioassay but dishes were left to dry for an hour before covering and exposing the wasps to the pesticide residues.\",\n \"On the cover, two small circular holes were drilled and used to provide the wasps with water and a solution of honey and sucrose.\",\n \"Mortality was recorded at 24 h and 48 h. Treatments The test product EC25%, a 25% essential oil EC formulation obtained from Codena Inc.\",\n \"Seven concentrations were prepared as follows: EC25% at 8% was prepared by mixing 3.2 ml of EC25% and 6.4 ml of tap water and successive dilutions of 4%, 2%, 1%, 0.5% and 0.125% were made from the stock solution.\",\n \"Commercially available insecticides were used at their respective recommended doses as positive controls: Trounce® (20.2% of fatty acids, Safer Ltd, Scarborough, Ont.)\",\n \"at the recommended concentration of 1%, the insect growth regulator Enstare (s-kinoprene) at the concentration of 0.065%; Avid® (abamectin 1.9% EC) at the concentrations of 0.0057% and 0.000855%, and Thiodan® (endosulfan 50 WP) at the concentration of 5%.\",\n \"The test product EC25% was used first, starting from the lowest to the highest concentration and followed by the water control and finally by Avid, Trounce, Enstar and Thiodan.\",\n \"The spray apparatus was rinsed three times between treatments using successively ethanol 95%, acetone, hexane, distilled water.\",\n \"Statistical Analysis Concentration was analysed as main effect and the weight of pesticide applied was tested as a covariate to correct for difference in quantity of applied pesticide.\",\n \"This covariate was deleted from the model when found not significant.\",\n \"Mortality regression lines were determined to estimate the lethal concentration to kill 10%, 50% and 90% of the parasitoid population using the POLO-PC program (LeOra, 1987).\",\n \"Toxicity values of LC50 are given as percent of active ingredient.\",\n \"Data were transformed to arcsine before analysis of variance but actual means were presented.\",\n \"Comparison between treatments were analysed using GLM procedure and means were separated by Fisher test at 5% probability (SAS, 1996).\",\n \"Effect of Treatments on Aphidius colemani Emergence from Mummies Myzus persicae mummies parasitized by Aphidius colemani females on leaves of cabbage (cv.\",\n \"Lennox) were used in this test.\",\n \"Portions of leaves bearing mummies were cut and placed in a Petri dish.\",\n \"The Petri dish was weighted and treated with a pesticide solution and immediately re-weighted to determine the amount of pesticide used.\",\n \"The treated Petri dish was then covered and sealed with parafilm.\",\n \"The cover of the Petri had a screened opening to enable ventilation and to prevent escape of emerging Aphidius adults.\",\n \"The incubation period lasted 7 days and all mummies that did not emergence as adult wasps were considered dead.\",\n \"Fecundity Assessment Females that survived the pesticide residual treatments were assessed for fecundity on wheat plants infested with aphids.\",\n \"Myzus persicae aphids reared on cabbage plants (c.v. Lennox) were brushed onto a pot containing 25 to 30 plants of wheat 6 days old.\",\n \"Soon after, the brushed aphids climbed the wheat plants and a density of at least 100 aphids per pot was required.\",\n \"Female wasps that survived the 48 h residual treatments were removed individually from the test arena by means of an aspirator and confined over pots of aphid-infested plants using ventilated transparent plastic cylinders for a period of 24 h. The females were then removed and the plant bearing parasitized aphids were incubated for a period of 10 days at 18° C. to 22° C. At the end of the incubation period, the wheat plant was cut and put in a Petri dish.\",\n \"The number of parasitized aphids were counted.\",\n \"Results Direct Contact Bioassay A total of 1174 adult wasps including 657 or 55.9% female parasitoids were tested.in the bioassay.\",\n \"The mean quantity of pesticide solutions applied was 4.58±1.36 mg/cm which was more than double the amount of 2.0±0.2 mg/cm recommended for the typical bioassay (Mead-Briggs et al., 2000).\",\n \"Mortality with EC25% at concentrations up to 1% was not significantly different than for the water control after 24 h. though at 48 h, results with EC25% treatments at the 0.5% and 1% concentrations significantly different from the control (FIG.\",\n \"39).\",\n \"At the 0.5% concentration of EC25%, recommended for field application, mortality varied from 18.6% to 35.2% at 24 h and 48 H after treatments respectively.\",\n \"Highest mortality was observed with the Avid treatments at concentrations of 0.0057% and 0.000855% and with the EC25% treatment at concentrations of 4% and 8%.\",\n \"Results in FIG.\",\n \"40 show that female wasps were relatively less sensitive to treatments than adult males.\",\n \"LC50 values for EC25% on A. colemani females (FIG.\",\n \"41) was equal to 1.28% which is more than twice the recommended concentration of 0.5% for field application.\",\n \"The LC50 for A. colemani males was lower at 0.77% but still above the 0.5% field recommended concentration of EC25%.\",\n \"However, the 95% confidence limits (CL 95%) of LD50% for both males and females were overlapping and therefore their LD50% were not differently significant (Robertson and Presisler, 1992).\",\n \"Residual Assay Results shown in FIGS.\",\n \"42 and 43.Effect of Treatments on Aphidius colemani Emergence from Treated Mummies FIG.\",\n \"44 showed that the effects of treatments on emergence of Aphidius colemani adults from treated mummies were significant (F=6.94, dl=16, P<0.0001).\",\n \"The emergence rate of A. colemani decreased steadily when EC25% concentration increased and there was no emergence at the concentration of 8%.\",\n \"At the recommended concentration for field application, i.e.\",\n \"0.5%, emergence was 86.4% and this result was not statistically different from that observed in the control.\",\n \"In the reference products tested, the highest emergence was observed in the Avid treatment with 96.1% and the lowest was Enstar at 35% emergence.\",\n \"Fecundity Assessment The results of FIG.\",\n \"45 indicated that females that survived the treatment were able to parasitize Myzus persicae hosts and that their reproductive functions did not seem to be affected.\",\n \"There was no enough surviving female to test for the EC25% concentration of 4% and 8%.\",\n \"The lowest fecundity rate was observed in the treatment of Avid with 9.1 mummies per plants compared to 23.9 mummies per plant recorded in the control treatment.\",\n \"The number of mummies produced from females treated with EC25% treatments at concentrations varying from 0.125 to 2% were not significantly different from the control.\",\n \"C. Direct Toxicity of the Essential Oil Extract on Predatory Minute Bug Orius insidiosus Say Various Orius species including Orius insidiosus Say (Heteroptera: Anthocoridae) are effective biological control agents of western flower thrips (WFT) Frankliniella occidentallis Pergrande (Thysanoptera: Thripidae) in sweet pepper, cucumber and other vegetable and ornamental crops (Veire de van et al., 1996).\",\n \"The present study was initiated to evaluate the side effects of EC25% on the predatory bug Orius insidiosus under laboratory conditions.\",\n \"Culture of Orius insidiosus Orius insidiosus stock culture was initiated with individuals obtained from a commercial supplier (Plant Prod Quebec, 3370 Le Corbusier, Laval, Quebec) and maintained in a laboratory growth chamber.\",\n \"Eggs of Ephestia spp were served as a food source and snaps beans of Phaseolus vulgaris as an oviposition substrate.\",\n \"The beans containing eggs were then incubated in folded brown paper until emergence.\",\n \"The folded paper was used to reduce cannibalism.\",\n \"Emerging nymphs were then transferred into one litre jars containing bean pods and fed with Ephestia eggs until the adult stage.\",\n \"The stock culture was renewed regularly.\",\n \"Direct Contact Bioassay The bioassays were carried out in small Petri dishes (5.5 cm in dia.)\",\n \"using a leaf disc method.\",\n \"A thin layer of agar 2% (2-3 mm) was poured into each Petri dish and a ring of apple leaf (cv.\",\n \"McIntosh, 3.5 cm in dia.)\",\n \"was cut and placed upside down on the surface of the agar.\",\n \"At least 10 Orius insidiosus 2nd nymph instar or adults were transferred carefully using an aspirator on the surface of the apple leaf disc.\",\n \"The Petri dish containing the nymphs or the adults bugs were dragged down to the bottom of the Petri dish by means of successive beats on the cover with a 15 cm long stick.\",\n \"The Petri dishes were weighted and immediately, they were treated immediately with 0.3 ml of pesticide solution at different concentrations using a paintbrush sprayer (Vega 2000, Thayer & chandler, Lake Bluff, Ill., USA) at 6 psi and set at 14.5 cm above the treated area.\",\n \"The Petri dishes were then re-weighted to determine the quantity of pesticide applied.\",\n \"The pesticide solutions were prepared on the day of treatment.\",\n \"The treated nymphs or adults were then transferred carefully to the surface of the apple leaf disc containing eggs of Ephestia spp as a source of food.\",\n \"To avoid contamination, a new camel brush is used for each concentration to transfer the treated nymphs or adults to the leaf discs.\",\n \"The Petri dishes were put in a tray and incubated in a growth chamber set at 25° C., 65% HR and 16 L Photoperiod.\",\n \"A fan was placed in front of the tray to provide continuous air flow.\",\n \"Mortality of nymphs was recorded at 1, 2, 5, 7 and 9 days after treatment when more than 80% of the nymphs became adults.\",\n \"Mortality of adult predators was recorded at 24 H and 48 H following treatment.\",\n \"Ten replicates were prepared per treatment and 12 treatments were evaluated on second instar nymphs and adults.\",\n \"Treatments The test product is EC25%, a 25% EC essential oil formulation obtained from Codena Inc.\",\n \"Seven concentrations were prepared as follow: EC25% at 8% was prepared by mixing 3.2 ml of EC25% and 6.4 ml of tap water and successive dilutions of 4%, 2%, 1%, 0.5% and 0.125% were made from the stock solution.\",\n \"EC25% was compared to the recommended doses of the following commercially available insecticides: Trounce® (20.2% potassium salts of fatty acids and 0.2% pyrethrins) at the recommended concentration of 1%; the insect growth regulator Enstar® (S-kinoprene), at the recommended concentration of 0.065% and Avide® (abamectin 1.9% EC) at the concentration of 0.000855%, Thiodane® (endosulfan 50 WP) at the concentration of 5% and Cygon® (dimethoate) at the concentration of 4%.\",\n \"Water was used as a negative control.\",\n \"The test product EC25% was sprayed first, starting from the lowest to the highest concentration followed by the water control treatment and finally by the reference products Avid, Cygon, Enstar, Thiodan and Trounce.\",\n \"The sprayer was rinsed three times between treatments using successively ethanol 95%, acetone, hexane and distilled water.\",\n \"Fecundity Assessment The potential sublethal effects of EC25% on Orius insidiosus female fecundity was monitored.\",\n \"Fecundity assessment was carried out on females that survived 48 h after the direct contact pesticide treatments.\",\n \"Surviving females were separated from males and put individually in a Petri dish filled with a 2 mm layer of agar used as a support and an apple ring (5.5 cm) placed upside down on the agar surface along with a 3 cm long pod of faba bean (Phaseolus vulgare).\",\n \"The apple leaf disc and the bean pod were used as oviposition substrates.\",\n \"The Petri dish was covered with the correspondent cover and sealed with parafilm.\",\n \"The Petri cover had an opening covered with fine muslin tissue for ventilation and air exchange.\",\n \"Females were left undisturbed for 48 H for oviposion and then were fed with sufficient numbers of Ephestia spp eggs.\",\n \"After the 48 h period, females were then transferred to another Petri dish for a second 48 H oviposition test.\",\n \"During both periods, the eggs laid were counted and left to hatch for 5 days.\",\n \"The eggs that do not hatch after 5 days were considered dead and not viable.\",\n \"Statistical Analysis LC50 values of EC25% were determined using probit analysis with POLO software (LeOra, 1987).\",\n \"Concentrations were analysed as main effects and the weight of pesticide applied was tested as a covariance to correct for difference in quantity of the applied pesticide.\",\n \"This covariance was deleted from the model when found not significant.\",\n \"Mortalities were analysed using General Linear model (GLM) procedure within SAS (SAS, 1996) and the number of individuals initially introduced were tested as a covariant.\",\n \"Means were adjusted for covariance when appropriate and separated using the Fisher test for means comparison.\",\n \"However, actual means were presented in the results section.\",\n \"Results Results show (FIG.\",\n \"46) that nine days following treatment application, with Orius nymphs, the most toxic treatments were in decreasing order, Trounce (99,5% mortality), Cygon (98% mortality), EC25% at 8% concentration (87.6% mortality), Avid (82.5% mortality) and EC25% at 4% concentration (79.6% mortality).\",\n \"All results were significantly different from that of the control treatment (3.6% mortality).\",\n \"Less than 50% mortality was obtained with the other treatments though only Thiodan (45.7%) and EC25% (35.1%) results were significantly different from the control.\",\n \"Results with EC25% at the recommended concentration for field application of 0.5% were not significantly different from results obtained with the control.\",\n \"Results show (FIG.\",\n \"47) that the effects of the 12 treatments expressed as percent mortality of adults was significantly different at 24 H after treatment (F=55.9, df=11, p<0.0001) and at 48 H after treatment (F=63.2, df=11, p<0.0001).\",\n \"The least toxic treatments of EC25% at concentration of 0.125% and 0.25% were not statistically different from the control treatment.\",\n \"The treatment of EC25% at the recommended field concentration of 0.5% was the least toxic of the remaining treatments causing a mortality of 28%.\",\n \"The most toxic group included Cygon (100% mortality), Trounce (98.9% mortality), EC25% at concentrations of 4 and 8% (94% and 94% respectively) and Avid (87.8%).\",\n \"Fecundity Assessment The results from the fedundity assessment assay (FIG.\",\n \"48) showed that almost all females tested had laid eggs.\",\n \"There were few surviving females to test for fecundity following treatments with Avid, Cygon, Trounce and EC25% at concentrations of 2, 4 and 8%.\",\n \"The mean number of eggs laid per female per day in the control treatment was 7.6 which was almost 4 times the minimum number of 2 eggs per female per day set by the IOBC standards for fecundity for Orius leavigatus, a closely related species of O. insidiosus.\",\n \"The lowest rate was 2.8 eggs per female per day obtained in the treatment with Thiodan followed by EC25% at 0.5% concentration with 3.6 eggs per female per day and both were significantly different from the rate obtained with the water control (7.6 eggs per female per day).\",\n \"The date rate of eggs laid in the EC25% treatments at concentrations of 0.25% (5 eggs) and 0.5% (5.4 eggs) were not significantly different from the number of eggs laid in the control.\",\n \"The eclosion rate varied from 28.5% in the Thiodan treatment to 53% in the control.\",\n \"There was 33.9% egg eclosion in the EC25% at 0.5% concentration treatment.\",\n \"LC50 values for Orius nymphs were 2.65%, 9 days following treatment with EC25% (FIG.\",\n \"49) and for adults were 1.14%, 2 days following treatment with EC25% (FIG.\",\n \"50).\",\n \"References: Ascher, K. R. S. and Cwilich, R. (1960) Laboratory Evaluation of Acaricides Against Tetranychus Telarius L. on Sugar Beet and on Beans.\",\n \"Israel J Agric.\",\n \"Res.\",\n \"10:159-163.ASTM.\",\n \"1987.Standard test methods for moisture, ash, and organic matter of peat and other organic soils.\",\n \"American Standards for Testing and Materials (ASTM) D 2974-87.ASTM.\",\n \"1989.Standard test method for pH of soils.\",\n \"American Standards for Testing and Materials (ASTM) D 4972-89.Bélanger et al., (1991) Extraction et Détermination de Composés Volatils de L'ail (Allium sativum), Riv.\",\n \"Ital.\",\n \"EPPOS 2:455-461.Busvine, J. R. (1980) Recommended Methods for Measurement of Pest Resistance to Pesticides.\",\n \"FAO Plant Production and Protection Paper.\",\n \"pp.\",\n \"49-53.Ceske, L. J. and P. B. Kaufman, (1999) Regulation of Metabolite Synthesis in Plants.\",\n \"In: Natural Products from Plants.\",\n \"Kaufman et al., eds.\",\n \"CRC Press.\",\n \"Boca Raton.\",\n \"pp.\",\n \"91-121.Chialva et al., (1983) Chemotaxonomy of Wormwood (Artemisia Absinthum L.).\",\n \"Z. Lebensm Unters Forsch 176:363-366.Dittrich, V. (1962) A Comparative Study of Toxicological Test Methods on a Population of the Two-spotted Spider Mite (Tetranychus telarius).\",\n \"J. Econ.\",\n \"Entomol.\",\n \"55:644-648.Duerbeck, K., et al., (1997) The Distillation of Essential Oils.\",\n \"Manufacturing and Plant Constuction Handbook.\",\n \"Protrade: Dept.\",\n \"of Foodstuffs and Agricultural Products.\",\n \"Eschborn, Germany.\",\n \"pp.\",\n \"21-25.Ebeling, W. and R. J. Pence.\",\n \"(1953) Pesticide Formulation: Influence of Formulation on Effectiveness.\",\n \"J. Agr.\",\n \"Food Chem.\",\n \"1:386-397.Feng, R. and M. B. Isman.\",\n \"(1995) Selection for resistance to azadirachtin in the green peach aphid, Myzus persicae, Experientia 51:831-833.Flint, M. L. (1990) Pests of the Garden and Small Farm.\",\n \"A Grower's Guide to Using Less Pesticide.\",\n \"Statewide IPM Project.\",\n \"U. of California.\",\n \"Publication 3332.pp.\",\n \"116-123.Foott, W. H. and H. R. Boyce.\",\n \"(1966) A Modification of the Leaf-Disc Technique for Acaricide Tests.\",\n \"Proc.\",\n \"Entomol.\",\n \"Soc.\",\n \"Ont.\",\n \"96:117-119.Georghiou, G. P. (1990) Overview of Insecticide Resistance.\",\n \"In: Managing Resistance to Agrochemicals.\",\n \"M. E. Green, H. M. Le Baron and W. K. Moberg, eds., ACS Symposium Series.\",\n \"Chapter 2.Vol.\",\n \"421:18-41.Gundersin et al., (1985).\",\n \"Effects of the Mint Monoterpene Pulegone on Spodoptera Eridania (Lepidoptera: Noctuidae) Environ.\",\n \"Entomol.\",\n \"14:859-863.Isman, M. (1999) Pesticides Based on Plant Essential Oils.\",\n \"Pesticide Outlook.\",\n \"pp.\",\n \"68-72.Jackson, S. A. L. and R. K. M. Hay.\",\n \"(1994) Characteristics of varieties of thyme (Thymus vulgaris L.) for use in the UK: Oil content, composition and related characters.\",\n \"J. Hort.\",\n \"Sci.\",\n \"69:275-281.LeOra Software.\",\n \"POLO-PC, (1994) Probit or Logit Analysis.\",\n \"LeOra Software, Berkeley Calif. pp.\",\n \"1-28 Lippold, P. (1963) Acaricidal Testing Techniques in the Two-spotted Spider Mite.\",\n \"Adv.\",\n \"Acarol.\",\n \"1:174-180.Mead-Briggs, et al., (2000) A laboratory test for evaluating the effects of plant protection products on the parasitic wasp, Aphidius rhopalosiphi (Destephani-Perez)(Hymenoptera: Braconidae).\",\n \"Pages 13-25.In.\",\n \"Guidelines to evaluate side effects of plants protection products to non target arthropods, IOBC, BART and EPPO Joint Initiative.\",\n \"Candolfi, M. P., Blumel, S., Forster, R. Bakker, F. M., Grimm, C., Hassan, S. A., Heimbach, U., Mead-Briggs, M. A., Reber, B, Schmuck, R., Vogt, H.\",\n \"(Edts.\",\n \"), Reinham, Germany.\",\n \"Meister, R. T. ed.\",\n \"(1999) Farm Chemicals Handbook.\",\n \"Vol.\",\n \"85.Meister Publishing Company, Willoughby, Ohio.\",\n \"Perez- Souto, N. et al., (1992) Use of high-performance liquid chromatographic peak deconvolution and peak labeling to identify antiparasitic components in plant extracts.\",\n \"J. of Chrom.\",\n \"593:209-215.Rembald, H. (1989) Azadirachtins.\",\n \"Their Structure and Mode of Action.\",\n \"In: Insecticides of Plant Origin.\",\n \"ACS Symposium Series No.\",\n \"387.pp.\",\n \"150-163.Roush, R. T. and J.\",\n \"A. McKenzie.\",\n \"(1987) Ecological Genetics of Insecticide and Acaricide Resistance.\",\n \"Ann.\",\n \"Rev.\",\n \"Entomol.\",\n \"32:361-380 The McGraw-Hill Dictionary of Chemical Terms (ed.\",\n \"Parker, S., 1985), McGraw-Hill, Sam Francisco.\",\n \"U.S. Pat.\",\n \"No.\",\n \"4,933,371 U.S. Pat.\",\n \"No.\",\n \"5,839,224\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10467696"}}},{"rowIdx":382,"cells":{"text":{"kind":"list like","value":[["Method and apparatus for treating renal disease with hemodialysis utilizing pulsatile pump","A method of removing toxins from blood from a patient in need of such toxin removal includes: providing a countercurrent dialysis filter (20) having a blood compartment and a dialysate compartment separated from the blood compartment by a semi-permeable membrane; conveying blood from the patient through the blood compartment of a countercurrent filter (20) an back to the patient; and drawing dialysate from a reservoir (32) through the dialysate compartment of the countercurrent filter (20).","At least one of the blood or dialysate experiences pulsatile flow.","The steps are carried out such that blood toxins are drawn from the blood compartment through the semi-permeable membrane into the dialysate compartment."],["1.A method of removing toxins from blood from a patient in need of such toxin removal, comprising: providing a countercurrent dialysis filter having a blood compartment and a dialysate compartment separated from said blood compartment by a semi-permeable membrane; conveying blood from the patient through the blood compartment of a filter an back to the patient; and drawing dialysate from a reservoir through the dialysate compartment of the filter such that blood toxins are drawn from said blood compartment through the semi-permeable membrane into the dialysate compartment; wherein at least one of the conveying and drawing steps is carried out with pulsatile flow.","2.The method defined in claim 1, wherein said drawing step comprises drawing the dialysate through the dialysate compartment with a pulsatile pump.","3.The method defined in claim 2, wherein said drawing step comprises drawing the dialysate through the dialysate compartment at a pulsatile pressure of greater than about 10 mm Hg.","4.The method defined in claim 1, wherein said drawing step comprises drawing the dialysate through the dialysate compartment at between 30 and 100 pulses per minute.","5.The method defined in claim 1, wherein said drawing step further comprises drawing the dialysate through the dialysate compartment with a roller head pump.","6.The method defined in claim 1, wherein said conveying step comprises conveying blood from the patient through the blood compartment with a constant flow pump.","7.The method defined in claim 1, wherein said providing step comprises providing a dialysis filter with a dialysate compartment comprising a dialysate casing and a blood compartment comprising a plurality of tubules that reside within the dialysate casing.","8.The method defined in claim 1, wherein said conveying step comprises conveying blood with a pulsatile pump.","9.The method defined in claim 1, wherein said conveying step comprises conveying the blood with a pulse pressure of greater than about 30 mm Hg.","10.An apparatus for performing hemodialysis on a subject in need of such treatment, comprising: a dialysis filter having a blood compartment and a dialysate compartment separated from said blood compartment by a semi-permeable membrane; a first pump fluidly connected with the blood compartment that conveys blood from the patient through a blood compartment of the filter and back to the subject; and a second pump fluidly connected to the dialysate compartment; wherein at least one of said first and second pumps is configured to induce pulsatile flow.","11.The apparatus defined in claim 10, wherein said second pump comprises a pulsatile pump.","12.The apparatus defined in claim 11, wherein said second pump is configured to induce a pulsatile pressure of greater than about 10 mm Hg in dialysate in the dialysate compartment.","13.The apparatus defined in claim 10, wherein said dialysate compartment comprises a dialysate casing and said blood compartment comprises a plurality of blood tubules residing within said dialysate casing.","14.The apparatus defined in claim 10, further comprising a third constant flow pump fluidly connected to said second pump.","15.The apparatus defined in claim 10, wherein said second pump is configured to provide pulsatile flow at a rate of between about 30 and 100 cycles per minute.","16.The apparatus defined in claim 10, wherein said first pump and said second pump are pulsatile pumps.","15.The apparatus defined in claim 10, wherein the pulsatile pressure induced by said first or second pump is at least 30 mm Hg.","16.The apparatus defined in claim 10, wherein the pulsatile pressure induced by said first pump is at least 30 mm Hg."],["<SOH> BACKGROUND OF THE INVENTION <EOH>Hemodialysis is a well-known treatment technique for ESRD, a condition in which the patient's renal system has essentially ceased to remove waste products and contaminants from the blood.","Hemodialysis is a process that involves removing blood from the vasculature of a patient (usually a shunt or vein), purifying it with dialysate (a fluid that helps to remove toxins and return electrolytes to the blood), and returning the blood to the patient (usually through another vein).","Hemodialysis machines typically operate with separate compartments for blood and dialysate.","In conventional systems, the compartments are separated by a semi-permeable membrane that allows selective diffusion; toxins are removed from the blood, and electrolytes are added to bring the electrolyte concentration of the blood to desired levels.","In many systems, the blood and dialysate compartments are arranged in a countercurrent flow exchange layout, with the blood traveling in one direction and the dialysate traveling in the opposite direction.","One arrangement employs a large tube within which reside many smaller diameter tubules.","Ordinarily, the large tube carries dialysate, and the smaller tubules carry blood.","Blood is typically pumped through the tubules with a positive pressure pump (exemplary is a roller head pump), and the dialysate is typically drawn through the large tube with a roller head pump.","There are some shortcomings with such an arrangement.","The relatively constant flow of the blood and dialysate can create “dead” regions (where flow essentially stops) and regions of laminar flow within the tube.","In each of these regions the component exchange between the blood and the dialysate can be adversely affected, as fluid in the dead regions and the regions of laminar flow tends to have less surface area contact with the membrane, thereby reducing the efficiency of exchange.","Also, the constant flow of dialysate can create “shunting” of dialysate in certain regions, which can lead to blood/dialysate mismatch.","As such, it would be desirable to provide a hemodialysis system with improved efficiency of exchange."],["<SOH> SUMMARY OF THE INVENTION <EOH>The present invention can address some of the shortcomings of prior systems by improving the efficiency of dialysis through the use of pulsatile flow.","As a first aspect, the invention includes a method of removing toxins from blood from a patient in need of such toxin removal, comprising: providing a dialysis filter having a blood compartment and a dialysate compartment separated from the blood compartment by a semi-permeable membrane; conveying blood from the patient through the blood compartment of a filter and back to the patient; and drawing dialysate from a reservoir through the dialysate compartment of the countercurrent filter.","At least one of the blood or dialysate experiences pulsatile flow.","The steps are carried out such that blood toxins are drawn from the blood compartment through the semi-permeable membrane into the dialysate compartment.","As a second aspect, the invention includes an apparatus for performing hemodialysis on a subject in need of such treatment.","The apparatus comprises: a dialysis filter having a blood compartment and a dialysate compartment separated from the blood compartment by a semi-permeable membrane; a first pump fluidly connected with the blood compartment that conveys blood from the patient through a blood compartment of a filter and back to the subject; and a second pump fluidly connected to the dialysate compartment.","At least one of the first and second pumps is configured to induce pulsatile flow.","It has been discovered that the use of pulsatile flow in the blood or dialysate circuits during hemodialysis can provide a number of advantages.","These include increased efficiency of transport, reduction in dead regions, and intermittent increased transmembrane pressure.","Objects of the present invention will be appreciated by those of ordinary skill in the art from a reading of the figures and the detailed description of the preferred embodiments which follow, such description being merely illustrative of the present invention."],["FIELD OF THE INVENTION The present invention is directed generally to treatment of renal disease, and more specifically to treatment of end stage renal disease (ESRD) with hemodialysis.","BACKGROUND OF THE INVENTION Hemodialysis is a well-known treatment technique for ESRD, a condition in which the patient's renal system has essentially ceased to remove waste products and contaminants from the blood.","Hemodialysis is a process that involves removing blood from the vasculature of a patient (usually a shunt or vein), purifying it with dialysate (a fluid that helps to remove toxins and return electrolytes to the blood), and returning the blood to the patient (usually through another vein).","Hemodialysis machines typically operate with separate compartments for blood and dialysate.","In conventional systems, the compartments are separated by a semi-permeable membrane that allows selective diffusion; toxins are removed from the blood, and electrolytes are added to bring the electrolyte concentration of the blood to desired levels.","In many systems, the blood and dialysate compartments are arranged in a countercurrent flow exchange layout, with the blood traveling in one direction and the dialysate traveling in the opposite direction.","One arrangement employs a large tube within which reside many smaller diameter tubules.","Ordinarily, the large tube carries dialysate, and the smaller tubules carry blood.","Blood is typically pumped through the tubules with a positive pressure pump (exemplary is a roller head pump), and the dialysate is typically drawn through the large tube with a roller head pump.","There are some shortcomings with such an arrangement.","The relatively constant flow of the blood and dialysate can create “dead” regions (where flow essentially stops) and regions of laminar flow within the tube.","In each of these regions the component exchange between the blood and the dialysate can be adversely affected, as fluid in the dead regions and the regions of laminar flow tends to have less surface area contact with the membrane, thereby reducing the efficiency of exchange.","Also, the constant flow of dialysate can create “shunting” of dialysate in certain regions, which can lead to blood/dialysate mismatch.","As such, it would be desirable to provide a hemodialysis system with improved efficiency of exchange.","SUMMARY OF THE INVENTION The present invention can address some of the shortcomings of prior systems by improving the efficiency of dialysis through the use of pulsatile flow.","As a first aspect, the invention includes a method of removing toxins from blood from a patient in need of such toxin removal, comprising: providing a dialysis filter having a blood compartment and a dialysate compartment separated from the blood compartment by a semi-permeable membrane; conveying blood from the patient through the blood compartment of a filter and back to the patient; and drawing dialysate from a reservoir through the dialysate compartment of the countercurrent filter.","At least one of the blood or dialysate experiences pulsatile flow.","The steps are carried out such that blood toxins are drawn from the blood compartment through the semi-permeable membrane into the dialysate compartment.","As a second aspect, the invention includes an apparatus for performing hemodialysis on a subject in need of such treatment.","The apparatus comprises: a dialysis filter having a blood compartment and a dialysate compartment separated from the blood compartment by a semi-permeable membrane; a first pump fluidly connected with the blood compartment that conveys blood from the patient through a blood compartment of a filter and back to the subject; and a second pump fluidly connected to the dialysate compartment.","At least one of the first and second pumps is configured to induce pulsatile flow.","It has been discovered that the use of pulsatile flow in the blood or dialysate circuits during hemodialysis can provide a number of advantages.","These include increased efficiency of transport, reduction in dead regions, and intermittent increased transmembrane pressure.","Objects of the present invention will be appreciated by those of ordinary skill in the art from a reading of the figures and the detailed description of the preferred embodiments which follow, such description being merely illustrative of the present invention.","BRIEF DESCRIPTION OF THE FIGURES The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, serve to explain principles of the invention.","FIG.","1 is a schematic diagram of a hemodialysis apparatus of the present invention.","FIG.","2 is an enlarged schematic view of a hollow fiber artificial kidney (HFAK) included in the hemodialysis apparatus of FIG.","1.FIG.","2A is a greatly enlarged schematic view of two blood tubules of the HFAK of FIG.","2.FIG.","3 is a graph plotting urea concentration as a function of time in a two pool model of a dialysate.","FIG.","4 is a graph plotting creatinine concentration as a function of time collected in a dialysis study on dogs.","FIG.","5 is a graph plotting BUN concentration as a function of time collected in a dialysis study on a dog.","DETAILED DESCRIPTION OF THE INVENTION The present invention now will be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments of the invention are shown and described.","This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.","Like numbers refer to like components throughout.","Referring now to FIG.","1, a hemodialysis apparatus, designated broadly at 10, is illustrated schematically.","The hemodialysis apparatus 10 comprises a blood subsystem 12 and a dialysate subsystem 30, each of which will be described in greater detail below.","The blood subsystem 12, which as the name implies conveys blood through the hemodialysis apparatus 10, includes a blood entry conduit 14 that leads from a patient's artery (usually in the wrist), a roller pump 16 fluidly connected with the blood entry conduit 14, an HFAK 20 of conventional construction, and a blood exit conduit 18 leading from the HFAK 20 back to the patient's vein.","A bubble trap 19 is located on the blood exit conduit 18 between the HFAK 20 and the vein of the patient to prevent bubbles created during the processing of the blood from entering the patient.","During operation, blood exits the patient through the blood entry conduit 14.The roller pump 16 forces the blood to the HFAK 20, wherein dialysis occurs as described below.","The dialyzed blood then exits the HFAK 20 in the blood exit conduit 18, passes through the bubble trap 19 and returns to the patient's body.","The dialysate subsystem 30, which conveys dialysate into and from the HFAK 20, includes a dialysate reservoir 32 that contains dialysate, a dialysate entry conduit 34 leading from the dialysate reservoir 32 to the HVAK 20, a dialysate exit conduit 36 leading from the HFAK 20, and an in-line pulsatile pump 38 located on the dialysate exit conduit 36.In operation, dialysate is pumped from the dialysate reservoir 32 through the dialysate entry conduit 34 and into the HFAK 20, wherein countercurrent exchange of components of the blood and dialysate.","The spent dialysate is then pumped via the pulsatile pump 38 back to the dialysate reservoir 32.An exemplary HVAK 20 is illustrated in FIG.","2.The HVAK 20 includes a plurality of narrow blood tubules 22 that are enclosed within a dialysate casing 24.As the name implies, blood is conveyed through the blood entry conduit 14 into the blood tubules 22, and dialysate is conveyed from the dialysate entry conduit 34 into the dialysate casing 24.The blood tubules 22 are formed of a semi-permeable membrane material, such as polysulfone, that enables countercurrent exchange of components to occur between the blood and dialysate.","The dialysate casing 24 is typically formed of an impervious material such as a plastic material.","The dialysate utilized in the dialysate subsystem 30 can be any dialysate known to those skilled in this art as being suitable for use in hemodialysis.","An exemplary dialysate is available under the trade name Neutralyte from Fresenius Medical Care, Lexington, Mass.","The use of the pulsatile pump 38 (which can be a piston pump, a modified roller head pump, or the like) in the dialysate subsystem 30 can have the effect of producing pulsatile flow of dialysate within the dialysate subsystem 30.As used herein, “pulsatile flow” means flow that has a pulse pressure of 10 mm Hg or greater, and preferably when applied to blood means a pulse pressure of 30 mm Hg or greater.","Preferably, the pulsatile flow is induced in the dialysate at a pulse rate of between about 30 and 100 cycles per minute, and more preferably at a rate of between about 50 and 80 cycles per minute.","Those skilled in this art will recognize that the pulsatile pump may be used alone or in combination with a more constant flow pump.","Such pulsatile flow within the dialysate subsystem 30 can generate significant turbulence within the dialysate casing 24, which can reduce or eliminate the number of “dead” regions, where flow stops and exchange is minimal.","A typical flow pattern in blood tubules 22 and the blood casing 24 is shown in FIG.","2A (darker areas represent regions of higher flow), with a dead region 25 being illustrated between two tubules 22.Turbulence can increase the exchange efficiency between the blood and dialysate by increasing the amount of surface area contact between the dialysate and the blood tubules 22.Also, the pulsation can produce bursts of increased transmembrane pressure, which additionally helps exchange.","The increased energy introduced into the system can also enhance transport.","Further, reduction of “dead” regions reduces the tendency of clotting, with secondary improvement in transport.","As a result of these combined effects, the overall efficiency of hemodialysis (for example, increased clearance of urea and creatinine) can be significantly increased over systems that lack a pulsatile dialysate flow.","It should also be noted that the pump 16 of the blood subsystem 12 may also be a pulsatile pump, with many of the advantages described above for the dialysate subsystem 30 also being achievable with pulsatile flow in the blood subsystem 12.If a hemodialysis apparatus includes pulsatile pumps in both the blood and dialysate subsystems, the pulsatile pumps may be synchronous, such that they pump at the same rate and with matching amplitudes, or they may be dissynchronous, such that they pump at different rates and/or with mismatched amplitudes.","Given that reduction of dead regions in the HFAK 20 can improve transport, it may be preferred to employ dissynchronous pulsatile pumps, as doing so may increase turbulence.","The invention and the advantages achievable therewith will now be described in greater detail in the following non-limiting examples.","Example 1 In Vitro Analysis of Pulsatile Flow of Dialysate In vitro dialysis experiments were conducted using a pair of pools filled with liquid and performing dialysis on these liquids.","Experimental blood and dialysate circuits were created that each included a two-liter reservoir connected to a roller head pump (Sarns 7400 MDX, available from Sarns, Inc., Ann Arbor, Mich.).","The roller head pump was connected to a dialysis filter, which was in turn connected to the two liter reservoir to form a closed loop.","All connections were made with ⅜ inch plastic tubing.","For the control apparatus, the dialysis machine used was a Fresenius 2008H dialysis machine (available from Fresenius USA, Lexington, Mass.","), which employs a relatively constant flow pump.","The experimental system employed a Fresenius F7NR dialysis filter (available from Fresenius USA, Lexington, Mass.)","and a Sarns roller head pump that operated at 50 cycles per minute and at a pulse pressure of 80 mm Hg.","For each system, the two liter reservoir was filled with a mixture of tap water and urea (2 liters water to 9.8-10 g of urea) to be used as experimental blood.","This mixture had a measured level of 95-100 mg/dL urea.","Tap water was used as the experimental dialysate.","Experimental blood and dialysate were then allowed to flow through the system at predetermined rates.","Samples of fluid were drawn from the reservoir at 5 minute intervals for 35 minutes, and the urea level was measured with an Olympus AU640 instrument (available from Olympus Corp., Dallas, Tex.).","The results of the procedures can be seen in Tables 1 and 2 below and FIG.","3.TABLE 1 Dialysate Flow -500 cc/min Pulsatile Experimental Blood Compartment Only Blood Flow Urea (cc/min) Procedure Baseline 5 min 10 min 15 min 20 min 200 Control Dialysis 94 77 53 40 28 (18.1%) (43.6%) (57.4%) (70.2%) Variable Speed 97 65 39 23 14 Dialysis (33%) (59.8%) (76.3%) (85.6%) % Greater Efficiency 45 27 22 18 250 Control Dialysis 97 64 42 25 15 (34.0%) (56.7%) (74.2%) (84.5%) Variable Speed 95 57 32 18 9 Dialysis (40.0%) (66.3%) (81.1%) (6.6%) % Greater Efficiency 15 9.6 8.5 6.6 330 Control Dialysis 97 61 33 18 10 (37.0%) (65.9%) (81.4%) (89.7%) Variable Speed 94 56 27 14 7 Dialysis (40.0%) (71.3%) (85.1%) (92.6%) % Greater Efficiency 7.5 7.6 3.7 3.0 400 Control Dialysis 113 75 53 33 20 (33.6%) (53.1%) (70.8%) (82.3%) Variable Speed 114 55 30 14 7 Dialysis (51.8%) (73.7%) (87.7%) (93.9%) % Greater Efficiency 35.1 20.6 19.3 12.4 TABLE 2 Dialysate Flow-190 cc/min Pulsatile Experimental Dialysate Compartment Only Blood Flow Urea (cc/min) Procedure Baseline 5 min 10 min 15 min 215 Control Dialysis 97 80 62 38 (17.5%) (36.0%) (60.8%) Variable Speed 96 73 54 30 Dialysis (23.9%) (43.7%) (68.8%) % Greater 26.7 17.6 11.6 Efficiency As can be seen from Tables 1 and 2 and FIG.","3, the efficiency of hemodialysis increases significantly (as much as 35 percent) with the employment of a variable speed pump for pumping blood or dialysate.","Example 2 The experimental apparatus used in Example 1 was employed again to perform dialysis on a dog with the following changes.","A HB 500 filter was employed with the Century System III dialysis machine (both available from Gambro Corp., Lakewood, Colo.).","Also, rather than tap water, a standard 3K dialysate was used to hemodialyze the dog.","Pulsatile flow was induced in the blood subsystem with a roller head pump operating at 50 cycles/minute.","In addition, both blood urea nitrogen (BUN) and creatinine levels were measured (BUN was measured with a Olympus AU640 instrument and creatinine was measured by a Jaffe assay).","The results of the procedure are shown in Tables 3 and 4 and FIGS.","4 and 5.TABLE 3 BUN Level Procedure Baseline 60 min 120 min Post 30 min Control Dialysis 110 110 (0.0%) 80 (27.3%) 97 Variable Speed Dialysis 126 84 (33.3%) 69 (45.3%) 73 % Greater Efficiency 39.8% TABLE 4 Creatinine Level Procedure Baseline 60 min 120 min Post 30 min Control Dialysis 10.2 8.7 (14.7%) 7.3 (28.5%) 9.1 Variable Speed 11.8 7.7 (34.7%) 6.3 (44.7%) 7.4 Dialysis % Greater 56.7% 36.3% Efficiency These results again indicate that the variable speed dialysis procedure is significantly more efficient than the control apparatus.","The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof.","The invention is defined by the following claims, with equivalents of the claims to be included therein."]],"string":"[\n [\n \"Method and apparatus for treating renal disease with hemodialysis utilizing pulsatile pump\",\n \"A method of removing toxins from blood from a patient in need of such toxin removal includes: providing a countercurrent dialysis filter (20) having a blood compartment and a dialysate compartment separated from the blood compartment by a semi-permeable membrane; conveying blood from the patient through the blood compartment of a countercurrent filter (20) an back to the patient; and drawing dialysate from a reservoir (32) through the dialysate compartment of the countercurrent filter (20).\",\n \"At least one of the blood or dialysate experiences pulsatile flow.\",\n \"The steps are carried out such that blood toxins are drawn from the blood compartment through the semi-permeable membrane into the dialysate compartment.\"\n ],\n [\n \"1.A method of removing toxins from blood from a patient in need of such toxin removal, comprising: providing a countercurrent dialysis filter having a blood compartment and a dialysate compartment separated from said blood compartment by a semi-permeable membrane; conveying blood from the patient through the blood compartment of a filter an back to the patient; and drawing dialysate from a reservoir through the dialysate compartment of the filter such that blood toxins are drawn from said blood compartment through the semi-permeable membrane into the dialysate compartment; wherein at least one of the conveying and drawing steps is carried out with pulsatile flow.\",\n \"2.The method defined in claim 1, wherein said drawing step comprises drawing the dialysate through the dialysate compartment with a pulsatile pump.\",\n \"3.The method defined in claim 2, wherein said drawing step comprises drawing the dialysate through the dialysate compartment at a pulsatile pressure of greater than about 10 mm Hg.\",\n \"4.The method defined in claim 1, wherein said drawing step comprises drawing the dialysate through the dialysate compartment at between 30 and 100 pulses per minute.\",\n \"5.The method defined in claim 1, wherein said drawing step further comprises drawing the dialysate through the dialysate compartment with a roller head pump.\",\n \"6.The method defined in claim 1, wherein said conveying step comprises conveying blood from the patient through the blood compartment with a constant flow pump.\",\n \"7.The method defined in claim 1, wherein said providing step comprises providing a dialysis filter with a dialysate compartment comprising a dialysate casing and a blood compartment comprising a plurality of tubules that reside within the dialysate casing.\",\n \"8.The method defined in claim 1, wherein said conveying step comprises conveying blood with a pulsatile pump.\",\n \"9.The method defined in claim 1, wherein said conveying step comprises conveying the blood with a pulse pressure of greater than about 30 mm Hg.\",\n \"10.An apparatus for performing hemodialysis on a subject in need of such treatment, comprising: a dialysis filter having a blood compartment and a dialysate compartment separated from said blood compartment by a semi-permeable membrane; a first pump fluidly connected with the blood compartment that conveys blood from the patient through a blood compartment of the filter and back to the subject; and a second pump fluidly connected to the dialysate compartment; wherein at least one of said first and second pumps is configured to induce pulsatile flow.\",\n \"11.The apparatus defined in claim 10, wherein said second pump comprises a pulsatile pump.\",\n \"12.The apparatus defined in claim 11, wherein said second pump is configured to induce a pulsatile pressure of greater than about 10 mm Hg in dialysate in the dialysate compartment.\",\n \"13.The apparatus defined in claim 10, wherein said dialysate compartment comprises a dialysate casing and said blood compartment comprises a plurality of blood tubules residing within said dialysate casing.\",\n \"14.The apparatus defined in claim 10, further comprising a third constant flow pump fluidly connected to said second pump.\",\n \"15.The apparatus defined in claim 10, wherein said second pump is configured to provide pulsatile flow at a rate of between about 30 and 100 cycles per minute.\",\n \"16.The apparatus defined in claim 10, wherein said first pump and said second pump are pulsatile pumps.\",\n \"15.The apparatus defined in claim 10, wherein the pulsatile pressure induced by said first or second pump is at least 30 mm Hg.\",\n \"16.The apparatus defined in claim 10, wherein the pulsatile pressure induced by said first pump is at least 30 mm Hg.\"\n ],\n [\n \"<SOH> BACKGROUND OF THE INVENTION <EOH>Hemodialysis is a well-known treatment technique for ESRD, a condition in which the patient's renal system has essentially ceased to remove waste products and contaminants from the blood.\",\n \"Hemodialysis is a process that involves removing blood from the vasculature of a patient (usually a shunt or vein), purifying it with dialysate (a fluid that helps to remove toxins and return electrolytes to the blood), and returning the blood to the patient (usually through another vein).\",\n \"Hemodialysis machines typically operate with separate compartments for blood and dialysate.\",\n \"In conventional systems, the compartments are separated by a semi-permeable membrane that allows selective diffusion; toxins are removed from the blood, and electrolytes are added to bring the electrolyte concentration of the blood to desired levels.\",\n \"In many systems, the blood and dialysate compartments are arranged in a countercurrent flow exchange layout, with the blood traveling in one direction and the dialysate traveling in the opposite direction.\",\n \"One arrangement employs a large tube within which reside many smaller diameter tubules.\",\n \"Ordinarily, the large tube carries dialysate, and the smaller tubules carry blood.\",\n \"Blood is typically pumped through the tubules with a positive pressure pump (exemplary is a roller head pump), and the dialysate is typically drawn through the large tube with a roller head pump.\",\n \"There are some shortcomings with such an arrangement.\",\n \"The relatively constant flow of the blood and dialysate can create “dead” regions (where flow essentially stops) and regions of laminar flow within the tube.\",\n \"In each of these regions the component exchange between the blood and the dialysate can be adversely affected, as fluid in the dead regions and the regions of laminar flow tends to have less surface area contact with the membrane, thereby reducing the efficiency of exchange.\",\n \"Also, the constant flow of dialysate can create “shunting” of dialysate in certain regions, which can lead to blood/dialysate mismatch.\",\n \"As such, it would be desirable to provide a hemodialysis system with improved efficiency of exchange.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>The present invention can address some of the shortcomings of prior systems by improving the efficiency of dialysis through the use of pulsatile flow.\",\n \"As a first aspect, the invention includes a method of removing toxins from blood from a patient in need of such toxin removal, comprising: providing a dialysis filter having a blood compartment and a dialysate compartment separated from the blood compartment by a semi-permeable membrane; conveying blood from the patient through the blood compartment of a filter and back to the patient; and drawing dialysate from a reservoir through the dialysate compartment of the countercurrent filter.\",\n \"At least one of the blood or dialysate experiences pulsatile flow.\",\n \"The steps are carried out such that blood toxins are drawn from the blood compartment through the semi-permeable membrane into the dialysate compartment.\",\n \"As a second aspect, the invention includes an apparatus for performing hemodialysis on a subject in need of such treatment.\",\n \"The apparatus comprises: a dialysis filter having a blood compartment and a dialysate compartment separated from the blood compartment by a semi-permeable membrane; a first pump fluidly connected with the blood compartment that conveys blood from the patient through a blood compartment of a filter and back to the subject; and a second pump fluidly connected to the dialysate compartment.\",\n \"At least one of the first and second pumps is configured to induce pulsatile flow.\",\n \"It has been discovered that the use of pulsatile flow in the blood or dialysate circuits during hemodialysis can provide a number of advantages.\",\n \"These include increased efficiency of transport, reduction in dead regions, and intermittent increased transmembrane pressure.\",\n \"Objects of the present invention will be appreciated by those of ordinary skill in the art from a reading of the figures and the detailed description of the preferred embodiments which follow, such description being merely illustrative of the present invention.\"\n ],\n [\n \"FIELD OF THE INVENTION The present invention is directed generally to treatment of renal disease, and more specifically to treatment of end stage renal disease (ESRD) with hemodialysis.\",\n \"BACKGROUND OF THE INVENTION Hemodialysis is a well-known treatment technique for ESRD, a condition in which the patient's renal system has essentially ceased to remove waste products and contaminants from the blood.\",\n \"Hemodialysis is a process that involves removing blood from the vasculature of a patient (usually a shunt or vein), purifying it with dialysate (a fluid that helps to remove toxins and return electrolytes to the blood), and returning the blood to the patient (usually through another vein).\",\n \"Hemodialysis machines typically operate with separate compartments for blood and dialysate.\",\n \"In conventional systems, the compartments are separated by a semi-permeable membrane that allows selective diffusion; toxins are removed from the blood, and electrolytes are added to bring the electrolyte concentration of the blood to desired levels.\",\n \"In many systems, the blood and dialysate compartments are arranged in a countercurrent flow exchange layout, with the blood traveling in one direction and the dialysate traveling in the opposite direction.\",\n \"One arrangement employs a large tube within which reside many smaller diameter tubules.\",\n \"Ordinarily, the large tube carries dialysate, and the smaller tubules carry blood.\",\n \"Blood is typically pumped through the tubules with a positive pressure pump (exemplary is a roller head pump), and the dialysate is typically drawn through the large tube with a roller head pump.\",\n \"There are some shortcomings with such an arrangement.\",\n \"The relatively constant flow of the blood and dialysate can create “dead” regions (where flow essentially stops) and regions of laminar flow within the tube.\",\n \"In each of these regions the component exchange between the blood and the dialysate can be adversely affected, as fluid in the dead regions and the regions of laminar flow tends to have less surface area contact with the membrane, thereby reducing the efficiency of exchange.\",\n \"Also, the constant flow of dialysate can create “shunting” of dialysate in certain regions, which can lead to blood/dialysate mismatch.\",\n \"As such, it would be desirable to provide a hemodialysis system with improved efficiency of exchange.\",\n \"SUMMARY OF THE INVENTION The present invention can address some of the shortcomings of prior systems by improving the efficiency of dialysis through the use of pulsatile flow.\",\n \"As a first aspect, the invention includes a method of removing toxins from blood from a patient in need of such toxin removal, comprising: providing a dialysis filter having a blood compartment and a dialysate compartment separated from the blood compartment by a semi-permeable membrane; conveying blood from the patient through the blood compartment of a filter and back to the patient; and drawing dialysate from a reservoir through the dialysate compartment of the countercurrent filter.\",\n \"At least one of the blood or dialysate experiences pulsatile flow.\",\n \"The steps are carried out such that blood toxins are drawn from the blood compartment through the semi-permeable membrane into the dialysate compartment.\",\n \"As a second aspect, the invention includes an apparatus for performing hemodialysis on a subject in need of such treatment.\",\n \"The apparatus comprises: a dialysis filter having a blood compartment and a dialysate compartment separated from the blood compartment by a semi-permeable membrane; a first pump fluidly connected with the blood compartment that conveys blood from the patient through a blood compartment of a filter and back to the subject; and a second pump fluidly connected to the dialysate compartment.\",\n \"At least one of the first and second pumps is configured to induce pulsatile flow.\",\n \"It has been discovered that the use of pulsatile flow in the blood or dialysate circuits during hemodialysis can provide a number of advantages.\",\n \"These include increased efficiency of transport, reduction in dead regions, and intermittent increased transmembrane pressure.\",\n \"Objects of the present invention will be appreciated by those of ordinary skill in the art from a reading of the figures and the detailed description of the preferred embodiments which follow, such description being merely illustrative of the present invention.\",\n \"BRIEF DESCRIPTION OF THE FIGURES The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, serve to explain principles of the invention.\",\n \"FIG.\",\n \"1 is a schematic diagram of a hemodialysis apparatus of the present invention.\",\n \"FIG.\",\n \"2 is an enlarged schematic view of a hollow fiber artificial kidney (HFAK) included in the hemodialysis apparatus of FIG.\",\n \"1.FIG.\",\n \"2A is a greatly enlarged schematic view of two blood tubules of the HFAK of FIG.\",\n \"2.FIG.\",\n \"3 is a graph plotting urea concentration as a function of time in a two pool model of a dialysate.\",\n \"FIG.\",\n \"4 is a graph plotting creatinine concentration as a function of time collected in a dialysis study on dogs.\",\n \"FIG.\",\n \"5 is a graph plotting BUN concentration as a function of time collected in a dialysis study on a dog.\",\n \"DETAILED DESCRIPTION OF THE INVENTION The present invention now will be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments of the invention are shown and described.\",\n \"This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.\",\n \"Like numbers refer to like components throughout.\",\n \"Referring now to FIG.\",\n \"1, a hemodialysis apparatus, designated broadly at 10, is illustrated schematically.\",\n \"The hemodialysis apparatus 10 comprises a blood subsystem 12 and a dialysate subsystem 30, each of which will be described in greater detail below.\",\n \"The blood subsystem 12, which as the name implies conveys blood through the hemodialysis apparatus 10, includes a blood entry conduit 14 that leads from a patient's artery (usually in the wrist), a roller pump 16 fluidly connected with the blood entry conduit 14, an HFAK 20 of conventional construction, and a blood exit conduit 18 leading from the HFAK 20 back to the patient's vein.\",\n \"A bubble trap 19 is located on the blood exit conduit 18 between the HFAK 20 and the vein of the patient to prevent bubbles created during the processing of the blood from entering the patient.\",\n \"During operation, blood exits the patient through the blood entry conduit 14.The roller pump 16 forces the blood to the HFAK 20, wherein dialysis occurs as described below.\",\n \"The dialyzed blood then exits the HFAK 20 in the blood exit conduit 18, passes through the bubble trap 19 and returns to the patient's body.\",\n \"The dialysate subsystem 30, which conveys dialysate into and from the HFAK 20, includes a dialysate reservoir 32 that contains dialysate, a dialysate entry conduit 34 leading from the dialysate reservoir 32 to the HVAK 20, a dialysate exit conduit 36 leading from the HFAK 20, and an in-line pulsatile pump 38 located on the dialysate exit conduit 36.In operation, dialysate is pumped from the dialysate reservoir 32 through the dialysate entry conduit 34 and into the HFAK 20, wherein countercurrent exchange of components of the blood and dialysate.\",\n \"The spent dialysate is then pumped via the pulsatile pump 38 back to the dialysate reservoir 32.An exemplary HVAK 20 is illustrated in FIG.\",\n \"2.The HVAK 20 includes a plurality of narrow blood tubules 22 that are enclosed within a dialysate casing 24.As the name implies, blood is conveyed through the blood entry conduit 14 into the blood tubules 22, and dialysate is conveyed from the dialysate entry conduit 34 into the dialysate casing 24.The blood tubules 22 are formed of a semi-permeable membrane material, such as polysulfone, that enables countercurrent exchange of components to occur between the blood and dialysate.\",\n \"The dialysate casing 24 is typically formed of an impervious material such as a plastic material.\",\n \"The dialysate utilized in the dialysate subsystem 30 can be any dialysate known to those skilled in this art as being suitable for use in hemodialysis.\",\n \"An exemplary dialysate is available under the trade name Neutralyte from Fresenius Medical Care, Lexington, Mass.\",\n \"The use of the pulsatile pump 38 (which can be a piston pump, a modified roller head pump, or the like) in the dialysate subsystem 30 can have the effect of producing pulsatile flow of dialysate within the dialysate subsystem 30.As used herein, “pulsatile flow” means flow that has a pulse pressure of 10 mm Hg or greater, and preferably when applied to blood means a pulse pressure of 30 mm Hg or greater.\",\n \"Preferably, the pulsatile flow is induced in the dialysate at a pulse rate of between about 30 and 100 cycles per minute, and more preferably at a rate of between about 50 and 80 cycles per minute.\",\n \"Those skilled in this art will recognize that the pulsatile pump may be used alone or in combination with a more constant flow pump.\",\n \"Such pulsatile flow within the dialysate subsystem 30 can generate significant turbulence within the dialysate casing 24, which can reduce or eliminate the number of “dead” regions, where flow stops and exchange is minimal.\",\n \"A typical flow pattern in blood tubules 22 and the blood casing 24 is shown in FIG.\",\n \"2A (darker areas represent regions of higher flow), with a dead region 25 being illustrated between two tubules 22.Turbulence can increase the exchange efficiency between the blood and dialysate by increasing the amount of surface area contact between the dialysate and the blood tubules 22.Also, the pulsation can produce bursts of increased transmembrane pressure, which additionally helps exchange.\",\n \"The increased energy introduced into the system can also enhance transport.\",\n \"Further, reduction of “dead” regions reduces the tendency of clotting, with secondary improvement in transport.\",\n \"As a result of these combined effects, the overall efficiency of hemodialysis (for example, increased clearance of urea and creatinine) can be significantly increased over systems that lack a pulsatile dialysate flow.\",\n \"It should also be noted that the pump 16 of the blood subsystem 12 may also be a pulsatile pump, with many of the advantages described above for the dialysate subsystem 30 also being achievable with pulsatile flow in the blood subsystem 12.If a hemodialysis apparatus includes pulsatile pumps in both the blood and dialysate subsystems, the pulsatile pumps may be synchronous, such that they pump at the same rate and with matching amplitudes, or they may be dissynchronous, such that they pump at different rates and/or with mismatched amplitudes.\",\n \"Given that reduction of dead regions in the HFAK 20 can improve transport, it may be preferred to employ dissynchronous pulsatile pumps, as doing so may increase turbulence.\",\n \"The invention and the advantages achievable therewith will now be described in greater detail in the following non-limiting examples.\",\n \"Example 1 In Vitro Analysis of Pulsatile Flow of Dialysate In vitro dialysis experiments were conducted using a pair of pools filled with liquid and performing dialysis on these liquids.\",\n \"Experimental blood and dialysate circuits were created that each included a two-liter reservoir connected to a roller head pump (Sarns 7400 MDX, available from Sarns, Inc., Ann Arbor, Mich.).\",\n \"The roller head pump was connected to a dialysis filter, which was in turn connected to the two liter reservoir to form a closed loop.\",\n \"All connections were made with ⅜ inch plastic tubing.\",\n \"For the control apparatus, the dialysis machine used was a Fresenius 2008H dialysis machine (available from Fresenius USA, Lexington, Mass.\",\n \"), which employs a relatively constant flow pump.\",\n \"The experimental system employed a Fresenius F7NR dialysis filter (available from Fresenius USA, Lexington, Mass.)\",\n \"and a Sarns roller head pump that operated at 50 cycles per minute and at a pulse pressure of 80 mm Hg.\",\n \"For each system, the two liter reservoir was filled with a mixture of tap water and urea (2 liters water to 9.8-10 g of urea) to be used as experimental blood.\",\n \"This mixture had a measured level of 95-100 mg/dL urea.\",\n \"Tap water was used as the experimental dialysate.\",\n \"Experimental blood and dialysate were then allowed to flow through the system at predetermined rates.\",\n \"Samples of fluid were drawn from the reservoir at 5 minute intervals for 35 minutes, and the urea level was measured with an Olympus AU640 instrument (available from Olympus Corp., Dallas, Tex.).\",\n \"The results of the procedures can be seen in Tables 1 and 2 below and FIG.\",\n \"3.TABLE 1 Dialysate Flow -500 cc/min Pulsatile Experimental Blood Compartment Only Blood Flow Urea (cc/min) Procedure Baseline 5 min 10 min 15 min 20 min 200 Control Dialysis 94 77 53 40 28 (18.1%) (43.6%) (57.4%) (70.2%) Variable Speed 97 65 39 23 14 Dialysis (33%) (59.8%) (76.3%) (85.6%) % Greater Efficiency 45 27 22 18 250 Control Dialysis 97 64 42 25 15 (34.0%) (56.7%) (74.2%) (84.5%) Variable Speed 95 57 32 18 9 Dialysis (40.0%) (66.3%) (81.1%) (6.6%) % Greater Efficiency 15 9.6 8.5 6.6 330 Control Dialysis 97 61 33 18 10 (37.0%) (65.9%) (81.4%) (89.7%) Variable Speed 94 56 27 14 7 Dialysis (40.0%) (71.3%) (85.1%) (92.6%) % Greater Efficiency 7.5 7.6 3.7 3.0 400 Control Dialysis 113 75 53 33 20 (33.6%) (53.1%) (70.8%) (82.3%) Variable Speed 114 55 30 14 7 Dialysis (51.8%) (73.7%) (87.7%) (93.9%) % Greater Efficiency 35.1 20.6 19.3 12.4 TABLE 2 Dialysate Flow-190 cc/min Pulsatile Experimental Dialysate Compartment Only Blood Flow Urea (cc/min) Procedure Baseline 5 min 10 min 15 min 215 Control Dialysis 97 80 62 38 (17.5%) (36.0%) (60.8%) Variable Speed 96 73 54 30 Dialysis (23.9%) (43.7%) (68.8%) % Greater 26.7 17.6 11.6 Efficiency As can be seen from Tables 1 and 2 and FIG.\",\n \"3, the efficiency of hemodialysis increases significantly (as much as 35 percent) with the employment of a variable speed pump for pumping blood or dialysate.\",\n \"Example 2 The experimental apparatus used in Example 1 was employed again to perform dialysis on a dog with the following changes.\",\n \"A HB 500 filter was employed with the Century System III dialysis machine (both available from Gambro Corp., Lakewood, Colo.).\",\n \"Also, rather than tap water, a standard 3K dialysate was used to hemodialyze the dog.\",\n \"Pulsatile flow was induced in the blood subsystem with a roller head pump operating at 50 cycles/minute.\",\n \"In addition, both blood urea nitrogen (BUN) and creatinine levels were measured (BUN was measured with a Olympus AU640 instrument and creatinine was measured by a Jaffe assay).\",\n \"The results of the procedure are shown in Tables 3 and 4 and FIGS.\",\n \"4 and 5.TABLE 3 BUN Level Procedure Baseline 60 min 120 min Post 30 min Control Dialysis 110 110 (0.0%) 80 (27.3%) 97 Variable Speed Dialysis 126 84 (33.3%) 69 (45.3%) 73 % Greater Efficiency 39.8% TABLE 4 Creatinine Level Procedure Baseline 60 min 120 min Post 30 min Control Dialysis 10.2 8.7 (14.7%) 7.3 (28.5%) 9.1 Variable Speed 11.8 7.7 (34.7%) 6.3 (44.7%) 7.4 Dialysis % Greater 56.7% 36.3% Efficiency These results again indicate that the variable speed dialysis procedure is significantly more efficient than the control apparatus.\",\n \"The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof.\",\n \"The invention is defined by the following claims, with equivalents of the claims to be included therein.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10467755"}}},{"rowIdx":383,"cells":{"text":{"kind":"list like","value":[["Dynamic management of access rights lists in a portable electronic object","Access rights lists, such as capacity or access control lists, are dynamically managed in a data processing element such as a smart card from an administrator server.","To access an access rights list from the server, the list is signed in the server so that a signature can be transmitted to the card.","The card compares the signature received from the server to signatures determined according to the access rights lists contained in the card and keys associated with those lists.","Server access to an identified list is only authorized when it corresponds to a signature which is found among the determined signatures in the card and which is identical to the received signature."],["1.A method for managing lists of rights of access between subjects and objects, stored in a data processing means from an external administrator entity, comprising the following steps: initially associating keys of administrator entities with access rights lists and storing a security algorithm in the data processing means, and subsequently accessing an access rights list from the entity by: signing the access rights list in the entity by applying determined data from the list and an associated key to the security algorithm in order to produce a signature, transmitting the signature from the entity to the data processing means, comparing the signature received in the data processing means with determined signatures according to applications of determined data in lists of access rights contained in the data processing means and of keys respectively associated with these lists to the security algorithm, and allowing access of the entity to an access rights list found only if its determined signature is identical to the received signature.","2.A method according to claim 1, wherein the association of the keys with the access rights lists is performed in advance in the processing means prior to bringing the data processing means into service.","3.A method according to claim 1, wherein the association of the keys with an access rights list to be added to the data processing means is performed by transmitting the list with the keys from the entity to the data processing means, and determination of signatures of the list received in the data processing means is performed by applying the determined data in the list received and the keys received to the algorithm, and validation of the recording of the list received in the data processing means occurs when one of the signatures determined is identical with the signature received.","4.A method according to claim 1, wherein the signing step is replaced by the reception in the entity of a signature transmitted by another entity.","5.A method according to claim 1, wherein delegation information is transmitted with the signature, and access of the entity to the list found is not authorised when the delegation information is detected in association with the list found in the data processing means.","6.A method according to claim 1, wherein the signatures to be compared with the signature received are determined in the data processing means prior to the transmitting step.","7.A method according to claim 1, further including the step before authorising access of the entity to the control list, of updating a duration of life parameter of the access rights list found in order to erase the access rights list found when the updated duration of life parameter exceeds a maximum limit and in order to authorise access to the access rights list found to the entity when the updated duration of life parameter is less than the maximum limit.","8.A method according to claim 7, wherein the duration of life parameter is a cumulative duration of sessions of use of the data processing means or a cumulative duration of absolute time.","9.A method according to claim 7, wherein the duration of life parameter is a number of sessions of use of the data processing means, or a number of sessions using the access rights list found.","10.A method according to claim 7, wherein the duration of life parameter is a number of commands received by the data processing means.","11.A method according to claim 7, wherein the duration of life parameter is a synchronisation value changed periodically in external administrator entities and transmitted with the signature of the access rights list.","12.A method according to claim 1, wherein the signatures of the access list associated with the keys of a list are again determined in the data processing means if the access rights list found has been modified by the entity.","13.A method according to claim 1, wherein the determined data of the list which are applied to the security algorithm depend on characteristics of at least one subject and/or at least one subject group and/or at least one object and/or at least one right of access of a subject to an object relating to the list.","14.A data processing means storing access rights lists managed from at least one external administrator entity comprising: a means for storing keys of administrator entities in association with access rights lists, a means for implementing a security algorithm, a means for determining signatures according to applications of determined data of the lists of access rights and keys respectively associated with said lists to the algorithm, a means for comparing a received signature of an access rights list which results, in the entity, from the application of determined data of the list and of the key of the entity to the algorithm and which is transmitted by the entity, to said determined signatures, and a means for authorising access of the entity to an access rights list found only in correspondence with a signature found amongst the determined signatures and identical to the received signature.","15.A data processing means according to claim 14, further including a means for storing a duration of life parameter and a maximum duration limit for each access rights list, and a means for updating the duration parameter of the list found in order to erase the access rights list found when the duration of life parameter updated exceeds the maximum limit and in order to authorise access to the access list found to the entity when the duration of life parameter updated is less than the maximum limit."],["The present invention relates in general to terms the management of rights of access, by subjects constituting users or software modules of a data processing means, to objects such as applications implemented in the data processing means.","More particularly, the invention relates to the administration of accesses to resources in a portable electronic object, such as a chip card, also known as a microcontroller card or integrated circuit card, constituting the data processing means, in particular when the chip card is a multiapplication card.","Because of the greater and greater number of more and more complex applications introduced into a chip card, the management of the applications constituting the principal resources of the chip card are more and more difficult to manage.","The difficulties in management are also due to the many partners participating in the allocation of access to the applications and whose interests sometimes diverge.","These partners may be the manufacturer of the chip card, the distributor or operator of the chip card, and the developers of the applications in the chip card.","Nevertheless, despite this complexity, access to the chip card resources must be controlled and made secure.","At the present time, access to a processing resource, such as an application, is made by transmitting, from a terminal accepting the chip card, at least one command constituting an application protocol data unit (APDU) which contains data or a reference to data present and to be processed in the card.","According to another variant, access to a resource in the card can be effected at a higher level by invoking a method of an object present in the card when the latter contains applications written in an object oriented high level programming language such as Java.","The coexistence and cooperation of several applications within the same chip card raises many problems from the point of view of security.","In particular, each application has its own data for which the developer of the application defines access rights peculiar to the application.","The access conditions are means of connection between external accesses which may be users of the card or software modules, such as user interfaces, and accesses internal to the card such as applications, possibly by means of other applications or other application software elements in the card.","The control of the access conditions is based on the identification of the subjects Su, such as the users, which are the “active” elements which manipulate information contained in objects Ob, such as applications, which are “passive” elements containing data.","The conditions for the access of the subjects Su to the objects Ob are governed by access control rules between the subjects and the objects.","Each rule comprises an access right, that is to say a link between a subject and an object in the form of an action which can be performed by the subject on the object.","It is known how to represent the conditions for access of subjects Su to objects Ob by an access matrix MA whose columns correspond to subjects and whose rows correspond to objects, as shown in FIG.","1.For example, the matrix MA relates to three subjects S1, S2 and S3, such as three users, and to three objects O1, O2 and O3, such as files and programs.","Each box in the matrix at the intersection of a row and column contains access rights, that is to say privileged actions which can be performed by the respective subject on the respective object.","The access rights may be positive in order to allow a predetermined action by a subject on an object, or may be negative in order to prevent a predetermined action by a subject or an object.","For example, the subject S2 can read and execute the object O2 but cannot write in this object, and the subject S3 can read the object O1 but cannot record and write to the object O1.As is known, the access control rules are generally dealt with according to two approaches.","The first approach consists of access control lists (ACL) corresponding to the rows in the access matrix MA and each specifying rights of access by subjects to the object associated with the row.","By way of example, in a multiapplication chip card of the Windows (registered trade mark) type, access control lists ACL define accesses by users to files included in the card.","Conversely, the second approach consists of capacities corresponding to the columns in the matrix MA and each specifying the access rights of the subject associated with the column over the objects.","For example, the access control relates to applet methods for multiapplication smart cards of the JavaCard type in which programs in Java language have been written.","The capacities are in the form of pointers making calls for accessing methods constituting objects, in predetermined applets constituting subjects.","For more simplicity, reference will be made hereinafter to the management of access control lists although the invention also relates to the management of capacities.","Access control lists and capacities are to be considered to be lists of rights of access between at least one subject and at least one object.","With a present-day chip card, the modification of access control lists is reserved for only one card administration authority.","After authentication of the administration authority by the card, the authority demands modifications to the access control lists, for example by adding or eliminating lists, adding or eliminating subjects in a list, or adding or eliminating access rights of a subject with respect to an object.","This single administration authority must of course comply with the requirements of the various partners participating in the production and management of the various application resources in the chip card.","The objective of the present invention is to make possible the dynamic management of access control lists or capacities in a portable electronic object of the chip card type in order to refine the management of such lists or capacities and thus permit an increase in the number of administrators authorised to act with regard to security on modifications to the access control lists or capacities.","To achieve this objective, a method for managing lists of rights of access between subjects and objects, stored in a data processing means from an external administrator entity, is characterised in that it comprises the following steps: initially associating keys of administrator entities with access rights lists and supplying a security algorithm in the data processing means, and in order to access an access rights list from the entity: signing the access rights list in the entity by applying determined data from the list and the key to the security algorithm in order to produce a signature, transmitting the signature from the entity to the data processing means, and comparing the signature received in the data processing means with determined signatures according to applications of determined data in lists of access rights contained in the data processing means and of keys respectively associated with these lists to the security algorithm, and allowing access of the entity to an access rights list found only in correspondence with a signature found amongst the determined signatures in the data processing means and identical to the received signature.","Thus, according to the invention, any authorised administrator accesses an access rights list constituting an access control list or a capacity in a data processing means consisting for example of the microcontroller of a portable electronic object.","An access right authorises or prohibits one or more subjects from performing an action on a subject, or a subject from performing an action on one or more objects.","This access control list or capacity is then managed dynamically by each administrator, by obliging it first to sign the access control list or the capacity to which it wishes to gain access, so as to be recognised in the electronic object by its signature of the access control list or of the capacity.","Management of the access control lists is thus decentralised in administrator entities external to the data processing means.","According to another characteristic of the invention, the duration of application of an access rights list is limited.","More precisely, before authorising access of the entity to the control list, a duration of life parameter of the access rights list found is updated in order to erase the access rights list found when the duration of life parameter updated exceeds a maximum limit and in order to allow access to the access rights list found to the entity when the duration of life parameter updated is less than the maximum limit.","The limited duration of life of an access rights list then prompts the subject or subjects affected by this list which has become out of date and is now erased to return to the administrator or administrators of this list in order to once again request access rights from them.","The invention also relates to a data processing means in particular in a portable electronic object, storing access rights lists managed from at least one external administrator entity, implementing the method of the invention.","It is characterised in that it comprises: a means for storing keys of administrator entities in association with access rights lists, a means for implementing a security algorithm, a means for determining signatures according to applications of determined data of the lists of access rights and keys respectively associated with these lists to the algorithm, a means for comparing a received signature of an access rights list which results, in the entity, from the application of determined data of the list and of the key of the entity to the algorithm and which is transmitted by the entity, to the said determined signatures, and a means for authorising access of the entity to an access rights list found only in correspondence with a signature found amongst the determined signatures and identical to the received signature.","The data processing means can also comprise a means for storing a duration of life parameter and a maximum duration limit for each access rights list, and a means for updating the duration parameter of the list found in order to erase the access rights list found when the duration of life parameter updated exceeds the maximum limit and in order to authorise access to the access list found to the entity when the duration of life parameter updated is less than the maximum limit.","Other characteristics and advantages of the present invention will emerge more clearly from a reading of the following description of several preferred embodiments of the invention with reference to the corresponding accompanying drawings, in which: FIG.","1 is a diagram showing a control matrix between three subjects and three objects, already commented on according to the prior art; FIG.","2 is a schematic block diagram of a telecommunications system between an administrator server and a portable electronic object of the chip card type for implementing the management method according to the invention; and FIG.","3 is an algorithm of steps of the method of dynamic management of access rights lists according to the invention.","With reference to FIG.","2, the data processing means is a controller, such as the microcontroller of a chip card CA which contains several applications constituting objects to which subjects, such as users or units or software means, can gain access according to access rights.","In a known manner, the microcontroller of the chip card CA comprises a microprocessor PR, a memory MO of the ROM type, a non-volatile memory MNV of the programmable and erasable type, such as a EEPROM memory, and a memory MA of the RAM type receiving in particular data from a terminal TE accepting the card.","In the memory MO there are included in particular an operating system OS of the card, communication, authentication and service applications AP constituting objects Ob, as well as a security algorithm AS used for recognising administrator signatures and an algorithm for managing access rights lists AG according to the invention.","The memory MNV contains data in particular relating to the possessor of the card and to the supplier of the card, as well as access control lists ACL(Ob), or capacities.","Each access control list relating to a respective object Ob, to which reference is made below as an access rights list, contains, as shown in FIG.","1, a list of subjects Su each associated with a list of positive access rights authorising and/or negative access rights prohibiting the performance of actions on the respective object Ob.","The memory MNV thus contains a subject set ES, an object set EO, that is to say a set referring to applications, and an access rule set ER.","The memory MNV preferably also contains a subject group set EG.","Each group Gp contains subjects Su each having at least one predetermined access to a predetermined object Ob; thus a subject in a group Gp has all the access rights granted to this group, and a subject can belong to one or more groups.","A rule R for access to an object Ob concerns an action, such as for example reading, writing, execution or recording, which is authorised or prohibited to a subject Su or to a subject group Gp on an object Ob.","Consequently an access control list ACL(Ob) relates to access rules for predetermined subjects and/or predetermined groups on the respective object Ob.","According to the invention, when the card is manufactured, or before the card CA is brought into service, that is to say before it is passed to the owner of the card, administrators of the lists ACL contained in the card are defined at an initial step ET01.Each administrator AD is defined by a respective administrator key KAD which is associated with one or more access control lists ACL to which the administrator AD has access.","Each access control list ACL is associated with an administrator sub-list consisting of the corresponding administrator keys KAD.","As will be seen hereinafter, the administrator key list KAD is preferably supplemented by an administrator signature list SGAD.","A signature represents the access control list ACL signed by the respective administrator according to the security algorithm AS.","Thus, according to the invention, the administrator keys KAD and preferably the administrator signatures SGAD are previously written in the non-volatile memory MNV of the chip card CA in correspondence with the access control lists ACL.","FIG.","2 also shows diagrammatically an administrator entity external to the card CA in the form of an administrator server SAD connected to the chip card through a telecommunication network RT and the accepting terminal TE.","The telecommunication network RT designates any type of telecommunication network, or combination of networks, such as a radiotelephony network, a switched telephone network, an integrated service digital network ISDN, a high-rate network of the ATM type, the Internet, a packet transmission network, etc.","The server SAD represents an administrator or several administrators.","Other administrators can be situated at other distant servers (not shown).","The administrator AD is for example the distributor of the chip card CA, or a partner commercially associated with this distributor, or the developer of one or more applications AP constituting objects Ob implemented in the chip card CA.","As a variant, an administrator himself possesses a chip card which is housed in an additional reader of the server SAD so that the server SAD reads in the chip card the references of the administrator such as an administrator identifier IAD and an administrator key KAD.","As shown schematically also in FIG.","2, the accepting terminal TE is provided with a keypad CL and a reader LE for receiving the chip card CA and connecting thereto by means of an electrical-contact connection LI according to this embodiment.","The accepting terminal TE is for example a chip card commissioning terminal, a bank terminal, or a point of sale terminal, or a mobile telephony terminal in which the chip card CA constitutes a removable subscriber identity module SIM or an additional chip card when the mobile terminal is provided with an additional card reader.","After the initial step ET01, the management method according to the invention preferably comprises a second initial step ET02 during which administrator signatures SGAD are determined.","Step ET02 is also implemented for a list ACL which has just been modified or introduced into the memory MNV in the card CA.","Each signature SGAD of an administrator SAD depends on the key KAD of the administrator and determined data in an access control list ACL with which the key KAD is associated.","The determined data in the access control list ACL depend essentially on the characteristics of the objects Ob and the subjects Su and/or groups of subjects Gp each having at least one right of access to the object Ob, as well as where applicable access rights characteristics.","The list ACL and the key KAD are applied to the security algorithm AS, the result of which constitutes the administrator signature SGAD.","The management method shown in FIG.","3 comprises principally steps ET2 to ET14 triggered by a first step ET1 during which the administrator server SAD requests the establishment of a call with the accepting terminal TE containing the chip card CA through the telecommunication network RT, so as to initiate an access control list management session with the chip card CA.","At the start of this session the card CA attempts to authenticate the administrator SAD at step ET2.The authentication is conventional and consists essentially in transmitting a random number by means of the chip card CA to the server SAD and comparing in the chip card CA the results of the application of this random number and of an authentication key prestored in the card CA and the server SAD, performed both in the card CA and the server SAD.","Conversely, the server SAD authenticates the chip card CA.","According to another variant, the authentication is mutual and comprises an authentication of the server SAD by the card CA and an authentication of the card CA by the server SAD.","If the authentication or one of the authentications at step ET2 gives different results in the server SAD and the chip card CA, the call is broken off at a final step ET14.In a variant, the management method comprises no authentication.","When the authentication has succeeded, the administrator server SAD selects an access control list ACL which it has under management locally in a table of access control lists, at the following step ET3, although the administrator can manage only one access control list ACL.","The server SAD next signs the selected access control list ACL by applying the determined data of the list ACL and the key KAD of the administrator server SAD to the security algorithm AS so as to produce an administrator signature SGAD at step ET4.In a variant, instead of itself signing the access rights list ACL and producing the administrator signature SGAD, the server SAD receives this signature SGAD transmitted by another main administrator editor, such as a main administrator server connected to the telecommunication network RT, after the main administrator server has authenticated the server SAD.","Then the signature SGAD constituting the selected access control list ACL signed is transmitted by the server SAD to the chip card CA through the network RT and the terminal TE in the form of an appropriate message at step ET5.This message can contain other data such as an administrator identifier IAD which designates a respective table of access control lists TACL in the chip card CA.","The table TACL contains identifiers of access control lists to which the administrator server SAD has access, and preferably contains signatures of these access control lists signed by the administrator server SAD.","At the following step ET6, in response to the administrator signature SGAD, the microprocessor PR in the chip card CA seeks an access control list which is able to be signed by the administrator server SAD.","This search can consist in determining all the administrator signatures respectively associated with the access control lists which the chip card CA contains, and then comparing the signatures determined with the signature SGAD received by the card CA.","However, as indicated at step ET02, the processor PR of the chip card CA has preferably, prior at least to step ET5, determined the signatures SGAD corresponding to all the administrators respectively associated with the access control lists ACL and has written them in EEPROM memory MNV.","The processor PR then compares the signature SGAD received at step ET5 only with the signatures previously determined and classified in the table TACL.","If the processor PR finds no administrator signature in memory MNV identical to the signature SGAD transmitted by the server SAD, the processor PR invites the accepting terminal TE to break off the communication at step ET14.In a variant, when the administrator identifiers IAD are provided in the memory MNV, the processor PR compares the received signature SGAD only with the signatures contained in the table TACL designated by the received identifier IAD and associated with the key KAD, that is to say only with the signatures associated with access control lists to which the administrator server SAD has access.","Thus, after step ET6, an access control list ACL corresponding to the received administrator signature SGAD is found in order to process it by means of the server SAD, as indicated at step ET7.Although, according to a simple variant, step ET7 is followed by a step ET10 allowing access of the administrator server SAD to the access control list ACL previously found, the invention provides intermediate steps ET8 and ET9 during which a duration of life parameter pdv of the access rights list ACL found is updated and compared with a maximum limit PDV.","The parameter pdv and the limit PDV are written in the memory MNV of the card.","The limit PDV expresses the duration of life of the access control list ACL found at step ET7 and is prestored in the memory MNV at the initial step ET01, when the card CA is manufactured or brought into service, or when there is an addition of the list ACL, as will be seen subsequently.","At the initial step ET01, the duration of life parameter pdv is zeroed so that it is incremented each time step ET8 is executed.","The duration of life parameter pdv can be a cumulative duration of sessions of use of the chip card CA expressed in hours and minutes like the limit PDV.","According to a second variant, the parameter pdv is a cumulative duration of absolute time expressed in terms of date and time, the limit PDV then designating a maximum predetermined duration or an expiry date of the determined list ACL.","The updating of the duration can be carried out in the card CA by interrogating a trustworthy time stamping device, for example included in the terminal TE, or in the administrator server SAD.","According to other variants, the duration of life parameter pdv is a number of sessions of use of the chip card CA, or a number of sessions using the list of access rights found ACL, the limit ACL being a maximum number of sessions.","According to yet another variant, the duration of life parameter pdv is a number of commands APDU received by the chip card CA, the limit PDV then being a maximum number of commands.","According to yet another variant, the duration of life parameter is a synchronisation value changed periodically in the administrator servers and transmitted to the card by any administrator server SAD in each message which contains the signature SGAD of the list of access rights sought ACL, at step ET5, the limit PDV being at the maximum number.","Step ET8 thus increments the duration of life parameter according to its nature, the increment being in particular a period or a difference in date, or a unit, or a number of commands.","If at step ET9 the duration of life parameter pdv exceeds the limit PDV, the processor PR erases the access control list ACL which was found at step ET7 and all the data associated with the list ACL at step ET13, and breaks off the communication with the server SAD at step ET14.In a variant, step ET13 only partially erases the list found, for example by erasing therein only the positive or negative access rights.","If the duration of life is not yet reached at step ET9, the administrator server SAD is authorised to effectively gain access to the access control list ACL found, at step ET10.The server SAD then proceeds with a modification of the access control list found ACL.","This modification consists for example in totally erasing the access control list ACL in the memory MNV, or modifying one of the data in the list found ACL, in particular eliminating or adding a subject Su in the access control list found with corresponding access rights R, or eliminating or adding at least one access right R relating to a subject Su included in the list found.","The modification can also consist in adding a new administrator key to be associated with the list found and included in the message transmitted at step ET5.If at step ET11 the access control list found ACL has not been modified or has been erased, the method passes directly to the breaking off of the communication at step ET14.On the other hand, if the access list found ACL has been modified by the server SAD, this modified list being designated by ACLm, the processor PR determines, at step ET12, new administrator signatures SGADm resulting from the application of the determined data in the modified access control list ACLm and respectively administrator keys KAD associated with the list, to the security algorithm AS.","The signatures SGADm replace the previous signatures SGAD so that, during a future list management session, the administrator server SAD can access the control list ACLm thus modified in the memory MNV of the card CA.","Then the management method ends with the breaking off of the communication between the server SAD and the card CA at step ET14.When an access control list established in the administrator server SAD is to be added in the memory MNV of the chip card CA, steps ET1 to ET5 are executed, the message transmitted at step ET5 also containing the list ACL to be added with the administrator keys KAD associated with the list.","Then, in place of steps ET6 to ET13, the processor PR determines and stores the signatures SGAD of the list received in the respective list tables TACL in the card CA.","For each administrator associated with the list, the respective signature SGAD results from application of the determined data of the list and of the respective key KAD received to the security algorithm AS.","The processor PR compares the signatures determined with the signature received, as shown at a step ET15 in dotted lines in FIG.","3.The recording of the list received and of the keys and associated administrator signatures in the memory MNV is validated at a step ET16 when one of the signatures determined is identical to the signature received.","Otherwise step ET14 breaks off communication between the server SAD and the card CA.","According to a variant of the embodiment described above, the server SAD is an entity delegated by another administrator server which has transmitted to it delegation rights information IDR.","Delegation rights information IDR was previously stored in the memory MNV of the chip card CA at the initial step ET01 and is transmitted in the message containing the administrator signature SGAD at step ET5.At step ET6, the chip card CA authorises access to the access control list ACL when both the signature SGAD is recognised in the table TACL associated with the key KAD of the server SAD and the delegation rights information IDR is detected in association with the list ACL recognised in the table TACL of the card.","Otherwise communication is broken off at step ET14.Although the invention has been described in relation to a chip card containing access control lists associated respectively with signatures, the chip card can also contain access control lists which are not signed.","Thus an administrator server SAD can decide to erase all the access control lists loaded in the chip card CA and associated with the key KAD of the server SAD, transmitting the corresponding signatures at step ET4.As already stated, everything which has been described above for access control lists in the chip card CA, or in any other portable electronic object such as a personal electronic assistant or organiser, an electronic purse, a token or a pocket calculator, is applicable to an access rights list making several subjects correspond to an object, such as a capacity."]],"string":"[\n [\n \"Dynamic management of access rights lists in a portable electronic object\",\n \"Access rights lists, such as capacity or access control lists, are dynamically managed in a data processing element such as a smart card from an administrator server.\",\n \"To access an access rights list from the server, the list is signed in the server so that a signature can be transmitted to the card.\",\n \"The card compares the signature received from the server to signatures determined according to the access rights lists contained in the card and keys associated with those lists.\",\n \"Server access to an identified list is only authorized when it corresponds to a signature which is found among the determined signatures in the card and which is identical to the received signature.\"\n ],\n [\n \"1.A method for managing lists of rights of access between subjects and objects, stored in a data processing means from an external administrator entity, comprising the following steps: initially associating keys of administrator entities with access rights lists and storing a security algorithm in the data processing means, and subsequently accessing an access rights list from the entity by: signing the access rights list in the entity by applying determined data from the list and an associated key to the security algorithm in order to produce a signature, transmitting the signature from the entity to the data processing means, comparing the signature received in the data processing means with determined signatures according to applications of determined data in lists of access rights contained in the data processing means and of keys respectively associated with these lists to the security algorithm, and allowing access of the entity to an access rights list found only if its determined signature is identical to the received signature.\",\n \"2.A method according to claim 1, wherein the association of the keys with the access rights lists is performed in advance in the processing means prior to bringing the data processing means into service.\",\n \"3.A method according to claim 1, wherein the association of the keys with an access rights list to be added to the data processing means is performed by transmitting the list with the keys from the entity to the data processing means, and determination of signatures of the list received in the data processing means is performed by applying the determined data in the list received and the keys received to the algorithm, and validation of the recording of the list received in the data processing means occurs when one of the signatures determined is identical with the signature received.\",\n \"4.A method according to claim 1, wherein the signing step is replaced by the reception in the entity of a signature transmitted by another entity.\",\n \"5.A method according to claim 1, wherein delegation information is transmitted with the signature, and access of the entity to the list found is not authorised when the delegation information is detected in association with the list found in the data processing means.\",\n \"6.A method according to claim 1, wherein the signatures to be compared with the signature received are determined in the data processing means prior to the transmitting step.\",\n \"7.A method according to claim 1, further including the step before authorising access of the entity to the control list, of updating a duration of life parameter of the access rights list found in order to erase the access rights list found when the updated duration of life parameter exceeds a maximum limit and in order to authorise access to the access rights list found to the entity when the updated duration of life parameter is less than the maximum limit.\",\n \"8.A method according to claim 7, wherein the duration of life parameter is a cumulative duration of sessions of use of the data processing means or a cumulative duration of absolute time.\",\n \"9.A method according to claim 7, wherein the duration of life parameter is a number of sessions of use of the data processing means, or a number of sessions using the access rights list found.\",\n \"10.A method according to claim 7, wherein the duration of life parameter is a number of commands received by the data processing means.\",\n \"11.A method according to claim 7, wherein the duration of life parameter is a synchronisation value changed periodically in external administrator entities and transmitted with the signature of the access rights list.\",\n \"12.A method according to claim 1, wherein the signatures of the access list associated with the keys of a list are again determined in the data processing means if the access rights list found has been modified by the entity.\",\n \"13.A method according to claim 1, wherein the determined data of the list which are applied to the security algorithm depend on characteristics of at least one subject and/or at least one subject group and/or at least one object and/or at least one right of access of a subject to an object relating to the list.\",\n \"14.A data processing means storing access rights lists managed from at least one external administrator entity comprising: a means for storing keys of administrator entities in association with access rights lists, a means for implementing a security algorithm, a means for determining signatures according to applications of determined data of the lists of access rights and keys respectively associated with said lists to the algorithm, a means for comparing a received signature of an access rights list which results, in the entity, from the application of determined data of the list and of the key of the entity to the algorithm and which is transmitted by the entity, to said determined signatures, and a means for authorising access of the entity to an access rights list found only in correspondence with a signature found amongst the determined signatures and identical to the received signature.\",\n \"15.A data processing means according to claim 14, further including a means for storing a duration of life parameter and a maximum duration limit for each access rights list, and a means for updating the duration parameter of the list found in order to erase the access rights list found when the duration of life parameter updated exceeds the maximum limit and in order to authorise access to the access list found to the entity when the duration of life parameter updated is less than the maximum limit.\"\n ],\n [\n \"The present invention relates in general to terms the management of rights of access, by subjects constituting users or software modules of a data processing means, to objects such as applications implemented in the data processing means.\",\n \"More particularly, the invention relates to the administration of accesses to resources in a portable electronic object, such as a chip card, also known as a microcontroller card or integrated circuit card, constituting the data processing means, in particular when the chip card is a multiapplication card.\",\n \"Because of the greater and greater number of more and more complex applications introduced into a chip card, the management of the applications constituting the principal resources of the chip card are more and more difficult to manage.\",\n \"The difficulties in management are also due to the many partners participating in the allocation of access to the applications and whose interests sometimes diverge.\",\n \"These partners may be the manufacturer of the chip card, the distributor or operator of the chip card, and the developers of the applications in the chip card.\",\n \"Nevertheless, despite this complexity, access to the chip card resources must be controlled and made secure.\",\n \"At the present time, access to a processing resource, such as an application, is made by transmitting, from a terminal accepting the chip card, at least one command constituting an application protocol data unit (APDU) which contains data or a reference to data present and to be processed in the card.\",\n \"According to another variant, access to a resource in the card can be effected at a higher level by invoking a method of an object present in the card when the latter contains applications written in an object oriented high level programming language such as Java.\",\n \"The coexistence and cooperation of several applications within the same chip card raises many problems from the point of view of security.\",\n \"In particular, each application has its own data for which the developer of the application defines access rights peculiar to the application.\",\n \"The access conditions are means of connection between external accesses which may be users of the card or software modules, such as user interfaces, and accesses internal to the card such as applications, possibly by means of other applications or other application software elements in the card.\",\n \"The control of the access conditions is based on the identification of the subjects Su, such as the users, which are the “active” elements which manipulate information contained in objects Ob, such as applications, which are “passive” elements containing data.\",\n \"The conditions for the access of the subjects Su to the objects Ob are governed by access control rules between the subjects and the objects.\",\n \"Each rule comprises an access right, that is to say a link between a subject and an object in the form of an action which can be performed by the subject on the object.\",\n \"It is known how to represent the conditions for access of subjects Su to objects Ob by an access matrix MA whose columns correspond to subjects and whose rows correspond to objects, as shown in FIG.\",\n \"1.For example, the matrix MA relates to three subjects S1, S2 and S3, such as three users, and to three objects O1, O2 and O3, such as files and programs.\",\n \"Each box in the matrix at the intersection of a row and column contains access rights, that is to say privileged actions which can be performed by the respective subject on the respective object.\",\n \"The access rights may be positive in order to allow a predetermined action by a subject on an object, or may be negative in order to prevent a predetermined action by a subject or an object.\",\n \"For example, the subject S2 can read and execute the object O2 but cannot write in this object, and the subject S3 can read the object O1 but cannot record and write to the object O1.As is known, the access control rules are generally dealt with according to two approaches.\",\n \"The first approach consists of access control lists (ACL) corresponding to the rows in the access matrix MA and each specifying rights of access by subjects to the object associated with the row.\",\n \"By way of example, in a multiapplication chip card of the Windows (registered trade mark) type, access control lists ACL define accesses by users to files included in the card.\",\n \"Conversely, the second approach consists of capacities corresponding to the columns in the matrix MA and each specifying the access rights of the subject associated with the column over the objects.\",\n \"For example, the access control relates to applet methods for multiapplication smart cards of the JavaCard type in which programs in Java language have been written.\",\n \"The capacities are in the form of pointers making calls for accessing methods constituting objects, in predetermined applets constituting subjects.\",\n \"For more simplicity, reference will be made hereinafter to the management of access control lists although the invention also relates to the management of capacities.\",\n \"Access control lists and capacities are to be considered to be lists of rights of access between at least one subject and at least one object.\",\n \"With a present-day chip card, the modification of access control lists is reserved for only one card administration authority.\",\n \"After authentication of the administration authority by the card, the authority demands modifications to the access control lists, for example by adding or eliminating lists, adding or eliminating subjects in a list, or adding or eliminating access rights of a subject with respect to an object.\",\n \"This single administration authority must of course comply with the requirements of the various partners participating in the production and management of the various application resources in the chip card.\",\n \"The objective of the present invention is to make possible the dynamic management of access control lists or capacities in a portable electronic object of the chip card type in order to refine the management of such lists or capacities and thus permit an increase in the number of administrators authorised to act with regard to security on modifications to the access control lists or capacities.\",\n \"To achieve this objective, a method for managing lists of rights of access between subjects and objects, stored in a data processing means from an external administrator entity, is characterised in that it comprises the following steps: initially associating keys of administrator entities with access rights lists and supplying a security algorithm in the data processing means, and in order to access an access rights list from the entity: signing the access rights list in the entity by applying determined data from the list and the key to the security algorithm in order to produce a signature, transmitting the signature from the entity to the data processing means, and comparing the signature received in the data processing means with determined signatures according to applications of determined data in lists of access rights contained in the data processing means and of keys respectively associated with these lists to the security algorithm, and allowing access of the entity to an access rights list found only in correspondence with a signature found amongst the determined signatures in the data processing means and identical to the received signature.\",\n \"Thus, according to the invention, any authorised administrator accesses an access rights list constituting an access control list or a capacity in a data processing means consisting for example of the microcontroller of a portable electronic object.\",\n \"An access right authorises or prohibits one or more subjects from performing an action on a subject, or a subject from performing an action on one or more objects.\",\n \"This access control list or capacity is then managed dynamically by each administrator, by obliging it first to sign the access control list or the capacity to which it wishes to gain access, so as to be recognised in the electronic object by its signature of the access control list or of the capacity.\",\n \"Management of the access control lists is thus decentralised in administrator entities external to the data processing means.\",\n \"According to another characteristic of the invention, the duration of application of an access rights list is limited.\",\n \"More precisely, before authorising access of the entity to the control list, a duration of life parameter of the access rights list found is updated in order to erase the access rights list found when the duration of life parameter updated exceeds a maximum limit and in order to allow access to the access rights list found to the entity when the duration of life parameter updated is less than the maximum limit.\",\n \"The limited duration of life of an access rights list then prompts the subject or subjects affected by this list which has become out of date and is now erased to return to the administrator or administrators of this list in order to once again request access rights from them.\",\n \"The invention also relates to a data processing means in particular in a portable electronic object, storing access rights lists managed from at least one external administrator entity, implementing the method of the invention.\",\n \"It is characterised in that it comprises: a means for storing keys of administrator entities in association with access rights lists, a means for implementing a security algorithm, a means for determining signatures according to applications of determined data of the lists of access rights and keys respectively associated with these lists to the algorithm, a means for comparing a received signature of an access rights list which results, in the entity, from the application of determined data of the list and of the key of the entity to the algorithm and which is transmitted by the entity, to the said determined signatures, and a means for authorising access of the entity to an access rights list found only in correspondence with a signature found amongst the determined signatures and identical to the received signature.\",\n \"The data processing means can also comprise a means for storing a duration of life parameter and a maximum duration limit for each access rights list, and a means for updating the duration parameter of the list found in order to erase the access rights list found when the duration of life parameter updated exceeds the maximum limit and in order to authorise access to the access list found to the entity when the duration of life parameter updated is less than the maximum limit.\",\n \"Other characteristics and advantages of the present invention will emerge more clearly from a reading of the following description of several preferred embodiments of the invention with reference to the corresponding accompanying drawings, in which: FIG.\",\n \"1 is a diagram showing a control matrix between three subjects and three objects, already commented on according to the prior art; FIG.\",\n \"2 is a schematic block diagram of a telecommunications system between an administrator server and a portable electronic object of the chip card type for implementing the management method according to the invention; and FIG.\",\n \"3 is an algorithm of steps of the method of dynamic management of access rights lists according to the invention.\",\n \"With reference to FIG.\",\n \"2, the data processing means is a controller, such as the microcontroller of a chip card CA which contains several applications constituting objects to which subjects, such as users or units or software means, can gain access according to access rights.\",\n \"In a known manner, the microcontroller of the chip card CA comprises a microprocessor PR, a memory MO of the ROM type, a non-volatile memory MNV of the programmable and erasable type, such as a EEPROM memory, and a memory MA of the RAM type receiving in particular data from a terminal TE accepting the card.\",\n \"In the memory MO there are included in particular an operating system OS of the card, communication, authentication and service applications AP constituting objects Ob, as well as a security algorithm AS used for recognising administrator signatures and an algorithm for managing access rights lists AG according to the invention.\",\n \"The memory MNV contains data in particular relating to the possessor of the card and to the supplier of the card, as well as access control lists ACL(Ob), or capacities.\",\n \"Each access control list relating to a respective object Ob, to which reference is made below as an access rights list, contains, as shown in FIG.\",\n \"1, a list of subjects Su each associated with a list of positive access rights authorising and/or negative access rights prohibiting the performance of actions on the respective object Ob.\",\n \"The memory MNV thus contains a subject set ES, an object set EO, that is to say a set referring to applications, and an access rule set ER.\",\n \"The memory MNV preferably also contains a subject group set EG.\",\n \"Each group Gp contains subjects Su each having at least one predetermined access to a predetermined object Ob; thus a subject in a group Gp has all the access rights granted to this group, and a subject can belong to one or more groups.\",\n \"A rule R for access to an object Ob concerns an action, such as for example reading, writing, execution or recording, which is authorised or prohibited to a subject Su or to a subject group Gp on an object Ob.\",\n \"Consequently an access control list ACL(Ob) relates to access rules for predetermined subjects and/or predetermined groups on the respective object Ob.\",\n \"According to the invention, when the card is manufactured, or before the card CA is brought into service, that is to say before it is passed to the owner of the card, administrators of the lists ACL contained in the card are defined at an initial step ET01.Each administrator AD is defined by a respective administrator key KAD which is associated with one or more access control lists ACL to which the administrator AD has access.\",\n \"Each access control list ACL is associated with an administrator sub-list consisting of the corresponding administrator keys KAD.\",\n \"As will be seen hereinafter, the administrator key list KAD is preferably supplemented by an administrator signature list SGAD.\",\n \"A signature represents the access control list ACL signed by the respective administrator according to the security algorithm AS.\",\n \"Thus, according to the invention, the administrator keys KAD and preferably the administrator signatures SGAD are previously written in the non-volatile memory MNV of the chip card CA in correspondence with the access control lists ACL.\",\n \"FIG.\",\n \"2 also shows diagrammatically an administrator entity external to the card CA in the form of an administrator server SAD connected to the chip card through a telecommunication network RT and the accepting terminal TE.\",\n \"The telecommunication network RT designates any type of telecommunication network, or combination of networks, such as a radiotelephony network, a switched telephone network, an integrated service digital network ISDN, a high-rate network of the ATM type, the Internet, a packet transmission network, etc.\",\n \"The server SAD represents an administrator or several administrators.\",\n \"Other administrators can be situated at other distant servers (not shown).\",\n \"The administrator AD is for example the distributor of the chip card CA, or a partner commercially associated with this distributor, or the developer of one or more applications AP constituting objects Ob implemented in the chip card CA.\",\n \"As a variant, an administrator himself possesses a chip card which is housed in an additional reader of the server SAD so that the server SAD reads in the chip card the references of the administrator such as an administrator identifier IAD and an administrator key KAD.\",\n \"As shown schematically also in FIG.\",\n \"2, the accepting terminal TE is provided with a keypad CL and a reader LE for receiving the chip card CA and connecting thereto by means of an electrical-contact connection LI according to this embodiment.\",\n \"The accepting terminal TE is for example a chip card commissioning terminal, a bank terminal, or a point of sale terminal, or a mobile telephony terminal in which the chip card CA constitutes a removable subscriber identity module SIM or an additional chip card when the mobile terminal is provided with an additional card reader.\",\n \"After the initial step ET01, the management method according to the invention preferably comprises a second initial step ET02 during which administrator signatures SGAD are determined.\",\n \"Step ET02 is also implemented for a list ACL which has just been modified or introduced into the memory MNV in the card CA.\",\n \"Each signature SGAD of an administrator SAD depends on the key KAD of the administrator and determined data in an access control list ACL with which the key KAD is associated.\",\n \"The determined data in the access control list ACL depend essentially on the characteristics of the objects Ob and the subjects Su and/or groups of subjects Gp each having at least one right of access to the object Ob, as well as where applicable access rights characteristics.\",\n \"The list ACL and the key KAD are applied to the security algorithm AS, the result of which constitutes the administrator signature SGAD.\",\n \"The management method shown in FIG.\",\n \"3 comprises principally steps ET2 to ET14 triggered by a first step ET1 during which the administrator server SAD requests the establishment of a call with the accepting terminal TE containing the chip card CA through the telecommunication network RT, so as to initiate an access control list management session with the chip card CA.\",\n \"At the start of this session the card CA attempts to authenticate the administrator SAD at step ET2.The authentication is conventional and consists essentially in transmitting a random number by means of the chip card CA to the server SAD and comparing in the chip card CA the results of the application of this random number and of an authentication key prestored in the card CA and the server SAD, performed both in the card CA and the server SAD.\",\n \"Conversely, the server SAD authenticates the chip card CA.\",\n \"According to another variant, the authentication is mutual and comprises an authentication of the server SAD by the card CA and an authentication of the card CA by the server SAD.\",\n \"If the authentication or one of the authentications at step ET2 gives different results in the server SAD and the chip card CA, the call is broken off at a final step ET14.In a variant, the management method comprises no authentication.\",\n \"When the authentication has succeeded, the administrator server SAD selects an access control list ACL which it has under management locally in a table of access control lists, at the following step ET3, although the administrator can manage only one access control list ACL.\",\n \"The server SAD next signs the selected access control list ACL by applying the determined data of the list ACL and the key KAD of the administrator server SAD to the security algorithm AS so as to produce an administrator signature SGAD at step ET4.In a variant, instead of itself signing the access rights list ACL and producing the administrator signature SGAD, the server SAD receives this signature SGAD transmitted by another main administrator editor, such as a main administrator server connected to the telecommunication network RT, after the main administrator server has authenticated the server SAD.\",\n \"Then the signature SGAD constituting the selected access control list ACL signed is transmitted by the server SAD to the chip card CA through the network RT and the terminal TE in the form of an appropriate message at step ET5.This message can contain other data such as an administrator identifier IAD which designates a respective table of access control lists TACL in the chip card CA.\",\n \"The table TACL contains identifiers of access control lists to which the administrator server SAD has access, and preferably contains signatures of these access control lists signed by the administrator server SAD.\",\n \"At the following step ET6, in response to the administrator signature SGAD, the microprocessor PR in the chip card CA seeks an access control list which is able to be signed by the administrator server SAD.\",\n \"This search can consist in determining all the administrator signatures respectively associated with the access control lists which the chip card CA contains, and then comparing the signatures determined with the signature SGAD received by the card CA.\",\n \"However, as indicated at step ET02, the processor PR of the chip card CA has preferably, prior at least to step ET5, determined the signatures SGAD corresponding to all the administrators respectively associated with the access control lists ACL and has written them in EEPROM memory MNV.\",\n \"The processor PR then compares the signature SGAD received at step ET5 only with the signatures previously determined and classified in the table TACL.\",\n \"If the processor PR finds no administrator signature in memory MNV identical to the signature SGAD transmitted by the server SAD, the processor PR invites the accepting terminal TE to break off the communication at step ET14.In a variant, when the administrator identifiers IAD are provided in the memory MNV, the processor PR compares the received signature SGAD only with the signatures contained in the table TACL designated by the received identifier IAD and associated with the key KAD, that is to say only with the signatures associated with access control lists to which the administrator server SAD has access.\",\n \"Thus, after step ET6, an access control list ACL corresponding to the received administrator signature SGAD is found in order to process it by means of the server SAD, as indicated at step ET7.Although, according to a simple variant, step ET7 is followed by a step ET10 allowing access of the administrator server SAD to the access control list ACL previously found, the invention provides intermediate steps ET8 and ET9 during which a duration of life parameter pdv of the access rights list ACL found is updated and compared with a maximum limit PDV.\",\n \"The parameter pdv and the limit PDV are written in the memory MNV of the card.\",\n \"The limit PDV expresses the duration of life of the access control list ACL found at step ET7 and is prestored in the memory MNV at the initial step ET01, when the card CA is manufactured or brought into service, or when there is an addition of the list ACL, as will be seen subsequently.\",\n \"At the initial step ET01, the duration of life parameter pdv is zeroed so that it is incremented each time step ET8 is executed.\",\n \"The duration of life parameter pdv can be a cumulative duration of sessions of use of the chip card CA expressed in hours and minutes like the limit PDV.\",\n \"According to a second variant, the parameter pdv is a cumulative duration of absolute time expressed in terms of date and time, the limit PDV then designating a maximum predetermined duration or an expiry date of the determined list ACL.\",\n \"The updating of the duration can be carried out in the card CA by interrogating a trustworthy time stamping device, for example included in the terminal TE, or in the administrator server SAD.\",\n \"According to other variants, the duration of life parameter pdv is a number of sessions of use of the chip card CA, or a number of sessions using the list of access rights found ACL, the limit ACL being a maximum number of sessions.\",\n \"According to yet another variant, the duration of life parameter pdv is a number of commands APDU received by the chip card CA, the limit PDV then being a maximum number of commands.\",\n \"According to yet another variant, the duration of life parameter is a synchronisation value changed periodically in the administrator servers and transmitted to the card by any administrator server SAD in each message which contains the signature SGAD of the list of access rights sought ACL, at step ET5, the limit PDV being at the maximum number.\",\n \"Step ET8 thus increments the duration of life parameter according to its nature, the increment being in particular a period or a difference in date, or a unit, or a number of commands.\",\n \"If at step ET9 the duration of life parameter pdv exceeds the limit PDV, the processor PR erases the access control list ACL which was found at step ET7 and all the data associated with the list ACL at step ET13, and breaks off the communication with the server SAD at step ET14.In a variant, step ET13 only partially erases the list found, for example by erasing therein only the positive or negative access rights.\",\n \"If the duration of life is not yet reached at step ET9, the administrator server SAD is authorised to effectively gain access to the access control list ACL found, at step ET10.The server SAD then proceeds with a modification of the access control list found ACL.\",\n \"This modification consists for example in totally erasing the access control list ACL in the memory MNV, or modifying one of the data in the list found ACL, in particular eliminating or adding a subject Su in the access control list found with corresponding access rights R, or eliminating or adding at least one access right R relating to a subject Su included in the list found.\",\n \"The modification can also consist in adding a new administrator key to be associated with the list found and included in the message transmitted at step ET5.If at step ET11 the access control list found ACL has not been modified or has been erased, the method passes directly to the breaking off of the communication at step ET14.On the other hand, if the access list found ACL has been modified by the server SAD, this modified list being designated by ACLm, the processor PR determines, at step ET12, new administrator signatures SGADm resulting from the application of the determined data in the modified access control list ACLm and respectively administrator keys KAD associated with the list, to the security algorithm AS.\",\n \"The signatures SGADm replace the previous signatures SGAD so that, during a future list management session, the administrator server SAD can access the control list ACLm thus modified in the memory MNV of the card CA.\",\n \"Then the management method ends with the breaking off of the communication between the server SAD and the card CA at step ET14.When an access control list established in the administrator server SAD is to be added in the memory MNV of the chip card CA, steps ET1 to ET5 are executed, the message transmitted at step ET5 also containing the list ACL to be added with the administrator keys KAD associated with the list.\",\n \"Then, in place of steps ET6 to ET13, the processor PR determines and stores the signatures SGAD of the list received in the respective list tables TACL in the card CA.\",\n \"For each administrator associated with the list, the respective signature SGAD results from application of the determined data of the list and of the respective key KAD received to the security algorithm AS.\",\n \"The processor PR compares the signatures determined with the signature received, as shown at a step ET15 in dotted lines in FIG.\",\n \"3.The recording of the list received and of the keys and associated administrator signatures in the memory MNV is validated at a step ET16 when one of the signatures determined is identical to the signature received.\",\n \"Otherwise step ET14 breaks off communication between the server SAD and the card CA.\",\n \"According to a variant of the embodiment described above, the server SAD is an entity delegated by another administrator server which has transmitted to it delegation rights information IDR.\",\n \"Delegation rights information IDR was previously stored in the memory MNV of the chip card CA at the initial step ET01 and is transmitted in the message containing the administrator signature SGAD at step ET5.At step ET6, the chip card CA authorises access to the access control list ACL when both the signature SGAD is recognised in the table TACL associated with the key KAD of the server SAD and the delegation rights information IDR is detected in association with the list ACL recognised in the table TACL of the card.\",\n \"Otherwise communication is broken off at step ET14.Although the invention has been described in relation to a chip card containing access control lists associated respectively with signatures, the chip card can also contain access control lists which are not signed.\",\n \"Thus an administrator server SAD can decide to erase all the access control lists loaded in the chip card CA and associated with the key KAD of the server SAD, transmitting the corresponding signatures at step ET4.As already stated, everything which has been described above for access control lists in the chip card CA, or in any other portable electronic object such as a personal electronic assistant or organiser, an electronic purse, a token or a pocket calculator, is applicable to an access rights list making several subjects correspond to an object, such as a capacity.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10467763"}}},{"rowIdx":384,"cells":{"text":{"kind":"list like","value":[["Use of type 4 phosphodiesterase inhibitors in myocardial diseases","The invention relates to the use of type 4 phosphodiesterase inhibitors to treat myocardial diseases."],["1.Use of a) compounds of formula I disclosed in EP 0763534 in which B is an aromatic heterocycle having 1 to 4 N, O and/or S atoms, bonded via N or C, which can be unsubstituted or mono-, di- or trisubstituted by HaI, A and/or OA, and can also be fused to a benzene or pyridine ring, Q is absent or is alkylene having 1-6 C atoms, X is CH2, S or O, R1 and R2 in each case independently of one another are H or A, R3 and R4 in each case independently of one another are —OH, OR5, —S—R5, —SO—R5, —SO2—R5, HaI, methylenedioxy, —NO2, —NH2, —NHR5 or —NR5R6, R5 and R6 in each case independently of one another are A, cycloalkyl having 3-7 C atoms, methylenecycloalkyl having 4-8 C atoms or alkenyl having 2-8 C atoms, A is alkyl having 1 to 10 C atoms, which can be substituted by 1 to 5 F and/or Cl atoms and HaI is F, Cl, Br or I and their stereoisomers and physiologically acceptable, salts and solvates; b) compounds of formula I disclosed in WO 99/65880 in which B is a phenyl ring which is unsubstituted or mono- or polysubstituted by R3, Q is absent or is alkylene having 1-4 C atoms, R1,R2 each independently of one another are —OR4, —S—R4, —SO—R4, —SO2—R4 or HaI, R1 and R2 together are also —O—CH2—O—, R3 is R4, HaI, OH, OR4, OPh, NO2, NHR4, N(R4)2, NHCOR4, NHSO2R4 or NHCOOR4, R4 is A, cycloalkyl having 3-7 C atoms, alkylenecycloalkyl having 5-10 C atoms or alkenyl having 2-8 C atoms, A is alkyl having 1 to 10 C atoms, which can be substituted by 1 to 5 F and/or Cl atoms and HaI is F, Cl, Br or I, and their physiologically acceptable salts and solvates; c) compounds of formula I disclosed in WO 00/26201 in which R1, R2 in each case independently of one another are —OH, OR5, —S—R5, —SO—R5, —SO2—R5 or HaI, R1 and R2 together are also —O—CH2—O—, R3 is NH2, NHA, NAA′ or a saturated heterocycle having 1 to 4 N, O and/or S atoms which can be unsubstituted or mono-, di- or tri-substituted by HaI, A and/or OA, Q is absent or is branched or unbranched alkylene having 1-10 C atoms, R5 is A, cycloalkyl having 3-7 C atoms, alkylenecycloalkyl having 4-8 C atoms or alkenyl having 2-8 C atoms, A, A′ in each case independently of one another are alkyl which has 1 to 10 C atoms and which can be substituted by 1 to 5 F and/or Cl atoms and HaI is F, Cl, Br or I, and the physiologically acceptable salts and solvates thereof; d) compounds of formula I disclosed in WO 98/06704 in which B is A, OA, NH2, NHA, NAA′ or an unsaturated heterocycle which has 1 to 4 N, O and/or S atoms and which can be unsubstituted or mono-, di- or trisubstituted by HaI, A and/or OA, Q is absent or is alkylene having 1-6 C atoms, R1, R2 in each case independently of one another are —OH, OR5, —S—R5, —SO—R5, —SO2—R5, HaI, —NO2, —NH2, —NHR5 or —NRR6, R1 and R2 together are also —O—CH2—O—, R3, R4 in each case independently of one another are H or A, R5, R6 in each case independently of one another are A, cycloalkyl having 3-7 C atoms, methylenecycloalkyl having 4-8 C atoms or alkenyl having 2-8 C atoms, A, A′ in each case independently of one another are alkyl which has 1 to 10 C atoms and which can be substituted by 1 to 5 F and/or Cl atoms and HaI is F, Cl, Br or I, and the stereoisomers and physiologically acceptable salts and solvates thereof; e) compounds disclosed in WO 00/59890 1-(4-ureidobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3-propoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-trifluoroacetamidobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-propoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-isopropoxycarbonylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-propoxycarbonylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3,4-dimethoxyphenyl)-4-ethyl-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3,4-dimethoxyphenyl)-4-ethyl-1,4,5,6-tetrahydropyridazine and 1-(4-acetamidobenzoyl)-3-(3,4-dimethoxyphenyl)-4-ethyl-1,4,5,6-tetrahydropyridazine, and their physiologically acceptable salts and solvates; f) compounds of formula I disclosed in DE 19604388 in which R1, R2 in each case independently of one another are H or A, R3, R4 in each case independently of one another are —OH, OA, —S-A, —SO-A, —SO2-A, HaI, methylenedioxy, —NO2, —NH2, —NHA or —NAA′, A, A′ in each case independently of one another are alkyl having 1 to 10 C-atoms, and which can be substituted by 1 to 5 F and/or Cl atoms, cycloalkyl having 3-7 C atoms or methylenecycloalkyl having 4-8 C atoms, B is —Y—R5 oder —O—Y—R5, Q is absent or is alkylene having 1-4 C atoms, Y is absent or is alkylene having 1-10 C atoms, X is CH2or S, R5 is NH2, NHA, NAA′ or is a saturated 3-8 membered heterocycle having at least one N atom, and wherein other CH2 groups optionally may be replaced by NH, NA, S or O, which can be unsubstituted or monosubstituted by A or OH, HaI is F, Cl, Br oder I and the stereoisomers and physiologically acceptable salts and solvates thereof; g) compounds of formula I disclosed in DE 19932315 in which R1, R2 in each case independently of one another are H, OH, OA, SA, SOA, SO2A, F, Cl or A′2N—(CH2)n—O—, R1 and R2 together are also —O—CH2—O—, R3, R4 in each case independently of one another are H, A, HaI, OH, OA, NO2, NHA, NA2, CN, COOH, COOA, NHCOA, NHSO2A or NHCOOA, R5, R6 in each case independently of one another are H or alkyl having 1 to 6 C atoms, A is alkyl having 1 to 10 C atoms, which can be substituted by 1 to 5 F and/or Cl atoms, is cycloalkyl having 3-7 C atoms, alkylenecycloalkyl having 5-10 C atoms or alkenyl having 2-8 C atoms, A′ is alkyl having 1, 2, 3, 4, 5 or 6 C atoms, n is 1, 2, 3 or 4, HaI is F, Cl, Br or I, and their physiologically acceptable salts and solvates; h) compounds of formula I disclosed in EP 0723962 in which R1 and R2 in each case independently of one another are H or A, R3 and R4 in each case independently of one another are —OH, —OR10, —S—R10, —SO—R10, —SO2R10, HaI, methylenedioxy, —NO2, —NH2, —NHR10 or —NR10R11, R5 is a phenyl radical which is unsubstituted or mono- or disubstituted by R6 and/or R7, Q is absent or is alkylene having 1-6 C atoms, R6 and R7 in each case independently of one another are —NH2, —NR8R9, —NHR10, —NR10R11, —NO2, HaI, —CN, —OA, —COOH or —COOA, R8 and R9 in each case independently of one another are H, acyl having 1-8 C atoms which can be substituted by 1-5 F and/or Cl atoms, —COOA, —S-A, —SO-A, —SO2A, —CONH2, —CONHA, —CONA2, —CO—COOH, —CO—COOA, —CO—CONH2, —CO—CONHA or —CO—CONA2, A is alkyl having 1 to 6 C atoms which can be substituted by 1-5 F and/or Cl atoms, R10 and R11 in each case independently of one another are A, cycloalkyl having 3-7 C atoms, methylenecycloalkyl having 4-8 C atoms or alkenyl having 2-8 C-atoms and HaI is F, Cl, Br or I, and their physiologically acceptable salts and solvates; i) compounds of formula I disclosed in EP 0738715 in which R1 and R2 in each case independently of one another are H or A, R3 and R4 in each case independently of one another are —OH, —OR10, —S—R10, —SO—R10, —SO2R10, HaI, methylenedioxy, —NO2, —NH2, —NHR10 or —NR10R11, R5 is a phenyl radical which is unsubstituted or mono- or disubstituted by R6 and/or R7, Q is absent or is alkylene having 1-6 C atoms, R6 and R7 in each case independently of one another are —NH2, —NR8R9, —NHR10, —NR10R11, —NO2, HaI, —CN, —OA, —COOH or —COOA, R8 and R9 in each case independently of one another are H, acyl having 1-8 C atoms which can be substituted by 1-5 F and/or Cl atoms, —COOA, —SO-A, —SO2A, —CONH2, —CONHA, —CONA2, —CO—COOH, —CO—COOA, —CO—CONH2, —CO—CONHA or —CO—CONA2, A is alkyl having 1 to 6 C atoms which can be substituted by 1-5 F and/or Cl atoms, R10 and R11 in each case independently of one another are A, cycloalkyl having 3-7 C atoms, methylenecycloalkyl having 4-8 C atoms or alkenyl having 2-8 C-atoms and HaI is F, Cl, Br or I, and their physiologically acceptable salts and solvates; for preparing a medicament for treating myocardial diseases.","2.Use according to claim 1 of a) compounds disclosed in EP 0763534: 2-(3-nicotinoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(2-nicotinoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(3-nicotinoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(2-nicotinoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-methoxy-4-trifluoromethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-methoxy-4-difluoromethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-methoxy-4-fluoromethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-difluoromethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-trifluoromethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-fluoromethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-methoxy-4-ethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-hydroxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(4-methylsulfonylphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(4-methyleneoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(3-nicotinoylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminophenethyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminophenethyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 3-(4-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(2-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(2-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-trifluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-difluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-fluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-difluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-trifluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-fluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-ethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-ethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-ethoxy-4-methoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-hydroxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(4-methysulfonylphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(4-methyleneoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-cyclopentyloxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3-cyclopentyloxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminophenethyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminophenethyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(2-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(2-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-trifluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-difluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-fluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-difluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-trifluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-fluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-ethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-ethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-hydroxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(4-methylsulfonylphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(4-methyleneoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-cyclopentyloxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3-cyclopentyloxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminophenethyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminophenethyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 2-(3-nicotinoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isonicotinoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pyrazinecarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-(isoxazole-5-carbonylamino)benzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3,4,-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, hydrochloride, and their stereoisomers and physiologically acceptable, salts and solvates; b) compounds disclosed in WO 99/65880 N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-methoxybenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-methylbenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)benzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-3,4-dichlorobenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-trifluoromethylbenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-3-chlorobenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-fluorobenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-butoxybenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-pentoxybenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-ethoxybenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-3,4-dimethoxybenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-3-methylbenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-3-methoxybenzoyl-3-carboxamide, and their physiologically acceptable salts and solvates; c) compounds disclosed in WO 00/26201 3-dimethylaminopropyl{4-[3-(3-ethoxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, N-methylpiperidin-4-yl-{4-[3-(3-ethoxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl{4-[3-(3-isopropoxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl{3-[3-(3-ethoxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl{3-[3-(3-cyclopentyloxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, N-methylpiperidin-4-yl-{3[3-(3-cyclopentyloxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl{3-[3-(3-propyloxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl{4-[3-(3,4-diethoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, N-methylpiperidin-4-yl-{4-[3-(3,4-diethoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl{3-[3-(3,4-dimethoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate 3-dimethylaminopropyl{4-[3-(3,4-dimethoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, and the physiologically acceptable salts and solvates thereof; d) compounds disclosed in WO 98/06704 1-(4-nicotinoylaminobenzoyl)-3-(3,4-dimethoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(3-nicotinoylaminobenzoyl)-3-(3,4-dimethoxy-phenyl)-1,4,5,6-tetrahydropyridazine hydrochloride, 1-(2-nicotinoylaminobenzoyl)-3-(3,4-dimethoxy-phenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(3-nicotinoylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3-cyclopentyloxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(3-nicotinoylaminobenzoyl)-3-(3-cyclopentyloxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3,4-methylene-dioxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3-methoxy-4-methylsulfonylphenyl)-1,4,5,6-tetrahydro-pyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3-trifluoro-methoxy-4-methoxyphenyl)-1,4,5,6-tetrahydro-pyridazine, 1-(4-ethoxy-carbonylaminobenzoyl)-3-(3,4-dimethoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(3-ethoxycarbonylaminobenzoyl)-3-(3,4-dimethoxy-phenyl)-1,4,5,6-tetrahydropyridazine, 1-(2-ethoxycarbonylaminobenzoyl)-3-(3,4-dimethoxy-phenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(3-ethoxycarbonylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-cyclo-pentyloxy-4-methoxyphenyl)-1,4,5,6-tetrahydro-pyridazine, 1-(3-ethoxycarbonylaminobenzoyl)-3-(3-cyclo-pentyloxy-4-methoxyphenyl)-1,4,5,6-tetrahydro-pyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3,4-methylene-dioxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-methoxy-4-methylsulfonylphenyl)-1,4,5,6-tetrahydro-pyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-trifluoro-methoxy-4-methoxyphenyl)-1,4,5,6-tetrahydro-pyridazine, and the stereoisomers and physiologically acceptable salts and solvates thereof; e) compounds disclosed in EP 0723962 3-(4-ethoxycarbonylaminobenzyl)-5-(3-ethoxy-4-methoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-ethoxycarbonylaminobenzyl)-5-(3-cyclopentyloxy-4-methoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, and their physiologically acceptable salts and solvates; f) compounds disclosed in EP 0738715 2-(4-butyrylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-acetamidobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-trifluoroacetamidobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methylsulfonamidobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-propionylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-tert-butylcarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isobutyrylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxycarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pivalylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-cyclopentylcarbamoylbenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ethoxycarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxalylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ureidobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentanoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-hexanoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropiridazin-3-one, 2-(4-pentafluoropropionylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-acetamidobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-trifluoroacetamidobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methylsulfonamidobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-propionylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-tert-butylcarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-butyrylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isobutyrylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxycarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pivalylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-cyclopentylcarbamoylbenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ethoxycarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxalylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ureidobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentanoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-hexanoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentafluoropropionylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-acetamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-trifluoroacetamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methylsulfonamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-propionylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-butyrylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isobutyrylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxycarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pivalylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-cyclopentylcarbamoylbenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ethoxycarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4, 5-tetrahydropyridazin-3-one; 2-(4-methoxalylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ureidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentanoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-hexanoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentafluoropropionylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-acetamidobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-trifluoroacetamidobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methylsulfonamidobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-propionylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-tert-butylcarbonylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-butyrylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isobutyrylaminobenzyl)-6(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxycarbonylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pivalylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-cyclopentylcarbamoylbenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ethoxycarbonylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxalylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ureidobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentanoylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-hexanoylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentafluoropropionylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-acetamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-trifluoroacetamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methylsulfonamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-propionylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-butyrylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isobutyrylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxycarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pivalylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-cyclopentylcarbamoylbenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ethoxycarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxalylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ureidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentanoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-hexanoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentafluoropropionylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, and their physiologically acceptable salts and solvates; for preparing a medicament for treating myocardial diseases.","3.Use according to claim 1 of compounds selected from 3-(4-nicotinoylaminobenzyl)-5-(3-ethoxy-4-methoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-methoxybenzoyl-3-carboxamide, 1-(4-nicotinoylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 2-(4-ethoxycarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, and their physiologically acceptable salts and solvates; for preparing a medicament for treating myocardial diseases.","4.Use according to claim 1 for preparing a medicament for treating myocardial diseases, where said myocardial diseases show inflammatory and immunological characteristics.","5.Use according to claim 1 for preparing a medicament for treating coronary artery disease, reversible or irreversible myocardial ischemia/reperfusion injury, acute or chronic heart failure and restenosis, including instent-restenosis and stent-in-stent-restenosis."],["The invention relates to the use of type 4 phosphodiesterase inhibitors to treat myocardial diseases.","Coronary artery disease is the most common cause of death in the western world.","In the presence of a critical stenosed coronary artery, a decrease of blood flow may result in myocardial ischemia.","Initiation of reperfusion results, depending on the severity of the preceding ischemic period, in a reversibly or irreversibly injured myocardium, which is characterized by a long-lasting depression or an irreversible loss of contractile function.","Depending on the size of the affected myocardial area, an acute or a chronic heart failure may develop.","A particular clinical problem in the above mentioned scenario is the development of restenosis after a primarily successful reperfusion intervention by PTCA, even after stent implantation, thrombolysis or coronary artery bypass grafting.","From experimental animal and clinical studies evidence exists, that in the different above mentioned heart diseases, i. e. coronary artery disease itself, reversible or irreversible myocardial ischemia/reperfusion injury, acute or chronic heart failure and restenosis, including instent-restenosis and stent-in-stent-restenosis, inflammatory processes play a casual role.","These inflammatory processes involve resident and invading macrophages as well as neutrophils and TH1 and TH2 helper cells.","This leukocyte response produces the characteristic cytokine pattern, involving TNF-α, IL-1β, IL-2, and IL-6, as well as IL-10 and IL-13 (Pulkki K J: Cytokines and cardiomyocyte death.","Ann.Med.","1997 29: 339-343.Birks E J, Yacoub M H: The role of nitric oxide and cytokines in heart failure.","Coron.Artery.Dis.","1997 8: 389-402).","The formation of these species has been demonstrated in human patients with myocardial ischemia.","Animal models show that cytokine production correlates with the invasion of peripheral macrophages and neutrophils which can spread the damage into the still intact myocardium.","The main player in the cytokine response, however, is TNF-a, which integrates inflammatory and pro-apoptotic responses and additionally has a direct negative ionotropic effect on cardiac myocytes (Ceconi C, Curello S, Bachetti T, Corti A, Ferrari R: Tumor necrosis factor in congestive heart failure: a mechanism of disease for the new millennium?","Prog.Cardiovasc.Dis.","1998 41: 25-30.Mann D L: The effect of tumor necrosis factor-alpha on cardiac structure and function: a tale of two cytokines.","J.Card.Fail.","1996 2: S165-S172.Squadrito F, Altavilla D, Zingarelli B, et al: Tumor necrosis factor involvement in myocardial ischaemia-reperfusion injury.","Eur.J.Pharmacol.","1993 237: 223-230).","It has been shown in animal models of myocardial infarction, that TNF-α is rapidly released during the reperfusion phase (Herskowitz A, Choi S, Ansari A A, Wesselingh S: Cytokine mRNA expression in postischemic/reperfused myocardium.","Am.J.Pathol.","1995 146: 419-428) and that the protective effects of drugs such as dexamethasone (Arras M, Strasser R, Mohri M, et al: Tumor necrosis factor-alpha is expressed by monocytes/macrophages following cardiac microembolization and is antagonized by cyclosporine.","Basic.Res.Cardiol.","1998 93: 97-107), cyclosporin A (Arras M, Strasser R, Mohri M, et al: Tumor necrosis factor-alpha is expressed by monocytes/macrophages following cardiac microembolization and is antagonized by cyclosporine.","Basic.Res.Cardiol.","1998 93: 97-107.Squadrito F, Altavilla D, Squadrito G, et al: Cyclosporin-A reduces leukocyte accumulation and protects against myocardial ischaemia reperfusion injury in rats.","Eur.J.Pharmacol.","1999 364: 159-168) or clorichromene (Squadrito F. Altavilla D, Zingarelli B, et al: The effect of cloricromene, a coumarine derivative, on leukocyte accumulation, myocardial necrosis and TNF-alpha production in myocardial ischaemia-reperfusion injury.","Life Sci.","1993 53: 341-355)correspond with a reduction of circulating TNF-α.","Preferred PDE4 inhibitors mentioned below are potent antagonists of macrophage and T-cell cytokine production.","They also inhibit the proliferation of T cells.","Consequently, PDE4 inhibition may have a beneficial effect in those myocardial diseases, which are causally linked to cytokine production and inflammatory processes.","As compared with PDE3 inhibitors and the early PDE4 inhibitor Rolipram, preferred PDE4 inhibitors are devoid of hemodynamic side effects, which can be dose limiting for the treatment of most cardiovascular disorders.","The invention was based on the object of discovering new uses of compounds having valuable properties, especially those which may be used to prepare medicaments.","It has been found that the preferred PDE IV inhibitors and their salts combine very valuable pharmacological properties with good tolerability for the treatment of myocardial diseases.","Accordingly, the invention provides in particular for the use of a) compounds of formula l disclosed in EP 0763534 in which B is an aromatic heterocycle having 1 to 4 N, O and/or S atoms, bonded via N or C, which can be unsubstituted or mono-, di- or trisubstituted by HaI, A and/or OA, and can also be fused to a benzene or pyridine ring, Q is absent or is alkylene having 1-6 C atoms, X is CH2, S or O, R1 and R2 in each case independently of one another are H or A, R3 and R4 in each case independently of one another are —OH, OR5, —S—R5, —SO—R5, —SO2—R5, HaI, methylenedioxy, —NO2, —NH2, —NHR5 or —NR5R6, R5 and R6 in each case independently of one another are A, cycloalkyl having 3-7 C atoms, methylenecycloalkyl having 4-8 C atoms or alkenyl having 2-8 C atoms, A is alkyl having 1 to 10 C atoms, which can be substituted by 1 to 5 F and/or Cl atoms and HaI is F, Cl, Br or I and their stereoisomers and physiologically acceptable, salts and solvates; b) compounds of formula I disclosed in WO 99/65880 in which B is a phenyl ring which is unsubstituted or mono- or polysubstituted by R3, Q is absent or is alkylene having 1-4 C atoms, R1,R2 each independently of one another are —OR4, —S—R4, —SO—R4, —SO2—R4 or HaI, R1 and R2 together are also —O—CH2—O—, R3 is R4, HaI, OH, OR4, OPh, NO2, NHR4, N(R4)2, NHCOR4, NHSO2R4 or NHCOOR4, R4 is A, cycloalkyl having 3-7 C atoms, alkylenecycloalkyl having 5-10 C atoms or alkenyl having 2-8 C atoms, A is alkyl having 1 to 10 C atoms, which can be substituted by 1 to 5 F and/or Cl atoms and HaI is F, Cl, Br or I, and their physiologically acceptable salts and solvates; c) compounds of formula I disclosed in WO 00/26201 in which R1, R2 in each case independently of one another are —OH, OR5, —S—R5, —SO—R5, —SO2—R5 or HaI, R1 and R2 together are also —O—CH2—O—, R3 is NH2, NHA, NAA′ or a saturated heterocycle having 1 to 4 N, O and/or S atoms which can be unsubstituted or mono-, di- or tri-substituted by HaI, A and/or OA, Q is absent or is branched or unbranched alkylene having 1-10 C atoms, R5 is A, cycloalkyl having 3-7 C atoms, alkylenecycloalkyl having 4-8 C atoms or alkenyl having 2-8 C atoms, A, A′ in each case independently of one another are alkyl which has 1 to 10 C atoms and which can be substituted by 1 to 5 F and/or Cl atoms and HaI is F, Cl, Br or I, and the physiologically acceptable salts and solvates thereof; d) compounds of formula I disclosed in WO 98/06704 in which B is A, OA, NH2, NHA, NAA′ or an unsaturated heterocycle which has 1 to 4 N, O and/or S atoms and which can be unsubstituted or mono-, di- or trisubstituted by HaI, A and/or OA, Q is absent or is alkylene having 1-6 C atoms, R1, R2 in each case independently of one another are —OH, OR5, —S—R5, —SO—R5, —SO2—R5, HaI, —NO2, —NH2, —NHR5 or —NR5R6, R1 and R2 together are also —O—CH2—O—, R3 R4 in each case independently of one another are H or A, R5, R6 in each case independently of one another are A, cycloalkyl having 3-7 C atoms, methylenecycloalkyl having 4-8 C atoms or alkenyl having 2-8 C atoms, A, A′ in each case independently of one another are alkyl which has 1 to 10 C atoms and which can be substituted by 1 to 5 F and/or Cl atoms and HaI is F, Cl, Br or I, and the stereoisomers and physiologically acceptable salts and solvates thereof; e) compounds disclosed in WO 00/59890 1-(4-ureidobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3-propoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-trifluoroacetamidobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-propoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-isopropoxycarbonylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-propoxycarbonylaminobenzoyl)-3-(3ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3,4-dimethoxyphenyl)-4-ethyl-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3,4-dimethoxyphenyl)-4-ethyl-1,4,5,6-tetrahydropyridazine and 1-(4-acetamidobenzoyl)-3-(3,4-dimethoxyphenyl)-4-ethyl-1,4,5,6-tetrahydropyridazine, and their physiologically acceptable salts and solvates; f) compounds of formula I disclosed in DE 19604388 in which R1, R2 in each case independently of one another are H or A, R3, R4 in each case independently of one another are —OH, OA, —S-A, —SO-A, —SO2-A, HaI, methylenedioxy, —NO2, —NH2, —NHA or —NAA′, A, A′ in each case independently of one another are alkyl having 1 to 10 C-atoms, and which can be substituted by 1 to 5 F and/or Cl atoms, cycloalkyl having 3-7 C atoms or methylenecycloalkyl having 4-8 C atoms, B is —Y—R5 oder —O—Y—R5, Q is absent or is alkylene having 1-4 C atoms, Y is absent or is alkylene having 1-10 C atoms, X is CH2 or S. R5 is NH2, NHA, NAA′ or is a saturated 3-8 membered heterocycle having at least one N atom, and wherein other CH2 groups optionally may be replaced by NH, NA, S or O, which can be unsubstituted or monosubstituted by A or OH, HaI is F, Cl, Br oder I and the stereoisomers and physiologically acceptable salts and solvates thereof; g) compounds of formula I disclosed in DE 19932315 in which R1, R2 in each case independently of one another are H, OH, OA, SA, SOA, SO2A, F, Cl or A′2N—(CH2)n—O—, R1 and R2 together are also —O—CH2—O—, R3, R4 in each case independently of one another are H, A, HaI, OH, OA, NO2, NHA, NA2, CN, COOH, COOA, NHCOA, NHSO2A or NHCOOA, R5, R6 in each case independently of one another are H or alkyl having 1 to 6 C atoms, A is alkyl having 1 to 10 C atoms, which can be substituted by 1 to 5 F and/or Cl atoms, is cycloalkyl having 3-7 C atoms, alkylenecycloalkyl having 5-10 C atoms or alkenyl having 2-8 C atoms, A′ is alkyl having 1, 2, 3, 4, 5 or 6 C atoms, n is 1, 2, 3 or 4, HaI is F, Cl, Br or 1, and their physiologically acceptable salts and solvates; h) compounds of formula I disclosed in EP 0723962 in which R1 and R2 in each case independently of one another are H or A, R3 and R4 in each case independently of one another are —OH, —OR10, —S—R10, —SO—R10, —SO2R1°, HaI, methylenedioxy, —NO2, —NH2, —NHR10 or —NR10R11, R5 is a phenyl radical which is unsubstituted or mono- or disubstituted by R6 and/or R7, Q is absent or is alkylene having 1-6 C atoms, R6 and R7 in each case independently of one another are —NH2, —NR8R9, —NHR10, —NR10R11, —NO2, HaI, —CN, —OA, —COOH or —COOA, R8 and R9 in each case independently of one another are H, acyl having 1-8 C atoms which can be substituted by 1-5 F and/or Cl atoms, —COOA, —S-A, —SO-A, —SO2A, —CONH2, —CONHA, —CONA2, —CO—COOH, —CO—COOA, —CO—CONH2, —CO—CONHA or —CO—CONA2.A is alkyl having 1 to 6 C atoms which can be substituted by 1-5 F and/or Cl atoms, R10 and R11 in each case independently of one another are A, cycloalkyl having 3-7 C atoms, methylenecycloalkyl having 4-8 C atoms or alkenyl having 2-8 C-atoms and HaI is F, Cl, Br or I, and their physiologically acceptable salts and solvates; i) compounds of formula I disclosed in EP 0738715 in which R1 and R2 in each case independently of one another are H or A, R3 and R4 in each case independently of one another are —OH, —OR10, —S—R10, —SO—R10, —SO2R10, HaI, methylenedioxy, —NO2, —NH2, —NHR10 or —NR10R11, R5 is a phenyl radical which is unsubstituted or mono- or disubstituted by R6 and/or R7, Q is absent or is alkylene having 1-6 C atoms, R6 and R7 in each case independently of one another are —NH2, —NR8R9, —NHR10, —NR10R11, —NO2, HaI, —CN, —OA, —COOH or —COOA, R8 and R9 in each case independently of one another are H, acyl having 1-8 C atoms which can be substituted by 1-5 F and/or Cl atoms, —COOA, —SO-A, —SO2A, —CONH2, —CONHA, —CONA2, —CO—COOH, —CO—COOA, —CO—CONH2, —CO—CONHA or —CO—CONA2, A is alkyl having 1 to 6 C atoms which can be substituted by 1-5 F and/or Cl atoms, R10 and R11 in each case independently of one another are A, cycloalkyl having 3-7 C atoms, methylenecycloalkyl having 4-8 C atoms or alkenyl having 2-8 C-atoms and HaI is F, Cl, Br or I, and their physiologically acceptable salts and solvates; for preparing a medicament for treating myocardial diseases.","Preferably, the invention provides for the use of a) compounds disclosed in EP 0763534: 2-(3-nicotinoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(2-nicotinoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(3-nicotinoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(2-nicotinoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-methoxy-4-trifluoromethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-methoxy-4-difluoromethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-methoxy-4-fluoromethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-difluoromethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-trifluoromethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-fluoromethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-methoxy-4-ethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-4-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-hydroxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(4-methylsulfonylphenyl)-5ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(4-methyleneoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(3-nicotinoylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminophenethyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminophenethyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 3-(4-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(2-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(2-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-trifluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-difluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyi)-5-(3-methoxy-4-fluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-difluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-trifluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-fluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-ethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-ethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-ethoxy-4-methoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-hydroxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(4-methysulfonylphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(4-methyleneoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-cyclopentyloxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3-cyclopentyloxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminophenethyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminophenethyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(2-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(2-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-trifluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-difluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-fluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-difluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-trifluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-fluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-ethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-ethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-hydroxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(4-methylsulfonylphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(4-methyleneoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-cyclopentyloxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3-cyclopentyloxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminophenethyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminophenethyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 2-(3-nicotinoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isonicotinoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pyrazinecarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-(isoxazole-5-carbonylamino)benzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3,4,-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, hydrochloride, and their stereoisomers and physiologically acceptable, salts and solvates; b) compounds disclosed in WO 99/65880 N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-methoxybenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-methylbenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)benzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-3,4-dichlorobenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-trifluoromethylbenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-3-chlorobenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-fluorobenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-butoxybenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-pentoxybenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-ethoxybenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-3,4-dimethoxybenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-3-methylbenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-3-methoxybenzoyl-3-carboxamide, and their physiologically acceptable salts and solvates; c) compounds disclosed in WO 00/26201 3-dimethylaminopropyl {4-[3-(3-ethoxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, N-methylpiperidin-4-yl-4-[3-(3-ethoxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl {4-[3-(3-isopropoxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl {3-[3-(3-ethoxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl{3-[3-(3-cyclopentyloxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, N-methylpiperidin-4-yl-3[3-(3-cyclopentyloxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl{3-[3-(3-propyloxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl{4-[3-(3,4-diethoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, N-methylpiperidin-4-yl-{4-[3-(3,4-diethoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl{3-[3-(3,4-dimethoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate 3-dimethylaminopropyl{4-[3-(3,4-dimethoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, and the physiologically acceptable salts and solvates thereof; d) compounds disclosed in WO 98/06704 1-(4-nicotinoylaminobenzoyl)-3-(3,4-dimethoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(3-nicotinoylaminobenzoyl)-3-(3,4-dimethoxy-phenyl)-1,4,5,6-tetrahydropyridazine hydrochloride, 1-(2-nicotinoylaminobenzoyl)-3-(3,4-dimethoxy-phenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(3-nicotinoylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3-cyclopentyloxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(3-nicotinoylaminobenzoyl)-3-(3-cyclopentyloxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3,4-methylene-dioxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3-methoxy-4-methylsulfonylphenyl)-1,4,5,6-tetrahydro-pyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3-trifluoro-methoxy-4-methoxyphenyl)-1,4,5,6-tetrahydro-pyridazine, 1-(4-ethoxy-carbonylaminobenzoyl)-3-(3,4-dimethoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(3-ethoxycarbonylaminobenzoyl)-3-(3,4-dimethoxy-phenyl)-1,4,5,6-tetrahydropyridazine, 1-(2-ethoxycarbonylaminobenzoyl)-3-(3,4-dimethoxy-phenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(3-ethoxycarbonylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-cyclo-pentyloxy-4-methoxyphenyl)-1,4,5,6-tetrahydro-pyridazine, 1-(3-ethoxycarbonylaminobenzoyl)-3-(3-cyclo-pentyloxy-4-methoxyphenyl)-1,4,5,6-tetrahydro-pyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3,4-methylene-dioxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-methoxy-4-methylsulfonylphenyl)-1,4,5,6-tetrahydro-pyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-trifluoro-methoxy-4-methoxyphenyl)-1,4,5,6-tetrahydro-pyridazine, and the stereoisomers and physiologically acceptable salts and solvates thereof; e) compounds disclosed in EP 0723962 3-(4-ethoxycarbonylaminobenzyl)-5-(3-ethoxy-4-methoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-ethoxycarbonylaminobenzyl)-5-(3-cyclopentyloxy-4-methoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, and their physiologically acceptable salts and solvates; f) compounds disclosed in EP 0738715 2-(4-butyrylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-acetamidobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-trifluoroacetamidobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methylsulfonamidobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-propionylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-tert-butylcarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isobutyrylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxycarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pivalylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2, 3,4,5-tetrahydropyridazin-3-one, 2-(4-cyclopentylcarbamoylbenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ethoxycarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2, 3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxalylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ureidobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentanoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-hexanoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropiridazin-3-one, 2-(4-pentafluoropropionylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-acetamidobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-trifluoroacetamidobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methylsulfonamidobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-propionylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-tert-butylcarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-butyrylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isobutyrylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxycarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pivalylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-cyclopentylcarbamoylbenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ethoxycarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxalylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ureidobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentanoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-hexanoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentafluoropropionylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-acetamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-trifluoroacetamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methylsulfonamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-propionylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-butyrylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isobutyrylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxycarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pivalylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-cyclopentylcarbamoylbenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ethoxycarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one; 2-(4-methoxalylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ureidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentanoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-hexanoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentafluoropropionylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-acetamidobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-trifluoroacetamidobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methylsulfonamidobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-propionylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-tert-butyloarbonylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-butyrylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isobutyrylaminobenzyl)-6(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxycarbonylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pivalylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-cyclopentylcarbamoylbenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ethoxycarbonylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxalylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ureidobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentanoylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-hexanoylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentafluoropropionylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-acetamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-trifluoroacetamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methylsulfonamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-propionylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-butyrylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isobutyrylaminobenzyl)-6-(3ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxycarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pivalylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-cyclopentylcarbamoylbenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ethoxycarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxalylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ureidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentanoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-hexanoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentafluoropropionylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, and their physiologically acceptable salts and solvates; for preparing a medicament for treating myocardial diseases.","Most preferably, the invention provides for the use of the following compounds 3-(4-nicotinoylaminobenzyl)-5-(3-ethoxy-4-methoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-methoxybenzoyl-3-carboxamide, 1-(4-nicotinoylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 2-(4-ethoxycarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, and their physiologically acceptable salts and solvates; for preparing a medicament for treating myocardial diseases.","The preferred compounds show a selective inhibition of phosphodiesterase IV, which is associated with an intracellular increase in cAMP (N. Sommer et al., Nature Medicine, 1, 244-248 (1995)).","The inhibition of PDE IV can be demonstrated, for example, analogously to C. W. Davis in Biochim.","Biophys.","Acta 797, 354-362 (1984).","The affinity of the compounds of the invention for phosphodiesterase IV is measured by determining their IC50 values (the concentration of inhibitor required to achieve 50% inhibition of the enzyme activity).","Preferably, the invention provides for the use of the compounds mentioned above for preparing a medicament for treating myocardial diseases, where said myocardial diseases show inflammatory and immunological characteristics.","Most preferably, the invention provides for the use of the compounds mentioned above for preparing a medicament for treating coronary artery disease, reversible or irreversible myocardial ischemia/reperfusion injury, acute or chronic heart failure and restenosis, including instent-restenosis and stent-in-stent-restenosis.","The preparations for the treatment of the mentioned diseases can be used as medicaments in human or veterinary medicine.","Possible excipients are organic or inorganic substances which are suitable for enteral (e.g.","oral) or parenteral administration or topical application and do not react with the novel compounds, for example water, vegetable oils, benzyl alcohols, alkylene glycols, polyethylene glycols, glycerol triacetate, gelatin, carbohydrates such as lactose or starch, magnesium stearate, talc and petroleum jelly.","In particular, tablets, pills, coated tablets, capsules, powders, granules, syrups, juices or drops are used for oral administration, suppositories are used for rectal administration, solutions, preferably oily or aqueous solutions, and furthermore suspensions, emulsions or implants, are used for parenteral administration, and ointments, creams or powders are used for topical application.","The novel compounds can also be lyophilized and the lyophilizates obtained used, for example, for the production of injection preparations.","The preparations indicated can be sterilized and/or can contain auxiliaries such as lubricants, preservatives, stabilizers and/or wetting agents, emulsifiers, salts for affecting the osmotic pressure, buffer substances, colourants, flavourings and/or one or more further active compounds, e.g.","one or more vitamins.","In these indications the substances are generally administered preferably in doses of between approximately 1 and 500 mg, in particular between 5 and 100 mg per dose unit.","The daily dose is preferably between approximately 0.02 and 10 mg/kg of body weight.","The specific dose for each patient depends, however, on a wide variety of factors, for example on the efficacy of the specific compound used, on age, body weight, general state of health, gender, on the diet, on the time and route of administration, on the excretion rate, medicament combination and severity of the particular disease to which the therapy is applied.","Oral administration is preferred.","EXAMPLE 1 Effect of PDE4 Inhibitors on T-Cell Proliferation Peripheral blood mononuclear cells (PBMC) were isolated from the blood of healthy donors by the Lymphoprep gradient method.","200000 PBMC/well were cultured in RPMI1640 culture medium containing 5% heat inactivated human serum (AB pool) for 5 days at 37° C. and 10% CO2 in 96 well flat bottom microtiter plates.","The T cells within the PBMC preparation were selectively stimulated with an monoclonal antibody to CD3.Cultures were set up as triplicates including a control group receiving no treatment.","PDE4 inhibitors were dissolved in DMSO at 10−2 M and diluted in culture medium.","Control cultures were treated with DMSO equivalent to the inhibitor concentration.","18 hrs before the end of the assay, 3H-thymidine was added to the cultures.","The incorporation of radioactivity into the cells was then measured in a beta-counter.","The data of at least three independent experiments were calculated as percent inhibition of the control (mean ±SEM) without inhibitor.","From this data the IC-50 value was determined.","Results: PDE4 inhibitors afforded a marked reduction of T-cell proliferation (see table 1).","TABLE 1 Effect of PDE4 inhibitors on T-cells and macrophages IL2 IFN-γ TNF-α IL10 IL12 T-cell T-cell macrophage macrophage macrophage T-cell prol.","IC50 [μM] IC50 [μM] IC50 [μM] EC50 [μM] IC50 [μM] IC50 [μM] Rolipram 0.7 1 0.6 0.05 >0.1 0.3 EMD 94360 0.3 0.05 0.03 0.001 0.006 0.02 EMD 95832 0.2 0.01 0.009 0.02 0.0005 0.05 EMD 125025 0.002 0.007 0.003 no effect <1E−13M 0.006 EMD 125059 0.06 0.008 0.03 0.008 <1E−13M 0.04 EMD 219901 0.003 0.005 0.002 0.001 <1E−13M 0.007 EMD 94360 (EP 0738715; Page 17, line 41): EMD 95832 (EP 0763534): EMD 125025 (WO 98/06704; page 13, lines 16-17): EMD 125059 (WO 98/06704; page 27, lines 2-3): EMD 219901 (WO 99/65880; page 14, lines 20-21) EXAMPLE 2 Effect of PDE4 Inhibitors on Cytokine Production in Human Peripheral Blood Monocytic Cells Peripheral blood mononuclear cells (PBMC) were isolated from the blood of healthy donors by the Lymphoprep gradient method.","200000 PBMC/well were cultured in RPMI1640 culture medium containing 5% heat inactivated human serum (AS pool).","at 37° C. and 10% CO2 in 96 well flat bottom microtiter plates.","Cultures were set up as triplicates including a control group.","Solutions of PDE4 inhibitors were prepared in DMSO at 10−2 M and diluted in culture medium.","Control cultures were treated with concentrations of DMSO equivalent to the inhibitor concentrations.","The cytokine of interest was stimulated as indicated in Table 2 The culture supernatants of three independent experiments were pooled and cytokine activity in the supernatant was measured with commercially available ELISA test kits.","TABLE 2 Activation of PBMC to form T-cell and macrophage specific cytokines cytokine activator incubation in culture IL-2 mab anti-CD3 24 h IL-10 LPS 48 h TNF-α LPS 48 h IFN-γ mab anti-CD3 48 h IL-12 SAC + IFNγ 48 h The data were calculated as percent inhibition/stimulation of the control without the compound and the IC50 value or EC50 value in case of stimulation was determined thereof.","Result PDE4 inhibitors afforded a marked reduction in the release of IL-2, IFN-γ, TNF-α and IL-12.The immunosuppressant cytokine IL-10, however, was stimulated by most PDE4 inhibitors (see Table 1).","EXAMPLE 3 Effect of PDE4 Inhibitors on Experimental Myocardial Infarction in Rats Compound 5, administered intraperitoneally with 1, 3, and 10 mg/kg, 1 hour before reversible occlusion of the left coronary artery in rats caused a significant dose dependent reduction of infarct size up to 38%.","In correspondence with this protection, a reduction of plasma TNF-□ levels was observed, as measured by ELISA.","EXAMPLE 4 Effect of PDE4 Inhibitors on Experimental Myocardial Infarction in Rabbits There was a cardioprotective effect by PDE4 inhibition in anaesthetised rabbits subjected to 30 minutes of coronary artery occlusion (side branch of the ramus circumflexus of the left coronary artery) followed by 120 minutes of reperfusion.","PDE4 inhibitors applied prior to the coronary occlusion reduced infarct size as compared with placebo treatment.","The areas at risk were comparable between verum and placebo groups.","The cardioprotective effect cannot be attributed to favorable hemodynamic effects, since heart rate and mean aortic pressure remained constant throughout the experimental protocol."]],"string":"[\n [\n \"Use of type 4 phosphodiesterase inhibitors in myocardial diseases\",\n \"The invention relates to the use of type 4 phosphodiesterase inhibitors to treat myocardial diseases.\"\n ],\n [\n \"1.Use of a) compounds of formula I disclosed in EP 0763534 in which B is an aromatic heterocycle having 1 to 4 N, O and/or S atoms, bonded via N or C, which can be unsubstituted or mono-, di- or trisubstituted by HaI, A and/or OA, and can also be fused to a benzene or pyridine ring, Q is absent or is alkylene having 1-6 C atoms, X is CH2, S or O, R1 and R2 in each case independently of one another are H or A, R3 and R4 in each case independently of one another are —OH, OR5, —S—R5, —SO—R5, —SO2—R5, HaI, methylenedioxy, —NO2, —NH2, —NHR5 or —NR5R6, R5 and R6 in each case independently of one another are A, cycloalkyl having 3-7 C atoms, methylenecycloalkyl having 4-8 C atoms or alkenyl having 2-8 C atoms, A is alkyl having 1 to 10 C atoms, which can be substituted by 1 to 5 F and/or Cl atoms and HaI is F, Cl, Br or I and their stereoisomers and physiologically acceptable, salts and solvates; b) compounds of formula I disclosed in WO 99/65880 in which B is a phenyl ring which is unsubstituted or mono- or polysubstituted by R3, Q is absent or is alkylene having 1-4 C atoms, R1,R2 each independently of one another are —OR4, —S—R4, —SO—R4, —SO2—R4 or HaI, R1 and R2 together are also —O—CH2—O—, R3 is R4, HaI, OH, OR4, OPh, NO2, NHR4, N(R4)2, NHCOR4, NHSO2R4 or NHCOOR4, R4 is A, cycloalkyl having 3-7 C atoms, alkylenecycloalkyl having 5-10 C atoms or alkenyl having 2-8 C atoms, A is alkyl having 1 to 10 C atoms, which can be substituted by 1 to 5 F and/or Cl atoms and HaI is F, Cl, Br or I, and their physiologically acceptable salts and solvates; c) compounds of formula I disclosed in WO 00/26201 in which R1, R2 in each case independently of one another are —OH, OR5, —S—R5, —SO—R5, —SO2—R5 or HaI, R1 and R2 together are also —O—CH2—O—, R3 is NH2, NHA, NAA′ or a saturated heterocycle having 1 to 4 N, O and/or S atoms which can be unsubstituted or mono-, di- or tri-substituted by HaI, A and/or OA, Q is absent or is branched or unbranched alkylene having 1-10 C atoms, R5 is A, cycloalkyl having 3-7 C atoms, alkylenecycloalkyl having 4-8 C atoms or alkenyl having 2-8 C atoms, A, A′ in each case independently of one another are alkyl which has 1 to 10 C atoms and which can be substituted by 1 to 5 F and/or Cl atoms and HaI is F, Cl, Br or I, and the physiologically acceptable salts and solvates thereof; d) compounds of formula I disclosed in WO 98/06704 in which B is A, OA, NH2, NHA, NAA′ or an unsaturated heterocycle which has 1 to 4 N, O and/or S atoms and which can be unsubstituted or mono-, di- or trisubstituted by HaI, A and/or OA, Q is absent or is alkylene having 1-6 C atoms, R1, R2 in each case independently of one another are —OH, OR5, —S—R5, —SO—R5, —SO2—R5, HaI, —NO2, —NH2, —NHR5 or —NRR6, R1 and R2 together are also —O—CH2—O—, R3, R4 in each case independently of one another are H or A, R5, R6 in each case independently of one another are A, cycloalkyl having 3-7 C atoms, methylenecycloalkyl having 4-8 C atoms or alkenyl having 2-8 C atoms, A, A′ in each case independently of one another are alkyl which has 1 to 10 C atoms and which can be substituted by 1 to 5 F and/or Cl atoms and HaI is F, Cl, Br or I, and the stereoisomers and physiologically acceptable salts and solvates thereof; e) compounds disclosed in WO 00/59890 1-(4-ureidobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3-propoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-trifluoroacetamidobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-propoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-isopropoxycarbonylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-propoxycarbonylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3,4-dimethoxyphenyl)-4-ethyl-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3,4-dimethoxyphenyl)-4-ethyl-1,4,5,6-tetrahydropyridazine and 1-(4-acetamidobenzoyl)-3-(3,4-dimethoxyphenyl)-4-ethyl-1,4,5,6-tetrahydropyridazine, and their physiologically acceptable salts and solvates; f) compounds of formula I disclosed in DE 19604388 in which R1, R2 in each case independently of one another are H or A, R3, R4 in each case independently of one another are —OH, OA, —S-A, —SO-A, —SO2-A, HaI, methylenedioxy, —NO2, —NH2, —NHA or —NAA′, A, A′ in each case independently of one another are alkyl having 1 to 10 C-atoms, and which can be substituted by 1 to 5 F and/or Cl atoms, cycloalkyl having 3-7 C atoms or methylenecycloalkyl having 4-8 C atoms, B is —Y—R5 oder —O—Y—R5, Q is absent or is alkylene having 1-4 C atoms, Y is absent or is alkylene having 1-10 C atoms, X is CH2or S, R5 is NH2, NHA, NAA′ or is a saturated 3-8 membered heterocycle having at least one N atom, and wherein other CH2 groups optionally may be replaced by NH, NA, S or O, which can be unsubstituted or monosubstituted by A or OH, HaI is F, Cl, Br oder I and the stereoisomers and physiologically acceptable salts and solvates thereof; g) compounds of formula I disclosed in DE 19932315 in which R1, R2 in each case independently of one another are H, OH, OA, SA, SOA, SO2A, F, Cl or A′2N—(CH2)n—O—, R1 and R2 together are also —O—CH2—O—, R3, R4 in each case independently of one another are H, A, HaI, OH, OA, NO2, NHA, NA2, CN, COOH, COOA, NHCOA, NHSO2A or NHCOOA, R5, R6 in each case independently of one another are H or alkyl having 1 to 6 C atoms, A is alkyl having 1 to 10 C atoms, which can be substituted by 1 to 5 F and/or Cl atoms, is cycloalkyl having 3-7 C atoms, alkylenecycloalkyl having 5-10 C atoms or alkenyl having 2-8 C atoms, A′ is alkyl having 1, 2, 3, 4, 5 or 6 C atoms, n is 1, 2, 3 or 4, HaI is F, Cl, Br or I, and their physiologically acceptable salts and solvates; h) compounds of formula I disclosed in EP 0723962 in which R1 and R2 in each case independently of one another are H or A, R3 and R4 in each case independently of one another are —OH, —OR10, —S—R10, —SO—R10, —SO2R10, HaI, methylenedioxy, —NO2, —NH2, —NHR10 or —NR10R11, R5 is a phenyl radical which is unsubstituted or mono- or disubstituted by R6 and/or R7, Q is absent or is alkylene having 1-6 C atoms, R6 and R7 in each case independently of one another are —NH2, —NR8R9, —NHR10, —NR10R11, —NO2, HaI, —CN, —OA, —COOH or —COOA, R8 and R9 in each case independently of one another are H, acyl having 1-8 C atoms which can be substituted by 1-5 F and/or Cl atoms, —COOA, —S-A, —SO-A, —SO2A, —CONH2, —CONHA, —CONA2, —CO—COOH, —CO—COOA, —CO—CONH2, —CO—CONHA or —CO—CONA2, A is alkyl having 1 to 6 C atoms which can be substituted by 1-5 F and/or Cl atoms, R10 and R11 in each case independently of one another are A, cycloalkyl having 3-7 C atoms, methylenecycloalkyl having 4-8 C atoms or alkenyl having 2-8 C-atoms and HaI is F, Cl, Br or I, and their physiologically acceptable salts and solvates; i) compounds of formula I disclosed in EP 0738715 in which R1 and R2 in each case independently of one another are H or A, R3 and R4 in each case independently of one another are —OH, —OR10, —S—R10, —SO—R10, —SO2R10, HaI, methylenedioxy, —NO2, —NH2, —NHR10 or —NR10R11, R5 is a phenyl radical which is unsubstituted or mono- or disubstituted by R6 and/or R7, Q is absent or is alkylene having 1-6 C atoms, R6 and R7 in each case independently of one another are —NH2, —NR8R9, —NHR10, —NR10R11, —NO2, HaI, —CN, —OA, —COOH or —COOA, R8 and R9 in each case independently of one another are H, acyl having 1-8 C atoms which can be substituted by 1-5 F and/or Cl atoms, —COOA, —SO-A, —SO2A, —CONH2, —CONHA, —CONA2, —CO—COOH, —CO—COOA, —CO—CONH2, —CO—CONHA or —CO—CONA2, A is alkyl having 1 to 6 C atoms which can be substituted by 1-5 F and/or Cl atoms, R10 and R11 in each case independently of one another are A, cycloalkyl having 3-7 C atoms, methylenecycloalkyl having 4-8 C atoms or alkenyl having 2-8 C-atoms and HaI is F, Cl, Br or I, and their physiologically acceptable salts and solvates; for preparing a medicament for treating myocardial diseases.\",\n \"2.Use according to claim 1 of a) compounds disclosed in EP 0763534: 2-(3-nicotinoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(2-nicotinoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(3-nicotinoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(2-nicotinoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-methoxy-4-trifluoromethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-methoxy-4-difluoromethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-methoxy-4-fluoromethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-difluoromethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-trifluoromethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-fluoromethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-methoxy-4-ethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-hydroxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(4-methylsulfonylphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(4-methyleneoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(3-nicotinoylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminophenethyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminophenethyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 3-(4-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(2-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(2-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-trifluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-difluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-fluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-difluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-trifluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-fluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-ethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-ethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-ethoxy-4-methoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-hydroxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(4-methysulfonylphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(4-methyleneoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-cyclopentyloxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3-cyclopentyloxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminophenethyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminophenethyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(2-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(2-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-trifluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-difluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-fluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-difluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-trifluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-fluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-ethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-ethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-hydroxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(4-methylsulfonylphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(4-methyleneoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-cyclopentyloxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3-cyclopentyloxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminophenethyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminophenethyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 2-(3-nicotinoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isonicotinoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pyrazinecarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-(isoxazole-5-carbonylamino)benzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3,4,-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, hydrochloride, and their stereoisomers and physiologically acceptable, salts and solvates; b) compounds disclosed in WO 99/65880 N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-methoxybenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-methylbenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)benzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-3,4-dichlorobenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-trifluoromethylbenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-3-chlorobenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-fluorobenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-butoxybenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-pentoxybenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-ethoxybenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-3,4-dimethoxybenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-3-methylbenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-3-methoxybenzoyl-3-carboxamide, and their physiologically acceptable salts and solvates; c) compounds disclosed in WO 00/26201 3-dimethylaminopropyl{4-[3-(3-ethoxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, N-methylpiperidin-4-yl-{4-[3-(3-ethoxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl{4-[3-(3-isopropoxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl{3-[3-(3-ethoxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl{3-[3-(3-cyclopentyloxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, N-methylpiperidin-4-yl-{3[3-(3-cyclopentyloxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl{3-[3-(3-propyloxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl{4-[3-(3,4-diethoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, N-methylpiperidin-4-yl-{4-[3-(3,4-diethoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl{3-[3-(3,4-dimethoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate 3-dimethylaminopropyl{4-[3-(3,4-dimethoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, and the physiologically acceptable salts and solvates thereof; d) compounds disclosed in WO 98/06704 1-(4-nicotinoylaminobenzoyl)-3-(3,4-dimethoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(3-nicotinoylaminobenzoyl)-3-(3,4-dimethoxy-phenyl)-1,4,5,6-tetrahydropyridazine hydrochloride, 1-(2-nicotinoylaminobenzoyl)-3-(3,4-dimethoxy-phenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(3-nicotinoylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3-cyclopentyloxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(3-nicotinoylaminobenzoyl)-3-(3-cyclopentyloxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3,4-methylene-dioxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3-methoxy-4-methylsulfonylphenyl)-1,4,5,6-tetrahydro-pyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3-trifluoro-methoxy-4-methoxyphenyl)-1,4,5,6-tetrahydro-pyridazine, 1-(4-ethoxy-carbonylaminobenzoyl)-3-(3,4-dimethoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(3-ethoxycarbonylaminobenzoyl)-3-(3,4-dimethoxy-phenyl)-1,4,5,6-tetrahydropyridazine, 1-(2-ethoxycarbonylaminobenzoyl)-3-(3,4-dimethoxy-phenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(3-ethoxycarbonylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-cyclo-pentyloxy-4-methoxyphenyl)-1,4,5,6-tetrahydro-pyridazine, 1-(3-ethoxycarbonylaminobenzoyl)-3-(3-cyclo-pentyloxy-4-methoxyphenyl)-1,4,5,6-tetrahydro-pyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3,4-methylene-dioxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-methoxy-4-methylsulfonylphenyl)-1,4,5,6-tetrahydro-pyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-trifluoro-methoxy-4-methoxyphenyl)-1,4,5,6-tetrahydro-pyridazine, and the stereoisomers and physiologically acceptable salts and solvates thereof; e) compounds disclosed in EP 0723962 3-(4-ethoxycarbonylaminobenzyl)-5-(3-ethoxy-4-methoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-ethoxycarbonylaminobenzyl)-5-(3-cyclopentyloxy-4-methoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, and their physiologically acceptable salts and solvates; f) compounds disclosed in EP 0738715 2-(4-butyrylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-acetamidobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-trifluoroacetamidobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methylsulfonamidobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-propionylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-tert-butylcarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isobutyrylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxycarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pivalylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-cyclopentylcarbamoylbenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ethoxycarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxalylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ureidobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentanoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-hexanoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropiridazin-3-one, 2-(4-pentafluoropropionylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-acetamidobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-trifluoroacetamidobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methylsulfonamidobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-propionylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-tert-butylcarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-butyrylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isobutyrylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxycarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pivalylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-cyclopentylcarbamoylbenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ethoxycarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxalylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ureidobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentanoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-hexanoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentafluoropropionylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-acetamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-trifluoroacetamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methylsulfonamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-propionylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-butyrylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isobutyrylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxycarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pivalylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-cyclopentylcarbamoylbenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ethoxycarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4, 5-tetrahydropyridazin-3-one; 2-(4-methoxalylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ureidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentanoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-hexanoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentafluoropropionylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-acetamidobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-trifluoroacetamidobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methylsulfonamidobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-propionylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-tert-butylcarbonylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-butyrylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isobutyrylaminobenzyl)-6(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxycarbonylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pivalylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-cyclopentylcarbamoylbenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ethoxycarbonylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxalylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ureidobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentanoylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-hexanoylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentafluoropropionylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-acetamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-trifluoroacetamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methylsulfonamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-propionylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-butyrylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isobutyrylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxycarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pivalylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-cyclopentylcarbamoylbenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ethoxycarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxalylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ureidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentanoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-hexanoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentafluoropropionylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, and their physiologically acceptable salts and solvates; for preparing a medicament for treating myocardial diseases.\",\n \"3.Use according to claim 1 of compounds selected from 3-(4-nicotinoylaminobenzyl)-5-(3-ethoxy-4-methoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-methoxybenzoyl-3-carboxamide, 1-(4-nicotinoylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 2-(4-ethoxycarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, and their physiologically acceptable salts and solvates; for preparing a medicament for treating myocardial diseases.\",\n \"4.Use according to claim 1 for preparing a medicament for treating myocardial diseases, where said myocardial diseases show inflammatory and immunological characteristics.\",\n \"5.Use according to claim 1 for preparing a medicament for treating coronary artery disease, reversible or irreversible myocardial ischemia/reperfusion injury, acute or chronic heart failure and restenosis, including instent-restenosis and stent-in-stent-restenosis.\"\n ],\n [\n \"The invention relates to the use of type 4 phosphodiesterase inhibitors to treat myocardial diseases.\",\n \"Coronary artery disease is the most common cause of death in the western world.\",\n \"In the presence of a critical stenosed coronary artery, a decrease of blood flow may result in myocardial ischemia.\",\n \"Initiation of reperfusion results, depending on the severity of the preceding ischemic period, in a reversibly or irreversibly injured myocardium, which is characterized by a long-lasting depression or an irreversible loss of contractile function.\",\n \"Depending on the size of the affected myocardial area, an acute or a chronic heart failure may develop.\",\n \"A particular clinical problem in the above mentioned scenario is the development of restenosis after a primarily successful reperfusion intervention by PTCA, even after stent implantation, thrombolysis or coronary artery bypass grafting.\",\n \"From experimental animal and clinical studies evidence exists, that in the different above mentioned heart diseases, i. e. coronary artery disease itself, reversible or irreversible myocardial ischemia/reperfusion injury, acute or chronic heart failure and restenosis, including instent-restenosis and stent-in-stent-restenosis, inflammatory processes play a casual role.\",\n \"These inflammatory processes involve resident and invading macrophages as well as neutrophils and TH1 and TH2 helper cells.\",\n \"This leukocyte response produces the characteristic cytokine pattern, involving TNF-α, IL-1β, IL-2, and IL-6, as well as IL-10 and IL-13 (Pulkki K J: Cytokines and cardiomyocyte death.\",\n \"Ann.Med.\",\n \"1997 29: 339-343.Birks E J, Yacoub M H: The role of nitric oxide and cytokines in heart failure.\",\n \"Coron.Artery.Dis.\",\n \"1997 8: 389-402).\",\n \"The formation of these species has been demonstrated in human patients with myocardial ischemia.\",\n \"Animal models show that cytokine production correlates with the invasion of peripheral macrophages and neutrophils which can spread the damage into the still intact myocardium.\",\n \"The main player in the cytokine response, however, is TNF-a, which integrates inflammatory and pro-apoptotic responses and additionally has a direct negative ionotropic effect on cardiac myocytes (Ceconi C, Curello S, Bachetti T, Corti A, Ferrari R: Tumor necrosis factor in congestive heart failure: a mechanism of disease for the new millennium?\",\n \"Prog.Cardiovasc.Dis.\",\n \"1998 41: 25-30.Mann D L: The effect of tumor necrosis factor-alpha on cardiac structure and function: a tale of two cytokines.\",\n \"J.Card.Fail.\",\n \"1996 2: S165-S172.Squadrito F, Altavilla D, Zingarelli B, et al: Tumor necrosis factor involvement in myocardial ischaemia-reperfusion injury.\",\n \"Eur.J.Pharmacol.\",\n \"1993 237: 223-230).\",\n \"It has been shown in animal models of myocardial infarction, that TNF-α is rapidly released during the reperfusion phase (Herskowitz A, Choi S, Ansari A A, Wesselingh S: Cytokine mRNA expression in postischemic/reperfused myocardium.\",\n \"Am.J.Pathol.\",\n \"1995 146: 419-428) and that the protective effects of drugs such as dexamethasone (Arras M, Strasser R, Mohri M, et al: Tumor necrosis factor-alpha is expressed by monocytes/macrophages following cardiac microembolization and is antagonized by cyclosporine.\",\n \"Basic.Res.Cardiol.\",\n \"1998 93: 97-107), cyclosporin A (Arras M, Strasser R, Mohri M, et al: Tumor necrosis factor-alpha is expressed by monocytes/macrophages following cardiac microembolization and is antagonized by cyclosporine.\",\n \"Basic.Res.Cardiol.\",\n \"1998 93: 97-107.Squadrito F, Altavilla D, Squadrito G, et al: Cyclosporin-A reduces leukocyte accumulation and protects against myocardial ischaemia reperfusion injury in rats.\",\n \"Eur.J.Pharmacol.\",\n \"1999 364: 159-168) or clorichromene (Squadrito F. Altavilla D, Zingarelli B, et al: The effect of cloricromene, a coumarine derivative, on leukocyte accumulation, myocardial necrosis and TNF-alpha production in myocardial ischaemia-reperfusion injury.\",\n \"Life Sci.\",\n \"1993 53: 341-355)correspond with a reduction of circulating TNF-α.\",\n \"Preferred PDE4 inhibitors mentioned below are potent antagonists of macrophage and T-cell cytokine production.\",\n \"They also inhibit the proliferation of T cells.\",\n \"Consequently, PDE4 inhibition may have a beneficial effect in those myocardial diseases, which are causally linked to cytokine production and inflammatory processes.\",\n \"As compared with PDE3 inhibitors and the early PDE4 inhibitor Rolipram, preferred PDE4 inhibitors are devoid of hemodynamic side effects, which can be dose limiting for the treatment of most cardiovascular disorders.\",\n \"The invention was based on the object of discovering new uses of compounds having valuable properties, especially those which may be used to prepare medicaments.\",\n \"It has been found that the preferred PDE IV inhibitors and their salts combine very valuable pharmacological properties with good tolerability for the treatment of myocardial diseases.\",\n \"Accordingly, the invention provides in particular for the use of a) compounds of formula l disclosed in EP 0763534 in which B is an aromatic heterocycle having 1 to 4 N, O and/or S atoms, bonded via N or C, which can be unsubstituted or mono-, di- or trisubstituted by HaI, A and/or OA, and can also be fused to a benzene or pyridine ring, Q is absent or is alkylene having 1-6 C atoms, X is CH2, S or O, R1 and R2 in each case independently of one another are H or A, R3 and R4 in each case independently of one another are —OH, OR5, —S—R5, —SO—R5, —SO2—R5, HaI, methylenedioxy, —NO2, —NH2, —NHR5 or —NR5R6, R5 and R6 in each case independently of one another are A, cycloalkyl having 3-7 C atoms, methylenecycloalkyl having 4-8 C atoms or alkenyl having 2-8 C atoms, A is alkyl having 1 to 10 C atoms, which can be substituted by 1 to 5 F and/or Cl atoms and HaI is F, Cl, Br or I and their stereoisomers and physiologically acceptable, salts and solvates; b) compounds of formula I disclosed in WO 99/65880 in which B is a phenyl ring which is unsubstituted or mono- or polysubstituted by R3, Q is absent or is alkylene having 1-4 C atoms, R1,R2 each independently of one another are —OR4, —S—R4, —SO—R4, —SO2—R4 or HaI, R1 and R2 together are also —O—CH2—O—, R3 is R4, HaI, OH, OR4, OPh, NO2, NHR4, N(R4)2, NHCOR4, NHSO2R4 or NHCOOR4, R4 is A, cycloalkyl having 3-7 C atoms, alkylenecycloalkyl having 5-10 C atoms or alkenyl having 2-8 C atoms, A is alkyl having 1 to 10 C atoms, which can be substituted by 1 to 5 F and/or Cl atoms and HaI is F, Cl, Br or I, and their physiologically acceptable salts and solvates; c) compounds of formula I disclosed in WO 00/26201 in which R1, R2 in each case independently of one another are —OH, OR5, —S—R5, —SO—R5, —SO2—R5 or HaI, R1 and R2 together are also —O—CH2—O—, R3 is NH2, NHA, NAA′ or a saturated heterocycle having 1 to 4 N, O and/or S atoms which can be unsubstituted or mono-, di- or tri-substituted by HaI, A and/or OA, Q is absent or is branched or unbranched alkylene having 1-10 C atoms, R5 is A, cycloalkyl having 3-7 C atoms, alkylenecycloalkyl having 4-8 C atoms or alkenyl having 2-8 C atoms, A, A′ in each case independently of one another are alkyl which has 1 to 10 C atoms and which can be substituted by 1 to 5 F and/or Cl atoms and HaI is F, Cl, Br or I, and the physiologically acceptable salts and solvates thereof; d) compounds of formula I disclosed in WO 98/06704 in which B is A, OA, NH2, NHA, NAA′ or an unsaturated heterocycle which has 1 to 4 N, O and/or S atoms and which can be unsubstituted or mono-, di- or trisubstituted by HaI, A and/or OA, Q is absent or is alkylene having 1-6 C atoms, R1, R2 in each case independently of one another are —OH, OR5, —S—R5, —SO—R5, —SO2—R5, HaI, —NO2, —NH2, —NHR5 or —NR5R6, R1 and R2 together are also —O—CH2—O—, R3 R4 in each case independently of one another are H or A, R5, R6 in each case independently of one another are A, cycloalkyl having 3-7 C atoms, methylenecycloalkyl having 4-8 C atoms or alkenyl having 2-8 C atoms, A, A′ in each case independently of one another are alkyl which has 1 to 10 C atoms and which can be substituted by 1 to 5 F and/or Cl atoms and HaI is F, Cl, Br or I, and the stereoisomers and physiologically acceptable salts and solvates thereof; e) compounds disclosed in WO 00/59890 1-(4-ureidobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3-propoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-trifluoroacetamidobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-propoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-isopropoxycarbonylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-propoxycarbonylaminobenzoyl)-3-(3ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3,4-dimethoxyphenyl)-4-ethyl-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3,4-dimethoxyphenyl)-4-ethyl-1,4,5,6-tetrahydropyridazine and 1-(4-acetamidobenzoyl)-3-(3,4-dimethoxyphenyl)-4-ethyl-1,4,5,6-tetrahydropyridazine, and their physiologically acceptable salts and solvates; f) compounds of formula I disclosed in DE 19604388 in which R1, R2 in each case independently of one another are H or A, R3, R4 in each case independently of one another are —OH, OA, —S-A, —SO-A, —SO2-A, HaI, methylenedioxy, —NO2, —NH2, —NHA or —NAA′, A, A′ in each case independently of one another are alkyl having 1 to 10 C-atoms, and which can be substituted by 1 to 5 F and/or Cl atoms, cycloalkyl having 3-7 C atoms or methylenecycloalkyl having 4-8 C atoms, B is —Y—R5 oder —O—Y—R5, Q is absent or is alkylene having 1-4 C atoms, Y is absent or is alkylene having 1-10 C atoms, X is CH2 or S. R5 is NH2, NHA, NAA′ or is a saturated 3-8 membered heterocycle having at least one N atom, and wherein other CH2 groups optionally may be replaced by NH, NA, S or O, which can be unsubstituted or monosubstituted by A or OH, HaI is F, Cl, Br oder I and the stereoisomers and physiologically acceptable salts and solvates thereof; g) compounds of formula I disclosed in DE 19932315 in which R1, R2 in each case independently of one another are H, OH, OA, SA, SOA, SO2A, F, Cl or A′2N—(CH2)n—O—, R1 and R2 together are also —O—CH2—O—, R3, R4 in each case independently of one another are H, A, HaI, OH, OA, NO2, NHA, NA2, CN, COOH, COOA, NHCOA, NHSO2A or NHCOOA, R5, R6 in each case independently of one another are H or alkyl having 1 to 6 C atoms, A is alkyl having 1 to 10 C atoms, which can be substituted by 1 to 5 F and/or Cl atoms, is cycloalkyl having 3-7 C atoms, alkylenecycloalkyl having 5-10 C atoms or alkenyl having 2-8 C atoms, A′ is alkyl having 1, 2, 3, 4, 5 or 6 C atoms, n is 1, 2, 3 or 4, HaI is F, Cl, Br or 1, and their physiologically acceptable salts and solvates; h) compounds of formula I disclosed in EP 0723962 in which R1 and R2 in each case independently of one another are H or A, R3 and R4 in each case independently of one another are —OH, —OR10, —S—R10, —SO—R10, —SO2R1°, HaI, methylenedioxy, —NO2, —NH2, —NHR10 or —NR10R11, R5 is a phenyl radical which is unsubstituted or mono- or disubstituted by R6 and/or R7, Q is absent or is alkylene having 1-6 C atoms, R6 and R7 in each case independently of one another are —NH2, —NR8R9, —NHR10, —NR10R11, —NO2, HaI, —CN, —OA, —COOH or —COOA, R8 and R9 in each case independently of one another are H, acyl having 1-8 C atoms which can be substituted by 1-5 F and/or Cl atoms, —COOA, —S-A, —SO-A, —SO2A, —CONH2, —CONHA, —CONA2, —CO—COOH, —CO—COOA, —CO—CONH2, —CO—CONHA or —CO—CONA2.A is alkyl having 1 to 6 C atoms which can be substituted by 1-5 F and/or Cl atoms, R10 and R11 in each case independently of one another are A, cycloalkyl having 3-7 C atoms, methylenecycloalkyl having 4-8 C atoms or alkenyl having 2-8 C-atoms and HaI is F, Cl, Br or I, and their physiologically acceptable salts and solvates; i) compounds of formula I disclosed in EP 0738715 in which R1 and R2 in each case independently of one another are H or A, R3 and R4 in each case independently of one another are —OH, —OR10, —S—R10, —SO—R10, —SO2R10, HaI, methylenedioxy, —NO2, —NH2, —NHR10 or —NR10R11, R5 is a phenyl radical which is unsubstituted or mono- or disubstituted by R6 and/or R7, Q is absent or is alkylene having 1-6 C atoms, R6 and R7 in each case independently of one another are —NH2, —NR8R9, —NHR10, —NR10R11, —NO2, HaI, —CN, —OA, —COOH or —COOA, R8 and R9 in each case independently of one another are H, acyl having 1-8 C atoms which can be substituted by 1-5 F and/or Cl atoms, —COOA, —SO-A, —SO2A, —CONH2, —CONHA, —CONA2, —CO—COOH, —CO—COOA, —CO—CONH2, —CO—CONHA or —CO—CONA2, A is alkyl having 1 to 6 C atoms which can be substituted by 1-5 F and/or Cl atoms, R10 and R11 in each case independently of one another are A, cycloalkyl having 3-7 C atoms, methylenecycloalkyl having 4-8 C atoms or alkenyl having 2-8 C-atoms and HaI is F, Cl, Br or I, and their physiologically acceptable salts and solvates; for preparing a medicament for treating myocardial diseases.\",\n \"Preferably, the invention provides for the use of a) compounds disclosed in EP 0763534: 2-(3-nicotinoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(2-nicotinoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(3-nicotinoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(2-nicotinoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-methoxy-4-trifluoromethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-methoxy-4-difluoromethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-methoxy-4-fluoromethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-difluoromethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-trifluoromethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-fluoromethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-methoxy-4-ethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-4-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-hydroxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(4-methylsulfonylphenyl)-5ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(4-methyleneoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(3-nicotinoylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminophenethyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminophenethyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 3-(4-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(2-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(2-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-trifluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-difluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyi)-5-(3-methoxy-4-fluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-difluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-trifluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-fluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-ethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-ethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-ethoxy-4-methoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-hydroxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(4-methysulfonylphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(4-methyleneoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-cyclopentyloxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3-cyclopentyloxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminophenethyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminophenethyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(2-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(2-nicotinoylaminobenzyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-trifluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-difluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-fluoromethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-difluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-trifluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-fluoromethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-methoxy-4-ethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-ethoxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-hydroxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(4-methylsulfonylphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(4-methyleneoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminobenzyl)-5-(3-cyclopentyloxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(3-nicotinoylaminobenzyl)-5-(3-cyclopentyloxy-4-methoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminophenethyl)-5-(3,4-dimethoxyphenyl)-3,6-dihydro-1,3,4-oxadiazin-2-one, 3-(4-nicotinoylaminophenethyl)-5-(3,4-dimethoxyphenyl)-6-ethyl-3,6-dihydro-1,3,4-oxadiazin-2-one, 2-(3-nicotinoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isonicotinoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pyrazinecarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-(isoxazole-5-carbonylamino)benzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-nicotinoylaminobenzyl)-6-(3,4,-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, hydrochloride, and their stereoisomers and physiologically acceptable, salts and solvates; b) compounds disclosed in WO 99/65880 N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-methoxybenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-methylbenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)benzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-3,4-dichlorobenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-trifluoromethylbenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-3-chlorobenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-fluorobenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-butoxybenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-pentoxybenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-ethoxybenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-3,4-dimethoxybenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-3-methylbenzoyl-3-carboxamide, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-3-methoxybenzoyl-3-carboxamide, and their physiologically acceptable salts and solvates; c) compounds disclosed in WO 00/26201 3-dimethylaminopropyl {4-[3-(3-ethoxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, N-methylpiperidin-4-yl-4-[3-(3-ethoxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl {4-[3-(3-isopropoxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl {3-[3-(3-ethoxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl{3-[3-(3-cyclopentyloxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, N-methylpiperidin-4-yl-3[3-(3-cyclopentyloxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl{3-[3-(3-propyloxy-4-methoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl{4-[3-(3,4-diethoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, N-methylpiperidin-4-yl-{4-[3-(3,4-diethoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, 3-dimethylaminopropyl{3-[3-(3,4-dimethoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate 3-dimethylaminopropyl{4-[3-(3,4-dimethoxyphenyl)-1,2,3,4-tetrahydropyridazin-1-ylcarbonyl]phenyl}carbamate, and the physiologically acceptable salts and solvates thereof; d) compounds disclosed in WO 98/06704 1-(4-nicotinoylaminobenzoyl)-3-(3,4-dimethoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(3-nicotinoylaminobenzoyl)-3-(3,4-dimethoxy-phenyl)-1,4,5,6-tetrahydropyridazine hydrochloride, 1-(2-nicotinoylaminobenzoyl)-3-(3,4-dimethoxy-phenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(3-nicotinoylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3-cyclopentyloxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(3-nicotinoylaminobenzoyl)-3-(3-cyclopentyloxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3,4-methylene-dioxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3-methoxy-4-methylsulfonylphenyl)-1,4,5,6-tetrahydro-pyridazine, 1-(4-nicotinoylaminobenzoyl)-3-(3-trifluoro-methoxy-4-methoxyphenyl)-1,4,5,6-tetrahydro-pyridazine, 1-(4-ethoxy-carbonylaminobenzoyl)-3-(3,4-dimethoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(3-ethoxycarbonylaminobenzoyl)-3-(3,4-dimethoxy-phenyl)-1,4,5,6-tetrahydropyridazine, 1-(2-ethoxycarbonylaminobenzoyl)-3-(3,4-dimethoxy-phenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(3-ethoxycarbonylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-cyclo-pentyloxy-4-methoxyphenyl)-1,4,5,6-tetrahydro-pyridazine, 1-(3-ethoxycarbonylaminobenzoyl)-3-(3-cyclo-pentyloxy-4-methoxyphenyl)-1,4,5,6-tetrahydro-pyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3,4-methylene-dioxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-methoxy-4-methylsulfonylphenyl)-1,4,5,6-tetrahydro-pyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-trifluoro-methoxy-4-methoxyphenyl)-1,4,5,6-tetrahydro-pyridazine, and the stereoisomers and physiologically acceptable salts and solvates thereof; e) compounds disclosed in EP 0723962 3-(4-ethoxycarbonylaminobenzyl)-5-(3-ethoxy-4-methoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, 3-(4-ethoxycarbonylaminobenzyl)-5-(3-cyclopentyloxy-4-methoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, and their physiologically acceptable salts and solvates; f) compounds disclosed in EP 0738715 2-(4-butyrylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-acetamidobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-trifluoroacetamidobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methylsulfonamidobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-propionylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-tert-butylcarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isobutyrylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxycarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pivalylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2, 3,4,5-tetrahydropyridazin-3-one, 2-(4-cyclopentylcarbamoylbenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ethoxycarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2, 3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxalylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ureidobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentanoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-hexanoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropiridazin-3-one, 2-(4-pentafluoropropionylaminobenzyl)-6-(3,4-dimethoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-acetamidobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-trifluoroacetamidobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methylsulfonamidobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-propionylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-tert-butylcarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-butyrylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isobutyrylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxycarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pivalylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-cyclopentylcarbamoylbenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ethoxycarbonylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxalylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ureidobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentanoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-hexanoylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentafluoropropionylaminobenzyl)-6-(3,4-dimethoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-acetamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-trifluoroacetamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methylsulfonamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-propionylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-butyrylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isobutyrylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxycarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pivalylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-cyclopentylcarbamoylbenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ethoxycarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one; 2-(4-methoxalylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ureidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentanoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-hexanoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentafluoropropionylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-acetamidobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-trifluoroacetamidobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methylsulfonamidobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-propionylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-tert-butyloarbonylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-butyrylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isobutyrylaminobenzyl)-6(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxycarbonylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pivalylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-cyclopentylcarbamoylbenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ethoxycarbonylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxalylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ureidobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentanoylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-hexanoylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentafluoropropionylaminobenzyl)-6-(3-cyclopentyloxy-4-methoxyphenyl)-5-ethyl-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-acetamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-trifluoroacetamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methylsulfonamidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-propionylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-butyrylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-isobutyrylaminobenzyl)-6-(3ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxycarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pivalylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-cyclopentylcarbamoylbenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ethoxycarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-methoxalylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-ureidobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentanoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-hexanoylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, 2-(4-pentafluoropropionylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, and their physiologically acceptable salts and solvates; for preparing a medicament for treating myocardial diseases.\",\n \"Most preferably, the invention provides for the use of the following compounds 3-(4-nicotinoylaminobenzyl)-5-(3-ethoxy-4-methoxyphenyl)-3,6-dihydro-1,3,4-thiadiazin-2-one, N-(3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazin-1-ylcarbonyl)phenyl)-4-methoxybenzoyl-3-carboxamide, 1-(4-nicotinoylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 1-(4-ethoxycarbonylaminobenzoyl)-3-(3-ethoxy-4-methoxyphenyl)-1,4,5,6-tetrahydropyridazine, 2-(4-ethoxycarbonylaminobenzyl)-6-(3-ethoxy-4-methoxyphenyl)-2,3,4,5-tetrahydropyridazin-3-one, and their physiologically acceptable salts and solvates; for preparing a medicament for treating myocardial diseases.\",\n \"The preferred compounds show a selective inhibition of phosphodiesterase IV, which is associated with an intracellular increase in cAMP (N. Sommer et al., Nature Medicine, 1, 244-248 (1995)).\",\n \"The inhibition of PDE IV can be demonstrated, for example, analogously to C. W. Davis in Biochim.\",\n \"Biophys.\",\n \"Acta 797, 354-362 (1984).\",\n \"The affinity of the compounds of the invention for phosphodiesterase IV is measured by determining their IC50 values (the concentration of inhibitor required to achieve 50% inhibition of the enzyme activity).\",\n \"Preferably, the invention provides for the use of the compounds mentioned above for preparing a medicament for treating myocardial diseases, where said myocardial diseases show inflammatory and immunological characteristics.\",\n \"Most preferably, the invention provides for the use of the compounds mentioned above for preparing a medicament for treating coronary artery disease, reversible or irreversible myocardial ischemia/reperfusion injury, acute or chronic heart failure and restenosis, including instent-restenosis and stent-in-stent-restenosis.\",\n \"The preparations for the treatment of the mentioned diseases can be used as medicaments in human or veterinary medicine.\",\n \"Possible excipients are organic or inorganic substances which are suitable for enteral (e.g.\",\n \"oral) or parenteral administration or topical application and do not react with the novel compounds, for example water, vegetable oils, benzyl alcohols, alkylene glycols, polyethylene glycols, glycerol triacetate, gelatin, carbohydrates such as lactose or starch, magnesium stearate, talc and petroleum jelly.\",\n \"In particular, tablets, pills, coated tablets, capsules, powders, granules, syrups, juices or drops are used for oral administration, suppositories are used for rectal administration, solutions, preferably oily or aqueous solutions, and furthermore suspensions, emulsions or implants, are used for parenteral administration, and ointments, creams or powders are used for topical application.\",\n \"The novel compounds can also be lyophilized and the lyophilizates obtained used, for example, for the production of injection preparations.\",\n \"The preparations indicated can be sterilized and/or can contain auxiliaries such as lubricants, preservatives, stabilizers and/or wetting agents, emulsifiers, salts for affecting the osmotic pressure, buffer substances, colourants, flavourings and/or one or more further active compounds, e.g.\",\n \"one or more vitamins.\",\n \"In these indications the substances are generally administered preferably in doses of between approximately 1 and 500 mg, in particular between 5 and 100 mg per dose unit.\",\n \"The daily dose is preferably between approximately 0.02 and 10 mg/kg of body weight.\",\n \"The specific dose for each patient depends, however, on a wide variety of factors, for example on the efficacy of the specific compound used, on age, body weight, general state of health, gender, on the diet, on the time and route of administration, on the excretion rate, medicament combination and severity of the particular disease to which the therapy is applied.\",\n \"Oral administration is preferred.\",\n \"EXAMPLE 1 Effect of PDE4 Inhibitors on T-Cell Proliferation Peripheral blood mononuclear cells (PBMC) were isolated from the blood of healthy donors by the Lymphoprep gradient method.\",\n \"200000 PBMC/well were cultured in RPMI1640 culture medium containing 5% heat inactivated human serum (AB pool) for 5 days at 37° C. and 10% CO2 in 96 well flat bottom microtiter plates.\",\n \"The T cells within the PBMC preparation were selectively stimulated with an monoclonal antibody to CD3.Cultures were set up as triplicates including a control group receiving no treatment.\",\n \"PDE4 inhibitors were dissolved in DMSO at 10−2 M and diluted in culture medium.\",\n \"Control cultures were treated with DMSO equivalent to the inhibitor concentration.\",\n \"18 hrs before the end of the assay, 3H-thymidine was added to the cultures.\",\n \"The incorporation of radioactivity into the cells was then measured in a beta-counter.\",\n \"The data of at least three independent experiments were calculated as percent inhibition of the control (mean ±SEM) without inhibitor.\",\n \"From this data the IC-50 value was determined.\",\n \"Results: PDE4 inhibitors afforded a marked reduction of T-cell proliferation (see table 1).\",\n \"TABLE 1 Effect of PDE4 inhibitors on T-cells and macrophages IL2 IFN-γ TNF-α IL10 IL12 T-cell T-cell macrophage macrophage macrophage T-cell prol.\",\n \"IC50 [μM] IC50 [μM] IC50 [μM] EC50 [μM] IC50 [μM] IC50 [μM] Rolipram 0.7 1 0.6 0.05 >0.1 0.3 EMD 94360 0.3 0.05 0.03 0.001 0.006 0.02 EMD 95832 0.2 0.01 0.009 0.02 0.0005 0.05 EMD 125025 0.002 0.007 0.003 no effect <1E−13M 0.006 EMD 125059 0.06 0.008 0.03 0.008 <1E−13M 0.04 EMD 219901 0.003 0.005 0.002 0.001 <1E−13M 0.007 EMD 94360 (EP 0738715; Page 17, line 41): EMD 95832 (EP 0763534): EMD 125025 (WO 98/06704; page 13, lines 16-17): EMD 125059 (WO 98/06704; page 27, lines 2-3): EMD 219901 (WO 99/65880; page 14, lines 20-21) EXAMPLE 2 Effect of PDE4 Inhibitors on Cytokine Production in Human Peripheral Blood Monocytic Cells Peripheral blood mononuclear cells (PBMC) were isolated from the blood of healthy donors by the Lymphoprep gradient method.\",\n \"200000 PBMC/well were cultured in RPMI1640 culture medium containing 5% heat inactivated human serum (AS pool).\",\n \"at 37° C. and 10% CO2 in 96 well flat bottom microtiter plates.\",\n \"Cultures were set up as triplicates including a control group.\",\n \"Solutions of PDE4 inhibitors were prepared in DMSO at 10−2 M and diluted in culture medium.\",\n \"Control cultures were treated with concentrations of DMSO equivalent to the inhibitor concentrations.\",\n \"The cytokine of interest was stimulated as indicated in Table 2 The culture supernatants of three independent experiments were pooled and cytokine activity in the supernatant was measured with commercially available ELISA test kits.\",\n \"TABLE 2 Activation of PBMC to form T-cell and macrophage specific cytokines cytokine activator incubation in culture IL-2 mab anti-CD3 24 h IL-10 LPS 48 h TNF-α LPS 48 h IFN-γ mab anti-CD3 48 h IL-12 SAC + IFNγ 48 h The data were calculated as percent inhibition/stimulation of the control without the compound and the IC50 value or EC50 value in case of stimulation was determined thereof.\",\n \"Result PDE4 inhibitors afforded a marked reduction in the release of IL-2, IFN-γ, TNF-α and IL-12.The immunosuppressant cytokine IL-10, however, was stimulated by most PDE4 inhibitors (see Table 1).\",\n \"EXAMPLE 3 Effect of PDE4 Inhibitors on Experimental Myocardial Infarction in Rats Compound 5, administered intraperitoneally with 1, 3, and 10 mg/kg, 1 hour before reversible occlusion of the left coronary artery in rats caused a significant dose dependent reduction of infarct size up to 38%.\",\n \"In correspondence with this protection, a reduction of plasma TNF-□ levels was observed, as measured by ELISA.\",\n \"EXAMPLE 4 Effect of PDE4 Inhibitors on Experimental Myocardial Infarction in Rabbits There was a cardioprotective effect by PDE4 inhibition in anaesthetised rabbits subjected to 30 minutes of coronary artery occlusion (side branch of the ramus circumflexus of the left coronary artery) followed by 120 minutes of reperfusion.\",\n \"PDE4 inhibitors applied prior to the coronary occlusion reduced infarct size as compared with placebo treatment.\",\n \"The areas at risk were comparable between verum and placebo groups.\",\n \"The cardioprotective effect cannot be attributed to favorable hemodynamic effects, since heart rate and mean aortic pressure remained constant throughout the experimental protocol.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10467793"}}},{"rowIdx":385,"cells":{"text":{"kind":"list like","value":[["Substrate processing method and substrate processing apparatus","In a substrate processing apparatus, a control electrode (131) separates a process space (11C) including a substrate to be processed and a plasma formation space (11B) not including the substrate.","The control electrode includes a conductive member formed in a processing vessel and having a plurality of apertures (131a) for passing plasma.","A surface of the control electrode is covered by an aluminum oxide or a conductive nitride.","In the substrate processing apparatus, a gas containing He and N2 is supplied into the processing vessel.","In the plasma formation space, there is formed plasma under a condition in which atomic state nitrogen N* are excited.","The atomic state nitrogen N* are used to nitride a surface of the substrate."],["1.A method of processing a substrate by using a substrate processing apparatus which has such a construction that a process space, in which a substrate to be processed is contained, is separated from plasma formation space, in which the substrate to be processed is not contained, by a control electrode in a processing vessel, characterized by the steps of: supplying a gas containing He and N2 to said processing vessel; forming plasma in said plasma formation space under a condition such that there is caused excitation of atomic state nitrogen N* in said plasma; and nitriding a surface of the substrate to be processed by said atomic state nitrogen N* in said process space.","2.The substrate processing method as claimed in claim 1, characterized in that said step of forming plasma is conducted such that an intermediate excitation state of the energy of 23 to 25 eV is realized.","3.The substrate processing method as claimed in claim 1, characterized in that said step of forming plasma comprises the step of supplying a microwave to said plasma formation space.","4.The substrate processing method as claimed in claim 3, characterized in that said step of supplying microwave is conducted by driving a radial line slot antenna.","5.The substrate processing method as claimed in claim 1, characterized in that said step of forming plasma comprises the step of forming an induction magnetic field in said plasma formation space.","6.The substrate processing method as claimed in claim 5, characterized in that said step of forming an induction magnetic field comprises the step of driving an induction coil wound around said processing vessel by a high frequency electric power.","7.The substrate processing method as claimed in claim 1, characterized in the said control electrode is grounded during said step of exciting plasma.","8.The substrate processing method as claimed in claim 1, characterized in that a negative potential is supplied to said control electrode in said step of forming plasma.","9.The substrate processing method as claimed in claim 1, wherein said gas supplied to said processing vessel further contains O2.10.A substrate processing apparatus, comprising: a processing vessel defined by an outer wall and having a stage for holding a substrate to be processed thereon; an evacuation system coupled to said processing vessel; a plasma gas supplying part supplying a plasma excitation gas and a process gas into said processing vessel; a microwave window provided on said processing vessel so as to face said substrate to be processed; and a control electrode provided between said substrate to be processed on said stage and said plasma gas supplying part so as to face said substrate to be procesed, and separating a plasma excitation space containing said microwave window and a process space containing said substrate to be processed, said control electrode comprising a conductive member having a plurality of apertures for passing plasma formed in said processing vessel therethrough, and a surface of said control electrode being covered by any of aluminum oxide or electrically conductive nitride.","11.The substrate processing apparatus as claimed in claim 10, characterized in that said control electrode has a lattice-shaped form and is grounded.","12.The substrate processing apparatus as claimed in claim 10, characterized in that said control electro has a form of a lattice-like shape and said substrate processing apparatus comprises a negative voltage source connected to said control electrode.","13.The substrate processing apparatus as claimed in claim 10, characterized in that an inner wall of said processing vessel is covered with an insulation film in said plasma excitation space.","14.The substrate processing apparatus as claimed in claim 10, characterized by further comprising a microwave antenna coupled to said microwave window at an outer side of said processing vessel.","15.A substrate processing apparatus, characterized by: a processing vessel defined by a wall of quartz glass and having a stage for holding a substrate to be processed; an evacuation system coupled to said processing vessel; a plasma gas supplying part supplying a plasma excitation gas and a process gas to said processing vessel; a control electrode provided so as to face said substrate to be processed on said stage and dividing an interior of said processing vessel into a process space containing said substrate to be processed and a plasma excitation space; and an induction coil provided outside said quartz glass wall in correspondence to said plasma excitation space, said control electrode comprising a conductive member having a plurality of apertures passing therethrough plasma formed in said processing vessel, and a surface of said control electrode being covered with any of aluminum oxide or electrically conductive nitride.","16.The substrate processing apparatus as claimed in claim 15, characterized in that said quartz glass wall defines a dome-like space.","17.The substrate processing apparatus as claimed in claim 15, characterized in that said control electrode is grounded.","18.The substrate processing apparatus as claimed in claim 15, characterized in that said control gate is connected to a negative voltage source."],["<SOH> BACKGROUND ART <EOH>FIG.","1 shows the schematic construction of a conventional induction-coupled plasma processing apparatus 1 .","Referring to FIG.","1 , the plasma processing apparatus 1 includes a processing vessel 2 of a quartz dome evacuated by an evacuation line 2 A, and there is provided a stage 3 in a process space 2 B defined by the processing vessel 2 such that the stage 2 is rotated by a rotating mechanism 3 A.","Further, a substrate 4 is held on the stage 3 .","Further, an inert gas such as Ar and a process gas such as oxygen or nitrogen are supplied to the process space 2 B via a process gas supply line 2 C. Further, there is provided a coil 5 around the top part of the processing vessel 2 at the outside thereof, and high-density plasma 2 D is inducted at the top part of the process space 2 B by driving the coil 5 by a d.c. power source.","In the plasma processing apparatus 1 of FIG.","1 , the radicals of the process gas formed with the high-density plasma 2 D reach the surface of the substrate 4 and the substrate processing such as oxidation or nitridation is achieved.","In such a conventional induction-coupled plasma processing apparatus 1 , on the other hand, there exists a drawback in that the high-density plasma 2 D is localized at the top part of the processing vessel and there appears an extremely non-uniform distribution in the radicals that are formed with the plasma.","Particularly, the non-uniformity of the radical concentration in the radial direction of the substrate is not resolved even when the stage 3 is rotated by the rotating mechanism 3 A.","Thus, in the conventional induction-coupled plasma processing apparatus 1 , the plasma processing apparatus was designed such that the substrate 4 is separated from the region in which the high-density plasma 2 D is formed with a large distance for realizing as uniform radical concentration distribution as possible at the surface of the substrate 4 .","As a result of such a construction, on the other hand, the overall size of the substrate processing apparatus 1 is increased inevitably.","Further, the amount of the radicals reaching the substrate 4 is reduced.","These problems become particularly serious in the technology of current trend of processing a large-diameter substrate.","On the other hand, there is a proposal of a microwave plasma processing apparatus that uses high-density plasma induced, not by an induction magnetic field but by a microwave electric field.","For example, there is proposed a plasma processing apparatus that uses a planar antenna (radial line slot antenna) having a large number of slots arranged so as to produce a uniform microwave, for emitting a microwave into a processing vessel.","In this apparatus, the microwave electric field thus induced is used to excite plasma by causing ionization in the gas in the vacuum vessel.","Reference should be made to Japanese Laid-Open Patent Application 9-63793.By using the microwave plasma excited according to such a process, it becomes possible to realize a high-plasma density over a wide area right underneath the antenna, and uniform plasma processing becomes possible with short time period.","Further, the microwave plasma thus excited has an advantageous feature of low electron temperature as a result of excitation of the plasma by using a microwave, and it becomes possible to avoid the problem of damages or metal contamination caused in the substrate.","Further, it becomes possible to excite uniform plasma over a substrate of large area, and thus, the plasma processing apparatus can easily handle the fabrication of semiconductor devices on a large-diameter semiconductor wafer or fabrication of large flat panel display devices.","FIG.","2 shows the construction of a microwave plasma processing apparatus 10 that uses such a radial line slot antenna as proposed before by the inventor of the present invention.","Referring to FIG.","2 , the microwave plasma processing apparatus 10 includes a processing chamber 11 evacuated at a plurality of evacuation ports 11 a , and there is provided a stage 13 inside the processing chamber 11 for supporting a substrate 12 to be processed.","In order to achieve uniform evacuation of the processing chamber 11 , there is provided a ring-shaped space 11 A around the stage 13 , and the processing chamber 11 is evacuated uniformly via the space 11 A and further via the evacuation ports 11 a by arranging the evacuation ports 11 a communicating with the space 11 A in axial symmetry with respect to the substrate.","On the processing chamber 11 , there is provided a plate-like shower plate 14 formed of a low-loss dielectric such as Al 2 O 3 or SiO 2 as a part of the outer wall of the processing chamber 11 at a location facing the substrate 12 held on the stage 13 , wherein the shower plate 14 is provided via a seal ring not illustrated and includes a number of apertures 14 A.","Further, a cover plate 15 also of a low-loss dielectric such as Al 2 O 3 or SiO 2 is provided at the outer side of the shower plate 14 via another seal ring not illustrated.","The shower plate 14 is provided with a gas passage 14 B at a top surface thereof, and each of the apertures 14 A are provided so as to communicate with the gas passage 14 B.","Further, there is provided a gas supply passage 14 C in the interior of the shower plate 14 in communication with a gas supply port lip provided at an outer wall of the processing vessel 11 .","Thus, the plasma-excitation gas such as Ar or Kr supplied to the gas supply port 11 p is forwarded to the apertures 11 A via the supply passage 14 C and further via the passage 14 B and is released to the process space 11 B right underneath the shower plate 14 inside the processing vessel 11 from the foregoing apertures 14 A.","On the processing vessel 11 , there is further provided a radial line slot antenna 20 at the outer side of the cover plate 15 with a separation of 4-5 mm from the cover plate 15 .","The radial line slot antenna 20 is connected to an external microwave source (not illustrated) via a coaxial waveguide 21 and causes excitation of the plasma-excitation gas released into the process space 11 B by the microwave from the microwave source.","It should be noted that the cover plate 15 and the radiation surface of the radial line slot antenna are contacted closely, and there is provided a cooling block 19 on the antenna 20 for cooling the antenna.","The cooling block 19 includes a cooling water passage 19 A.","The radial line slot antenna 20 is formed of a flat, disk-shaped antenna body 17 connected to an outer waveguide tube 21 A of the coaxial waveguide 21 and a radiation plate 16 provided at the opening of the antenna body 17 , wherein the radiation plate 16 is formed with a number of slots and a retardation plate of a dielectric plate having a constant thickness is interposed between the antenna body 17 and the radiation plate 16 .","In the radial line slot antenna 20 having such a construction, the microwave fed thereto from the coaxial waveguide 21 propagates along a path between the disk-shaped antenna body 17 and the radiation plate 16 in the radial direction, wherein the microwave thus propagating undergoes compression of wavelength as a result of the existence of the retardation plate 18 .","Thus, by forming the slots concentrically in correspondence to the wavelength of the microwave thus propagating in the radial direction, and by forming the slots so as to form a perpendicular angle with each other, it becomes possible to emit a plane wave having a circular polarization from the radial line slot antenna 20 in the direction substantially perpendicular to the radiation plate 16 .","By using such a radial line slot antenna 20 , there is formed uniform high-density plasma in the process space 11 B right underneath the shower plate 14 .","The high-density plasma thus formed has a feature of low electron temperature and the occurrence of damages in the substrate 12 to be processed is avoided.","Further, there occurs no metal contamination caused by sputtering of the chamber wall of the processing vessel 11 .","Thus, by supplying a process gas, such as an O 2 gas, an NH 3 gas, or a mixed gas of an N 2 gas and an H 2 gas, to the gas inlet port 11 p of the substrate processing apparatus 10 of FIG.","2 in addition to the plasma-excitation gas such as Ar or Kr, there is caused an excitation of active species such as atomic state oxygen O* or hydrogen nitride radicals NH* in the process space 11 B by the high-density plasma, and it becomes possible to conduct oxidation processing, nitridation processing or oxynitridation processing on the surface of the substrate 12 .","Further, there is proposed a substrate processing apparatus 10 A shown in FIG.","3 having a construction similar to the substrate processing apparatus 10 of FIG.","2 except that there is provided a lower shower plate 31 at the lower side of the shower plate 14 .","The lower shower plate 31 is provided with a process gas passage 31 A communicating with a process gas inlet port 11 r formed at the surface of the processing vessel 1 and a large number of process gas inlet nozzle openings 31 B are formed in communication with the process gas passage 31 A.","Further, the lower shower plate 31 is provided with large apertures for passing the process gas radicals formed in the space 11 B.","Thus, in the substrate processing apparatus 10 A of FIG.","3 , there is defined another process space 11 C underneath the lower shower plate 31 .","By forming the lower shower plate 31 by a conductive material such as a stainless steel having a passivation surface by aluminum oxide (Al 2 O 3 ) in such an apparatus, it becomes possible to block the penetration of microwave to the process space 11 C. Thereby, the excitation of plasma is limited in the process space 11 B right underneath the upper shower plate 14 , and the radicals Kr* of Kr or Ar* of Ar penetrate into the process space 11 C through the large apertures formed in the shower plate 31 after excitation in the space 11 B.","The radicals Kr* or Ar* thus penetrated into the process space 11 C cause activation of the process gas released from the nozzle apertures 31 B, and the processing of the substrate 12 is achieved by the process gas radicals thus activated.","In the substrate processing apparatus 10 A of FIG.","3 , it should be noted that the microwave is expelled from the process space 11 C by forming the lower shower plate 31 by a conductive material, and the damaging of the substrate by microwave is avoided.","In the substrate processing apparatus 10 A of FIG.","3 , it is also possible to conduct a plasma CVD process by introducing a CVD source gas from the lower shower plate 31 .","Further, it is possible to conduct a dry etching process by introducing a dry etching gas from the lower shower plate 31 and applying a high-frequency bias to the stage 13 .","Thus, in the substrate processing apparatus of FIG.","2 of FIG.","3 , Kr radicals (Kr*) of intermediate excitation state having an energy of about 10 eV are excited at the time of conducting an oxidation processing, by introducing a Kr gas and an oxygen gas into the process space 11 B.","The Kr radicals thus excited cause efficient excitation of atomic state oxygen O* according to the reaction in-line-formulae description=\"In-line Formulae\" end=\"lead\"?","O 2 →O*+O*, in-line-formulae description=\"In-line Formulae\" end=\"tail\"?","while the atomic state oxygen O* thus excited cause the desired oxidation of the surface of the substrate 12 .","In the case of conducting a nitridation processing of the substrate 12 , a Kr gas and an ammonia gas, or a Kr gas and a nitrogen gas and a hydrogen gas are introduced.","In this case, the excited Kr radicals (Kr*) or Ar radicals (Ar*) cause the excitation of hydrogen nitride radicals NH* according to the reaction in-line-formulae description=\"In-line Formulae\" end=\"lead\"?","NH 3 →NH*+2H*+ e − , in-line-formulae description=\"In-line Formulae\" end=\"tail\"?","in-line-formulae description=\"In-line Formulae\" end=\"lead\"?","or in-line-formulae description=\"In-line Formulae\" end=\"tail\"?","in-line-formulae description=\"In-line Formulae\" end=\"lead\"?","N 2 +H 2 →NH*+NH*, in-line-formulae description=\"In-line Formulae\" end=\"tail\"?","wherein the hydrogen nitride radicals thus excited cause the desired nitridation processing of the substrate of the surface 12 .","Meanwhile, there are cases in which it is preferable to use atomic state nitrogen (N*), free from hydrogen and having a strong nitriding power, at the time of the nitridation processing of the substrate.","The atomic state nitrogen N* are formed according to the reaction in-line-formulae description=\"In-line Formulae\" end=\"lead\"?","N 2 →N*+N*, in-line-formulae description=\"In-line Formulae\" end=\"tail\"?","wherein it should be noted that such a reaction requires the energy of 23-25 eV.","This means that it is not possible to excite the atomic state nitrogen N* according to the foregoing reaction, as long as Kr or Ar plasma is used.","As noted previously, the energy of the Kr radicals or Ar radicals obtained by the Kr or Ar plasma is merely in the order of 10 eV.","Thus, even when there is made an attempt to supply a nitrogen gas in the substrate processing apparatus of FIG.","2 or FIG.","3 in place of the Kr gas or the Ar gas, merely the reaction in-line-formulae description=\"In-line Formulae\" end=\"lead\"?","N 2 →N 2 + +e − , in-line-formulae description=\"In-line Formulae\" end=\"tail\"?","is obtained, and there is caused no desired atomic state oxygen N*.","FIG.","4 shows the relationship between the state density of the Kr plasma and the excitation energy of the atomic state nitrogen N*, hydrogen nitride radicals NH* and nitrogen atoms N 2 + .","Referring to FIG.","4 , it can be seen that the state density of the Kr plasma is large at the low energy side, while the state density shows a rapid decrease with increase of the energy.","Such a plasma cannot achieve efficient excitation of the desired nitrogen radicals."],["<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.","1 is a diagram showing the construction of a conventional induction coupled plasma processing apparatus; FIG.","2 is a diagram showing the construction of a previously proposed microwave substrate processing apparatus; FIG.","3 is a diagram showing the construction of another previously proposed microwave substrate processing apparatus; FIG.","4 is a diagram explaining the characteristics of plasma excitation in the microwave substrate processing apparatus of FIG.","2 or FIG.","3 ; FIG.","5 is a diagram showing the construction of a microwave substrate processing apparatus according to a first embodiment of the present invention; FIG.","6 is a diagram showing a part of the microwave substrate processing apparatus of FIG.","5 ; FIG.","7 is a diagram showing the characteristics of plasma excitation in the microwave substrate processing apparatus of FIG.","5 ; FIG.","8 is a diagram showing a modification of the microwave plasma processing apparatus of FIG.","5 ; FIG.","9 is a diagram showing the construction of a microwave plasma processing apparatus according to a second embodiment of the present invention; and FIG.","10 is a diagram showing the construction of an induction coupled plasma processing apparatus according to a third embodiment of the present invention.","detailed-description description=\"Detailed Description\" end=\"lead\"?"],["TECHNICAL FIELD The present invention generally relates to plasma processing apparatuses and more particularly to a microwave plasma processing apparatus.","Plasma process and plasma processing apparatus constitute indispensable technology for fabricating ultrafine semiconductor devices such as the one called deep submicron device or deep sub-quarter micron device having a gate length near 0.1 μm or less, or for fabricating high-resolution flat panel display device including a liquid crystal display device.","Conventionally, various plasma excitation methods have been employed in the plasma processing apparatus used for fabricating semiconductor devices or liquid crystal display devices.","Particularly, high-frequency plasma apparatuses of parallel plate type or induction-coupled type plasma apparatus are used commonly.","However, such a conventional plasma processing apparatuses suffers from the problem of non-uniform plasma formation in that the region in which high electron density is achieved is substantially limited, and there has been a difficulty in conducting a uniform processing over the entire surface of the substrate with a large processing rate or throughput.","This problem becomes particularly serious in the case of processing a substrate of large diameter.","Further, such a conventional plasma processing apparatus has inherent problems, associated with its high electron temperature, in that damages are caused in the semiconductor devices formed on the substrate.","Further, severe metal contamination may be caused as a result of sputtering of the chamber wall.","Thus, it is becoming difficult with conventional plasma processing apparatuses to satisfy the stringent demand of further miniaturization and further improvement of productivity of semiconductor devices or flat display devices.","Meanwhile, there has been a proposal of a microwave plasma processing apparatus that uses high-density plasma excited, not by d.c. magnetic field, but by a microwave electric field.","For example, there is a proposal of a plasma processing apparatus that excites plasma by emitting a microwave into a processing vessel from a planar antenna (radial line slot antenna) having a number of slots arranged so as to produce a uniform microwave, for emitting a microwave into a processing vessel.","In this plasma processing apparatus, the microwave electric field induces plasma by causing ionization in the gas in the vacuum vessel.","Reference should be made to Japanese Laid-Open Patent Application 9-63793.By using the microwave plasma excited according to such a process, it becomes possible to realize a high-plasma density over a wide area right underneath the antenna, and uniform plasma processing becomes possible with short time period.","Further, the microwave plasma thus excited has an advantageous feature of low electron temperature as a result of excitation of the plasma by using a microwave, and it becomes possible to avoid the problem of damages or metal contamination caused in the substrate.","Further, it becomes possible to excite uniform plasma over a substrate of large area, and thus, the plasma processing apparatus can easily handle the fabrication of semiconductor devices on a large-diameter semiconductor wafer or fabrication of large flat panel display devices.","BACKGROUND ART FIG.","1 shows the schematic construction of a conventional induction-coupled plasma processing apparatus 1.Referring to FIG.","1, the plasma processing apparatus 1 includes a processing vessel 2 of a quartz dome evacuated by an evacuation line 2A, and there is provided a stage 3 in a process space 2B defined by the processing vessel 2 such that the stage 2 is rotated by a rotating mechanism 3A.","Further, a substrate 4 is held on the stage 3.Further, an inert gas such as Ar and a process gas such as oxygen or nitrogen are supplied to the process space 2B via a process gas supply line 2C.","Further, there is provided a coil 5 around the top part of the processing vessel 2 at the outside thereof, and high-density plasma 2D is inducted at the top part of the process space 2B by driving the coil 5 by a d.c. power source.","In the plasma processing apparatus 1 of FIG.","1, the radicals of the process gas formed with the high-density plasma 2D reach the surface of the substrate 4 and the substrate processing such as oxidation or nitridation is achieved.","In such a conventional induction-coupled plasma processing apparatus 1, on the other hand, there exists a drawback in that the high-density plasma 2D is localized at the top part of the processing vessel and there appears an extremely non-uniform distribution in the radicals that are formed with the plasma.","Particularly, the non-uniformity of the radical concentration in the radial direction of the substrate is not resolved even when the stage 3 is rotated by the rotating mechanism 3A.","Thus, in the conventional induction-coupled plasma processing apparatus 1, the plasma processing apparatus was designed such that the substrate 4 is separated from the region in which the high-density plasma 2D is formed with a large distance for realizing as uniform radical concentration distribution as possible at the surface of the substrate 4.As a result of such a construction, on the other hand, the overall size of the substrate processing apparatus 1 is increased inevitably.","Further, the amount of the radicals reaching the substrate 4 is reduced.","These problems become particularly serious in the technology of current trend of processing a large-diameter substrate.","On the other hand, there is a proposal of a microwave plasma processing apparatus that uses high-density plasma induced, not by an induction magnetic field but by a microwave electric field.","For example, there is proposed a plasma processing apparatus that uses a planar antenna (radial line slot antenna) having a large number of slots arranged so as to produce a uniform microwave, for emitting a microwave into a processing vessel.","In this apparatus, the microwave electric field thus induced is used to excite plasma by causing ionization in the gas in the vacuum vessel.","Reference should be made to Japanese Laid-Open Patent Application 9-63793.By using the microwave plasma excited according to such a process, it becomes possible to realize a high-plasma density over a wide area right underneath the antenna, and uniform plasma processing becomes possible with short time period.","Further, the microwave plasma thus excited has an advantageous feature of low electron temperature as a result of excitation of the plasma by using a microwave, and it becomes possible to avoid the problem of damages or metal contamination caused in the substrate.","Further, it becomes possible to excite uniform plasma over a substrate of large area, and thus, the plasma processing apparatus can easily handle the fabrication of semiconductor devices on a large-diameter semiconductor wafer or fabrication of large flat panel display devices.","FIG.","2 shows the construction of a microwave plasma processing apparatus 10 that uses such a radial line slot antenna as proposed before by the inventor of the present invention.","Referring to FIG.","2, the microwave plasma processing apparatus 10 includes a processing chamber 11 evacuated at a plurality of evacuation ports 11a, and there is provided a stage 13 inside the processing chamber 11 for supporting a substrate 12 to be processed.","In order to achieve uniform evacuation of the processing chamber 11, there is provided a ring-shaped space 11A around the stage 13, and the processing chamber 11 is evacuated uniformly via the space 11A and further via the evacuation ports 11a by arranging the evacuation ports 11a communicating with the space 11A in axial symmetry with respect to the substrate.","On the processing chamber 11, there is provided a plate-like shower plate 14 formed of a low-loss dielectric such as Al2O3 or SiO2 as a part of the outer wall of the processing chamber 11 at a location facing the substrate 12 held on the stage 13, wherein the shower plate 14 is provided via a seal ring not illustrated and includes a number of apertures 14A.","Further, a cover plate 15 also of a low-loss dielectric such as Al2O3 or SiO2 is provided at the outer side of the shower plate 14 via another seal ring not illustrated.","The shower plate 14 is provided with a gas passage 14B at a top surface thereof, and each of the apertures 14A are provided so as to communicate with the gas passage 14B.","Further, there is provided a gas supply passage 14C in the interior of the shower plate 14 in communication with a gas supply port lip provided at an outer wall of the processing vessel 11.Thus, the plasma-excitation gas such as Ar or Kr supplied to the gas supply port 11p is forwarded to the apertures 11A via the supply passage 14C and further via the passage 14B and is released to the process space 11B right underneath the shower plate 14 inside the processing vessel 11 from the foregoing apertures 14A.","On the processing vessel 11, there is further provided a radial line slot antenna 20 at the outer side of the cover plate 15 with a separation of 4-5 mm from the cover plate 15.The radial line slot antenna 20 is connected to an external microwave source (not illustrated) via a coaxial waveguide 21 and causes excitation of the plasma-excitation gas released into the process space 11B by the microwave from the microwave source.","It should be noted that the cover plate 15 and the radiation surface of the radial line slot antenna are contacted closely, and there is provided a cooling block 19 on the antenna 20 for cooling the antenna.","The cooling block 19 includes a cooling water passage 19A.","The radial line slot antenna 20 is formed of a flat, disk-shaped antenna body 17 connected to an outer waveguide tube 21A of the coaxial waveguide 21 and a radiation plate 16 provided at the opening of the antenna body 17, wherein the radiation plate 16 is formed with a number of slots and a retardation plate of a dielectric plate having a constant thickness is interposed between the antenna body 17 and the radiation plate 16.In the radial line slot antenna 20 having such a construction, the microwave fed thereto from the coaxial waveguide 21 propagates along a path between the disk-shaped antenna body 17 and the radiation plate 16 in the radial direction, wherein the microwave thus propagating undergoes compression of wavelength as a result of the existence of the retardation plate 18.Thus, by forming the slots concentrically in correspondence to the wavelength of the microwave thus propagating in the radial direction, and by forming the slots so as to form a perpendicular angle with each other, it becomes possible to emit a plane wave having a circular polarization from the radial line slot antenna 20 in the direction substantially perpendicular to the radiation plate 16.By using such a radial line slot antenna 20, there is formed uniform high-density plasma in the process space 11B right underneath the shower plate 14.The high-density plasma thus formed has a feature of low electron temperature and the occurrence of damages in the substrate 12 to be processed is avoided.","Further, there occurs no metal contamination caused by sputtering of the chamber wall of the processing vessel 11.Thus, by supplying a process gas, such as an O2 gas, an NH3 gas, or a mixed gas of an N2 gas and an H2 gas, to the gas inlet port 11p of the substrate processing apparatus 10 of FIG.","2 in addition to the plasma-excitation gas such as Ar or Kr, there is caused an excitation of active species such as atomic state oxygen O* or hydrogen nitride radicals NH* in the process space 11B by the high-density plasma, and it becomes possible to conduct oxidation processing, nitridation processing or oxynitridation processing on the surface of the substrate 12.Further, there is proposed a substrate processing apparatus 10A shown in FIG.","3 having a construction similar to the substrate processing apparatus 10 of FIG.","2 except that there is provided a lower shower plate 31 at the lower side of the shower plate 14.The lower shower plate 31 is provided with a process gas passage 31A communicating with a process gas inlet port 11r formed at the surface of the processing vessel 1 and a large number of process gas inlet nozzle openings 31B are formed in communication with the process gas passage 31A.","Further, the lower shower plate 31 is provided with large apertures for passing the process gas radicals formed in the space 11B.","Thus, in the substrate processing apparatus 10A of FIG.","3, there is defined another process space 11C underneath the lower shower plate 31.By forming the lower shower plate 31 by a conductive material such as a stainless steel having a passivation surface by aluminum oxide (Al2O3) in such an apparatus, it becomes possible to block the penetration of microwave to the process space 11C.","Thereby, the excitation of plasma is limited in the process space 11B right underneath the upper shower plate 14, and the radicals Kr* of Kr or Ar* of Ar penetrate into the process space 11C through the large apertures formed in the shower plate 31 after excitation in the space 11B.","The radicals Kr* or Ar* thus penetrated into the process space 11C cause activation of the process gas released from the nozzle apertures 31B, and the processing of the substrate 12 is achieved by the process gas radicals thus activated.","In the substrate processing apparatus 10A of FIG.","3, it should be noted that the microwave is expelled from the process space 11C by forming the lower shower plate 31 by a conductive material, and the damaging of the substrate by microwave is avoided.","In the substrate processing apparatus 10A of FIG.","3, it is also possible to conduct a plasma CVD process by introducing a CVD source gas from the lower shower plate 31.Further, it is possible to conduct a dry etching process by introducing a dry etching gas from the lower shower plate 31 and applying a high-frequency bias to the stage 13.Thus, in the substrate processing apparatus of FIG.","2 of FIG.","3, Kr radicals (Kr*) of intermediate excitation state having an energy of about 10 eV are excited at the time of conducting an oxidation processing, by introducing a Kr gas and an oxygen gas into the process space 11B.","The Kr radicals thus excited cause efficient excitation of atomic state oxygen O* according to the reaction O2→O*+O*, while the atomic state oxygen O* thus excited cause the desired oxidation of the surface of the substrate 12.In the case of conducting a nitridation processing of the substrate 12, a Kr gas and an ammonia gas, or a Kr gas and a nitrogen gas and a hydrogen gas are introduced.","In this case, the excited Kr radicals (Kr*) or Ar radicals (Ar*) cause the excitation of hydrogen nitride radicals NH* according to the reaction NH3→NH*+2H*+e−, or N2+H2→NH*+NH*, wherein the hydrogen nitride radicals thus excited cause the desired nitridation processing of the substrate of the surface 12.Meanwhile, there are cases in which it is preferable to use atomic state nitrogen (N*), free from hydrogen and having a strong nitriding power, at the time of the nitridation processing of the substrate.","The atomic state nitrogen N* are formed according to the reaction N2→N*+N*, wherein it should be noted that such a reaction requires the energy of 23-25 eV.","This means that it is not possible to excite the atomic state nitrogen N* according to the foregoing reaction, as long as Kr or Ar plasma is used.","As noted previously, the energy of the Kr radicals or Ar radicals obtained by the Kr or Ar plasma is merely in the order of 10 eV.","Thus, even when there is made an attempt to supply a nitrogen gas in the substrate processing apparatus of FIG.","2 or FIG.","3 in place of the Kr gas or the Ar gas, merely the reaction N2→N2++e−, is obtained, and there is caused no desired atomic state oxygen N*.","FIG.","4 shows the relationship between the state density of the Kr plasma and the excitation energy of the atomic state nitrogen N*, hydrogen nitride radicals NH* and nitrogen atoms N2+.","Referring to FIG.","4, it can be seen that the state density of the Kr plasma is large at the low energy side, while the state density shows a rapid decrease with increase of the energy.","Such a plasma cannot achieve efficient excitation of the desired nitrogen radicals.","DISCLOSURE OF THE INVENTION Accordingly, it is a general object of the present invention to provide a novel and useful substrate processing apparatus wherein the foregoing problems are eliminated.","Another and more specific object of the present invention is to provide a substrate processing method and apparatus capable of forming nitrogen radicals N* efficiently.","Another object of the present invention is to provide a method of processing a substrate by using a substrate processing apparatus which has such a construction that a process space, in which a substrate to be processed is contained, is separated from a plasma formation space, in which the substrate to be processed is not contained, by a control electrode in a processing vessel, characterized by the steps of: supplying a gas containing He and N2 to said processing vessel; forming plasma in said plasma formation space under a condition such that there is caused excitation of atomic state nitrogen N* in said plasma; and nitriding a surface of the substrate to be processed by said atomic state nitrogen N* in said process space.","Another object of the present invention is.","to provide a substrate processing apparatus, comprising: a processing vessel defined by an outer wall and having a stage for holding a substrate to be processed thereon; an evacuation system coupled to said processing vessel; a plasma gas supplying part supplying a plasma excitation gas and a process gas into said processing vessel; a microwave window provided on said processing vessel so as to face said substrate to be processed; and a control electrode provided between said substrate to be processed on said stage and said plasma gas supplying part so as to face said substrate to be processed and separating a plasma excitation space containing said microwave window and a process space containing said substrate to be processed, said control electrode comprising a conductive member having a plurality of apertures for passing plasma formed in said processing vessel therethrough, and a surface of said control electrode being covered by any of aluminum oxide or electrically conductive nitride.","Another object of the present invention is to provide a substrate processing apparatus, characterized by: a processing vessel defined by a wall of quartz glass and having a stage for holding a substrate to be processed; an evacuation system coupled to said processing vessel; a plasma gas supplying part supplying a plasma excitation gas and a process gas to said processing vessel; a control electrode provided so as to face said substrate to be processed on said stage and dividing an interior of said processing vessel into a process space containing said substrate to be processed and a plasma excitation space; and an induction coil provided outside said quartz glass wall in correspondence to said plasma excitation space, said control electrode comprising a conductive member having a plurality of apertures passing therethrough plasma formed in said processing vessel, and a surface of said control electrode being covered with any of aluminum oxide or electrically conductive nitride.","According to the present invention, it becomes possible to form plasma having the energy sufficient for causing excitation of atomic state nitrogen N* in the substrate processing apparatus by using He for the plasma excitation gas, and it becomes possible to conduct an efficient nitridation of the substrate by using the atomic state nitrogen N* thus excited.","By separating the plasma excitation space in which the high-density plasma is excited from the process space in which the substrate is included by means of the control electrode, it becomes possible to reduce the plasma energy in the process space to the level suitable for substrate processing.","Further, it becomes possible to trap the positive ions formed in the plasma excitation space.","In the case of applying the present invention to the substrate processing apparatus that uses microwave-excited plasma, it becomes possible to avoid excessive increase of the plasma energy by conducting the plasma excitation by using a microwave having the frequency of about 28 GHz or more.","BRIEF DESCRIPTION OF THE DRAWINGS FIG.","1 is a diagram showing the construction of a conventional induction coupled plasma processing apparatus; FIG.","2 is a diagram showing the construction of a previously proposed microwave substrate processing apparatus; FIG.","3 is a diagram showing the construction of another previously proposed microwave substrate processing apparatus; FIG.","4 is a diagram explaining the characteristics of plasma excitation in the microwave substrate processing apparatus of FIG.","2 or FIG.","3; FIG.","5 is a diagram showing the construction of a microwave substrate processing apparatus according to a first embodiment of the present invention; FIG.","6 is a diagram showing a part of the microwave substrate processing apparatus of FIG.","5; FIG.","7 is a diagram showing the characteristics of plasma excitation in the microwave substrate processing apparatus of FIG.","5; FIG.","8 is a diagram showing a modification of the microwave plasma processing apparatus of FIG.","5; FIG.","9 is a diagram showing the construction of a microwave plasma processing apparatus according to a second embodiment of the present invention; and FIG.","10 is a diagram showing the construction of an induction coupled plasma processing apparatus according to a third embodiment of the present invention.","DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [First Embodiment] FIG.","5 shows the construction of a substrate processing apparatus 100 according to a first embodiment of the present invention.","In FIG.","5, those parts corresponding to the parts described previously are designated by the same reference numerals and the description thereof will be omitted.","Referring to FIG.","5, the shower plate 14 is mounted on the processing vessel 11 via a seal 11s, and the cover plate 15 is mounted on the shower plate 14 via a seal 11t.","Further, the radial line slot antenna 20 is mounted on the processing vessel 11 via a seal 11u.","Further, in the substrate processing apparatus 100 of FIG.","5, the interface between the emission plate 16 and the cover plate 15 is evacuated via a ring-shaped groove 11g formed at the top part of the processing vessel 11 in the region where the processing vessel makes an engagement with the emission plate and further via an evacuation port 11G communicating with the ring-shaped groove 11g.","After evacuation, a He gas is introduced into the foregoing interface with a pressure of about 0.8 atmospheres as a thermal conducting medium.","The He gas thus introduced is confined therein by closing the valve 11V.","In the substrate processing apparatus 100 of FIG.","5, it should be noted that the lower shower plate 31 used in the substrate processing apparatus 10A of FIG.","3 is removed and a control electrode 131 of a conductive member is formed, wherein the control electrode 31 has a lattice shape as represented in FIG.","6 and is formed so as to separate the plasma excitation space 11B and the process space 11C.","Referring to FIG.","6, the lattice-shaped control electrode 131 is formed with large number of apertures 132 having a size set such that there occurs free passage of the radicals excited in the plasma excitation spate 11B, and thus, the plasma excited in the plasma excitation space 11B cause diffusion freely into the process space 11C through the control electrode 131.In the construction of FIG.","5, it should be noted that the lattice-shaped control electrode 131 is grounded, and thus, the microwave introduced into the plasma excitation space 11B from the radial line slot antenna 11B is reflected by the lattice shaped control electrode 131, and there is caused no invasion of the microwave into the process space 11C.","Thus, the problem of the microwave causing damages in the substrate 12 is not caused in the substrate processing apparatus 100 of FIG.","5.It should be noted that the lattice-shaped control electrode 131 can be formed by W, Ti, or the like, wherein it is possible to increase the resistance against plasma irradiation by forming a layer 131a of a conductive nitride such as WN or TiN on the surface thereof.","Further, it is possible to form such a lattice-shaped control electrode 131 by using a quartz glass and provide the conductive nitride layer 131a on the surface thereof.","Further, in the substrate processing apparatus 100, it should be noted that the sidewall surface of the processing vessel 11 is covered by a quartz liner 11D for the part corresponding to the plasma excitation space 11B In the substrate processing apparatus 100 of FIG.","5, a He gas and an N2 gas are introduced to the process gas inlet port 11p, and a microwave of about 28 GHz is supplied to the radial line slot antenna.","Typically, the process pressure in the processing vessel 11 is set to the range of 66.5-266 Pa (0.5-2 Torr), and nitridation processing or oxynitridation processing of the substrate 12 is conducted in the temperature range of 200-500° C. FIG.","7 shows the state density of the plasma excited in the substrate processing apparatus 100 of FIG.","5 for the case He is used for the plasma gas.","Referring to FIG.","7, it should be noted that the use of He having a characteristically small collision cross-section for the plasma gas causes significant acceleration in the excited He radicals He* with the microwave electric field, and as a result, there is caused significant increase of plasma energy to the level suitable for excitation of the atomic state nitrogen N*.","On the other hand, it can be seen that the efficiency of excitation of the hydrogen nitride radicals NH* or nitrogen ions N2+, which are excited efficiently in the case Kr is used for the plasma gas, is reduced significantly.","Thus, in the present invention, efficient excitation of the atomic state nitrogen N* is achieved in the substrate processing apparatus 100 at the high plasma energy of 23-25 eV by using He for the plasma gas.","In order to avoid excessive increase of the electron temperature in the plasma, the present invention uses a microwave source 22 that produce a microwave of the frequency higher than the previously proposed frequency, such as about 28 GHz or more, for driving the radial line slot antenna 20.Thereby, it is possible to select the frequency of the microwave source from the frequencies such as about 2.4 GHz or about 8.3 GHz.","Further, by separating the plasma excitation space 1lB and the process space 11C by the control electrode 131, it is possible to reduce the electron temperature and the plasma energy to a level suitable for substrate processing.","Particularly, it should be noted that the control electrode is protected effectively from the high-energy plasma by forming a conductive nitride such as an Al2O3 passivation film on the surface of the control electrode 131 as explained already.","Further, the problem of sputtering of the inner wall of the processing vessel by the high-energy plasma and the associated problem of contamination of the substrate are avoided by covering the inner wall of the processing vessel 11 by a quartz liner 11D for the part corresponding to the plasma excitation region 11B.","FIG.","8 shows the construction of a substrate processing apparatus 100A according to a modification of the present embodiment.","Referring to FIG.","8, it becomes possible in the substrate processing apparatus 100A to capture the nitrogen ions N2+ excited in the plasma excitation space 11B with the positive electric charge, by controlling the potential of the control electrode 31 to a suitable negative potential value.","Thereby, penetration of the nitrogen ions N2+ into the process space 11C is avoided.","In the substrate processing apparatus 100 or 100A of the present embodiment, it is possible to conduct an oxynitridation processing of the substrate 12 by supplying a He gas, an N2 gas and an O2 gas to the plasma gas supply port 11p.","[Second Embodiment] FIG.","9 shows the construction of a substrate processing apparatus 200 according to a second embodiment of the present invention.","In FIG.","9, those parts corresponding to the parts described previously are designated by the same reference numerals and the description thereof will be omitted.","Referring to FIG.","9, it should be noted that the shower plate 14 is removed in the present embodiment, and in place of this, there are provided a plurality of process gas inlet ports lip on the processing vessel 11 such that the process gas inlet ports 11P are disposed with a symmetric relationship with respect to the substrate 12.As a result, therefore, the cover plate 15 constituting the dielectric window is exposed at the top part of the plasma excitation space 11B.","Further, the sidewall surface of the processing vessel is covered by the quartz liner 11D for the part corresponding to the plasma excitation space 11B similarly to the previous embodiment.","According to the present embodiment, the construction of the substrate processing apparatus 11 is simplified, and it becomes possible to conduct the nitridation processing of the substrate 12 efficiently with low cost by using the atomic state nitrogen N*, by supplying a He gas and an N2 gas to the plasma gas supplying port lip and by supplying the microwave of about 28 GHz to the radial line slot antenna 20.Further, it is possible to conduct an oxynitridation processing by supplying a He gas, an N2 gas and an O2 gas to the plasma gas supplying port 11p.","[Third Embodiment] FIG.","10 shows the construction of a substrate processing apparatus 300 according to a third embodiment of the present invention.","In FIG.","10, those parts corresponding to the parts described previously are designated by the same reference numerals and the description thereof will be omitted.","Referring to FIG.","10, the substrate processing apparatus 300 has a construction similar to the substrate processing apparatus 1 explained before with reference to FIG.","1, except that a control electrode 6 similar to the control electrode 31 is provided in the quartz vessel 2, and the space inside the quartz vessel 2 is divided by the control electrode 6 into a plasma excitation space 2B1 in which the high-density plasma 2D is excited and a process space 2B2 that includes the substrate 4 to be processed.","In the present embodiment, a He gas and an N2 gas are introduced into the plasma excitation space 2B1 via the process gas supply line 2C, and there is formed high-density plasma 2D having a high electron temperature and plasma energy sufficient for exciting atomic state nitrogen N* in the plasma excitation space 2B1.The atomic state nitrogen N* thus formed cause diffusion into the process space 2C through the control electrode 6, and the surface of the substrate 4 undergoes nitridation.","In such a construction, it should be noted that the plasma has a very high electron temperature and energy in the plasma excitation space 2B1, while the electron temperature and the energy of the plasma are reduced to the level suitable for processing the substrate 4 in the process space 2B2.In the present embodiment, too, it becomes possible to remove the low energy positive ions such as N2+ formed in the plasma excitation space 2B1 from the process space 2B2 by trapping the same, by controlling the potential of the control electrode 6 by the voltage source 6A.","Further, it becomes possible to control the state of the high-density plasma 2D in the plasma excitation space 2B1 by controlling the potential of the control electrode 6.In the substrate processing apparatus 200 of the present embodiment, it is also possible to conduct an oxynitridation processing of the substrate 4 in the process space 2B2 by introducing a He gas and an N2 gas and an O2 gas from the process gas supply line 2C.","Further, the present invention is not limited to the specific preferred embodiments described heretofore, but various variations and modifications may be made without departing from the scope of the invention recited in the claims.","INDUSTRIAL APPLICABILITY According to the present invention, it becomes possible to form plasma having the energy sufficient for causing excitation of atomic state nitrogen N* in the substrate processing apparatus by using He for the plasma excitation gas, and it becomes possible to conduct an efficient nitridation of the substrate by using the atomic state nitrogen N* thus excited.","By separating the plasma excitation space in which the high-density plasma is excited from the process space in which the substrate is included by means of the control electrode, it becomes possible to reduce the plasma energy in the process space to the level suitable for substrate processing.","Further, it becomes possible to trap the positive ions formed in the plasma excitation space.","In the case of applying the present invention to the substrate processing apparatus that uses microwave-excited plasma, it becomes possible to avoid excessive increase of the plasma energy by conducting the plasma excitation by using a microwave having the frequency of about 28 GHz or more."]],"string":"[\n [\n \"Substrate processing method and substrate processing apparatus\",\n \"In a substrate processing apparatus, a control electrode (131) separates a process space (11C) including a substrate to be processed and a plasma formation space (11B) not including the substrate.\",\n \"The control electrode includes a conductive member formed in a processing vessel and having a plurality of apertures (131a) for passing plasma.\",\n \"A surface of the control electrode is covered by an aluminum oxide or a conductive nitride.\",\n \"In the substrate processing apparatus, a gas containing He and N2 is supplied into the processing vessel.\",\n \"In the plasma formation space, there is formed plasma under a condition in which atomic state nitrogen N* are excited.\",\n \"The atomic state nitrogen N* are used to nitride a surface of the substrate.\"\n ],\n [\n \"1.A method of processing a substrate by using a substrate processing apparatus which has such a construction that a process space, in which a substrate to be processed is contained, is separated from plasma formation space, in which the substrate to be processed is not contained, by a control electrode in a processing vessel, characterized by the steps of: supplying a gas containing He and N2 to said processing vessel; forming plasma in said plasma formation space under a condition such that there is caused excitation of atomic state nitrogen N* in said plasma; and nitriding a surface of the substrate to be processed by said atomic state nitrogen N* in said process space.\",\n \"2.The substrate processing method as claimed in claim 1, characterized in that said step of forming plasma is conducted such that an intermediate excitation state of the energy of 23 to 25 eV is realized.\",\n \"3.The substrate processing method as claimed in claim 1, characterized in that said step of forming plasma comprises the step of supplying a microwave to said plasma formation space.\",\n \"4.The substrate processing method as claimed in claim 3, characterized in that said step of supplying microwave is conducted by driving a radial line slot antenna.\",\n \"5.The substrate processing method as claimed in claim 1, characterized in that said step of forming plasma comprises the step of forming an induction magnetic field in said plasma formation space.\",\n \"6.The substrate processing method as claimed in claim 5, characterized in that said step of forming an induction magnetic field comprises the step of driving an induction coil wound around said processing vessel by a high frequency electric power.\",\n \"7.The substrate processing method as claimed in claim 1, characterized in the said control electrode is grounded during said step of exciting plasma.\",\n \"8.The substrate processing method as claimed in claim 1, characterized in that a negative potential is supplied to said control electrode in said step of forming plasma.\",\n \"9.The substrate processing method as claimed in claim 1, wherein said gas supplied to said processing vessel further contains O2.10.A substrate processing apparatus, comprising: a processing vessel defined by an outer wall and having a stage for holding a substrate to be processed thereon; an evacuation system coupled to said processing vessel; a plasma gas supplying part supplying a plasma excitation gas and a process gas into said processing vessel; a microwave window provided on said processing vessel so as to face said substrate to be processed; and a control electrode provided between said substrate to be processed on said stage and said plasma gas supplying part so as to face said substrate to be procesed, and separating a plasma excitation space containing said microwave window and a process space containing said substrate to be processed, said control electrode comprising a conductive member having a plurality of apertures for passing plasma formed in said processing vessel therethrough, and a surface of said control electrode being covered by any of aluminum oxide or electrically conductive nitride.\",\n \"11.The substrate processing apparatus as claimed in claim 10, characterized in that said control electrode has a lattice-shaped form and is grounded.\",\n \"12.The substrate processing apparatus as claimed in claim 10, characterized in that said control electro has a form of a lattice-like shape and said substrate processing apparatus comprises a negative voltage source connected to said control electrode.\",\n \"13.The substrate processing apparatus as claimed in claim 10, characterized in that an inner wall of said processing vessel is covered with an insulation film in said plasma excitation space.\",\n \"14.The substrate processing apparatus as claimed in claim 10, characterized by further comprising a microwave antenna coupled to said microwave window at an outer side of said processing vessel.\",\n \"15.A substrate processing apparatus, characterized by: a processing vessel defined by a wall of quartz glass and having a stage for holding a substrate to be processed; an evacuation system coupled to said processing vessel; a plasma gas supplying part supplying a plasma excitation gas and a process gas to said processing vessel; a control electrode provided so as to face said substrate to be processed on said stage and dividing an interior of said processing vessel into a process space containing said substrate to be processed and a plasma excitation space; and an induction coil provided outside said quartz glass wall in correspondence to said plasma excitation space, said control electrode comprising a conductive member having a plurality of apertures passing therethrough plasma formed in said processing vessel, and a surface of said control electrode being covered with any of aluminum oxide or electrically conductive nitride.\",\n \"16.The substrate processing apparatus as claimed in claim 15, characterized in that said quartz glass wall defines a dome-like space.\",\n \"17.The substrate processing apparatus as claimed in claim 15, characterized in that said control electrode is grounded.\",\n \"18.The substrate processing apparatus as claimed in claim 15, characterized in that said control gate is connected to a negative voltage source.\"\n ],\n [\n \"<SOH> BACKGROUND ART <EOH>FIG.\",\n \"1 shows the schematic construction of a conventional induction-coupled plasma processing apparatus 1 .\",\n \"Referring to FIG.\",\n \"1 , the plasma processing apparatus 1 includes a processing vessel 2 of a quartz dome evacuated by an evacuation line 2 A, and there is provided a stage 3 in a process space 2 B defined by the processing vessel 2 such that the stage 2 is rotated by a rotating mechanism 3 A.\",\n \"Further, a substrate 4 is held on the stage 3 .\",\n \"Further, an inert gas such as Ar and a process gas such as oxygen or nitrogen are supplied to the process space 2 B via a process gas supply line 2 C. Further, there is provided a coil 5 around the top part of the processing vessel 2 at the outside thereof, and high-density plasma 2 D is inducted at the top part of the process space 2 B by driving the coil 5 by a d.c. power source.\",\n \"In the plasma processing apparatus 1 of FIG.\",\n \"1 , the radicals of the process gas formed with the high-density plasma 2 D reach the surface of the substrate 4 and the substrate processing such as oxidation or nitridation is achieved.\",\n \"In such a conventional induction-coupled plasma processing apparatus 1 , on the other hand, there exists a drawback in that the high-density plasma 2 D is localized at the top part of the processing vessel and there appears an extremely non-uniform distribution in the radicals that are formed with the plasma.\",\n \"Particularly, the non-uniformity of the radical concentration in the radial direction of the substrate is not resolved even when the stage 3 is rotated by the rotating mechanism 3 A.\",\n \"Thus, in the conventional induction-coupled plasma processing apparatus 1 , the plasma processing apparatus was designed such that the substrate 4 is separated from the region in which the high-density plasma 2 D is formed with a large distance for realizing as uniform radical concentration distribution as possible at the surface of the substrate 4 .\",\n \"As a result of such a construction, on the other hand, the overall size of the substrate processing apparatus 1 is increased inevitably.\",\n \"Further, the amount of the radicals reaching the substrate 4 is reduced.\",\n \"These problems become particularly serious in the technology of current trend of processing a large-diameter substrate.\",\n \"On the other hand, there is a proposal of a microwave plasma processing apparatus that uses high-density plasma induced, not by an induction magnetic field but by a microwave electric field.\",\n \"For example, there is proposed a plasma processing apparatus that uses a planar antenna (radial line slot antenna) having a large number of slots arranged so as to produce a uniform microwave, for emitting a microwave into a processing vessel.\",\n \"In this apparatus, the microwave electric field thus induced is used to excite plasma by causing ionization in the gas in the vacuum vessel.\",\n \"Reference should be made to Japanese Laid-Open Patent Application 9-63793.By using the microwave plasma excited according to such a process, it becomes possible to realize a high-plasma density over a wide area right underneath the antenna, and uniform plasma processing becomes possible with short time period.\",\n \"Further, the microwave plasma thus excited has an advantageous feature of low electron temperature as a result of excitation of the plasma by using a microwave, and it becomes possible to avoid the problem of damages or metal contamination caused in the substrate.\",\n \"Further, it becomes possible to excite uniform plasma over a substrate of large area, and thus, the plasma processing apparatus can easily handle the fabrication of semiconductor devices on a large-diameter semiconductor wafer or fabrication of large flat panel display devices.\",\n \"FIG.\",\n \"2 shows the construction of a microwave plasma processing apparatus 10 that uses such a radial line slot antenna as proposed before by the inventor of the present invention.\",\n \"Referring to FIG.\",\n \"2 , the microwave plasma processing apparatus 10 includes a processing chamber 11 evacuated at a plurality of evacuation ports 11 a , and there is provided a stage 13 inside the processing chamber 11 for supporting a substrate 12 to be processed.\",\n \"In order to achieve uniform evacuation of the processing chamber 11 , there is provided a ring-shaped space 11 A around the stage 13 , and the processing chamber 11 is evacuated uniformly via the space 11 A and further via the evacuation ports 11 a by arranging the evacuation ports 11 a communicating with the space 11 A in axial symmetry with respect to the substrate.\",\n \"On the processing chamber 11 , there is provided a plate-like shower plate 14 formed of a low-loss dielectric such as Al 2 O 3 or SiO 2 as a part of the outer wall of the processing chamber 11 at a location facing the substrate 12 held on the stage 13 , wherein the shower plate 14 is provided via a seal ring not illustrated and includes a number of apertures 14 A.\",\n \"Further, a cover plate 15 also of a low-loss dielectric such as Al 2 O 3 or SiO 2 is provided at the outer side of the shower plate 14 via another seal ring not illustrated.\",\n \"The shower plate 14 is provided with a gas passage 14 B at a top surface thereof, and each of the apertures 14 A are provided so as to communicate with the gas passage 14 B.\",\n \"Further, there is provided a gas supply passage 14 C in the interior of the shower plate 14 in communication with a gas supply port lip provided at an outer wall of the processing vessel 11 .\",\n \"Thus, the plasma-excitation gas such as Ar or Kr supplied to the gas supply port 11 p is forwarded to the apertures 11 A via the supply passage 14 C and further via the passage 14 B and is released to the process space 11 B right underneath the shower plate 14 inside the processing vessel 11 from the foregoing apertures 14 A.\",\n \"On the processing vessel 11 , there is further provided a radial line slot antenna 20 at the outer side of the cover plate 15 with a separation of 4-5 mm from the cover plate 15 .\",\n \"The radial line slot antenna 20 is connected to an external microwave source (not illustrated) via a coaxial waveguide 21 and causes excitation of the plasma-excitation gas released into the process space 11 B by the microwave from the microwave source.\",\n \"It should be noted that the cover plate 15 and the radiation surface of the radial line slot antenna are contacted closely, and there is provided a cooling block 19 on the antenna 20 for cooling the antenna.\",\n \"The cooling block 19 includes a cooling water passage 19 A.\",\n \"The radial line slot antenna 20 is formed of a flat, disk-shaped antenna body 17 connected to an outer waveguide tube 21 A of the coaxial waveguide 21 and a radiation plate 16 provided at the opening of the antenna body 17 , wherein the radiation plate 16 is formed with a number of slots and a retardation plate of a dielectric plate having a constant thickness is interposed between the antenna body 17 and the radiation plate 16 .\",\n \"In the radial line slot antenna 20 having such a construction, the microwave fed thereto from the coaxial waveguide 21 propagates along a path between the disk-shaped antenna body 17 and the radiation plate 16 in the radial direction, wherein the microwave thus propagating undergoes compression of wavelength as a result of the existence of the retardation plate 18 .\",\n \"Thus, by forming the slots concentrically in correspondence to the wavelength of the microwave thus propagating in the radial direction, and by forming the slots so as to form a perpendicular angle with each other, it becomes possible to emit a plane wave having a circular polarization from the radial line slot antenna 20 in the direction substantially perpendicular to the radiation plate 16 .\",\n \"By using such a radial line slot antenna 20 , there is formed uniform high-density plasma in the process space 11 B right underneath the shower plate 14 .\",\n \"The high-density plasma thus formed has a feature of low electron temperature and the occurrence of damages in the substrate 12 to be processed is avoided.\",\n \"Further, there occurs no metal contamination caused by sputtering of the chamber wall of the processing vessel 11 .\",\n \"Thus, by supplying a process gas, such as an O 2 gas, an NH 3 gas, or a mixed gas of an N 2 gas and an H 2 gas, to the gas inlet port 11 p of the substrate processing apparatus 10 of FIG.\",\n \"2 in addition to the plasma-excitation gas such as Ar or Kr, there is caused an excitation of active species such as atomic state oxygen O* or hydrogen nitride radicals NH* in the process space 11 B by the high-density plasma, and it becomes possible to conduct oxidation processing, nitridation processing or oxynitridation processing on the surface of the substrate 12 .\",\n \"Further, there is proposed a substrate processing apparatus 10 A shown in FIG.\",\n \"3 having a construction similar to the substrate processing apparatus 10 of FIG.\",\n \"2 except that there is provided a lower shower plate 31 at the lower side of the shower plate 14 .\",\n \"The lower shower plate 31 is provided with a process gas passage 31 A communicating with a process gas inlet port 11 r formed at the surface of the processing vessel 1 and a large number of process gas inlet nozzle openings 31 B are formed in communication with the process gas passage 31 A.\",\n \"Further, the lower shower plate 31 is provided with large apertures for passing the process gas radicals formed in the space 11 B.\",\n \"Thus, in the substrate processing apparatus 10 A of FIG.\",\n \"3 , there is defined another process space 11 C underneath the lower shower plate 31 .\",\n \"By forming the lower shower plate 31 by a conductive material such as a stainless steel having a passivation surface by aluminum oxide (Al 2 O 3 ) in such an apparatus, it becomes possible to block the penetration of microwave to the process space 11 C. Thereby, the excitation of plasma is limited in the process space 11 B right underneath the upper shower plate 14 , and the radicals Kr* of Kr or Ar* of Ar penetrate into the process space 11 C through the large apertures formed in the shower plate 31 after excitation in the space 11 B.\",\n \"The radicals Kr* or Ar* thus penetrated into the process space 11 C cause activation of the process gas released from the nozzle apertures 31 B, and the processing of the substrate 12 is achieved by the process gas radicals thus activated.\",\n \"In the substrate processing apparatus 10 A of FIG.\",\n \"3 , it should be noted that the microwave is expelled from the process space 11 C by forming the lower shower plate 31 by a conductive material, and the damaging of the substrate by microwave is avoided.\",\n \"In the substrate processing apparatus 10 A of FIG.\",\n \"3 , it is also possible to conduct a plasma CVD process by introducing a CVD source gas from the lower shower plate 31 .\",\n \"Further, it is possible to conduct a dry etching process by introducing a dry etching gas from the lower shower plate 31 and applying a high-frequency bias to the stage 13 .\",\n \"Thus, in the substrate processing apparatus of FIG.\",\n \"2 of FIG.\",\n \"3 , Kr radicals (Kr*) of intermediate excitation state having an energy of about 10 eV are excited at the time of conducting an oxidation processing, by introducing a Kr gas and an oxygen gas into the process space 11 B.\",\n \"The Kr radicals thus excited cause efficient excitation of atomic state oxygen O* according to the reaction in-line-formulae description=\\\"In-line Formulae\\\" end=\\\"lead\\\"?\",\n \"O 2 →O*+O*, in-line-formulae description=\\\"In-line Formulae\\\" end=\\\"tail\\\"?\",\n \"while the atomic state oxygen O* thus excited cause the desired oxidation of the surface of the substrate 12 .\",\n \"In the case of conducting a nitridation processing of the substrate 12 , a Kr gas and an ammonia gas, or a Kr gas and a nitrogen gas and a hydrogen gas are introduced.\",\n \"In this case, the excited Kr radicals (Kr*) or Ar radicals (Ar*) cause the excitation of hydrogen nitride radicals NH* according to the reaction in-line-formulae description=\\\"In-line Formulae\\\" end=\\\"lead\\\"?\",\n \"NH 3 →NH*+2H*+ e − , in-line-formulae description=\\\"In-line Formulae\\\" end=\\\"tail\\\"?\",\n \"in-line-formulae description=\\\"In-line Formulae\\\" end=\\\"lead\\\"?\",\n \"or in-line-formulae description=\\\"In-line Formulae\\\" end=\\\"tail\\\"?\",\n \"in-line-formulae description=\\\"In-line Formulae\\\" end=\\\"lead\\\"?\",\n \"N 2 +H 2 →NH*+NH*, in-line-formulae description=\\\"In-line Formulae\\\" end=\\\"tail\\\"?\",\n \"wherein the hydrogen nitride radicals thus excited cause the desired nitridation processing of the substrate of the surface 12 .\",\n \"Meanwhile, there are cases in which it is preferable to use atomic state nitrogen (N*), free from hydrogen and having a strong nitriding power, at the time of the nitridation processing of the substrate.\",\n \"The atomic state nitrogen N* are formed according to the reaction in-line-formulae description=\\\"In-line Formulae\\\" end=\\\"lead\\\"?\",\n \"N 2 →N*+N*, in-line-formulae description=\\\"In-line Formulae\\\" end=\\\"tail\\\"?\",\n \"wherein it should be noted that such a reaction requires the energy of 23-25 eV.\",\n \"This means that it is not possible to excite the atomic state nitrogen N* according to the foregoing reaction, as long as Kr or Ar plasma is used.\",\n \"As noted previously, the energy of the Kr radicals or Ar radicals obtained by the Kr or Ar plasma is merely in the order of 10 eV.\",\n \"Thus, even when there is made an attempt to supply a nitrogen gas in the substrate processing apparatus of FIG.\",\n \"2 or FIG.\",\n \"3 in place of the Kr gas or the Ar gas, merely the reaction in-line-formulae description=\\\"In-line Formulae\\\" end=\\\"lead\\\"?\",\n \"N 2 →N 2 + +e − , in-line-formulae description=\\\"In-line Formulae\\\" end=\\\"tail\\\"?\",\n \"is obtained, and there is caused no desired atomic state oxygen N*.\",\n \"FIG.\",\n \"4 shows the relationship between the state density of the Kr plasma and the excitation energy of the atomic state nitrogen N*, hydrogen nitride radicals NH* and nitrogen atoms N 2 + .\",\n \"Referring to FIG.\",\n \"4 , it can be seen that the state density of the Kr plasma is large at the low energy side, while the state density shows a rapid decrease with increase of the energy.\",\n \"Such a plasma cannot achieve efficient excitation of the desired nitrogen radicals.\"\n ],\n [\n \"<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.\",\n \"1 is a diagram showing the construction of a conventional induction coupled plasma processing apparatus; FIG.\",\n \"2 is a diagram showing the construction of a previously proposed microwave substrate processing apparatus; FIG.\",\n \"3 is a diagram showing the construction of another previously proposed microwave substrate processing apparatus; FIG.\",\n \"4 is a diagram explaining the characteristics of plasma excitation in the microwave substrate processing apparatus of FIG.\",\n \"2 or FIG.\",\n \"3 ; FIG.\",\n \"5 is a diagram showing the construction of a microwave substrate processing apparatus according to a first embodiment of the present invention; FIG.\",\n \"6 is a diagram showing a part of the microwave substrate processing apparatus of FIG.\",\n \"5 ; FIG.\",\n \"7 is a diagram showing the characteristics of plasma excitation in the microwave substrate processing apparatus of FIG.\",\n \"5 ; FIG.\",\n \"8 is a diagram showing a modification of the microwave plasma processing apparatus of FIG.\",\n \"5 ; FIG.\",\n \"9 is a diagram showing the construction of a microwave plasma processing apparatus according to a second embodiment of the present invention; and FIG.\",\n \"10 is a diagram showing the construction of an induction coupled plasma processing apparatus according to a third embodiment of the present invention.\",\n \"detailed-description description=\\\"Detailed Description\\\" end=\\\"lead\\\"?\"\n ],\n [\n \"TECHNICAL FIELD The present invention generally relates to plasma processing apparatuses and more particularly to a microwave plasma processing apparatus.\",\n \"Plasma process and plasma processing apparatus constitute indispensable technology for fabricating ultrafine semiconductor devices such as the one called deep submicron device or deep sub-quarter micron device having a gate length near 0.1 μm or less, or for fabricating high-resolution flat panel display device including a liquid crystal display device.\",\n \"Conventionally, various plasma excitation methods have been employed in the plasma processing apparatus used for fabricating semiconductor devices or liquid crystal display devices.\",\n \"Particularly, high-frequency plasma apparatuses of parallel plate type or induction-coupled type plasma apparatus are used commonly.\",\n \"However, such a conventional plasma processing apparatuses suffers from the problem of non-uniform plasma formation in that the region in which high electron density is achieved is substantially limited, and there has been a difficulty in conducting a uniform processing over the entire surface of the substrate with a large processing rate or throughput.\",\n \"This problem becomes particularly serious in the case of processing a substrate of large diameter.\",\n \"Further, such a conventional plasma processing apparatus has inherent problems, associated with its high electron temperature, in that damages are caused in the semiconductor devices formed on the substrate.\",\n \"Further, severe metal contamination may be caused as a result of sputtering of the chamber wall.\",\n \"Thus, it is becoming difficult with conventional plasma processing apparatuses to satisfy the stringent demand of further miniaturization and further improvement of productivity of semiconductor devices or flat display devices.\",\n \"Meanwhile, there has been a proposal of a microwave plasma processing apparatus that uses high-density plasma excited, not by d.c. magnetic field, but by a microwave electric field.\",\n \"For example, there is a proposal of a plasma processing apparatus that excites plasma by emitting a microwave into a processing vessel from a planar antenna (radial line slot antenna) having a number of slots arranged so as to produce a uniform microwave, for emitting a microwave into a processing vessel.\",\n \"In this plasma processing apparatus, the microwave electric field induces plasma by causing ionization in the gas in the vacuum vessel.\",\n \"Reference should be made to Japanese Laid-Open Patent Application 9-63793.By using the microwave plasma excited according to such a process, it becomes possible to realize a high-plasma density over a wide area right underneath the antenna, and uniform plasma processing becomes possible with short time period.\",\n \"Further, the microwave plasma thus excited has an advantageous feature of low electron temperature as a result of excitation of the plasma by using a microwave, and it becomes possible to avoid the problem of damages or metal contamination caused in the substrate.\",\n \"Further, it becomes possible to excite uniform plasma over a substrate of large area, and thus, the plasma processing apparatus can easily handle the fabrication of semiconductor devices on a large-diameter semiconductor wafer or fabrication of large flat panel display devices.\",\n \"BACKGROUND ART FIG.\",\n \"1 shows the schematic construction of a conventional induction-coupled plasma processing apparatus 1.Referring to FIG.\",\n \"1, the plasma processing apparatus 1 includes a processing vessel 2 of a quartz dome evacuated by an evacuation line 2A, and there is provided a stage 3 in a process space 2B defined by the processing vessel 2 such that the stage 2 is rotated by a rotating mechanism 3A.\",\n \"Further, a substrate 4 is held on the stage 3.Further, an inert gas such as Ar and a process gas such as oxygen or nitrogen are supplied to the process space 2B via a process gas supply line 2C.\",\n \"Further, there is provided a coil 5 around the top part of the processing vessel 2 at the outside thereof, and high-density plasma 2D is inducted at the top part of the process space 2B by driving the coil 5 by a d.c. power source.\",\n \"In the plasma processing apparatus 1 of FIG.\",\n \"1, the radicals of the process gas formed with the high-density plasma 2D reach the surface of the substrate 4 and the substrate processing such as oxidation or nitridation is achieved.\",\n \"In such a conventional induction-coupled plasma processing apparatus 1, on the other hand, there exists a drawback in that the high-density plasma 2D is localized at the top part of the processing vessel and there appears an extremely non-uniform distribution in the radicals that are formed with the plasma.\",\n \"Particularly, the non-uniformity of the radical concentration in the radial direction of the substrate is not resolved even when the stage 3 is rotated by the rotating mechanism 3A.\",\n \"Thus, in the conventional induction-coupled plasma processing apparatus 1, the plasma processing apparatus was designed such that the substrate 4 is separated from the region in which the high-density plasma 2D is formed with a large distance for realizing as uniform radical concentration distribution as possible at the surface of the substrate 4.As a result of such a construction, on the other hand, the overall size of the substrate processing apparatus 1 is increased inevitably.\",\n \"Further, the amount of the radicals reaching the substrate 4 is reduced.\",\n \"These problems become particularly serious in the technology of current trend of processing a large-diameter substrate.\",\n \"On the other hand, there is a proposal of a microwave plasma processing apparatus that uses high-density plasma induced, not by an induction magnetic field but by a microwave electric field.\",\n \"For example, there is proposed a plasma processing apparatus that uses a planar antenna (radial line slot antenna) having a large number of slots arranged so as to produce a uniform microwave, for emitting a microwave into a processing vessel.\",\n \"In this apparatus, the microwave electric field thus induced is used to excite plasma by causing ionization in the gas in the vacuum vessel.\",\n \"Reference should be made to Japanese Laid-Open Patent Application 9-63793.By using the microwave plasma excited according to such a process, it becomes possible to realize a high-plasma density over a wide area right underneath the antenna, and uniform plasma processing becomes possible with short time period.\",\n \"Further, the microwave plasma thus excited has an advantageous feature of low electron temperature as a result of excitation of the plasma by using a microwave, and it becomes possible to avoid the problem of damages or metal contamination caused in the substrate.\",\n \"Further, it becomes possible to excite uniform plasma over a substrate of large area, and thus, the plasma processing apparatus can easily handle the fabrication of semiconductor devices on a large-diameter semiconductor wafer or fabrication of large flat panel display devices.\",\n \"FIG.\",\n \"2 shows the construction of a microwave plasma processing apparatus 10 that uses such a radial line slot antenna as proposed before by the inventor of the present invention.\",\n \"Referring to FIG.\",\n \"2, the microwave plasma processing apparatus 10 includes a processing chamber 11 evacuated at a plurality of evacuation ports 11a, and there is provided a stage 13 inside the processing chamber 11 for supporting a substrate 12 to be processed.\",\n \"In order to achieve uniform evacuation of the processing chamber 11, there is provided a ring-shaped space 11A around the stage 13, and the processing chamber 11 is evacuated uniformly via the space 11A and further via the evacuation ports 11a by arranging the evacuation ports 11a communicating with the space 11A in axial symmetry with respect to the substrate.\",\n \"On the processing chamber 11, there is provided a plate-like shower plate 14 formed of a low-loss dielectric such as Al2O3 or SiO2 as a part of the outer wall of the processing chamber 11 at a location facing the substrate 12 held on the stage 13, wherein the shower plate 14 is provided via a seal ring not illustrated and includes a number of apertures 14A.\",\n \"Further, a cover plate 15 also of a low-loss dielectric such as Al2O3 or SiO2 is provided at the outer side of the shower plate 14 via another seal ring not illustrated.\",\n \"The shower plate 14 is provided with a gas passage 14B at a top surface thereof, and each of the apertures 14A are provided so as to communicate with the gas passage 14B.\",\n \"Further, there is provided a gas supply passage 14C in the interior of the shower plate 14 in communication with a gas supply port lip provided at an outer wall of the processing vessel 11.Thus, the plasma-excitation gas such as Ar or Kr supplied to the gas supply port 11p is forwarded to the apertures 11A via the supply passage 14C and further via the passage 14B and is released to the process space 11B right underneath the shower plate 14 inside the processing vessel 11 from the foregoing apertures 14A.\",\n \"On the processing vessel 11, there is further provided a radial line slot antenna 20 at the outer side of the cover plate 15 with a separation of 4-5 mm from the cover plate 15.The radial line slot antenna 20 is connected to an external microwave source (not illustrated) via a coaxial waveguide 21 and causes excitation of the plasma-excitation gas released into the process space 11B by the microwave from the microwave source.\",\n \"It should be noted that the cover plate 15 and the radiation surface of the radial line slot antenna are contacted closely, and there is provided a cooling block 19 on the antenna 20 for cooling the antenna.\",\n \"The cooling block 19 includes a cooling water passage 19A.\",\n \"The radial line slot antenna 20 is formed of a flat, disk-shaped antenna body 17 connected to an outer waveguide tube 21A of the coaxial waveguide 21 and a radiation plate 16 provided at the opening of the antenna body 17, wherein the radiation plate 16 is formed with a number of slots and a retardation plate of a dielectric plate having a constant thickness is interposed between the antenna body 17 and the radiation plate 16.In the radial line slot antenna 20 having such a construction, the microwave fed thereto from the coaxial waveguide 21 propagates along a path between the disk-shaped antenna body 17 and the radiation plate 16 in the radial direction, wherein the microwave thus propagating undergoes compression of wavelength as a result of the existence of the retardation plate 18.Thus, by forming the slots concentrically in correspondence to the wavelength of the microwave thus propagating in the radial direction, and by forming the slots so as to form a perpendicular angle with each other, it becomes possible to emit a plane wave having a circular polarization from the radial line slot antenna 20 in the direction substantially perpendicular to the radiation plate 16.By using such a radial line slot antenna 20, there is formed uniform high-density plasma in the process space 11B right underneath the shower plate 14.The high-density plasma thus formed has a feature of low electron temperature and the occurrence of damages in the substrate 12 to be processed is avoided.\",\n \"Further, there occurs no metal contamination caused by sputtering of the chamber wall of the processing vessel 11.Thus, by supplying a process gas, such as an O2 gas, an NH3 gas, or a mixed gas of an N2 gas and an H2 gas, to the gas inlet port 11p of the substrate processing apparatus 10 of FIG.\",\n \"2 in addition to the plasma-excitation gas such as Ar or Kr, there is caused an excitation of active species such as atomic state oxygen O* or hydrogen nitride radicals NH* in the process space 11B by the high-density plasma, and it becomes possible to conduct oxidation processing, nitridation processing or oxynitridation processing on the surface of the substrate 12.Further, there is proposed a substrate processing apparatus 10A shown in FIG.\",\n \"3 having a construction similar to the substrate processing apparatus 10 of FIG.\",\n \"2 except that there is provided a lower shower plate 31 at the lower side of the shower plate 14.The lower shower plate 31 is provided with a process gas passage 31A communicating with a process gas inlet port 11r formed at the surface of the processing vessel 1 and a large number of process gas inlet nozzle openings 31B are formed in communication with the process gas passage 31A.\",\n \"Further, the lower shower plate 31 is provided with large apertures for passing the process gas radicals formed in the space 11B.\",\n \"Thus, in the substrate processing apparatus 10A of FIG.\",\n \"3, there is defined another process space 11C underneath the lower shower plate 31.By forming the lower shower plate 31 by a conductive material such as a stainless steel having a passivation surface by aluminum oxide (Al2O3) in such an apparatus, it becomes possible to block the penetration of microwave to the process space 11C.\",\n \"Thereby, the excitation of plasma is limited in the process space 11B right underneath the upper shower plate 14, and the radicals Kr* of Kr or Ar* of Ar penetrate into the process space 11C through the large apertures formed in the shower plate 31 after excitation in the space 11B.\",\n \"The radicals Kr* or Ar* thus penetrated into the process space 11C cause activation of the process gas released from the nozzle apertures 31B, and the processing of the substrate 12 is achieved by the process gas radicals thus activated.\",\n \"In the substrate processing apparatus 10A of FIG.\",\n \"3, it should be noted that the microwave is expelled from the process space 11C by forming the lower shower plate 31 by a conductive material, and the damaging of the substrate by microwave is avoided.\",\n \"In the substrate processing apparatus 10A of FIG.\",\n \"3, it is also possible to conduct a plasma CVD process by introducing a CVD source gas from the lower shower plate 31.Further, it is possible to conduct a dry etching process by introducing a dry etching gas from the lower shower plate 31 and applying a high-frequency bias to the stage 13.Thus, in the substrate processing apparatus of FIG.\",\n \"2 of FIG.\",\n \"3, Kr radicals (Kr*) of intermediate excitation state having an energy of about 10 eV are excited at the time of conducting an oxidation processing, by introducing a Kr gas and an oxygen gas into the process space 11B.\",\n \"The Kr radicals thus excited cause efficient excitation of atomic state oxygen O* according to the reaction O2→O*+O*, while the atomic state oxygen O* thus excited cause the desired oxidation of the surface of the substrate 12.In the case of conducting a nitridation processing of the substrate 12, a Kr gas and an ammonia gas, or a Kr gas and a nitrogen gas and a hydrogen gas are introduced.\",\n \"In this case, the excited Kr radicals (Kr*) or Ar radicals (Ar*) cause the excitation of hydrogen nitride radicals NH* according to the reaction NH3→NH*+2H*+e−, or N2+H2→NH*+NH*, wherein the hydrogen nitride radicals thus excited cause the desired nitridation processing of the substrate of the surface 12.Meanwhile, there are cases in which it is preferable to use atomic state nitrogen (N*), free from hydrogen and having a strong nitriding power, at the time of the nitridation processing of the substrate.\",\n \"The atomic state nitrogen N* are formed according to the reaction N2→N*+N*, wherein it should be noted that such a reaction requires the energy of 23-25 eV.\",\n \"This means that it is not possible to excite the atomic state nitrogen N* according to the foregoing reaction, as long as Kr or Ar plasma is used.\",\n \"As noted previously, the energy of the Kr radicals or Ar radicals obtained by the Kr or Ar plasma is merely in the order of 10 eV.\",\n \"Thus, even when there is made an attempt to supply a nitrogen gas in the substrate processing apparatus of FIG.\",\n \"2 or FIG.\",\n \"3 in place of the Kr gas or the Ar gas, merely the reaction N2→N2++e−, is obtained, and there is caused no desired atomic state oxygen N*.\",\n \"FIG.\",\n \"4 shows the relationship between the state density of the Kr plasma and the excitation energy of the atomic state nitrogen N*, hydrogen nitride radicals NH* and nitrogen atoms N2+.\",\n \"Referring to FIG.\",\n \"4, it can be seen that the state density of the Kr plasma is large at the low energy side, while the state density shows a rapid decrease with increase of the energy.\",\n \"Such a plasma cannot achieve efficient excitation of the desired nitrogen radicals.\",\n \"DISCLOSURE OF THE INVENTION Accordingly, it is a general object of the present invention to provide a novel and useful substrate processing apparatus wherein the foregoing problems are eliminated.\",\n \"Another and more specific object of the present invention is to provide a substrate processing method and apparatus capable of forming nitrogen radicals N* efficiently.\",\n \"Another object of the present invention is to provide a method of processing a substrate by using a substrate processing apparatus which has such a construction that a process space, in which a substrate to be processed is contained, is separated from a plasma formation space, in which the substrate to be processed is not contained, by a control electrode in a processing vessel, characterized by the steps of: supplying a gas containing He and N2 to said processing vessel; forming plasma in said plasma formation space under a condition such that there is caused excitation of atomic state nitrogen N* in said plasma; and nitriding a surface of the substrate to be processed by said atomic state nitrogen N* in said process space.\",\n \"Another object of the present invention is.\",\n \"to provide a substrate processing apparatus, comprising: a processing vessel defined by an outer wall and having a stage for holding a substrate to be processed thereon; an evacuation system coupled to said processing vessel; a plasma gas supplying part supplying a plasma excitation gas and a process gas into said processing vessel; a microwave window provided on said processing vessel so as to face said substrate to be processed; and a control electrode provided between said substrate to be processed on said stage and said plasma gas supplying part so as to face said substrate to be processed and separating a plasma excitation space containing said microwave window and a process space containing said substrate to be processed, said control electrode comprising a conductive member having a plurality of apertures for passing plasma formed in said processing vessel therethrough, and a surface of said control electrode being covered by any of aluminum oxide or electrically conductive nitride.\",\n \"Another object of the present invention is to provide a substrate processing apparatus, characterized by: a processing vessel defined by a wall of quartz glass and having a stage for holding a substrate to be processed; an evacuation system coupled to said processing vessel; a plasma gas supplying part supplying a plasma excitation gas and a process gas to said processing vessel; a control electrode provided so as to face said substrate to be processed on said stage and dividing an interior of said processing vessel into a process space containing said substrate to be processed and a plasma excitation space; and an induction coil provided outside said quartz glass wall in correspondence to said plasma excitation space, said control electrode comprising a conductive member having a plurality of apertures passing therethrough plasma formed in said processing vessel, and a surface of said control electrode being covered with any of aluminum oxide or electrically conductive nitride.\",\n \"According to the present invention, it becomes possible to form plasma having the energy sufficient for causing excitation of atomic state nitrogen N* in the substrate processing apparatus by using He for the plasma excitation gas, and it becomes possible to conduct an efficient nitridation of the substrate by using the atomic state nitrogen N* thus excited.\",\n \"By separating the plasma excitation space in which the high-density plasma is excited from the process space in which the substrate is included by means of the control electrode, it becomes possible to reduce the plasma energy in the process space to the level suitable for substrate processing.\",\n \"Further, it becomes possible to trap the positive ions formed in the plasma excitation space.\",\n \"In the case of applying the present invention to the substrate processing apparatus that uses microwave-excited plasma, it becomes possible to avoid excessive increase of the plasma energy by conducting the plasma excitation by using a microwave having the frequency of about 28 GHz or more.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS FIG.\",\n \"1 is a diagram showing the construction of a conventional induction coupled plasma processing apparatus; FIG.\",\n \"2 is a diagram showing the construction of a previously proposed microwave substrate processing apparatus; FIG.\",\n \"3 is a diagram showing the construction of another previously proposed microwave substrate processing apparatus; FIG.\",\n \"4 is a diagram explaining the characteristics of plasma excitation in the microwave substrate processing apparatus of FIG.\",\n \"2 or FIG.\",\n \"3; FIG.\",\n \"5 is a diagram showing the construction of a microwave substrate processing apparatus according to a first embodiment of the present invention; FIG.\",\n \"6 is a diagram showing a part of the microwave substrate processing apparatus of FIG.\",\n \"5; FIG.\",\n \"7 is a diagram showing the characteristics of plasma excitation in the microwave substrate processing apparatus of FIG.\",\n \"5; FIG.\",\n \"8 is a diagram showing a modification of the microwave plasma processing apparatus of FIG.\",\n \"5; FIG.\",\n \"9 is a diagram showing the construction of a microwave plasma processing apparatus according to a second embodiment of the present invention; and FIG.\",\n \"10 is a diagram showing the construction of an induction coupled plasma processing apparatus according to a third embodiment of the present invention.\",\n \"DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [First Embodiment] FIG.\",\n \"5 shows the construction of a substrate processing apparatus 100 according to a first embodiment of the present invention.\",\n \"In FIG.\",\n \"5, those parts corresponding to the parts described previously are designated by the same reference numerals and the description thereof will be omitted.\",\n \"Referring to FIG.\",\n \"5, the shower plate 14 is mounted on the processing vessel 11 via a seal 11s, and the cover plate 15 is mounted on the shower plate 14 via a seal 11t.\",\n \"Further, the radial line slot antenna 20 is mounted on the processing vessel 11 via a seal 11u.\",\n \"Further, in the substrate processing apparatus 100 of FIG.\",\n \"5, the interface between the emission plate 16 and the cover plate 15 is evacuated via a ring-shaped groove 11g formed at the top part of the processing vessel 11 in the region where the processing vessel makes an engagement with the emission plate and further via an evacuation port 11G communicating with the ring-shaped groove 11g.\",\n \"After evacuation, a He gas is introduced into the foregoing interface with a pressure of about 0.8 atmospheres as a thermal conducting medium.\",\n \"The He gas thus introduced is confined therein by closing the valve 11V.\",\n \"In the substrate processing apparatus 100 of FIG.\",\n \"5, it should be noted that the lower shower plate 31 used in the substrate processing apparatus 10A of FIG.\",\n \"3 is removed and a control electrode 131 of a conductive member is formed, wherein the control electrode 31 has a lattice shape as represented in FIG.\",\n \"6 and is formed so as to separate the plasma excitation space 11B and the process space 11C.\",\n \"Referring to FIG.\",\n \"6, the lattice-shaped control electrode 131 is formed with large number of apertures 132 having a size set such that there occurs free passage of the radicals excited in the plasma excitation spate 11B, and thus, the plasma excited in the plasma excitation space 11B cause diffusion freely into the process space 11C through the control electrode 131.In the construction of FIG.\",\n \"5, it should be noted that the lattice-shaped control electrode 131 is grounded, and thus, the microwave introduced into the plasma excitation space 11B from the radial line slot antenna 11B is reflected by the lattice shaped control electrode 131, and there is caused no invasion of the microwave into the process space 11C.\",\n \"Thus, the problem of the microwave causing damages in the substrate 12 is not caused in the substrate processing apparatus 100 of FIG.\",\n \"5.It should be noted that the lattice-shaped control electrode 131 can be formed by W, Ti, or the like, wherein it is possible to increase the resistance against plasma irradiation by forming a layer 131a of a conductive nitride such as WN or TiN on the surface thereof.\",\n \"Further, it is possible to form such a lattice-shaped control electrode 131 by using a quartz glass and provide the conductive nitride layer 131a on the surface thereof.\",\n \"Further, in the substrate processing apparatus 100, it should be noted that the sidewall surface of the processing vessel 11 is covered by a quartz liner 11D for the part corresponding to the plasma excitation space 11B In the substrate processing apparatus 100 of FIG.\",\n \"5, a He gas and an N2 gas are introduced to the process gas inlet port 11p, and a microwave of about 28 GHz is supplied to the radial line slot antenna.\",\n \"Typically, the process pressure in the processing vessel 11 is set to the range of 66.5-266 Pa (0.5-2 Torr), and nitridation processing or oxynitridation processing of the substrate 12 is conducted in the temperature range of 200-500° C. FIG.\",\n \"7 shows the state density of the plasma excited in the substrate processing apparatus 100 of FIG.\",\n \"5 for the case He is used for the plasma gas.\",\n \"Referring to FIG.\",\n \"7, it should be noted that the use of He having a characteristically small collision cross-section for the plasma gas causes significant acceleration in the excited He radicals He* with the microwave electric field, and as a result, there is caused significant increase of plasma energy to the level suitable for excitation of the atomic state nitrogen N*.\",\n \"On the other hand, it can be seen that the efficiency of excitation of the hydrogen nitride radicals NH* or nitrogen ions N2+, which are excited efficiently in the case Kr is used for the plasma gas, is reduced significantly.\",\n \"Thus, in the present invention, efficient excitation of the atomic state nitrogen N* is achieved in the substrate processing apparatus 100 at the high plasma energy of 23-25 eV by using He for the plasma gas.\",\n \"In order to avoid excessive increase of the electron temperature in the plasma, the present invention uses a microwave source 22 that produce a microwave of the frequency higher than the previously proposed frequency, such as about 28 GHz or more, for driving the radial line slot antenna 20.Thereby, it is possible to select the frequency of the microwave source from the frequencies such as about 2.4 GHz or about 8.3 GHz.\",\n \"Further, by separating the plasma excitation space 1lB and the process space 11C by the control electrode 131, it is possible to reduce the electron temperature and the plasma energy to a level suitable for substrate processing.\",\n \"Particularly, it should be noted that the control electrode is protected effectively from the high-energy plasma by forming a conductive nitride such as an Al2O3 passivation film on the surface of the control electrode 131 as explained already.\",\n \"Further, the problem of sputtering of the inner wall of the processing vessel by the high-energy plasma and the associated problem of contamination of the substrate are avoided by covering the inner wall of the processing vessel 11 by a quartz liner 11D for the part corresponding to the plasma excitation region 11B.\",\n \"FIG.\",\n \"8 shows the construction of a substrate processing apparatus 100A according to a modification of the present embodiment.\",\n \"Referring to FIG.\",\n \"8, it becomes possible in the substrate processing apparatus 100A to capture the nitrogen ions N2+ excited in the plasma excitation space 11B with the positive electric charge, by controlling the potential of the control electrode 31 to a suitable negative potential value.\",\n \"Thereby, penetration of the nitrogen ions N2+ into the process space 11C is avoided.\",\n \"In the substrate processing apparatus 100 or 100A of the present embodiment, it is possible to conduct an oxynitridation processing of the substrate 12 by supplying a He gas, an N2 gas and an O2 gas to the plasma gas supply port 11p.\",\n \"[Second Embodiment] FIG.\",\n \"9 shows the construction of a substrate processing apparatus 200 according to a second embodiment of the present invention.\",\n \"In FIG.\",\n \"9, those parts corresponding to the parts described previously are designated by the same reference numerals and the description thereof will be omitted.\",\n \"Referring to FIG.\",\n \"9, it should be noted that the shower plate 14 is removed in the present embodiment, and in place of this, there are provided a plurality of process gas inlet ports lip on the processing vessel 11 such that the process gas inlet ports 11P are disposed with a symmetric relationship with respect to the substrate 12.As a result, therefore, the cover plate 15 constituting the dielectric window is exposed at the top part of the plasma excitation space 11B.\",\n \"Further, the sidewall surface of the processing vessel is covered by the quartz liner 11D for the part corresponding to the plasma excitation space 11B similarly to the previous embodiment.\",\n \"According to the present embodiment, the construction of the substrate processing apparatus 11 is simplified, and it becomes possible to conduct the nitridation processing of the substrate 12 efficiently with low cost by using the atomic state nitrogen N*, by supplying a He gas and an N2 gas to the plasma gas supplying port lip and by supplying the microwave of about 28 GHz to the radial line slot antenna 20.Further, it is possible to conduct an oxynitridation processing by supplying a He gas, an N2 gas and an O2 gas to the plasma gas supplying port 11p.\",\n \"[Third Embodiment] FIG.\",\n \"10 shows the construction of a substrate processing apparatus 300 according to a third embodiment of the present invention.\",\n \"In FIG.\",\n \"10, those parts corresponding to the parts described previously are designated by the same reference numerals and the description thereof will be omitted.\",\n \"Referring to FIG.\",\n \"10, the substrate processing apparatus 300 has a construction similar to the substrate processing apparatus 1 explained before with reference to FIG.\",\n \"1, except that a control electrode 6 similar to the control electrode 31 is provided in the quartz vessel 2, and the space inside the quartz vessel 2 is divided by the control electrode 6 into a plasma excitation space 2B1 in which the high-density plasma 2D is excited and a process space 2B2 that includes the substrate 4 to be processed.\",\n \"In the present embodiment, a He gas and an N2 gas are introduced into the plasma excitation space 2B1 via the process gas supply line 2C, and there is formed high-density plasma 2D having a high electron temperature and plasma energy sufficient for exciting atomic state nitrogen N* in the plasma excitation space 2B1.The atomic state nitrogen N* thus formed cause diffusion into the process space 2C through the control electrode 6, and the surface of the substrate 4 undergoes nitridation.\",\n \"In such a construction, it should be noted that the plasma has a very high electron temperature and energy in the plasma excitation space 2B1, while the electron temperature and the energy of the plasma are reduced to the level suitable for processing the substrate 4 in the process space 2B2.In the present embodiment, too, it becomes possible to remove the low energy positive ions such as N2+ formed in the plasma excitation space 2B1 from the process space 2B2 by trapping the same, by controlling the potential of the control electrode 6 by the voltage source 6A.\",\n \"Further, it becomes possible to control the state of the high-density plasma 2D in the plasma excitation space 2B1 by controlling the potential of the control electrode 6.In the substrate processing apparatus 200 of the present embodiment, it is also possible to conduct an oxynitridation processing of the substrate 4 in the process space 2B2 by introducing a He gas and an N2 gas and an O2 gas from the process gas supply line 2C.\",\n \"Further, the present invention is not limited to the specific preferred embodiments described heretofore, but various variations and modifications may be made without departing from the scope of the invention recited in the claims.\",\n \"INDUSTRIAL APPLICABILITY According to the present invention, it becomes possible to form plasma having the energy sufficient for causing excitation of atomic state nitrogen N* in the substrate processing apparatus by using He for the plasma excitation gas, and it becomes possible to conduct an efficient nitridation of the substrate by using the atomic state nitrogen N* thus excited.\",\n \"By separating the plasma excitation space in which the high-density plasma is excited from the process space in which the substrate is included by means of the control electrode, it becomes possible to reduce the plasma energy in the process space to the level suitable for substrate processing.\",\n \"Further, it becomes possible to trap the positive ions formed in the plasma excitation space.\",\n \"In the case of applying the present invention to the substrate processing apparatus that uses microwave-excited plasma, it becomes possible to avoid excessive increase of the plasma energy by conducting the plasma excitation by using a microwave having the frequency of about 28 GHz or more.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10467820"}}},{"rowIdx":386,"cells":{"text":{"kind":"list like","value":[["Immunogenic compositions comprising an antigen and a purified m protein from respiratory syncytial virus","Methods and compositions for enhancing an immune response to an antigen in a host are provided.","Immunogenic composition comprising an antigen and an amount of purified M protein from respiratory syncytial virus are provided in a pre-selected amount to provide an enhanced immune response to said antigen in a host having a pre-existing respiratory syncytial virus M-specific immune response.","The antigen can be an antigen from Respiratory syncytial virus."],["1.An immunogenic composition comprising an antigen and an amount of purified M protein from respiratory syncytial virus or at least one immunoeffective fragment thereof, wherein the amount of said M protein or immunoeffective fragment is provided in a pre-selected amount to provide an enhanced immune response to said antigen in a host having a pre-existing respiratory syncytial virus M-specific immune response.","2.The immunogenic composition of claim 1 wherein said antigen is from a pathogen.","3.The immunogenic composition of claim 1 formulated as a vaccine.","4.The immunogenic composition of claim 1 wherein said antigen or at least one immunogenic fragment thereof, is a protein antigen selected from the group consisting of, a viral antigen, a bacterial antigen, a respiratory syncytial virus antigen, a respiratory syncytial virus G antigen substantially free from respiratory syncytial virus F antigen, respiratory syncytial virus F antigen substantially free from respiratory syncytial virus G antigen, or a respiratory syncytial virus M22 antigen.","5.The immunogenic composition of claim 1 wherein said pre-existing respiratory syncytial virus M-specific immune response is a T-cell immune response.","6.The immunogenic composition of claim 1 wherein the amount of purified M protein is about 0.1 μg to about 1,000 μg per dose.","7.The immunogenic composition of claim 6 wherein the amount of purified M protein is about 1 μg to about 100 μg per dose.","8.The immunogenic composition of claim 7 wherein the amount of purified M protein is about 1 μg to about 50 μg per dose.","9.The immunogenic composition of claim 1 wherein the purified M protein is greater than 50% pure as measured by SDS-PAGE analysis or in an M-specific ELISA assay.","10.The immunogenic composition of claim 9 wherein the purity is greater that 70%.","11.The immunogenic composition of claim 10 wherein the purity is greater that 90%.","12.A method of making a immunogenic composition comprising providing an antigen and an amount of purified M protein from from respiratory syncytial virus or at least one immunoeffective fragment thereof, wherein the amount of said M protein or immunoeffective fragment is provided in a pre-selected amount to provide an enhanced immune response to said antigen in a host having a pre-existing respiratory syncytial virus M-specific immune response.","13.The method of claim 12 further formulated with a pharmaceutically acceptable carrier to provide a vaccine.","14.A method of immunizing a host comprising administering the immunogenic composition of claim 1 to said host.","15.A method of enhancing an immune response to an antigen in a host having a pre-existing immune response to respiratory syncytial virus M protein comprising the steps of: i) purifying M protein of respiratory syncytial virus; ii) mixing a pre-selected amount of said purified M protein with a different antigen; iii) formulating said mixture as a vaccine; and iv) administering said vaccine to a host.","16.The immunogenic composition of claim 1 wherein the antigen is encoded by a nucleic acid vector comprising an antigen encoding portion and a promoter to effect expression of the antigen encoding portion in the host.","17.The use of a pre-selected amount of purified M from respiratory syncytial virus or immunoeffective fragments thereof, to enhance the immune response to an antigen in a host having a pre-existing respiratory syncytial virus M-specific immune response."],["<SOH> BACKGROUND OF THE INVENTION <EOH>Adjuvants have been used for many years to improve the host immune response to antigens of interest in vaccines, especially subunit or component vaccines comprised of recombinant proteins.","Adjuvants are immunomodulators that are typically non-covalently linked to antigens and are formulated to enhance the host immune response.","Examples include aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum).","While little or no systemic toxicity is observed with alum, its use is associated with local reactions such as erythema, subcutaneous nodules, contact hypersensitivity and granulomatous inflammation.","Such local reactions may be of particular concern in the context of frequent, e.g., annual immunizations.","Adjuvants enhance the immunogenicity of an immunogen but are not necessarily immunogenic themselves.","Adjuvants have been identified that enhance the immune response to antigens delivered parenterally.","Some of these adjuvants are toxic, however, and can cause undesirable side-effects making them unsuitable for use in humans and many animals.","Indeed, only aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum) are routinely used as adjuvants in human and veterinary vaccines.","To efficiently induce humoral immune responses (HIR) and cell-mediated immunity (CMI), immunogens (antigens) are often emulsified in adjuvants.","Many adjuvants are toxic, inducing granulomas, acute and chronic inflammations (Freund's complete adjuvant, FCA), cytolysis (saponins and pluronic polymers) and pyrogenicity, arthritis and anterior uveitis (LPS and MDP).","Although FCA is a potent adjuvant and widely used in research, it is not licensed for use in human or veterinary vaccines because of its toxicity.","Human Respiratory Syncytial Virus (RSV) is a major cause of respiratory tract infections.","Globally, 65 million infections occur every year resulting in 160,000 deaths (ref.","1; a list of references appears at the end of the disclosure and each of the references in the list is incorporated herein by reference thereto.)","In the USA alone 100,000 children, may require hospitalization for pneumonia and bronchiolitis caused by RSV in a single year (refs.","2).","Providing inpatient and ambulatory care for children with RSV infections costs in excess of $340 million annually in the USA.","RSV is a major cause of serious lower respiratory illness in elderly and immunocompromised adults (refs.","3).","Outbreaks in nursing or retirement homes are well documented (ref.","3) and a significant proportion of disease involving the lower respiratory tract in outbreaks were associated with mortality.","Approximately 7% of hospitalized community acquired pneumonias have been attributed to RSV (ref.","4).","Mortality due to RSV may exceed that due to influenza by 60 to 80% (ref.","5) The annual costs attributed to hospitalizations for RSV pneumonia in the elderly in the USA has been conservatively estimated at between $150 to $680 million (ref.","6).","An RSV vaccine could therefore play an important role in lessening morbidity and mortality in the elderly and decreasing health care costs.","RSV is an enveloped RNA virus of the family paramyxoviridae and of the genus pneumovirus.","The structure and composition of RSV has been elucidated and is described in detail in the textbook “Fields Virology”, Fields, B.N.","Raven Press, N.Y. (1996), pp 1313-1351 “Respiratory Syncytial Virus” by Collins, P., McIntosh, K., and Chanock, R. M. (ref.","7).","Cross neutralization studies have shown that RSV isolates can be classified into two major antigenic groups, designated A and B.","(ref.","8) The G glycoprotein shows the greatest divergence between groups showing 53% amino acid homology between RSV A and B.","(ref.","9) The two major protective antigens of RSV are the envelope fusion (F) and the attachment (G) glycoproteins (ref.","10).","The F protein is synthesized as an about 68 kDa precursor molecule (Fo) which is proteolytically cleaved into disulfide-linked F1 (about 48 kDa) and F2 (about 20 kDa) polypeptide fragments (ref.","11).","The G protein (about 33 kDa) is heavily O-glycosylated giving rise to a glycoprotein of apparent molecular weight of about 90 kDa (ref.","12).","Two broad subtypes of RSV have been defined A and B (ref.","13).","The major antigenic differences between these subtypes are found in the G glycoprotein while the F glycoprotein is more conserved (refs.","14).","Antibodies directed against the F protein or against the G protein can neutralize the virus.","Antibodies to the F protein block the spread of the virus between cells.","In addition to the antibody response generated by the F and G glycoproteins, human cytotoxic T cells produced by RSV infection have been shown to recognize the RSV F protein, matrix protein (M), 22 k protein (M22) nucleoprotein (N), small hydrophobic protein (SH), and the nonstructural protein (lb.)","(ref 15).","The human RSV M gene coding for the matrix protein has been sequenced (ref 18).","It would be desirable to enhance the immune response to an antigen, in particular an RSV antigen to provide a more efficacious vaccine."],["<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides methods and compositions of enhancing the response to vaccines.","Accordingly, in one aspect of the present invention there is provided an immunogenic composition comprising an antigen and an amount of purified M protein from respiratory syncytial virus or at least one immunogenic fragment thereof, wherein the amount of said M protein or immunogenic fragment is provided in a pre-selected amount to provide an enhanced immune response to said antigen in a host having a pre-existing respiratory syncytial virus M-specific immune response.","In accordance with another aspect of the present invention, there is provided a method of making a immunogenic composition comprising providing an antigen and an amount of purified M protein from from respiratory syncytial virus or at least one immunogenic fragment thereof, wherein the amount of said M protein or immunogenic fragment is provided in a pre-selected amount to provide an enhanced immune response to said antigen in a host having a pre-existing respiratory syncytial virus M-specific immune response.","In a further aspect of the present invention there is provided the use of a pre-selected amount of purified M from respiratory syncytial virus or immunogenic fragments thereof, to enhance the immune response to an antigen in a host having a pre-existing respiratory syncytial virus M-specific immune response.","The hosts protected against disease caused by RSV include humans and the invention includes methods of immunization and protection of hosts against disease caused by infection by RSV by administering the immunogenic and preparations and vaccines as provided herein to susceptible hosts.","The hosts may be elderly humans or other humans previously exposed to RSV M protein and immunologically primed to respond to the immunization."],["FIELD OF THE INVENTION The present invention relates to the field of immunology and is particularly concerned with enhancing an immune response to an antigen.","BACKGROUND OF THE INVENTION Adjuvants have been used for many years to improve the host immune response to antigens of interest in vaccines, especially subunit or component vaccines comprised of recombinant proteins.","Adjuvants are immunomodulators that are typically non-covalently linked to antigens and are formulated to enhance the host immune response.","Examples include aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum).","While little or no systemic toxicity is observed with alum, its use is associated with local reactions such as erythema, subcutaneous nodules, contact hypersensitivity and granulomatous inflammation.","Such local reactions may be of particular concern in the context of frequent, e.g., annual immunizations.","Adjuvants enhance the immunogenicity of an immunogen but are not necessarily immunogenic themselves.","Adjuvants have been identified that enhance the immune response to antigens delivered parenterally.","Some of these adjuvants are toxic, however, and can cause undesirable side-effects making them unsuitable for use in humans and many animals.","Indeed, only aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum) are routinely used as adjuvants in human and veterinary vaccines.","To efficiently induce humoral immune responses (HIR) and cell-mediated immunity (CMI), immunogens (antigens) are often emulsified in adjuvants.","Many adjuvants are toxic, inducing granulomas, acute and chronic inflammations (Freund's complete adjuvant, FCA), cytolysis (saponins and pluronic polymers) and pyrogenicity, arthritis and anterior uveitis (LPS and MDP).","Although FCA is a potent adjuvant and widely used in research, it is not licensed for use in human or veterinary vaccines because of its toxicity.","Human Respiratory Syncytial Virus (RSV) is a major cause of respiratory tract infections.","Globally, 65 million infections occur every year resulting in 160,000 deaths (ref.","1; a list of references appears at the end of the disclosure and each of the references in the list is incorporated herein by reference thereto.)","In the USA alone 100,000 children, may require hospitalization for pneumonia and bronchiolitis caused by RSV in a single year (refs.","2).","Providing inpatient and ambulatory care for children with RSV infections costs in excess of $340 million annually in the USA.","RSV is a major cause of serious lower respiratory illness in elderly and immunocompromised adults (refs.","3).","Outbreaks in nursing or retirement homes are well documented (ref.","3) and a significant proportion of disease involving the lower respiratory tract in outbreaks were associated with mortality.","Approximately 7% of hospitalized community acquired pneumonias have been attributed to RSV (ref.","4).","Mortality due to RSV may exceed that due to influenza by 60 to 80% (ref.","5) The annual costs attributed to hospitalizations for RSV pneumonia in the elderly in the USA has been conservatively estimated at between $150 to $680 million (ref.","6).","An RSV vaccine could therefore play an important role in lessening morbidity and mortality in the elderly and decreasing health care costs.","RSV is an enveloped RNA virus of the family paramyxoviridae and of the genus pneumovirus.","The structure and composition of RSV has been elucidated and is described in detail in the textbook “Fields Virology”, Fields, B.N.","Raven Press, N.Y. (1996), pp 1313-1351 “Respiratory Syncytial Virus” by Collins, P., McIntosh, K., and Chanock, R. M. (ref.","7).","Cross neutralization studies have shown that RSV isolates can be classified into two major antigenic groups, designated A and B.","(ref.","8) The G glycoprotein shows the greatest divergence between groups showing 53% amino acid homology between RSV A and B.","(ref.","9) The two major protective antigens of RSV are the envelope fusion (F) and the attachment (G) glycoproteins (ref.","10).","The F protein is synthesized as an about 68 kDa precursor molecule (Fo) which is proteolytically cleaved into disulfide-linked F1 (about 48 kDa) and F2 (about 20 kDa) polypeptide fragments (ref.","11).","The G protein (about 33 kDa) is heavily O-glycosylated giving rise to a glycoprotein of apparent molecular weight of about 90 kDa (ref.","12).","Two broad subtypes of RSV have been defined A and B (ref.","13).","The major antigenic differences between these subtypes are found in the G glycoprotein while the F glycoprotein is more conserved (refs.","14).","Antibodies directed against the F protein or against the G protein can neutralize the virus.","Antibodies to the F protein block the spread of the virus between cells.","In addition to the antibody response generated by the F and G glycoproteins, human cytotoxic T cells produced by RSV infection have been shown to recognize the RSV F protein, matrix protein (M), 22 k protein (M22) nucleoprotein (N), small hydrophobic protein (SH), and the nonstructural protein (lb.)","(ref 15).","The human RSV M gene coding for the matrix protein has been sequenced (ref 18).","It would be desirable to enhance the immune response to an antigen, in particular an RSV antigen to provide a more efficacious vaccine.","SUMMARY OF THE INVENTION The present invention provides methods and compositions of enhancing the response to vaccines.","Accordingly, in one aspect of the present invention there is provided an immunogenic composition comprising an antigen and an amount of purified M protein from respiratory syncytial virus or at least one immunogenic fragment thereof, wherein the amount of said M protein or immunogenic fragment is provided in a pre-selected amount to provide an enhanced immune response to said antigen in a host having a pre-existing respiratory syncytial virus M-specific immune response.","In accordance with another aspect of the present invention, there is provided a method of making a immunogenic composition comprising providing an antigen and an amount of purified M protein from from respiratory syncytial virus or at least one immunogenic fragment thereof, wherein the amount of said M protein or immunogenic fragment is provided in a pre-selected amount to provide an enhanced immune response to said antigen in a host having a pre-existing respiratory syncytial virus M-specific immune response.","In a further aspect of the present invention there is provided the use of a pre-selected amount of purified M from respiratory syncytial virus or immunogenic fragments thereof, to enhance the immune response to an antigen in a host having a pre-existing respiratory syncytial virus M-specific immune response.","The hosts protected against disease caused by RSV include humans and the invention includes methods of immunization and protection of hosts against disease caused by infection by RSV by administering the immunogenic and preparations and vaccines as provided herein to susceptible hosts.","The hosts may be elderly humans or other humans previously exposed to RSV M protein and immunologically primed to respond to the immunization.","BRIEF DESCRIPTION OF DRAWINGS The present invention will be further understood from the following description with reference to the drawings, in which: FIG.","1 is a graphical representation of the IFN-γ secretion from spleen cells of mice primed with live RSV and boosted with various RSV antigens encoded by a viral vector (NYVAC).","FIG.","2 is a graphical representation of the CTL response from the viral vectors of FIG.","1, showing the percentage specific lysis of target cells.","FIG.","3 shows the T-cell response in mice to other RSV antigens when RSV M protein is co-administered.","FIG.","4 shows the enhanced antibody response in mice to other RSV antigens when RSV M protein is co-administered.","GENERAL DESCRIPTION OF THE INVENTION As discussed above, the present invention provides an enhanced response to antigens in sub-unit vaccines by the use of respiratory syncytial virus M protein.","While the inventors have not identified the exact mechanism of the enhanced immune response to an antigen due to the RSV M protein, it is thought that the M protein mobilizes T-cell help.","In a preferred embodiment, proteins to be included in the sub-unit vaccines may include the RSV F, G and M22 proteins.","The proteins can be isolated from RSV by, for example, immunoaffinity purification, ion-exchange or other biochemical procedures as described in, for example, International Patent Application No.","WO 94/27636 of Hancock, published Dec. 8, 1994 or by the procedure described in U.S. Pat.","No.","5,194,595.","(Each of the cited patent documents are incorporated herein by reference thereto) The RSV proteins and immunogenic fragments thereof can be isolated from recombinant organisms that express the proteins or immunogenic fragments.","The gene encoding the F protein is described in ref.","16.The gene encoding the G protein is described in ref.","17 and the gene encoding the M protein is described in ref.","18.The production of recombinant organisms expressing the RSV proteins or immunogenic fragments thereof and the identification and purification of the expressed gene products is described in, for example, U.S. Pat.","No.","5,223,254 (and incorporated herein by reference thereto).","Such recombinants include any bacterial transformants, yeast transformants, cultured insect cells infected with recombinant baculoviruses or cultured mammalian cells as known in the art, for example, Chinese hamster ovary cells that express the RSV virus proteins or immunogenic fragments thereof.","The RSV proteins and immunogenic fragments thereof can also be chemically synthesized.","The fusion (F) protein may comprise multimeric fusion (F) proteins which may include, when analyzed under nonreducing conditions, heterodimers of molecular weight approximately 70 kDa and dimeric and trimeric forms thereof: The attachment (G) protein may comprise, when analyzed under non-reducing conditions, oligomeric G protein, G protein of molecular weight approximately 95 kDa and G protein of molecular weight approximately 55 kDa.","The matrix (M) protein may comprise, when analyzed under non-reducing conditions, protein of molecular weight approximately 28 to 34 kDa The immunogenic compositions provided herein may be formulated as a vaccine for in vivo administration to a host, which may be a primate, most preferably a human host, to confer protection against disease caused by RSV.","The immunogenic compositions and vaccines provided herein may comprise at least one further immunogenic material which may be an antigen from a pathogen other than RSV, such as a bacterial or viral antigen to provide a combination vaccine for protection against a plurality of diseases.","The immunogenic compositions may be formulated as a vaccine wherein a non-RSV antigen is provided.","The non-RSV antigen may be a bacterial or viral antigen such that the RSV M protein enhances the host immune response to the non-RSV antigen.","In this specification RSV M antigen is defined as purified and isolated RSV M protein or immunoeffective fragments thereof, or vectors delivering RSV M protein.","Immunoeffective fragments of RSV M protein includes fragments or peptides derived from RSV M protein that are capable of eliciting the immunologic enhancement to another antigen.","Purified in this specification means isolated away from other RSV proteins such that the RSV M protein represents the dominant material as assayed by SDS-PAGE or M-specific ELISA assays for example.","Preferably the M protein should be greater than about 50% pure, more preferably greater than about 70% pure and most preferably greater than about 90% pure.","Pre-selected amounts of RSV M protein refers to defined amounts of the M protein, for example as measured by M-specific ELISA, that are added to another antigen such that there is an enhanced immune response to the other antigen in the host when compared to the same composition without the RSV M protein.","Enhanced immune response can be measured by interferon gamma (IFN γ) secretion, a CTL response as determined by specific cell lysis, IL-5 secretion, or antibody responses as measured by specific ELISA.","Immune response can also be measured by any other assay available to one skilled in the art and is not limited to those exemplified here.","A further measure can be by enhanced efficacy against disease in a relevant model system.","Pre-selected amounts may differ depending upon the host to which it is administered.","Examples of RSV M amounts can be from about 0.1 μg to about 1,000 μg, or about 0.1 μg to about 100 μg.","In other situations it may be preferable to administer between about 1 μg to about 50 μg of RSV M protein.","Pre-existing RSV M-specific response is defined as any detectable immune response to RSV M protein in the host.","This includes T-cell responses as measured by cytokine production, for example IFNγ from CD8+ T-cells, as detected by cytokine specific-ELISA assay or ELISPOT assay, both well known assays in the art.","RSV M protein pre-priming induces cytokine responses to heterologous proteins including viral derived from RSV or any other virus, bacterial or tumor antigens, CTL responses to heterologous antigens delivered by a vector (bacterial, viral or nucleic acid, or antigens formulated to deliver antigens for class I presentation such as liposomes) and antibody responses in naive or previously RSVimmune population.","The antigen to be used with the RSV M protein can be a purified protein antigen, isolated from the disease causing organism (pathogen), or can be recombinantly produced in any of the well known bacterial or eukaryotic expression systems.","The antigen can also be delivered by a nucleic acid vector such as non-replicating plasmid expression vectors encoding the antigen of choice, and containing a promoter to effect expression of said antigen in the host.","Alternatively the nucleic acid vector can be a viral vector.","Examples of non-replicating in the host viral vectors used to effect expression of said antigen are the pox vectors NYVAC and ALVAC (Ref 20) Viral vectors can also be replicating viral vectors such as recombinant alphavirus vectors as described in U.S. Pat.","No.","6,224,879 and U.S. Pat.","No.","6,015,686.Vaccine Preparation and Use Immunogenic compositions, suitable to be used as vaccines, may be prepared from mixtures comprising M protein and an antigen.","The immunogenic composition elicits an immune response which produces antibodies, and/or cell mediated responses such as cytotoxic T-cell response to the specific antigen.","Immunogenic compositions including vaccines may be prepared as injectables, as liquid solutions, suspensions or emulsions.","The active immunogenic ingredients may be mixed with pharmaceutically acceptable excipients which are compatible therewith.","Such excipients may include water, saline, dextrose, glycerol, ethanol and combinations thereof.","The immunogenic compositions and vaccines may further contain auxiliary substances, such as, wetting or emulsifying agents, pH buffering agents, or adjuvants to enhance the effectiveness thereof.","Immunogenic compositions and vaccines may be administered parentally, by injection subcutaneous, intradermal or intramuscularly injection.","Alternatively, the immunogenic compositions formulated according to the present invention, may be formulated and delivered in a manner to evoke an immune response at mucosal surfaces.","Thus, the immunogenic composition may be administered to mucosal surfaces by, for example, the nasal or oral (intragastric) routes.","Alternatively, other modes of administration including suppositories and oral formulations may be desirable.","For suppositories, binders and carriers may include, for example, polyalkalene glycols or triglycerides.","Such suppositories may be formed from mixtures containing the active immunogenic ingredient(s) in the range of about 10%, preferably about 1 to 2%.","Oral formulations may include normally employed carriers, such as, pharmaceutical grades of saccharine, cellulose and magnesium carbonate.","These compositions can take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 1 to 95% of the active ingredients, preferably about 20 to 75%.","The immunogenic preparations and vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective, immunogenic and protective.","The quantity to be administered depends on the subject to be treated, including, for example, the capacity of the individual's immune system to synthesize antibodies, and, if needed, to produce a cell-mediated immune response.","Precise amount of active ingredients required to be administered depend on the judgment of the practitioner.","However, suitable dosage ranges are readily determinable by one skilled in the art and may be of the order of micrograms to milligrams of the active ingredients) per vaccination.","Suitable regimes for initial administration and booster doses are also variable, but may include an initial administration followed by subsequent booster administrations.","The dosage may also depend on the route of administration and will vary according to the size of the host.","The concentration of the active ingredients in an immunogenic composition according to the invention is in general about 1 to 95%.","A vaccine which contains antigenic material of only one pathogen is a monovalent vaccine.","EXAMPLES The above disclosure generally describes the present invention.","A more complete understanding can be obtained by reference to the following specific Examples.","These Examples are described solely for purposes of illustration and are not intended to limit the scope of the invention.","Changes in form, and substitution of equivalents are contemplated as circumstances may suggest or render expedient.","Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.","Methods of determining tissue culture infectious dose50 (TCID50/mL), plaque and neutralization titres, not explicitly described in this disclosure are amply reported in the scientific literature and well within the scope of those skilled in the art.","Protein concentrations were determined by the bicinchoninie acid (BCA) method as described in the Pierce Manual (23220, 23225; Pierce Chemical company, U.S.A.), incorporated herein by reference.","CMRL 1969 culture medium was used for cell culture and virus growth.","The cells used in this study are vaccine quality African green monkey kidney cells (VERO lot M6) obtained from Institut Merieux.","The RS viruses used were the RS virus subtype A (Long and A2 strains) obtained from the American Type culture Collection (ATCC) for use in the virus neutralization assay and a recent subtype A clinical isolate for viral protein purification.","Example 1 This example illustrates a method of purifying RSV M protein.","An RSV concentrate was pelleted by centrifugation for 30 minutes at 5,000×g and the viral pellet was extracted with 2% Triton® X-100 in 1 mM sodium phosphate pH 6.8, 300 mM NaCl by stirring for 1 hour at room temperature.","The growth and harvest conditions for RSV can be found in U.S. Pat.","No.","6,020,182.The extract was centrifuged for 30 minutes at 15,000×g and the supernatant was collected.","The soluble supernatant was diluted two-fold with 1 mM sodium phosphate pH 6.8, 2% Triton® X-100 and then applied to a ceramic hydroxyapatite type II (Bio-Rad Laboratories) column equilibrated with 1 mM sodium phosphate, pH 6.8, 50 mM NaCl and 0.02% Triton® X-100.The column was washed with the same buffer and then an RSV M-rich fraction was collected by elution with the 1 mM sodium phosphate pH 6.8, 300 mM NaCl and 0.02% Triton® X-100.The elution pool was concentrated using an Amicon stirred-cell concentrator and a YM-10 ultrafiltration membrane and then dialyzed overnight against 20 mM Tris, pH 8.0, 600 mM NaCl, 0.02% Triton® X-100.The dialyzed concentrate was applied to a Sephacryl® S-200 size exclusion column equilibrated with 20 mM Tris, pH 8.0, 600 mM NaCl, 0.02% Triton® X-100 and the M-rich peak was collected.","Trace amounts of RSV F and RSV G glycoproteins were removed by passing the M-rich pool consecutively over an anti-F affinity column and a jacalin-Sepharose® column.","The purified RSV M was concentrated using an Amicon stirred-cell concentrator and a YM-10 ultrafiltration membrane and then frozen at −70° C. Example 2 This example illustrates the T-cell response in mice to other RSV antigens when delivered by viral vector immunization.","Eight-week old BALB/c mice used in this study were primed with either PBS or 103 pfu of live RSV intranasally.","They were boosted (i.m.)","4 weeks later with 107 pfu of NYVAC-M22 or NYVAC-F or NYVAC with or without RSV-M protein at 1 μg/mouse in PBS as outlined in Table 1 below.","Sera samples were collected to measure anti-RSV F titers.","Spleens were collected 4 weeks after boosting to look for CTL activity and cytokine production.","Spleen cells were restimulated in vitro with BC or BCH4 cells (persistantly infected with RSV).","Supematants were collected at 72 h and tested for IFN-γ and IL-5.For IFN-γ detection in the culture supernatants, wells of Nunc-Immulon-Maxisorp plates were coated with 50 mL of rat anti-mouse rIFN-γ antibody (Biosource) at 2 mg/mL in 0.05 M Carbonate/Bicarbonate buffer (pH 9.6), overnight at room temperature.","Wells were blocked with 200 mL of 10% FBS in PBS for 1 hour at room temperature, followed by 2 washes in 0.05% Tween 20 in PBS (washing buffer).","Samples and reference standards were diluted in sample buffer (0.05% Tween 20 in RPMI 1640) and 100 mL of each sample was added to the wells.","Plates were incubated at 37° C. for 2 hours, followed by 5 washes in washing buffer.","One hundred mL of biotinylated rat anti-mouse rIFN-g antibody diluted in sample buffer to 2 mg/mL, were added to each well and plates were incubated at 37° C. for 1 hr followed by 5 washes with washing buffer.","To each well, 100 mL of avidin-HRP (1/2000 dilution) were then added and plates were incubated for one hour at room temperature, followed by 5 washes with washing buffer.","Color reaction was developed by adding 100 mL of tetra methyl benzidine (TMB) /H2O2 and the reaction was terminated after 15 minutes by adding 50 mL of H2SO4.The plates were read at 450 nm in a Multiscan plate reader and the results were calculated using the Titersoft data reduction program.","For IL-5 detection, similar protocol was followed with appropriate antibodies and stanadards from Pharmingen and Biosource International.","Splenocytes were restimulated with BC or BCH4 cells at day 7 and then tested for CTL activity after 12 days.","CTL assays were carried out as follows.","Splenocytes (2.5 to 3×106 cells/mL) were incubated in complete RPMI containing 10 U/mL IL-2 and 2.5-3×106 cells/mL γ-irradiated (3,000 rads) syngeneic splenocytes which had been infected with 1 pfu/cell RSV for 2 hr at 37° C. After 5 days of culture, cells were washed and incubated (4 hr/37° C.) at varying effector cell dilutions with 2×103 51Cr-labeled target cells (persistently RSV-infected BCH4 fibroblasts).","Uninfected BALB/c fibroblasts (BC cells) served as a control.","Spontaneous and total chromium releases were measured in supernatants of target cells suspended in medium or 2.5% Triton-X 100.The percent of specific lysis was calculated as (counts−spontaneous counts)/(total counts−spontaneous counts)×100.Results are reported as mean specific lysis from triplicate assays.","Each experiment was performed 3 times.","These experiments show that boosting with RSV M protein antigen enhances CD8.T-cell responses as measured by IFN-γ shown in FIG.","1, and CTL activity shown in FIG.","2.RSV M antigen can enhance the specific response to at least two RSV antigens tested, RSV F and M22.Example 3 This example illustrates the T-cell response in mice to other RSV antigens when RSV M protein is co-administered.","Groups of BALB/c mice were primed with 103 pfu of live RSV intranassally as before.","Four weeks after priming the mice were boosted with PBS, purified RSV F protein (50 ng) in aluminum phosphate or purified RSV F plus purified RSV M (1 μg) in aluminum phosphate.","Four weeks after boosting the spleens were harvested as before, cultured and incubated in vitro with purified RSV F, G or M proteins (0.5 μg/ml final).","The cells were plated at 3×106 cells/ml and were stimulated with the same number of syngeneic spleen cells y-irradiated at 3000 rads.","Supematants were collected at 72 h and were assayed for IFN-γ as described in Example 2.The results of this assay are shown in FIG.","3.No detectable levels of anti-F, G or M responses were found in mice that were not boosted (PBS).","Mice that were boosted with F plus M (F+M) responded to F, M and also G. The anti-G response is attributable to trace amounts of G present in the purified F. The mice that received only F boost (F) did not produce significant IFN-γ to any of the proteins.","The IFN-γ response to RSV F in the F+M group of mice was enhanced when compared to the RSV F only boosted group.","Example 4 This example illustrates the antibody response to other RSV proteins in mice when RSV M is co-administered.","Groups of BALB/c mice were primed with 103 pfu of live RSV intranassally as before.","Four weeks later the mice were boosted with either PBS, or FI-RSV (formalin-inactivated) 5 ng of F with 1 μg of M in alum (aluminum phosphate) or 5 ng of F in alum.","The animals were boosted again four weeks later and the sera samples were collected to determine anti-F responses by F-specific ELISA.","The spleens were also collected and were restimulated with 500 ng/ml of F, M, G or 106PFU of RSV.","The culture supemates were collected 72 hrs later for IFN-gamma and IL-5 determinations.","The antibody results shown in FIG.","4, show that the inclusion of the RSV M antigen enhances the antibody response (antibody titre) to RSV F antigen by two logs when compared to the RSV F only boost.","TABLE 1 Groups Primary Boost 1 Total # of mice 1 RSV 103 pfu PBS 5 2 RSV 103 pfu M (1 ug) + NYVAC-F 5 3 RSV 103 pfu M (1 ug) + NYVAC-M22 5 4 RSV 103 pfu NYVAC-F 5 5 RSV 103 pfu NYVAC-M22 5 6 RSV 103 pfu M (1 ug) + NYVAC 5 7 PBS PBS 5 8 PBS M (1 ug) + NYVAC-F 5 9 PBS M (1 ug) + NYVAC-M22 5 10 PBS NYVAC-F 5 11 PBS NYVAC-M22 5 12 PBS M (1 ug) + NYVAC 5 Summary of the Disclosure In summary of this disclosure, the present invention provides compositions and methods for enhancing an immune response to an antigen, in particular RSV antigens.","The methods provide formulating immunogenic preparations to contain purified RSV M protein and at least one antigen from RSV or another antigen from a different infectious organism, or at least one immunogenic fragment thereof, in pre-selected amounts to elicit said enhanced immune response.","Modifications are possible within the scope of the invention.","REFERENCES 1.Robbins, A., and Freeman, P. (1988).","Sci.","Am.","259:126-133.2.Hall C B.","Prospects for a respiratory syncytial virus vaccine.","(1994) Science 265:1393-1394.3.Falsey A R, Cunningham C K, Barker W H, et al.","Respiratory syncytial virus and influenza A infections in the hospitalized elderly.","(1995) J. Infect.","Dis.","172:389-394.4.Nicholson K G, Kent J, Hammersley V, Cancio E. Acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, population based study of disease burden.","(1997) BMJ 315:1060-1064.5.Nicholson K G. Impact of influenza and respiratory syncytial virus on mortality in England and Wales from January 1975 to December 1990.Epidemiol Infect 1996;116:51-63.6.Han L. L., Alexander J. P_, Anderson L. J.","(1999) Vaccine 179:25-30.7.Collins, P., McIntosh, K., and Chanock, R. M., in “Fields of Virology” ed_ Fields, B. M., Knipe, D. M_ and Howley P. M., Lippincott-Raven Press New York, (1996) pp.","1313-1351.8.Coates, H. N., et al.","(1966).","Am.","J. Epidemeol.","83259-313.9.Johnson, R R., et al.","(1987).","J. Virol.","61:3163-3166.10.Walsh, E. E., Hall, C. B., Briselli, M., Braudiss, M. W., and Schlesinger, J. J.","(1987).","J. Infec.","Dis.","155:1198-1204.11.Walsh, E. E., Hruska, J.","(1983).","J. Virol.","47—171-177.12.Levine, S., Kleiber-France, R, and Paradiso, P. R. (1987) J. Gen- Virol.","69:2521-2524.13.Anderson, L. J., Hierholzer, J C., Tsou, C., Hendry, R. M., Femie, B. F., Stone Y_ and McIntosh, K. (1985) J. Infec.","Dis.151:626-633.14.Wertz, G. W., Sullender, W. M. (1992) Biotech 20:151-176.15.Cherrie, A. H., Anderson, K., Wertz, G. W. and Openshaw, P. J. M. (1992) J. Virol.","66:2102-2110.16.Collins, P. L., Huang, Y. T. and Wertz, G. W. (1984).","Proc.","Nat).","Acad.","Sci.","81:7683-7687.17.Wertz, G. W., Collins P. L., Huang, Y., Gruber C., Levine S., and Ball, L. A.","(1985) Proc.","Natl.","Acad.","Sci.","82:4075-4079.18.Satake, M. and Venkatesan S. (1984).","J.Virol.","50:92-99.19.Levine S, Diliman T R, and Montgomery P C (1989).","Proc.","Soc.","Exp.","Biol Med 190:349-356.20.Paoletti E, Tartaglia J, Taylor J.","(1994) Dev Biol Stand 82:65-69."]],"string":"[\n [\n \"Immunogenic compositions comprising an antigen and a purified m protein from respiratory syncytial virus\",\n \"Methods and compositions for enhancing an immune response to an antigen in a host are provided.\",\n \"Immunogenic composition comprising an antigen and an amount of purified M protein from respiratory syncytial virus are provided in a pre-selected amount to provide an enhanced immune response to said antigen in a host having a pre-existing respiratory syncytial virus M-specific immune response.\",\n \"The antigen can be an antigen from Respiratory syncytial virus.\"\n ],\n [\n \"1.An immunogenic composition comprising an antigen and an amount of purified M protein from respiratory syncytial virus or at least one immunoeffective fragment thereof, wherein the amount of said M protein or immunoeffective fragment is provided in a pre-selected amount to provide an enhanced immune response to said antigen in a host having a pre-existing respiratory syncytial virus M-specific immune response.\",\n \"2.The immunogenic composition of claim 1 wherein said antigen is from a pathogen.\",\n \"3.The immunogenic composition of claim 1 formulated as a vaccine.\",\n \"4.The immunogenic composition of claim 1 wherein said antigen or at least one immunogenic fragment thereof, is a protein antigen selected from the group consisting of, a viral antigen, a bacterial antigen, a respiratory syncytial virus antigen, a respiratory syncytial virus G antigen substantially free from respiratory syncytial virus F antigen, respiratory syncytial virus F antigen substantially free from respiratory syncytial virus G antigen, or a respiratory syncytial virus M22 antigen.\",\n \"5.The immunogenic composition of claim 1 wherein said pre-existing respiratory syncytial virus M-specific immune response is a T-cell immune response.\",\n \"6.The immunogenic composition of claim 1 wherein the amount of purified M protein is about 0.1 μg to about 1,000 μg per dose.\",\n \"7.The immunogenic composition of claim 6 wherein the amount of purified M protein is about 1 μg to about 100 μg per dose.\",\n \"8.The immunogenic composition of claim 7 wherein the amount of purified M protein is about 1 μg to about 50 μg per dose.\",\n \"9.The immunogenic composition of claim 1 wherein the purified M protein is greater than 50% pure as measured by SDS-PAGE analysis or in an M-specific ELISA assay.\",\n \"10.The immunogenic composition of claim 9 wherein the purity is greater that 70%.\",\n \"11.The immunogenic composition of claim 10 wherein the purity is greater that 90%.\",\n \"12.A method of making a immunogenic composition comprising providing an antigen and an amount of purified M protein from from respiratory syncytial virus or at least one immunoeffective fragment thereof, wherein the amount of said M protein or immunoeffective fragment is provided in a pre-selected amount to provide an enhanced immune response to said antigen in a host having a pre-existing respiratory syncytial virus M-specific immune response.\",\n \"13.The method of claim 12 further formulated with a pharmaceutically acceptable carrier to provide a vaccine.\",\n \"14.A method of immunizing a host comprising administering the immunogenic composition of claim 1 to said host.\",\n \"15.A method of enhancing an immune response to an antigen in a host having a pre-existing immune response to respiratory syncytial virus M protein comprising the steps of: i) purifying M protein of respiratory syncytial virus; ii) mixing a pre-selected amount of said purified M protein with a different antigen; iii) formulating said mixture as a vaccine; and iv) administering said vaccine to a host.\",\n \"16.The immunogenic composition of claim 1 wherein the antigen is encoded by a nucleic acid vector comprising an antigen encoding portion and a promoter to effect expression of the antigen encoding portion in the host.\",\n \"17.The use of a pre-selected amount of purified M from respiratory syncytial virus or immunoeffective fragments thereof, to enhance the immune response to an antigen in a host having a pre-existing respiratory syncytial virus M-specific immune response.\"\n ],\n [\n \"<SOH> BACKGROUND OF THE INVENTION <EOH>Adjuvants have been used for many years to improve the host immune response to antigens of interest in vaccines, especially subunit or component vaccines comprised of recombinant proteins.\",\n \"Adjuvants are immunomodulators that are typically non-covalently linked to antigens and are formulated to enhance the host immune response.\",\n \"Examples include aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum).\",\n \"While little or no systemic toxicity is observed with alum, its use is associated with local reactions such as erythema, subcutaneous nodules, contact hypersensitivity and granulomatous inflammation.\",\n \"Such local reactions may be of particular concern in the context of frequent, e.g., annual immunizations.\",\n \"Adjuvants enhance the immunogenicity of an immunogen but are not necessarily immunogenic themselves.\",\n \"Adjuvants have been identified that enhance the immune response to antigens delivered parenterally.\",\n \"Some of these adjuvants are toxic, however, and can cause undesirable side-effects making them unsuitable for use in humans and many animals.\",\n \"Indeed, only aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum) are routinely used as adjuvants in human and veterinary vaccines.\",\n \"To efficiently induce humoral immune responses (HIR) and cell-mediated immunity (CMI), immunogens (antigens) are often emulsified in adjuvants.\",\n \"Many adjuvants are toxic, inducing granulomas, acute and chronic inflammations (Freund's complete adjuvant, FCA), cytolysis (saponins and pluronic polymers) and pyrogenicity, arthritis and anterior uveitis (LPS and MDP).\",\n \"Although FCA is a potent adjuvant and widely used in research, it is not licensed for use in human or veterinary vaccines because of its toxicity.\",\n \"Human Respiratory Syncytial Virus (RSV) is a major cause of respiratory tract infections.\",\n \"Globally, 65 million infections occur every year resulting in 160,000 deaths (ref.\",\n \"1; a list of references appears at the end of the disclosure and each of the references in the list is incorporated herein by reference thereto.)\",\n \"In the USA alone 100,000 children, may require hospitalization for pneumonia and bronchiolitis caused by RSV in a single year (refs.\",\n \"2).\",\n \"Providing inpatient and ambulatory care for children with RSV infections costs in excess of $340 million annually in the USA.\",\n \"RSV is a major cause of serious lower respiratory illness in elderly and immunocompromised adults (refs.\",\n \"3).\",\n \"Outbreaks in nursing or retirement homes are well documented (ref.\",\n \"3) and a significant proportion of disease involving the lower respiratory tract in outbreaks were associated with mortality.\",\n \"Approximately 7% of hospitalized community acquired pneumonias have been attributed to RSV (ref.\",\n \"4).\",\n \"Mortality due to RSV may exceed that due to influenza by 60 to 80% (ref.\",\n \"5) The annual costs attributed to hospitalizations for RSV pneumonia in the elderly in the USA has been conservatively estimated at between $150 to $680 million (ref.\",\n \"6).\",\n \"An RSV vaccine could therefore play an important role in lessening morbidity and mortality in the elderly and decreasing health care costs.\",\n \"RSV is an enveloped RNA virus of the family paramyxoviridae and of the genus pneumovirus.\",\n \"The structure and composition of RSV has been elucidated and is described in detail in the textbook “Fields Virology”, Fields, B.N.\",\n \"Raven Press, N.Y. (1996), pp 1313-1351 “Respiratory Syncytial Virus” by Collins, P., McIntosh, K., and Chanock, R. M. (ref.\",\n \"7).\",\n \"Cross neutralization studies have shown that RSV isolates can be classified into two major antigenic groups, designated A and B.\",\n \"(ref.\",\n \"8) The G glycoprotein shows the greatest divergence between groups showing 53% amino acid homology between RSV A and B.\",\n \"(ref.\",\n \"9) The two major protective antigens of RSV are the envelope fusion (F) and the attachment (G) glycoproteins (ref.\",\n \"10).\",\n \"The F protein is synthesized as an about 68 kDa precursor molecule (Fo) which is proteolytically cleaved into disulfide-linked F1 (about 48 kDa) and F2 (about 20 kDa) polypeptide fragments (ref.\",\n \"11).\",\n \"The G protein (about 33 kDa) is heavily O-glycosylated giving rise to a glycoprotein of apparent molecular weight of about 90 kDa (ref.\",\n \"12).\",\n \"Two broad subtypes of RSV have been defined A and B (ref.\",\n \"13).\",\n \"The major antigenic differences between these subtypes are found in the G glycoprotein while the F glycoprotein is more conserved (refs.\",\n \"14).\",\n \"Antibodies directed against the F protein or against the G protein can neutralize the virus.\",\n \"Antibodies to the F protein block the spread of the virus between cells.\",\n \"In addition to the antibody response generated by the F and G glycoproteins, human cytotoxic T cells produced by RSV infection have been shown to recognize the RSV F protein, matrix protein (M), 22 k protein (M22) nucleoprotein (N), small hydrophobic protein (SH), and the nonstructural protein (lb.)\",\n \"(ref 15).\",\n \"The human RSV M gene coding for the matrix protein has been sequenced (ref 18).\",\n \"It would be desirable to enhance the immune response to an antigen, in particular an RSV antigen to provide a more efficacious vaccine.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides methods and compositions of enhancing the response to vaccines.\",\n \"Accordingly, in one aspect of the present invention there is provided an immunogenic composition comprising an antigen and an amount of purified M protein from respiratory syncytial virus or at least one immunogenic fragment thereof, wherein the amount of said M protein or immunogenic fragment is provided in a pre-selected amount to provide an enhanced immune response to said antigen in a host having a pre-existing respiratory syncytial virus M-specific immune response.\",\n \"In accordance with another aspect of the present invention, there is provided a method of making a immunogenic composition comprising providing an antigen and an amount of purified M protein from from respiratory syncytial virus or at least one immunogenic fragment thereof, wherein the amount of said M protein or immunogenic fragment is provided in a pre-selected amount to provide an enhanced immune response to said antigen in a host having a pre-existing respiratory syncytial virus M-specific immune response.\",\n \"In a further aspect of the present invention there is provided the use of a pre-selected amount of purified M from respiratory syncytial virus or immunogenic fragments thereof, to enhance the immune response to an antigen in a host having a pre-existing respiratory syncytial virus M-specific immune response.\",\n \"The hosts protected against disease caused by RSV include humans and the invention includes methods of immunization and protection of hosts against disease caused by infection by RSV by administering the immunogenic and preparations and vaccines as provided herein to susceptible hosts.\",\n \"The hosts may be elderly humans or other humans previously exposed to RSV M protein and immunologically primed to respond to the immunization.\"\n ],\n [\n \"FIELD OF THE INVENTION The present invention relates to the field of immunology and is particularly concerned with enhancing an immune response to an antigen.\",\n \"BACKGROUND OF THE INVENTION Adjuvants have been used for many years to improve the host immune response to antigens of interest in vaccines, especially subunit or component vaccines comprised of recombinant proteins.\",\n \"Adjuvants are immunomodulators that are typically non-covalently linked to antigens and are formulated to enhance the host immune response.\",\n \"Examples include aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum).\",\n \"While little or no systemic toxicity is observed with alum, its use is associated with local reactions such as erythema, subcutaneous nodules, contact hypersensitivity and granulomatous inflammation.\",\n \"Such local reactions may be of particular concern in the context of frequent, e.g., annual immunizations.\",\n \"Adjuvants enhance the immunogenicity of an immunogen but are not necessarily immunogenic themselves.\",\n \"Adjuvants have been identified that enhance the immune response to antigens delivered parenterally.\",\n \"Some of these adjuvants are toxic, however, and can cause undesirable side-effects making them unsuitable for use in humans and many animals.\",\n \"Indeed, only aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum) are routinely used as adjuvants in human and veterinary vaccines.\",\n \"To efficiently induce humoral immune responses (HIR) and cell-mediated immunity (CMI), immunogens (antigens) are often emulsified in adjuvants.\",\n \"Many adjuvants are toxic, inducing granulomas, acute and chronic inflammations (Freund's complete adjuvant, FCA), cytolysis (saponins and pluronic polymers) and pyrogenicity, arthritis and anterior uveitis (LPS and MDP).\",\n \"Although FCA is a potent adjuvant and widely used in research, it is not licensed for use in human or veterinary vaccines because of its toxicity.\",\n \"Human Respiratory Syncytial Virus (RSV) is a major cause of respiratory tract infections.\",\n \"Globally, 65 million infections occur every year resulting in 160,000 deaths (ref.\",\n \"1; a list of references appears at the end of the disclosure and each of the references in the list is incorporated herein by reference thereto.)\",\n \"In the USA alone 100,000 children, may require hospitalization for pneumonia and bronchiolitis caused by RSV in a single year (refs.\",\n \"2).\",\n \"Providing inpatient and ambulatory care for children with RSV infections costs in excess of $340 million annually in the USA.\",\n \"RSV is a major cause of serious lower respiratory illness in elderly and immunocompromised adults (refs.\",\n \"3).\",\n \"Outbreaks in nursing or retirement homes are well documented (ref.\",\n \"3) and a significant proportion of disease involving the lower respiratory tract in outbreaks were associated with mortality.\",\n \"Approximately 7% of hospitalized community acquired pneumonias have been attributed to RSV (ref.\",\n \"4).\",\n \"Mortality due to RSV may exceed that due to influenza by 60 to 80% (ref.\",\n \"5) The annual costs attributed to hospitalizations for RSV pneumonia in the elderly in the USA has been conservatively estimated at between $150 to $680 million (ref.\",\n \"6).\",\n \"An RSV vaccine could therefore play an important role in lessening morbidity and mortality in the elderly and decreasing health care costs.\",\n \"RSV is an enveloped RNA virus of the family paramyxoviridae and of the genus pneumovirus.\",\n \"The structure and composition of RSV has been elucidated and is described in detail in the textbook “Fields Virology”, Fields, B.N.\",\n \"Raven Press, N.Y. (1996), pp 1313-1351 “Respiratory Syncytial Virus” by Collins, P., McIntosh, K., and Chanock, R. M. (ref.\",\n \"7).\",\n \"Cross neutralization studies have shown that RSV isolates can be classified into two major antigenic groups, designated A and B.\",\n \"(ref.\",\n \"8) The G glycoprotein shows the greatest divergence between groups showing 53% amino acid homology between RSV A and B.\",\n \"(ref.\",\n \"9) The two major protective antigens of RSV are the envelope fusion (F) and the attachment (G) glycoproteins (ref.\",\n \"10).\",\n \"The F protein is synthesized as an about 68 kDa precursor molecule (Fo) which is proteolytically cleaved into disulfide-linked F1 (about 48 kDa) and F2 (about 20 kDa) polypeptide fragments (ref.\",\n \"11).\",\n \"The G protein (about 33 kDa) is heavily O-glycosylated giving rise to a glycoprotein of apparent molecular weight of about 90 kDa (ref.\",\n \"12).\",\n \"Two broad subtypes of RSV have been defined A and B (ref.\",\n \"13).\",\n \"The major antigenic differences between these subtypes are found in the G glycoprotein while the F glycoprotein is more conserved (refs.\",\n \"14).\",\n \"Antibodies directed against the F protein or against the G protein can neutralize the virus.\",\n \"Antibodies to the F protein block the spread of the virus between cells.\",\n \"In addition to the antibody response generated by the F and G glycoproteins, human cytotoxic T cells produced by RSV infection have been shown to recognize the RSV F protein, matrix protein (M), 22 k protein (M22) nucleoprotein (N), small hydrophobic protein (SH), and the nonstructural protein (lb.)\",\n \"(ref 15).\",\n \"The human RSV M gene coding for the matrix protein has been sequenced (ref 18).\",\n \"It would be desirable to enhance the immune response to an antigen, in particular an RSV antigen to provide a more efficacious vaccine.\",\n \"SUMMARY OF THE INVENTION The present invention provides methods and compositions of enhancing the response to vaccines.\",\n \"Accordingly, in one aspect of the present invention there is provided an immunogenic composition comprising an antigen and an amount of purified M protein from respiratory syncytial virus or at least one immunogenic fragment thereof, wherein the amount of said M protein or immunogenic fragment is provided in a pre-selected amount to provide an enhanced immune response to said antigen in a host having a pre-existing respiratory syncytial virus M-specific immune response.\",\n \"In accordance with another aspect of the present invention, there is provided a method of making a immunogenic composition comprising providing an antigen and an amount of purified M protein from from respiratory syncytial virus or at least one immunogenic fragment thereof, wherein the amount of said M protein or immunogenic fragment is provided in a pre-selected amount to provide an enhanced immune response to said antigen in a host having a pre-existing respiratory syncytial virus M-specific immune response.\",\n \"In a further aspect of the present invention there is provided the use of a pre-selected amount of purified M from respiratory syncytial virus or immunogenic fragments thereof, to enhance the immune response to an antigen in a host having a pre-existing respiratory syncytial virus M-specific immune response.\",\n \"The hosts protected against disease caused by RSV include humans and the invention includes methods of immunization and protection of hosts against disease caused by infection by RSV by administering the immunogenic and preparations and vaccines as provided herein to susceptible hosts.\",\n \"The hosts may be elderly humans or other humans previously exposed to RSV M protein and immunologically primed to respond to the immunization.\",\n \"BRIEF DESCRIPTION OF DRAWINGS The present invention will be further understood from the following description with reference to the drawings, in which: FIG.\",\n \"1 is a graphical representation of the IFN-γ secretion from spleen cells of mice primed with live RSV and boosted with various RSV antigens encoded by a viral vector (NYVAC).\",\n \"FIG.\",\n \"2 is a graphical representation of the CTL response from the viral vectors of FIG.\",\n \"1, showing the percentage specific lysis of target cells.\",\n \"FIG.\",\n \"3 shows the T-cell response in mice to other RSV antigens when RSV M protein is co-administered.\",\n \"FIG.\",\n \"4 shows the enhanced antibody response in mice to other RSV antigens when RSV M protein is co-administered.\",\n \"GENERAL DESCRIPTION OF THE INVENTION As discussed above, the present invention provides an enhanced response to antigens in sub-unit vaccines by the use of respiratory syncytial virus M protein.\",\n \"While the inventors have not identified the exact mechanism of the enhanced immune response to an antigen due to the RSV M protein, it is thought that the M protein mobilizes T-cell help.\",\n \"In a preferred embodiment, proteins to be included in the sub-unit vaccines may include the RSV F, G and M22 proteins.\",\n \"The proteins can be isolated from RSV by, for example, immunoaffinity purification, ion-exchange or other biochemical procedures as described in, for example, International Patent Application No.\",\n \"WO 94/27636 of Hancock, published Dec. 8, 1994 or by the procedure described in U.S. Pat.\",\n \"No.\",\n \"5,194,595.\",\n \"(Each of the cited patent documents are incorporated herein by reference thereto) The RSV proteins and immunogenic fragments thereof can be isolated from recombinant organisms that express the proteins or immunogenic fragments.\",\n \"The gene encoding the F protein is described in ref.\",\n \"16.The gene encoding the G protein is described in ref.\",\n \"17 and the gene encoding the M protein is described in ref.\",\n \"18.The production of recombinant organisms expressing the RSV proteins or immunogenic fragments thereof and the identification and purification of the expressed gene products is described in, for example, U.S. Pat.\",\n \"No.\",\n \"5,223,254 (and incorporated herein by reference thereto).\",\n \"Such recombinants include any bacterial transformants, yeast transformants, cultured insect cells infected with recombinant baculoviruses or cultured mammalian cells as known in the art, for example, Chinese hamster ovary cells that express the RSV virus proteins or immunogenic fragments thereof.\",\n \"The RSV proteins and immunogenic fragments thereof can also be chemically synthesized.\",\n \"The fusion (F) protein may comprise multimeric fusion (F) proteins which may include, when analyzed under nonreducing conditions, heterodimers of molecular weight approximately 70 kDa and dimeric and trimeric forms thereof: The attachment (G) protein may comprise, when analyzed under non-reducing conditions, oligomeric G protein, G protein of molecular weight approximately 95 kDa and G protein of molecular weight approximately 55 kDa.\",\n \"The matrix (M) protein may comprise, when analyzed under non-reducing conditions, protein of molecular weight approximately 28 to 34 kDa The immunogenic compositions provided herein may be formulated as a vaccine for in vivo administration to a host, which may be a primate, most preferably a human host, to confer protection against disease caused by RSV.\",\n \"The immunogenic compositions and vaccines provided herein may comprise at least one further immunogenic material which may be an antigen from a pathogen other than RSV, such as a bacterial or viral antigen to provide a combination vaccine for protection against a plurality of diseases.\",\n \"The immunogenic compositions may be formulated as a vaccine wherein a non-RSV antigen is provided.\",\n \"The non-RSV antigen may be a bacterial or viral antigen such that the RSV M protein enhances the host immune response to the non-RSV antigen.\",\n \"In this specification RSV M antigen is defined as purified and isolated RSV M protein or immunoeffective fragments thereof, or vectors delivering RSV M protein.\",\n \"Immunoeffective fragments of RSV M protein includes fragments or peptides derived from RSV M protein that are capable of eliciting the immunologic enhancement to another antigen.\",\n \"Purified in this specification means isolated away from other RSV proteins such that the RSV M protein represents the dominant material as assayed by SDS-PAGE or M-specific ELISA assays for example.\",\n \"Preferably the M protein should be greater than about 50% pure, more preferably greater than about 70% pure and most preferably greater than about 90% pure.\",\n \"Pre-selected amounts of RSV M protein refers to defined amounts of the M protein, for example as measured by M-specific ELISA, that are added to another antigen such that there is an enhanced immune response to the other antigen in the host when compared to the same composition without the RSV M protein.\",\n \"Enhanced immune response can be measured by interferon gamma (IFN γ) secretion, a CTL response as determined by specific cell lysis, IL-5 secretion, or antibody responses as measured by specific ELISA.\",\n \"Immune response can also be measured by any other assay available to one skilled in the art and is not limited to those exemplified here.\",\n \"A further measure can be by enhanced efficacy against disease in a relevant model system.\",\n \"Pre-selected amounts may differ depending upon the host to which it is administered.\",\n \"Examples of RSV M amounts can be from about 0.1 μg to about 1,000 μg, or about 0.1 μg to about 100 μg.\",\n \"In other situations it may be preferable to administer between about 1 μg to about 50 μg of RSV M protein.\",\n \"Pre-existing RSV M-specific response is defined as any detectable immune response to RSV M protein in the host.\",\n \"This includes T-cell responses as measured by cytokine production, for example IFNγ from CD8+ T-cells, as detected by cytokine specific-ELISA assay or ELISPOT assay, both well known assays in the art.\",\n \"RSV M protein pre-priming induces cytokine responses to heterologous proteins including viral derived from RSV or any other virus, bacterial or tumor antigens, CTL responses to heterologous antigens delivered by a vector (bacterial, viral or nucleic acid, or antigens formulated to deliver antigens for class I presentation such as liposomes) and antibody responses in naive or previously RSVimmune population.\",\n \"The antigen to be used with the RSV M protein can be a purified protein antigen, isolated from the disease causing organism (pathogen), or can be recombinantly produced in any of the well known bacterial or eukaryotic expression systems.\",\n \"The antigen can also be delivered by a nucleic acid vector such as non-replicating plasmid expression vectors encoding the antigen of choice, and containing a promoter to effect expression of said antigen in the host.\",\n \"Alternatively the nucleic acid vector can be a viral vector.\",\n \"Examples of non-replicating in the host viral vectors used to effect expression of said antigen are the pox vectors NYVAC and ALVAC (Ref 20) Viral vectors can also be replicating viral vectors such as recombinant alphavirus vectors as described in U.S. Pat.\",\n \"No.\",\n \"6,224,879 and U.S. Pat.\",\n \"No.\",\n \"6,015,686.Vaccine Preparation and Use Immunogenic compositions, suitable to be used as vaccines, may be prepared from mixtures comprising M protein and an antigen.\",\n \"The immunogenic composition elicits an immune response which produces antibodies, and/or cell mediated responses such as cytotoxic T-cell response to the specific antigen.\",\n \"Immunogenic compositions including vaccines may be prepared as injectables, as liquid solutions, suspensions or emulsions.\",\n \"The active immunogenic ingredients may be mixed with pharmaceutically acceptable excipients which are compatible therewith.\",\n \"Such excipients may include water, saline, dextrose, glycerol, ethanol and combinations thereof.\",\n \"The immunogenic compositions and vaccines may further contain auxiliary substances, such as, wetting or emulsifying agents, pH buffering agents, or adjuvants to enhance the effectiveness thereof.\",\n \"Immunogenic compositions and vaccines may be administered parentally, by injection subcutaneous, intradermal or intramuscularly injection.\",\n \"Alternatively, the immunogenic compositions formulated according to the present invention, may be formulated and delivered in a manner to evoke an immune response at mucosal surfaces.\",\n \"Thus, the immunogenic composition may be administered to mucosal surfaces by, for example, the nasal or oral (intragastric) routes.\",\n \"Alternatively, other modes of administration including suppositories and oral formulations may be desirable.\",\n \"For suppositories, binders and carriers may include, for example, polyalkalene glycols or triglycerides.\",\n \"Such suppositories may be formed from mixtures containing the active immunogenic ingredient(s) in the range of about 10%, preferably about 1 to 2%.\",\n \"Oral formulations may include normally employed carriers, such as, pharmaceutical grades of saccharine, cellulose and magnesium carbonate.\",\n \"These compositions can take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 1 to 95% of the active ingredients, preferably about 20 to 75%.\",\n \"The immunogenic preparations and vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective, immunogenic and protective.\",\n \"The quantity to be administered depends on the subject to be treated, including, for example, the capacity of the individual's immune system to synthesize antibodies, and, if needed, to produce a cell-mediated immune response.\",\n \"Precise amount of active ingredients required to be administered depend on the judgment of the practitioner.\",\n \"However, suitable dosage ranges are readily determinable by one skilled in the art and may be of the order of micrograms to milligrams of the active ingredients) per vaccination.\",\n \"Suitable regimes for initial administration and booster doses are also variable, but may include an initial administration followed by subsequent booster administrations.\",\n \"The dosage may also depend on the route of administration and will vary according to the size of the host.\",\n \"The concentration of the active ingredients in an immunogenic composition according to the invention is in general about 1 to 95%.\",\n \"A vaccine which contains antigenic material of only one pathogen is a monovalent vaccine.\",\n \"EXAMPLES The above disclosure generally describes the present invention.\",\n \"A more complete understanding can be obtained by reference to the following specific Examples.\",\n \"These Examples are described solely for purposes of illustration and are not intended to limit the scope of the invention.\",\n \"Changes in form, and substitution of equivalents are contemplated as circumstances may suggest or render expedient.\",\n \"Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.\",\n \"Methods of determining tissue culture infectious dose50 (TCID50/mL), plaque and neutralization titres, not explicitly described in this disclosure are amply reported in the scientific literature and well within the scope of those skilled in the art.\",\n \"Protein concentrations were determined by the bicinchoninie acid (BCA) method as described in the Pierce Manual (23220, 23225; Pierce Chemical company, U.S.A.), incorporated herein by reference.\",\n \"CMRL 1969 culture medium was used for cell culture and virus growth.\",\n \"The cells used in this study are vaccine quality African green monkey kidney cells (VERO lot M6) obtained from Institut Merieux.\",\n \"The RS viruses used were the RS virus subtype A (Long and A2 strains) obtained from the American Type culture Collection (ATCC) for use in the virus neutralization assay and a recent subtype A clinical isolate for viral protein purification.\",\n \"Example 1 This example illustrates a method of purifying RSV M protein.\",\n \"An RSV concentrate was pelleted by centrifugation for 30 minutes at 5,000×g and the viral pellet was extracted with 2% Triton® X-100 in 1 mM sodium phosphate pH 6.8, 300 mM NaCl by stirring for 1 hour at room temperature.\",\n \"The growth and harvest conditions for RSV can be found in U.S. Pat.\",\n \"No.\",\n \"6,020,182.The extract was centrifuged for 30 minutes at 15,000×g and the supernatant was collected.\",\n \"The soluble supernatant was diluted two-fold with 1 mM sodium phosphate pH 6.8, 2% Triton® X-100 and then applied to a ceramic hydroxyapatite type II (Bio-Rad Laboratories) column equilibrated with 1 mM sodium phosphate, pH 6.8, 50 mM NaCl and 0.02% Triton® X-100.The column was washed with the same buffer and then an RSV M-rich fraction was collected by elution with the 1 mM sodium phosphate pH 6.8, 300 mM NaCl and 0.02% Triton® X-100.The elution pool was concentrated using an Amicon stirred-cell concentrator and a YM-10 ultrafiltration membrane and then dialyzed overnight against 20 mM Tris, pH 8.0, 600 mM NaCl, 0.02% Triton® X-100.The dialyzed concentrate was applied to a Sephacryl® S-200 size exclusion column equilibrated with 20 mM Tris, pH 8.0, 600 mM NaCl, 0.02% Triton® X-100 and the M-rich peak was collected.\",\n \"Trace amounts of RSV F and RSV G glycoproteins were removed by passing the M-rich pool consecutively over an anti-F affinity column and a jacalin-Sepharose® column.\",\n \"The purified RSV M was concentrated using an Amicon stirred-cell concentrator and a YM-10 ultrafiltration membrane and then frozen at −70° C. Example 2 This example illustrates the T-cell response in mice to other RSV antigens when delivered by viral vector immunization.\",\n \"Eight-week old BALB/c mice used in this study were primed with either PBS or 103 pfu of live RSV intranasally.\",\n \"They were boosted (i.m.)\",\n \"4 weeks later with 107 pfu of NYVAC-M22 or NYVAC-F or NYVAC with or without RSV-M protein at 1 μg/mouse in PBS as outlined in Table 1 below.\",\n \"Sera samples were collected to measure anti-RSV F titers.\",\n \"Spleens were collected 4 weeks after boosting to look for CTL activity and cytokine production.\",\n \"Spleen cells were restimulated in vitro with BC or BCH4 cells (persistantly infected with RSV).\",\n \"Supematants were collected at 72 h and tested for IFN-γ and IL-5.For IFN-γ detection in the culture supernatants, wells of Nunc-Immulon-Maxisorp plates were coated with 50 mL of rat anti-mouse rIFN-γ antibody (Biosource) at 2 mg/mL in 0.05 M Carbonate/Bicarbonate buffer (pH 9.6), overnight at room temperature.\",\n \"Wells were blocked with 200 mL of 10% FBS in PBS for 1 hour at room temperature, followed by 2 washes in 0.05% Tween 20 in PBS (washing buffer).\",\n \"Samples and reference standards were diluted in sample buffer (0.05% Tween 20 in RPMI 1640) and 100 mL of each sample was added to the wells.\",\n \"Plates were incubated at 37° C. for 2 hours, followed by 5 washes in washing buffer.\",\n \"One hundred mL of biotinylated rat anti-mouse rIFN-g antibody diluted in sample buffer to 2 mg/mL, were added to each well and plates were incubated at 37° C. for 1 hr followed by 5 washes with washing buffer.\",\n \"To each well, 100 mL of avidin-HRP (1/2000 dilution) were then added and plates were incubated for one hour at room temperature, followed by 5 washes with washing buffer.\",\n \"Color reaction was developed by adding 100 mL of tetra methyl benzidine (TMB) /H2O2 and the reaction was terminated after 15 minutes by adding 50 mL of H2SO4.The plates were read at 450 nm in a Multiscan plate reader and the results were calculated using the Titersoft data reduction program.\",\n \"For IL-5 detection, similar protocol was followed with appropriate antibodies and stanadards from Pharmingen and Biosource International.\",\n \"Splenocytes were restimulated with BC or BCH4 cells at day 7 and then tested for CTL activity after 12 days.\",\n \"CTL assays were carried out as follows.\",\n \"Splenocytes (2.5 to 3×106 cells/mL) were incubated in complete RPMI containing 10 U/mL IL-2 and 2.5-3×106 cells/mL γ-irradiated (3,000 rads) syngeneic splenocytes which had been infected with 1 pfu/cell RSV for 2 hr at 37° C. After 5 days of culture, cells were washed and incubated (4 hr/37° C.) at varying effector cell dilutions with 2×103 51Cr-labeled target cells (persistently RSV-infected BCH4 fibroblasts).\",\n \"Uninfected BALB/c fibroblasts (BC cells) served as a control.\",\n \"Spontaneous and total chromium releases were measured in supernatants of target cells suspended in medium or 2.5% Triton-X 100.The percent of specific lysis was calculated as (counts−spontaneous counts)/(total counts−spontaneous counts)×100.Results are reported as mean specific lysis from triplicate assays.\",\n \"Each experiment was performed 3 times.\",\n \"These experiments show that boosting with RSV M protein antigen enhances CD8.T-cell responses as measured by IFN-γ shown in FIG.\",\n \"1, and CTL activity shown in FIG.\",\n \"2.RSV M antigen can enhance the specific response to at least two RSV antigens tested, RSV F and M22.Example 3 This example illustrates the T-cell response in mice to other RSV antigens when RSV M protein is co-administered.\",\n \"Groups of BALB/c mice were primed with 103 pfu of live RSV intranassally as before.\",\n \"Four weeks after priming the mice were boosted with PBS, purified RSV F protein (50 ng) in aluminum phosphate or purified RSV F plus purified RSV M (1 μg) in aluminum phosphate.\",\n \"Four weeks after boosting the spleens were harvested as before, cultured and incubated in vitro with purified RSV F, G or M proteins (0.5 μg/ml final).\",\n \"The cells were plated at 3×106 cells/ml and were stimulated with the same number of syngeneic spleen cells y-irradiated at 3000 rads.\",\n \"Supematants were collected at 72 h and were assayed for IFN-γ as described in Example 2.The results of this assay are shown in FIG.\",\n \"3.No detectable levels of anti-F, G or M responses were found in mice that were not boosted (PBS).\",\n \"Mice that were boosted with F plus M (F+M) responded to F, M and also G. The anti-G response is attributable to trace amounts of G present in the purified F. The mice that received only F boost (F) did not produce significant IFN-γ to any of the proteins.\",\n \"The IFN-γ response to RSV F in the F+M group of mice was enhanced when compared to the RSV F only boosted group.\",\n \"Example 4 This example illustrates the antibody response to other RSV proteins in mice when RSV M is co-administered.\",\n \"Groups of BALB/c mice were primed with 103 pfu of live RSV intranassally as before.\",\n \"Four weeks later the mice were boosted with either PBS, or FI-RSV (formalin-inactivated) 5 ng of F with 1 μg of M in alum (aluminum phosphate) or 5 ng of F in alum.\",\n \"The animals were boosted again four weeks later and the sera samples were collected to determine anti-F responses by F-specific ELISA.\",\n \"The spleens were also collected and were restimulated with 500 ng/ml of F, M, G or 106PFU of RSV.\",\n \"The culture supemates were collected 72 hrs later for IFN-gamma and IL-5 determinations.\",\n \"The antibody results shown in FIG.\",\n \"4, show that the inclusion of the RSV M antigen enhances the antibody response (antibody titre) to RSV F antigen by two logs when compared to the RSV F only boost.\",\n \"TABLE 1 Groups Primary Boost 1 Total # of mice 1 RSV 103 pfu PBS 5 2 RSV 103 pfu M (1 ug) + NYVAC-F 5 3 RSV 103 pfu M (1 ug) + NYVAC-M22 5 4 RSV 103 pfu NYVAC-F 5 5 RSV 103 pfu NYVAC-M22 5 6 RSV 103 pfu M (1 ug) + NYVAC 5 7 PBS PBS 5 8 PBS M (1 ug) + NYVAC-F 5 9 PBS M (1 ug) + NYVAC-M22 5 10 PBS NYVAC-F 5 11 PBS NYVAC-M22 5 12 PBS M (1 ug) + NYVAC 5 Summary of the Disclosure In summary of this disclosure, the present invention provides compositions and methods for enhancing an immune response to an antigen, in particular RSV antigens.\",\n \"The methods provide formulating immunogenic preparations to contain purified RSV M protein and at least one antigen from RSV or another antigen from a different infectious organism, or at least one immunogenic fragment thereof, in pre-selected amounts to elicit said enhanced immune response.\",\n \"Modifications are possible within the scope of the invention.\",\n \"REFERENCES 1.Robbins, A., and Freeman, P. (1988).\",\n \"Sci.\",\n \"Am.\",\n \"259:126-133.2.Hall C B.\",\n \"Prospects for a respiratory syncytial virus vaccine.\",\n \"(1994) Science 265:1393-1394.3.Falsey A R, Cunningham C K, Barker W H, et al.\",\n \"Respiratory syncytial virus and influenza A infections in the hospitalized elderly.\",\n \"(1995) J. Infect.\",\n \"Dis.\",\n \"172:389-394.4.Nicholson K G, Kent J, Hammersley V, Cancio E. Acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, population based study of disease burden.\",\n \"(1997) BMJ 315:1060-1064.5.Nicholson K G. Impact of influenza and respiratory syncytial virus on mortality in England and Wales from January 1975 to December 1990.Epidemiol Infect 1996;116:51-63.6.Han L. L., Alexander J. P_, Anderson L. J.\",\n \"(1999) Vaccine 179:25-30.7.Collins, P., McIntosh, K., and Chanock, R. M., in “Fields of Virology” ed_ Fields, B. M., Knipe, D. M_ and Howley P. M., Lippincott-Raven Press New York, (1996) pp.\",\n \"1313-1351.8.Coates, H. N., et al.\",\n \"(1966).\",\n \"Am.\",\n \"J. Epidemeol.\",\n \"83259-313.9.Johnson, R R., et al.\",\n \"(1987).\",\n \"J. Virol.\",\n \"61:3163-3166.10.Walsh, E. E., Hall, C. B., Briselli, M., Braudiss, M. W., and Schlesinger, J. J.\",\n \"(1987).\",\n \"J. Infec.\",\n \"Dis.\",\n \"155:1198-1204.11.Walsh, E. E., Hruska, J.\",\n \"(1983).\",\n \"J. Virol.\",\n \"47—171-177.12.Levine, S., Kleiber-France, R, and Paradiso, P. R. (1987) J. Gen- Virol.\",\n \"69:2521-2524.13.Anderson, L. J., Hierholzer, J C., Tsou, C., Hendry, R. M., Femie, B. F., Stone Y_ and McIntosh, K. (1985) J. Infec.\",\n \"Dis.151:626-633.14.Wertz, G. W., Sullender, W. M. (1992) Biotech 20:151-176.15.Cherrie, A. H., Anderson, K., Wertz, G. W. and Openshaw, P. J. M. (1992) J. Virol.\",\n \"66:2102-2110.16.Collins, P. L., Huang, Y. T. and Wertz, G. W. (1984).\",\n \"Proc.\",\n \"Nat).\",\n \"Acad.\",\n \"Sci.\",\n \"81:7683-7687.17.Wertz, G. W., Collins P. L., Huang, Y., Gruber C., Levine S., and Ball, L. A.\",\n \"(1985) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"82:4075-4079.18.Satake, M. and Venkatesan S. (1984).\",\n \"J.Virol.\",\n \"50:92-99.19.Levine S, Diliman T R, and Montgomery P C (1989).\",\n \"Proc.\",\n \"Soc.\",\n \"Exp.\",\n \"Biol Med 190:349-356.20.Paoletti E, Tartaglia J, Taylor J.\",\n \"(1994) Dev Biol Stand 82:65-69.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10467828"}}},{"rowIdx":387,"cells":{"text":{"kind":"list like","value":[["Sequences involved in phenomena of tumour suppression, tumour reversion, apoptosis and/or virus resistance and their use as medicines","This invention is directed to sequences that are involved in the molecular pathways of tumor suppression, tumor revision, apoptosis and/or virus resistance.","The use of the compounds having these sequences, or encoded by them, in treating cancer, neurodegenerative diseases or viral diseases, and screening methods for compounds having such therapeutic properties are also encompassed."],["1.An isolated nucleotide sequence comprising a nucleotide sequence chosen from the group comprising: a) SEQ.","ID NO.","72, b) a nucleotide sequence of at least 15 consecutive nucleotides of a sequence as defined in a), c) a nucleotide sequence having a percentage identity of al least 80%, after optimal alignment, with a sequence defined in a) or b), d) a nucleotide sequence which hybridizes under high stringency conditions with a sequence defined in a) or b), and e) a complementary nucleotide sequence or the RNA sequence corresponding to a sequence as defined in a), b), c) or d).","2-30.","(canceled)"],["The present invention relates to the identification of genes involved in the molecular pathways of tumour suppression, tumour reversion, apoptosis and/or resistance to viruses.","The present invention was made possible by the isolation of cDNA corresponding to messenger RNAs expressed or repressed during tumour suppression, tumour reversion and/or during the process of apoptosis.","In order to isolate the genes activated or inhibited during tumour reversion, overall screening for the expression of the genes in a malignant cell line (U937) and a derived cell line (US4) was carried out with suppression of the malignant phenotype.","Comparison of the expressed genes (messenger RNAs expressed in both types of cell) made it possible to identify genes expressed differentially, that is to say expressed in one of the cells while they are not in the other (the genes may be activated or inhibited).","It is easily deduced therefrom that these genes are at least involved in the cancerization process, in one case by their absence, and in the other case by their presence.","For this differential study, the method used is the method described in 2000 by Brenner et al.","(Proc.","Natl.","Acad.","Sci.","USA, 97 (4), 1665-70).","In order to produce a model, the inventors made the following hypotheses: if it were possible to select, from a tumour which is sensitive to the cytopathic effect of the parvovirus H-1, cells which are resistant, then this resistance could be due to a change in their malignant phenotype.","It has been possible to demonstrate this for US4 cells selected from the U937 cancer cells.","Unlike the U937 parental line, the US4 clones (but also US3 clones which will not be dealt with in the present invention) are resistant to the cytopathic effect of the parvovirus H-1.At the molecular level, it has been possible to observe that this suppression of the malignant phenotype was accompanied by activation of the expression of the p21waf1 gene, this being independently of the expression of the p53 gene.","The approach to the problem according to the present invention made it possible to isolate sequences directly linked to several precise functions.","Accordingly, unlike the random sequencing of ESTs, the sequences are sequences whose function is known since they are involved in the process of suppression of the malignant phenotype, of tumour reversion, of apoptosis and/or in resistance to viruses.","Tumour reversion is distinguishable from tumour suppression by the fact that it covers a broader domain than that of tumour suppressor genes.","In other words, tumour reversion is achieved by the use of metabolic and/or molecular pathways which are not limited to the metabolic and molecular pathways in which the tumour suppressor genes are involved.","Thus, the present invention relates in particular to novel sequences and to the use of these sequences in the diagnostic field and for carrying out methods for screening test compounds.","The invention also relates to methods for the detection and/or assay of the sequences of the invention or of their product(s) of expression in a biological sample.","The present invention first of all relates to an isolated nucleotide sequence comprising a nucleotide sequence chosen from the group comprising: a) SEQ ID No.","1 to SEQ ID No.","1020, preferably SEQ ID No.","72, b) a nucleotide sequence of at least 15 consecutive nucleotides of a sequence as defined in a), c) a nucleotide sequence exhibiting a percentage identity of at least 80%, after optimum alignment, with a sequence defined in a) or b), d) a nucleotide sequence which hybridizes under high stringency conditions with a sequence defined in a) or b), and e) a complementary nucleotide sequence where the RNA sequence corresponding to a sequence as defined in a), b), c) or d).","The nucleotide sequence according to the invention defined in c) exhibits a percentage identity of at least 80% after optimum alignment with a sequence as defined in a) or b) above, preferably of at least 90%, more preferably of at least 98%.","In the context of the present invention, SEQ ID No.","72 is the preferred nucleotide sequence and corresponds to the TPT1 gene also called TCTP.","This gene is involved in particular in the phenomenon of tumour reversion and has been the subject of more thorough experiments on the part of the inventors as indicated below.","The expressions nucleotide sequence, nucleic acid, nucleic or nucleic acid sequence, polynucleotide, oligonucleotide and polynucleotide sequence, terms which will be used without distinction in the present description, are understood to mean a precise succession of nucleotides, modified or unmodified, which makes it possible to define a fragment or a region of a nucleic acid, containing, or otherwise, unnatural nucleotides, and being capable of corresponding at the same time to a double-stranded DNA, a single-stranded DNA and transcriptional products of the said DNAs.","Thus, the nucleic sequences according to the invention also cover PNAs (Peptide Nucleic Acids), or the like.","The fragments of the nucleotide sequences of the invention comprise at least 15 consecutive nucleotides.","Preferably, they comprise at least 20 consecutive nucleotides and still more preferably they comprise at least 30 consecutive nucleotides.","It should be understood that the present invention does not relate to the nucleotide sequences in their natural chromosomal environment, that is to say in the natural state.","It involves sequences which have been isolated and/or purified, that is to say that they were collected directly or indirectly, for example by copying, their environment having been at least partially modified.","The nucleic acids obtained by chemical synthesis are understood to also be thereby designated.","The expression “percentage identity” between two nucleic acid or amino acid sequences for the purposes of the present invention is understood to mean a percentage of identical nucleotides or amino acid residues between the two sequences to be compared, which is obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being randomly distributed and over their entire length.","The expression “best alignment” or “optimum alignment” is understood to mean the alignment for which the percentage identity determined as below is the highest.","Sequence comparisons between two nucleic acid or amino acid sequences are traditionally carried out by comparing these sequences after having optimally aligned them, the said comparison being carried out per segment or per “comparison window” in order to identify and compare the local regions with sequence similarity.","Optimum alignment of the sequences for comparison may be carried out, apart from manually, by means of the Smith and Waterman local homology algorithm (1981), by means of the Neddleman and Wunsch local homology algorithm (1970), by means of the Pearson and Lipman search for similarity methods (1988), by means of computer software packages using these algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.).","In order to obtain optimum alignment, the BLAST program is preferably used, with the BLOSUM 62 matrix.","The PAM or PAM 250 matrices can also be used.","The percentage identity between two nucleic acid or amino acid sequences is determined by comparing these two sequences aligned optimally, it being possible for the nucleic acid or amino acid sequence to be compared to comprise additions or deletions in relation to the reference sequence for an optimum alignment between these two sequences.","The percentage identity is calculated by determining the number of identical positions for which the nucleotide or amino acid residue is identical between the two sequences, by dividing this number of identical positions by the total number of positions compared and by multiplying the result obtained by 100 in order to obtain the percentage identity between these two sequences.","The expression nucleic sequences exhibiting a percentage identity of at least 80%, preferably of at least 90%, more preferably of at least 98%, after optimum alignment with a reference sequence, is understood to mean the nucleic sequences exhibiting, in relation to the reference nucleic sequence, certain modifications such as in particular a deletion, truncation, alignment, chimeric fusion and/or substitution, in particular point substitution, and whose nucleic sequence exhibits at least 80%, preferably at least 90%, more preferably at least 98%, identity after optimum alignment with the reference nucleic sequence.","Preferably, the specific or high stringency hybridization conditions will be such that they ensure at least 80%, preferably at least 90%, more preferably at least 98% identity after optimum alignment between one of the two sequences and the sequence complementary to the other.","Hybridization under high stringency conditions means that the temperature and ionic strength conditions are chosen so that they allow hybridization to be maintained between two complementary nucleic acid fragments.","By way of illustration, high stringency conditions of the hybridization step for the purposes of defining the nucleotide sequences described above, will be advantageously the following.","The DNA-DNA or DNA-RNA hybridization is carried out in two steps: (1) prehybridization at 42° C. for 3 hours in phosphate buffer (20 mM, pH 7.5) containing 5×SSC (1×SSC corresponds to a solution containing 0.15 M NaCl+0.015 M sodium citrate), 50% formamide, 7% sodium dodecyl sulphate (SDS), 10×Denhardt's, 5% dextran sulphate and 1% salmon sperm DNA; (2) hybridization proper for 20 hours at a temperature dependent on the size of the probe (i.e.",": 42° C., for a probe of size>100 nucleotides) followed by 2 washes of 20 minutes at 20° C. in 2×SSC+2% SDS, 1 wash of 20 minutes at 20° C. in 0.1×SSC+0.1% SDS.","The final wash is performed in 0.1×SSC+0.1% SDS for 30 minutes at 60° C. for a probe of size>100 nucleotides.","The high stringency hybridization conditions described above for a polynucleotide of defined size may be adjusted by persons skilled in the art for larger- or smaller-size oligonucleotides, according to the teaching of Sambrook et al., 1989.Among the nucleotide sequences exhibiting a percentage identity of at least 80%, preferably of at least 90%, more preferably of at least 98%, after optimum alignment with the sequences according to the invention, preference is also given to the variant nucleic sequences of the sequences of the invention, or of fragments thereof, that is to say all the nucleic sequences corresponding to allelic variants, that is to say individual variations of the sequences of the invention.","The expression “variant nucleotide sequence” is understood to mean any RNA or cDNA resulting from a mutation and/or variation of a splice site of the genomic DNA corresponding to the nucleotide sequences of the invention.","The subject of the present invention is also a polypeptide encoded by a nucleotide sequence in accordance with the invention.","The expression “polypeptide” is understood to mean, for the purposes of the present invention, either proteins or peptides.","According to a particular embodiment, the polypeptides in accordance with the invention comprise a polypeptide chosen from: a) a polypeptide encoded by a nucleotide sequence in accordance with the invention, b) a polypeptide exhibiting at least 80% identity with a polypeptide as defined in a), c) a fragment of at least 5 amino acids of a polypeptide as defined in a) orb), d) a biologically active fragment of a polypeptide as defined in a), b) or c), and e) a modified polypeptide of a polypeptide as defined in a), b), c) or d).","The percentage identity is understood to be evaluated after optimum alignment of the relevant sequences.","The expression polypeptide whose amino acid sequence exhibits a percentage identity of at least 80%, preferably of at least 90%, more preferably of at least 98% after optimum alignment with a reference sequence, is understood to mean the polypeptides exhibiting certain modifications in relation to the reference polypeptide, such as in particular one or more deletions or truncations, an extension, a chimeric fusion and/or one or more substitutions.","Among the polypeptides whose amino acid sequence exhibits a percentage identity of at least 80%, preferably of at least 90% and more preferably of at least 98%, after optimum alignment with a reference sequence such as a polypeptide in accordance with the present invention or with one of its fragments, preference is given to the variant polypeptides encoded by the variant nucleotide sequences as previously defined, in particular the polypeptides whose amino acid sequence exhibits at least one mutation corresponding in particular to a truncation, deletion, substitution and/or addition of at least one residue in relation to the polypeptide sequences of the invention or with one of their fragments.","The present invention also relates to a cell cloning and/or expression vector, characterized in that it comprises a nucleotide sequence according to the invention or that it encodes a polypeptide according to the invention.","Such a vector may also contain the elements necessary for the expression and possibly for the secretion of the polypeptide in a host cell.","Such a host cell is also a subject of the present invention.","The vectors comprising promoter and/or regulatory sequences also form part of the present invention.","The said vectors preferably contain a promoter, signals for initiation and termination of translation, and appropriate regions for regulation of transcription.","They should be able to be stably maintained in the cell and they may also possess particular signals so as to allow the secretion of the translated protein.","These different control signals are chosen according to the cellular host used.","To this effect, the nucleic acid sequences according to the invention may be inserted into vectors which replicate autonomously in the chosen host or into integrating vectors of the chosen host.","Among the autonomously replicating systems, use is preferably made, according to the host cell, of the plasmid or viral type systems, it being possible for the viral vectors in particular to be adenoviruses (5), retroviruses, lentiviruses, poxviruses or herpesviruses (5a).","Persons skilled in the art know the technologies which can be used for each of these systems.","When the integration of the sequence into the chromosomes of the host cell is desired, it is possible to use, for example, systems of the plasmid or viral type; such viruses are, for example, retroviruses (6) or AAVs (7).","Advantageously, the vectors in accordance with the invention contain a sequence allowing tissue-specific targeting and/or expression.","Among the nonviral vectors, preference is given to naked polynucleotides such as naked DNA or naked RNA according to the technique developed by the company VICAL, bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC) for expression in yeast, mouse artificial chromosomes (MAC) for expression in murine cells and preferably human artificial chromosomes (HAC) for expression in human cells.","Such vectors are prepared according to methods commonly used by persons skilled in the art, and the clones resulting therefrom may be introduced into an appropriate host by standard methods such as for example lipofection, electroporation, heat shock, transformation after chemical permeabilization of the membrane, cell fusion.","The invention additionally comprises the host cells, in particular the eukaryotic and prokaryotic cells, transformed with the vectors according to the invention and the transgenic animals, preferably the mammals, except humans, comprising one of the said transformed cells according to the invention.","These animals may be used as models, for studying the aetiology of inflammatory and/or immune diseases, and in particular inflammatory diseases of the digestive tube, or for studying cancers.","Among the cells which can be used for the purposes of the present invention, there may be mentioned bacterial cells (8), but also yeast cells (9), as well as animal cells, in particular mammalian cell cultures (10), and in particular Chinese hamster ovary (CHO) cells.","There may also be mentioned insect cells in which it is possible to use methods using for example baculoviruses (11).","A preferred cellular host for the expression of the proteins of the invention consists of COS cells.","Among the mammals according to the invention, preference is given to animals such as rodents, in particular mice, rats or rabbits, expressing a polypeptide according to the invention.","These transgenic animals are obtained, for example, by homologous recombination on embryonic stem cells, transfer of these stem cells into embryos, selection of the chimeras affected at the level of the reproductive lines, and growth of the said chimeras.","The transgenic animals according to the invention can thus overexpress the gene encoding the protein according to the invention, or their homologous gene, or may express the said gene into which a mutation is introduced.","These transgenic animals, in particular mice, are obtained for example by transfection of a copy of this gene under the control of a strong promoter of a ubiquitous nature, or which is selective for a type of tissue, or after viral transcription.","The cells and mammals according to the invention can be used in a method for producing a polypeptide according to the invention, as described below, and can likewise serve as a model for analysis.","The transformed cells or mammals as described above may also be used as models in order to study the interactions between the polypeptides according to the invention and the chemical or protein compounds involved directly or indirectly in the activities of the polypeptides according to the invention, this being in order to study the different mechanisms and interactions involved.","They can in particular be used for the selection of products interacting with the polypeptides according to the invention, or their [lacuna] as a cofactor, or as an inhibitor, in particular competitive, or having an agonist or antagonist activity on the activity of the polypeptides according to the invention.","Preferably, the said transformed cells or transgenic animals are used as a model in particular for the selection of products which make it possible to control pathologies linked to abnormal expression of this gene.","The subject of the present invention is also a monoclonal or polyclonal antibody, a fragment of this antibody or a chimeric antibody capable of specifically recognizing a polypeptide in accordance with the present invention.","The specific monoclonal antibodies may be obtained according to the conventional hybridoma culture method well known to persons skilled in the art.","The antibodies according to the invention are for example humanized antibodies, Fab or F(ab′)2 fragments.","They may also be provided in the form of immunoconjugates or antibodies labelled so as to obtain a detectable and/or quantifiable signal.","The antibodies in accordance with the invention, but also the immunoconjugates, are therefore capable of specifically recognizing a polypeptide according to the present invention.","The specific polyclonal antibodies may be obtained from the serum of an animal immunized against the polypeptides of the invention, in particular produced by genetic recombination or by peptide synthesis, according to the customary procedures.","The importance of antibodies specifically recognizing the polypeptides, their variants or their immunogenic fragments, according to the invention,: should be noted in particular.","The subject of the invention is also the use of a nucleotide sequence in accordance with the present invention as a probe or a primer for the detection, identification, assay and/or amplification of nucleic acid sequences.","According to the invention, the nucleotide sequences which may be used as a probe or as a primer in methods for the detection, identification, assay and/or amplification of a nucleic acid sequence have a minimum size of 15 bases, preferably of 20 bases, or even better from 25 to 30 bases.","The probes and primers according to the invention may be directly or indirectly labelled with a radioactive or nonradioactive compound by methods well known to persons skilled in the art, so as to obtain a detectable and/or quantifiable signal.","The nonlabelled nucleic sequences according to the invention may be used directly as a probe or a primer.","The sequences are generally labelled in order to obtain sequences which can be used for numerous applications.","The labelling of the primers or of the probes according to the invention is carried out with radioactive elements or with nonradioactive molecules.","Among the radioactive isotopes used, there may be mentioned 32P, 33P, 35S, 3H or 125I.","The nonradioactive entities are selected from ligands such as biotin, avidin, streptavidin, dioxygenin, haptens, colourings, luminescent agents such as radioluminescent, chemoluminescent, bioluminescent, fluorescent and phosphorescent agents.","The nucleotide sequences according to the invention may thus be used as a primer and/or a probe in methods using in particular the PCR (polymerase chain reaction) technique (11a).","This technique requires the choice of pairs of oligonucleotide primers flanking the fragment which has to be amplified.","Reference may be made, for example, to the technique described in American Patent U.S. Pat.","No.","4,683,202.The amplified fragments may be identified, for example after agarose or polyacrylamide gel electrophoresis, or after a chromatography technique such as gel filtration or ion-exchange chromatography, and then sequenced.","The specificity of the amplification may be checked, using as primers, the nucleotide sequences of the invention and, as templates, plasmids containing these sequences or the derived products of amplification.","The amplified nucleotide fragments may be used as reagents in hybridization reactions in order to demonstrate the presence, in a biological sample, of a target nucleic acid having a sequence complementary to that of the said amplified nucleotide fragments.","The invention also relates to the nucleic acids which can be obtained by amplification using primers according to the invention.","Other techniques for amplifying the target nucleic acid may be advantageously used as an alternative to PCR (PCR-like) using pairs of primers of nucleotide sequences according to the invention.","The expression PCR-like is understood to mean any of the methods using direct or indirect reproductions of the nucleic acid sequences, or in which the labelling systems were amplified, these techniques are of course known.","In general, they involve the amplification of DNA by a polymerase, when the original sample is an RNA, a reverse transcription should be carried out beforehand.","A great number of methods allowing this amplification currently exist, such as for example the SDA (Strand Displacement Amplification) technique (12), the TAS (Transcription-based Amplification System) technique described by (13), the 3SR (Self-Sustained Sequence Replication) technique described by (14), the NASBA (Nucleic Acid Sequence Based Amplification) technique described by (15), the TMA (Transcription Mediated Amplification) technique, the LCR (Ligase Chain Reaction) technique described by (16), the RCR (Repair Chain Reaction) technique described by (17), the CPR (Cycling Probing Reaction) technique described by (18), the Q-beta-replicase amplification technique described by (19).","Some of these techniques have since been improved.","In the case where the target polynucleotide to be detected is an mRNA, an enzyme of the reverse transcriptase type is advantageously used, prior to the use of an amplification reaction with the aid of the primers according to the invention or to the use of a method of detection with the aid of the probes of the invention, in order to obtain a cDNA from the mRNA contained in the biological sample.","The cDNA obtained will then serve as a target for the primers or probes used in the method of amplification or detection according to the invention.","The probe hybridization technique may be carried out in various ways (20).","The method most generally used consists in immobilizing the nucleic acid extracted from the cells of different tissues or cells in culture on a support (such as nitrocellulose, nylon, polystyrene) and in incubating, under well defined conditions, the immobilized target nucleic acid with the probe.","After hybridization, the excess probe is removed and the hybrid molecules formed are detected by the appropriate method (measurement of the radioactivity, the fluorescence or the enzymatic activity linked to the probe).","According to another embodiment of the nucleic probes according to the invention, they may be used as capture probes.","In this case, a probe, called “capture probe”, is immobilized on a support and serves to capture, through specific hybridization, the target nucleic acid obtained from the biological sample to be tested and the target nucleic acid is then detected by means of a second probe, called “detection probe”, labelled with an easily detectable element.","The nucleotide sequences according to the invention may moreover be of interest when they are used as antisense nucleotides, that is to say whose structure brings about, through hybridization with the target sequence, inhibition of the expression of the corresponding product.","They may also be used as sense nucleotides which, through interaction with proteins involved in the regulation of the expression of the corresponding product, will induce either an inhibition, or an activation of this expression.","The subject of the invention is also the use of a nucleotide sequence according to the present invention for the production or the synthesis of a recombinant polypeptide.","The method of producing a polypeptide of the invention in recombinant form, which is itself included in the present invention, is characterized in that the transformed cells, in particular the cells or mammals of the present invention, are cultured under conditions allowing the expression of a recombinant polypeptide encoded by a nucleotide sequence according to the invention, and that the said recombinant polypeptide is recovered.","The recombinant polypeptides, characterized in that they can be obtained by the said method of production, also form part of the invention.","The recombinant polypeptides obtained as indicated above may be provided either in glycosylated or nonglycosylated form and may or may not have the natural tertiary structure.","The sequences of the recombinant polypeptides may also be modified in order to improve their solubility, in particular in aqueous solvents.","Such modifications are known to persons skilled in the art, such as for example the deletion of hydrophobic domains or the replacement of hydrophobic amino acids with hydrophilic amino acids.","These polypeptides may be produced from the nucleic acid sequences defined above, according to techniques for producing recombinant polypeptides known to persons skilled in the art.","In this case, the nucleic acid sequence used is placed under the control of signals allowing its expression in a cellular host.","An effective system for producing a recombinant polypeptide requires having a vector and a host cell according to the invention.","These cells may be obtained by introducing into host cells a nucleotide sequence inserted into a vector as defined above, and then culturing the said cells under conditions allowing the replication and/or the expression of the transfected nucleotide sequence.","The methods used for purifying a recombinant polypeptide are known to persons skilled in the art.","The recombinant polypeptide may be purified from cell lysates and extracts, from the supernatant of the culture medium, by methods used individually or in combination, such as fractionation, chromatography methods, immunoaffinity methods using specific monoclonal or polyclonal antibodies, and the like.","The polypeptides according to the present invention may also be obtained by chemical synthesis using one of the numerous known peptide syntheses, for example the techniques using solid phases (21) or techniques using partial solid phases, by condensation of fragments or by conventional synthesis in solution.","The polypeptides obtained by chemical synthesis and which may contain corresponding unnatural amino acids are also included in the invention.","The subject of the present invention is also a DNA chip, characterized in that it contains at least one nucleotide sequence in accordance with the present invention.","Actually, the nucleotide sequences according to the invention which it is envisaged to use as probe or as primer for the detection, identification, assay and/or amplification of nucleic acid sequences may be covalently or noncovalently immobilized on a support, this support being a DNA chip or a high density filter.","The expression “DNA chip” or “high density filter” is understood to mean a support to which DNA sequences are attached, it being possible for each of them to be located by its geographic localization.","These chips or filters differ mainly in their size, the support material, and possibly the number of sequences attached thereto.","In particular, it is possible to carry out a synthesis in situ by photochemical targeting or by ink jet targeting.","Other techniques consist in carrying out a synthesis ex situ or in attaching the probes to the support of the DNA chip by ink jet, electronic or mechanical addressing.","These different methods are well known to persons skilled in the art.","The subject of the invention is also a protein chip comprising a polypeptide or antibody according to the invention.","Such a protein chip allows the study of interactions between the polypeptides according to the invention and other proteins or chemical compounds and can thus be useful for screening compounds which interact with the polypeptides according to the invention.","It may also be possible to use the protein chips according to the invention for detecting the presence of antibodies directed against the polypeptides according to the invention in the serum of patients to be tested.","It may also be possible to use a protein chip comprising an antibody according to the invention, this time to detect the presence of polypeptides in the serum of patients which are capable of being recognized by the said antibodies.","The subject of the present invention is also the use of a compound chosen from a nucleotide sequence, a polypeptide, a vector, a cell or an antibody according to the invention for the preparation of a medicament.","The pathologies more specifically targeted are viral diseases and diseases characterized by the development of tumour cells or cellular degeneration such as Alzheimer's disease or schizophrenia.","Thus, the abovementioned medicament is intended for the prevention and/or the treatment of these diseases.","In particular, the targeted disease is cancer.","One of the benefits of the present invention is that it has demonstrated the involvement of a large number of nucleotide sequences in the phenomena of tumour suppression, tumour reversion, apoptosis and/or viral resistance.","These sequences are therefore expressed differentially when one of these abovementioned processes is triggered.","Consequently, in the presence of a patient in whom the onset of one of these processes is suspected or for whom it is desired to check the absence of such an onset, it is useful to be able to determine, or even quantify, the expression of one or more sequences in accordance with the invention from a biological sample from the said patient.","Optionally, analysis of the expression of one or more of the said sequences may be accompanied by a comparison with a reference level of expression corresponding to that of a healthy individual.","Consequently, the invention therefore also comprises a method for the diagnosis and/or prognostic evaluation of a viral disease or a disease characterized by tumour development or cell degeneration comprising the analysis of the expression of at least one sequence of the invention from a biological sample from a patient to be tested.","According to a preferred embodiment, the said method comprises the following steps: isolation of the messenger RNA from a biological sample obtained from a patient to be tested, preparation of the complementary cDNA from the said messenger RNA, optionally, amplification of a portion of the complementary DNA corresponding to at least one sequence of the invention, and detection of the optionally amplified complementary DNA.","In particular, analysis of the expression of the sequence may be carried out by means of a DNA chip as described above.","Among the sequences of the present invention, some exhibit the characteristic of being receptors expressed at the surface of cells and in order to understand the mechanism thereof in the context of the abovementioned processes, it is useful to search for compounds capable of interacting with this receptor, that is to say of interacting with a polypeptide in accordance with the invention, it is also necessary to predict the secreted protein.","This also applies to the polypeptides in accordance with the invention corresponding to secreted proteins (at the surface or outside the cells), hormone-like proteins, and the like.","Consequently, the subject of the present invention is also a method for screening compounds capable of binding to a peptide in accordance with the invention and comprising the following steps: bringing a polypeptide or a cell according to the invention into contact with a candidate compound, and detection of the formation of a complex between the said candidate compound and the said polypeptide or the said cell.","A method for screening compounds may also be useful in relation to compounds capable of interacting with a nucleotide sequence according to the invention, or even with the sequences necessary for the expression or the regulation of these sequences.","Indeed, the compounds are capable of interacting with the said sequences, the effect being to reduce, inhibit or on the contrary potentiate the expression of the sequences in question.","Such a method comprises the following steps: bringing a nucleotide sequence or a cell according to the invention into contact with a candidate compound, and detection of the formation of a complex between the said candidate compound and the said nucleotide sequence or the said cell.","For reasons mentioned above, it may therefore be useful to search for and/or assay, in a biological specimen or sample from a patient to be tested, the presence of a nucleotide sequence according to the invention.","Such a method of detection and/or assay comprises the following steps: bringing a labelled nucleotide sequence according to the invention into contact with the biological sample to be tested, under the conditions necessary for the formation of a hybrid, and detection and/or assay of the hybrid which may be formed between the said nucleotide sequence and the nucleic acid present in the said biological sample.","This method may additionally comprise a step for amplifying the nucleic acid of the said biological sample with the aid of primers chosen from the nucleotide sequences according to the invention.","In particular, this method may be carried out by means of the DNA chip described above.","Persons skilled in the art know how to carry out such a method and may in particular use a reagent kit comprising: a) a nucleotide sequence according to the invention, used as a probe, b) the reagents necessary for carrying out a hybridization reaction between the said probe and the nucleic acid of the biological sample, c) the reagents necessary for the detection and/or assay of the hybrid formed between the said probe and the nucleic acid of the biological sample.","Such a kit may also contain positive or negative controls in order to ensure the quality of the results obtained.","Likewise, the detection and/or the assay of a polypeptide according to the invention can also be envisaged in the context of the present invention and this method therefore comprises the following steps: bringing the biological sample into contact with a labelled antibody according to the invention, and detection and/or assay of the complex formed by the said antibody of polypeptide present in the said sample.","Advantageously, this method may be carried out by means of the protein chip as described above.","Here again, persons skilled in the art know how to carry out such a method and may in particular use a reagent kit comprising: a) a monoclonal or polyclonal antibody according to the invention; b) optionally reagents for preparing a medium favourable for the antigen/antibody reaction; c) the reagents allowing the detection of the antigen/antibody complex.","Finally, the subject of the present invention is also a computer readable support or a computer support on which are recorded at least one nucleotide sequence according to the Claim 1 and/or at least one polypeptide sequence as defined in Claim 3 or 4.In particular, this support is chosen from the group comprising: a) a floppy disk, b) a hard disk, c) a random access memory (RAM), d) a read only memory (ROM), e) a CD ROM.","The invention is not limited to the present description but encompasses on the contrary all the variants and will be understood more clearly in the light of the experimental data below.","FIGURES FIG.","1 represents a curve for tumour growth for the U937 and US4 groups in SCID mice.","FIG.","2 represents a curve for the body weight of mice carrying a U937 or US4 tumour.","FIG.","3 illustrates: a/ the analysis of the expression of the TPT1/TCTP gene in the U937/US4.2 lines and in the MCF7/MCF7+SIAH1 lines by Northern blotting.","The quantities of total transcripts are checked by monitoring GAPDH; b/ Western blot analysis of the expression of the TCTP protein in the U937/US4.2 model, in the MCF7/MCF7+siah model and analysis of the expression of the murine homologue of TCTP, TCTP_MOUSE, in the M1/LTR6 system after 20 h of induction at 32° C.; c/ the analysis of the expression of TCTP in U937 cells transfected with the antisense TPT1/TCTP.","The clones I and III are compared with the control, U937 cells with the vector alone.","On the right hand panel, detection of the cleavage of PARP, a marker for apoptosis, in the clones I and III, U937 cells transfected with the antisense TPT1/TCTP.","Jurkat cells treated with an anti-Fas (anti-CD95) antibody, an inducer of apoptosis, serve as a control; d/ characterization of the apoptotic cells by double labelling FITC-Annexin V and propidium iodide (PI).","The U937 cells+control vector and the U937 cells+antisense TPT1/TCTP are simultaneously labelled with annexin V coupled to FITC (which binds to the phosphatidylserine of the apoptotic cells in the early phase) and PI (which only labels necrotic cells whose plasma membrane is damaged).","Cytofluorometric analysis makes it possible to determine the percentage of apoptotic cells (Annexin+, PI−), necrotic cells (Annexin+, PI+ or Annexin−, PI+) and intact cells (Annexin−, PI−); e/ evaluation of apoptosis in the cells transfected with the antisense TPT1/TCTP, by in situ labelling of the free 3′ ends of the nuclear DNA: TUNEL (“Terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labelling”) method.","This labelling makes it possible to demonstrate in situ the fragmentation of nuclear DNA resulting from the activation of endonucleases during apoptosis.","The apoptotic cells appear in green.","f/ the study of the in vivo tumorigenicity of the U937 cells transfected with: the vector alone (top curve—91 tumours/100), an antisense presenilin 1 (second curve from the top—12 tumours/20), SIAH1 (middle curve—12 tumours/20) and an antisense TPT1/TCTP, clone I and III (the bottom 2 curves—8 tumours/20 and 4 tumours/20).","10 million cells injected into each flank and into each shoulder.","FIG.","4 represents cells cultured on an extra-cellular tumour matrix (Matrigel).","The 184B5 line, mammary epithelial cells transformed with benzo(a)pyrene, is used as a control for the formation of acini.","The tumour cells T47D and MCF7 form irregular colonies.","The MCF7 cells transfected with SIAH1 partially recover an organized structure.","The MCF7 and T47D cells transfected with the antisense TPT1/TCTP result in a cellular reorganization comparable to the normal formation of the acini.","The cells which appear green (light spots in the figure) are labelled with an anti-E_Cadherin antibody coupled to FITC and the nuclei labelled with PI appear red (darker spots).","Western blot analysis of the expression of TCTP in the MCF7 and T47D cells transfected with the antisense TPT1/TCTP.","1—DATA RELATING TO THE U937 and US4 CELLS As seen above, the present invention uses the parental cells U937 and the “derived” cells US4.Indeed, the US4 cells and the US3 cells (which are not involved in the context of the present invention) share certain characteristics.","Their mode of production and their properties are presented below.","Selection and Characterization of US3 and US4 Cells The U937 cells were subjected to two series of limiting dilution until a single clonal population is obtained.","These cells were infected with the H-1 parvovirus.","The cytopathic effect of the virus creates massive cell death which spares two resistant clones which are US3 and US4 after three months of continuous culture.","The survival of the cells is defined as the relative number of viable cells in the culture infected with the H-1 virus compared with the untreated culture, as measured four days after reinfection.","To measure the tumorigenicity, 107 U937, US3 and US4 cells were subcutaneously injected into scid/scid mice (4 or 5 weeks old).","The tumorigenicity is expressed by the number of tumours developed by the mice within two months following the injection.","The approach was as follows: using clonal populations of malignant cells, subclones were derived with a suppressed tumorigenic phenotype.","This selection made by means of the H-1 parvovirus is carried out by the elimination of the tumour cells which are preferentially killed while sparing the normal cells.","The selection of the cells resistant to the cytopathic effect of the H-1 parvovirus outside a sensitive tumour can give rise to cells which have a reduced malignant phenotype.","On this basis, a clonal population of U937 cells was isolated, these cells are sensitive to the cytopathic effect of H-1 parvovirus and the US3 and US4 clones are, for their part, resistant to the virus.","The US3 and US4 clones have a strong suppressed tumorigenic phenotype while the parental U937 cells develop tumours in 80% of cases among the scid/scid mice which were infected with the parvovirus, the US3 cells form only a single tumour and the US4 cells develop one tumour per 20 inoculations with 107 cells.","The results are assembled in the table below.","Resistance to the H-1 Parvovirus and Tumorigenicity of the U397, US3 and US4 Cells Survival of the Tumorigenicity cells to in infection with the scid/scid Cell lines the H-1 virus mice U937 0.4 16/20 US3 96 0/10 US4 89 1/20 2—MATERIALS AND METHODS The aim is to compare the growth of subcutaneous tumours in SCID mice induced by subcutaneous injection of transfected U937 and US4 human leukaemic cell lines.","A. Leukaemic Cell Lines and Culture Conditions All the injected cells of the U937 (ATCC) and US4 cell lines were provided in the form of cell suspensions in bottles supplemented with RPMI-1640 culture medium supplemented with 2 mM L-glutamine, 10% foetal calf serum and gentamycin.","The U937 cell line is a CD4+ human monocytic cell line derived from a patient having a diffuse histiocytic lymphoma (1).","The cells were counted in a haemocytometer and their viability was tested with exclusions of trypan blue dyes at 0.25%.","The viability was 95.5% and 90.5% for the U937 cells and the US4 cells, respectively.","The U937 and US4 cells were centrifuged and then resuspended in an RPMI medium before being injected into SCID mice.","B.","The animals 10 female SCID mice in good health (CB17/IcrHsd), 31 weeks old and weighing between 20 and 25 g, were supplied by Harlan France (Gannat, France).","The animals were observed for 7 days in a specific room belonging to the Applicant which is the unit for treating specific-pathogen-free animals before their treatment.","The unit for treating animals (INRA, Dijon, France) is authorized by the French Ministers of Agriculture and Research (authorization No.","A21100).","The animal experiments are carried out according to the European Ethical Directives for the well-being of animals in the context of experimental neoplasia (3).","B.1.Environment The animals were kept in rooms under controlled conditions of temperature (24±1° C.), humidity (55±1%), light period (12 h of light/12 h of darkness) and air renewal.","The animals were kept under SPF conditions and the temperature and the humidity of the room were continuously monitored.","The aeration system was programmed to give rise to 14 air renewals per hour without recirculation.","Fresh air from outside passes inside a series of filters before being uniformly diffused into each room.","A high pressure (2 mm) was maintained in the experimentation rooms in order to prevent contamination or diffusion of pathogens within a mouse colony.","All the staff working under SPF conditions followed specific directives as regards hygiene and clothing when they were in the breeding area.","B.2.Breeding The animals were housed in polycarbonate cages (UAR, Epinay sur Orge, France) which were equipped so as to provide them with food and water.","The standard size of the cages used is 637 cm2 for 10 mice according to the standard internal operating procedures.","The litter for animals and made up of sterile wood shavings (UAR) and is replaced twice per week.","B.3.Food and Drink The animal food was bought from Extralabo (Provins, France).","The food was provided ad libitum and was placed on the metal lid at the top of the cage.","Water was also provided ad libitum from bottles of water equipped with rubber taps.","The water bottles were washed, sterilized and replaced once per week.","The water supply was sterilized by filtration with a 0.2 μm absolute filter.","B.4.Identification of the Animal and the Cage After random distribution, the animals were identified with 2 different numbers engraved on the two ears.","Each cage was marked with a specific code.","C—Experimental Data and Treatments C.1.Induction of Tumours in SCID Mice Before cell injection, the SCID mice were randomly divided into two groups, in an amount of 5 mice per group.","107 US4 or U937 tumour cells in 0.2 ml of RPMI medium were subcutaneously inoculated at time 0 for each site of injection into SCID mice.","Each animal received 4 injections of tumour cells located in different areas, one in each flank and one in each shoulder.","C.2.Collection of the Tumours When the tumours reached a volume of 1 500 mm3, the mice were killed and the tumours were collected, weighed and frozen in liquid nitrogen, stored at −80° C. and then specifically labelled.","C.3.Monitoring of the Mice Isoflurane forene (Minerve, Bondouble, France) was used to anaesthetize the animals before injection of cells for sacrificing.","After injection of tumour cells, the mice were observed for 5 hours.","The viability, behaviour, body weight of the mice and the growth of the subcutaneous tumour were recorded twice per week.","During the period of experimentation, the animals were killed under anaesthetic with isoflurane by cervical dislocation if one of the following signs appears: sign of suffering (cachexia, weakness, difficulty in moving or eating), tumour growth up to 10% of the body weight, tumour ulceration and persistently becoming nude, position of the tumour interfering with movement and/or feeding, loss of weight of 20% for three consecutive days.","An autopsy was performed on each animal to detect the possible presence of metastases or morphological abnormalities.","D—Presentation of the Data D.1.Monitoring Parameters The calculation for the median and mean survival time was expressed as follows: Mean survival time=S1/(S2−NT) With: S1=sum of the daily survivors from day 0 up to the end of the experiment (without the survivors “not taken into account”*) S2=number of animals at the beginning NT=number of animals “not taken into consideration”* *“not taken into consideration”: they are animals with tumours which are smaller than the predetermined limit considered as resulting from a defect in implantation of the tumour.","D.2.System for Inhibition of the Tumour The tumour size was measured twice per week with a compass (and the tumour volume in mm3) will be estimated according to the formula: (length×width2/2 (4).","The experiments were stopped when the tumour sizes in the mice reached 1 500 mm3.After sacrificing, the tumours were excised and weighed.","The curve for tumour growth for the US4 and U937 groups was noted using the mean of the tumour volumes.","The tumour doubling time for the US4 and U937 groups was defined as the period required to reach a mean tumour volume of 200% during the growth period.","The specific growth period over one or two doubling times (DT) from injections of cells is defined as follows: specific growth period=(DT US4−DT U937)/DT U937.The growth period is calculated as the difference between the median growth time for the US4 group and the U937 group in order to reach the same tumour size.","D.3.Statistical Tests All the statistical analyses were carried out with the StatView® software (Abacus Concept, Berkeley, USA).","The statistical analyses of the changes in mean body weight, the tumour doubling time and the time to reach “V” were performed using the Bonferroni/Dunn test.","A value p<0.05 was considered to be significant.","All the groups were compared with each other.","E—Results The curves for mean tumour volumes and body weights are shown in FIGS.","1 and 2 respectively.","No significant loss of body weight in SCID mice from the two groups was observed between day 8 and day 19.A significant difference is observed as regards the time to reach “V” between the 2 groups of SCID mice whereas no significant difference was observed for the change in mean body weight (day 19−day 8) and for the doubling time.","The autopsies performed did not show the presence of metastases or of developments of suspicious nodosity.","The tumours were collected on animals sacrificed after anaesthetic and cervical dislocation.","The tumours were immediately placed in tubes, frozen in liquid nitrogen and stored at −80° C. The excised tumours had an ovoid shape, a moderate consistency and a pinkish colour.","The interactions of the tumour with its environment (skin and muscle tissue) were limited and superficial.","F—Conclusions The US4 cell line showed an uptake level significantly lower compared with the U937 cell line in the SCID mice.","The retardation of growth between the US4 and U937 tumours was 23.5 days and the doubling time was equivalent.","3—INVOLVEMENT OF TPT1/TCPT IN TUMOUR REVERSION In order to study the involvement of TPT1/TCPT (Translationally controlled Tumour Protein encoding the Histamine Release Factor) in tumour reversion, the expression of the gene (FIG.","3a) and the expression of the protein (FIG.","3b) in various cellular models of tumour reversion: U937/US4.2, MCF7/MCF7+SIAH1 and M1/LTR6, were analysed.","The level of expression of TPT1/TCTP is highly reduced in the revertant cells compared with the tumour cells.","To deal with the physiological aspect of this reduction, the U937 tumour cells were transfected with the antisense TPT1/TCTP.","After having checked the drop in the expression of TPT1/TCTP (FIG.","3c, left panel), the inventors detected its consequences in apoptosis by three different methods: detection of the cleavage of PARP, labelling with Annexin V and TUNEL method (FIG.","3c, right panel; FIG.","3d; FIG.","3e, respectively).","The reduction in the expression of TPT1/TCTP in tumour cells in culture increases apoptosis.","The inventors then expressed in vivo the physiological consequences of this loss of TPT1/TCTP functions.","We injected U937 cells transfected with antisense TPT1/TCTP into mice and observed a drastic reduction in the number of tumours formed when the expression of TPT1/TCTP is reduced (FIG.","3f).","In parallel with these results, the reduction of TPT1/TCTP, obtained by transfection of double-stranded RNA specific for TPT1/TCTP (siRNA), into MCF7 and T47D cells, cultured on an extracellular matrix, leads to a cellular reorganization of acini which is comparable to a normal structure (FIG.","4).","REFERENCES (1) Sundström C. and Nilsson K., Int.","J.","Cancer, 17, 565, 1976.","(2) Principe d'éthique de l'expérimentation animale [Principle of ethics of animal experimentation], Directive No.","86/609 EEC of 24 Nov. 1986, Decree No.","87/848 of 19 Oct. 1987, Application Order of 19 Apr.","1988.","(3) United Kingdom co-ordinating committee on cancer research guidelines for welfare of animals in experimental neoplasia, Br.","J.","Cancer, 77: 1-10, 1998.","(4) Bissery M. C. et al., Bull.","Cancer, 78: 587, 1991.","(5) Perricaudet et al.","(1991).","La Recherche 23: 471.","(5a) Epstein (1992), Médecine/Sciences, 8, 902.","(6) Temin (1986), Retrovirus vectors for gene transfer.","In Kucherlapati R., ed.","Gene.","(7) Carter (1993), Curr.","Op.","Biotechnology 3, 533.","(8) Olins and Lee (1993), Curr.","Op.","Biotechnology 4: 520.","(9) Buckholz (1993), Curr.","Op.","Biotechnology 4, 538.","(10) Edwards and Aruffo (1993), Curr.","Op.","Biotechnology 4, 558.","(11) Luckow (1993), Curr.","Op.","Biotechnology 4, 564.","(11a) Rolfs, A. et al.","(1991), Berlin: Springer-Verlag.","(12) Walker (1992), Nucleic Acids Res.","20: 1691.","(13) Kwoh et al.","(1989), Proc.","Natl.","Acad.","Sci.","USA, 86, 1173.","(14) Guatelli et al.","(1990), Proc.","Natl.","Acad.","Sci.","USA, 87, 1874.","(15) Kievitis et al.","(1991), J. Virol.","Methods, 35, 273.","(16) Landegren et al.","(1988) Science 241, 1077.","(17) Segev (1992), Kessler C. Springer Verlag, Berlin, New York, 197-205.","(18) Duck et al.","(1990), Biotechniques, 9, 142.","(19) Miele et al.","(1983), J. Mol.","Biol., 171, 281.","(20) Matthews et al.","(1988), Anal.","Biochem., 169, 1-25.","(21) Stewart and Yound (1984), Solid phase peptides synthesis, Pierce Chem.","Company, Rockford, 111, 2nd ed.","(1984)."]],"string":"[\n [\n \"Sequences involved in phenomena of tumour suppression, tumour reversion, apoptosis and/or virus resistance and their use as medicines\",\n \"This invention is directed to sequences that are involved in the molecular pathways of tumor suppression, tumor revision, apoptosis and/or virus resistance.\",\n \"The use of the compounds having these sequences, or encoded by them, in treating cancer, neurodegenerative diseases or viral diseases, and screening methods for compounds having such therapeutic properties are also encompassed.\"\n ],\n [\n \"1.An isolated nucleotide sequence comprising a nucleotide sequence chosen from the group comprising: a) SEQ.\",\n \"ID NO.\",\n \"72, b) a nucleotide sequence of at least 15 consecutive nucleotides of a sequence as defined in a), c) a nucleotide sequence having a percentage identity of al least 80%, after optimal alignment, with a sequence defined in a) or b), d) a nucleotide sequence which hybridizes under high stringency conditions with a sequence defined in a) or b), and e) a complementary nucleotide sequence or the RNA sequence corresponding to a sequence as defined in a), b), c) or d).\",\n \"2-30.\",\n \"(canceled)\"\n ],\n [\n \"The present invention relates to the identification of genes involved in the molecular pathways of tumour suppression, tumour reversion, apoptosis and/or resistance to viruses.\",\n \"The present invention was made possible by the isolation of cDNA corresponding to messenger RNAs expressed or repressed during tumour suppression, tumour reversion and/or during the process of apoptosis.\",\n \"In order to isolate the genes activated or inhibited during tumour reversion, overall screening for the expression of the genes in a malignant cell line (U937) and a derived cell line (US4) was carried out with suppression of the malignant phenotype.\",\n \"Comparison of the expressed genes (messenger RNAs expressed in both types of cell) made it possible to identify genes expressed differentially, that is to say expressed in one of the cells while they are not in the other (the genes may be activated or inhibited).\",\n \"It is easily deduced therefrom that these genes are at least involved in the cancerization process, in one case by their absence, and in the other case by their presence.\",\n \"For this differential study, the method used is the method described in 2000 by Brenner et al.\",\n \"(Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA, 97 (4), 1665-70).\",\n \"In order to produce a model, the inventors made the following hypotheses: if it were possible to select, from a tumour which is sensitive to the cytopathic effect of the parvovirus H-1, cells which are resistant, then this resistance could be due to a change in their malignant phenotype.\",\n \"It has been possible to demonstrate this for US4 cells selected from the U937 cancer cells.\",\n \"Unlike the U937 parental line, the US4 clones (but also US3 clones which will not be dealt with in the present invention) are resistant to the cytopathic effect of the parvovirus H-1.At the molecular level, it has been possible to observe that this suppression of the malignant phenotype was accompanied by activation of the expression of the p21waf1 gene, this being independently of the expression of the p53 gene.\",\n \"The approach to the problem according to the present invention made it possible to isolate sequences directly linked to several precise functions.\",\n \"Accordingly, unlike the random sequencing of ESTs, the sequences are sequences whose function is known since they are involved in the process of suppression of the malignant phenotype, of tumour reversion, of apoptosis and/or in resistance to viruses.\",\n \"Tumour reversion is distinguishable from tumour suppression by the fact that it covers a broader domain than that of tumour suppressor genes.\",\n \"In other words, tumour reversion is achieved by the use of metabolic and/or molecular pathways which are not limited to the metabolic and molecular pathways in which the tumour suppressor genes are involved.\",\n \"Thus, the present invention relates in particular to novel sequences and to the use of these sequences in the diagnostic field and for carrying out methods for screening test compounds.\",\n \"The invention also relates to methods for the detection and/or assay of the sequences of the invention or of their product(s) of expression in a biological sample.\",\n \"The present invention first of all relates to an isolated nucleotide sequence comprising a nucleotide sequence chosen from the group comprising: a) SEQ ID No.\",\n \"1 to SEQ ID No.\",\n \"1020, preferably SEQ ID No.\",\n \"72, b) a nucleotide sequence of at least 15 consecutive nucleotides of a sequence as defined in a), c) a nucleotide sequence exhibiting a percentage identity of at least 80%, after optimum alignment, with a sequence defined in a) or b), d) a nucleotide sequence which hybridizes under high stringency conditions with a sequence defined in a) or b), and e) a complementary nucleotide sequence where the RNA sequence corresponding to a sequence as defined in a), b), c) or d).\",\n \"The nucleotide sequence according to the invention defined in c) exhibits a percentage identity of at least 80% after optimum alignment with a sequence as defined in a) or b) above, preferably of at least 90%, more preferably of at least 98%.\",\n \"In the context of the present invention, SEQ ID No.\",\n \"72 is the preferred nucleotide sequence and corresponds to the TPT1 gene also called TCTP.\",\n \"This gene is involved in particular in the phenomenon of tumour reversion and has been the subject of more thorough experiments on the part of the inventors as indicated below.\",\n \"The expressions nucleotide sequence, nucleic acid, nucleic or nucleic acid sequence, polynucleotide, oligonucleotide and polynucleotide sequence, terms which will be used without distinction in the present description, are understood to mean a precise succession of nucleotides, modified or unmodified, which makes it possible to define a fragment or a region of a nucleic acid, containing, or otherwise, unnatural nucleotides, and being capable of corresponding at the same time to a double-stranded DNA, a single-stranded DNA and transcriptional products of the said DNAs.\",\n \"Thus, the nucleic sequences according to the invention also cover PNAs (Peptide Nucleic Acids), or the like.\",\n \"The fragments of the nucleotide sequences of the invention comprise at least 15 consecutive nucleotides.\",\n \"Preferably, they comprise at least 20 consecutive nucleotides and still more preferably they comprise at least 30 consecutive nucleotides.\",\n \"It should be understood that the present invention does not relate to the nucleotide sequences in their natural chromosomal environment, that is to say in the natural state.\",\n \"It involves sequences which have been isolated and/or purified, that is to say that they were collected directly or indirectly, for example by copying, their environment having been at least partially modified.\",\n \"The nucleic acids obtained by chemical synthesis are understood to also be thereby designated.\",\n \"The expression “percentage identity” between two nucleic acid or amino acid sequences for the purposes of the present invention is understood to mean a percentage of identical nucleotides or amino acid residues between the two sequences to be compared, which is obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being randomly distributed and over their entire length.\",\n \"The expression “best alignment” or “optimum alignment” is understood to mean the alignment for which the percentage identity determined as below is the highest.\",\n \"Sequence comparisons between two nucleic acid or amino acid sequences are traditionally carried out by comparing these sequences after having optimally aligned them, the said comparison being carried out per segment or per “comparison window” in order to identify and compare the local regions with sequence similarity.\",\n \"Optimum alignment of the sequences for comparison may be carried out, apart from manually, by means of the Smith and Waterman local homology algorithm (1981), by means of the Neddleman and Wunsch local homology algorithm (1970), by means of the Pearson and Lipman search for similarity methods (1988), by means of computer software packages using these algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.).\",\n \"In order to obtain optimum alignment, the BLAST program is preferably used, with the BLOSUM 62 matrix.\",\n \"The PAM or PAM 250 matrices can also be used.\",\n \"The percentage identity between two nucleic acid or amino acid sequences is determined by comparing these two sequences aligned optimally, it being possible for the nucleic acid or amino acid sequence to be compared to comprise additions or deletions in relation to the reference sequence for an optimum alignment between these two sequences.\",\n \"The percentage identity is calculated by determining the number of identical positions for which the nucleotide or amino acid residue is identical between the two sequences, by dividing this number of identical positions by the total number of positions compared and by multiplying the result obtained by 100 in order to obtain the percentage identity between these two sequences.\",\n \"The expression nucleic sequences exhibiting a percentage identity of at least 80%, preferably of at least 90%, more preferably of at least 98%, after optimum alignment with a reference sequence, is understood to mean the nucleic sequences exhibiting, in relation to the reference nucleic sequence, certain modifications such as in particular a deletion, truncation, alignment, chimeric fusion and/or substitution, in particular point substitution, and whose nucleic sequence exhibits at least 80%, preferably at least 90%, more preferably at least 98%, identity after optimum alignment with the reference nucleic sequence.\",\n \"Preferably, the specific or high stringency hybridization conditions will be such that they ensure at least 80%, preferably at least 90%, more preferably at least 98% identity after optimum alignment between one of the two sequences and the sequence complementary to the other.\",\n \"Hybridization under high stringency conditions means that the temperature and ionic strength conditions are chosen so that they allow hybridization to be maintained between two complementary nucleic acid fragments.\",\n \"By way of illustration, high stringency conditions of the hybridization step for the purposes of defining the nucleotide sequences described above, will be advantageously the following.\",\n \"The DNA-DNA or DNA-RNA hybridization is carried out in two steps: (1) prehybridization at 42° C. for 3 hours in phosphate buffer (20 mM, pH 7.5) containing 5×SSC (1×SSC corresponds to a solution containing 0.15 M NaCl+0.015 M sodium citrate), 50% formamide, 7% sodium dodecyl sulphate (SDS), 10×Denhardt's, 5% dextran sulphate and 1% salmon sperm DNA; (2) hybridization proper for 20 hours at a temperature dependent on the size of the probe (i.e.\",\n \": 42° C., for a probe of size>100 nucleotides) followed by 2 washes of 20 minutes at 20° C. in 2×SSC+2% SDS, 1 wash of 20 minutes at 20° C. in 0.1×SSC+0.1% SDS.\",\n \"The final wash is performed in 0.1×SSC+0.1% SDS for 30 minutes at 60° C. for a probe of size>100 nucleotides.\",\n \"The high stringency hybridization conditions described above for a polynucleotide of defined size may be adjusted by persons skilled in the art for larger- or smaller-size oligonucleotides, according to the teaching of Sambrook et al., 1989.Among the nucleotide sequences exhibiting a percentage identity of at least 80%, preferably of at least 90%, more preferably of at least 98%, after optimum alignment with the sequences according to the invention, preference is also given to the variant nucleic sequences of the sequences of the invention, or of fragments thereof, that is to say all the nucleic sequences corresponding to allelic variants, that is to say individual variations of the sequences of the invention.\",\n \"The expression “variant nucleotide sequence” is understood to mean any RNA or cDNA resulting from a mutation and/or variation of a splice site of the genomic DNA corresponding to the nucleotide sequences of the invention.\",\n \"The subject of the present invention is also a polypeptide encoded by a nucleotide sequence in accordance with the invention.\",\n \"The expression “polypeptide” is understood to mean, for the purposes of the present invention, either proteins or peptides.\",\n \"According to a particular embodiment, the polypeptides in accordance with the invention comprise a polypeptide chosen from: a) a polypeptide encoded by a nucleotide sequence in accordance with the invention, b) a polypeptide exhibiting at least 80% identity with a polypeptide as defined in a), c) a fragment of at least 5 amino acids of a polypeptide as defined in a) orb), d) a biologically active fragment of a polypeptide as defined in a), b) or c), and e) a modified polypeptide of a polypeptide as defined in a), b), c) or d).\",\n \"The percentage identity is understood to be evaluated after optimum alignment of the relevant sequences.\",\n \"The expression polypeptide whose amino acid sequence exhibits a percentage identity of at least 80%, preferably of at least 90%, more preferably of at least 98% after optimum alignment with a reference sequence, is understood to mean the polypeptides exhibiting certain modifications in relation to the reference polypeptide, such as in particular one or more deletions or truncations, an extension, a chimeric fusion and/or one or more substitutions.\",\n \"Among the polypeptides whose amino acid sequence exhibits a percentage identity of at least 80%, preferably of at least 90% and more preferably of at least 98%, after optimum alignment with a reference sequence such as a polypeptide in accordance with the present invention or with one of its fragments, preference is given to the variant polypeptides encoded by the variant nucleotide sequences as previously defined, in particular the polypeptides whose amino acid sequence exhibits at least one mutation corresponding in particular to a truncation, deletion, substitution and/or addition of at least one residue in relation to the polypeptide sequences of the invention or with one of their fragments.\",\n \"The present invention also relates to a cell cloning and/or expression vector, characterized in that it comprises a nucleotide sequence according to the invention or that it encodes a polypeptide according to the invention.\",\n \"Such a vector may also contain the elements necessary for the expression and possibly for the secretion of the polypeptide in a host cell.\",\n \"Such a host cell is also a subject of the present invention.\",\n \"The vectors comprising promoter and/or regulatory sequences also form part of the present invention.\",\n \"The said vectors preferably contain a promoter, signals for initiation and termination of translation, and appropriate regions for regulation of transcription.\",\n \"They should be able to be stably maintained in the cell and they may also possess particular signals so as to allow the secretion of the translated protein.\",\n \"These different control signals are chosen according to the cellular host used.\",\n \"To this effect, the nucleic acid sequences according to the invention may be inserted into vectors which replicate autonomously in the chosen host or into integrating vectors of the chosen host.\",\n \"Among the autonomously replicating systems, use is preferably made, according to the host cell, of the plasmid or viral type systems, it being possible for the viral vectors in particular to be adenoviruses (5), retroviruses, lentiviruses, poxviruses or herpesviruses (5a).\",\n \"Persons skilled in the art know the technologies which can be used for each of these systems.\",\n \"When the integration of the sequence into the chromosomes of the host cell is desired, it is possible to use, for example, systems of the plasmid or viral type; such viruses are, for example, retroviruses (6) or AAVs (7).\",\n \"Advantageously, the vectors in accordance with the invention contain a sequence allowing tissue-specific targeting and/or expression.\",\n \"Among the nonviral vectors, preference is given to naked polynucleotides such as naked DNA or naked RNA according to the technique developed by the company VICAL, bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC) for expression in yeast, mouse artificial chromosomes (MAC) for expression in murine cells and preferably human artificial chromosomes (HAC) for expression in human cells.\",\n \"Such vectors are prepared according to methods commonly used by persons skilled in the art, and the clones resulting therefrom may be introduced into an appropriate host by standard methods such as for example lipofection, electroporation, heat shock, transformation after chemical permeabilization of the membrane, cell fusion.\",\n \"The invention additionally comprises the host cells, in particular the eukaryotic and prokaryotic cells, transformed with the vectors according to the invention and the transgenic animals, preferably the mammals, except humans, comprising one of the said transformed cells according to the invention.\",\n \"These animals may be used as models, for studying the aetiology of inflammatory and/or immune diseases, and in particular inflammatory diseases of the digestive tube, or for studying cancers.\",\n \"Among the cells which can be used for the purposes of the present invention, there may be mentioned bacterial cells (8), but also yeast cells (9), as well as animal cells, in particular mammalian cell cultures (10), and in particular Chinese hamster ovary (CHO) cells.\",\n \"There may also be mentioned insect cells in which it is possible to use methods using for example baculoviruses (11).\",\n \"A preferred cellular host for the expression of the proteins of the invention consists of COS cells.\",\n \"Among the mammals according to the invention, preference is given to animals such as rodents, in particular mice, rats or rabbits, expressing a polypeptide according to the invention.\",\n \"These transgenic animals are obtained, for example, by homologous recombination on embryonic stem cells, transfer of these stem cells into embryos, selection of the chimeras affected at the level of the reproductive lines, and growth of the said chimeras.\",\n \"The transgenic animals according to the invention can thus overexpress the gene encoding the protein according to the invention, or their homologous gene, or may express the said gene into which a mutation is introduced.\",\n \"These transgenic animals, in particular mice, are obtained for example by transfection of a copy of this gene under the control of a strong promoter of a ubiquitous nature, or which is selective for a type of tissue, or after viral transcription.\",\n \"The cells and mammals according to the invention can be used in a method for producing a polypeptide according to the invention, as described below, and can likewise serve as a model for analysis.\",\n \"The transformed cells or mammals as described above may also be used as models in order to study the interactions between the polypeptides according to the invention and the chemical or protein compounds involved directly or indirectly in the activities of the polypeptides according to the invention, this being in order to study the different mechanisms and interactions involved.\",\n \"They can in particular be used for the selection of products interacting with the polypeptides according to the invention, or their [lacuna] as a cofactor, or as an inhibitor, in particular competitive, or having an agonist or antagonist activity on the activity of the polypeptides according to the invention.\",\n \"Preferably, the said transformed cells or transgenic animals are used as a model in particular for the selection of products which make it possible to control pathologies linked to abnormal expression of this gene.\",\n \"The subject of the present invention is also a monoclonal or polyclonal antibody, a fragment of this antibody or a chimeric antibody capable of specifically recognizing a polypeptide in accordance with the present invention.\",\n \"The specific monoclonal antibodies may be obtained according to the conventional hybridoma culture method well known to persons skilled in the art.\",\n \"The antibodies according to the invention are for example humanized antibodies, Fab or F(ab′)2 fragments.\",\n \"They may also be provided in the form of immunoconjugates or antibodies labelled so as to obtain a detectable and/or quantifiable signal.\",\n \"The antibodies in accordance with the invention, but also the immunoconjugates, are therefore capable of specifically recognizing a polypeptide according to the present invention.\",\n \"The specific polyclonal antibodies may be obtained from the serum of an animal immunized against the polypeptides of the invention, in particular produced by genetic recombination or by peptide synthesis, according to the customary procedures.\",\n \"The importance of antibodies specifically recognizing the polypeptides, their variants or their immunogenic fragments, according to the invention,: should be noted in particular.\",\n \"The subject of the invention is also the use of a nucleotide sequence in accordance with the present invention as a probe or a primer for the detection, identification, assay and/or amplification of nucleic acid sequences.\",\n \"According to the invention, the nucleotide sequences which may be used as a probe or as a primer in methods for the detection, identification, assay and/or amplification of a nucleic acid sequence have a minimum size of 15 bases, preferably of 20 bases, or even better from 25 to 30 bases.\",\n \"The probes and primers according to the invention may be directly or indirectly labelled with a radioactive or nonradioactive compound by methods well known to persons skilled in the art, so as to obtain a detectable and/or quantifiable signal.\",\n \"The nonlabelled nucleic sequences according to the invention may be used directly as a probe or a primer.\",\n \"The sequences are generally labelled in order to obtain sequences which can be used for numerous applications.\",\n \"The labelling of the primers or of the probes according to the invention is carried out with radioactive elements or with nonradioactive molecules.\",\n \"Among the radioactive isotopes used, there may be mentioned 32P, 33P, 35S, 3H or 125I.\",\n \"The nonradioactive entities are selected from ligands such as biotin, avidin, streptavidin, dioxygenin, haptens, colourings, luminescent agents such as radioluminescent, chemoluminescent, bioluminescent, fluorescent and phosphorescent agents.\",\n \"The nucleotide sequences according to the invention may thus be used as a primer and/or a probe in methods using in particular the PCR (polymerase chain reaction) technique (11a).\",\n \"This technique requires the choice of pairs of oligonucleotide primers flanking the fragment which has to be amplified.\",\n \"Reference may be made, for example, to the technique described in American Patent U.S. Pat.\",\n \"No.\",\n \"4,683,202.The amplified fragments may be identified, for example after agarose or polyacrylamide gel electrophoresis, or after a chromatography technique such as gel filtration or ion-exchange chromatography, and then sequenced.\",\n \"The specificity of the amplification may be checked, using as primers, the nucleotide sequences of the invention and, as templates, plasmids containing these sequences or the derived products of amplification.\",\n \"The amplified nucleotide fragments may be used as reagents in hybridization reactions in order to demonstrate the presence, in a biological sample, of a target nucleic acid having a sequence complementary to that of the said amplified nucleotide fragments.\",\n \"The invention also relates to the nucleic acids which can be obtained by amplification using primers according to the invention.\",\n \"Other techniques for amplifying the target nucleic acid may be advantageously used as an alternative to PCR (PCR-like) using pairs of primers of nucleotide sequences according to the invention.\",\n \"The expression PCR-like is understood to mean any of the methods using direct or indirect reproductions of the nucleic acid sequences, or in which the labelling systems were amplified, these techniques are of course known.\",\n \"In general, they involve the amplification of DNA by a polymerase, when the original sample is an RNA, a reverse transcription should be carried out beforehand.\",\n \"A great number of methods allowing this amplification currently exist, such as for example the SDA (Strand Displacement Amplification) technique (12), the TAS (Transcription-based Amplification System) technique described by (13), the 3SR (Self-Sustained Sequence Replication) technique described by (14), the NASBA (Nucleic Acid Sequence Based Amplification) technique described by (15), the TMA (Transcription Mediated Amplification) technique, the LCR (Ligase Chain Reaction) technique described by (16), the RCR (Repair Chain Reaction) technique described by (17), the CPR (Cycling Probing Reaction) technique described by (18), the Q-beta-replicase amplification technique described by (19).\",\n \"Some of these techniques have since been improved.\",\n \"In the case where the target polynucleotide to be detected is an mRNA, an enzyme of the reverse transcriptase type is advantageously used, prior to the use of an amplification reaction with the aid of the primers according to the invention or to the use of a method of detection with the aid of the probes of the invention, in order to obtain a cDNA from the mRNA contained in the biological sample.\",\n \"The cDNA obtained will then serve as a target for the primers or probes used in the method of amplification or detection according to the invention.\",\n \"The probe hybridization technique may be carried out in various ways (20).\",\n \"The method most generally used consists in immobilizing the nucleic acid extracted from the cells of different tissues or cells in culture on a support (such as nitrocellulose, nylon, polystyrene) and in incubating, under well defined conditions, the immobilized target nucleic acid with the probe.\",\n \"After hybridization, the excess probe is removed and the hybrid molecules formed are detected by the appropriate method (measurement of the radioactivity, the fluorescence or the enzymatic activity linked to the probe).\",\n \"According to another embodiment of the nucleic probes according to the invention, they may be used as capture probes.\",\n \"In this case, a probe, called “capture probe”, is immobilized on a support and serves to capture, through specific hybridization, the target nucleic acid obtained from the biological sample to be tested and the target nucleic acid is then detected by means of a second probe, called “detection probe”, labelled with an easily detectable element.\",\n \"The nucleotide sequences according to the invention may moreover be of interest when they are used as antisense nucleotides, that is to say whose structure brings about, through hybridization with the target sequence, inhibition of the expression of the corresponding product.\",\n \"They may also be used as sense nucleotides which, through interaction with proteins involved in the regulation of the expression of the corresponding product, will induce either an inhibition, or an activation of this expression.\",\n \"The subject of the invention is also the use of a nucleotide sequence according to the present invention for the production or the synthesis of a recombinant polypeptide.\",\n \"The method of producing a polypeptide of the invention in recombinant form, which is itself included in the present invention, is characterized in that the transformed cells, in particular the cells or mammals of the present invention, are cultured under conditions allowing the expression of a recombinant polypeptide encoded by a nucleotide sequence according to the invention, and that the said recombinant polypeptide is recovered.\",\n \"The recombinant polypeptides, characterized in that they can be obtained by the said method of production, also form part of the invention.\",\n \"The recombinant polypeptides obtained as indicated above may be provided either in glycosylated or nonglycosylated form and may or may not have the natural tertiary structure.\",\n \"The sequences of the recombinant polypeptides may also be modified in order to improve their solubility, in particular in aqueous solvents.\",\n \"Such modifications are known to persons skilled in the art, such as for example the deletion of hydrophobic domains or the replacement of hydrophobic amino acids with hydrophilic amino acids.\",\n \"These polypeptides may be produced from the nucleic acid sequences defined above, according to techniques for producing recombinant polypeptides known to persons skilled in the art.\",\n \"In this case, the nucleic acid sequence used is placed under the control of signals allowing its expression in a cellular host.\",\n \"An effective system for producing a recombinant polypeptide requires having a vector and a host cell according to the invention.\",\n \"These cells may be obtained by introducing into host cells a nucleotide sequence inserted into a vector as defined above, and then culturing the said cells under conditions allowing the replication and/or the expression of the transfected nucleotide sequence.\",\n \"The methods used for purifying a recombinant polypeptide are known to persons skilled in the art.\",\n \"The recombinant polypeptide may be purified from cell lysates and extracts, from the supernatant of the culture medium, by methods used individually or in combination, such as fractionation, chromatography methods, immunoaffinity methods using specific monoclonal or polyclonal antibodies, and the like.\",\n \"The polypeptides according to the present invention may also be obtained by chemical synthesis using one of the numerous known peptide syntheses, for example the techniques using solid phases (21) or techniques using partial solid phases, by condensation of fragments or by conventional synthesis in solution.\",\n \"The polypeptides obtained by chemical synthesis and which may contain corresponding unnatural amino acids are also included in the invention.\",\n \"The subject of the present invention is also a DNA chip, characterized in that it contains at least one nucleotide sequence in accordance with the present invention.\",\n \"Actually, the nucleotide sequences according to the invention which it is envisaged to use as probe or as primer for the detection, identification, assay and/or amplification of nucleic acid sequences may be covalently or noncovalently immobilized on a support, this support being a DNA chip or a high density filter.\",\n \"The expression “DNA chip” or “high density filter” is understood to mean a support to which DNA sequences are attached, it being possible for each of them to be located by its geographic localization.\",\n \"These chips or filters differ mainly in their size, the support material, and possibly the number of sequences attached thereto.\",\n \"In particular, it is possible to carry out a synthesis in situ by photochemical targeting or by ink jet targeting.\",\n \"Other techniques consist in carrying out a synthesis ex situ or in attaching the probes to the support of the DNA chip by ink jet, electronic or mechanical addressing.\",\n \"These different methods are well known to persons skilled in the art.\",\n \"The subject of the invention is also a protein chip comprising a polypeptide or antibody according to the invention.\",\n \"Such a protein chip allows the study of interactions between the polypeptides according to the invention and other proteins or chemical compounds and can thus be useful for screening compounds which interact with the polypeptides according to the invention.\",\n \"It may also be possible to use the protein chips according to the invention for detecting the presence of antibodies directed against the polypeptides according to the invention in the serum of patients to be tested.\",\n \"It may also be possible to use a protein chip comprising an antibody according to the invention, this time to detect the presence of polypeptides in the serum of patients which are capable of being recognized by the said antibodies.\",\n \"The subject of the present invention is also the use of a compound chosen from a nucleotide sequence, a polypeptide, a vector, a cell or an antibody according to the invention for the preparation of a medicament.\",\n \"The pathologies more specifically targeted are viral diseases and diseases characterized by the development of tumour cells or cellular degeneration such as Alzheimer's disease or schizophrenia.\",\n \"Thus, the abovementioned medicament is intended for the prevention and/or the treatment of these diseases.\",\n \"In particular, the targeted disease is cancer.\",\n \"One of the benefits of the present invention is that it has demonstrated the involvement of a large number of nucleotide sequences in the phenomena of tumour suppression, tumour reversion, apoptosis and/or viral resistance.\",\n \"These sequences are therefore expressed differentially when one of these abovementioned processes is triggered.\",\n \"Consequently, in the presence of a patient in whom the onset of one of these processes is suspected or for whom it is desired to check the absence of such an onset, it is useful to be able to determine, or even quantify, the expression of one or more sequences in accordance with the invention from a biological sample from the said patient.\",\n \"Optionally, analysis of the expression of one or more of the said sequences may be accompanied by a comparison with a reference level of expression corresponding to that of a healthy individual.\",\n \"Consequently, the invention therefore also comprises a method for the diagnosis and/or prognostic evaluation of a viral disease or a disease characterized by tumour development or cell degeneration comprising the analysis of the expression of at least one sequence of the invention from a biological sample from a patient to be tested.\",\n \"According to a preferred embodiment, the said method comprises the following steps: isolation of the messenger RNA from a biological sample obtained from a patient to be tested, preparation of the complementary cDNA from the said messenger RNA, optionally, amplification of a portion of the complementary DNA corresponding to at least one sequence of the invention, and detection of the optionally amplified complementary DNA.\",\n \"In particular, analysis of the expression of the sequence may be carried out by means of a DNA chip as described above.\",\n \"Among the sequences of the present invention, some exhibit the characteristic of being receptors expressed at the surface of cells and in order to understand the mechanism thereof in the context of the abovementioned processes, it is useful to search for compounds capable of interacting with this receptor, that is to say of interacting with a polypeptide in accordance with the invention, it is also necessary to predict the secreted protein.\",\n \"This also applies to the polypeptides in accordance with the invention corresponding to secreted proteins (at the surface or outside the cells), hormone-like proteins, and the like.\",\n \"Consequently, the subject of the present invention is also a method for screening compounds capable of binding to a peptide in accordance with the invention and comprising the following steps: bringing a polypeptide or a cell according to the invention into contact with a candidate compound, and detection of the formation of a complex between the said candidate compound and the said polypeptide or the said cell.\",\n \"A method for screening compounds may also be useful in relation to compounds capable of interacting with a nucleotide sequence according to the invention, or even with the sequences necessary for the expression or the regulation of these sequences.\",\n \"Indeed, the compounds are capable of interacting with the said sequences, the effect being to reduce, inhibit or on the contrary potentiate the expression of the sequences in question.\",\n \"Such a method comprises the following steps: bringing a nucleotide sequence or a cell according to the invention into contact with a candidate compound, and detection of the formation of a complex between the said candidate compound and the said nucleotide sequence or the said cell.\",\n \"For reasons mentioned above, it may therefore be useful to search for and/or assay, in a biological specimen or sample from a patient to be tested, the presence of a nucleotide sequence according to the invention.\",\n \"Such a method of detection and/or assay comprises the following steps: bringing a labelled nucleotide sequence according to the invention into contact with the biological sample to be tested, under the conditions necessary for the formation of a hybrid, and detection and/or assay of the hybrid which may be formed between the said nucleotide sequence and the nucleic acid present in the said biological sample.\",\n \"This method may additionally comprise a step for amplifying the nucleic acid of the said biological sample with the aid of primers chosen from the nucleotide sequences according to the invention.\",\n \"In particular, this method may be carried out by means of the DNA chip described above.\",\n \"Persons skilled in the art know how to carry out such a method and may in particular use a reagent kit comprising: a) a nucleotide sequence according to the invention, used as a probe, b) the reagents necessary for carrying out a hybridization reaction between the said probe and the nucleic acid of the biological sample, c) the reagents necessary for the detection and/or assay of the hybrid formed between the said probe and the nucleic acid of the biological sample.\",\n \"Such a kit may also contain positive or negative controls in order to ensure the quality of the results obtained.\",\n \"Likewise, the detection and/or the assay of a polypeptide according to the invention can also be envisaged in the context of the present invention and this method therefore comprises the following steps: bringing the biological sample into contact with a labelled antibody according to the invention, and detection and/or assay of the complex formed by the said antibody of polypeptide present in the said sample.\",\n \"Advantageously, this method may be carried out by means of the protein chip as described above.\",\n \"Here again, persons skilled in the art know how to carry out such a method and may in particular use a reagent kit comprising: a) a monoclonal or polyclonal antibody according to the invention; b) optionally reagents for preparing a medium favourable for the antigen/antibody reaction; c) the reagents allowing the detection of the antigen/antibody complex.\",\n \"Finally, the subject of the present invention is also a computer readable support or a computer support on which are recorded at least one nucleotide sequence according to the Claim 1 and/or at least one polypeptide sequence as defined in Claim 3 or 4.In particular, this support is chosen from the group comprising: a) a floppy disk, b) a hard disk, c) a random access memory (RAM), d) a read only memory (ROM), e) a CD ROM.\",\n \"The invention is not limited to the present description but encompasses on the contrary all the variants and will be understood more clearly in the light of the experimental data below.\",\n \"FIGURES FIG.\",\n \"1 represents a curve for tumour growth for the U937 and US4 groups in SCID mice.\",\n \"FIG.\",\n \"2 represents a curve for the body weight of mice carrying a U937 or US4 tumour.\",\n \"FIG.\",\n \"3 illustrates: a/ the analysis of the expression of the TPT1/TCTP gene in the U937/US4.2 lines and in the MCF7/MCF7+SIAH1 lines by Northern blotting.\",\n \"The quantities of total transcripts are checked by monitoring GAPDH; b/ Western blot analysis of the expression of the TCTP protein in the U937/US4.2 model, in the MCF7/MCF7+siah model and analysis of the expression of the murine homologue of TCTP, TCTP_MOUSE, in the M1/LTR6 system after 20 h of induction at 32° C.; c/ the analysis of the expression of TCTP in U937 cells transfected with the antisense TPT1/TCTP.\",\n \"The clones I and III are compared with the control, U937 cells with the vector alone.\",\n \"On the right hand panel, detection of the cleavage of PARP, a marker for apoptosis, in the clones I and III, U937 cells transfected with the antisense TPT1/TCTP.\",\n \"Jurkat cells treated with an anti-Fas (anti-CD95) antibody, an inducer of apoptosis, serve as a control; d/ characterization of the apoptotic cells by double labelling FITC-Annexin V and propidium iodide (PI).\",\n \"The U937 cells+control vector and the U937 cells+antisense TPT1/TCTP are simultaneously labelled with annexin V coupled to FITC (which binds to the phosphatidylserine of the apoptotic cells in the early phase) and PI (which only labels necrotic cells whose plasma membrane is damaged).\",\n \"Cytofluorometric analysis makes it possible to determine the percentage of apoptotic cells (Annexin+, PI−), necrotic cells (Annexin+, PI+ or Annexin−, PI+) and intact cells (Annexin−, PI−); e/ evaluation of apoptosis in the cells transfected with the antisense TPT1/TCTP, by in situ labelling of the free 3′ ends of the nuclear DNA: TUNEL (“Terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labelling”) method.\",\n \"This labelling makes it possible to demonstrate in situ the fragmentation of nuclear DNA resulting from the activation of endonucleases during apoptosis.\",\n \"The apoptotic cells appear in green.\",\n \"f/ the study of the in vivo tumorigenicity of the U937 cells transfected with: the vector alone (top curve—91 tumours/100), an antisense presenilin 1 (second curve from the top—12 tumours/20), SIAH1 (middle curve—12 tumours/20) and an antisense TPT1/TCTP, clone I and III (the bottom 2 curves—8 tumours/20 and 4 tumours/20).\",\n \"10 million cells injected into each flank and into each shoulder.\",\n \"FIG.\",\n \"4 represents cells cultured on an extra-cellular tumour matrix (Matrigel).\",\n \"The 184B5 line, mammary epithelial cells transformed with benzo(a)pyrene, is used as a control for the formation of acini.\",\n \"The tumour cells T47D and MCF7 form irregular colonies.\",\n \"The MCF7 cells transfected with SIAH1 partially recover an organized structure.\",\n \"The MCF7 and T47D cells transfected with the antisense TPT1/TCTP result in a cellular reorganization comparable to the normal formation of the acini.\",\n \"The cells which appear green (light spots in the figure) are labelled with an anti-E_Cadherin antibody coupled to FITC and the nuclei labelled with PI appear red (darker spots).\",\n \"Western blot analysis of the expression of TCTP in the MCF7 and T47D cells transfected with the antisense TPT1/TCTP.\",\n \"1—DATA RELATING TO THE U937 and US4 CELLS As seen above, the present invention uses the parental cells U937 and the “derived” cells US4.Indeed, the US4 cells and the US3 cells (which are not involved in the context of the present invention) share certain characteristics.\",\n \"Their mode of production and their properties are presented below.\",\n \"Selection and Characterization of US3 and US4 Cells The U937 cells were subjected to two series of limiting dilution until a single clonal population is obtained.\",\n \"These cells were infected with the H-1 parvovirus.\",\n \"The cytopathic effect of the virus creates massive cell death which spares two resistant clones which are US3 and US4 after three months of continuous culture.\",\n \"The survival of the cells is defined as the relative number of viable cells in the culture infected with the H-1 virus compared with the untreated culture, as measured four days after reinfection.\",\n \"To measure the tumorigenicity, 107 U937, US3 and US4 cells were subcutaneously injected into scid/scid mice (4 or 5 weeks old).\",\n \"The tumorigenicity is expressed by the number of tumours developed by the mice within two months following the injection.\",\n \"The approach was as follows: using clonal populations of malignant cells, subclones were derived with a suppressed tumorigenic phenotype.\",\n \"This selection made by means of the H-1 parvovirus is carried out by the elimination of the tumour cells which are preferentially killed while sparing the normal cells.\",\n \"The selection of the cells resistant to the cytopathic effect of the H-1 parvovirus outside a sensitive tumour can give rise to cells which have a reduced malignant phenotype.\",\n \"On this basis, a clonal population of U937 cells was isolated, these cells are sensitive to the cytopathic effect of H-1 parvovirus and the US3 and US4 clones are, for their part, resistant to the virus.\",\n \"The US3 and US4 clones have a strong suppressed tumorigenic phenotype while the parental U937 cells develop tumours in 80% of cases among the scid/scid mice which were infected with the parvovirus, the US3 cells form only a single tumour and the US4 cells develop one tumour per 20 inoculations with 107 cells.\",\n \"The results are assembled in the table below.\",\n \"Resistance to the H-1 Parvovirus and Tumorigenicity of the U397, US3 and US4 Cells Survival of the Tumorigenicity cells to in infection with the scid/scid Cell lines the H-1 virus mice U937 0.4 16/20 US3 96 0/10 US4 89 1/20 2—MATERIALS AND METHODS The aim is to compare the growth of subcutaneous tumours in SCID mice induced by subcutaneous injection of transfected U937 and US4 human leukaemic cell lines.\",\n \"A. Leukaemic Cell Lines and Culture Conditions All the injected cells of the U937 (ATCC) and US4 cell lines were provided in the form of cell suspensions in bottles supplemented with RPMI-1640 culture medium supplemented with 2 mM L-glutamine, 10% foetal calf serum and gentamycin.\",\n \"The U937 cell line is a CD4+ human monocytic cell line derived from a patient having a diffuse histiocytic lymphoma (1).\",\n \"The cells were counted in a haemocytometer and their viability was tested with exclusions of trypan blue dyes at 0.25%.\",\n \"The viability was 95.5% and 90.5% for the U937 cells and the US4 cells, respectively.\",\n \"The U937 and US4 cells were centrifuged and then resuspended in an RPMI medium before being injected into SCID mice.\",\n \"B.\",\n \"The animals 10 female SCID mice in good health (CB17/IcrHsd), 31 weeks old and weighing between 20 and 25 g, were supplied by Harlan France (Gannat, France).\",\n \"The animals were observed for 7 days in a specific room belonging to the Applicant which is the unit for treating specific-pathogen-free animals before their treatment.\",\n \"The unit for treating animals (INRA, Dijon, France) is authorized by the French Ministers of Agriculture and Research (authorization No.\",\n \"A21100).\",\n \"The animal experiments are carried out according to the European Ethical Directives for the well-being of animals in the context of experimental neoplasia (3).\",\n \"B.1.Environment The animals were kept in rooms under controlled conditions of temperature (24±1° C.), humidity (55±1%), light period (12 h of light/12 h of darkness) and air renewal.\",\n \"The animals were kept under SPF conditions and the temperature and the humidity of the room were continuously monitored.\",\n \"The aeration system was programmed to give rise to 14 air renewals per hour without recirculation.\",\n \"Fresh air from outside passes inside a series of filters before being uniformly diffused into each room.\",\n \"A high pressure (2 mm) was maintained in the experimentation rooms in order to prevent contamination or diffusion of pathogens within a mouse colony.\",\n \"All the staff working under SPF conditions followed specific directives as regards hygiene and clothing when they were in the breeding area.\",\n \"B.2.Breeding The animals were housed in polycarbonate cages (UAR, Epinay sur Orge, France) which were equipped so as to provide them with food and water.\",\n \"The standard size of the cages used is 637 cm2 for 10 mice according to the standard internal operating procedures.\",\n \"The litter for animals and made up of sterile wood shavings (UAR) and is replaced twice per week.\",\n \"B.3.Food and Drink The animal food was bought from Extralabo (Provins, France).\",\n \"The food was provided ad libitum and was placed on the metal lid at the top of the cage.\",\n \"Water was also provided ad libitum from bottles of water equipped with rubber taps.\",\n \"The water bottles were washed, sterilized and replaced once per week.\",\n \"The water supply was sterilized by filtration with a 0.2 μm absolute filter.\",\n \"B.4.Identification of the Animal and the Cage After random distribution, the animals were identified with 2 different numbers engraved on the two ears.\",\n \"Each cage was marked with a specific code.\",\n \"C—Experimental Data and Treatments C.1.Induction of Tumours in SCID Mice Before cell injection, the SCID mice were randomly divided into two groups, in an amount of 5 mice per group.\",\n \"107 US4 or U937 tumour cells in 0.2 ml of RPMI medium were subcutaneously inoculated at time 0 for each site of injection into SCID mice.\",\n \"Each animal received 4 injections of tumour cells located in different areas, one in each flank and one in each shoulder.\",\n \"C.2.Collection of the Tumours When the tumours reached a volume of 1 500 mm3, the mice were killed and the tumours were collected, weighed and frozen in liquid nitrogen, stored at −80° C. and then specifically labelled.\",\n \"C.3.Monitoring of the Mice Isoflurane forene (Minerve, Bondouble, France) was used to anaesthetize the animals before injection of cells for sacrificing.\",\n \"After injection of tumour cells, the mice were observed for 5 hours.\",\n \"The viability, behaviour, body weight of the mice and the growth of the subcutaneous tumour were recorded twice per week.\",\n \"During the period of experimentation, the animals were killed under anaesthetic with isoflurane by cervical dislocation if one of the following signs appears: sign of suffering (cachexia, weakness, difficulty in moving or eating), tumour growth up to 10% of the body weight, tumour ulceration and persistently becoming nude, position of the tumour interfering with movement and/or feeding, loss of weight of 20% for three consecutive days.\",\n \"An autopsy was performed on each animal to detect the possible presence of metastases or morphological abnormalities.\",\n \"D—Presentation of the Data D.1.Monitoring Parameters The calculation for the median and mean survival time was expressed as follows: Mean survival time=S1/(S2−NT) With: S1=sum of the daily survivors from day 0 up to the end of the experiment (without the survivors “not taken into account”*) S2=number of animals at the beginning NT=number of animals “not taken into consideration”* *“not taken into consideration”: they are animals with tumours which are smaller than the predetermined limit considered as resulting from a defect in implantation of the tumour.\",\n \"D.2.System for Inhibition of the Tumour The tumour size was measured twice per week with a compass (and the tumour volume in mm3) will be estimated according to the formula: (length×width2/2 (4).\",\n \"The experiments were stopped when the tumour sizes in the mice reached 1 500 mm3.After sacrificing, the tumours were excised and weighed.\",\n \"The curve for tumour growth for the US4 and U937 groups was noted using the mean of the tumour volumes.\",\n \"The tumour doubling time for the US4 and U937 groups was defined as the period required to reach a mean tumour volume of 200% during the growth period.\",\n \"The specific growth period over one or two doubling times (DT) from injections of cells is defined as follows: specific growth period=(DT US4−DT U937)/DT U937.The growth period is calculated as the difference between the median growth time for the US4 group and the U937 group in order to reach the same tumour size.\",\n \"D.3.Statistical Tests All the statistical analyses were carried out with the StatView® software (Abacus Concept, Berkeley, USA).\",\n \"The statistical analyses of the changes in mean body weight, the tumour doubling time and the time to reach “V” were performed using the Bonferroni/Dunn test.\",\n \"A value p<0.05 was considered to be significant.\",\n \"All the groups were compared with each other.\",\n \"E—Results The curves for mean tumour volumes and body weights are shown in FIGS.\",\n \"1 and 2 respectively.\",\n \"No significant loss of body weight in SCID mice from the two groups was observed between day 8 and day 19.A significant difference is observed as regards the time to reach “V” between the 2 groups of SCID mice whereas no significant difference was observed for the change in mean body weight (day 19−day 8) and for the doubling time.\",\n \"The autopsies performed did not show the presence of metastases or of developments of suspicious nodosity.\",\n \"The tumours were collected on animals sacrificed after anaesthetic and cervical dislocation.\",\n \"The tumours were immediately placed in tubes, frozen in liquid nitrogen and stored at −80° C. The excised tumours had an ovoid shape, a moderate consistency and a pinkish colour.\",\n \"The interactions of the tumour with its environment (skin and muscle tissue) were limited and superficial.\",\n \"F—Conclusions The US4 cell line showed an uptake level significantly lower compared with the U937 cell line in the SCID mice.\",\n \"The retardation of growth between the US4 and U937 tumours was 23.5 days and the doubling time was equivalent.\",\n \"3—INVOLVEMENT OF TPT1/TCPT IN TUMOUR REVERSION In order to study the involvement of TPT1/TCPT (Translationally controlled Tumour Protein encoding the Histamine Release Factor) in tumour reversion, the expression of the gene (FIG.\",\n \"3a) and the expression of the protein (FIG.\",\n \"3b) in various cellular models of tumour reversion: U937/US4.2, MCF7/MCF7+SIAH1 and M1/LTR6, were analysed.\",\n \"The level of expression of TPT1/TCTP is highly reduced in the revertant cells compared with the tumour cells.\",\n \"To deal with the physiological aspect of this reduction, the U937 tumour cells were transfected with the antisense TPT1/TCTP.\",\n \"After having checked the drop in the expression of TPT1/TCTP (FIG.\",\n \"3c, left panel), the inventors detected its consequences in apoptosis by three different methods: detection of the cleavage of PARP, labelling with Annexin V and TUNEL method (FIG.\",\n \"3c, right panel; FIG.\",\n \"3d; FIG.\",\n \"3e, respectively).\",\n \"The reduction in the expression of TPT1/TCTP in tumour cells in culture increases apoptosis.\",\n \"The inventors then expressed in vivo the physiological consequences of this loss of TPT1/TCTP functions.\",\n \"We injected U937 cells transfected with antisense TPT1/TCTP into mice and observed a drastic reduction in the number of tumours formed when the expression of TPT1/TCTP is reduced (FIG.\",\n \"3f).\",\n \"In parallel with these results, the reduction of TPT1/TCTP, obtained by transfection of double-stranded RNA specific for TPT1/TCTP (siRNA), into MCF7 and T47D cells, cultured on an extracellular matrix, leads to a cellular reorganization of acini which is comparable to a normal structure (FIG.\",\n \"4).\",\n \"REFERENCES (1) Sundström C. and Nilsson K., Int.\",\n \"J.\",\n \"Cancer, 17, 565, 1976.\",\n \"(2) Principe d'éthique de l'expérimentation animale [Principle of ethics of animal experimentation], Directive No.\",\n \"86/609 EEC of 24 Nov. 1986, Decree No.\",\n \"87/848 of 19 Oct. 1987, Application Order of 19 Apr.\",\n \"1988.\",\n \"(3) United Kingdom co-ordinating committee on cancer research guidelines for welfare of animals in experimental neoplasia, Br.\",\n \"J.\",\n \"Cancer, 77: 1-10, 1998.\",\n \"(4) Bissery M. C. et al., Bull.\",\n \"Cancer, 78: 587, 1991.\",\n \"(5) Perricaudet et al.\",\n \"(1991).\",\n \"La Recherche 23: 471.\",\n \"(5a) Epstein (1992), Médecine/Sciences, 8, 902.\",\n \"(6) Temin (1986), Retrovirus vectors for gene transfer.\",\n \"In Kucherlapati R., ed.\",\n \"Gene.\",\n \"(7) Carter (1993), Curr.\",\n \"Op.\",\n \"Biotechnology 3, 533.\",\n \"(8) Olins and Lee (1993), Curr.\",\n \"Op.\",\n \"Biotechnology 4: 520.\",\n \"(9) Buckholz (1993), Curr.\",\n \"Op.\",\n \"Biotechnology 4, 538.\",\n \"(10) Edwards and Aruffo (1993), Curr.\",\n \"Op.\",\n \"Biotechnology 4, 558.\",\n \"(11) Luckow (1993), Curr.\",\n \"Op.\",\n \"Biotechnology 4, 564.\",\n \"(11a) Rolfs, A. et al.\",\n \"(1991), Berlin: Springer-Verlag.\",\n \"(12) Walker (1992), Nucleic Acids Res.\",\n \"20: 1691.\",\n \"(13) Kwoh et al.\",\n \"(1989), Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA, 86, 1173.\",\n \"(14) Guatelli et al.\",\n \"(1990), Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA, 87, 1874.\",\n \"(15) Kievitis et al.\",\n \"(1991), J. Virol.\",\n \"Methods, 35, 273.\",\n \"(16) Landegren et al.\",\n \"(1988) Science 241, 1077.\",\n \"(17) Segev (1992), Kessler C. Springer Verlag, Berlin, New York, 197-205.\",\n \"(18) Duck et al.\",\n \"(1990), Biotechniques, 9, 142.\",\n \"(19) Miele et al.\",\n \"(1983), J. Mol.\",\n \"Biol., 171, 281.\",\n \"(20) Matthews et al.\",\n \"(1988), Anal.\",\n \"Biochem., 169, 1-25.\",\n \"(21) Stewart and Yound (1984), Solid phase peptides synthesis, Pierce Chem.\",\n \"Company, Rockford, 111, 2nd ed.\",\n \"(1984).\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10467851"}}},{"rowIdx":388,"cells":{"text":{"kind":"list like","value":[["System and method for an auction of multiple types of items","An improved system and method for a computer-implemented auction in which multiple types of items are auctioned together without imposing a particular division of supply or demand among the individual types of items.","In some embodiments the auction of the present invention provides a means or method for establishing prices for the types of items, wherein the prices maintain a relationship.","In other embodiments, the present invention provides a means or method for implying prices from price parameters in the bids received form bidders, based on a relation among the prices for the types of items.","Market clearing may be defined by the condition that the aggregate quantity bid for all types of items is less than or equal to the available quantity of all types of items.","The division among the types of items within is thus determined flexibly, based on the bids at the associated prices.","In other embodiments, market clearing is defined by the condition that the quantity bid for one selected type of item is less than or equal to the available quantity of the selected type of item.","The quantities of the other types of items are thus determined flexibly, based on the bids at the associated prices."],["1.A computer system for conducting an auction of a plurality of types of items among a plurality of bidders, wherein bids are received and an allocation of the items among the bidders is determined by the computer system based on the bids, comprising: establishing means for establishing prices for the plurality of types of items, wherein the prices maintain a relationship, receiving means for receiving bids associated with the established prices, and processing means for processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, otherwise enabling the establishing means to alter the prices established for the plurality of types of items.","2.A system as recited in claim 1 which further comprises means for constraining the changes in a bid from a given bidder as the established prices are altered by considering, in the processing means, only bids which satisfy a constraint enforced by the means for constraining.","3.A system as recited in claim 2 wherein the constraining means includes summation means for calculating a sum of quantity parameters for a group of types of items.","4.A system as recited in claim 3 wherein the constraining means includes comparing means for comparing the calculated sum with a previous calculated sum for the same group of types of items.","5.A system as recited in claim 1 wherein each received bid comprises a quantity parameter for at least one of the types of items in the auction.","6.A system as recited in claim 5 wherein the establishing means establishes a price for a first type of item and then determines prices for each other type of item based on the price for the first type of item.","7.A system as recited in claim 6 wherein the establishing means determines prices from a predetermined schedule.","8.A system as recited in claim 7 wherein the establishing means includes interpolation means for interpolating prices among prices on the predetermined schedule.","9.A system as recited in claim 6 wherein the processing means includes summation means for calculating a sum of the quantity parameters for the first type of item.","10.A system as recited in claim 9 wherein the processing means includes comparing means for comparing the calculated sum with an available quantity parameter.","11.A system as recited in claim 1 wherein the plurality of types of items are contracts for provision of a commodity covering different time periods, wherein the time periods of at least some of the contracts have at least some overlap.","12.A system as recited in claim 5 wherein the plurality of types of items are contracts for provision of a commodity covering different time periods, wherein the time periods of at least some of the contracts have at least some overlap.","13.A system as recited in claim 12 wherein the processing means includes summation means for calculating a sum of the quantity parameters for the overlapping contracts.","14.A system as recited in claim 13 wherein the processing means includes comparing means for comparing the calculated sum with an available quantity parameter.","15.A system as recited in claim 1 wherein the relationship of the prices established by the establishing means is determined by a schedule.","16.A system as recited in claim 1 wherein the relationship of the prices established by the establishing means is determined by an algorithm.","17.A computer implemented method for conducting an auction of a plurality of types of items among a plurality of bidders, wherein bids are received and an allocation of the items among the bidders is determined based on the bids, comprising: establishing prices for the plurality of types of items, wherein the prices maintain a relationship, receiving bids associated with the established prices, and processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, otherwise altering the established prices for the plurality of types of items.","18.A method as recited in claim 17 which further comprises constraining the changes in a bid from a given bidder as the established prices are altered by considering, in the processing, only bids which satisfy a constraint.","19.A method as recited in claim 18 wherein the constraining includes calculating a sum of quantity parameters for a group of types of items.","20.A method as recited in claim 19 wherein the constraining includes comparing the calculated sum with a previous calculated sum for the same group of types of items.","21.A method as recited in claim 17 wherein each received bid comprises a quantity parameter for at least one of the types of items in the auction.","22.A method as recited in claim 21 wherein a price is established for a first type of item and then prices for each other type of item are determined based on the price for the first type of item.","23.A method as recited in claim 22 wherein the determined prices are determined from a predetermined schedule.","24.A method as recited in claim 23 wherein establishing prices includes interpolating prices among prices included in the predetermined schedule.","25.A method as recited in claim 22 wherein processing the received bids includes calculating a sum of the quantity parameters for the first type of item.","26.A method as recited in claim 25 wherein processing the received bids includes comparing the calculated sum with an available quantity parameter.","27.A method as recited in claim 17 wherein the plurality of types of items are contracts for provision of a commodity covering different time periods, wherein the time periods of at least some of the contracts have at least some overlap.","28.A method as recited in claim 21 wherein the plurality of types of items are contracts for provision of a commodity covering different time periods, wherein the time periods of at least some of the contracts have at least some overlap.","29.A method as recited in claim 28 wherein processing the received bids includes calculating a sum of the quantity parameters for the overlapping contracts.","30.A method as recited in claim 29 wherein processing the received bids includes comparing the calculated sum with an available quantity parameter.","31.A method as recited in claim 17 wherein the relationship of the prices is based on a schedule.","32.A method as recited in claim 17 wherein the relationship of the prices is based on an algorithm.","33.A computer system for conducting an auction of a plurality of types of items among a plurality of bidders, wherein bids are received and an allocation of the items among the bidders is determined based on the bids, comprising: receiving means for receiving bids comprising a quantity parameter for at least one of the types of items in the auction and a price parameter, and processing means for processing the received bids to determine an allocation of the items among the bidders that is consistent with the bids in the event that an allocation consistent with the bids is possible, wherein the processing means includes implying means for implying prices from price parameters in the received bids based on a fixed relation among the prices of the plurality of types of items.","34.A system as recited in claim 33 which includes means for inviting additional bids in the event that an allocation of items consistent with the received bids is not possible, and means for applying the additional bids to the processing means for processing the additional bids to determine an allocation of the items among the bidders consistent with the additional bids in the event that an allocation consistent with the additional bids is possible.","35.A computer system as recited in claim 34 which further comprises means for constraining the additional bids from a given bidder by limiting the processing to only additional bids which satisfy a constraint.","36.A system as recited in claim 35 wherein the constraining means includes summation means for calculating a sum of quantity parameters for a group of types of items.","37.A system as recited in claim, 36 wherein the constraining means includes comparing means for comparing the calculated sum with a previous calculated sum for the same group of types of items.","38.A system as recited in claim 33 wherein the plurality of types of items are contracts for provision of a commodity covering different time periods, wherein the time periods for at least some of the contracts have at least some overlap.","39.A system as recited in claim 35 wherein the processing means includes summation means for calculating a sum of the quantity parameters for at least some of the overlapping contracts.","40.A system as recited in claim 39 wherein the processing means includes comparing means for comparing the calculated sum with an available quantity parameter.","41.A system as recited in claim 33 wherein the fixed relation of the implying means is based on a schedule.","42.A system as recited in claim 33 wherein the fixed relation of the implying means is based on an algorithm.","43.A computer implemented method for conducting an auction of a plurality of types of items among a plurality of bidders, wherein bids are received and an allocation of the items among the bidders is determined based on the bids, comprising: receiving bids comprising a quantity parameter for at least one of the types of items in the auction and a price parameter, and processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, wherein the processing includes implying prices from price parameters in the received bids based on a fixed relation among the prices of the plurality of types of items.","44.A method as recited in claim 43 which includes inviting additional bids in the event that an allocation of items consistent with the received bids is not possible, and processing the additional bids to determine an allocation of the items among the bidders consistent with the additional bids in the event that an allocation consistent with the additional bids is possible.","45.A method as recited in claim 44 which further comprises constraining the additional bids from a given bidder by considering, in the processing, only additional bids which satisfy a constraint.","46.A method as recited in claim 45 wherein the constraining includes calculating a sum of quantity parameters for a group of types of items.","47.A method as recited in claim 46 wherein the constraining includes comparing the calculated sum with a previous calculated sum for the same group of types of items.","48.A method as recited in claim 43 wherein the plurality of types of items are contracts for provision of a commodity covering different time periods, wherein the time periods for at least some of the contracts have at least some overlap.","49.A method as recited in claim 48 wherein the processing includes calculating a sum of the quantity parameters for at least some of the overlapping contracts.","50.A method as recited in claim 49 wherein the processing includes comparing the calculated sum with an available quantity parameter.","51.A method as recited in claim 43 wherein the fixed relation of the processing is determined by a schedule.","52.A method as recited in claim 43 wherein the fixed relation of the processing is determined by an algorithm.","53.A computer system for conducting an auction of at least one type of item among a plurality of bidders in a plurality of rounds, wherein bids are received and an allocation of the items among the bidders is determined by the computer system based on the bids, comprising: establishing means for establishing starting prices and ending prices for each type of item in a plurality of rounds, receiving means for receiving bids, wherein a bid comprises a quantity for at least one type of item and a price parameter signifying a percentage of the distance from the established starting prices to the established ending prices, and processing means for processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, otherwise enabling the establishing means to alter the prices established for at least one type of item.","54.A system as recited in claim 53 where the establishing means includes means to maintain a relation among the established prices.","55.A system as recited in claim 54 wherein the means to maintain a relation among the established prices employs a schedule of prices.","56.A system as recited in claim 54 wherein the means to maintain a relation among the established prices employs an algorithm.","57.A system as recited in claim 53 which further comprises means for constraining the changes in a bid from a given bidder as the established prices are altered by considering, in the processing means, only bids which satisfy a constraint enforced by the means for constraining.","58.A system as recited in claim 58 wherein the constraining means includes summation means for calculating a sum of quantities bid for a group of types of items.","59.A system as recited in claim 59 wherein the constraining means includes comparing means for comparing the calculated sum with a previous calculated sum for the same group of types of items.","60.A computer implemented method for conducting an auction of at least one type of item among a plurality of bidders in a plurality of rounds, wherein bids are received and an allocation of the items among the bidders is determined based on the bids, comprising: establishing starting prices and ending prices for each type of item in a plurality of rounds, receiving bids, wherein a bid comprises a quantity for at least one type of item and a price parameter signifying a percentage of the distance from the established starting prices to the established ending prices, and processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, otherwise altering the established prices for at least one type of item.","61.A method as recited in claim 61 where the establishing includes maintaining a relation among the established prices.","62.A method as recited in claim 62 wherein the relation among the established prices is determined by a schedule.","63.A method as recited in claim 62 wherein the relation among the established prices is determined by an algorithm.","64.A method as recited in claim 61 which further comprises constraining the changes in a bid from a given bidder as the established prices are altered by considering, in the processing, only bids which satisfy a constraint.","65.A method as recited in claim 65 wherein the constraining includes calculating a sum of quantities bid for a group of types of items.","66.A method as recited in claim 66 wherein the constraining includes comparing the calculated sum with a previous calculated sum for the same group of types of items."],["<SOH> BACKGROUND OF THE INVENTION <EOH>Sellers and buyers of commodities, financial securities, and other goods and services are increasingly turning to auctions to assist in the sale or purchase of a wide range of items.","Auctions are useful for selling a unique item (e.g., a painting), a single type of item available in multiple units (e.g., 10 tons of gold), and multiple types of items available in multiple units (e.g., 10 tons of gold and 20 tons of silver).","And just as auctions are useful for efficiently selling items at maximum price, they are also useful for efficiently procuring items at minimum cost.","Auction formats in the art tend generally to be of the sealed-bid or ascending-bid variety.","In standard sealed-bid auctions, bidders—in one single bidding round—simultaneously and independently submit bids to the auctioneer, who then determines the auction outcome.","In standard ascending-bid auctions, bidders—in a dynamic bidding process—submit bids in real time until no more bids are forthcoming.","Sealed-bid auction formats offer the advantage of speed of the auction process.","Ascending-bid auction formats offer the advantage that there is feedback among participants' bids, which tends to result in more aggressive bidding and in more efficient auction outcomes.","When auction systems and methods in the art are used to sell (or buy) multiple types of items, the auctioneer generally specifies an available supply (or demand) for each type of item.","For example, the auctioneer announces a supply of 10 tons of gold and 20 tons of silver, and then solicits bids, in a sealed-bid or ascending-bid procedure, in order to obtain market-clearing prices for each of the two types of commodities.","However, consider a situation where the types of items are related, for example, contracts for provision of a commodity covering different time periods.","For example, a government may wish to sell three types of financial securities: 3-month Treasury bills; 6-month Treasury bills; and 12-month Treasury bills.","The government's only constraint may be that it needs to sell $50 billion worth of new debt securities at this auction, since this is the quantity of old debt securities that are maturing in the current calendar quarter.","However, the government may have no preference as to how the required $50 billion is divided among 3-month bills, 6-month bills, and 12-month bills, respectively, and the government may simply wish to “let the market decide” the division among the various durations Auction systems and methods in the art provide effective ways for the government to sell bonds with a fixed division among the various durations.","However, new and better systems and methods can be devised for “letting the market decide” the division among the various durations.","The same problem also occurs in real life in the sale of contracts for electricity and all kinds of other commodities, as well as in the procurement of related items.","In several preferred embodiments, the present invention provides a system and method for the government in the above example to simply auction $50 billion of Treasury bills at the lowest possible cost (relative to the market's yield curve), without imposing a particular division among the various durations."],["<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is an improved system and method for a computer-implemented auction, particularly for a computer-implemented auction in which multiple types of items are auctioned together.","In several embodiments, the present invention provides a system and method to conduct an auction of a group of related types of items, without imposing a particular division of supply or demand among the individual types of items within the group.","In other embodiments, the present invention provides a system and method to conduct an auction of a group of related types of items, without imposing a particular division of supply or demand on some of the individual types of items within the group.","Instead, in some embodiments, the present invention provides a means or method for establishing prices for the types of items within a group, wherein the prices maintain a relationship.","In other embodiments, the present invention provides a means or method for implying prices from price parameters in the bids received from bidders, based on a relation among the prices for the types of items within a group.","In some embodiments, market clearing is defined by the condition that the aggregate quantity bid for all types of items within a group is less than or equal to the available quantity of all types of items within the group.","The division among the types of items within the group is thus determined flexibly, based on the bids at the associated prices.","In other embodiments, market clearing is defined by the condition that the quantity bid for one selected type of item within a group is less than or equal to the available quantity of the selected type of item.","The quantities of the other types of items within the group are thus determined flexibly, based on the bids at the associated prices.","In a first preferred embodiment, the present invention is: A computer implemented method for conducting an auction of a plurality of types of items among a plurality of bidders, wherein bids are received and an allocation of the items among the bidders is determined based on the bids, comprising: establishing prices for the plurality of types of items, wherein the prices maintain a relationship, receiving bids associated with the established prices, and processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, otherwise altering the established prices for the plurality of types of items.","In a second preferred embodiment, the present invention is a computer system for conducting an auction according to the method of the first preferred embodiment.","The system includes an establishing means, a receiving means, and a processing means.","In both the first and second preferred embodiments, the relationship or relation among the prices may involve a schedule or algorithm.","In the event that the relationship or relation is based on a schedule, determining prices may involve looking up and possibly interpolating numbers that are contained in a table.","Alternatively the relationship or relation may be represented as an algorithm or a formula that provides a relationship among the prices.","The data table may reflect a market beyond the auction (for example, it may reflect price differentials in a separate commodities market or exchange) and the data table may change during the course of the auction (for example, if price differentials change in the separate commodities market or exchange, or if a significant news event occurs during the course of the auction).","Similarly, the data set to which the algorithm or formula are applied may reflect a market beyond the auction and the data set may change during the course of the auction.","In a third preferred embodiment, the present invention is: A computer implemented method for conducting an auction of a plurality of types of items among a plurality of bidders, wherein bids are received and an allocation of the items among the bidders is determined based on the bids, comprising: receiving bids comprising a quantity parameter for at least one of the types of items in the auction and a price parameter, and processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, wherein the processing includes implying prices from price parameters in the received bids based on a fixed relation among the prices of the plurality of types of items.","In a fourth preferred embodiment, the present invention is a computer system for conducting an auction according to the method of the third preferred embodiment.","The system includes a receiving means and a processing means.","In both the third and fourth preferred embodiments, the “price parameters” in the received bids that are used for “implying prices” could be prices for one or more selected types of items.","(For example, the price parameter could be an actual price for 3-month Treasury bills, and the prices for 6-month and 12-month Treasury bills could be implied, based on a fixed relation among the prices.)","The “price parameters” could also be neutral parameters or indicators (e.g., in the nature of an index) of a price.","Indeed, in a fifth preferred embodiment, bids include price parameters that may be a particularly useful form of neutral parameter.","For example, an auctioneer may announce a starting price vector of (4.00, 4.50, 4.75) and an ending price vector of (8.00, 8.50, 8.75).","If a bidder enters a price parameter of 25%, this may signify that the bidder is indicating a price which is 25% of the distance from the starting price to the ending price, i.e., an implied price vector of (5.00, 5.50, 5.75).","In the fifth preferred embodiment, the present invention is: A computer implemented method for conducting an auction of at least one type of item among a plurality of bidders in a plurality of rounds, wherein bids are received and an allocation of the items among the bidders is determined based on the bids, comprising: establishing starting prices and ending prices for each type of item in a plurality of rounds, receiving bids, wherein a bid comprises a quantity for at least one type of item and a price parameter signifying a percentage of the distance from the established starting prices to the established ending prices, and processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, otherwise altering the established prices for at least one type of item.","In a sixth preferred embodiment, the present invention is a computer system for conducting an auction according to the method of the fifth preferred embodiment.","The system includes an establishing means, a receiving means, and a processing means.","Various preferred embodiments of the present invention resolve a number of important limitations to the systems and methods for computer-implemented auctions in the art.","Thus, the present invention offers the further technical effect of improving computer-implemented auctions, making them operate more efficiently.","The present invention improves the ability of bidders to readily participate in the auction and to express their needs and preferences.","The present invention improves the ability of the auctioneer to sell at higher prices the items being sold in the auction or to obtain at lower cost the items being procured in the auction.","The present invention improves the efficiency of the auction process by enabling the auction to operate in shorter time, expending less computer time, fewer communications resources, and less bidder and auctioneer time.","Finally, the present invention improves the allocative efficiency of the auction process by better allocating items to bidders who value them the highest (or, in a procurement auction, to bidders who can provide them at lowest cost).","Certain constraints are desirable in order for the present invention to operate optimally and to reach an economically efficient outcome.","In many auctions, it is desirable to impose constraints on bidders that require them to bid significantly on items early in the auction in order for them to be allowed to bid on items later in the auction.","Otherwise, one may find oneself in a situation where bidders refrain from submitting serious bids until near the very end of the auction, defeating the purpose of a dynamic auction.","One exemplary constraint is an activity rule which constrains a bidder not to increase his quantity, summed over all of the types of items within a group, from one round to the next.","Another exemplary constraint is a more stringent activity rule which constrains a bidder not to increase his quantity, individually on each of the individual types of items, from one bid or one round to the next.","A third exemplary constraint is a reduction rule which constrains a bidder not to decrease his quantity, for any single type of item, beyond the point where the sum of the quantities bid for this type of item by all bidders equals the sum of the quantities being auctioned.","(If, in a given round, two or more bidders simultaneously attempt to decrease their quantities, for any single type of item, having the effect of reducing bids beyond the point where the sum of the quantities bid for this type of item by all bidders equals the sum of the quantities being auctioned, the auction procedure will resolve this discrepancy.","For example, the auctioneer may honor these attempts to decrease in order of time priority, or may ration these simultaneous attempts to decrease in proportion to the attempted reductions.)","The present invention includes treating the case of auctioning a set of items which includes two (or more) items that are neither identical nor perfect substitutes to one another.","Henceforth, this will be described for short as a situation with “multiple types of items,” or simply “heterogeneous items” or “heterogeneous objects.” Often, but not always, the heterogeneous items auctioned together will bear some relationship to one another: for example, they may be securities issued by the same entity but with different durations to maturity; or contracts for the same goods or services but with different durations.","One particularly salient use for the present invention is as follows.","A national government or a private corporation may wish to simultaneously sell 3-month, 6-month and 12-month bonds or notes.","More generally, these may be contracts for provision of any commodity; the contracts are for the same commodity, but covering different but possibly overlapping time periods.","For the government's or corporation's purposes, the allocation of sales among the 3-month, 6-month and 12-month bonds or notes may not be critical; the main issue is that the total quantity of the bonds or notes of the three durations add up to the desired dollar figure.","Meanwhile, the current “yield curve” on financial markets may have 6-month bonds or notes yielding interest rates 0.50% higher than 3-month bonds or notes, and the current “yield curve” on financial markets may have 12-month bonds or notes yielding interest rates 0.25% higher than 6-month bonds or notes.","Under these circumstances, the government or corporation may utilize the present invention by beginning the auction with an (interest rate) vector of (6.00%, 6.50%, 6.75%) for the three respective durations.","If the aggregate quantity of the bonds or notes desired by all the bidders at these interest rates is greater than the quantity of bonds or notes that the government or corporation wishes to sell, the government or corporation might then continue the auction by announcing a new vector of (5.99%, 6.49%, 6.74%) for the three respective durations.","If the aggregate quantity of the bonds or notes desired by all the bidders at these interest rates is still greater than the quantity of bonds or notes that the government or corporation wishes to sell, the government or corporation might then continue the auction by announcing a new vector of (5.98%, 6.48%, 6.73%) for the three respective durations.","The auction continues in this way, with the government or corporation always following the yield curve in setting.","the relative interest rates for the various durations.","Meanwhile, bidders are held to the exemplary activity rule which constrains a bidder not to increase his quantity, summed over the m types of items, from one round to the next.","That is, the bidder is allowed to change the mix of 3-month, 6-month and 12-month bonds or notes that he demands, but his total quantity demanded of bonds or notes may not be increased as the interest rate goes down.","A second use for the present invention is as follows.","An electric utility may wish to sell contracts to provide a given quantity of electricity for 3-month, 6-month and 12-month durations.","For the electric utility's purposes, the allocation of sales among the 3-month, 6-month and 12-month contracts may not be critical; the main issue may be that the total quantity of the contracts add up to a desired amount of electricity.","Meanwhile, current energy prices may imply that a 6-month contract should be priced at $2 per-unit higher than a 3-month contract, and that a 12-month contract should be priced at $1 per unit higher than a 6-month contract.","Under these circumstances, the electric utility may utilize the present invention by beginning the auction with a price vector of ($10.50, $12.50, $13.50) for the three respective durations.","If the aggregate quantity of contracts desired by all the bidders at these prices is greater than the quantity of contracts that the electric utility wishes to sell, the electric utility might then continue the auction by announcing a new price vector of ($10.60, $12.60, $13.60) for the three respective durations.","If the aggregate quantity of contracts desired by all the bidders at these prices is still greater than the quantity of contracts that the electric utility wishes to sell, the electric utility might then continue the auction by announcing a new price vector of ($10.70, $12.70, $13.70) for the three respective durations.","The auction continues in this way, with the electric utility always following the implied price differential in setting the relative prices for the various durations.","Meanwhile, bidders are held to the exemplary activity rule which constrains a bidder not to increase his quantity, summed over the types of items within a group, from one round to the next.","That is, the bidder is allowed to change the mix of 3-month, 6-month and 12-month contracts that he demands, but his total quantity demanded of contracts may not be increased as the price goes up.","In addition, the present invention may also be used to auction two or more groups of types of items.","For example, the previous electric utility example could be complicated by the presence of two groups of electricity contracts: base and peak.","(Base-load quantity is produced all of the time while peak-load quantity is only produced at times of peak demand.)","Within each group, there might be three durations of contracts.","Thus, in total, there could be six contracts for sale: 3-month, 6-month and 12-month base contracts; and 3-month, 6-month and 12-month peak contracts.","To continue the example, current energy prices may imply that a 6-month base contract should be priced at $2 per unit higher than a 3-month base contract, and that a 12-month base contract should be priced at $1 per unit higher than a 6-month base contract.","Meanwhile, current energy prices may imply that a 6-month peak contract should be priced at $3 per unit higher than a 3-month peak contract, and that a 12-month peak contract should be priced at $2 per unit higher than a 6-month peak contract.","Under these circumstances, the electric utility may utilize the present invention by beginning the auction with a price vector of ($10.50, $12.50, $13.50) for the three respective base durations and a price vector of ($26.50, $29.50, $31.50) for the three respective peak durations.","If the aggregate quantity of base contracts desired by all the bidders at these prices is slightly greater than the quantity of base contracts that the electric utility wishes to sell, but the aggregate quantity of peak contracts desired by all the bidders at these prices is much greater than the quantity of peak contracts that the electric utility wishes to sell, the electric utility might then continue the auction by announcing a new price vector of ($10.60, $12.60, $13.60) for the three respective base durations and a price vector of ($29.20, $32.20, $34.20) for the three respective peak durations.","If the aggregate quantity of base contracts desired by all the bidders at these prices is still slightly greater than the quantity of base contracts that the electric utility wishes to sell, and the aggregate quantity of peak contracts desired by all the bidders at these prices is still somewhat greater than the quantity of peak contracts that the electric utility wishes to sell, the electric utility might then continue the auction by announcing a new price vector of ($10.60, $12.60, $13.60) for the three respective base durations and a price vector of ($29.40, $32.40, $34.40) for the three respective peak durations.","The auction continues in this way, with the electric utility always following the implied price differential in setting the relative prices for the various durations within a group.","(However, between groups, the prices may go up at very different rates.)","Meanwhile, bidders are held to the exemplary activity rule which constrains a bidder not to increase his quantity, summed over the types of items within a group, from one round to the next.","That is, the bidder is allowed to change the mix of 3-month, 6-month and 12-month base contracts that he demands, but his total quantity demanded of base contracts may not be increased as the price goes up.","Similarly, the bidder is allowed to change the mix of 3-month, 6-month and 12-month peak contracts that he demands, but his total quantity demanded of peak contracts may not be increased as the price goes up.","However, in this exemplary embodiment, the bidder is not allowed to substitute any quantity between the base group and the peak group.","In many of the preferred embodiments, the auction with two or more groups of types of items ends at the first moment in time that the aggregate quantity of items bid for by all the bidders within each group is less than or equal to the quantity of items being auctioned within each group.","An auction in accordance with an embodiment of the present invention could also allow various improvements to the bidding rules.","In the previous example of base contracts and peak contracts, it may be the case that even if the price of only one group goes up, a bidder may be permitted to reduce his demands in both product groups.","For example, if the base price vector is held constant and the peak price vector goes up, a bidder may be permitted to reduce his quantity bid on both base and peak products.","The reason for allowing this is that the base and peak product may be complements (loosely speaking, products that are useful together, such as left shoes and right shoes), so an increased price for one product could reduce a bidder's demand for both.","Throughout this document, the terms “objects”, “items”, “units” and “goods” are used essentially interchangeably.","The inventive system and method may be used both for tangible objects, such as real or personal property, and intangible items, such as telecommunications licenses or electric power.","The inventive system and method may be used in auctions where the auctioneer is a seller, buyer or broker, the bidders are buyers, sellers or brokers, and for auction-like activities which cannot be interpreted as selling or buying.","The inventive system and method may be used for items including, but not restricted to, the following: public-sector bonds, bills, notes, stocks, and other securities or derivatives; private-sector bonds, bills, notes, stocks, and other securities or derivatives; communication licenses and spectrum rights; clearing, relocation or other rights concerning encumbrances of spectrum licenses; electric power and other commodity items; rights for terminal, entry, exit or transmission capacities or other rights in gas pipeline systems; airport landing rights; emission allowances and pollution permits; and other goods, services, objects, items or other property, tangible or intangible.","It may also be used for option contracts on any of the above.","It may be used in initial public offerings, secondary offerings, and in secondary or resale markets.","The present invention is useful for “reverse auctions” conducted by or for buyers to acquire various kinds of items or resources, “standard auctions” conducted by sellers in which items are offered for sale, and “exchanges” in which both buyers and sellers place bids.","Although terms such as “items or quantities demanded” (by a bidder) and “demand curve” (of a bidder) are used to describe the present invention, the terms “items or quantities offered” (by a bidder) and “supply curve” (of a bidder) are equally applicable.","In some cases, this is made explicit by the use of both terms, or by the use of the terms “items or quantities transacted” (by a bidder) and “transaction curve” (of a bidder).","The term “items or quantities transacted” includes both “items or quantities demanded” and “items or quantities offered”.","The term “bid” includes both offers to sell and offers to buy.","The term “transaction curve” includes both “demand curve” and “supply curve”.","Moreover, any references to “items or quantities being offered” includes both “items or quantities being sold” by the auctioneer, in the case this is a standard auction for selling items, as well as “items or quantities being bought or procured” by the auctioneer, in the case this is a reverse auction for buying items or procuring items.","Moreover, while standard auctions to sell typically involve ascending prices and reverse auctions to buy typically involve descending prices, the present invention may utilize prices that ascend and/or descend.","While an auction method following this description could be conducted manually, computerized implementation of the auction method on an auction system allows the auction to be conducted with all bidding information taken into account, while carefully controlling the degree to which the information itself is disclosed to the participants.","In one embodiment, all bidding information is displayed to the bidders.","In another embodiment, no bidding information is displayed to the bidders; only the results of the auction are displayed.","A number of intermediate embodiments are also possible, in which some but not all bidding information is displayed to the bidders.","For example, in one preferred embodiment, the auctioneer discloses only the aggregate quantity bid for each type of item in each round, as opposed to disclosing each individual bid.","Computerized implementation of the auction also allows the auction to be conducted swiftly and reliably, even if bidders are not located on-site.","The auctioneer can receive large numbers of bids, determine the auction outcome, and transmit the results to bidders with minimal labor and in a very short span of time.","In particular, sophisticated dynamic auction methods become feasible with a computer implementation; it becomes practical to operate iterative auction procedures in which there may be ten or more auction rounds, yet bidders can participate from different parts of the globe and the entire process can conclude in a single day.","The present invention comprises a computer that receives bids in a static or dynamic bidding process and assigns the items to bidders, and a method for receiving bids in a static or dynamic bidding process and assigning the items to bidders.","In one embodiment, the invention comprises a Bidding Information Processor (BIP), a plurality of Bidding Terminals (BT's) communicatively coupled to the BIP, and an Auctioneer's Terminal (AT) communicatively coupled to the BIP.","Bidders at the BT's enter bids in multiple rounds, and may observe displayed auction information.","The BIP, BT's and AT communicate and process information in order to conduct an auction.","The network used, if any, can be any system capable of providing the necessary communication to/from a Bidding Information Processor (BIP), a Bidding Terminal (BT), and an Auctioneer's Terminal (AT).","The network may be a local or wide area network such as, for example, Ethernet, token ring, the Internet, the World Wide Web, the information superhighway, an intranet or a virtual private network, or alternatively a telephone system, either private or public, a facsimile system, an electronic mail system, or a wireless communications system, or combinations of the foregoing.","The following patents and published applications are related to the present invention: Ausubel, Lawrence M., U.S. Pat.","No.","5,905,975, May 1999.Ausubel, Lawrence M., U.S. Pat.","No.","6,021,398, Feb. 2000.Ausubel, Lawrence M., U.S. Pat.","No.","6,026,383, Feb. 2000.Ausubel, Lawrence M., Application No.","00304195.1 at the European Patent Office, May 2000.The following other references may be related to the present invention: Arrow, Kenneth, H. D. Block and Leonid Hurwicz (1959), “On the Stability of Competitive Equilibrium, II,” Econometrica 27: 82-109.Ausubel, Lawrence M. (1997a), “An Efficient Ascending-Bid Auction for Multiple Objects,” Working Paper 97-06, University of Maryland, downloadable at www-ausubel-com.","Ausubel, Lawrence M. (1997b), “On Generalizing the English Auction,” working paper, downloadable at www-ausubel-com.","Ausubel, Lawrence M. (2000), “An Efficient Dynamic Auction for Heterogeneous Commodities,” working paper, University of Maryland, downloadable at www-ausubel-com.","Ausubel, Lawrence M. and Peter Cramton (1996), “Demand Reduction and Inefficiency in Multi-Unit Auctions,” Working Paper No.","96-07, downloadable at www-ausubel-com.","Ausubel, Lawrence M. and Peter C. Cramton (1999), “The Optimality of Being Efficient,” mimeo, University of Maryland, June 1999, downloadable at www-ausubel-com.","Ausubel, Lawrence M., Peter Cramton, R. Preston McAfee, and John McMillan (1997), “Synergies in Wireless Telephony: Evidence from the Broadband PCS Auctions,” Journal of Economics and Management Strategy, 6:3, 497-527.Ausubel, Lawrence M. and Jesse Schwartz (1999), “The Ascending Auction Paradox,” Mimeo, University of Maryland and Vanderbilt University, July 1999.Banks, Jeffrey S., John O. Ledyard and David P. Porter, “Allocating Uncertain and Unresponsive Resources: An Experimental Approach,” Rand Journal of Economics, 20: 1-25.Bernheim, B. Douglas and Michael Whinston (1986), “Menu Auctions, Resource Allocation and Economic Influence,” Quarterly Journal of Economics, 101: 1-31.Bikhchandani, Sushil, “Auctions of Heterogeneous Objects.” Mimeo, UCLA, March 1996.Bikhchandani, Sushil and John W. Mamer (1997), “Competitive Equilibrium in an Exchange Economy with Indivisibilities,” Journal of Economic Theory, June, 74(2), pp.","385-413.Bikhchandani, Sushil and Joseph M. Ostroy (1999), “The Package Assignment Problem,” working paper, UCLA.","Cassady, Ralph (1967).","Auctions and Auctioneering .","Berkeley: University of California Press.","Charles River Associates and Market Design Inc. (1997), “Package Bidding for Spectrum Licenses,” Report to the Federal Communications Commission, downloadable at www-fcc-gov.","Clarke, E. H. (1971), “Multipart Pricing of Public Goods,” Public Choice, XI, 17-33.Computer Reseller News, “Onsale Joins Fray as Online Shopping Picks Up Speed: Internet Booms,” Jun.","5, 1995, p. 73; Dialog: File 16, Acc#05649796.Cramton, Peter C., “Money Out of Thin Air: The Nationwide Narrowband PCS Auction.” Journal of Economics and Management Strategy , Summer 1995, 4(2), pp.","267-343.Cybernomics (2000), “An Experimental Comparison of the Simultaneous Multiple Round Auction and the CRA Combinatorial Auction.” Report to the Federal Communications Commission, downloadable at www-fcc-gov.","Dasgupta, Partha and Eric S. Maskin (2000), “Efficient Auctions,” Quarterly Journal of Economics , May, 115(2), pp.","341-388.DasVarma, Gopal (2000), “The Pareto Optimality of Disclosing Bidder Identities,” working paper, Duke University.","Demange, Gabrielle, David Gale, and Marilda Sotomayor, “Multi-Item Auctions.” Journal of Political Economy , August 1986, 94(4), pp.","863-872.DeMartini, Christine, Anthony Kwasnica, John Ledyard, and David Porter (1999), “A New and Improved Design for Multi-Object Iterative Auctions,” Cal Tech working paper.","DeVries, Sven and Rakesh Vohra (2001), “Combinatorial Auctions: A Survey.” Working paper.","Available at www-m9.ma.tum-de.","Engelbrecht-Wiggans, Richard and Charles M. Kahn, “Protecting the Winner: Second-Price versus Oral Auctions.” Economics Letters , March 1991, 35(3), pp.","243-248.Engelbrecht-Wiggans, Richard and Charles M. Kahn (1998), “Multi-Unit Auctions with Uniform Prices,” Economic Theory , September, 12(2), pp.","227-258.Federal Communications Commission (1995), “Bidder Information Packet for FCC Auction Scheduled for Aug. 2, 1995.” Federal Communications Commission (1995), “Bidder Information Packet for FCC Auction Scheduled for Dec. 11, 1995.” Federal Communications Commission (2001), “Bidder Information Packet for FCC Auction No.","31.” Groves, Theodore (1973), “Incentives in Teams,” Econometrica 61: 617-31.Gul, Faruk and Ennio Stacchetti (1999), “Walrasian Equilibrium with Gross Substitutes,” Journal of Economic Theory, 87, 9-124.Gul, Faruk and Ennio Stacchetti (2000), “The English Auction with Differentiated Commodities,” Journal of Economic Theory, May, 92(1), pp.","66-95.Hahn, Frank H. (1982), “Stability,” in Kenneth J. Arrow and Michael D. Intriligator, eds., Handbook of Mathematical Economics, Volume II , Amsterdam: North-Holland,.","pp.","745-793.Jehiel, Philippe and Benny Moldovanu (1999), “Efficient Auctions with Interdependent Valuations,” Working Paper No.","99-74, University of Mannheim, July.","Jehiel, Philippe and Benny Moldovanu (2000), “A Critique of the Planned Rules for the German UMTS/IMT-2000 License Auction,” mimeo, University of Mannheim, March.","Jehiel, Philippe and Benny Moldovanu (2001), “The European UMTS/IMT-2000 License Auctions,” working paper.","Kagel, John H., Ronald M. Harstad, and Dan Levin, “Information Impact and Allocation Rules in Auctions with Affiliated Private Values: A Laboratory Study.” Econometrica , November 1987, 55(6), pp.","1275-1304.Kagel, John H., Scott Kinross, and Dan Levin (2001), “Comparing Efficient Multi-Object Auction Institutions,” mimeo, Ohio State University.","Kagel, John H. and Dan Levin (2001), “Behavior in Multi_Unit Demand Auctions: Experiments with Uniform Price and Dynamic Vickrey Auctions,” Econometrica, pp 413-454.Kelso, Alexander S. and Vincent P. Crawford (1982), “Job Matching, Coalition Formation, and Gross Substitutes,” Econometrica 50: 1483-1504.Krishna, Vijay and Motty Perry, “Efficient Mechanism Design.” Mimeo, Penn State University and Hebrew University, March 1997.Ledyard, John, David Porter and Antonio Rangel (1997), “Experiments Testing Multi-object Allocation Mechanisms,” Journal of Economics and Management Strategy, 6: 639-675.Manelli, Alejandro M., Martin Sefton and Benjamin S. Wilner, “Multi-Unit Auctions: A Comparison of Static and Dynamic Mechanisms.” Mimeo, Arizona State University, University of Newcastle and LECG/Navigant Consulting, October 1999.Maskin, Eric S. (1992), “Auctions and Privatization,” in Horst Siebert, ed., Privatization: Symposium in Honor of Herbert Giersch , Tubingen: Mohr (Siebeck), pp.","115-136.Maskin, Eric S. and John G. Riley, “Optimal Multi-unit Auctions,” in Frank Hahn, ed., The Economics of Missing Markets, Information, and Games .","Oxford: Oxford University Press, 1989, pp.","312-35.McAfee, R. Preston and John McMillan, “Auctions and Bidding.” Journal of Economic Literature , June 1987, 25(2), pp.","699-738.McCabe, Kevin A., Stephen J. Rassenti, and Vernon L. Smith, “Auction Institutional Design: Theory and Behavior of Simultaneous Multiple-Unit Generalizations of the Dutch and English Auctions.” American Economic Review , December 1990, 80(5), pp.","1276-1283.K.","A. McCabe, S. J. Rassenti and V. L. Smith, “Testing Vickrey's and Other Simultaneous Multiple Unit Versions of the English Auction,” Research in Experimental Economics, vol.","4, Greenwich, Conn.: JAI Press, 1991, pp.","45-79.McMillan, John (1994), “Selling Spectrum Rights,” Journal of Economics Perspectives, 8: 145-162.F.","M. Menezes, Four Essays on Auction Theory , University of Illinois doctoral dissertation, February 1993, pp.","1-97 and 143-152.Milgrom, Paul (1981), “Rational Expectations, Information Acquisition, and Competitive Bidding,” Econometrica, 49: 921-43.Milgrom, Paul (1987), “Auction Theory,” in Truman F. Bewley, ed., Advances in Economic Theory Fifth World Congress .","Cambridge: Cambridge University Press, 1987, pp.","1-32.Milgrom, Paul (1996), “Auctioning the Radio Spectrum,” downloadable at www-market-design.com.","Milgrom, Paul (2000).","“Putting Auction Theory to Work: The Simultaneous Ascending Auction,” Journal of Political Economy, 108:2, 245-272.Milgrom, Paul and John Roberts (1991), “Adaptive and Sophisticated Learning in Repeated * Normal Form Games,” Games and Economic Behavior, 3: 82-100.Milgrom, Paul and Robert Weber (1982), “A Theory of Auctions and Competitive Bidding,” Econometrica 50: 1089-1122.Milgrom, Paul R. and Robert J. Weber, “A Theory of Auctions and Competitive Bidding, II.” Unpublished manuscript, Northwestern University, 1982.Nisan, Noam (1999), “Bidding and Allocation in Combinatorial Auctions,” working paper, www-cs-huji-ac-il.","Onsale release, “Onsale Brings Thrill of Auctions and Bargain Hunting Online; Unique Internet retail service debuts with week-long charity auction for The Computer Museum in Boston”; Dialog Abstract: File 610, Acc#0489267; May 24, 1995.D.","C. Parkes (1999), “iBundle: An Efficient Ascending Price Bundle Auction”, Proceedings ACM Conference on Electronic Commerce (EC-99), Denver, 148-157.Perry, Motty and Philip J. Reny (1999a), “An Ex-Post Efficient Auction,” Working Paper, Hebrew University and University of Chicago, September.","Perry, Motty and Philip J. Reny (1999b), “An Ex-Post Efficient Multi-Unit Ascending Auction,” Working Paper, Hebrew University and University of Chicago, September.","Plott, Charles (1997), “Laboratory Experimental Testbeds: Application to the PCS Auction,” Journal of Economics and Management Strategy, 6: 605-638.Rassenti, S. J., V. L. Smith, and R. L. Bulfin (1982), “A Combinatorial Auction Mechanism for Airport Time Slot Allocation,” Bell Journal of Economics 13: 402-417.M.","H. Rothkopf, A. Pekec and R. M. Harstad (1998), “Computationally Manageable Combinatorial Auctions”, Management Science , Vol.","44, No.","8, 1131-1147.Rothkopf, Michael, Thomas Teisberg and Edward Kahn (1990), “Why Are Vickrey Auctions Rare?” Journal of Political Economy, 98: 94-109.Sholtz & Associates, LLC, “ACE Market Operations Guide,” Jan. 3, 1996, pp.","1-5.Siegmann, “Nowhere to Go But Up.","(Onsale CEO Jerry Kaplan) (PC Week Inside) (Inside People)” ; PC Week; v12 n42; pA5 (1); Oct. 23, 1995; Dialog: File 148, Acc#08222496.U.S.","Department of the Treasury, U.S. Securities and Exchange Commission, and Board of Governors of the Federal Reserve System, Joint Report on the Government Securities Market , Washington, D.C.: U.S.G.P.O., January 1992.Vickrey, William (1961), “Counterspeculation, Auctions and Competitive Sealed Tenders,” Journal of Finance , March, 16(1), pp.","8-37.Vickrey, William (1962), “Auctions and Bidding Games,” Recent Advances in Game Theory, Princeton: Princeton University Conference, 1962, pp.","15-29.Vickrey, William (1976), “Auctions, Markets, and Optimal Allocation,” Bidding and Auctioning for Procurement and Allocation, New York: New York University Press 1976, pp.","13-20.Weber, Robert J.","(1983), “Multiple-Object Auctions,” Auctions, Bidding, and Contracting: Uses and Theory, New York: New York University Press, 1983, pp.","165-191.Weber, Robert J.","(1997), “Making More from Less: Strategic Demand Reduction in the FCC Spectrum Auctions,” Journal of Economics and Management Strategy, 6:529-548.Williams, Steven (1999), “A Characterization of Efficient, Bayesian Incentive Compatible Mechanisms,” Economic Theory 14: 155-180.Wilson, Robert (1979), “Auctions of Shares,” Quarterly Journal of Economics , vol.","94, 1979, pp.675-689."],["RELATED APPLICATIONS This application claims the benefit of the filing dates of the following co-pending U.S. applications: Ausubel, “SYSTEM AND METHOD FOR AN EFFICIENT DYNAMIC AUCTION FOR MULTIPLE TYPES OF ITEMS”, U.S.","Provisional Patent Application, Ser.","No.","60/294,246 Filed: May 31, 2001.Ausubel, “SYSTEM AND METHOD FOR AN EFFICIENT DYNAMIC MULTI-UNIT AUCTION”, U.S. patent application, Ser.","No.","09/898,483 Filed: Jul.","05, 2001.FIELD OF THE INVENTION The present invention relates to improving computer-implemented auctions and, more particularly, to computer implementation of an auction in which multiple types of items are auctioned together.","BACKGROUND OF THE INVENTION Sellers and buyers of commodities, financial securities, and other goods and services are increasingly turning to auctions to assist in the sale or purchase of a wide range of items.","Auctions are useful for selling a unique item (e.g., a painting), a single type of item available in multiple units (e.g., 10 tons of gold), and multiple types of items available in multiple units (e.g., 10 tons of gold and 20 tons of silver).","And just as auctions are useful for efficiently selling items at maximum price, they are also useful for efficiently procuring items at minimum cost.","Auction formats in the art tend generally to be of the sealed-bid or ascending-bid variety.","In standard sealed-bid auctions, bidders—in one single bidding round—simultaneously and independently submit bids to the auctioneer, who then determines the auction outcome.","In standard ascending-bid auctions, bidders—in a dynamic bidding process—submit bids in real time until no more bids are forthcoming.","Sealed-bid auction formats offer the advantage of speed of the auction process.","Ascending-bid auction formats offer the advantage that there is feedback among participants' bids, which tends to result in more aggressive bidding and in more efficient auction outcomes.","When auction systems and methods in the art are used to sell (or buy) multiple types of items, the auctioneer generally specifies an available supply (or demand) for each type of item.","For example, the auctioneer announces a supply of 10 tons of gold and 20 tons of silver, and then solicits bids, in a sealed-bid or ascending-bid procedure, in order to obtain market-clearing prices for each of the two types of commodities.","However, consider a situation where the types of items are related, for example, contracts for provision of a commodity covering different time periods.","For example, a government may wish to sell three types of financial securities: 3-month Treasury bills; 6-month Treasury bills; and 12-month Treasury bills.","The government's only constraint may be that it needs to sell $50 billion worth of new debt securities at this auction, since this is the quantity of old debt securities that are maturing in the current calendar quarter.","However, the government may have no preference as to how the required $50 billion is divided among 3-month bills, 6-month bills, and 12-month bills, respectively, and the government may simply wish to “let the market decide” the division among the various durations Auction systems and methods in the art provide effective ways for the government to sell bonds with a fixed division among the various durations.","However, new and better systems and methods can be devised for “letting the market decide” the division among the various durations.","The same problem also occurs in real life in the sale of contracts for electricity and all kinds of other commodities, as well as in the procurement of related items.","In several preferred embodiments, the present invention provides a system and method for the government in the above example to simply auction $50 billion of Treasury bills at the lowest possible cost (relative to the market's yield curve), without imposing a particular division among the various durations.","SUMMARY OF THE INVENTION The present invention is an improved system and method for a computer-implemented auction, particularly for a computer-implemented auction in which multiple types of items are auctioned together.","In several embodiments, the present invention provides a system and method to conduct an auction of a group of related types of items, without imposing a particular division of supply or demand among the individual types of items within the group.","In other embodiments, the present invention provides a system and method to conduct an auction of a group of related types of items, without imposing a particular division of supply or demand on some of the individual types of items within the group.","Instead, in some embodiments, the present invention provides a means or method for establishing prices for the types of items within a group, wherein the prices maintain a relationship.","In other embodiments, the present invention provides a means or method for implying prices from price parameters in the bids received from bidders, based on a relation among the prices for the types of items within a group.","In some embodiments, market clearing is defined by the condition that the aggregate quantity bid for all types of items within a group is less than or equal to the available quantity of all types of items within the group.","The division among the types of items within the group is thus determined flexibly, based on the bids at the associated prices.","In other embodiments, market clearing is defined by the condition that the quantity bid for one selected type of item within a group is less than or equal to the available quantity of the selected type of item.","The quantities of the other types of items within the group are thus determined flexibly, based on the bids at the associated prices.","In a first preferred embodiment, the present invention is: A computer implemented method for conducting an auction of a plurality of types of items among a plurality of bidders, wherein bids are received and an allocation of the items among the bidders is determined based on the bids, comprising: establishing prices for the plurality of types of items, wherein the prices maintain a relationship, receiving bids associated with the established prices, and processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, otherwise altering the established prices for the plurality of types of items.","In a second preferred embodiment, the present invention is a computer system for conducting an auction according to the method of the first preferred embodiment.","The system includes an establishing means, a receiving means, and a processing means.","In both the first and second preferred embodiments, the relationship or relation among the prices may involve a schedule or algorithm.","In the event that the relationship or relation is based on a schedule, determining prices may involve looking up and possibly interpolating numbers that are contained in a table.","Alternatively the relationship or relation may be represented as an algorithm or a formula that provides a relationship among the prices.","The data table may reflect a market beyond the auction (for example, it may reflect price differentials in a separate commodities market or exchange) and the data table may change during the course of the auction (for example, if price differentials change in the separate commodities market or exchange, or if a significant news event occurs during the course of the auction).","Similarly, the data set to which the algorithm or formula are applied may reflect a market beyond the auction and the data set may change during the course of the auction.","In a third preferred embodiment, the present invention is: A computer implemented method for conducting an auction of a plurality of types of items among a plurality of bidders, wherein bids are received and an allocation of the items among the bidders is determined based on the bids, comprising: receiving bids comprising a quantity parameter for at least one of the types of items in the auction and a price parameter, and processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, wherein the processing includes implying prices from price parameters in the received bids based on a fixed relation among the prices of the plurality of types of items.","In a fourth preferred embodiment, the present invention is a computer system for conducting an auction according to the method of the third preferred embodiment.","The system includes a receiving means and a processing means.","In both the third and fourth preferred embodiments, the “price parameters” in the received bids that are used for “implying prices” could be prices for one or more selected types of items.","(For example, the price parameter could be an actual price for 3-month Treasury bills, and the prices for 6-month and 12-month Treasury bills could be implied, based on a fixed relation among the prices.)","The “price parameters” could also be neutral parameters or indicators (e.g., in the nature of an index) of a price.","Indeed, in a fifth preferred embodiment, bids include price parameters that may be a particularly useful form of neutral parameter.","For example, an auctioneer may announce a starting price vector of (4.00, 4.50, 4.75) and an ending price vector of (8.00, 8.50, 8.75).","If a bidder enters a price parameter of 25%, this may signify that the bidder is indicating a price which is 25% of the distance from the starting price to the ending price, i.e., an implied price vector of (5.00, 5.50, 5.75).","In the fifth preferred embodiment, the present invention is: A computer implemented method for conducting an auction of at least one type of item among a plurality of bidders in a plurality of rounds, wherein bids are received and an allocation of the items among the bidders is determined based on the bids, comprising: establishing starting prices and ending prices for each type of item in a plurality of rounds, receiving bids, wherein a bid comprises a quantity for at least one type of item and a price parameter signifying a percentage of the distance from the established starting prices to the established ending prices, and processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, otherwise altering the established prices for at least one type of item.","In a sixth preferred embodiment, the present invention is a computer system for conducting an auction according to the method of the fifth preferred embodiment.","The system includes an establishing means, a receiving means, and a processing means.","Various preferred embodiments of the present invention resolve a number of important limitations to the systems and methods for computer-implemented auctions in the art.","Thus, the present invention offers the further technical effect of improving computer-implemented auctions, making them operate more efficiently.","The present invention improves the ability of bidders to readily participate in the auction and to express their needs and preferences.","The present invention improves the ability of the auctioneer to sell at higher prices the items being sold in the auction or to obtain at lower cost the items being procured in the auction.","The present invention improves the efficiency of the auction process by enabling the auction to operate in shorter time, expending less computer time, fewer communications resources, and less bidder and auctioneer time.","Finally, the present invention improves the allocative efficiency of the auction process by better allocating items to bidders who value them the highest (or, in a procurement auction, to bidders who can provide them at lowest cost).","Certain constraints are desirable in order for the present invention to operate optimally and to reach an economically efficient outcome.","In many auctions, it is desirable to impose constraints on bidders that require them to bid significantly on items early in the auction in order for them to be allowed to bid on items later in the auction.","Otherwise, one may find oneself in a situation where bidders refrain from submitting serious bids until near the very end of the auction, defeating the purpose of a dynamic auction.","One exemplary constraint is an activity rule which constrains a bidder not to increase his quantity, summed over all of the types of items within a group, from one round to the next.","Another exemplary constraint is a more stringent activity rule which constrains a bidder not to increase his quantity, individually on each of the individual types of items, from one bid or one round to the next.","A third exemplary constraint is a reduction rule which constrains a bidder not to decrease his quantity, for any single type of item, beyond the point where the sum of the quantities bid for this type of item by all bidders equals the sum of the quantities being auctioned.","(If, in a given round, two or more bidders simultaneously attempt to decrease their quantities, for any single type of item, having the effect of reducing bids beyond the point where the sum of the quantities bid for this type of item by all bidders equals the sum of the quantities being auctioned, the auction procedure will resolve this discrepancy.","For example, the auctioneer may honor these attempts to decrease in order of time priority, or may ration these simultaneous attempts to decrease in proportion to the attempted reductions.)","The present invention includes treating the case of auctioning a set of items which includes two (or more) items that are neither identical nor perfect substitutes to one another.","Henceforth, this will be described for short as a situation with “multiple types of items,” or simply “heterogeneous items” or “heterogeneous objects.” Often, but not always, the heterogeneous items auctioned together will bear some relationship to one another: for example, they may be securities issued by the same entity but with different durations to maturity; or contracts for the same goods or services but with different durations.","One particularly salient use for the present invention is as follows.","A national government or a private corporation may wish to simultaneously sell 3-month, 6-month and 12-month bonds or notes.","More generally, these may be contracts for provision of any commodity; the contracts are for the same commodity, but covering different but possibly overlapping time periods.","For the government's or corporation's purposes, the allocation of sales among the 3-month, 6-month and 12-month bonds or notes may not be critical; the main issue is that the total quantity of the bonds or notes of the three durations add up to the desired dollar figure.","Meanwhile, the current “yield curve” on financial markets may have 6-month bonds or notes yielding interest rates 0.50% higher than 3-month bonds or notes, and the current “yield curve” on financial markets may have 12-month bonds or notes yielding interest rates 0.25% higher than 6-month bonds or notes.","Under these circumstances, the government or corporation may utilize the present invention by beginning the auction with an (interest rate) vector of (6.00%, 6.50%, 6.75%) for the three respective durations.","If the aggregate quantity of the bonds or notes desired by all the bidders at these interest rates is greater than the quantity of bonds or notes that the government or corporation wishes to sell, the government or corporation might then continue the auction by announcing a new vector of (5.99%, 6.49%, 6.74%) for the three respective durations.","If the aggregate quantity of the bonds or notes desired by all the bidders at these interest rates is still greater than the quantity of bonds or notes that the government or corporation wishes to sell, the government or corporation might then continue the auction by announcing a new vector of (5.98%, 6.48%, 6.73%) for the three respective durations.","The auction continues in this way, with the government or corporation always following the yield curve in setting.","the relative interest rates for the various durations.","Meanwhile, bidders are held to the exemplary activity rule which constrains a bidder not to increase his quantity, summed over the m types of items, from one round to the next.","That is, the bidder is allowed to change the mix of 3-month, 6-month and 12-month bonds or notes that he demands, but his total quantity demanded of bonds or notes may not be increased as the interest rate goes down.","A second use for the present invention is as follows.","An electric utility may wish to sell contracts to provide a given quantity of electricity for 3-month, 6-month and 12-month durations.","For the electric utility's purposes, the allocation of sales among the 3-month, 6-month and 12-month contracts may not be critical; the main issue may be that the total quantity of the contracts add up to a desired amount of electricity.","Meanwhile, current energy prices may imply that a 6-month contract should be priced at $2 per-unit higher than a 3-month contract, and that a 12-month contract should be priced at $1 per unit higher than a 6-month contract.","Under these circumstances, the electric utility may utilize the present invention by beginning the auction with a price vector of ($10.50, $12.50, $13.50) for the three respective durations.","If the aggregate quantity of contracts desired by all the bidders at these prices is greater than the quantity of contracts that the electric utility wishes to sell, the electric utility might then continue the auction by announcing a new price vector of ($10.60, $12.60, $13.60) for the three respective durations.","If the aggregate quantity of contracts desired by all the bidders at these prices is still greater than the quantity of contracts that the electric utility wishes to sell, the electric utility might then continue the auction by announcing a new price vector of ($10.70, $12.70, $13.70) for the three respective durations.","The auction continues in this way, with the electric utility always following the implied price differential in setting the relative prices for the various durations.","Meanwhile, bidders are held to the exemplary activity rule which constrains a bidder not to increase his quantity, summed over the types of items within a group, from one round to the next.","That is, the bidder is allowed to change the mix of 3-month, 6-month and 12-month contracts that he demands, but his total quantity demanded of contracts may not be increased as the price goes up.","In addition, the present invention may also be used to auction two or more groups of types of items.","For example, the previous electric utility example could be complicated by the presence of two groups of electricity contracts: base and peak.","(Base-load quantity is produced all of the time while peak-load quantity is only produced at times of peak demand.)","Within each group, there might be three durations of contracts.","Thus, in total, there could be six contracts for sale: 3-month, 6-month and 12-month base contracts; and 3-month, 6-month and 12-month peak contracts.","To continue the example, current energy prices may imply that a 6-month base contract should be priced at $2 per unit higher than a 3-month base contract, and that a 12-month base contract should be priced at $1 per unit higher than a 6-month base contract.","Meanwhile, current energy prices may imply that a 6-month peak contract should be priced at $3 per unit higher than a 3-month peak contract, and that a 12-month peak contract should be priced at $2 per unit higher than a 6-month peak contract.","Under these circumstances, the electric utility may utilize the present invention by beginning the auction with a price vector of ($10.50, $12.50, $13.50) for the three respective base durations and a price vector of ($26.50, $29.50, $31.50) for the three respective peak durations.","If the aggregate quantity of base contracts desired by all the bidders at these prices is slightly greater than the quantity of base contracts that the electric utility wishes to sell, but the aggregate quantity of peak contracts desired by all the bidders at these prices is much greater than the quantity of peak contracts that the electric utility wishes to sell, the electric utility might then continue the auction by announcing a new price vector of ($10.60, $12.60, $13.60) for the three respective base durations and a price vector of ($29.20, $32.20, $34.20) for the three respective peak durations.","If the aggregate quantity of base contracts desired by all the bidders at these prices is still slightly greater than the quantity of base contracts that the electric utility wishes to sell, and the aggregate quantity of peak contracts desired by all the bidders at these prices is still somewhat greater than the quantity of peak contracts that the electric utility wishes to sell, the electric utility might then continue the auction by announcing a new price vector of ($10.60, $12.60, $13.60) for the three respective base durations and a price vector of ($29.40, $32.40, $34.40) for the three respective peak durations.","The auction continues in this way, with the electric utility always following the implied price differential in setting the relative prices for the various durations within a group.","(However, between groups, the prices may go up at very different rates.)","Meanwhile, bidders are held to the exemplary activity rule which constrains a bidder not to increase his quantity, summed over the types of items within a group, from one round to the next.","That is, the bidder is allowed to change the mix of 3-month, 6-month and 12-month base contracts that he demands, but his total quantity demanded of base contracts may not be increased as the price goes up.","Similarly, the bidder is allowed to change the mix of 3-month, 6-month and 12-month peak contracts that he demands, but his total quantity demanded of peak contracts may not be increased as the price goes up.","However, in this exemplary embodiment, the bidder is not allowed to substitute any quantity between the base group and the peak group.","In many of the preferred embodiments, the auction with two or more groups of types of items ends at the first moment in time that the aggregate quantity of items bid for by all the bidders within each group is less than or equal to the quantity of items being auctioned within each group.","An auction in accordance with an embodiment of the present invention could also allow various improvements to the bidding rules.","In the previous example of base contracts and peak contracts, it may be the case that even if the price of only one group goes up, a bidder may be permitted to reduce his demands in both product groups.","For example, if the base price vector is held constant and the peak price vector goes up, a bidder may be permitted to reduce his quantity bid on both base and peak products.","The reason for allowing this is that the base and peak product may be complements (loosely speaking, products that are useful together, such as left shoes and right shoes), so an increased price for one product could reduce a bidder's demand for both.","Throughout this document, the terms “objects”, “items”, “units” and “goods” are used essentially interchangeably.","The inventive system and method may be used both for tangible objects, such as real or personal property, and intangible items, such as telecommunications licenses or electric power.","The inventive system and method may be used in auctions where the auctioneer is a seller, buyer or broker, the bidders are buyers, sellers or brokers, and for auction-like activities which cannot be interpreted as selling or buying.","The inventive system and method may be used for items including, but not restricted to, the following: public-sector bonds, bills, notes, stocks, and other securities or derivatives; private-sector bonds, bills, notes, stocks, and other securities or derivatives; communication licenses and spectrum rights; clearing, relocation or other rights concerning encumbrances of spectrum licenses; electric power and other commodity items; rights for terminal, entry, exit or transmission capacities or other rights in gas pipeline systems; airport landing rights; emission allowances and pollution permits; and other goods, services, objects, items or other property, tangible or intangible.","It may also be used for option contracts on any of the above.","It may be used in initial public offerings, secondary offerings, and in secondary or resale markets.","The present invention is useful for “reverse auctions” conducted by or for buyers to acquire various kinds of items or resources, “standard auctions” conducted by sellers in which items are offered for sale, and “exchanges” in which both buyers and sellers place bids.","Although terms such as “items or quantities demanded” (by a bidder) and “demand curve” (of a bidder) are used to describe the present invention, the terms “items or quantities offered” (by a bidder) and “supply curve” (of a bidder) are equally applicable.","In some cases, this is made explicit by the use of both terms, or by the use of the terms “items or quantities transacted” (by a bidder) and “transaction curve” (of a bidder).","The term “items or quantities transacted” includes both “items or quantities demanded” and “items or quantities offered”.","The term “bid” includes both offers to sell and offers to buy.","The term “transaction curve” includes both “demand curve” and “supply curve”.","Moreover, any references to “items or quantities being offered” includes both “items or quantities being sold” by the auctioneer, in the case this is a standard auction for selling items, as well as “items or quantities being bought or procured” by the auctioneer, in the case this is a reverse auction for buying items or procuring items.","Moreover, while standard auctions to sell typically involve ascending prices and reverse auctions to buy typically involve descending prices, the present invention may utilize prices that ascend and/or descend.","While an auction method following this description could be conducted manually, computerized implementation of the auction method on an auction system allows the auction to be conducted with all bidding information taken into account, while carefully controlling the degree to which the information itself is disclosed to the participants.","In one embodiment, all bidding information is displayed to the bidders.","In another embodiment, no bidding information is displayed to the bidders; only the results of the auction are displayed.","A number of intermediate embodiments are also possible, in which some but not all bidding information is displayed to the bidders.","For example, in one preferred embodiment, the auctioneer discloses only the aggregate quantity bid for each type of item in each round, as opposed to disclosing each individual bid.","Computerized implementation of the auction also allows the auction to be conducted swiftly and reliably, even if bidders are not located on-site.","The auctioneer can receive large numbers of bids, determine the auction outcome, and transmit the results to bidders with minimal labor and in a very short span of time.","In particular, sophisticated dynamic auction methods become feasible with a computer implementation; it becomes practical to operate iterative auction procedures in which there may be ten or more auction rounds, yet bidders can participate from different parts of the globe and the entire process can conclude in a single day.","The present invention comprises a computer that receives bids in a static or dynamic bidding process and assigns the items to bidders, and a method for receiving bids in a static or dynamic bidding process and assigning the items to bidders.","In one embodiment, the invention comprises a Bidding Information Processor (BIP), a plurality of Bidding Terminals (BT's) communicatively coupled to the BIP, and an Auctioneer's Terminal (AT) communicatively coupled to the BIP.","Bidders at the BT's enter bids in multiple rounds, and may observe displayed auction information.","The BIP, BT's and AT communicate and process information in order to conduct an auction.","The network used, if any, can be any system capable of providing the necessary communication to/from a Bidding Information Processor (BIP), a Bidding Terminal (BT), and an Auctioneer's Terminal (AT).","The network may be a local or wide area network such as, for example, Ethernet, token ring, the Internet, the World Wide Web, the information superhighway, an intranet or a virtual private network, or alternatively a telephone system, either private or public, a facsimile system, an electronic mail system, or a wireless communications system, or combinations of the foregoing.","The following patents and published applications are related to the present invention: Ausubel, Lawrence M., U.S. Pat.","No.","5,905,975, May 1999.Ausubel, Lawrence M., U.S. Pat.","No.","6,021,398, Feb. 2000.Ausubel, Lawrence M., U.S. Pat.","No.","6,026,383, Feb. 2000.Ausubel, Lawrence M., Application No.","00304195.1 at the European Patent Office, May 2000.The following other references may be related to the present invention: Arrow, Kenneth, H. D. Block and Leonid Hurwicz (1959), “On the Stability of Competitive Equilibrium, II,” Econometrica 27: 82-109.Ausubel, Lawrence M. (1997a), “An Efficient Ascending-Bid Auction for Multiple Objects,” Working Paper 97-06, University of Maryland, downloadable at www-ausubel-com.","Ausubel, Lawrence M. (1997b), “On Generalizing the English Auction,” working paper, downloadable at www-ausubel-com.","Ausubel, Lawrence M. (2000), “An Efficient Dynamic Auction for Heterogeneous Commodities,” working paper, University of Maryland, downloadable at www-ausubel-com.","Ausubel, Lawrence M. and Peter Cramton (1996), “Demand Reduction and Inefficiency in Multi-Unit Auctions,” Working Paper No.","96-07, downloadable at www-ausubel-com.","Ausubel, Lawrence M. and Peter C. Cramton (1999), “The Optimality of Being Efficient,” mimeo, University of Maryland, June 1999, downloadable at www-ausubel-com.","Ausubel, Lawrence M., Peter Cramton, R. Preston McAfee, and John McMillan (1997), “Synergies in Wireless Telephony: Evidence from the Broadband PCS Auctions,” Journal of Economics and Management Strategy, 6:3, 497-527.Ausubel, Lawrence M. and Jesse Schwartz (1999), “The Ascending Auction Paradox,” Mimeo, University of Maryland and Vanderbilt University, July 1999.Banks, Jeffrey S., John O. Ledyard and David P. Porter, “Allocating Uncertain and Unresponsive Resources: An Experimental Approach,” Rand Journal of Economics, 20: 1-25.Bernheim, B. Douglas and Michael Whinston (1986), “Menu Auctions, Resource Allocation and Economic Influence,” Quarterly Journal of Economics, 101: 1-31.Bikhchandani, Sushil, “Auctions of Heterogeneous Objects.” Mimeo, UCLA, March 1996.Bikhchandani, Sushil and John W. Mamer (1997), “Competitive Equilibrium in an Exchange Economy with Indivisibilities,” Journal of Economic Theory, June, 74(2), pp.","385-413.Bikhchandani, Sushil and Joseph M. Ostroy (1999), “The Package Assignment Problem,” working paper, UCLA.","Cassady, Ralph (1967).","Auctions and Auctioneering.","Berkeley: University of California Press.","Charles River Associates and Market Design Inc. (1997), “Package Bidding for Spectrum Licenses,” Report to the Federal Communications Commission, downloadable at www-fcc-gov.","Clarke, E. H. (1971), “Multipart Pricing of Public Goods,” Public Choice, XI, 17-33.Computer Reseller News, “Onsale Joins Fray as Online Shopping Picks Up Speed: Internet Booms,” Jun.","5, 1995, p. 73; Dialog: File 16, Acc#05649796.Cramton, Peter C., “Money Out of Thin Air: The Nationwide Narrowband PCS Auction.” Journal of Economics and Management Strategy, Summer 1995, 4(2), pp.","267-343.Cybernomics (2000), “An Experimental Comparison of the Simultaneous Multiple Round Auction and the CRA Combinatorial Auction.” Report to the Federal Communications Commission, downloadable at www-fcc-gov.","Dasgupta, Partha and Eric S. Maskin (2000), “Efficient Auctions,” Quarterly Journal of Economics, May, 115(2), pp.","341-388.DasVarma, Gopal (2000), “The Pareto Optimality of Disclosing Bidder Identities,” working paper, Duke University.","Demange, Gabrielle, David Gale, and Marilda Sotomayor, “Multi-Item Auctions.” Journal of Political Economy, August 1986, 94(4), pp.","863-872.DeMartini, Christine, Anthony Kwasnica, John Ledyard, and David Porter (1999), “A New and Improved Design for Multi-Object Iterative Auctions,” Cal Tech working paper.","DeVries, Sven and Rakesh Vohra (2001), “Combinatorial Auctions: A Survey.” Working paper.","Available at www-m9.ma.tum-de.","Engelbrecht-Wiggans, Richard and Charles M. Kahn, “Protecting the Winner: Second-Price versus Oral Auctions.” Economics Letters, March 1991, 35(3), pp.","243-248.Engelbrecht-Wiggans, Richard and Charles M. Kahn (1998), “Multi-Unit Auctions with Uniform Prices,” Economic Theory, September, 12(2), pp.","227-258.Federal Communications Commission (1995), “Bidder Information Packet for FCC Auction Scheduled for Aug. 2, 1995.” Federal Communications Commission (1995), “Bidder Information Packet for FCC Auction Scheduled for Dec. 11, 1995.” Federal Communications Commission (2001), “Bidder Information Packet for FCC Auction No.","31.” Groves, Theodore (1973), “Incentives in Teams,” Econometrica 61: 617-31.Gul, Faruk and Ennio Stacchetti (1999), “Walrasian Equilibrium with Gross Substitutes,” Journal of Economic Theory, 87, 9-124.Gul, Faruk and Ennio Stacchetti (2000), “The English Auction with Differentiated Commodities,” Journal of Economic Theory, May, 92(1), pp.","66-95.Hahn, Frank H. (1982), “Stability,” in Kenneth J. Arrow and Michael D. Intriligator, eds., Handbook of Mathematical Economics, Volume II, Amsterdam: North-Holland,.","pp.","745-793.Jehiel, Philippe and Benny Moldovanu (1999), “Efficient Auctions with Interdependent Valuations,” Working Paper No.","99-74, University of Mannheim, July.","Jehiel, Philippe and Benny Moldovanu (2000), “A Critique of the Planned Rules for the German UMTS/IMT-2000 License Auction,” mimeo, University of Mannheim, March.","Jehiel, Philippe and Benny Moldovanu (2001), “The European UMTS/IMT-2000 License Auctions,” working paper.","Kagel, John H., Ronald M. Harstad, and Dan Levin, “Information Impact and Allocation Rules in Auctions with Affiliated Private Values: A Laboratory Study.” Econometrica, November 1987, 55(6), pp.","1275-1304.Kagel, John H., Scott Kinross, and Dan Levin (2001), “Comparing Efficient Multi-Object Auction Institutions,” mimeo, Ohio State University.","Kagel, John H. and Dan Levin (2001), “Behavior in Multi_Unit Demand Auctions: Experiments with Uniform Price and Dynamic Vickrey Auctions,” Econometrica, pp 413-454.Kelso, Alexander S. and Vincent P. Crawford (1982), “Job Matching, Coalition Formation, and Gross Substitutes,” Econometrica 50: 1483-1504.Krishna, Vijay and Motty Perry, “Efficient Mechanism Design.” Mimeo, Penn State University and Hebrew University, March 1997.Ledyard, John, David Porter and Antonio Rangel (1997), “Experiments Testing Multi-object Allocation Mechanisms,” Journal of Economics and Management Strategy, 6: 639-675.Manelli, Alejandro M., Martin Sefton and Benjamin S. Wilner, “Multi-Unit Auctions: A Comparison of Static and Dynamic Mechanisms.” Mimeo, Arizona State University, University of Newcastle and LECG/Navigant Consulting, October 1999.Maskin, Eric S. (1992), “Auctions and Privatization,” in Horst Siebert, ed., Privatization: Symposium in Honor of Herbert Giersch, Tubingen: Mohr (Siebeck), pp.","115-136.Maskin, Eric S. and John G. Riley, “Optimal Multi-unit Auctions,” in Frank Hahn, ed., The Economics of Missing Markets, Information, and Games.","Oxford: Oxford University Press, 1989, pp.","312-35.McAfee, R. Preston and John McMillan, “Auctions and Bidding.” Journal of Economic Literature, June 1987, 25(2), pp.","699-738.McCabe, Kevin A., Stephen J. Rassenti, and Vernon L. Smith, “Auction Institutional Design: Theory and Behavior of Simultaneous Multiple-Unit Generalizations of the Dutch and English Auctions.” American Economic Review, December 1990, 80(5), pp.","1276-1283.K.","A. McCabe, S. J. Rassenti and V. L. Smith, “Testing Vickrey's and Other Simultaneous Multiple Unit Versions of the English Auction,” Research in Experimental Economics, vol.","4, Greenwich, Conn.: JAI Press, 1991, pp.","45-79.McMillan, John (1994), “Selling Spectrum Rights,” Journal of Economics Perspectives, 8: 145-162.F.","M. Menezes, Four Essays on Auction Theory, University of Illinois doctoral dissertation, February 1993, pp.","1-97 and 143-152.Milgrom, Paul (1981), “Rational Expectations, Information Acquisition, and Competitive Bidding,” Econometrica, 49: 921-43.Milgrom, Paul (1987), “Auction Theory,” in Truman F. Bewley, ed., Advances in Economic Theory Fifth World Congress.","Cambridge: Cambridge University Press, 1987, pp.","1-32.Milgrom, Paul (1996), “Auctioning the Radio Spectrum,” downloadable at www-market-design.com.","Milgrom, Paul (2000).","“Putting Auction Theory to Work: The Simultaneous Ascending Auction,” Journal of Political Economy, 108:2, 245-272.Milgrom, Paul and John Roberts (1991), “Adaptive and Sophisticated Learning in Repeated * Normal Form Games,” Games and Economic Behavior, 3: 82-100.Milgrom, Paul and Robert Weber (1982), “A Theory of Auctions and Competitive Bidding,” Econometrica 50: 1089-1122.Milgrom, Paul R. and Robert J. Weber, “A Theory of Auctions and Competitive Bidding, II.” Unpublished manuscript, Northwestern University, 1982.Nisan, Noam (1999), “Bidding and Allocation in Combinatorial Auctions,” working paper, www-cs-huji-ac-il.","Onsale release, “Onsale Brings Thrill of Auctions and Bargain Hunting Online; Unique Internet retail service debuts with week-long charity auction for The Computer Museum in Boston”; Dialog Abstract: File 610, Acc#0489267; May 24, 1995.D.","C. Parkes (1999), “iBundle: An Efficient Ascending Price Bundle Auction”, Proceedings ACM Conference on Electronic Commerce (EC-99), Denver, 148-157.Perry, Motty and Philip J. Reny (1999a), “An Ex-Post Efficient Auction,” Working Paper, Hebrew University and University of Chicago, September.","Perry, Motty and Philip J. Reny (1999b), “An Ex-Post Efficient Multi-Unit Ascending Auction,” Working Paper, Hebrew University and University of Chicago, September.","Plott, Charles (1997), “Laboratory Experimental Testbeds: Application to the PCS Auction,” Journal of Economics and Management Strategy, 6: 605-638.Rassenti, S. J., V. L. Smith, and R. L. Bulfin (1982), “A Combinatorial Auction Mechanism for Airport Time Slot Allocation,” Bell Journal of Economics 13: 402-417.M.","H. Rothkopf, A. Pekec and R. M. Harstad (1998), “Computationally Manageable Combinatorial Auctions”, Management Science, Vol.","44, No.","8, 1131-1147.Rothkopf, Michael, Thomas Teisberg and Edward Kahn (1990), “Why Are Vickrey Auctions Rare?” Journal of Political Economy, 98: 94-109.Sholtz & Associates, LLC, “ACE Market Operations Guide,” Jan. 3, 1996, pp.","1-5.Siegmann, “Nowhere to Go But Up.","(Onsale CEO Jerry Kaplan) (PC Week Inside) (Inside People)” ; PC Week; v12 n42; pA5 (1); Oct. 23, 1995; Dialog: File 148, Acc#08222496.U.S.","Department of the Treasury, U.S. Securities and Exchange Commission, and Board of Governors of the Federal Reserve System, Joint Report on the Government Securities Market, Washington, D.C.: U.S.G.P.O., January 1992.Vickrey, William (1961), “Counterspeculation, Auctions and Competitive Sealed Tenders,” Journal of Finance, March, 16(1), pp.","8-37.Vickrey, William (1962), “Auctions and Bidding Games,” Recent Advances in Game Theory, Princeton: Princeton University Conference, 1962, pp.","15-29.Vickrey, William (1976), “Auctions, Markets, and Optimal Allocation,” Bidding and Auctioning for Procurement and Allocation, New York: New York University Press 1976, pp.","13-20.Weber, Robert J.","(1983), “Multiple-Object Auctions,” Auctions, Bidding, and Contracting: Uses and Theory, New York: New York University Press, 1983, pp.","165-191.Weber, Robert J.","(1997), “Making More from Less: Strategic Demand Reduction in the FCC Spectrum Auctions,” Journal of Economics and Management Strategy, 6:529-548.Williams, Steven (1999), “A Characterization of Efficient, Bayesian Incentive Compatible Mechanisms,” Economic Theory 14: 155-180.Wilson, Robert (1979), “Auctions of Shares,” Quarterly Journal of Economics, vol.","94, 1979, pp.675-689.BRIEF DESCRIPTION OF THE DRAWINGS FIG.","1 is a graphical depiction of the architecture of an exemplary computer system in accordance with an embodiment of the invention; FIG.","2 is a graphical depiction of another exemplary computer system in accordance with an embodiment of the invention; FIG.","3 is a detail of one element of the computer system of FIG.","2; FIG.","4 is a flow diagram of an exemplary auction process in accordance with one embodiment of the invention; FIGS.","5a and 5b are flow diagrams illustrating, in greater detail, elements of the flow diagram of FIG.","4; FIGS.","6a and 6b are flow diagrams illustrating, in greater detail, elements of the flow diagram of FIG.","4; FIG.","7 is a flow diagram illustrating, in greater detail, an element of the flow diagram of FIG.","4; and FIGS.","8a, 8b and 8c are flow diagrams illustrating, in greater detail, elements of the flow diagram of FIG.","4.DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Overall Structure of Auction System Earlier auction methods and systems are described in U.S. Pat.","Nos.","5,905,975, 6,021,398 and 6,026,383.The following description will detail the flow of the novel features of the preferred embodiments of the present method and system for an auction of multiple types of items.","Before describing the auction process in detail, reference is made to FIG.","1 to describe the architecture of an exemplary computer system in accordance with an embodiment of the present invention.","In the graphical depiction of FIG.","1, the computer system consists of multiple bidder and auctioneer computers or terminals 20a-n and 30 communicating with the server (or auction computer) 10 over a network 40.The computers or terminals 20a-n are employed by bidders, the computer or terminal 30 is employed by the auctioneer, and the server 10 is the auction computer.","The server 10 consists of a CPU 11, memory 12, a data storage device 13, a communications interface 14, a clock 15, an operating system 16, and an auction program 17.In one embodiment, the system architecture is as a client-server system: the auction computer is a server; and the bidder and auctioneer computers are clients.","FIG.","2 is another graphical depiction of an exemplary computer system in accordance with an embodiment of the present invention.","The auction system of FIG.","2 includes an auction computer 60 (sometimes also referred to as a Bidding Information Processor or BIP), a plurality of user systems 70a, 70b and so on (sometimes also referred to as Bidder Terminal or BT), each user system 70a-n representing an individual bidder, and a user system 80 (sometimes also referred to as an Auctioneer Terminal or AT).","The systems 60, 70a-n, and 80 communicate over a network 90.The network represents any system capable of providing the necessary communication to/from BIP, BT, and AT.","The network may be a local or wide area network such as, for example, Ethernet, token ring, the Internet, the World Wide Web, the information superhighway, an intranet or a virtual private network, or alternatively a telephone system, either private or public, a facsimile system, an electronic mail system, or a wireless communications system.","Each of the systems 60, 70a-n, and 80 may include a typical user interface 65, 75a-n, 85 for input/output which may include a conventional keyboard, display, and other input/output devices.","Within each of the systems, the user interface (65, 75a-n, 85) is coupled to a network interface (64, 74a-n, 84), which in turn communicates via the network 90.Both the user interface and network interface connect, at each system, to a CPU (62, 72a-n, 82).","Each system includes a memory (66, 76a-n, 86).","The BIP 60 also includes a clock 61 and a data storage device 63, which will ordinarily contain a database.","(However, in some embodiments the database might instead be stored in memory 66.)","The memory 66 of the BIP 60 can further be broken down into a program 67, data 68 and an operating system 69.The memory (76a-n, 86) of the BT's 70a-n and the AT 80 may include a web browser (for example, Internet Explorer or Netscape) (79, 89) or other general-purpose software, but not necessarily any computer program specific to the auction process.","In each system the CPU (62, 72a-n, 82) represents a source of intelligence when executing instructions from the memory (66, 76a-n, 86) so that appropriate input/output operations via the user interface and the network interface take place as is conventional in the art.","The particular steps used in implementing the inventive auction system and method are described in more detail below.","In one embodiment, each of the user systems is a personal computer or workstation.","FIG.","3 is a more detailed illustration of an exemplary BIP 60 showing details of the database.","As discussed for FIG.","2, the database is ordinarily stored on a data storage device 63, although in some embodiments it might instead be stored in memory 66.As depicted in FIG.","3, the database includes provision for creating, storing, and retrieving records representing Items in the Auction 63-1, Status of the Items in the Auction 63-2, Auction Timetable 63-3, Current Price(s) 63-4, List of Bidder ID's 63-5, List of Passwords 63-6, Bidding History 63-7, and Constraints on Bids 63-8.The particular set of data required for any particular auction and the format of that datum or data (such as scalar, vector, list, etc.)","is more particularly specified by the detailed description of that auction.","Definitions Concerning “Types” of Items and “Groups” of Types of Items Many of the most useful embodiments of the present invention apply in situations where an entity wishes to sell or buy multiple “types” of items or commodities.","Furthermore, related types of items may be usefully organized as “groups” of items.","In order to describe the preferred embodiments, it will be helpful for us to define some terminology.","DEFINITION 1: A type of item comprises a (nonempty) subset of the set of all items being auctioned, such that any two items within the same type are identical items or close substitutes.","Meanwhile, types are defined so that any two items of different types exhibit significant differences in time, location or any other product characteristics.","Typically, there are multiple units of each type of item.","In what follows, we will assume that m (m≧1) types of items are being auctioned, and that there are n (n≧1) bidders participating in the auction.","Whenever we state that there is a “plurality of types of items,” we are restricting attention to the case where m≧2.The items in the auction may also be said to be “homogeneous” in the case where m=1 and to be “heterogeneous” in the case where m≧2.Whenever we state that there is a “plurality of bidders,” we are restricting attention to the case where n≧2.DEFINITION 2: A group G of types comprises a (nonempty) subset of {1, .",".",".",", m}, the set of all types of items.","In other words, a group is one or more types of items that are usefully treated together.","Typically, the reason that two types of items are included in the same group is that they are related, for example, they may be contracts for provision of the same commodity, covering different but overlapping time periods.","In what follows, we will assume that the items in the auction are organized so that there are h (h≧1) groups of types of items.","There is no requirement that each group contains the same number of types of items, but this often happens to be the case.","Whenever we need notation indicating the number of types of items contained in the exemplary group G, our notation will be that there are g (g≧1) types of items in group G. In some of the preferred embodiments of the present invention, the prices for the various types of items within the same group are established so as to maintain a relationship according to a schedule.","In others of the preferred embodiments of the present invention, a bidder submits a bid that includes the price for one type of item within a group, and a fixed relation among these prices implies the prices of the other types of item within the same group.","In others of the preferred embodiments of the present invention, a bidder submits a bid that includes a one-dimensional price parameter, and this price parameter implies prices for all of the types of items within the group based on a fixed relation among the prices of the types of items.","Some examples of “types” and “groups,” in situations where there may be significant commercial possibilities for embodiments of the present invention, include the following: Treasury bills or other securities: A government or central bank may wish to auction 3-month, 6-month and 12-month Treasury securities (for example, with the same starting date) together.","Thus, there is one group of types of items (h=1), and it contains three types of items (g=3).","In total, there are three types of items (m=3).","Electricity contracts: An electric generating company may wish to simultaneously auction some forward contracts or options contracts for base-load and peak-load electricity generation, with durations of 2 months, 3 months, 6 months, 12 months, 24 months and 36 months, respectively.","Thus, there are two groups of types of items (h=2), each containing six types of items (g=6).","In total there are 2×6=12 types of items (m=12).","Two unrelated, heterogeneous consumer commodities (e.g., apples and oranges).","There are two groups of types of items (h=2), each containing just one type of item (g=1), for a total of two types of items (m=2).","An auction in accordance with an embodiment of the present invention proceeds as follows.","First, the auctioneer (i.e., the auctioneer terminal) determines a starting price vector, (P1, .",".",".",",Pm), and transmits it to the bidding information processor, which in turn transmits it to bidders (i.e., bidder terminals).","Second, a bidder responds with a bid vector indicating the quantity of each respective type of item that the bidder wishes to transact at the current price vector.","Let the bidders be superscripted by i, where (i=1, .",".",".",", n).","The bid vector for bidder i is denoted by (Q1i, .",".",".",",Qmi).","The following definitions are helpful in describing the process associated with a first embodiment of the present invention.","Other Definitions.","The available quantities may, in principle, be specified for each group of types of items or for each type of items.","In the text that follows, we will usually specify the available quantity for the group.","The available quantity for group G will be denoted {overscore (Q)}G and this refers, in the case of an auction to sell, to the overall quantity of items in group G to be offered for sale in the auction or, in the case of an auction to buy (i.e., a procurement auction or a “reverse auction”), to the overall quantity of items in group G to be bought in the auction.","The vector of available quantities for all groups will be denoted ({overscore (Q)}1, .",".",".",", {overscore (Q)}h) Optionally, the available quantities may be allowed to depend on the prices, or otherwise be contingent on the progress of the auction.","The current prices comprise a vector, (P1, .",".",".",",Pm), whose components represent the prices for the m respective types of items.","The current bid of bidder i comprises a vector, (Q1i, .",".",".",",Qmi), whose components represent the quantities that bidder i is willing to buy (in the case of an auction to sell) or to sell (in the case of an auction to buy) at the current prices for the m respective types of items.","The current bids comprise the collection of vectors, {Q1i, .",".",".",",Qmi}i=1n consisting of the current bid of bidder i for every bidder (i=1.. .",", n) in the auction.","The bidding history comprises the current prices and the current bids associated with the present time and all earlier times in the current auction.","A clock auction is a dynamic auction procedure whereby: the auctioneer announces the current prices to bidders; the bidders respond with current bids; the auctioneer determines whether the auction should continue based on the bidding history; the auctioneer updates the current prices based on the bidding history and the process repeats, if it is determined that the auction should continue; and the auctioneer allocates the items among the bidders and assesses payments among the bidders based on the bidding history, if it is determined that the auction should not continue.","Observe that a “clock auction” differs from a standard ascending-bid electronic auction in the following important sense.","In standard ascending-bid electronic auctions—such as in the Federal Communications Commission auctions for radio communications spectrum or in eBay auctions—the bidders name prices (and, perhaps also, quantities) that they propose to pay for the items being auctioned, in an iterative process.","In a standard clock auction, the auctioneer sets the pace for price increases, and bidders respond only with quantities in an iterative process.","(however, in the discussion of Intra-Round Bids, below, it will be seen that there still may be a role for bidders naming prices—to a limited extent—in a clock auction.)","FIG.","4 is a flow diagram of a clock auction in accordance with one embodiment of the present invention.","The process starts with step 102, in which memory locations of a computer are initialized.","In one preferred embodiment, the appropriate memory locations of the bidding information processor (auction computer) are initialized with information such as the types of items in the auction, the available quantity of each type of item in the auction, the data table or algorithm reflecting the “yield curve” schedule, an initial price parameter, an auction timetable, a list of bidder ID's, and a list of passwords.","In step 104, a computer establishes the initial prices (P1, .",".",".",",Pm).","One preferred embodiment of the process of step 104 is shown in greater detail in FIG.","5b.","The process proceeds to step 106, in which a computer outputs auction information, including the current price vector (P1, .",".",".",",Pm).","In one preferred embodiment, the bidding information processor outputs the auction information through its network interface and transmits it via the network.","The bidder terminals then receive the auction information through their network interfaces and display the information to bidders through their user interfaces.","In step 108, a computer receives bids (Q1i, .",".",".",",Qmi) from bidders.","In one preferred embodiment, a bidder inputs his bids through the user interface of the bidder terminal, which then outputs the auction information through its network interface and transmits it via the network.","The bidding information processor then receives the bids through its network interface for use in the next step.","One preferred embodiment of the process of step 108, utilizing Intra-Round Bids, is shown in greater detail in FIG.","8a.","In step 110, a computer applies constraints, if any, to the received bids, and enters only those bids that satisfy said constraints.","This process is illustrated in greater detail in FIGS.","6a and 6b.","In one preferred embodiment, the constraints are applied at the bidding information processor, although they may also easily be applied at the bidder terminals.","In step 112, a computer processes the received bids and determines whether the auction should continue.","Exemplary processes of step 112 are illustrated in greater detail in FIGS.","7 and 8b.","In some preferred embodiments, this determination occurs at the bidding information processor.","If the auction should continue, the process goes to step 114, in which a computer establishes an updated price vector (P1, .",".",".",",Pm).","One preferred embodiment of the process of step 114 is shown in greater detail in FIG.","5a.","Then, at step 116, a computer updates other auction information, if any.","In one preferred embodiment, the bidding information processor automatically generates a suggested revised price vector, outputs the suggested revised price vector through its network interface, and transmits it via the network.","The auctioneer terminal then receives the suggested revised price vector through its network interface and displays it to the auctioneer through its user interface.","The auctioneer either approves or modifies the revised price vector through the user interface of the auctioneer terminal, which then outputs the revised price vector through its network interface and transmits it via the network.","The bidding information processor then receives the revised price vector through its network interface for use in subsequent steps.","The process then loops to step 106.If the auction should not continue, the process goes to step 118, in which a computer outputs a final message, including the allocation of items among bidders and the payments of the bidders.","In one preferred embodiment, the bidding information processor takes the allocation of items among bidders to be their final bids and takes the payment of each bidder i to be the dot product of the final price vector and bidder i's final quantity vector: (P1, .",".",".",",Pm·(Q1i, .",".",".",", Qmi).","The bidding information processor outputs the allocation and payment outcome through its network interface and transmits it via the network.","The bidder terminals and auctioneer terminal then receive the allocation and payment outcome through their network interfaces and display the information to bidders and the auctioneer through their user interfaces.","One preferred embodiment of the process of step 118, utilizing Intra-Round Bids, is shown in greater detail in FIG.","8c.","The process then ends.","Embodiments of the Invention Concerned with Establishing or Implying Prices FIG.","5a is a flow diagram illustrating an exemplary process by which updated prices are established.","Thus, FIG.","5a illustrates, in greater detail, step 114 of FIG.","4.The process begins with step 114-1, in which a computer selects a group G of types of items not yet considered.","In step 114-2, a computer calculates the excess demand ZG for group G, which is given by: Z G = - Q _ G + ∑ i = 1 n ∑ k ∈ G Q k i .","Thus, ZG represents the amount by which the bids for all types of items in group G, by all bidders, exceeds the available quantity {overscore (Q)}G of all items in group G. In step 114-3, a computer selects a type of item included in group G, whose “old” price is denoted by {overscore (P)}1G.","In step 114-4, a computer sets a “new” price P1G, where P1G equals the {overscore (P)}1G plus an increment Δ.","The increment Δ is given by Δ=c ZG, where c is a positive constant and ZG was computed in step 114-2.","(In other embodiments, other rules for incrementing P1 are used.)","In step 114-5, a computer looks up the new P1G in a data table reflecting the “yield curve” schedule.","The following Table 1 is an example of such a data table for a group G containing three types of items (corresponding to the base group of the earlier example containing base and peak groups of electricity contracts): TABLE 1 P1G P2G P3G 0.00 2.00 3.00 5.00 7.00 8.00 10.00 12.00 13.00 15.00 17.00 18.00 20.00 22.00 23.00 The process continues with step 114-6 in which a computer determines whether the new P1G is an entry in the data table.","If it is, the process continues with step 114-7 in which a computer reads across the row for the new P1G in the data table and thereby determines the new prices (P2G, .",".",".",", PgG), where g denotes the number of types of items within group G. For example, with the data of Table 1, if P1G equaled 15.00, then the computer would determine “Yes” at step 114-6, and the computer would determine P2G=17.00 and P3G=18.00 at step 114-7.The process then proceeds to step 114-10.If the new P1G is not an entry in the data table, the process continues with step 114-8, in which a computer retrieves P1low, the largest entry in the data table less than P1G, and a computer retrieves P1high, the smallest entry in the data table greater than P1G.","The process then continues with step 114-9, in which a computer determines the new prices (P2G, .",".",".",", PgG) by linear interpolation, reading across the rows in the data table corresponding to P1low and P1high, and applying the new P1G.","For example, with the data of Table 1, if P1G equaled 10.50, then the computer would determine “No” at step 114-6.The computer would then retrieve P1low=10.00 and P1high=15.00 at step 114-8, and the computer would determine P2G=12.50 and P3G=13.50 at step 114-9.The process then proceeds to step 114-10, in which a computer determines whether all groups G have been considered.","If not, the process returns to step 114-1.However, if all groups G have been considered, then the entire step 114 has been completed, and the process continues with step 116 of FIG.","4.Three observations should be made at this point.","First, rather than determining prices by looking up and possibly interpolating of numbers that are contained in a data table, the relationship or relation among the prices may instead be an algorithm or a formula.","For example, instead of using the data of Table 1, another preferred embodiment would be obtained by equivalently applying the formulae P2G=P1G+2 and P3G=P1G+3.Second, there is no particular reason that the relationship or relation among the prices need be linear, as it happens to be in Table 1 and in some applications.","The relationship or relation (data table, or algorithm or formula) could be a nonlinear relationship between (P2G, .",".",".",", PgG) and P1G.","Third, in some embodiments, the relationship or relation (whether schedule or data table, or algorithm or formula) among prices is predetermined and fixed, and in other embodiments, the relationship or relation can be based on data which may be revised during the course of the auction.","In typical applications, the price relationship or relation may be determined from market data external to the auction, for example, from the “yield curve” in the government securities market, expressing the differential among prices or interest rates for securities of varying durations.","If the market data external to the auction were to markedly change during the course of the auction, then the auctioneer might wish to revise the price relationship or relation accordingly.","FIG.","5b is a flow diagram illustrating an exemplary process by which initial prices are established.","Thus, FIG.","5b illustrates, in greater detail, step 104 of FIG.","4.The process begins with step 104-1, in which a computer selects a group G of types of items not yet considered.","In step 104-2, a computer selects a type of item included in group G, whose price is denoted by P1G.","In step 104-3, a computer looks up the initial value for the selected type of item in group G among the memory locations that were initialized in step 102, and uses this value as the initial P1G.","In step 104-4, a computer looks up the initial P1G in a data table reflecting the “yield curve” schedule.","The previous Table 1 serves as an example of such a data table, as in the description of FIG.","5a.","The process continues with step 104-5 in which a computer determines whether the initial P1G is an entry in the data table.","If it is, the process continues with step 104-6 in which a computer reads across the row for the initial P1G in the data table and thereby determines the initial prices (P2G, .",".",".",", PgG), where g denotes the number of types of items within group G. The process then proceeds to step 104-9.If the initial P1G is not an entry in the data table, the process continues with step 104-7, in which a computer retrieves P1low, the largest entry in the data table less than P1G, and a computer retrieves P1high, the smallest entry in the data table greater than P1G.","The process then continues with step 104-8, in which the computer determines the initial prices (P2G, .",".",".",", PgG) by linear interpolation, reading across the rows in the data table corresponding to P1low and P1high, and applying the initial P1G.","The process then proceeds to step 104-9, in which a computer determines whether all groups G have been considered.","If not, the process returns to step 104-1.However, if all groups G have been considered, then the entire step 104 has been completed, and the process continues with step 106 of FIG.","4.The first two observations made after FIG.","5a also apply to the process of FIG.","5b.","Elements of the Invention Concerned with Applying Constraints to Bids FIGS.","6a and 6b are flow diagrams of two exemplary subprocesses of step 110.The process of FIG.","6a begins with step 110a-1, in which a bidder i who has not yet been considered is selected.","In step 110a-2, a bid (Qki,t)kεG by bidder i which has not yet been considered is selected.","The bid (Qki,t)kεG is a bid for group G of item types at time t in the auction.","In step I10a-3, it is checked whether each quantity Qki,t in the selected bid is a nonnegative integer.","If each component of the bid is a nonnegative integer, the process goes to step 110a-4.In step 110a-4, it is checked whether the selected bid is consistent with bidder i's initial eligibility, that is, whether: ∑ k ∈ G Q k i , t ≤ Q _ G i , where bidder i's initial eligibility, {overscore (Q)}Gi, for group G may, for example, be determined by the level of financial guarantee posted by bidder i.","If the selected bid is consistent with bidder i's initial eligibility, the process goes to step 110a-5, where bidder i's most recent previously-processed bid for group G, denoted (Qki-t-1)kεG, is recalled.","In step 110a-6, it is checked whether the selected bid is consistent with the auction's activity rule, that is, whether the constraint: ∑ k ∈ G Q k i , t ≤ ∑ k ∈ G Q k i , t - 1 , is satisfied.","If it is, the process continues to step 110a-7, where the selected bid (Qki,t)kεG is entered as a valid bid by bidder i on group G. Optionally, bidder i is sent a message confirming to him that the bid is valid.","The process then goes to step 110a-8, where it is determined whether all bids by bidder i have been considered.","If not, the process loops back to step 110a-2.If all bids by bidder i have been considered, the process continues to step 110a-9, where it is determined whether all bidders have been considered.","If not, the process loops back to step 110a-1.If all bidders have been considered, the process goes to step 112 of FIG.","4.If the selected bid fails any of the checks at steps 110a-3, 110a-4 or 110a-6, the process instead goes to step 110a-10, where a message is outputted to bidder i that the selected bid is invalid.","The selected bid then is not entered as a valid bid.","The process then goes to step 110a-8, where it is determined whether all bids by bidder i have been considered.","If not, the process loops back to step 110a-2.If all bids by bidder i have been considered, the process continues to step 110a-9, where it is determined whether all bidders have been considered.","If not, the process loops back to step 110a-1.If all bidders have been considered, the process goes to step 112 of FIG.","4.The process of FIG.","6b begins with step 110b-1, in which a bidder i who has not yet been considered is selected.","In step 110b-2, a bid (Qki,t)kεG by bidder i which has not yet been considered is selected.","The bid (Qki,t)kεG is a bid for group G of item types at time t in the auction.","In step 110b-3, it is checked whether each quantity Qki,t in the selected bid satisfies the constraint: ∑ k ∈ G C k i , t Q k i , t ≤ C _ G i , t , where Cki,t and {overscore (C)}Gi,t are arbitrary constants.","If the constraint of step 110b-3 is satisfied, the process goes to step 110b-4.In step 110b-4, it is checked whether each quantity Qki,t in the selected bid satisfies the constraint: ∑ k ∈ G C k ′ i , t Q k i , t ≥ C ^ G i , t , where Ck′i,t and ĈGi,t are arbitrary constants.","If the constraint of step 110b-4 is satisfied, the process goes to step 110b-5, where it is checked whether the selected bid was submitted at a time no earlier than the starting time of the current round.","If it was, the process goes to step 110b-6, where it is checked whether the selected bid was submitted at a time no later than the ending time of the current round.","If it was, the process continues to step 110b-7, where the selected bid (Qki,t)kεG is entered as a valid bid by bidder i on group G. Optionally, bidder i is sent a message confirming to him that the bid is valid.","The process then goes to step 110b-8, where it is determined whether all bids by bidder i have been considered.","If not, the process loops back to step 110b-2.If all bids by bidder i have been considered, the process continues to step 110b-9, where it is determined whether all bidders have been considered.","If not, the process loops back to step 110b-1.If all bidders have been considered, the process goes to step 112 of FIG.","4.If the selected bid fails any of the checks at steps 110b-3, 110b-4, 110b-5 or 110b-6, the process instead goes to step 110b-10, where a message is outputted to bidder i that the selected bid is invalid.","The selected bid then is not entered as a valid bid.","The process then goes to step 110b-8, where it is determined whether all bids by bidder i have been considered.","If not, the process loops back to step 110b-2.If all bids by bidder i have been considered, the process continues to step 110b-9, where it is determined whether all bidders have been considered.","If not, the process loops back to step 110b-1.If all bidders have been considered, the process goes to step 112 of FIG.","4.Embodiments of the Invention Concerned with Whether the Auction Should Continue FIG.","7 is a flow diagram of a subprocess of step 112 of FIG.","4.It illustrates an exemplary process by which a computer may determine whether the auction should continue.","Related to this will also be FIG.","8b, below, which illustrates an exemplary process by which a computer determines whether the auction should continue, in a system where bidders are permitted to submit Intra-Round Bids.","FIG.","7 begins with step 112a-1, in which a group G of types of items not yet considered is selected.","In step 112a-2, a computer determines whether the excess demand for group G of types of items is within {overscore (C)}G of the available quantity, that is, whether: Q _ G - ∑ i = 1 n · ∑ k ∈ G Q k l ≤ C _ G .","The constant, {overscore (C)}G, has the interpretation that this is the tolerance to which the auctioneer is allowing oversell or undersell to occur.","If the auctioneer needs to sell exactly the available quantity of the group G of item types, then {overscore (C)}G=0.If this inequality is not satisfied, then group G of item types has not yet cleared, and so the auction should continue.","The process thus jumps immediately to step 114 of FIG.","4.If the inequality of step 112a-2 is satisfied, the process then goes to step 112a-3, where it is determined whether all groups G of types of items have been considered.","If not, the process loops back to step 112a-1.However, if all groups G of types of items have already been considered, then it has been found that all groups G of types of items have cleared within a tolerance of {overscore (C)}G, and so the auction should not continue.","The process proceeds to step 118 of FIG.","4, where the final message is generated.","Embodiments of the Invention Concerned with Intra-Round Bids In many of the leading dynamic electronic auctions in the prior art, bidders submit bids in a sequence of discrete rounds.","For example, in the Federal Communications Commission auctions for radio communications spectrum or in the recent UMTS auctions held by European nations, the following would be a typical bidding schedule for an auction: Round 1: 9:00-9:45 Round 2: 10:00-10:45 Round 3: 11:00-11:45 Round 4: 12:00-12:45 Round 5: 13:00-13:45 Round 6: 14:00-14:45 Round 7: 15:00-15:45 Round 8: 16:00-16:45 This bidding schedule would have the following interpretation.","During the specified time period of each round, a bidder would be required to submit a new bid or new collection of bids (unless this bidder was already the standing high bidder on an item after the bidding of the previous round).","If a bidder who was required to submit a new bid failed to submit a new bid, then (except for provisions in the rules concerning automatic waivers) the bidder would be eliminated from the auction.","By contrast, some other electronic auctions in the prior art—for example, online auctions at eBay—allow bidding to occur continuously.","Rather than adhering to any rigid round schedule, bidders may submit bids at any times that they like up to a specified closing time.","Related to this, there is no sense that a bidder is required to bid a certain amount by any particular time in order to retain eligibility to bid at a later time in the auction.","Many or most electronic auctions for high-valued items utilize a discrete round structure, rather than allowing bidding to occur continuously.","There appear to be several reasons for this.","First, a discrete round structure has desirable information properties.","The auction can be easily structured so that the results of Round t are disseminated to bidders before the bids of Round t+1 need to be submitted.","Second, a discrete round structure is especially conducive to enforcing “activity rules,” in which a bidder is required to be active (i.e., either be the standing high bidder or place a new high bid) on a given number of items in an earlier round of the auction in order to continue to bid on a given number of items in a later round of the auction.","This forces bidders to effectively disclose to their opponents (through their bidding) the values that they attach to the items, helping to mitigate the well-known “Winner's Curse” present in auctions.","Third, a discrete round structure requires a bidder to repeatedly affirm, in successive rounds, his willingness to pay a given price for an item in the auction—which may be especially desirable when items such as communications licenses may sell for millions or billions of dollars or euros.","At the same time, the desirable properties of a discrete round structure may come at some considerable cost.","It will typically be reasonable to hold only something like 8 to 12 rounds of bidding in a given day.","As a result, the auctioneer must accept at least one of several problems: (1) The auction may be required to last a very long time: in some North American and European spectrum auctions, the bidding extended more than 20 business days.","Such a lengthy auction may be rather onerous for bidders and for the seller.","In particular, it may discourage bidder participation, causing the seller to forgo substantial revenues.","(2) The bid increment between successive rounds may be required to be rather substantial: in some North American and European spectrum auctions, the bid increment between successive rounds never was allowed to drop below five percent of the previous bid.","It can be argued that a seller suffers an expected revenue loss which is directly proportional to the minimum bid increment, so this may cost a seller millions of dollars or euros.","(3) The starting price may be required to be very near to the expected closing price.","This may discourage bidder participation, as well as potentially eliminating the possibility of bidders getting caught up in the excitement of the auction and bidding very high prices (which is one of the advantages of conducting a dynamic auction).","This also runs the risk that the auction will fail: that is, quantities bid at the starting price being less than the available quantity at the auction.","Moreover, in a clock auction, problem (2) above, a large bid increment, may lead to a heightened risk of “undersell”.","Consider an auction with an available quantity of 100 units of an item, and suppose a bid increment of five percent.","It is quite plausible that, at a price of $1,000,000 per unit, the aggregate quantity bid by all bidders would equal 110 units, but at the next price of $1,050,000 per unit, the aggregate quantity bid by all bidders would decline to only 60 units.","The auctioneer then faces the unattractive alternatives of: selling only 60 units out of the available quantity of 100 units at a price of $1,050,000 each; rationing bidders so that only 100 units, out of the 110 demanded, are sold at $1,000,000; or restarting the auction at $1,000,000.Observe however that the “undersell” problem would in all likelihood have been substantially avoided, had a much smaller bid increment been possible.","One embodiment of the present invention is a system and method for “Intra-Round Bids.” A discrete round structure—with all of its many advantages—is preserved for a clock auction.","However, in each round of the clock auction, a “starting price” and “ending price” is established for each type of item.","Bidders are permitted to submit bids at prices between the starting price and the ending price.","In a preferred embodiment, a bidder submits a price parameter for group G representing a percentage of the distance from the starting price vector for group G and the ending price vector for group G. Bidders have every incentive to utilize Intra-Round Bids, and to the extent that bidders utilize them, the seller should be expected to attain higher auction revenues and to reduce the probability of undersell.","Thus, a system and method for Intra-Round Bids improves upon the prior art for auction systems and methods, and has immediate practical application for dynamic auctions of radio communications spectrum, securities and other financial products, electric power, etc.","While the previous and following description of Intra-Round Bids is framed largely in terms of regular auctions to sell (where bidders are buyers), the invention is equally applicable for reverse or procurement auctions to buy (where bidders are sellers).","For the sake of brevity, this specification will not run through the process a second time with the roles of selling and buying reversed, but it should be clear to anybody skilled in the art that the technology can be equally used in both situations.","Here is an example illustrating the usefulness and exact meaning of Intra-Round Bids.","Suppose that, in a clock auction with an available quantity of 100 units, the ending price per unit associated with Round 4 is $1,000,000, and the ending price per unit associated with Round 5 is $1,050,000.In an auction with discrete bidding rounds, Bidder 1 might submit a bid quantity of 55 units for Round 4 and a bid quantity of 30 units for Round 5.If there also exists a Bidder 2 who submits the same bid quantities, then we would have exactly the “undersell” problem described above: an aggregate quantity bid by all bidders of 110 units in Round 4 but only 60 units in Round 5 (with available quantity of 100 units).","With an auction system and method with Intra-Round Bidding, the ending price for Round 4 may be taken to be the starting price for Round 5, i.e., the starting price for Round 5 is $1,000,000.Here is an example of the bids that Bidder 1 might submit for Auction Round 5: 53 units at $1,010,000 per unit; 51 units at $1,020,000 per unit; 49 units at $1,030,000 per unit; 45 units at $1,035,000 per unit; 40 units at $1,040,000 per unit; and 30 units at $1,045,000 per unit.","These bids have the following exact meaning: the parameters corresponding to price indicate the price at which Bidder 1 wishes to change his quantity demanded as compared to his “previous” (that is, next lower price) bid.","Thus, in this example: Bidder 1 is willing to purchase 55 units (his previous bid from Round 4) at prices of $1,000,001-$1,009,999; Bidder 1 is willing to purchase 53 units at prices of $1,010,000-$1,019,999; Bidder 1 is willing to purchase 51 units at prices of $1,020,000-$1,029,999; Bidder 1 is willing to purchase 49 units at prices of $1,030,000-$1,034,999; Bidder 1 is willing to purchase 45 units at prices of $1,035,000-$1,039,999; Bidder 1 is willing to purchase 40 units at prices of $1,040,000-$1,044,999; and Bidder 1 is willing to purchase 30 units at prices of $1,045,000-$1,050,000.If there also exists a Bidder 2 who submits the same bid quantities, then the auctioneer would be able to declare the auction over at a price between $1,030,000 and $1,034,999, with 98 out of the 100 available units sold.","The auction revenues are improved, and the undersell problem is greatly reduced.","FIG.","8a is a flow diagram of a subprocess of step 108 of FIG.","4.It illustrates an exemplary process by which a particular bidder i may submit Intra-Round Bids.","FIG.","8a begins with step 108-1, in which bidder i selects a group, G, of item types on which he wishes to place a bid.","In step 108-2, bidder i selects a price parameter for group G representing a percentage of the distance from the starting price vector for group G and the ending price vector for group G in the current round.","For example, in a group containing three types of items, if the starting price vector is (4.00, 4.50, 4.75), if the ending price vector is (8.00, 8.50, 8.75), and if a bidder enters a price parameter of 25%, this signifies that the bidder is indicating an implied price vector of (5.00, 5.50, 5.75).","In step 108-3, bidder i selects quantities of the item types of group G that he would like to take effect as bids at the price vector implied by the selected price parameter.","In step 108-4, bidder i expresses whether he wishes to enter more bids.","If so, the process loops back to step 108-1.If not, the process continues to step 108-5.In step 108-5, a computer determines whether bidder i has submitted at least one bid for each group G of item types.","If not, the process loops back to step 108-1, and optionally a computer prompts bidder i to submit bids on the groups G of item types on which bidder i has not submitted at least one valid bid in the current round.","If so, the process goes to step 110 of FIG.","4.FIG.","8b is a flow diagram of a subprocess of step 112 of FIG.","4.It illustrates an exemplary process by which a computer determines whether the auction should continue, in a system where bidders are permitted to submit Intra-Round Bids.","FIG.","8b begins with step 112b-1, in which a group G of item types not yet considered is selected.","In step 112b-2, a computer sorts all bids entered for group G in the current round.","The sorting is done: first, by bidder ID; second, by price parameter in the entered bid (in descending order); and third, by time stamp of submission (in descending order).","In step 112b-3, a computer selects, for each bidder i, the bid, QG,i, for group G with the highest price parameter (and then the latest time stamp).","In step 112b-4, a computer determines whether the aggregate quantity bid for group G is no greater than the available quantity, that is, whether: ∑ i = 1 n ∑ k ∈ G Q k G , i ≤ Q _ G .","If this inequality is not satisfied, then group G of item types has not yet cleared, and so the auction should continue.","The process thus jumps immediately to step 114 of FIG.","4.If the inequality of step 112b-4 is satisfied, the process then goes to step 112b-5, where it is determined whether all groups G of item types have been considered.","If not, the process loops back to step 112b-1.However, if all groups G of item types have already been considered, then it has been found that all groups G of item types have cleared, and so the auction should not continue.","The process proceeds to step 118 of FIG.","4, where the final message is generated.","FIG.","8c is a flow diagram of a subprocess of step 118 of FIG.","4.It illustrates an exemplary process by which a computer determines final allocations and payments, in a system where bidders are permitted to submit Intra-Round Bids.","FIG.","8c begins with step 118-1, in which for all bids entered in the current (i.e., final) round, a computer sorts the price parameters from smallest to largest, and denotes them λ1<π2<.",".",".","<πR.","In step 118-2, a computer initializes the price parameter subscript to r=1, so that in the first iteration of the remaining steps, the computer considers the smallest value, π1.In step 118-3, a group G of item types not yet considered is selected.","In step 118-4, a computer sorts all bids entered for group G in the current round.","The sorting is done: first, by bidder ID; second, by price parameter in the entered bid (in descending order); and third, by time stamp of submission (in descending order).","In step 118-5, a computer selects, for each bidder i, the bid, QG,i, for group G with the highest price parameter that is less than or equal to πr (and then with the latest time stamp).","In step 118-6, a computer determines whether the aggregate quantity bid for group G is no greater than the available quantity, that is, whether: ∑ i = 1 n ∑ k ∈ G Q k G , i ≤ Q _ G .","If this inequality is not satisfied, then group G of item types has not yet cleared at price parameter πr, and so r needs to be incremented.","The process thus goes to step 118-8, where the price parameter subscript r is advanced by 1, so that in the next iteration of these steps, the computer considers the next price parameter, πr+1.The process then loops back to step 118-3, using the new higher value of πr+1 and starting over for groups G of item types.","If the inequality of step 118-6 is satisfied, the process continues to step 118-7, where it is determined whether all groups G of item types have been considered.","If not, the process loops back to step 118-3.However, if all groups G of item types have already been considered, then it has been found that all groups G of item types have cleared at price parameter ;r,.","Thus, the price parameter A, implies market-clearing prices for the auction.","The process proceeds to calculate the price vector implied by price parameter ;r, to note the quantities bid by all bidders at this price vector, and to incorporate these computations into a final message that is outputted from a machine.","Auction-Like Optimization Problems and Machine-Generated Bids In the course of this application, a method and apparatus for implementing auctions of multiple items has been described.","The methods and apparatus described allow users to participate in various auctions with a level of attention which varies from continuous, down to the input of information on a single occasion.","It should also be apparent that the required level of attention by the “auctioneer” may vary from continuous to essentially zero—aside from initiating the auction.","Thus, for all intents and purposes, once the basic auction description is selected and the users input desired information, the auction implemented by the invention can be essentially automatic, i.e.","devoid of human interaction.","Because in the past auctions have generally been considered to be processes engaged in by persons, the feature of an automatic auction may be, by itself, considered relatively new.","There are, however, many other automatic systems which interact in a way that is entirely analogous to an auction and to which the present invention could be applied.","Hence, the present invention can be applied to improve the efficiency of computers which are used to operate the automatic systems, by economizing on the collection of informational inputs needed for the system and to speed the computational of optimal resource assignments.","At the same time, many optimization problems encountered in the field of operations research have similar mathematical structures to the problem of determining the winners of an auction.","Hence, the present invention can be applied to improve the efficiency of computer systems which are used to solve the similar optimization problems, by enabling the computations to be implemented on a system with parallel processing or generally by speeding the computation of solutions.","For example, the air conditioning plant in an office building allocates cool air among individual offices in the building via a dynamic auction.","Periodically, the central computer of the air conditioning system serves the role of the “auction computer” in a clock auction, while computers interfaced with the thermostats in each suite of offices serve the role of “bidder terminals.” Each thermostat is programmed to send back bids consisting of a desired quantity of cooled air based on: the current temperature reading of the thermostat, the desired temperature in the office, and the use (if any) to which the office is currently being put.","Based on the parameters to which it has been programmed, the central computer of the air conditioning system then provides the results of the auction in its allocation of cooled air among the bidding offices.","In another context, a communications, transportation or energy transmission network faces the technical problem of how to allocate its scarce network resources.","The optimization problem in allocating its network resources (e.g., bandwidth, switches, etc.)","has a very similar structure to the auction problem.","Moreover, clock auctions are again well suited to the problem, since a network provider needing multiple types of network resources is able to iteratively bid on a vector of network resources, always knowing the price of each.","Hence, the present invention can be usefully applied to improving the solution to this technical problem.","In another context, computational resources on a distributed computer system can be allocated via a clock auction.","Whenever a new job requiring a given quantity of CPU time enters the system, an auction is conducted.","Each member of the distributed computer system indicates the quantity of CPU time which it can make available at a given priority level or a given price.","In this case, the “auctioneer computer” selects and allocates the resources to be applied to the new job in accordance with some programmed timetable and hence in this fashion provides the results of the auction.","The several examples described herein are exemplary of the invention, whose scope is not limited thereby but rather is indicated in the attached claims."]],"string":"[\n [\n \"System and method for an auction of multiple types of items\",\n \"An improved system and method for a computer-implemented auction in which multiple types of items are auctioned together without imposing a particular division of supply or demand among the individual types of items.\",\n \"In some embodiments the auction of the present invention provides a means or method for establishing prices for the types of items, wherein the prices maintain a relationship.\",\n \"In other embodiments, the present invention provides a means or method for implying prices from price parameters in the bids received form bidders, based on a relation among the prices for the types of items.\",\n \"Market clearing may be defined by the condition that the aggregate quantity bid for all types of items is less than or equal to the available quantity of all types of items.\",\n \"The division among the types of items within is thus determined flexibly, based on the bids at the associated prices.\",\n \"In other embodiments, market clearing is defined by the condition that the quantity bid for one selected type of item is less than or equal to the available quantity of the selected type of item.\",\n \"The quantities of the other types of items are thus determined flexibly, based on the bids at the associated prices.\"\n ],\n [\n \"1.A computer system for conducting an auction of a plurality of types of items among a plurality of bidders, wherein bids are received and an allocation of the items among the bidders is determined by the computer system based on the bids, comprising: establishing means for establishing prices for the plurality of types of items, wherein the prices maintain a relationship, receiving means for receiving bids associated with the established prices, and processing means for processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, otherwise enabling the establishing means to alter the prices established for the plurality of types of items.\",\n \"2.A system as recited in claim 1 which further comprises means for constraining the changes in a bid from a given bidder as the established prices are altered by considering, in the processing means, only bids which satisfy a constraint enforced by the means for constraining.\",\n \"3.A system as recited in claim 2 wherein the constraining means includes summation means for calculating a sum of quantity parameters for a group of types of items.\",\n \"4.A system as recited in claim 3 wherein the constraining means includes comparing means for comparing the calculated sum with a previous calculated sum for the same group of types of items.\",\n \"5.A system as recited in claim 1 wherein each received bid comprises a quantity parameter for at least one of the types of items in the auction.\",\n \"6.A system as recited in claim 5 wherein the establishing means establishes a price for a first type of item and then determines prices for each other type of item based on the price for the first type of item.\",\n \"7.A system as recited in claim 6 wherein the establishing means determines prices from a predetermined schedule.\",\n \"8.A system as recited in claim 7 wherein the establishing means includes interpolation means for interpolating prices among prices on the predetermined schedule.\",\n \"9.A system as recited in claim 6 wherein the processing means includes summation means for calculating a sum of the quantity parameters for the first type of item.\",\n \"10.A system as recited in claim 9 wherein the processing means includes comparing means for comparing the calculated sum with an available quantity parameter.\",\n \"11.A system as recited in claim 1 wherein the plurality of types of items are contracts for provision of a commodity covering different time periods, wherein the time periods of at least some of the contracts have at least some overlap.\",\n \"12.A system as recited in claim 5 wherein the plurality of types of items are contracts for provision of a commodity covering different time periods, wherein the time periods of at least some of the contracts have at least some overlap.\",\n \"13.A system as recited in claim 12 wherein the processing means includes summation means for calculating a sum of the quantity parameters for the overlapping contracts.\",\n \"14.A system as recited in claim 13 wherein the processing means includes comparing means for comparing the calculated sum with an available quantity parameter.\",\n \"15.A system as recited in claim 1 wherein the relationship of the prices established by the establishing means is determined by a schedule.\",\n \"16.A system as recited in claim 1 wherein the relationship of the prices established by the establishing means is determined by an algorithm.\",\n \"17.A computer implemented method for conducting an auction of a plurality of types of items among a plurality of bidders, wherein bids are received and an allocation of the items among the bidders is determined based on the bids, comprising: establishing prices for the plurality of types of items, wherein the prices maintain a relationship, receiving bids associated with the established prices, and processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, otherwise altering the established prices for the plurality of types of items.\",\n \"18.A method as recited in claim 17 which further comprises constraining the changes in a bid from a given bidder as the established prices are altered by considering, in the processing, only bids which satisfy a constraint.\",\n \"19.A method as recited in claim 18 wherein the constraining includes calculating a sum of quantity parameters for a group of types of items.\",\n \"20.A method as recited in claim 19 wherein the constraining includes comparing the calculated sum with a previous calculated sum for the same group of types of items.\",\n \"21.A method as recited in claim 17 wherein each received bid comprises a quantity parameter for at least one of the types of items in the auction.\",\n \"22.A method as recited in claim 21 wherein a price is established for a first type of item and then prices for each other type of item are determined based on the price for the first type of item.\",\n \"23.A method as recited in claim 22 wherein the determined prices are determined from a predetermined schedule.\",\n \"24.A method as recited in claim 23 wherein establishing prices includes interpolating prices among prices included in the predetermined schedule.\",\n \"25.A method as recited in claim 22 wherein processing the received bids includes calculating a sum of the quantity parameters for the first type of item.\",\n \"26.A method as recited in claim 25 wherein processing the received bids includes comparing the calculated sum with an available quantity parameter.\",\n \"27.A method as recited in claim 17 wherein the plurality of types of items are contracts for provision of a commodity covering different time periods, wherein the time periods of at least some of the contracts have at least some overlap.\",\n \"28.A method as recited in claim 21 wherein the plurality of types of items are contracts for provision of a commodity covering different time periods, wherein the time periods of at least some of the contracts have at least some overlap.\",\n \"29.A method as recited in claim 28 wherein processing the received bids includes calculating a sum of the quantity parameters for the overlapping contracts.\",\n \"30.A method as recited in claim 29 wherein processing the received bids includes comparing the calculated sum with an available quantity parameter.\",\n \"31.A method as recited in claim 17 wherein the relationship of the prices is based on a schedule.\",\n \"32.A method as recited in claim 17 wherein the relationship of the prices is based on an algorithm.\",\n \"33.A computer system for conducting an auction of a plurality of types of items among a plurality of bidders, wherein bids are received and an allocation of the items among the bidders is determined based on the bids, comprising: receiving means for receiving bids comprising a quantity parameter for at least one of the types of items in the auction and a price parameter, and processing means for processing the received bids to determine an allocation of the items among the bidders that is consistent with the bids in the event that an allocation consistent with the bids is possible, wherein the processing means includes implying means for implying prices from price parameters in the received bids based on a fixed relation among the prices of the plurality of types of items.\",\n \"34.A system as recited in claim 33 which includes means for inviting additional bids in the event that an allocation of items consistent with the received bids is not possible, and means for applying the additional bids to the processing means for processing the additional bids to determine an allocation of the items among the bidders consistent with the additional bids in the event that an allocation consistent with the additional bids is possible.\",\n \"35.A computer system as recited in claim 34 which further comprises means for constraining the additional bids from a given bidder by limiting the processing to only additional bids which satisfy a constraint.\",\n \"36.A system as recited in claim 35 wherein the constraining means includes summation means for calculating a sum of quantity parameters for a group of types of items.\",\n \"37.A system as recited in claim, 36 wherein the constraining means includes comparing means for comparing the calculated sum with a previous calculated sum for the same group of types of items.\",\n \"38.A system as recited in claim 33 wherein the plurality of types of items are contracts for provision of a commodity covering different time periods, wherein the time periods for at least some of the contracts have at least some overlap.\",\n \"39.A system as recited in claim 35 wherein the processing means includes summation means for calculating a sum of the quantity parameters for at least some of the overlapping contracts.\",\n \"40.A system as recited in claim 39 wherein the processing means includes comparing means for comparing the calculated sum with an available quantity parameter.\",\n \"41.A system as recited in claim 33 wherein the fixed relation of the implying means is based on a schedule.\",\n \"42.A system as recited in claim 33 wherein the fixed relation of the implying means is based on an algorithm.\",\n \"43.A computer implemented method for conducting an auction of a plurality of types of items among a plurality of bidders, wherein bids are received and an allocation of the items among the bidders is determined based on the bids, comprising: receiving bids comprising a quantity parameter for at least one of the types of items in the auction and a price parameter, and processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, wherein the processing includes implying prices from price parameters in the received bids based on a fixed relation among the prices of the plurality of types of items.\",\n \"44.A method as recited in claim 43 which includes inviting additional bids in the event that an allocation of items consistent with the received bids is not possible, and processing the additional bids to determine an allocation of the items among the bidders consistent with the additional bids in the event that an allocation consistent with the additional bids is possible.\",\n \"45.A method as recited in claim 44 which further comprises constraining the additional bids from a given bidder by considering, in the processing, only additional bids which satisfy a constraint.\",\n \"46.A method as recited in claim 45 wherein the constraining includes calculating a sum of quantity parameters for a group of types of items.\",\n \"47.A method as recited in claim 46 wherein the constraining includes comparing the calculated sum with a previous calculated sum for the same group of types of items.\",\n \"48.A method as recited in claim 43 wherein the plurality of types of items are contracts for provision of a commodity covering different time periods, wherein the time periods for at least some of the contracts have at least some overlap.\",\n \"49.A method as recited in claim 48 wherein the processing includes calculating a sum of the quantity parameters for at least some of the overlapping contracts.\",\n \"50.A method as recited in claim 49 wherein the processing includes comparing the calculated sum with an available quantity parameter.\",\n \"51.A method as recited in claim 43 wherein the fixed relation of the processing is determined by a schedule.\",\n \"52.A method as recited in claim 43 wherein the fixed relation of the processing is determined by an algorithm.\",\n \"53.A computer system for conducting an auction of at least one type of item among a plurality of bidders in a plurality of rounds, wherein bids are received and an allocation of the items among the bidders is determined by the computer system based on the bids, comprising: establishing means for establishing starting prices and ending prices for each type of item in a plurality of rounds, receiving means for receiving bids, wherein a bid comprises a quantity for at least one type of item and a price parameter signifying a percentage of the distance from the established starting prices to the established ending prices, and processing means for processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, otherwise enabling the establishing means to alter the prices established for at least one type of item.\",\n \"54.A system as recited in claim 53 where the establishing means includes means to maintain a relation among the established prices.\",\n \"55.A system as recited in claim 54 wherein the means to maintain a relation among the established prices employs a schedule of prices.\",\n \"56.A system as recited in claim 54 wherein the means to maintain a relation among the established prices employs an algorithm.\",\n \"57.A system as recited in claim 53 which further comprises means for constraining the changes in a bid from a given bidder as the established prices are altered by considering, in the processing means, only bids which satisfy a constraint enforced by the means for constraining.\",\n \"58.A system as recited in claim 58 wherein the constraining means includes summation means for calculating a sum of quantities bid for a group of types of items.\",\n \"59.A system as recited in claim 59 wherein the constraining means includes comparing means for comparing the calculated sum with a previous calculated sum for the same group of types of items.\",\n \"60.A computer implemented method for conducting an auction of at least one type of item among a plurality of bidders in a plurality of rounds, wherein bids are received and an allocation of the items among the bidders is determined based on the bids, comprising: establishing starting prices and ending prices for each type of item in a plurality of rounds, receiving bids, wherein a bid comprises a quantity for at least one type of item and a price parameter signifying a percentage of the distance from the established starting prices to the established ending prices, and processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, otherwise altering the established prices for at least one type of item.\",\n \"61.A method as recited in claim 61 where the establishing includes maintaining a relation among the established prices.\",\n \"62.A method as recited in claim 62 wherein the relation among the established prices is determined by a schedule.\",\n \"63.A method as recited in claim 62 wherein the relation among the established prices is determined by an algorithm.\",\n \"64.A method as recited in claim 61 which further comprises constraining the changes in a bid from a given bidder as the established prices are altered by considering, in the processing, only bids which satisfy a constraint.\",\n \"65.A method as recited in claim 65 wherein the constraining includes calculating a sum of quantities bid for a group of types of items.\",\n \"66.A method as recited in claim 66 wherein the constraining includes comparing the calculated sum with a previous calculated sum for the same group of types of items.\"\n ],\n [\n \"<SOH> BACKGROUND OF THE INVENTION <EOH>Sellers and buyers of commodities, financial securities, and other goods and services are increasingly turning to auctions to assist in the sale or purchase of a wide range of items.\",\n \"Auctions are useful for selling a unique item (e.g., a painting), a single type of item available in multiple units (e.g., 10 tons of gold), and multiple types of items available in multiple units (e.g., 10 tons of gold and 20 tons of silver).\",\n \"And just as auctions are useful for efficiently selling items at maximum price, they are also useful for efficiently procuring items at minimum cost.\",\n \"Auction formats in the art tend generally to be of the sealed-bid or ascending-bid variety.\",\n \"In standard sealed-bid auctions, bidders—in one single bidding round—simultaneously and independently submit bids to the auctioneer, who then determines the auction outcome.\",\n \"In standard ascending-bid auctions, bidders—in a dynamic bidding process—submit bids in real time until no more bids are forthcoming.\",\n \"Sealed-bid auction formats offer the advantage of speed of the auction process.\",\n \"Ascending-bid auction formats offer the advantage that there is feedback among participants' bids, which tends to result in more aggressive bidding and in more efficient auction outcomes.\",\n \"When auction systems and methods in the art are used to sell (or buy) multiple types of items, the auctioneer generally specifies an available supply (or demand) for each type of item.\",\n \"For example, the auctioneer announces a supply of 10 tons of gold and 20 tons of silver, and then solicits bids, in a sealed-bid or ascending-bid procedure, in order to obtain market-clearing prices for each of the two types of commodities.\",\n \"However, consider a situation where the types of items are related, for example, contracts for provision of a commodity covering different time periods.\",\n \"For example, a government may wish to sell three types of financial securities: 3-month Treasury bills; 6-month Treasury bills; and 12-month Treasury bills.\",\n \"The government's only constraint may be that it needs to sell $50 billion worth of new debt securities at this auction, since this is the quantity of old debt securities that are maturing in the current calendar quarter.\",\n \"However, the government may have no preference as to how the required $50 billion is divided among 3-month bills, 6-month bills, and 12-month bills, respectively, and the government may simply wish to “let the market decide” the division among the various durations Auction systems and methods in the art provide effective ways for the government to sell bonds with a fixed division among the various durations.\",\n \"However, new and better systems and methods can be devised for “letting the market decide” the division among the various durations.\",\n \"The same problem also occurs in real life in the sale of contracts for electricity and all kinds of other commodities, as well as in the procurement of related items.\",\n \"In several preferred embodiments, the present invention provides a system and method for the government in the above example to simply auction $50 billion of Treasury bills at the lowest possible cost (relative to the market's yield curve), without imposing a particular division among the various durations.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is an improved system and method for a computer-implemented auction, particularly for a computer-implemented auction in which multiple types of items are auctioned together.\",\n \"In several embodiments, the present invention provides a system and method to conduct an auction of a group of related types of items, without imposing a particular division of supply or demand among the individual types of items within the group.\",\n \"In other embodiments, the present invention provides a system and method to conduct an auction of a group of related types of items, without imposing a particular division of supply or demand on some of the individual types of items within the group.\",\n \"Instead, in some embodiments, the present invention provides a means or method for establishing prices for the types of items within a group, wherein the prices maintain a relationship.\",\n \"In other embodiments, the present invention provides a means or method for implying prices from price parameters in the bids received from bidders, based on a relation among the prices for the types of items within a group.\",\n \"In some embodiments, market clearing is defined by the condition that the aggregate quantity bid for all types of items within a group is less than or equal to the available quantity of all types of items within the group.\",\n \"The division among the types of items within the group is thus determined flexibly, based on the bids at the associated prices.\",\n \"In other embodiments, market clearing is defined by the condition that the quantity bid for one selected type of item within a group is less than or equal to the available quantity of the selected type of item.\",\n \"The quantities of the other types of items within the group are thus determined flexibly, based on the bids at the associated prices.\",\n \"In a first preferred embodiment, the present invention is: A computer implemented method for conducting an auction of a plurality of types of items among a plurality of bidders, wherein bids are received and an allocation of the items among the bidders is determined based on the bids, comprising: establishing prices for the plurality of types of items, wherein the prices maintain a relationship, receiving bids associated with the established prices, and processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, otherwise altering the established prices for the plurality of types of items.\",\n \"In a second preferred embodiment, the present invention is a computer system for conducting an auction according to the method of the first preferred embodiment.\",\n \"The system includes an establishing means, a receiving means, and a processing means.\",\n \"In both the first and second preferred embodiments, the relationship or relation among the prices may involve a schedule or algorithm.\",\n \"In the event that the relationship or relation is based on a schedule, determining prices may involve looking up and possibly interpolating numbers that are contained in a table.\",\n \"Alternatively the relationship or relation may be represented as an algorithm or a formula that provides a relationship among the prices.\",\n \"The data table may reflect a market beyond the auction (for example, it may reflect price differentials in a separate commodities market or exchange) and the data table may change during the course of the auction (for example, if price differentials change in the separate commodities market or exchange, or if a significant news event occurs during the course of the auction).\",\n \"Similarly, the data set to which the algorithm or formula are applied may reflect a market beyond the auction and the data set may change during the course of the auction.\",\n \"In a third preferred embodiment, the present invention is: A computer implemented method for conducting an auction of a plurality of types of items among a plurality of bidders, wherein bids are received and an allocation of the items among the bidders is determined based on the bids, comprising: receiving bids comprising a quantity parameter for at least one of the types of items in the auction and a price parameter, and processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, wherein the processing includes implying prices from price parameters in the received bids based on a fixed relation among the prices of the plurality of types of items.\",\n \"In a fourth preferred embodiment, the present invention is a computer system for conducting an auction according to the method of the third preferred embodiment.\",\n \"The system includes a receiving means and a processing means.\",\n \"In both the third and fourth preferred embodiments, the “price parameters” in the received bids that are used for “implying prices” could be prices for one or more selected types of items.\",\n \"(For example, the price parameter could be an actual price for 3-month Treasury bills, and the prices for 6-month and 12-month Treasury bills could be implied, based on a fixed relation among the prices.)\",\n \"The “price parameters” could also be neutral parameters or indicators (e.g., in the nature of an index) of a price.\",\n \"Indeed, in a fifth preferred embodiment, bids include price parameters that may be a particularly useful form of neutral parameter.\",\n \"For example, an auctioneer may announce a starting price vector of (4.00, 4.50, 4.75) and an ending price vector of (8.00, 8.50, 8.75).\",\n \"If a bidder enters a price parameter of 25%, this may signify that the bidder is indicating a price which is 25% of the distance from the starting price to the ending price, i.e., an implied price vector of (5.00, 5.50, 5.75).\",\n \"In the fifth preferred embodiment, the present invention is: A computer implemented method for conducting an auction of at least one type of item among a plurality of bidders in a plurality of rounds, wherein bids are received and an allocation of the items among the bidders is determined based on the bids, comprising: establishing starting prices and ending prices for each type of item in a plurality of rounds, receiving bids, wherein a bid comprises a quantity for at least one type of item and a price parameter signifying a percentage of the distance from the established starting prices to the established ending prices, and processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, otherwise altering the established prices for at least one type of item.\",\n \"In a sixth preferred embodiment, the present invention is a computer system for conducting an auction according to the method of the fifth preferred embodiment.\",\n \"The system includes an establishing means, a receiving means, and a processing means.\",\n \"Various preferred embodiments of the present invention resolve a number of important limitations to the systems and methods for computer-implemented auctions in the art.\",\n \"Thus, the present invention offers the further technical effect of improving computer-implemented auctions, making them operate more efficiently.\",\n \"The present invention improves the ability of bidders to readily participate in the auction and to express their needs and preferences.\",\n \"The present invention improves the ability of the auctioneer to sell at higher prices the items being sold in the auction or to obtain at lower cost the items being procured in the auction.\",\n \"The present invention improves the efficiency of the auction process by enabling the auction to operate in shorter time, expending less computer time, fewer communications resources, and less bidder and auctioneer time.\",\n \"Finally, the present invention improves the allocative efficiency of the auction process by better allocating items to bidders who value them the highest (or, in a procurement auction, to bidders who can provide them at lowest cost).\",\n \"Certain constraints are desirable in order for the present invention to operate optimally and to reach an economically efficient outcome.\",\n \"In many auctions, it is desirable to impose constraints on bidders that require them to bid significantly on items early in the auction in order for them to be allowed to bid on items later in the auction.\",\n \"Otherwise, one may find oneself in a situation where bidders refrain from submitting serious bids until near the very end of the auction, defeating the purpose of a dynamic auction.\",\n \"One exemplary constraint is an activity rule which constrains a bidder not to increase his quantity, summed over all of the types of items within a group, from one round to the next.\",\n \"Another exemplary constraint is a more stringent activity rule which constrains a bidder not to increase his quantity, individually on each of the individual types of items, from one bid or one round to the next.\",\n \"A third exemplary constraint is a reduction rule which constrains a bidder not to decrease his quantity, for any single type of item, beyond the point where the sum of the quantities bid for this type of item by all bidders equals the sum of the quantities being auctioned.\",\n \"(If, in a given round, two or more bidders simultaneously attempt to decrease their quantities, for any single type of item, having the effect of reducing bids beyond the point where the sum of the quantities bid for this type of item by all bidders equals the sum of the quantities being auctioned, the auction procedure will resolve this discrepancy.\",\n \"For example, the auctioneer may honor these attempts to decrease in order of time priority, or may ration these simultaneous attempts to decrease in proportion to the attempted reductions.)\",\n \"The present invention includes treating the case of auctioning a set of items which includes two (or more) items that are neither identical nor perfect substitutes to one another.\",\n \"Henceforth, this will be described for short as a situation with “multiple types of items,” or simply “heterogeneous items” or “heterogeneous objects.” Often, but not always, the heterogeneous items auctioned together will bear some relationship to one another: for example, they may be securities issued by the same entity but with different durations to maturity; or contracts for the same goods or services but with different durations.\",\n \"One particularly salient use for the present invention is as follows.\",\n \"A national government or a private corporation may wish to simultaneously sell 3-month, 6-month and 12-month bonds or notes.\",\n \"More generally, these may be contracts for provision of any commodity; the contracts are for the same commodity, but covering different but possibly overlapping time periods.\",\n \"For the government's or corporation's purposes, the allocation of sales among the 3-month, 6-month and 12-month bonds or notes may not be critical; the main issue is that the total quantity of the bonds or notes of the three durations add up to the desired dollar figure.\",\n \"Meanwhile, the current “yield curve” on financial markets may have 6-month bonds or notes yielding interest rates 0.50% higher than 3-month bonds or notes, and the current “yield curve” on financial markets may have 12-month bonds or notes yielding interest rates 0.25% higher than 6-month bonds or notes.\",\n \"Under these circumstances, the government or corporation may utilize the present invention by beginning the auction with an (interest rate) vector of (6.00%, 6.50%, 6.75%) for the three respective durations.\",\n \"If the aggregate quantity of the bonds or notes desired by all the bidders at these interest rates is greater than the quantity of bonds or notes that the government or corporation wishes to sell, the government or corporation might then continue the auction by announcing a new vector of (5.99%, 6.49%, 6.74%) for the three respective durations.\",\n \"If the aggregate quantity of the bonds or notes desired by all the bidders at these interest rates is still greater than the quantity of bonds or notes that the government or corporation wishes to sell, the government or corporation might then continue the auction by announcing a new vector of (5.98%, 6.48%, 6.73%) for the three respective durations.\",\n \"The auction continues in this way, with the government or corporation always following the yield curve in setting.\",\n \"the relative interest rates for the various durations.\",\n \"Meanwhile, bidders are held to the exemplary activity rule which constrains a bidder not to increase his quantity, summed over the m types of items, from one round to the next.\",\n \"That is, the bidder is allowed to change the mix of 3-month, 6-month and 12-month bonds or notes that he demands, but his total quantity demanded of bonds or notes may not be increased as the interest rate goes down.\",\n \"A second use for the present invention is as follows.\",\n \"An electric utility may wish to sell contracts to provide a given quantity of electricity for 3-month, 6-month and 12-month durations.\",\n \"For the electric utility's purposes, the allocation of sales among the 3-month, 6-month and 12-month contracts may not be critical; the main issue may be that the total quantity of the contracts add up to a desired amount of electricity.\",\n \"Meanwhile, current energy prices may imply that a 6-month contract should be priced at $2 per-unit higher than a 3-month contract, and that a 12-month contract should be priced at $1 per unit higher than a 6-month contract.\",\n \"Under these circumstances, the electric utility may utilize the present invention by beginning the auction with a price vector of ($10.50, $12.50, $13.50) for the three respective durations.\",\n \"If the aggregate quantity of contracts desired by all the bidders at these prices is greater than the quantity of contracts that the electric utility wishes to sell, the electric utility might then continue the auction by announcing a new price vector of ($10.60, $12.60, $13.60) for the three respective durations.\",\n \"If the aggregate quantity of contracts desired by all the bidders at these prices is still greater than the quantity of contracts that the electric utility wishes to sell, the electric utility might then continue the auction by announcing a new price vector of ($10.70, $12.70, $13.70) for the three respective durations.\",\n \"The auction continues in this way, with the electric utility always following the implied price differential in setting the relative prices for the various durations.\",\n \"Meanwhile, bidders are held to the exemplary activity rule which constrains a bidder not to increase his quantity, summed over the types of items within a group, from one round to the next.\",\n \"That is, the bidder is allowed to change the mix of 3-month, 6-month and 12-month contracts that he demands, but his total quantity demanded of contracts may not be increased as the price goes up.\",\n \"In addition, the present invention may also be used to auction two or more groups of types of items.\",\n \"For example, the previous electric utility example could be complicated by the presence of two groups of electricity contracts: base and peak.\",\n \"(Base-load quantity is produced all of the time while peak-load quantity is only produced at times of peak demand.)\",\n \"Within each group, there might be three durations of contracts.\",\n \"Thus, in total, there could be six contracts for sale: 3-month, 6-month and 12-month base contracts; and 3-month, 6-month and 12-month peak contracts.\",\n \"To continue the example, current energy prices may imply that a 6-month base contract should be priced at $2 per unit higher than a 3-month base contract, and that a 12-month base contract should be priced at $1 per unit higher than a 6-month base contract.\",\n \"Meanwhile, current energy prices may imply that a 6-month peak contract should be priced at $3 per unit higher than a 3-month peak contract, and that a 12-month peak contract should be priced at $2 per unit higher than a 6-month peak contract.\",\n \"Under these circumstances, the electric utility may utilize the present invention by beginning the auction with a price vector of ($10.50, $12.50, $13.50) for the three respective base durations and a price vector of ($26.50, $29.50, $31.50) for the three respective peak durations.\",\n \"If the aggregate quantity of base contracts desired by all the bidders at these prices is slightly greater than the quantity of base contracts that the electric utility wishes to sell, but the aggregate quantity of peak contracts desired by all the bidders at these prices is much greater than the quantity of peak contracts that the electric utility wishes to sell, the electric utility might then continue the auction by announcing a new price vector of ($10.60, $12.60, $13.60) for the three respective base durations and a price vector of ($29.20, $32.20, $34.20) for the three respective peak durations.\",\n \"If the aggregate quantity of base contracts desired by all the bidders at these prices is still slightly greater than the quantity of base contracts that the electric utility wishes to sell, and the aggregate quantity of peak contracts desired by all the bidders at these prices is still somewhat greater than the quantity of peak contracts that the electric utility wishes to sell, the electric utility might then continue the auction by announcing a new price vector of ($10.60, $12.60, $13.60) for the three respective base durations and a price vector of ($29.40, $32.40, $34.40) for the three respective peak durations.\",\n \"The auction continues in this way, with the electric utility always following the implied price differential in setting the relative prices for the various durations within a group.\",\n \"(However, between groups, the prices may go up at very different rates.)\",\n \"Meanwhile, bidders are held to the exemplary activity rule which constrains a bidder not to increase his quantity, summed over the types of items within a group, from one round to the next.\",\n \"That is, the bidder is allowed to change the mix of 3-month, 6-month and 12-month base contracts that he demands, but his total quantity demanded of base contracts may not be increased as the price goes up.\",\n \"Similarly, the bidder is allowed to change the mix of 3-month, 6-month and 12-month peak contracts that he demands, but his total quantity demanded of peak contracts may not be increased as the price goes up.\",\n \"However, in this exemplary embodiment, the bidder is not allowed to substitute any quantity between the base group and the peak group.\",\n \"In many of the preferred embodiments, the auction with two or more groups of types of items ends at the first moment in time that the aggregate quantity of items bid for by all the bidders within each group is less than or equal to the quantity of items being auctioned within each group.\",\n \"An auction in accordance with an embodiment of the present invention could also allow various improvements to the bidding rules.\",\n \"In the previous example of base contracts and peak contracts, it may be the case that even if the price of only one group goes up, a bidder may be permitted to reduce his demands in both product groups.\",\n \"For example, if the base price vector is held constant and the peak price vector goes up, a bidder may be permitted to reduce his quantity bid on both base and peak products.\",\n \"The reason for allowing this is that the base and peak product may be complements (loosely speaking, products that are useful together, such as left shoes and right shoes), so an increased price for one product could reduce a bidder's demand for both.\",\n \"Throughout this document, the terms “objects”, “items”, “units” and “goods” are used essentially interchangeably.\",\n \"The inventive system and method may be used both for tangible objects, such as real or personal property, and intangible items, such as telecommunications licenses or electric power.\",\n \"The inventive system and method may be used in auctions where the auctioneer is a seller, buyer or broker, the bidders are buyers, sellers or brokers, and for auction-like activities which cannot be interpreted as selling or buying.\",\n \"The inventive system and method may be used for items including, but not restricted to, the following: public-sector bonds, bills, notes, stocks, and other securities or derivatives; private-sector bonds, bills, notes, stocks, and other securities or derivatives; communication licenses and spectrum rights; clearing, relocation or other rights concerning encumbrances of spectrum licenses; electric power and other commodity items; rights for terminal, entry, exit or transmission capacities or other rights in gas pipeline systems; airport landing rights; emission allowances and pollution permits; and other goods, services, objects, items or other property, tangible or intangible.\",\n \"It may also be used for option contracts on any of the above.\",\n \"It may be used in initial public offerings, secondary offerings, and in secondary or resale markets.\",\n \"The present invention is useful for “reverse auctions” conducted by or for buyers to acquire various kinds of items or resources, “standard auctions” conducted by sellers in which items are offered for sale, and “exchanges” in which both buyers and sellers place bids.\",\n \"Although terms such as “items or quantities demanded” (by a bidder) and “demand curve” (of a bidder) are used to describe the present invention, the terms “items or quantities offered” (by a bidder) and “supply curve” (of a bidder) are equally applicable.\",\n \"In some cases, this is made explicit by the use of both terms, or by the use of the terms “items or quantities transacted” (by a bidder) and “transaction curve” (of a bidder).\",\n \"The term “items or quantities transacted” includes both “items or quantities demanded” and “items or quantities offered”.\",\n \"The term “bid” includes both offers to sell and offers to buy.\",\n \"The term “transaction curve” includes both “demand curve” and “supply curve”.\",\n \"Moreover, any references to “items or quantities being offered” includes both “items or quantities being sold” by the auctioneer, in the case this is a standard auction for selling items, as well as “items or quantities being bought or procured” by the auctioneer, in the case this is a reverse auction for buying items or procuring items.\",\n \"Moreover, while standard auctions to sell typically involve ascending prices and reverse auctions to buy typically involve descending prices, the present invention may utilize prices that ascend and/or descend.\",\n \"While an auction method following this description could be conducted manually, computerized implementation of the auction method on an auction system allows the auction to be conducted with all bidding information taken into account, while carefully controlling the degree to which the information itself is disclosed to the participants.\",\n \"In one embodiment, all bidding information is displayed to the bidders.\",\n \"In another embodiment, no bidding information is displayed to the bidders; only the results of the auction are displayed.\",\n \"A number of intermediate embodiments are also possible, in which some but not all bidding information is displayed to the bidders.\",\n \"For example, in one preferred embodiment, the auctioneer discloses only the aggregate quantity bid for each type of item in each round, as opposed to disclosing each individual bid.\",\n \"Computerized implementation of the auction also allows the auction to be conducted swiftly and reliably, even if bidders are not located on-site.\",\n \"The auctioneer can receive large numbers of bids, determine the auction outcome, and transmit the results to bidders with minimal labor and in a very short span of time.\",\n \"In particular, sophisticated dynamic auction methods become feasible with a computer implementation; it becomes practical to operate iterative auction procedures in which there may be ten or more auction rounds, yet bidders can participate from different parts of the globe and the entire process can conclude in a single day.\",\n \"The present invention comprises a computer that receives bids in a static or dynamic bidding process and assigns the items to bidders, and a method for receiving bids in a static or dynamic bidding process and assigning the items to bidders.\",\n \"In one embodiment, the invention comprises a Bidding Information Processor (BIP), a plurality of Bidding Terminals (BT's) communicatively coupled to the BIP, and an Auctioneer's Terminal (AT) communicatively coupled to the BIP.\",\n \"Bidders at the BT's enter bids in multiple rounds, and may observe displayed auction information.\",\n \"The BIP, BT's and AT communicate and process information in order to conduct an auction.\",\n \"The network used, if any, can be any system capable of providing the necessary communication to/from a Bidding Information Processor (BIP), a Bidding Terminal (BT), and an Auctioneer's Terminal (AT).\",\n \"The network may be a local or wide area network such as, for example, Ethernet, token ring, the Internet, the World Wide Web, the information superhighway, an intranet or a virtual private network, or alternatively a telephone system, either private or public, a facsimile system, an electronic mail system, or a wireless communications system, or combinations of the foregoing.\",\n \"The following patents and published applications are related to the present invention: Ausubel, Lawrence M., U.S. Pat.\",\n \"No.\",\n \"5,905,975, May 1999.Ausubel, Lawrence M., U.S. Pat.\",\n \"No.\",\n \"6,021,398, Feb. 2000.Ausubel, Lawrence M., U.S. Pat.\",\n \"No.\",\n \"6,026,383, Feb. 2000.Ausubel, Lawrence M., Application No.\",\n \"00304195.1 at the European Patent Office, May 2000.The following other references may be related to the present invention: Arrow, Kenneth, H. D. Block and Leonid Hurwicz (1959), “On the Stability of Competitive Equilibrium, II,” Econometrica 27: 82-109.Ausubel, Lawrence M. (1997a), “An Efficient Ascending-Bid Auction for Multiple Objects,” Working Paper 97-06, University of Maryland, downloadable at www-ausubel-com.\",\n \"Ausubel, Lawrence M. (1997b), “On Generalizing the English Auction,” working paper, downloadable at www-ausubel-com.\",\n \"Ausubel, Lawrence M. (2000), “An Efficient Dynamic Auction for Heterogeneous Commodities,” working paper, University of Maryland, downloadable at www-ausubel-com.\",\n \"Ausubel, Lawrence M. and Peter Cramton (1996), “Demand Reduction and Inefficiency in Multi-Unit Auctions,” Working Paper No.\",\n \"96-07, downloadable at www-ausubel-com.\",\n \"Ausubel, Lawrence M. and Peter C. Cramton (1999), “The Optimality of Being Efficient,” mimeo, University of Maryland, June 1999, downloadable at www-ausubel-com.\",\n \"Ausubel, Lawrence M., Peter Cramton, R. Preston McAfee, and John McMillan (1997), “Synergies in Wireless Telephony: Evidence from the Broadband PCS Auctions,” Journal of Economics and Management Strategy, 6:3, 497-527.Ausubel, Lawrence M. and Jesse Schwartz (1999), “The Ascending Auction Paradox,” Mimeo, University of Maryland and Vanderbilt University, July 1999.Banks, Jeffrey S., John O. Ledyard and David P. Porter, “Allocating Uncertain and Unresponsive Resources: An Experimental Approach,” Rand Journal of Economics, 20: 1-25.Bernheim, B. Douglas and Michael Whinston (1986), “Menu Auctions, Resource Allocation and Economic Influence,” Quarterly Journal of Economics, 101: 1-31.Bikhchandani, Sushil, “Auctions of Heterogeneous Objects.” Mimeo, UCLA, March 1996.Bikhchandani, Sushil and John W. Mamer (1997), “Competitive Equilibrium in an Exchange Economy with Indivisibilities,” Journal of Economic Theory, June, 74(2), pp.\",\n \"385-413.Bikhchandani, Sushil and Joseph M. Ostroy (1999), “The Package Assignment Problem,” working paper, UCLA.\",\n \"Cassady, Ralph (1967).\",\n \"Auctions and Auctioneering .\",\n \"Berkeley: University of California Press.\",\n \"Charles River Associates and Market Design Inc. (1997), “Package Bidding for Spectrum Licenses,” Report to the Federal Communications Commission, downloadable at www-fcc-gov.\",\n \"Clarke, E. H. (1971), “Multipart Pricing of Public Goods,” Public Choice, XI, 17-33.Computer Reseller News, “Onsale Joins Fray as Online Shopping Picks Up Speed: Internet Booms,” Jun.\",\n \"5, 1995, p. 73; Dialog: File 16, Acc#05649796.Cramton, Peter C., “Money Out of Thin Air: The Nationwide Narrowband PCS Auction.” Journal of Economics and Management Strategy , Summer 1995, 4(2), pp.\",\n \"267-343.Cybernomics (2000), “An Experimental Comparison of the Simultaneous Multiple Round Auction and the CRA Combinatorial Auction.” Report to the Federal Communications Commission, downloadable at www-fcc-gov.\",\n \"Dasgupta, Partha and Eric S. Maskin (2000), “Efficient Auctions,” Quarterly Journal of Economics , May, 115(2), pp.\",\n \"341-388.DasVarma, Gopal (2000), “The Pareto Optimality of Disclosing Bidder Identities,” working paper, Duke University.\",\n \"Demange, Gabrielle, David Gale, and Marilda Sotomayor, “Multi-Item Auctions.” Journal of Political Economy , August 1986, 94(4), pp.\",\n \"863-872.DeMartini, Christine, Anthony Kwasnica, John Ledyard, and David Porter (1999), “A New and Improved Design for Multi-Object Iterative Auctions,” Cal Tech working paper.\",\n \"DeVries, Sven and Rakesh Vohra (2001), “Combinatorial Auctions: A Survey.” Working paper.\",\n \"Available at www-m9.ma.tum-de.\",\n \"Engelbrecht-Wiggans, Richard and Charles M. Kahn, “Protecting the Winner: Second-Price versus Oral Auctions.” Economics Letters , March 1991, 35(3), pp.\",\n \"243-248.Engelbrecht-Wiggans, Richard and Charles M. Kahn (1998), “Multi-Unit Auctions with Uniform Prices,” Economic Theory , September, 12(2), pp.\",\n \"227-258.Federal Communications Commission (1995), “Bidder Information Packet for FCC Auction Scheduled for Aug. 2, 1995.” Federal Communications Commission (1995), “Bidder Information Packet for FCC Auction Scheduled for Dec. 11, 1995.” Federal Communications Commission (2001), “Bidder Information Packet for FCC Auction No.\",\n \"31.” Groves, Theodore (1973), “Incentives in Teams,” Econometrica 61: 617-31.Gul, Faruk and Ennio Stacchetti (1999), “Walrasian Equilibrium with Gross Substitutes,” Journal of Economic Theory, 87, 9-124.Gul, Faruk and Ennio Stacchetti (2000), “The English Auction with Differentiated Commodities,” Journal of Economic Theory, May, 92(1), pp.\",\n \"66-95.Hahn, Frank H. (1982), “Stability,” in Kenneth J. Arrow and Michael D. Intriligator, eds., Handbook of Mathematical Economics, Volume II , Amsterdam: North-Holland,.\",\n \"pp.\",\n \"745-793.Jehiel, Philippe and Benny Moldovanu (1999), “Efficient Auctions with Interdependent Valuations,” Working Paper No.\",\n \"99-74, University of Mannheim, July.\",\n \"Jehiel, Philippe and Benny Moldovanu (2000), “A Critique of the Planned Rules for the German UMTS/IMT-2000 License Auction,” mimeo, University of Mannheim, March.\",\n \"Jehiel, Philippe and Benny Moldovanu (2001), “The European UMTS/IMT-2000 License Auctions,” working paper.\",\n \"Kagel, John H., Ronald M. Harstad, and Dan Levin, “Information Impact and Allocation Rules in Auctions with Affiliated Private Values: A Laboratory Study.” Econometrica , November 1987, 55(6), pp.\",\n \"1275-1304.Kagel, John H., Scott Kinross, and Dan Levin (2001), “Comparing Efficient Multi-Object Auction Institutions,” mimeo, Ohio State University.\",\n \"Kagel, John H. and Dan Levin (2001), “Behavior in Multi_Unit Demand Auctions: Experiments with Uniform Price and Dynamic Vickrey Auctions,” Econometrica, pp 413-454.Kelso, Alexander S. and Vincent P. Crawford (1982), “Job Matching, Coalition Formation, and Gross Substitutes,” Econometrica 50: 1483-1504.Krishna, Vijay and Motty Perry, “Efficient Mechanism Design.” Mimeo, Penn State University and Hebrew University, March 1997.Ledyard, John, David Porter and Antonio Rangel (1997), “Experiments Testing Multi-object Allocation Mechanisms,” Journal of Economics and Management Strategy, 6: 639-675.Manelli, Alejandro M., Martin Sefton and Benjamin S. Wilner, “Multi-Unit Auctions: A Comparison of Static and Dynamic Mechanisms.” Mimeo, Arizona State University, University of Newcastle and LECG/Navigant Consulting, October 1999.Maskin, Eric S. (1992), “Auctions and Privatization,” in Horst Siebert, ed., Privatization: Symposium in Honor of Herbert Giersch , Tubingen: Mohr (Siebeck), pp.\",\n \"115-136.Maskin, Eric S. and John G. Riley, “Optimal Multi-unit Auctions,” in Frank Hahn, ed., The Economics of Missing Markets, Information, and Games .\",\n \"Oxford: Oxford University Press, 1989, pp.\",\n \"312-35.McAfee, R. Preston and John McMillan, “Auctions and Bidding.” Journal of Economic Literature , June 1987, 25(2), pp.\",\n \"699-738.McCabe, Kevin A., Stephen J. Rassenti, and Vernon L. Smith, “Auction Institutional Design: Theory and Behavior of Simultaneous Multiple-Unit Generalizations of the Dutch and English Auctions.” American Economic Review , December 1990, 80(5), pp.\",\n \"1276-1283.K.\",\n \"A. McCabe, S. J. Rassenti and V. L. Smith, “Testing Vickrey's and Other Simultaneous Multiple Unit Versions of the English Auction,” Research in Experimental Economics, vol.\",\n \"4, Greenwich, Conn.: JAI Press, 1991, pp.\",\n \"45-79.McMillan, John (1994), “Selling Spectrum Rights,” Journal of Economics Perspectives, 8: 145-162.F.\",\n \"M. Menezes, Four Essays on Auction Theory , University of Illinois doctoral dissertation, February 1993, pp.\",\n \"1-97 and 143-152.Milgrom, Paul (1981), “Rational Expectations, Information Acquisition, and Competitive Bidding,” Econometrica, 49: 921-43.Milgrom, Paul (1987), “Auction Theory,” in Truman F. Bewley, ed., Advances in Economic Theory Fifth World Congress .\",\n \"Cambridge: Cambridge University Press, 1987, pp.\",\n \"1-32.Milgrom, Paul (1996), “Auctioning the Radio Spectrum,” downloadable at www-market-design.com.\",\n \"Milgrom, Paul (2000).\",\n \"“Putting Auction Theory to Work: The Simultaneous Ascending Auction,” Journal of Political Economy, 108:2, 245-272.Milgrom, Paul and John Roberts (1991), “Adaptive and Sophisticated Learning in Repeated * Normal Form Games,” Games and Economic Behavior, 3: 82-100.Milgrom, Paul and Robert Weber (1982), “A Theory of Auctions and Competitive Bidding,” Econometrica 50: 1089-1122.Milgrom, Paul R. and Robert J. Weber, “A Theory of Auctions and Competitive Bidding, II.” Unpublished manuscript, Northwestern University, 1982.Nisan, Noam (1999), “Bidding and Allocation in Combinatorial Auctions,” working paper, www-cs-huji-ac-il.\",\n \"Onsale release, “Onsale Brings Thrill of Auctions and Bargain Hunting Online; Unique Internet retail service debuts with week-long charity auction for The Computer Museum in Boston”; Dialog Abstract: File 610, Acc#0489267; May 24, 1995.D.\",\n \"C. Parkes (1999), “iBundle: An Efficient Ascending Price Bundle Auction”, Proceedings ACM Conference on Electronic Commerce (EC-99), Denver, 148-157.Perry, Motty and Philip J. Reny (1999a), “An Ex-Post Efficient Auction,” Working Paper, Hebrew University and University of Chicago, September.\",\n \"Perry, Motty and Philip J. Reny (1999b), “An Ex-Post Efficient Multi-Unit Ascending Auction,” Working Paper, Hebrew University and University of Chicago, September.\",\n \"Plott, Charles (1997), “Laboratory Experimental Testbeds: Application to the PCS Auction,” Journal of Economics and Management Strategy, 6: 605-638.Rassenti, S. J., V. L. Smith, and R. L. Bulfin (1982), “A Combinatorial Auction Mechanism for Airport Time Slot Allocation,” Bell Journal of Economics 13: 402-417.M.\",\n \"H. Rothkopf, A. Pekec and R. M. Harstad (1998), “Computationally Manageable Combinatorial Auctions”, Management Science , Vol.\",\n \"44, No.\",\n \"8, 1131-1147.Rothkopf, Michael, Thomas Teisberg and Edward Kahn (1990), “Why Are Vickrey Auctions Rare?” Journal of Political Economy, 98: 94-109.Sholtz & Associates, LLC, “ACE Market Operations Guide,” Jan. 3, 1996, pp.\",\n \"1-5.Siegmann, “Nowhere to Go But Up.\",\n \"(Onsale CEO Jerry Kaplan) (PC Week Inside) (Inside People)” ; PC Week; v12 n42; pA5 (1); Oct. 23, 1995; Dialog: File 148, Acc#08222496.U.S.\",\n \"Department of the Treasury, U.S. Securities and Exchange Commission, and Board of Governors of the Federal Reserve System, Joint Report on the Government Securities Market , Washington, D.C.: U.S.G.P.O., January 1992.Vickrey, William (1961), “Counterspeculation, Auctions and Competitive Sealed Tenders,” Journal of Finance , March, 16(1), pp.\",\n \"8-37.Vickrey, William (1962), “Auctions and Bidding Games,” Recent Advances in Game Theory, Princeton: Princeton University Conference, 1962, pp.\",\n \"15-29.Vickrey, William (1976), “Auctions, Markets, and Optimal Allocation,” Bidding and Auctioning for Procurement and Allocation, New York: New York University Press 1976, pp.\",\n \"13-20.Weber, Robert J.\",\n \"(1983), “Multiple-Object Auctions,” Auctions, Bidding, and Contracting: Uses and Theory, New York: New York University Press, 1983, pp.\",\n \"165-191.Weber, Robert J.\",\n \"(1997), “Making More from Less: Strategic Demand Reduction in the FCC Spectrum Auctions,” Journal of Economics and Management Strategy, 6:529-548.Williams, Steven (1999), “A Characterization of Efficient, Bayesian Incentive Compatible Mechanisms,” Economic Theory 14: 155-180.Wilson, Robert (1979), “Auctions of Shares,” Quarterly Journal of Economics , vol.\",\n \"94, 1979, pp.675-689.\"\n ],\n [\n \"RELATED APPLICATIONS This application claims the benefit of the filing dates of the following co-pending U.S. applications: Ausubel, “SYSTEM AND METHOD FOR AN EFFICIENT DYNAMIC AUCTION FOR MULTIPLE TYPES OF ITEMS”, U.S.\",\n \"Provisional Patent Application, Ser.\",\n \"No.\",\n \"60/294,246 Filed: May 31, 2001.Ausubel, “SYSTEM AND METHOD FOR AN EFFICIENT DYNAMIC MULTI-UNIT AUCTION”, U.S. patent application, Ser.\",\n \"No.\",\n \"09/898,483 Filed: Jul.\",\n \"05, 2001.FIELD OF THE INVENTION The present invention relates to improving computer-implemented auctions and, more particularly, to computer implementation of an auction in which multiple types of items are auctioned together.\",\n \"BACKGROUND OF THE INVENTION Sellers and buyers of commodities, financial securities, and other goods and services are increasingly turning to auctions to assist in the sale or purchase of a wide range of items.\",\n \"Auctions are useful for selling a unique item (e.g., a painting), a single type of item available in multiple units (e.g., 10 tons of gold), and multiple types of items available in multiple units (e.g., 10 tons of gold and 20 tons of silver).\",\n \"And just as auctions are useful for efficiently selling items at maximum price, they are also useful for efficiently procuring items at minimum cost.\",\n \"Auction formats in the art tend generally to be of the sealed-bid or ascending-bid variety.\",\n \"In standard sealed-bid auctions, bidders—in one single bidding round—simultaneously and independently submit bids to the auctioneer, who then determines the auction outcome.\",\n \"In standard ascending-bid auctions, bidders—in a dynamic bidding process—submit bids in real time until no more bids are forthcoming.\",\n \"Sealed-bid auction formats offer the advantage of speed of the auction process.\",\n \"Ascending-bid auction formats offer the advantage that there is feedback among participants' bids, which tends to result in more aggressive bidding and in more efficient auction outcomes.\",\n \"When auction systems and methods in the art are used to sell (or buy) multiple types of items, the auctioneer generally specifies an available supply (or demand) for each type of item.\",\n \"For example, the auctioneer announces a supply of 10 tons of gold and 20 tons of silver, and then solicits bids, in a sealed-bid or ascending-bid procedure, in order to obtain market-clearing prices for each of the two types of commodities.\",\n \"However, consider a situation where the types of items are related, for example, contracts for provision of a commodity covering different time periods.\",\n \"For example, a government may wish to sell three types of financial securities: 3-month Treasury bills; 6-month Treasury bills; and 12-month Treasury bills.\",\n \"The government's only constraint may be that it needs to sell $50 billion worth of new debt securities at this auction, since this is the quantity of old debt securities that are maturing in the current calendar quarter.\",\n \"However, the government may have no preference as to how the required $50 billion is divided among 3-month bills, 6-month bills, and 12-month bills, respectively, and the government may simply wish to “let the market decide” the division among the various durations Auction systems and methods in the art provide effective ways for the government to sell bonds with a fixed division among the various durations.\",\n \"However, new and better systems and methods can be devised for “letting the market decide” the division among the various durations.\",\n \"The same problem also occurs in real life in the sale of contracts for electricity and all kinds of other commodities, as well as in the procurement of related items.\",\n \"In several preferred embodiments, the present invention provides a system and method for the government in the above example to simply auction $50 billion of Treasury bills at the lowest possible cost (relative to the market's yield curve), without imposing a particular division among the various durations.\",\n \"SUMMARY OF THE INVENTION The present invention is an improved system and method for a computer-implemented auction, particularly for a computer-implemented auction in which multiple types of items are auctioned together.\",\n \"In several embodiments, the present invention provides a system and method to conduct an auction of a group of related types of items, without imposing a particular division of supply or demand among the individual types of items within the group.\",\n \"In other embodiments, the present invention provides a system and method to conduct an auction of a group of related types of items, without imposing a particular division of supply or demand on some of the individual types of items within the group.\",\n \"Instead, in some embodiments, the present invention provides a means or method for establishing prices for the types of items within a group, wherein the prices maintain a relationship.\",\n \"In other embodiments, the present invention provides a means or method for implying prices from price parameters in the bids received from bidders, based on a relation among the prices for the types of items within a group.\",\n \"In some embodiments, market clearing is defined by the condition that the aggregate quantity bid for all types of items within a group is less than or equal to the available quantity of all types of items within the group.\",\n \"The division among the types of items within the group is thus determined flexibly, based on the bids at the associated prices.\",\n \"In other embodiments, market clearing is defined by the condition that the quantity bid for one selected type of item within a group is less than or equal to the available quantity of the selected type of item.\",\n \"The quantities of the other types of items within the group are thus determined flexibly, based on the bids at the associated prices.\",\n \"In a first preferred embodiment, the present invention is: A computer implemented method for conducting an auction of a plurality of types of items among a plurality of bidders, wherein bids are received and an allocation of the items among the bidders is determined based on the bids, comprising: establishing prices for the plurality of types of items, wherein the prices maintain a relationship, receiving bids associated with the established prices, and processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, otherwise altering the established prices for the plurality of types of items.\",\n \"In a second preferred embodiment, the present invention is a computer system for conducting an auction according to the method of the first preferred embodiment.\",\n \"The system includes an establishing means, a receiving means, and a processing means.\",\n \"In both the first and second preferred embodiments, the relationship or relation among the prices may involve a schedule or algorithm.\",\n \"In the event that the relationship or relation is based on a schedule, determining prices may involve looking up and possibly interpolating numbers that are contained in a table.\",\n \"Alternatively the relationship or relation may be represented as an algorithm or a formula that provides a relationship among the prices.\",\n \"The data table may reflect a market beyond the auction (for example, it may reflect price differentials in a separate commodities market or exchange) and the data table may change during the course of the auction (for example, if price differentials change in the separate commodities market or exchange, or if a significant news event occurs during the course of the auction).\",\n \"Similarly, the data set to which the algorithm or formula are applied may reflect a market beyond the auction and the data set may change during the course of the auction.\",\n \"In a third preferred embodiment, the present invention is: A computer implemented method for conducting an auction of a plurality of types of items among a plurality of bidders, wherein bids are received and an allocation of the items among the bidders is determined based on the bids, comprising: receiving bids comprising a quantity parameter for at least one of the types of items in the auction and a price parameter, and processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, wherein the processing includes implying prices from price parameters in the received bids based on a fixed relation among the prices of the plurality of types of items.\",\n \"In a fourth preferred embodiment, the present invention is a computer system for conducting an auction according to the method of the third preferred embodiment.\",\n \"The system includes a receiving means and a processing means.\",\n \"In both the third and fourth preferred embodiments, the “price parameters” in the received bids that are used for “implying prices” could be prices for one or more selected types of items.\",\n \"(For example, the price parameter could be an actual price for 3-month Treasury bills, and the prices for 6-month and 12-month Treasury bills could be implied, based on a fixed relation among the prices.)\",\n \"The “price parameters” could also be neutral parameters or indicators (e.g., in the nature of an index) of a price.\",\n \"Indeed, in a fifth preferred embodiment, bids include price parameters that may be a particularly useful form of neutral parameter.\",\n \"For example, an auctioneer may announce a starting price vector of (4.00, 4.50, 4.75) and an ending price vector of (8.00, 8.50, 8.75).\",\n \"If a bidder enters a price parameter of 25%, this may signify that the bidder is indicating a price which is 25% of the distance from the starting price to the ending price, i.e., an implied price vector of (5.00, 5.50, 5.75).\",\n \"In the fifth preferred embodiment, the present invention is: A computer implemented method for conducting an auction of at least one type of item among a plurality of bidders in a plurality of rounds, wherein bids are received and an allocation of the items among the bidders is determined based on the bids, comprising: establishing starting prices and ending prices for each type of item in a plurality of rounds, receiving bids, wherein a bid comprises a quantity for at least one type of item and a price parameter signifying a percentage of the distance from the established starting prices to the established ending prices, and processing the received bids to determine an allocation of the items among the bidders consistent with the bids in the event that an allocation consistent with the bids is possible, otherwise altering the established prices for at least one type of item.\",\n \"In a sixth preferred embodiment, the present invention is a computer system for conducting an auction according to the method of the fifth preferred embodiment.\",\n \"The system includes an establishing means, a receiving means, and a processing means.\",\n \"Various preferred embodiments of the present invention resolve a number of important limitations to the systems and methods for computer-implemented auctions in the art.\",\n \"Thus, the present invention offers the further technical effect of improving computer-implemented auctions, making them operate more efficiently.\",\n \"The present invention improves the ability of bidders to readily participate in the auction and to express their needs and preferences.\",\n \"The present invention improves the ability of the auctioneer to sell at higher prices the items being sold in the auction or to obtain at lower cost the items being procured in the auction.\",\n \"The present invention improves the efficiency of the auction process by enabling the auction to operate in shorter time, expending less computer time, fewer communications resources, and less bidder and auctioneer time.\",\n \"Finally, the present invention improves the allocative efficiency of the auction process by better allocating items to bidders who value them the highest (or, in a procurement auction, to bidders who can provide them at lowest cost).\",\n \"Certain constraints are desirable in order for the present invention to operate optimally and to reach an economically efficient outcome.\",\n \"In many auctions, it is desirable to impose constraints on bidders that require them to bid significantly on items early in the auction in order for them to be allowed to bid on items later in the auction.\",\n \"Otherwise, one may find oneself in a situation where bidders refrain from submitting serious bids until near the very end of the auction, defeating the purpose of a dynamic auction.\",\n \"One exemplary constraint is an activity rule which constrains a bidder not to increase his quantity, summed over all of the types of items within a group, from one round to the next.\",\n \"Another exemplary constraint is a more stringent activity rule which constrains a bidder not to increase his quantity, individually on each of the individual types of items, from one bid or one round to the next.\",\n \"A third exemplary constraint is a reduction rule which constrains a bidder not to decrease his quantity, for any single type of item, beyond the point where the sum of the quantities bid for this type of item by all bidders equals the sum of the quantities being auctioned.\",\n \"(If, in a given round, two or more bidders simultaneously attempt to decrease their quantities, for any single type of item, having the effect of reducing bids beyond the point where the sum of the quantities bid for this type of item by all bidders equals the sum of the quantities being auctioned, the auction procedure will resolve this discrepancy.\",\n \"For example, the auctioneer may honor these attempts to decrease in order of time priority, or may ration these simultaneous attempts to decrease in proportion to the attempted reductions.)\",\n \"The present invention includes treating the case of auctioning a set of items which includes two (or more) items that are neither identical nor perfect substitutes to one another.\",\n \"Henceforth, this will be described for short as a situation with “multiple types of items,” or simply “heterogeneous items” or “heterogeneous objects.” Often, but not always, the heterogeneous items auctioned together will bear some relationship to one another: for example, they may be securities issued by the same entity but with different durations to maturity; or contracts for the same goods or services but with different durations.\",\n \"One particularly salient use for the present invention is as follows.\",\n \"A national government or a private corporation may wish to simultaneously sell 3-month, 6-month and 12-month bonds or notes.\",\n \"More generally, these may be contracts for provision of any commodity; the contracts are for the same commodity, but covering different but possibly overlapping time periods.\",\n \"For the government's or corporation's purposes, the allocation of sales among the 3-month, 6-month and 12-month bonds or notes may not be critical; the main issue is that the total quantity of the bonds or notes of the three durations add up to the desired dollar figure.\",\n \"Meanwhile, the current “yield curve” on financial markets may have 6-month bonds or notes yielding interest rates 0.50% higher than 3-month bonds or notes, and the current “yield curve” on financial markets may have 12-month bonds or notes yielding interest rates 0.25% higher than 6-month bonds or notes.\",\n \"Under these circumstances, the government or corporation may utilize the present invention by beginning the auction with an (interest rate) vector of (6.00%, 6.50%, 6.75%) for the three respective durations.\",\n \"If the aggregate quantity of the bonds or notes desired by all the bidders at these interest rates is greater than the quantity of bonds or notes that the government or corporation wishes to sell, the government or corporation might then continue the auction by announcing a new vector of (5.99%, 6.49%, 6.74%) for the three respective durations.\",\n \"If the aggregate quantity of the bonds or notes desired by all the bidders at these interest rates is still greater than the quantity of bonds or notes that the government or corporation wishes to sell, the government or corporation might then continue the auction by announcing a new vector of (5.98%, 6.48%, 6.73%) for the three respective durations.\",\n \"The auction continues in this way, with the government or corporation always following the yield curve in setting.\",\n \"the relative interest rates for the various durations.\",\n \"Meanwhile, bidders are held to the exemplary activity rule which constrains a bidder not to increase his quantity, summed over the m types of items, from one round to the next.\",\n \"That is, the bidder is allowed to change the mix of 3-month, 6-month and 12-month bonds or notes that he demands, but his total quantity demanded of bonds or notes may not be increased as the interest rate goes down.\",\n \"A second use for the present invention is as follows.\",\n \"An electric utility may wish to sell contracts to provide a given quantity of electricity for 3-month, 6-month and 12-month durations.\",\n \"For the electric utility's purposes, the allocation of sales among the 3-month, 6-month and 12-month contracts may not be critical; the main issue may be that the total quantity of the contracts add up to a desired amount of electricity.\",\n \"Meanwhile, current energy prices may imply that a 6-month contract should be priced at $2 per-unit higher than a 3-month contract, and that a 12-month contract should be priced at $1 per unit higher than a 6-month contract.\",\n \"Under these circumstances, the electric utility may utilize the present invention by beginning the auction with a price vector of ($10.50, $12.50, $13.50) for the three respective durations.\",\n \"If the aggregate quantity of contracts desired by all the bidders at these prices is greater than the quantity of contracts that the electric utility wishes to sell, the electric utility might then continue the auction by announcing a new price vector of ($10.60, $12.60, $13.60) for the three respective durations.\",\n \"If the aggregate quantity of contracts desired by all the bidders at these prices is still greater than the quantity of contracts that the electric utility wishes to sell, the electric utility might then continue the auction by announcing a new price vector of ($10.70, $12.70, $13.70) for the three respective durations.\",\n \"The auction continues in this way, with the electric utility always following the implied price differential in setting the relative prices for the various durations.\",\n \"Meanwhile, bidders are held to the exemplary activity rule which constrains a bidder not to increase his quantity, summed over the types of items within a group, from one round to the next.\",\n \"That is, the bidder is allowed to change the mix of 3-month, 6-month and 12-month contracts that he demands, but his total quantity demanded of contracts may not be increased as the price goes up.\",\n \"In addition, the present invention may also be used to auction two or more groups of types of items.\",\n \"For example, the previous electric utility example could be complicated by the presence of two groups of electricity contracts: base and peak.\",\n \"(Base-load quantity is produced all of the time while peak-load quantity is only produced at times of peak demand.)\",\n \"Within each group, there might be three durations of contracts.\",\n \"Thus, in total, there could be six contracts for sale: 3-month, 6-month and 12-month base contracts; and 3-month, 6-month and 12-month peak contracts.\",\n \"To continue the example, current energy prices may imply that a 6-month base contract should be priced at $2 per unit higher than a 3-month base contract, and that a 12-month base contract should be priced at $1 per unit higher than a 6-month base contract.\",\n \"Meanwhile, current energy prices may imply that a 6-month peak contract should be priced at $3 per unit higher than a 3-month peak contract, and that a 12-month peak contract should be priced at $2 per unit higher than a 6-month peak contract.\",\n \"Under these circumstances, the electric utility may utilize the present invention by beginning the auction with a price vector of ($10.50, $12.50, $13.50) for the three respective base durations and a price vector of ($26.50, $29.50, $31.50) for the three respective peak durations.\",\n \"If the aggregate quantity of base contracts desired by all the bidders at these prices is slightly greater than the quantity of base contracts that the electric utility wishes to sell, but the aggregate quantity of peak contracts desired by all the bidders at these prices is much greater than the quantity of peak contracts that the electric utility wishes to sell, the electric utility might then continue the auction by announcing a new price vector of ($10.60, $12.60, $13.60) for the three respective base durations and a price vector of ($29.20, $32.20, $34.20) for the three respective peak durations.\",\n \"If the aggregate quantity of base contracts desired by all the bidders at these prices is still slightly greater than the quantity of base contracts that the electric utility wishes to sell, and the aggregate quantity of peak contracts desired by all the bidders at these prices is still somewhat greater than the quantity of peak contracts that the electric utility wishes to sell, the electric utility might then continue the auction by announcing a new price vector of ($10.60, $12.60, $13.60) for the three respective base durations and a price vector of ($29.40, $32.40, $34.40) for the three respective peak durations.\",\n \"The auction continues in this way, with the electric utility always following the implied price differential in setting the relative prices for the various durations within a group.\",\n \"(However, between groups, the prices may go up at very different rates.)\",\n \"Meanwhile, bidders are held to the exemplary activity rule which constrains a bidder not to increase his quantity, summed over the types of items within a group, from one round to the next.\",\n \"That is, the bidder is allowed to change the mix of 3-month, 6-month and 12-month base contracts that he demands, but his total quantity demanded of base contracts may not be increased as the price goes up.\",\n \"Similarly, the bidder is allowed to change the mix of 3-month, 6-month and 12-month peak contracts that he demands, but his total quantity demanded of peak contracts may not be increased as the price goes up.\",\n \"However, in this exemplary embodiment, the bidder is not allowed to substitute any quantity between the base group and the peak group.\",\n \"In many of the preferred embodiments, the auction with two or more groups of types of items ends at the first moment in time that the aggregate quantity of items bid for by all the bidders within each group is less than or equal to the quantity of items being auctioned within each group.\",\n \"An auction in accordance with an embodiment of the present invention could also allow various improvements to the bidding rules.\",\n \"In the previous example of base contracts and peak contracts, it may be the case that even if the price of only one group goes up, a bidder may be permitted to reduce his demands in both product groups.\",\n \"For example, if the base price vector is held constant and the peak price vector goes up, a bidder may be permitted to reduce his quantity bid on both base and peak products.\",\n \"The reason for allowing this is that the base and peak product may be complements (loosely speaking, products that are useful together, such as left shoes and right shoes), so an increased price for one product could reduce a bidder's demand for both.\",\n \"Throughout this document, the terms “objects”, “items”, “units” and “goods” are used essentially interchangeably.\",\n \"The inventive system and method may be used both for tangible objects, such as real or personal property, and intangible items, such as telecommunications licenses or electric power.\",\n \"The inventive system and method may be used in auctions where the auctioneer is a seller, buyer or broker, the bidders are buyers, sellers or brokers, and for auction-like activities which cannot be interpreted as selling or buying.\",\n \"The inventive system and method may be used for items including, but not restricted to, the following: public-sector bonds, bills, notes, stocks, and other securities or derivatives; private-sector bonds, bills, notes, stocks, and other securities or derivatives; communication licenses and spectrum rights; clearing, relocation or other rights concerning encumbrances of spectrum licenses; electric power and other commodity items; rights for terminal, entry, exit or transmission capacities or other rights in gas pipeline systems; airport landing rights; emission allowances and pollution permits; and other goods, services, objects, items or other property, tangible or intangible.\",\n \"It may also be used for option contracts on any of the above.\",\n \"It may be used in initial public offerings, secondary offerings, and in secondary or resale markets.\",\n \"The present invention is useful for “reverse auctions” conducted by or for buyers to acquire various kinds of items or resources, “standard auctions” conducted by sellers in which items are offered for sale, and “exchanges” in which both buyers and sellers place bids.\",\n \"Although terms such as “items or quantities demanded” (by a bidder) and “demand curve” (of a bidder) are used to describe the present invention, the terms “items or quantities offered” (by a bidder) and “supply curve” (of a bidder) are equally applicable.\",\n \"In some cases, this is made explicit by the use of both terms, or by the use of the terms “items or quantities transacted” (by a bidder) and “transaction curve” (of a bidder).\",\n \"The term “items or quantities transacted” includes both “items or quantities demanded” and “items or quantities offered”.\",\n \"The term “bid” includes both offers to sell and offers to buy.\",\n \"The term “transaction curve” includes both “demand curve” and “supply curve”.\",\n \"Moreover, any references to “items or quantities being offered” includes both “items or quantities being sold” by the auctioneer, in the case this is a standard auction for selling items, as well as “items or quantities being bought or procured” by the auctioneer, in the case this is a reverse auction for buying items or procuring items.\",\n \"Moreover, while standard auctions to sell typically involve ascending prices and reverse auctions to buy typically involve descending prices, the present invention may utilize prices that ascend and/or descend.\",\n \"While an auction method following this description could be conducted manually, computerized implementation of the auction method on an auction system allows the auction to be conducted with all bidding information taken into account, while carefully controlling the degree to which the information itself is disclosed to the participants.\",\n \"In one embodiment, all bidding information is displayed to the bidders.\",\n \"In another embodiment, no bidding information is displayed to the bidders; only the results of the auction are displayed.\",\n \"A number of intermediate embodiments are also possible, in which some but not all bidding information is displayed to the bidders.\",\n \"For example, in one preferred embodiment, the auctioneer discloses only the aggregate quantity bid for each type of item in each round, as opposed to disclosing each individual bid.\",\n \"Computerized implementation of the auction also allows the auction to be conducted swiftly and reliably, even if bidders are not located on-site.\",\n \"The auctioneer can receive large numbers of bids, determine the auction outcome, and transmit the results to bidders with minimal labor and in a very short span of time.\",\n \"In particular, sophisticated dynamic auction methods become feasible with a computer implementation; it becomes practical to operate iterative auction procedures in which there may be ten or more auction rounds, yet bidders can participate from different parts of the globe and the entire process can conclude in a single day.\",\n \"The present invention comprises a computer that receives bids in a static or dynamic bidding process and assigns the items to bidders, and a method for receiving bids in a static or dynamic bidding process and assigning the items to bidders.\",\n \"In one embodiment, the invention comprises a Bidding Information Processor (BIP), a plurality of Bidding Terminals (BT's) communicatively coupled to the BIP, and an Auctioneer's Terminal (AT) communicatively coupled to the BIP.\",\n \"Bidders at the BT's enter bids in multiple rounds, and may observe displayed auction information.\",\n \"The BIP, BT's and AT communicate and process information in order to conduct an auction.\",\n \"The network used, if any, can be any system capable of providing the necessary communication to/from a Bidding Information Processor (BIP), a Bidding Terminal (BT), and an Auctioneer's Terminal (AT).\",\n \"The network may be a local or wide area network such as, for example, Ethernet, token ring, the Internet, the World Wide Web, the information superhighway, an intranet or a virtual private network, or alternatively a telephone system, either private or public, a facsimile system, an electronic mail system, or a wireless communications system, or combinations of the foregoing.\",\n \"The following patents and published applications are related to the present invention: Ausubel, Lawrence M., U.S. Pat.\",\n \"No.\",\n \"5,905,975, May 1999.Ausubel, Lawrence M., U.S. Pat.\",\n \"No.\",\n \"6,021,398, Feb. 2000.Ausubel, Lawrence M., U.S. Pat.\",\n \"No.\",\n \"6,026,383, Feb. 2000.Ausubel, Lawrence M., Application No.\",\n \"00304195.1 at the European Patent Office, May 2000.The following other references may be related to the present invention: Arrow, Kenneth, H. D. Block and Leonid Hurwicz (1959), “On the Stability of Competitive Equilibrium, II,” Econometrica 27: 82-109.Ausubel, Lawrence M. (1997a), “An Efficient Ascending-Bid Auction for Multiple Objects,” Working Paper 97-06, University of Maryland, downloadable at www-ausubel-com.\",\n \"Ausubel, Lawrence M. (1997b), “On Generalizing the English Auction,” working paper, downloadable at www-ausubel-com.\",\n \"Ausubel, Lawrence M. (2000), “An Efficient Dynamic Auction for Heterogeneous Commodities,” working paper, University of Maryland, downloadable at www-ausubel-com.\",\n \"Ausubel, Lawrence M. and Peter Cramton (1996), “Demand Reduction and Inefficiency in Multi-Unit Auctions,” Working Paper No.\",\n \"96-07, downloadable at www-ausubel-com.\",\n \"Ausubel, Lawrence M. and Peter C. Cramton (1999), “The Optimality of Being Efficient,” mimeo, University of Maryland, June 1999, downloadable at www-ausubel-com.\",\n \"Ausubel, Lawrence M., Peter Cramton, R. Preston McAfee, and John McMillan (1997), “Synergies in Wireless Telephony: Evidence from the Broadband PCS Auctions,” Journal of Economics and Management Strategy, 6:3, 497-527.Ausubel, Lawrence M. and Jesse Schwartz (1999), “The Ascending Auction Paradox,” Mimeo, University of Maryland and Vanderbilt University, July 1999.Banks, Jeffrey S., John O. Ledyard and David P. Porter, “Allocating Uncertain and Unresponsive Resources: An Experimental Approach,” Rand Journal of Economics, 20: 1-25.Bernheim, B. Douglas and Michael Whinston (1986), “Menu Auctions, Resource Allocation and Economic Influence,” Quarterly Journal of Economics, 101: 1-31.Bikhchandani, Sushil, “Auctions of Heterogeneous Objects.” Mimeo, UCLA, March 1996.Bikhchandani, Sushil and John W. Mamer (1997), “Competitive Equilibrium in an Exchange Economy with Indivisibilities,” Journal of Economic Theory, June, 74(2), pp.\",\n \"385-413.Bikhchandani, Sushil and Joseph M. Ostroy (1999), “The Package Assignment Problem,” working paper, UCLA.\",\n \"Cassady, Ralph (1967).\",\n \"Auctions and Auctioneering.\",\n \"Berkeley: University of California Press.\",\n \"Charles River Associates and Market Design Inc. (1997), “Package Bidding for Spectrum Licenses,” Report to the Federal Communications Commission, downloadable at www-fcc-gov.\",\n \"Clarke, E. H. (1971), “Multipart Pricing of Public Goods,” Public Choice, XI, 17-33.Computer Reseller News, “Onsale Joins Fray as Online Shopping Picks Up Speed: Internet Booms,” Jun.\",\n \"5, 1995, p. 73; Dialog: File 16, Acc#05649796.Cramton, Peter C., “Money Out of Thin Air: The Nationwide Narrowband PCS Auction.” Journal of Economics and Management Strategy, Summer 1995, 4(2), pp.\",\n \"267-343.Cybernomics (2000), “An Experimental Comparison of the Simultaneous Multiple Round Auction and the CRA Combinatorial Auction.” Report to the Federal Communications Commission, downloadable at www-fcc-gov.\",\n \"Dasgupta, Partha and Eric S. Maskin (2000), “Efficient Auctions,” Quarterly Journal of Economics, May, 115(2), pp.\",\n \"341-388.DasVarma, Gopal (2000), “The Pareto Optimality of Disclosing Bidder Identities,” working paper, Duke University.\",\n \"Demange, Gabrielle, David Gale, and Marilda Sotomayor, “Multi-Item Auctions.” Journal of Political Economy, August 1986, 94(4), pp.\",\n \"863-872.DeMartini, Christine, Anthony Kwasnica, John Ledyard, and David Porter (1999), “A New and Improved Design for Multi-Object Iterative Auctions,” Cal Tech working paper.\",\n \"DeVries, Sven and Rakesh Vohra (2001), “Combinatorial Auctions: A Survey.” Working paper.\",\n \"Available at www-m9.ma.tum-de.\",\n \"Engelbrecht-Wiggans, Richard and Charles M. Kahn, “Protecting the Winner: Second-Price versus Oral Auctions.” Economics Letters, March 1991, 35(3), pp.\",\n \"243-248.Engelbrecht-Wiggans, Richard and Charles M. Kahn (1998), “Multi-Unit Auctions with Uniform Prices,” Economic Theory, September, 12(2), pp.\",\n \"227-258.Federal Communications Commission (1995), “Bidder Information Packet for FCC Auction Scheduled for Aug. 2, 1995.” Federal Communications Commission (1995), “Bidder Information Packet for FCC Auction Scheduled for Dec. 11, 1995.” Federal Communications Commission (2001), “Bidder Information Packet for FCC Auction No.\",\n \"31.” Groves, Theodore (1973), “Incentives in Teams,” Econometrica 61: 617-31.Gul, Faruk and Ennio Stacchetti (1999), “Walrasian Equilibrium with Gross Substitutes,” Journal of Economic Theory, 87, 9-124.Gul, Faruk and Ennio Stacchetti (2000), “The English Auction with Differentiated Commodities,” Journal of Economic Theory, May, 92(1), pp.\",\n \"66-95.Hahn, Frank H. (1982), “Stability,” in Kenneth J. Arrow and Michael D. Intriligator, eds., Handbook of Mathematical Economics, Volume II, Amsterdam: North-Holland,.\",\n \"pp.\",\n \"745-793.Jehiel, Philippe and Benny Moldovanu (1999), “Efficient Auctions with Interdependent Valuations,” Working Paper No.\",\n \"99-74, University of Mannheim, July.\",\n \"Jehiel, Philippe and Benny Moldovanu (2000), “A Critique of the Planned Rules for the German UMTS/IMT-2000 License Auction,” mimeo, University of Mannheim, March.\",\n \"Jehiel, Philippe and Benny Moldovanu (2001), “The European UMTS/IMT-2000 License Auctions,” working paper.\",\n \"Kagel, John H., Ronald M. Harstad, and Dan Levin, “Information Impact and Allocation Rules in Auctions with Affiliated Private Values: A Laboratory Study.” Econometrica, November 1987, 55(6), pp.\",\n \"1275-1304.Kagel, John H., Scott Kinross, and Dan Levin (2001), “Comparing Efficient Multi-Object Auction Institutions,” mimeo, Ohio State University.\",\n \"Kagel, John H. and Dan Levin (2001), “Behavior in Multi_Unit Demand Auctions: Experiments with Uniform Price and Dynamic Vickrey Auctions,” Econometrica, pp 413-454.Kelso, Alexander S. and Vincent P. Crawford (1982), “Job Matching, Coalition Formation, and Gross Substitutes,” Econometrica 50: 1483-1504.Krishna, Vijay and Motty Perry, “Efficient Mechanism Design.” Mimeo, Penn State University and Hebrew University, March 1997.Ledyard, John, David Porter and Antonio Rangel (1997), “Experiments Testing Multi-object Allocation Mechanisms,” Journal of Economics and Management Strategy, 6: 639-675.Manelli, Alejandro M., Martin Sefton and Benjamin S. Wilner, “Multi-Unit Auctions: A Comparison of Static and Dynamic Mechanisms.” Mimeo, Arizona State University, University of Newcastle and LECG/Navigant Consulting, October 1999.Maskin, Eric S. (1992), “Auctions and Privatization,” in Horst Siebert, ed., Privatization: Symposium in Honor of Herbert Giersch, Tubingen: Mohr (Siebeck), pp.\",\n \"115-136.Maskin, Eric S. and John G. Riley, “Optimal Multi-unit Auctions,” in Frank Hahn, ed., The Economics of Missing Markets, Information, and Games.\",\n \"Oxford: Oxford University Press, 1989, pp.\",\n \"312-35.McAfee, R. Preston and John McMillan, “Auctions and Bidding.” Journal of Economic Literature, June 1987, 25(2), pp.\",\n \"699-738.McCabe, Kevin A., Stephen J. Rassenti, and Vernon L. Smith, “Auction Institutional Design: Theory and Behavior of Simultaneous Multiple-Unit Generalizations of the Dutch and English Auctions.” American Economic Review, December 1990, 80(5), pp.\",\n \"1276-1283.K.\",\n \"A. McCabe, S. J. Rassenti and V. L. Smith, “Testing Vickrey's and Other Simultaneous Multiple Unit Versions of the English Auction,” Research in Experimental Economics, vol.\",\n \"4, Greenwich, Conn.: JAI Press, 1991, pp.\",\n \"45-79.McMillan, John (1994), “Selling Spectrum Rights,” Journal of Economics Perspectives, 8: 145-162.F.\",\n \"M. Menezes, Four Essays on Auction Theory, University of Illinois doctoral dissertation, February 1993, pp.\",\n \"1-97 and 143-152.Milgrom, Paul (1981), “Rational Expectations, Information Acquisition, and Competitive Bidding,” Econometrica, 49: 921-43.Milgrom, Paul (1987), “Auction Theory,” in Truman F. Bewley, ed., Advances in Economic Theory Fifth World Congress.\",\n \"Cambridge: Cambridge University Press, 1987, pp.\",\n \"1-32.Milgrom, Paul (1996), “Auctioning the Radio Spectrum,” downloadable at www-market-design.com.\",\n \"Milgrom, Paul (2000).\",\n \"“Putting Auction Theory to Work: The Simultaneous Ascending Auction,” Journal of Political Economy, 108:2, 245-272.Milgrom, Paul and John Roberts (1991), “Adaptive and Sophisticated Learning in Repeated * Normal Form Games,” Games and Economic Behavior, 3: 82-100.Milgrom, Paul and Robert Weber (1982), “A Theory of Auctions and Competitive Bidding,” Econometrica 50: 1089-1122.Milgrom, Paul R. and Robert J. Weber, “A Theory of Auctions and Competitive Bidding, II.” Unpublished manuscript, Northwestern University, 1982.Nisan, Noam (1999), “Bidding and Allocation in Combinatorial Auctions,” working paper, www-cs-huji-ac-il.\",\n \"Onsale release, “Onsale Brings Thrill of Auctions and Bargain Hunting Online; Unique Internet retail service debuts with week-long charity auction for The Computer Museum in Boston”; Dialog Abstract: File 610, Acc#0489267; May 24, 1995.D.\",\n \"C. Parkes (1999), “iBundle: An Efficient Ascending Price Bundle Auction”, Proceedings ACM Conference on Electronic Commerce (EC-99), Denver, 148-157.Perry, Motty and Philip J. Reny (1999a), “An Ex-Post Efficient Auction,” Working Paper, Hebrew University and University of Chicago, September.\",\n \"Perry, Motty and Philip J. Reny (1999b), “An Ex-Post Efficient Multi-Unit Ascending Auction,” Working Paper, Hebrew University and University of Chicago, September.\",\n \"Plott, Charles (1997), “Laboratory Experimental Testbeds: Application to the PCS Auction,” Journal of Economics and Management Strategy, 6: 605-638.Rassenti, S. J., V. L. Smith, and R. L. Bulfin (1982), “A Combinatorial Auction Mechanism for Airport Time Slot Allocation,” Bell Journal of Economics 13: 402-417.M.\",\n \"H. Rothkopf, A. Pekec and R. M. Harstad (1998), “Computationally Manageable Combinatorial Auctions”, Management Science, Vol.\",\n \"44, No.\",\n \"8, 1131-1147.Rothkopf, Michael, Thomas Teisberg and Edward Kahn (1990), “Why Are Vickrey Auctions Rare?” Journal of Political Economy, 98: 94-109.Sholtz & Associates, LLC, “ACE Market Operations Guide,” Jan. 3, 1996, pp.\",\n \"1-5.Siegmann, “Nowhere to Go But Up.\",\n \"(Onsale CEO Jerry Kaplan) (PC Week Inside) (Inside People)” ; PC Week; v12 n42; pA5 (1); Oct. 23, 1995; Dialog: File 148, Acc#08222496.U.S.\",\n \"Department of the Treasury, U.S. Securities and Exchange Commission, and Board of Governors of the Federal Reserve System, Joint Report on the Government Securities Market, Washington, D.C.: U.S.G.P.O., January 1992.Vickrey, William (1961), “Counterspeculation, Auctions and Competitive Sealed Tenders,” Journal of Finance, March, 16(1), pp.\",\n \"8-37.Vickrey, William (1962), “Auctions and Bidding Games,” Recent Advances in Game Theory, Princeton: Princeton University Conference, 1962, pp.\",\n \"15-29.Vickrey, William (1976), “Auctions, Markets, and Optimal Allocation,” Bidding and Auctioning for Procurement and Allocation, New York: New York University Press 1976, pp.\",\n \"13-20.Weber, Robert J.\",\n \"(1983), “Multiple-Object Auctions,” Auctions, Bidding, and Contracting: Uses and Theory, New York: New York University Press, 1983, pp.\",\n \"165-191.Weber, Robert J.\",\n \"(1997), “Making More from Less: Strategic Demand Reduction in the FCC Spectrum Auctions,” Journal of Economics and Management Strategy, 6:529-548.Williams, Steven (1999), “A Characterization of Efficient, Bayesian Incentive Compatible Mechanisms,” Economic Theory 14: 155-180.Wilson, Robert (1979), “Auctions of Shares,” Quarterly Journal of Economics, vol.\",\n \"94, 1979, pp.675-689.BRIEF DESCRIPTION OF THE DRAWINGS FIG.\",\n \"1 is a graphical depiction of the architecture of an exemplary computer system in accordance with an embodiment of the invention; FIG.\",\n \"2 is a graphical depiction of another exemplary computer system in accordance with an embodiment of the invention; FIG.\",\n \"3 is a detail of one element of the computer system of FIG.\",\n \"2; FIG.\",\n \"4 is a flow diagram of an exemplary auction process in accordance with one embodiment of the invention; FIGS.\",\n \"5a and 5b are flow diagrams illustrating, in greater detail, elements of the flow diagram of FIG.\",\n \"4; FIGS.\",\n \"6a and 6b are flow diagrams illustrating, in greater detail, elements of the flow diagram of FIG.\",\n \"4; FIG.\",\n \"7 is a flow diagram illustrating, in greater detail, an element of the flow diagram of FIG.\",\n \"4; and FIGS.\",\n \"8a, 8b and 8c are flow diagrams illustrating, in greater detail, elements of the flow diagram of FIG.\",\n \"4.DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Overall Structure of Auction System Earlier auction methods and systems are described in U.S. Pat.\",\n \"Nos.\",\n \"5,905,975, 6,021,398 and 6,026,383.The following description will detail the flow of the novel features of the preferred embodiments of the present method and system for an auction of multiple types of items.\",\n \"Before describing the auction process in detail, reference is made to FIG.\",\n \"1 to describe the architecture of an exemplary computer system in accordance with an embodiment of the present invention.\",\n \"In the graphical depiction of FIG.\",\n \"1, the computer system consists of multiple bidder and auctioneer computers or terminals 20a-n and 30 communicating with the server (or auction computer) 10 over a network 40.The computers or terminals 20a-n are employed by bidders, the computer or terminal 30 is employed by the auctioneer, and the server 10 is the auction computer.\",\n \"The server 10 consists of a CPU 11, memory 12, a data storage device 13, a communications interface 14, a clock 15, an operating system 16, and an auction program 17.In one embodiment, the system architecture is as a client-server system: the auction computer is a server; and the bidder and auctioneer computers are clients.\",\n \"FIG.\",\n \"2 is another graphical depiction of an exemplary computer system in accordance with an embodiment of the present invention.\",\n \"The auction system of FIG.\",\n \"2 includes an auction computer 60 (sometimes also referred to as a Bidding Information Processor or BIP), a plurality of user systems 70a, 70b and so on (sometimes also referred to as Bidder Terminal or BT), each user system 70a-n representing an individual bidder, and a user system 80 (sometimes also referred to as an Auctioneer Terminal or AT).\",\n \"The systems 60, 70a-n, and 80 communicate over a network 90.The network represents any system capable of providing the necessary communication to/from BIP, BT, and AT.\",\n \"The network may be a local or wide area network such as, for example, Ethernet, token ring, the Internet, the World Wide Web, the information superhighway, an intranet or a virtual private network, or alternatively a telephone system, either private or public, a facsimile system, an electronic mail system, or a wireless communications system.\",\n \"Each of the systems 60, 70a-n, and 80 may include a typical user interface 65, 75a-n, 85 for input/output which may include a conventional keyboard, display, and other input/output devices.\",\n \"Within each of the systems, the user interface (65, 75a-n, 85) is coupled to a network interface (64, 74a-n, 84), which in turn communicates via the network 90.Both the user interface and network interface connect, at each system, to a CPU (62, 72a-n, 82).\",\n \"Each system includes a memory (66, 76a-n, 86).\",\n \"The BIP 60 also includes a clock 61 and a data storage device 63, which will ordinarily contain a database.\",\n \"(However, in some embodiments the database might instead be stored in memory 66.)\",\n \"The memory 66 of the BIP 60 can further be broken down into a program 67, data 68 and an operating system 69.The memory (76a-n, 86) of the BT's 70a-n and the AT 80 may include a web browser (for example, Internet Explorer or Netscape) (79, 89) or other general-purpose software, but not necessarily any computer program specific to the auction process.\",\n \"In each system the CPU (62, 72a-n, 82) represents a source of intelligence when executing instructions from the memory (66, 76a-n, 86) so that appropriate input/output operations via the user interface and the network interface take place as is conventional in the art.\",\n \"The particular steps used in implementing the inventive auction system and method are described in more detail below.\",\n \"In one embodiment, each of the user systems is a personal computer or workstation.\",\n \"FIG.\",\n \"3 is a more detailed illustration of an exemplary BIP 60 showing details of the database.\",\n \"As discussed for FIG.\",\n \"2, the database is ordinarily stored on a data storage device 63, although in some embodiments it might instead be stored in memory 66.As depicted in FIG.\",\n \"3, the database includes provision for creating, storing, and retrieving records representing Items in the Auction 63-1, Status of the Items in the Auction 63-2, Auction Timetable 63-3, Current Price(s) 63-4, List of Bidder ID's 63-5, List of Passwords 63-6, Bidding History 63-7, and Constraints on Bids 63-8.The particular set of data required for any particular auction and the format of that datum or data (such as scalar, vector, list, etc.)\",\n \"is more particularly specified by the detailed description of that auction.\",\n \"Definitions Concerning “Types” of Items and “Groups” of Types of Items Many of the most useful embodiments of the present invention apply in situations where an entity wishes to sell or buy multiple “types” of items or commodities.\",\n \"Furthermore, related types of items may be usefully organized as “groups” of items.\",\n \"In order to describe the preferred embodiments, it will be helpful for us to define some terminology.\",\n \"DEFINITION 1: A type of item comprises a (nonempty) subset of the set of all items being auctioned, such that any two items within the same type are identical items or close substitutes.\",\n \"Meanwhile, types are defined so that any two items of different types exhibit significant differences in time, location or any other product characteristics.\",\n \"Typically, there are multiple units of each type of item.\",\n \"In what follows, we will assume that m (m≧1) types of items are being auctioned, and that there are n (n≧1) bidders participating in the auction.\",\n \"Whenever we state that there is a “plurality of types of items,” we are restricting attention to the case where m≧2.The items in the auction may also be said to be “homogeneous” in the case where m=1 and to be “heterogeneous” in the case where m≧2.Whenever we state that there is a “plurality of bidders,” we are restricting attention to the case where n≧2.DEFINITION 2: A group G of types comprises a (nonempty) subset of {1, .\",\n \".\",\n \".\",\n \", m}, the set of all types of items.\",\n \"In other words, a group is one or more types of items that are usefully treated together.\",\n \"Typically, the reason that two types of items are included in the same group is that they are related, for example, they may be contracts for provision of the same commodity, covering different but overlapping time periods.\",\n \"In what follows, we will assume that the items in the auction are organized so that there are h (h≧1) groups of types of items.\",\n \"There is no requirement that each group contains the same number of types of items, but this often happens to be the case.\",\n \"Whenever we need notation indicating the number of types of items contained in the exemplary group G, our notation will be that there are g (g≧1) types of items in group G. In some of the preferred embodiments of the present invention, the prices for the various types of items within the same group are established so as to maintain a relationship according to a schedule.\",\n \"In others of the preferred embodiments of the present invention, a bidder submits a bid that includes the price for one type of item within a group, and a fixed relation among these prices implies the prices of the other types of item within the same group.\",\n \"In others of the preferred embodiments of the present invention, a bidder submits a bid that includes a one-dimensional price parameter, and this price parameter implies prices for all of the types of items within the group based on a fixed relation among the prices of the types of items.\",\n \"Some examples of “types” and “groups,” in situations where there may be significant commercial possibilities for embodiments of the present invention, include the following: Treasury bills or other securities: A government or central bank may wish to auction 3-month, 6-month and 12-month Treasury securities (for example, with the same starting date) together.\",\n \"Thus, there is one group of types of items (h=1), and it contains three types of items (g=3).\",\n \"In total, there are three types of items (m=3).\",\n \"Electricity contracts: An electric generating company may wish to simultaneously auction some forward contracts or options contracts for base-load and peak-load electricity generation, with durations of 2 months, 3 months, 6 months, 12 months, 24 months and 36 months, respectively.\",\n \"Thus, there are two groups of types of items (h=2), each containing six types of items (g=6).\",\n \"In total there are 2×6=12 types of items (m=12).\",\n \"Two unrelated, heterogeneous consumer commodities (e.g., apples and oranges).\",\n \"There are two groups of types of items (h=2), each containing just one type of item (g=1), for a total of two types of items (m=2).\",\n \"An auction in accordance with an embodiment of the present invention proceeds as follows.\",\n \"First, the auctioneer (i.e., the auctioneer terminal) determines a starting price vector, (P1, .\",\n \".\",\n \".\",\n \",Pm), and transmits it to the bidding information processor, which in turn transmits it to bidders (i.e., bidder terminals).\",\n \"Second, a bidder responds with a bid vector indicating the quantity of each respective type of item that the bidder wishes to transact at the current price vector.\",\n \"Let the bidders be superscripted by i, where (i=1, .\",\n \".\",\n \".\",\n \", n).\",\n \"The bid vector for bidder i is denoted by (Q1i, .\",\n \".\",\n \".\",\n \",Qmi).\",\n \"The following definitions are helpful in describing the process associated with a first embodiment of the present invention.\",\n \"Other Definitions.\",\n \"The available quantities may, in principle, be specified for each group of types of items or for each type of items.\",\n \"In the text that follows, we will usually specify the available quantity for the group.\",\n \"The available quantity for group G will be denoted {overscore (Q)}G and this refers, in the case of an auction to sell, to the overall quantity of items in group G to be offered for sale in the auction or, in the case of an auction to buy (i.e., a procurement auction or a “reverse auction”), to the overall quantity of items in group G to be bought in the auction.\",\n \"The vector of available quantities for all groups will be denoted ({overscore (Q)}1, .\",\n \".\",\n \".\",\n \", {overscore (Q)}h) Optionally, the available quantities may be allowed to depend on the prices, or otherwise be contingent on the progress of the auction.\",\n \"The current prices comprise a vector, (P1, .\",\n \".\",\n \".\",\n \",Pm), whose components represent the prices for the m respective types of items.\",\n \"The current bid of bidder i comprises a vector, (Q1i, .\",\n \".\",\n \".\",\n \",Qmi), whose components represent the quantities that bidder i is willing to buy (in the case of an auction to sell) or to sell (in the case of an auction to buy) at the current prices for the m respective types of items.\",\n \"The current bids comprise the collection of vectors, {Q1i, .\",\n \".\",\n \".\",\n \",Qmi}i=1n consisting of the current bid of bidder i for every bidder (i=1.. .\",\n \", n) in the auction.\",\n \"The bidding history comprises the current prices and the current bids associated with the present time and all earlier times in the current auction.\",\n \"A clock auction is a dynamic auction procedure whereby: the auctioneer announces the current prices to bidders; the bidders respond with current bids; the auctioneer determines whether the auction should continue based on the bidding history; the auctioneer updates the current prices based on the bidding history and the process repeats, if it is determined that the auction should continue; and the auctioneer allocates the items among the bidders and assesses payments among the bidders based on the bidding history, if it is determined that the auction should not continue.\",\n \"Observe that a “clock auction” differs from a standard ascending-bid electronic auction in the following important sense.\",\n \"In standard ascending-bid electronic auctions—such as in the Federal Communications Commission auctions for radio communications spectrum or in eBay auctions—the bidders name prices (and, perhaps also, quantities) that they propose to pay for the items being auctioned, in an iterative process.\",\n \"In a standard clock auction, the auctioneer sets the pace for price increases, and bidders respond only with quantities in an iterative process.\",\n \"(however, in the discussion of Intra-Round Bids, below, it will be seen that there still may be a role for bidders naming prices—to a limited extent—in a clock auction.)\",\n \"FIG.\",\n \"4 is a flow diagram of a clock auction in accordance with one embodiment of the present invention.\",\n \"The process starts with step 102, in which memory locations of a computer are initialized.\",\n \"In one preferred embodiment, the appropriate memory locations of the bidding information processor (auction computer) are initialized with information such as the types of items in the auction, the available quantity of each type of item in the auction, the data table or algorithm reflecting the “yield curve” schedule, an initial price parameter, an auction timetable, a list of bidder ID's, and a list of passwords.\",\n \"In step 104, a computer establishes the initial prices (P1, .\",\n \".\",\n \".\",\n \",Pm).\",\n \"One preferred embodiment of the process of step 104 is shown in greater detail in FIG.\",\n \"5b.\",\n \"The process proceeds to step 106, in which a computer outputs auction information, including the current price vector (P1, .\",\n \".\",\n \".\",\n \",Pm).\",\n \"In one preferred embodiment, the bidding information processor outputs the auction information through its network interface and transmits it via the network.\",\n \"The bidder terminals then receive the auction information through their network interfaces and display the information to bidders through their user interfaces.\",\n \"In step 108, a computer receives bids (Q1i, .\",\n \".\",\n \".\",\n \",Qmi) from bidders.\",\n \"In one preferred embodiment, a bidder inputs his bids through the user interface of the bidder terminal, which then outputs the auction information through its network interface and transmits it via the network.\",\n \"The bidding information processor then receives the bids through its network interface for use in the next step.\",\n \"One preferred embodiment of the process of step 108, utilizing Intra-Round Bids, is shown in greater detail in FIG.\",\n \"8a.\",\n \"In step 110, a computer applies constraints, if any, to the received bids, and enters only those bids that satisfy said constraints.\",\n \"This process is illustrated in greater detail in FIGS.\",\n \"6a and 6b.\",\n \"In one preferred embodiment, the constraints are applied at the bidding information processor, although they may also easily be applied at the bidder terminals.\",\n \"In step 112, a computer processes the received bids and determines whether the auction should continue.\",\n \"Exemplary processes of step 112 are illustrated in greater detail in FIGS.\",\n \"7 and 8b.\",\n \"In some preferred embodiments, this determination occurs at the bidding information processor.\",\n \"If the auction should continue, the process goes to step 114, in which a computer establishes an updated price vector (P1, .\",\n \".\",\n \".\",\n \",Pm).\",\n \"One preferred embodiment of the process of step 114 is shown in greater detail in FIG.\",\n \"5a.\",\n \"Then, at step 116, a computer updates other auction information, if any.\",\n \"In one preferred embodiment, the bidding information processor automatically generates a suggested revised price vector, outputs the suggested revised price vector through its network interface, and transmits it via the network.\",\n \"The auctioneer terminal then receives the suggested revised price vector through its network interface and displays it to the auctioneer through its user interface.\",\n \"The auctioneer either approves or modifies the revised price vector through the user interface of the auctioneer terminal, which then outputs the revised price vector through its network interface and transmits it via the network.\",\n \"The bidding information processor then receives the revised price vector through its network interface for use in subsequent steps.\",\n \"The process then loops to step 106.If the auction should not continue, the process goes to step 118, in which a computer outputs a final message, including the allocation of items among bidders and the payments of the bidders.\",\n \"In one preferred embodiment, the bidding information processor takes the allocation of items among bidders to be their final bids and takes the payment of each bidder i to be the dot product of the final price vector and bidder i's final quantity vector: (P1, .\",\n \".\",\n \".\",\n \",Pm·(Q1i, .\",\n \".\",\n \".\",\n \", Qmi).\",\n \"The bidding information processor outputs the allocation and payment outcome through its network interface and transmits it via the network.\",\n \"The bidder terminals and auctioneer terminal then receive the allocation and payment outcome through their network interfaces and display the information to bidders and the auctioneer through their user interfaces.\",\n \"One preferred embodiment of the process of step 118, utilizing Intra-Round Bids, is shown in greater detail in FIG.\",\n \"8c.\",\n \"The process then ends.\",\n \"Embodiments of the Invention Concerned with Establishing or Implying Prices FIG.\",\n \"5a is a flow diagram illustrating an exemplary process by which updated prices are established.\",\n \"Thus, FIG.\",\n \"5a illustrates, in greater detail, step 114 of FIG.\",\n \"4.The process begins with step 114-1, in which a computer selects a group G of types of items not yet considered.\",\n \"In step 114-2, a computer calculates the excess demand ZG for group G, which is given by: Z G = - Q _ G + ∑ i = 1 n ∑ k ∈ G Q k i .\",\n \"Thus, ZG represents the amount by which the bids for all types of items in group G, by all bidders, exceeds the available quantity {overscore (Q)}G of all items in group G. In step 114-3, a computer selects a type of item included in group G, whose “old” price is denoted by {overscore (P)}1G.\",\n \"In step 114-4, a computer sets a “new” price P1G, where P1G equals the {overscore (P)}1G plus an increment Δ.\",\n \"The increment Δ is given by Δ=c ZG, where c is a positive constant and ZG was computed in step 114-2.\",\n \"(In other embodiments, other rules for incrementing P1 are used.)\",\n \"In step 114-5, a computer looks up the new P1G in a data table reflecting the “yield curve” schedule.\",\n \"The following Table 1 is an example of such a data table for a group G containing three types of items (corresponding to the base group of the earlier example containing base and peak groups of electricity contracts): TABLE 1 P1G P2G P3G 0.00 2.00 3.00 5.00 7.00 8.00 10.00 12.00 13.00 15.00 17.00 18.00 20.00 22.00 23.00 The process continues with step 114-6 in which a computer determines whether the new P1G is an entry in the data table.\",\n \"If it is, the process continues with step 114-7 in which a computer reads across the row for the new P1G in the data table and thereby determines the new prices (P2G, .\",\n \".\",\n \".\",\n \", PgG), where g denotes the number of types of items within group G. For example, with the data of Table 1, if P1G equaled 15.00, then the computer would determine “Yes” at step 114-6, and the computer would determine P2G=17.00 and P3G=18.00 at step 114-7.The process then proceeds to step 114-10.If the new P1G is not an entry in the data table, the process continues with step 114-8, in which a computer retrieves P1low, the largest entry in the data table less than P1G, and a computer retrieves P1high, the smallest entry in the data table greater than P1G.\",\n \"The process then continues with step 114-9, in which a computer determines the new prices (P2G, .\",\n \".\",\n \".\",\n \", PgG) by linear interpolation, reading across the rows in the data table corresponding to P1low and P1high, and applying the new P1G.\",\n \"For example, with the data of Table 1, if P1G equaled 10.50, then the computer would determine “No” at step 114-6.The computer would then retrieve P1low=10.00 and P1high=15.00 at step 114-8, and the computer would determine P2G=12.50 and P3G=13.50 at step 114-9.The process then proceeds to step 114-10, in which a computer determines whether all groups G have been considered.\",\n \"If not, the process returns to step 114-1.However, if all groups G have been considered, then the entire step 114 has been completed, and the process continues with step 116 of FIG.\",\n \"4.Three observations should be made at this point.\",\n \"First, rather than determining prices by looking up and possibly interpolating of numbers that are contained in a data table, the relationship or relation among the prices may instead be an algorithm or a formula.\",\n \"For example, instead of using the data of Table 1, another preferred embodiment would be obtained by equivalently applying the formulae P2G=P1G+2 and P3G=P1G+3.Second, there is no particular reason that the relationship or relation among the prices need be linear, as it happens to be in Table 1 and in some applications.\",\n \"The relationship or relation (data table, or algorithm or formula) could be a nonlinear relationship between (P2G, .\",\n \".\",\n \".\",\n \", PgG) and P1G.\",\n \"Third, in some embodiments, the relationship or relation (whether schedule or data table, or algorithm or formula) among prices is predetermined and fixed, and in other embodiments, the relationship or relation can be based on data which may be revised during the course of the auction.\",\n \"In typical applications, the price relationship or relation may be determined from market data external to the auction, for example, from the “yield curve” in the government securities market, expressing the differential among prices or interest rates for securities of varying durations.\",\n \"If the market data external to the auction were to markedly change during the course of the auction, then the auctioneer might wish to revise the price relationship or relation accordingly.\",\n \"FIG.\",\n \"5b is a flow diagram illustrating an exemplary process by which initial prices are established.\",\n \"Thus, FIG.\",\n \"5b illustrates, in greater detail, step 104 of FIG.\",\n \"4.The process begins with step 104-1, in which a computer selects a group G of types of items not yet considered.\",\n \"In step 104-2, a computer selects a type of item included in group G, whose price is denoted by P1G.\",\n \"In step 104-3, a computer looks up the initial value for the selected type of item in group G among the memory locations that were initialized in step 102, and uses this value as the initial P1G.\",\n \"In step 104-4, a computer looks up the initial P1G in a data table reflecting the “yield curve” schedule.\",\n \"The previous Table 1 serves as an example of such a data table, as in the description of FIG.\",\n \"5a.\",\n \"The process continues with step 104-5 in which a computer determines whether the initial P1G is an entry in the data table.\",\n \"If it is, the process continues with step 104-6 in which a computer reads across the row for the initial P1G in the data table and thereby determines the initial prices (P2G, .\",\n \".\",\n \".\",\n \", PgG), where g denotes the number of types of items within group G. The process then proceeds to step 104-9.If the initial P1G is not an entry in the data table, the process continues with step 104-7, in which a computer retrieves P1low, the largest entry in the data table less than P1G, and a computer retrieves P1high, the smallest entry in the data table greater than P1G.\",\n \"The process then continues with step 104-8, in which the computer determines the initial prices (P2G, .\",\n \".\",\n \".\",\n \", PgG) by linear interpolation, reading across the rows in the data table corresponding to P1low and P1high, and applying the initial P1G.\",\n \"The process then proceeds to step 104-9, in which a computer determines whether all groups G have been considered.\",\n \"If not, the process returns to step 104-1.However, if all groups G have been considered, then the entire step 104 has been completed, and the process continues with step 106 of FIG.\",\n \"4.The first two observations made after FIG.\",\n \"5a also apply to the process of FIG.\",\n \"5b.\",\n \"Elements of the Invention Concerned with Applying Constraints to Bids FIGS.\",\n \"6a and 6b are flow diagrams of two exemplary subprocesses of step 110.The process of FIG.\",\n \"6a begins with step 110a-1, in which a bidder i who has not yet been considered is selected.\",\n \"In step 110a-2, a bid (Qki,t)kεG by bidder i which has not yet been considered is selected.\",\n \"The bid (Qki,t)kεG is a bid for group G of item types at time t in the auction.\",\n \"In step I10a-3, it is checked whether each quantity Qki,t in the selected bid is a nonnegative integer.\",\n \"If each component of the bid is a nonnegative integer, the process goes to step 110a-4.In step 110a-4, it is checked whether the selected bid is consistent with bidder i's initial eligibility, that is, whether: ∑ k ∈ G Q k i , t ≤ Q _ G i , where bidder i's initial eligibility, {overscore (Q)}Gi, for group G may, for example, be determined by the level of financial guarantee posted by bidder i.\",\n \"If the selected bid is consistent with bidder i's initial eligibility, the process goes to step 110a-5, where bidder i's most recent previously-processed bid for group G, denoted (Qki-t-1)kεG, is recalled.\",\n \"In step 110a-6, it is checked whether the selected bid is consistent with the auction's activity rule, that is, whether the constraint: ∑ k ∈ G Q k i , t ≤ ∑ k ∈ G Q k i , t - 1 , is satisfied.\",\n \"If it is, the process continues to step 110a-7, where the selected bid (Qki,t)kεG is entered as a valid bid by bidder i on group G. Optionally, bidder i is sent a message confirming to him that the bid is valid.\",\n \"The process then goes to step 110a-8, where it is determined whether all bids by bidder i have been considered.\",\n \"If not, the process loops back to step 110a-2.If all bids by bidder i have been considered, the process continues to step 110a-9, where it is determined whether all bidders have been considered.\",\n \"If not, the process loops back to step 110a-1.If all bidders have been considered, the process goes to step 112 of FIG.\",\n \"4.If the selected bid fails any of the checks at steps 110a-3, 110a-4 or 110a-6, the process instead goes to step 110a-10, where a message is outputted to bidder i that the selected bid is invalid.\",\n \"The selected bid then is not entered as a valid bid.\",\n \"The process then goes to step 110a-8, where it is determined whether all bids by bidder i have been considered.\",\n \"If not, the process loops back to step 110a-2.If all bids by bidder i have been considered, the process continues to step 110a-9, where it is determined whether all bidders have been considered.\",\n \"If not, the process loops back to step 110a-1.If all bidders have been considered, the process goes to step 112 of FIG.\",\n \"4.The process of FIG.\",\n \"6b begins with step 110b-1, in which a bidder i who has not yet been considered is selected.\",\n \"In step 110b-2, a bid (Qki,t)kεG by bidder i which has not yet been considered is selected.\",\n \"The bid (Qki,t)kεG is a bid for group G of item types at time t in the auction.\",\n \"In step 110b-3, it is checked whether each quantity Qki,t in the selected bid satisfies the constraint: ∑ k ∈ G C k i , t Q k i , t ≤ C _ G i , t , where Cki,t and {overscore (C)}Gi,t are arbitrary constants.\",\n \"If the constraint of step 110b-3 is satisfied, the process goes to step 110b-4.In step 110b-4, it is checked whether each quantity Qki,t in the selected bid satisfies the constraint: ∑ k ∈ G C k ′ i , t Q k i , t ≥ C ^ G i , t , where Ck′i,t and ĈGi,t are arbitrary constants.\",\n \"If the constraint of step 110b-4 is satisfied, the process goes to step 110b-5, where it is checked whether the selected bid was submitted at a time no earlier than the starting time of the current round.\",\n \"If it was, the process goes to step 110b-6, where it is checked whether the selected bid was submitted at a time no later than the ending time of the current round.\",\n \"If it was, the process continues to step 110b-7, where the selected bid (Qki,t)kεG is entered as a valid bid by bidder i on group G. Optionally, bidder i is sent a message confirming to him that the bid is valid.\",\n \"The process then goes to step 110b-8, where it is determined whether all bids by bidder i have been considered.\",\n \"If not, the process loops back to step 110b-2.If all bids by bidder i have been considered, the process continues to step 110b-9, where it is determined whether all bidders have been considered.\",\n \"If not, the process loops back to step 110b-1.If all bidders have been considered, the process goes to step 112 of FIG.\",\n \"4.If the selected bid fails any of the checks at steps 110b-3, 110b-4, 110b-5 or 110b-6, the process instead goes to step 110b-10, where a message is outputted to bidder i that the selected bid is invalid.\",\n \"The selected bid then is not entered as a valid bid.\",\n \"The process then goes to step 110b-8, where it is determined whether all bids by bidder i have been considered.\",\n \"If not, the process loops back to step 110b-2.If all bids by bidder i have been considered, the process continues to step 110b-9, where it is determined whether all bidders have been considered.\",\n \"If not, the process loops back to step 110b-1.If all bidders have been considered, the process goes to step 112 of FIG.\",\n \"4.Embodiments of the Invention Concerned with Whether the Auction Should Continue FIG.\",\n \"7 is a flow diagram of a subprocess of step 112 of FIG.\",\n \"4.It illustrates an exemplary process by which a computer may determine whether the auction should continue.\",\n \"Related to this will also be FIG.\",\n \"8b, below, which illustrates an exemplary process by which a computer determines whether the auction should continue, in a system where bidders are permitted to submit Intra-Round Bids.\",\n \"FIG.\",\n \"7 begins with step 112a-1, in which a group G of types of items not yet considered is selected.\",\n \"In step 112a-2, a computer determines whether the excess demand for group G of types of items is within {overscore (C)}G of the available quantity, that is, whether: Q _ G - ∑ i = 1 n · ∑ k ∈ G Q k l ≤ C _ G .\",\n \"The constant, {overscore (C)}G, has the interpretation that this is the tolerance to which the auctioneer is allowing oversell or undersell to occur.\",\n \"If the auctioneer needs to sell exactly the available quantity of the group G of item types, then {overscore (C)}G=0.If this inequality is not satisfied, then group G of item types has not yet cleared, and so the auction should continue.\",\n \"The process thus jumps immediately to step 114 of FIG.\",\n \"4.If the inequality of step 112a-2 is satisfied, the process then goes to step 112a-3, where it is determined whether all groups G of types of items have been considered.\",\n \"If not, the process loops back to step 112a-1.However, if all groups G of types of items have already been considered, then it has been found that all groups G of types of items have cleared within a tolerance of {overscore (C)}G, and so the auction should not continue.\",\n \"The process proceeds to step 118 of FIG.\",\n \"4, where the final message is generated.\",\n \"Embodiments of the Invention Concerned with Intra-Round Bids In many of the leading dynamic electronic auctions in the prior art, bidders submit bids in a sequence of discrete rounds.\",\n \"For example, in the Federal Communications Commission auctions for radio communications spectrum or in the recent UMTS auctions held by European nations, the following would be a typical bidding schedule for an auction: Round 1: 9:00-9:45 Round 2: 10:00-10:45 Round 3: 11:00-11:45 Round 4: 12:00-12:45 Round 5: 13:00-13:45 Round 6: 14:00-14:45 Round 7: 15:00-15:45 Round 8: 16:00-16:45 This bidding schedule would have the following interpretation.\",\n \"During the specified time period of each round, a bidder would be required to submit a new bid or new collection of bids (unless this bidder was already the standing high bidder on an item after the bidding of the previous round).\",\n \"If a bidder who was required to submit a new bid failed to submit a new bid, then (except for provisions in the rules concerning automatic waivers) the bidder would be eliminated from the auction.\",\n \"By contrast, some other electronic auctions in the prior art—for example, online auctions at eBay—allow bidding to occur continuously.\",\n \"Rather than adhering to any rigid round schedule, bidders may submit bids at any times that they like up to a specified closing time.\",\n \"Related to this, there is no sense that a bidder is required to bid a certain amount by any particular time in order to retain eligibility to bid at a later time in the auction.\",\n \"Many or most electronic auctions for high-valued items utilize a discrete round structure, rather than allowing bidding to occur continuously.\",\n \"There appear to be several reasons for this.\",\n \"First, a discrete round structure has desirable information properties.\",\n \"The auction can be easily structured so that the results of Round t are disseminated to bidders before the bids of Round t+1 need to be submitted.\",\n \"Second, a discrete round structure is especially conducive to enforcing “activity rules,” in which a bidder is required to be active (i.e., either be the standing high bidder or place a new high bid) on a given number of items in an earlier round of the auction in order to continue to bid on a given number of items in a later round of the auction.\",\n \"This forces bidders to effectively disclose to their opponents (through their bidding) the values that they attach to the items, helping to mitigate the well-known “Winner's Curse” present in auctions.\",\n \"Third, a discrete round structure requires a bidder to repeatedly affirm, in successive rounds, his willingness to pay a given price for an item in the auction—which may be especially desirable when items such as communications licenses may sell for millions or billions of dollars or euros.\",\n \"At the same time, the desirable properties of a discrete round structure may come at some considerable cost.\",\n \"It will typically be reasonable to hold only something like 8 to 12 rounds of bidding in a given day.\",\n \"As a result, the auctioneer must accept at least one of several problems: (1) The auction may be required to last a very long time: in some North American and European spectrum auctions, the bidding extended more than 20 business days.\",\n \"Such a lengthy auction may be rather onerous for bidders and for the seller.\",\n \"In particular, it may discourage bidder participation, causing the seller to forgo substantial revenues.\",\n \"(2) The bid increment between successive rounds may be required to be rather substantial: in some North American and European spectrum auctions, the bid increment between successive rounds never was allowed to drop below five percent of the previous bid.\",\n \"It can be argued that a seller suffers an expected revenue loss which is directly proportional to the minimum bid increment, so this may cost a seller millions of dollars or euros.\",\n \"(3) The starting price may be required to be very near to the expected closing price.\",\n \"This may discourage bidder participation, as well as potentially eliminating the possibility of bidders getting caught up in the excitement of the auction and bidding very high prices (which is one of the advantages of conducting a dynamic auction).\",\n \"This also runs the risk that the auction will fail: that is, quantities bid at the starting price being less than the available quantity at the auction.\",\n \"Moreover, in a clock auction, problem (2) above, a large bid increment, may lead to a heightened risk of “undersell”.\",\n \"Consider an auction with an available quantity of 100 units of an item, and suppose a bid increment of five percent.\",\n \"It is quite plausible that, at a price of $1,000,000 per unit, the aggregate quantity bid by all bidders would equal 110 units, but at the next price of $1,050,000 per unit, the aggregate quantity bid by all bidders would decline to only 60 units.\",\n \"The auctioneer then faces the unattractive alternatives of: selling only 60 units out of the available quantity of 100 units at a price of $1,050,000 each; rationing bidders so that only 100 units, out of the 110 demanded, are sold at $1,000,000; or restarting the auction at $1,000,000.Observe however that the “undersell” problem would in all likelihood have been substantially avoided, had a much smaller bid increment been possible.\",\n \"One embodiment of the present invention is a system and method for “Intra-Round Bids.” A discrete round structure—with all of its many advantages—is preserved for a clock auction.\",\n \"However, in each round of the clock auction, a “starting price” and “ending price” is established for each type of item.\",\n \"Bidders are permitted to submit bids at prices between the starting price and the ending price.\",\n \"In a preferred embodiment, a bidder submits a price parameter for group G representing a percentage of the distance from the starting price vector for group G and the ending price vector for group G. Bidders have every incentive to utilize Intra-Round Bids, and to the extent that bidders utilize them, the seller should be expected to attain higher auction revenues and to reduce the probability of undersell.\",\n \"Thus, a system and method for Intra-Round Bids improves upon the prior art for auction systems and methods, and has immediate practical application for dynamic auctions of radio communications spectrum, securities and other financial products, electric power, etc.\",\n \"While the previous and following description of Intra-Round Bids is framed largely in terms of regular auctions to sell (where bidders are buyers), the invention is equally applicable for reverse or procurement auctions to buy (where bidders are sellers).\",\n \"For the sake of brevity, this specification will not run through the process a second time with the roles of selling and buying reversed, but it should be clear to anybody skilled in the art that the technology can be equally used in both situations.\",\n \"Here is an example illustrating the usefulness and exact meaning of Intra-Round Bids.\",\n \"Suppose that, in a clock auction with an available quantity of 100 units, the ending price per unit associated with Round 4 is $1,000,000, and the ending price per unit associated with Round 5 is $1,050,000.In an auction with discrete bidding rounds, Bidder 1 might submit a bid quantity of 55 units for Round 4 and a bid quantity of 30 units for Round 5.If there also exists a Bidder 2 who submits the same bid quantities, then we would have exactly the “undersell” problem described above: an aggregate quantity bid by all bidders of 110 units in Round 4 but only 60 units in Round 5 (with available quantity of 100 units).\",\n \"With an auction system and method with Intra-Round Bidding, the ending price for Round 4 may be taken to be the starting price for Round 5, i.e., the starting price for Round 5 is $1,000,000.Here is an example of the bids that Bidder 1 might submit for Auction Round 5: 53 units at $1,010,000 per unit; 51 units at $1,020,000 per unit; 49 units at $1,030,000 per unit; 45 units at $1,035,000 per unit; 40 units at $1,040,000 per unit; and 30 units at $1,045,000 per unit.\",\n \"These bids have the following exact meaning: the parameters corresponding to price indicate the price at which Bidder 1 wishes to change his quantity demanded as compared to his “previous” (that is, next lower price) bid.\",\n \"Thus, in this example: Bidder 1 is willing to purchase 55 units (his previous bid from Round 4) at prices of $1,000,001-$1,009,999; Bidder 1 is willing to purchase 53 units at prices of $1,010,000-$1,019,999; Bidder 1 is willing to purchase 51 units at prices of $1,020,000-$1,029,999; Bidder 1 is willing to purchase 49 units at prices of $1,030,000-$1,034,999; Bidder 1 is willing to purchase 45 units at prices of $1,035,000-$1,039,999; Bidder 1 is willing to purchase 40 units at prices of $1,040,000-$1,044,999; and Bidder 1 is willing to purchase 30 units at prices of $1,045,000-$1,050,000.If there also exists a Bidder 2 who submits the same bid quantities, then the auctioneer would be able to declare the auction over at a price between $1,030,000 and $1,034,999, with 98 out of the 100 available units sold.\",\n \"The auction revenues are improved, and the undersell problem is greatly reduced.\",\n \"FIG.\",\n \"8a is a flow diagram of a subprocess of step 108 of FIG.\",\n \"4.It illustrates an exemplary process by which a particular bidder i may submit Intra-Round Bids.\",\n \"FIG.\",\n \"8a begins with step 108-1, in which bidder i selects a group, G, of item types on which he wishes to place a bid.\",\n \"In step 108-2, bidder i selects a price parameter for group G representing a percentage of the distance from the starting price vector for group G and the ending price vector for group G in the current round.\",\n \"For example, in a group containing three types of items, if the starting price vector is (4.00, 4.50, 4.75), if the ending price vector is (8.00, 8.50, 8.75), and if a bidder enters a price parameter of 25%, this signifies that the bidder is indicating an implied price vector of (5.00, 5.50, 5.75).\",\n \"In step 108-3, bidder i selects quantities of the item types of group G that he would like to take effect as bids at the price vector implied by the selected price parameter.\",\n \"In step 108-4, bidder i expresses whether he wishes to enter more bids.\",\n \"If so, the process loops back to step 108-1.If not, the process continues to step 108-5.In step 108-5, a computer determines whether bidder i has submitted at least one bid for each group G of item types.\",\n \"If not, the process loops back to step 108-1, and optionally a computer prompts bidder i to submit bids on the groups G of item types on which bidder i has not submitted at least one valid bid in the current round.\",\n \"If so, the process goes to step 110 of FIG.\",\n \"4.FIG.\",\n \"8b is a flow diagram of a subprocess of step 112 of FIG.\",\n \"4.It illustrates an exemplary process by which a computer determines whether the auction should continue, in a system where bidders are permitted to submit Intra-Round Bids.\",\n \"FIG.\",\n \"8b begins with step 112b-1, in which a group G of item types not yet considered is selected.\",\n \"In step 112b-2, a computer sorts all bids entered for group G in the current round.\",\n \"The sorting is done: first, by bidder ID; second, by price parameter in the entered bid (in descending order); and third, by time stamp of submission (in descending order).\",\n \"In step 112b-3, a computer selects, for each bidder i, the bid, QG,i, for group G with the highest price parameter (and then the latest time stamp).\",\n \"In step 112b-4, a computer determines whether the aggregate quantity bid for group G is no greater than the available quantity, that is, whether: ∑ i = 1 n ∑ k ∈ G Q k G , i ≤ Q _ G .\",\n \"If this inequality is not satisfied, then group G of item types has not yet cleared, and so the auction should continue.\",\n \"The process thus jumps immediately to step 114 of FIG.\",\n \"4.If the inequality of step 112b-4 is satisfied, the process then goes to step 112b-5, where it is determined whether all groups G of item types have been considered.\",\n \"If not, the process loops back to step 112b-1.However, if all groups G of item types have already been considered, then it has been found that all groups G of item types have cleared, and so the auction should not continue.\",\n \"The process proceeds to step 118 of FIG.\",\n \"4, where the final message is generated.\",\n \"FIG.\",\n \"8c is a flow diagram of a subprocess of step 118 of FIG.\",\n \"4.It illustrates an exemplary process by which a computer determines final allocations and payments, in a system where bidders are permitted to submit Intra-Round Bids.\",\n \"FIG.\",\n \"8c begins with step 118-1, in which for all bids entered in the current (i.e., final) round, a computer sorts the price parameters from smallest to largest, and denotes them λ1<π2<.\",\n \".\",\n \".\",\n \"<πR.\",\n \"In step 118-2, a computer initializes the price parameter subscript to r=1, so that in the first iteration of the remaining steps, the computer considers the smallest value, π1.In step 118-3, a group G of item types not yet considered is selected.\",\n \"In step 118-4, a computer sorts all bids entered for group G in the current round.\",\n \"The sorting is done: first, by bidder ID; second, by price parameter in the entered bid (in descending order); and third, by time stamp of submission (in descending order).\",\n \"In step 118-5, a computer selects, for each bidder i, the bid, QG,i, for group G with the highest price parameter that is less than or equal to πr (and then with the latest time stamp).\",\n \"In step 118-6, a computer determines whether the aggregate quantity bid for group G is no greater than the available quantity, that is, whether: ∑ i = 1 n ∑ k ∈ G Q k G , i ≤ Q _ G .\",\n \"If this inequality is not satisfied, then group G of item types has not yet cleared at price parameter πr, and so r needs to be incremented.\",\n \"The process thus goes to step 118-8, where the price parameter subscript r is advanced by 1, so that in the next iteration of these steps, the computer considers the next price parameter, πr+1.The process then loops back to step 118-3, using the new higher value of πr+1 and starting over for groups G of item types.\",\n \"If the inequality of step 118-6 is satisfied, the process continues to step 118-7, where it is determined whether all groups G of item types have been considered.\",\n \"If not, the process loops back to step 118-3.However, if all groups G of item types have already been considered, then it has been found that all groups G of item types have cleared at price parameter ;r,.\",\n \"Thus, the price parameter A, implies market-clearing prices for the auction.\",\n \"The process proceeds to calculate the price vector implied by price parameter ;r, to note the quantities bid by all bidders at this price vector, and to incorporate these computations into a final message that is outputted from a machine.\",\n \"Auction-Like Optimization Problems and Machine-Generated Bids In the course of this application, a method and apparatus for implementing auctions of multiple items has been described.\",\n \"The methods and apparatus described allow users to participate in various auctions with a level of attention which varies from continuous, down to the input of information on a single occasion.\",\n \"It should also be apparent that the required level of attention by the “auctioneer” may vary from continuous to essentially zero—aside from initiating the auction.\",\n \"Thus, for all intents and purposes, once the basic auction description is selected and the users input desired information, the auction implemented by the invention can be essentially automatic, i.e.\",\n \"devoid of human interaction.\",\n \"Because in the past auctions have generally been considered to be processes engaged in by persons, the feature of an automatic auction may be, by itself, considered relatively new.\",\n \"There are, however, many other automatic systems which interact in a way that is entirely analogous to an auction and to which the present invention could be applied.\",\n \"Hence, the present invention can be applied to improve the efficiency of computers which are used to operate the automatic systems, by economizing on the collection of informational inputs needed for the system and to speed the computational of optimal resource assignments.\",\n \"At the same time, many optimization problems encountered in the field of operations research have similar mathematical structures to the problem of determining the winners of an auction.\",\n \"Hence, the present invention can be applied to improve the efficiency of computer systems which are used to solve the similar optimization problems, by enabling the computations to be implemented on a system with parallel processing or generally by speeding the computation of solutions.\",\n \"For example, the air conditioning plant in an office building allocates cool air among individual offices in the building via a dynamic auction.\",\n \"Periodically, the central computer of the air conditioning system serves the role of the “auction computer” in a clock auction, while computers interfaced with the thermostats in each suite of offices serve the role of “bidder terminals.” Each thermostat is programmed to send back bids consisting of a desired quantity of cooled air based on: the current temperature reading of the thermostat, the desired temperature in the office, and the use (if any) to which the office is currently being put.\",\n \"Based on the parameters to which it has been programmed, the central computer of the air conditioning system then provides the results of the auction in its allocation of cooled air among the bidding offices.\",\n \"In another context, a communications, transportation or energy transmission network faces the technical problem of how to allocate its scarce network resources.\",\n \"The optimization problem in allocating its network resources (e.g., bandwidth, switches, etc.)\",\n \"has a very similar structure to the auction problem.\",\n \"Moreover, clock auctions are again well suited to the problem, since a network provider needing multiple types of network resources is able to iteratively bid on a vector of network resources, always knowing the price of each.\",\n \"Hence, the present invention can be usefully applied to improving the solution to this technical problem.\",\n \"In another context, computational resources on a distributed computer system can be allocated via a clock auction.\",\n \"Whenever a new job requiring a given quantity of CPU time enters the system, an auction is conducted.\",\n \"Each member of the distributed computer system indicates the quantity of CPU time which it can make available at a given priority level or a given price.\",\n \"In this case, the “auctioneer computer” selects and allocates the resources to be applied to the new job in accordance with some programmed timetable and hence in this fashion provides the results of the auction.\",\n \"The several examples described herein are exemplary of the invention, whose scope is not limited thereby but rather is indicated in the attached claims.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10467868"}}},{"rowIdx":389,"cells":{"text":{"kind":"list like","value":[["Packet data recording method and system","The present invention provides for a data recording system including data recording means having a plurality of network interface cards in which a plurality of network interface cards can be employed within a single recording mean and each card can be provided with a plurality of network connecting ports then, for example, for each RTP packet stream being recorded, the system can advantageously note the sequence number for the most recently accepted packet and any packet received with a lower sequence number can be readily discarded."],["1.A data recording system including data recording means having a plurality of network interface cards.","2.A system as claimed in claim 1 and including a plurality of network interface cards within a single recording means.","3.A system as claimed in claim 2, wherein each Network interface card has multiple network connection ports.","4.A system as claimed in claim 1, 2 or 3, wherein each individual recording means is arranged for mutual communication so as to advise each other of the presence of particular packet streams.","5.A system as claimed in any one of claims 1 to 4, and arranged such that each recorder is advised of the addresses to be monitored; when a recorder identifies a packet with the required addresses it begins recording it and advises all other recorders to stand down; and other recorders are arranged to note these addresses as being recorded elsewhere.","6.A system as claimed in claim 5 and further arranged such that the notification sent to other recorders include within it the sequence number of an RTP packet that was first received and the IP address of the receiving recorder; that in the event that more than one recorder receives packets prior to hearing notification from another recorder that it has commenced recording, the recorder which received the earliest packet can maintain responsibility for recording and the other recorder shall cease recording; that should more than one recorder receive the same packet and send notification of this, then the recorder with highest IP address can maintain its recording and the other(s) will stand down; and that when recording begins, a recorder will not create a record in the database of recordings until a pre-defined time has elapsed in case it receives, during this period, a notification from another recorder that it too has started recording.","Should it receive such notification and, according to the algorithms defined in (d) to (f) above, it is to stand down, it will abandon its recording without having altered the database of recordings.","7.A system as claimed in claim 6 and further arranged such that should a packet be lost an indication of the sequence number(s) of the lost packets can be maintained as part of the recording control structure for that IP address; and that when packets are received that are determined to be earlier than the most recently accepted packet for that address, their sequence number can subsequently be compared against the list of recently missed packets and, if found, can be stored at the appropriate offset within the recording buffer overwriting the padding that was inserted when the later packet was received and loss of packet(s) identified.","8.A method of recording a data employing a plurality of network interface cards and including the steps carried out by the system of any one or more of claims 2 to 7.9.A data packet recording system arranged for determining an IP address of an application and including means for comparing at least a sample of received or transmitted packets with at least a set of pre-programmed signature packets.","10.A system as claimed in claim 9 and arranged for comparing all packets received from or sent to each destination against a set of pre-programmed signature packets.","11.A system as claimed in claim 9 and arranged such that the sustained absence of such packets is employed as an indicator of an error condition with the device that was previously active on that address.","12.A method of recording a data packet for determining an IP address of an application and including the step of comparing at least a sample of received or transmitted packets with at least a set of pre-programmed signature packets.","13.A data recording system arranged such that the data packets are arranged to be transmitted via a proxy server and further arranged such that the IP address of at least one party to the conversation is altered.","14.A system as claimed in claim 13, and arranged to determine the presence and IP address of the proxy servers, to allow for recorders passing their knowledge of current mappings to other recorders allowing them to refine their filtering algorithms; and to pass examples of specific packets identified as having been mapped by the proxy server to other recorders.","15.A data packet recording system including packet filtering means arranged for filtering on the basis of an IP address.","16.A data packet recording system as claimed in claim 15, and arranged for filtering on the basis of an IP port number of both source and/or destination address(es).","17.A system as claimed in claim 15 or 16, and arranged to analyse the sequence number and timestamp to determine the next packet in the expected sequence based on whether one or more packets have been missed such that performance counters can be incremented appropriately; inserting additional data in the recorded stream to pad-out the space that should have been taken up by the missing packet(s) and whether the payload of the RTP packet can be appended to a buffer and the remainder of the packet discarded.","18.A system as claimed in claim 15 or 16 and arranged such that a signature data pattern is appended to a data buffer collecting packets to/from the specific destination; a timestamp indicating the time at which the packet was received to the buffer; a length indicator specifying the number of bytes in the recorded packet is appended to the buffer; and that the contents of the packet are appended to the buffer.","19.A data recording system for IP telephones controlled by an IP-PBX application and wherein the controlling application is arranged to request that each phone advise it of the port number for which it can receive incoming audio data.","20.A system as claimed in claim 19, and arranged for the identification and storage of only the payloads of successive RTP messages.","21.A system as claimed in any one of the preceding claims, and arranged to compare the sequence number of the received packet with that of the previously received packet and to identify if a difference exists.","22.A data recording system as claimed in any one of the preceding claims and arranged such that recordings stored independently for each direction of transmission."],["The present invention relates to a packet data recording method and related system.","The integration of computers into office communication systems has enabled many functions previously performed by separate devices to be combined into a single management system.","For example, computer-based voice logging systems enable a computer to: receive voice communication through a hardware connection to the regular telephony network; to record either a conversation, in which at least two parties converse, or a message from at least one party to one or more parties; and to replay such recorded conversations or messages upon request.","It is therefore appreciated that commercial entities perform substantial amounts of business via telephone or Internet contact with their customers.","The analysis of such contact can help businesses improve the quality and efficiency of their services, and assist with customer retention and, ultimately, profitability.","Attempts have been made previously to achieve such analysis in a satisfactory manner and to a satisfactory degree.","For example, many businesses have, for some time, recorded some of their staff/customer interactions.","Traditionally this was done to satisfy regulatory requirements or to help resolve disputes.","More recently, the emphasis has moved towards the reviewing of these interactions from a quality perspective.","This is intended to identify good and bad aspects of particular interaction calls with a view to improving the level of customer service.","Recently, for example, recording the activity on a PC screen has been undertaken to improve the completeness of the review procedure with the reviewer able to see how accurately staff are entering information received via the telephone.","Also, it has been known to employ Call Detail Recording (CDR) systems to prevent perceived abuse of telephone systems and to apportion costs to the department or individual making the calls.","Originally such records were printed out directly from the Private Automatic Branch Exchange (PABX) onto a line printer.","Later, systems were designed to store this information in a database allowing more sophisticated reporting and searching fro calls on the basis of one or more of the stored call details.","More recently, Computer Telephony Integration (CTI) interfaces have been provided that give this information in real-time, during the call.","Further, several systems currently exist that use call recording in combination with CDR or CTI and a database application in order to perform routine monitoring of calls with the intention of identifying weaknesses in individual Customer Service Representatives (CSR).","Typically a small percentage of the CSR calls are reviewed and “scored” against a set of predetermined criteria to give an indication of the quality of that particular member of staff.","Within a call-centre environment, it should also be noted that rather than simply using standard PC office automation applications when dealing with customers, staff in most call centres use increasingly sophisticated applications that help them to handle the calls more efficiently and effectively.","Help desk applications and telemarketing call scripting applications are examples of such applications.","U.S. Pat.","No.","6,122,665 discloses a method and a system for: the management of communication sessions for computer network-based telephone communication, and in particular for the identification of packets containing audio and/or video data; the storage of these packets; and for the reconstruction of selected communication sessions for audio and/or video display as needed.","In particular, this document teaches the provision of a system and a method arranged to: record communication sessions performed over a computer network; to provide such a system and method for analysing data transmitted over the computer network in order to detect audio and video data transmitted over the computer network in order to detect audio and video data for recording; to provide such as system and method for displaying recorded video and audio data upon request; and to provide such a system and method for analysing, recording and displaying communication session conducted with a LAN-based telephone system.","As will be appreciated, U.S. Pat.","No.","6,122,665 serves to illustrate a general move towards Voice over IP (VoIP) Communications as bringing together voice and data environments.","However, there remains a need to record interactions over data networks irrespective of whether these are voice communication sessions, video or data interaction between people and systems, systems and systems or people and people.","While it is noted that systems produced by, e.g.","Hewlett Packard, have long been able to monitor data networks and store details and contents of the packets they observe, the primary function of such devices has normally been for network performance analysis and diagnostics.","Disadvantageously they do not provide for the management of large volumes of recordings, and related archival and indexing.","Thus, such systems, and related methods, do exhibit limitations and disadvantages.","As a further illustration U.S. Pat.","No.","6,122,665 merely serves to address the recording of a single network segment.","However, with the prevalence of Ethernet switches and routers, it is rare for a system of any size to be implemented as a single network segment.","Whenever multiple segments are introduced, the packets are routed between these segments as required in order to reach their destination.","There is therefore no single point at which all packets can be observed.","It would therefore prove advantageous to provide a system which can be extended to tap into the network at however many points are needed to achieve the required coverage.","Unfortunately, merely extending a simple system designed for a single tap-point will result in highly redundant recordings as many of the packets to be recorded will arise at more than one tap point.","Further, known systems require knowledge of network addresses to operate properly.","To determine what, and indeed how to record, some knowledge of the identity of specific IP addresses is often required.","For example, knowing which IP address hosts an IP-PBX will allow better filtering of information and the analysis of call control packets involving that node.","Unfortunately, many IP addresses are dynamically assigned, e.g.","via DHCP.","Also network configurations are regularly changed and updated as networks expand and evolve.","Neither do known systems address the use of proxy addresses.","In many IP networks, packets are readdressed en route by Proxy Servers.","This makes it difficult for the controlling software to identify which packets should be recorded as a single IP address is often only appropriate for packets transmitted over the leg of the journey from which they emanate, or which they finally reach having regard to the network node having that IP address.","The Known systems also do not support the recording of packet data streams along with voice/video.","The system disclosed in U.S. Pat.","No.","6,122,665 filters packets out that are not part of a voice or video stream.","This prohibits its use in cases where the recording of a combination of data streams is required.","With regard to packet filtering, known systems are far from optimal.","It is important that a recording system is able to process all the packets it receives and eliminate those that are not required as efficiently as possible.","Often only a small proportion of received traffic is to be recorded.","The described system performs its filtering initially by determining the type of traffic contained within the packet, for example voice or video versus other.","In a large voice/video system with many concurrent sessions occurring, there will be many thousands of packets per second being received at the network tap point.","In many cases only a small proportion of these will be required.","Known systems will not filter out any of these at the first level and will pass all packets on to the following stage.","Such manner of operation proves to be sub-optimal.","Disadvantageously, the integration of such a system with the systems controlling call placement is not generally achievable.","Also, the determination of which IP ports to record on the basis of H323 signalling information also received via the network tap point is often superfluous.","Rather, better information regarding the contacts and the requirement to record them can be obtained by interfacing to real-time event outputs, i.e.","CTI ports, of the systems that are controlling the placement of calls.","These ACDs or PABXs will often include additional information regarding the calls that cannot be deduced by observing the session control messages.","For example, the linkage of one call to the next as calls are transferred between people is not always available from the session control protocol.","Also, tolerance issues such as the loss of critical packets is not addressed in known systems.","However, in situations where session control information can be gleaned from analysis of session control packets, it can be disadvantageously adversely affected by the loss of a single packet.","This can readily lead to a call not being recorded.","In many systems, there can prove to be multiple ways to deduce such information, thereby making the system tolerant to the loss of certain packets.","As with filtering, known systems prove to be sub-optimal with regard to the storage of received packet data.","The described system does not disclose in detail how the packets are stored and it appears that these are simply kept as raw, independent packets such that a significant potential for storage overhead has been found to arise.","Importantly, the known systems do not integrate well with traditional (non VoIP) storage and replay mechanisms.","Again, the described system appears to store the recorded packets as packets and replays these by retransmitting them to a device similar to that which would have received them originally.","In many systems, however, only part of the voice system is packet-based and certainly the replay mechanisms are often not packet based.","Hence there is a need to store, retrieve and replay conversations in a way that is both efficient and compatible with existing circuit-switched voice systems.","Still further, such known systems do not address the specific requirements of testing and monitoring, nor do they attempt performance measure.","In any packet system, there is the chance of the loss of a particular packet and the system described makes no reference to a determination of how well it is performing and whether any packets not recorded were lost by the network or at the recording device's network interface.","It is now appreciated that with simple extensions, such recording systems can be used to record data steams in a form suitable for use in simulated loading tests.","The capability for advanced recording options such as stereo and multi-party (conference call) recording are also disadvantageously absent from known systems.","In particular, the system described does not address the added value that can be obtained by recording conversations so that they may be replayed in “stereo” with multiple tracks allowing the replayer to separate the audio travelling in each direction.","Nor does it describe the extension of this technique into the recording of conference calls where the separation and identification of individual speakers is of great benefit in aiding the listener to follow the flow of conversation.","The present invention seeks to provide for a data recording system and method and exhibiting an advantage over such known systems.","According to one aspect of the present invention, there is provided a data recording system including data recording means having a plurality of network interface cards.","Of course, alternatively, where more than one segment must be tapped in order to receive all required packets, this can be done either by providing multiple systems, each tapping a single segment but this can result in the same recordings being made by multiple recorders.","A preferred embodiment is to use multiple network interface cards (NICs) within a single recorder.","Advantageously, each NIC may have multiple network connection ports, such as typically are used to provide fault tolerant connection to multiple Ethernet switches.","In this case, the filtering mechanism is enhanced to allow rapid elimination of duplicate packets.","For each RTP packet stream being recorded, the system can advantageously note the sequence number of the most recently accepted packet.","Any packet received with a lower sequence number (account being taken of sequence numbers wrapping as they overflow their assigned binary range) will immediately be discarded.","In this way, the system advantageously need not actively keep track of network topology and will operate regardless of how many copies of each packet it receives.","In larger or physically separated systems which require multiple recorders, it can prove impractical to bring all packets into a single recorder.","In such cases, the individual recorders can be arranged to communicate with each other so as to advise each other of the presence of particular packet streams.","In a prepared example, it is desired to record packets travelling between a specific pair of addresses: a) each recorder is advised of the addresses to be monitored; b) when a recorder identifies a packet with the required addresses it begins recording it and advises all other recorders to stand down; and c) other recorders are arranged to note these addresses as being recorded elsewhere.","Further advantageous refinements to such a scheme can seek to ensure that only one recorder records each call or session in that: d) the notification sent to other recorders call include within it the sequence number (or timestamp) of the RTP packet that was first received and the IP address (or other identifier) of the receiving recorder; e) in the event that more than one recorder receives packets prior to hearing notification from another recorder that it has commenced recording, the recorder which received the earliest packet can maintain responsibility for recording and the other recorder shall cease recording; f) should more than one recorder receive the same packet and send notification of this, then the recorder with highest IP address (or other algorithm allowing unique determination of priority) can maintain its recording and the other(s) will stand down; g) when recording begins, a recorder will not create a record in the database of recordings until a pre-defined time has elapsed in case it receives, during this period, a notification from another recorder that it too has started recording.","Should it receive such notification and, according to the algorithms defined in (d) to (f) above, it is to stand down, it will abandon its recording without having altered the database of recordings.","The following further refinements can be employed to ensure that packet loss is minimized: h) should a packet be lost (as can be determined from a gap in the sequence number of received packets) an indication of the sequence number(s) of the lost packets can be maintained as part of the recording control structure for that IP address; and i) when packets are received that are determined to be earlier than the most recently accepted packet for that address, their sequence number can subsequently be compared against the list of recently missed packets and, if found, can be stored at the appropriate offset within the recording buffer overwriting the padding that was inserted when the later packet was received and loss of packet(s) identified.","It should be appreciated that the invention also provides for a data recording method employing a plurality of network interface cards and as described above.","According to another aspect of the present invention, there is provided a data packet recording system arranged for determining an IP address of an application and including means for comparing at least a sample of received or transmitted packets with at least a set of pre-programmed signature packets.","The system can then advantageously determine the IP addresses of significant applications by comparing all packets received from or sent to each destination against a set of pre-programmed signature packets.","These packets are preferably chosen to be examples of the traffic expected to involve the node being sought.","For example, in many IP-PBX systems, each IP-phone regularly heartbeats by sending a simple packet to the IP-PBX control application every minute or so.","By monitoring for these packets it is possible to determine the location of the IP-PBXes in the system being monitored.","Preferably, the sustained absence of such packets can also be used as an indicator of an error condition with the device that was previously active on that address and can trigger an alarm condition and/or fallback mode of operation.","It should be appreciated that the invention also provides for a data packet recording method including a comparison step as described above.","According to yet another aspect of the present invention, a data recording system is provided wherein data packets are arranged to be transmitted via a Proxy server and further arranged such that the IP address of at least one party to the conversation is altered.","This advantageously allows for security and re-use of address space although difficulties can be experienced in determining which packets should be recorded.","The invention can preferably address this by being arranged to: a) determine the presence and IP address of such Proxy servers either by explicit configuration information or, preferably, by analysing the pattern of packets entering and leaving the IP node.","This can be achieved for example by comparing the contents of packets transmitted by each node with the contents of those received by that node.","Should it find packets that are identical bar the IP address, it can both deduce that this node is performing a Proxy function and determine the current mapping of IP addresses that it is performing; b) allow for recorders passing their knowledge of current mappings to other recorders allowing them to refine their filtering algorithms; and c) pass examples of specific packets identified as having been mapped by the Proxy server to other recorders.","This advantageously allows recorders to differentiate between overlapping address ranges.","For example, two different Proxy servers may both be mapping addresses in a given range.","By passing occasional packets to each other, along with their pre-mapping addresses, recorders can determine whether the stream of data they are observing is indeed that for this address or a completely different stream mapped by a different Proxy server.","As before, the invention also provides for a corresponding method including IP address alteration.","According to still another aspect of the present invention, there is provided a data packet recording system including packet filtering means arranged to perform on the basis of an IP address and preferably, an IP port number of both source and/or destination address(es).","Having determined that the packet is one that is to be recorded, the system can preferably then store the packet in the most appropriate way.","For example, for RTP streaming data: a) The system is arranged to analyse the sequence number and timestamp to determine whether this is the next packet in the expected sequence; b) if one or more packets have been missed then performance counters can be incremented appropriately; c) additional data can preferably be inserted in the recorded stream to pad out the space that should have been taken up by the missing packet(s).","This advantageously avoids gradual drift between the recording and real-time which would otherwise build up and result in the call being shorter when replayed than it had been originally; and d) the payload of the RTP packet can be appended to a buffer and the remainder of the packet discarded; and e) optionally, information can be flagged from the packet header so as to be retained where this is not the default header, i.e.","it cannot be fully deduced from knowledge of the previous header.","As an example for other packet oriented data: a) optionally, a signature data pattern (e.g.","well known particular 4 byte value) can be appended to the data buffer collecting packets to/from the specific destination.","This advantageously ensures that any application reading the recording subsequently can re-synch with these inter-packet boundaries should the recording become corrupted or the start of the recording cannot be accessed; b) a timestamp indicating the time at which the packet was received is appended to said buffer.","This will typically be precise to at least millisecond and can often achieve micro-nanosecond precision; c) a length indicator specifying the number of bytes in the recorded packet is appended to said buffer; and d) finally, the contents of the packet can be appended to said buffer Again, the invention also provides for a related method.","In an alternative data filtering arrangement and method filtering according solely to address data, or at least before looking at packet content type can be provided.","In some cases a given IP port might only be transmitting one type of data and so filtering on its address alone can advantageously prove sufficient in whether or not to record the packet.","A preferred arrangement can involve looking up the address of the IP destination of each packet, or equally source data, as the primary filter.","A “map” of known addresses is maintained and each entry serves to identify whether data going to (or from) that address should be recorded.","Advantageously, by utilizing the IP address as the primary key into this map, extremely fast look-up can be achieved.","Further preferred refinements to this scheme include: a) the pre-filtering of packets on IP address so as to eliminate packets sent to or from the recorder itself.","This allows the same network interface card to be used for normal IP communications (e.g.","with clients searching and replaying previously recorded calls) whilst it is also being used a promiscuous mode tap.","This simple filtering out on a single (32-bit) integer IP address avoids the need to look up the address map repeatedly for all packets that are directed at other sockets on this node.","With the introduction of IP Version 6, this will become a 128-bit integer comparison; b) in preference to hashing or sorted table indexing, the map of required addresses can be implemented as a memory array.","By using the fact that most IP addresses required will lie within a narrow range of possible values it is faster to compare the high order bytes of the IP address with the sub net or nets on which all the target nodes are located.","The remainder of the address can then be used as an index into a (typically sparsely populated) memory map in which each address to be recorded is represented by a binary 1 and the remainder as 0.For a Class B address as described here, only 64K bits of information are required to achieve this though the use of 65,536×32 bit words allows for each entry in the map to be either null (0x00000000) or a pointer to the structure describing the known address and its recording details e.g.","buffer storage location.","It should be noted that a similar scheme can be used in IP Version 6 albeit with a much smaller portion of the overall numbering range being covered by such a table; c) in RTP based systems, many of the data streams being transmitted are constant bit rate.","In these cases, many sessions will be transmitting packets at a regular rate resulting in a strong correlation between the order in which packets are received.","This is especially true in cases where all transmitters are of the same type e.g.","an IP-PBX with many identical IP-phones, all configured with the same packet interval.","This can be used to advantage in filtering the incoming packets.","If a record is maintained of the recently received packet addresses, the following optimized search algorithm can be deployed I. note the IP destination address of the first RTP packet received at the head of a list of such addresses.","Look up the known destination for this address and include a pointer to it at the head of a list of such pointers, II.","as subsequent packets are received, compare their address against the packet at the head of the list, III.","if they are different, append their addresses to the list; look up their known destination descriptor and append it to the list of such pointers, IV.","if it matches the address at the head of the list, use the pointer to the known destination at the head of such list.","Note that the next packet should be compared to the following entry in the list, V. subsequent packets should be compared to the next entry in the list.","If they match, repeat step IV, if not, insert them into the list at this point.","d) The algorithm of c above can preferably be further refined to accommodate the cessation of transmission from any address as happens when a call terminates.","By comparing the new packets address against the following item in the list, we can determine that the previous address has probably ceased transmitting and it can be removed from the list.","e) To allow for the case where two streams cease in one cycle of the list, it is necessary to look two addresses ahead if the new packet s address is not found at the head or subsequent entries in the list.","This is a common case as both directions of the call will typically terminate at or about the same time.","However, given a typical interval between transmission of 50 ms it is rare that more than one call terminates in any given cycle of the list.","As will be appreciated, traditional telephone recording systems are often controlled by or determine call details by interacting with the telephony switch via a Computer Telephony Integration (CTI) interface.","Such an interface typically advises the recording system of call setup and teardown as well as associated details such as dialled digits, calling line identifier (CLI) etc.","As such systems move to packet transmission of voice, many are doing so incrementally and hence supporting a mix of packet and traditional calls.","Where such an interface exists, it is beneficial to retain the existing investment in its design and also to allow support of VoIP and traditional calls with a single interface rather than having to support a further interface.","The present system and related method advantageously supports the use of existing interfaces, provided that where such interface previously provided call identification in the form of telecoms circuit and timeslot information allowing the matching of recorded data stream with call details, it is enhanced to provide equivalent information in the form of IP address and, preferably IP port number.","Ideally the addresses of both parties to the conversation (be they IP phone and/or gateway) are provided.","However, in some cases, the telephony system is only aware of the IP address of one or other party involved in the call.","It is a preferred feature of the present invention that the instruction to record a specified address (source or destination) can optionally be extended with the instruction to automatically start recording of the counterpart with which that address starts to exchange data.","In the case of an IP destination being recorded, this is achieved by noting the source of the packet that is recorded and creating a corresponding known destination record for that address.","In this way, subsequent packets sent to the newly identified address will also be recorded.","Obviously the converse applies should packet source be specified originally.","Further, where call control packets are analysed to determine which addresses are to be recorded, there is a danger that the failure to receive such a packet could result in the subsequent failure to record the entire conversation e.g.","if the packet lost contained the extension number of the IP phone and hence it was not recognized that a call to a recorded extension had started.","Fortunately, many such protocols are redundant and the required information can be deduced from several of the packets.","According to a further aspect of the present invention, the system can specifically address the case where IP telephones are controlled by an IP-PBX application.","In such cases it is typically possible to deduce the IP port address to be used for transmission in multiple ways.","In one case (Cisco CallManager) the controlling application requests that each phone advise it of the port number it can receive incoming audio data.","It then advises the other phone in the call of that number.","Hence the recorder can determine the port number to record should it receive either of these interactions.","Should it receive both, the redundant information can easily be recognized as such and ignored.","Yet further, when recording RTP streams, the header of these packets can be almost entirely derived from that of the previous packet and hence contains little of value.","By advantageously arranging for the identification and storage of only the payloads of successive RTP messages, the storage requirements for recording a session are dramatically reduced.","This is particularly so in the case of compressed audio transmission where the audio payload may be as little as twenty bytes in a packet of over one hundred bytes in total.","An embodiment of the present invention also addresses the issue of packet loss by being arranged to compare the sequence number of the received packet with that of the previously received packet.","Should these differ by more than one, packet loss can be deduced.","In addition to noting the loss in system performance counters, the missing packets can be “padded” out in the buffer being used to receive the recording.","Aspects of the present invention can also address the use of silence suppression which results in breaks in the transmission stream.","In such cases, the timestamps of successive packets indicate the extent of the suppressed silence whilst the successive sequence numbers let us differentiate between deliberate silence suppression and the accidental loss of packets as described above.","When packets are suppressed in this way, the present invention provides for either: the insertion of the appropriate amount of “padding” data which may be pure silence or “noise” derived according to a compression scheme; or the insertion of explicit indication of the silent period into the recorded stream.","Such as scheme is described by Microsoft in the definition of the “.WAV” file format.","This provides more efficient storage of such silence periods but unfortunately, most “.WAV” file players do not support this mechanism.","The present invention can advantageously further optimize storage by supporting a range of compression formats and acting on the received data according to user defined rules.","These can include: compressing data received in a specified format (e.g.","G.711 mu-law) to a specified second format (e.g.","G.726 16 kbps); leaving data received in a compressed format in that format (to avoid the inherent quality reduction associated with decompression and further compression; optionally mixing the two halves of a communication session into a single, mono, recording (preferably) prior to compression; or optionally saving to non-volatile storage (e.g.","disk) a summary representation of the audio levels on each of the two channels prior to compression and/or mixing.","This Energy Envelope representation allows a compact (20 samples per second, 2 byte values) representation that permits the graphical display of the call showing which party was speaking at what level even though the two halves of the call are subsequently mixed and only available as a mono rather than stereo signal.","Within aspects of the present invention, by storing the packet payload in a standard .WAV file, the recorded audio can be replayed alongside traditional recordings using any existing replay mechanism.","Also, given the ability of the system to record packet data with timestamps for each packet received, the system may be run in a special mode in which all packets recorded are processed and stored as described in above for the aforesaid other packet oriented data.","By storing all packets, including RTP streams, in this way, the recordings made can subsequently be used advantageously as known test cases for exercising and testing the invention.","By replaying recordings made in this way over the network, recorders under test will receive exactly the same packet stream as the recorder that made the original recording did.","Hence known test cases can be run automatically and the output of the recorders compared with that of previous test runs.","It is a further feature of the present invention that said recording files can be arranged to be processed offline, so as to multiply the traffic and hence simulate larger systems than were actually recorded.","By replicating the packets in the recording file and, for example, incrementing the IP port numbers for each replication, large volumes of traffic can be simulated from a single recording.","In another aspect of the invention, it is important that such recorders do not lose data packets as this will reflect in loss of audio and/or loss of control information and hence possibly missed calls.","The present invention therefore can be arranged to monitor its performance in the following way: 1.the gaps in the received packet stream can be determined as described above; 2.by analysing the RTCP packets received alongside the RTP packet streams it is possible to see the loss statistics being experienced by the actual participants in the call; 3.by comparing the above two figures an estimate of the packet losses occurring somewhere other than en route between the end points can be determined.","Some of this will be due to the recorder and can be used as an indication of the quality of the recording; In yet another aspect and as discussed above, recordings may advantageously be stored independently for each direction of transmission or combined into a single, mixed recording.","The former is highly advantageous should analysis of who was talking be required e.g.","to identify angry callers from high interruption levels or in performing speech recognition on the files.","The latter however is both less accurate and less useful when performed on a mixed recording since a single speaker model cannot be used and it is not clear who said each word.","On conference calls it is particularly beneficial to retain a recording of each party involved in the call since with many speakers, it can be difficult to differentiate between speakers with similar voices, and there is often background noise from one or more parties that makes it difficult to hear the principal speaker It is a preferred feature of the present invention that the recordings of a multiparty interaction can be kept as independent streams or mixed together to optimize storage space.","As mentioned above, it is possible to retain a summary record of the level of audio from each speaker even though the actual audio signal has been mixed.","It is a further preferred feature of the present invention that when such conferences are handled by a conference bridge, which mixes the signals, storage of the conversations can be limited to the minimum set of data streams needed to reconstruct the conference audio.","This advantageous aspect is achieved as follows for a conference between parties A, B and C using conference bridge X A, B and C each transmit audio to X X transmits audio from (B+C) to A X transmits audio from (A+C) to B X transmits audio from (A+B) to C The recorder may simply be instructed not to record packet streams coming from the conference bridge X and hence only records the pure audio from each of the contributing parties.","The required mixed signals can be reconstructed at replay time if required from these three streams.","Of course, it should be appreciated that the invention can comprise any one or more of the aspects and embodiments described above in combination or alone.","The invention is described further hereinafter, by way of example only, with reference to the accompanying drawings in which: FIG.","1 is a block diagram of a recording sub-system embodying the present invention; FIG.","2 is a schematic block diagram showing the operation of an IP address filter embodying an aspect of the present invention; FIG.","3 is a schematic block diagram showing the operation of a protocol filter embodying an aspect of the present invention; FIG.","4 is an illustration of packet data buffering as arising in an embodiment of the present invention; FIG.","5 is an illustration of RTP/PTCP Packet Buffering as arising in an embodiment of the present invention; and FIGS.","6A-6C illustrate the structure of IP packets, RTP packets and RTCP packets as arising within the present invention.","Turning now to FIG.","1, there is illustrated a recording sub-system embodying the present invention and in which a recorder is connected to a network from which calls are to be recorded by connecting into allocated ports on one or network routers, switches or hubs such as the 10/100 Mbps Ethernet hubs [1], [2], [3].","In the case of intelligent switches, which only transmit data packets to those ports that need to receive the data, a “SPAN” setting is normally applied to the port used for the recorder.","This setting is normally used for network diagnostics and forces the switch to copy packets destined for one or more of its other ports to this port as well.","Each router/switch/hub is connected to an appropriate form of network interface card (NIC), for example 10/100 Mbps Ethernet, Token Ring, Gigabit Ethernet [4], [5].","The NIC [5] is an example of a multiport NIC which allows connection to more than one network access point.","This allows efficient recording of larger networks where calls are spread across several network concentrator devices, without the need for a separate recorder for each of these.","Within the operating system on the Recorder, such as Windows 2000 there will be installed one or more drivers that allow applications to communicate with the NIC(s) installed.","A common NDIS driver (6) is illustrated.","This typically allows other applications running on the same computer to share access to the NICs without having to be aware of each other.","Hence this diagram only shows the recording sub-system.","Note the archival, search and retrieval functions typical of a bulk voice recorder can still be hosted on the same PC providing of course that there are sufficient memory, processor and disk resources for these to co-exist.","The system described communicates with the NDIS driver [6] and establishes a data channel which it places in promiscuous mode.","This overrides the normal behaviour in which only data packets destined for this application will be delivered and instead requests that all data packets received via the network access point are passed through to an IP Address Filter mechanism [7].","The IP Address Filter mechanism [7] is described in more detail below with reference to FIG.","2.In summary, it compares the IP address and port number of each received IP packet against a list of known destinations.","This could be equally well be applied to known sources rather than destinations.","A rapid decision is made as to whether this packet is to be recorded or not.","If the answer is yes, it will pass the packet on to a Protocol Filter [8].","This second stage filter [8] can therefore now use both knowledge of the destination to which the packet was sent and information from the IP packet itself to determine whether the packet contains RTP or RTCP data or other packet data.","The filter [8] passes the packet on to either a Packet Data Buffering module [9] or a RTP Packet Buffering module [10].","Each of these two buffering modules [9], [10] applies schemes appropriate to the data type being recorded before passing on data, in relatively large, contiguous blocks, to the Record Thread Manager [14a] which, running at slightly lower priority than the previous components will write this data to the appropriate files on the hard disk.","This results in efficient large data writes to the permanent storage such as a hard disk [11].","The end result is typically a pair of files one containing the actual content of the data stream, for example a .WAV audio file for RTP transmitted telephony, and the other containing details of the recording such as a .XML file containing start time, duration, IP address etc.","Alternatively the details of the recordings may be inserted directly into a database.","In buffering the RTP data streams, the RTP Packet Buffering module analyses the sequence numbers and timestamps of the received packets.","It compares the packet loss rates it is experiencing against those reported by the participants in the streaming interaction and can hence deduce performance levels which it logs for example to a file [14].","It could equally well pass this information into a network management tool such as via Simple Network Management Protocol (SNMP).","The set of known destinations that are to be recorded can be fixed in a variety of ways.","For example, it could be achieved by direct, local configuration of the IP Address Filter [7], through blanket rules such as recording all streams; or it can be instructed explicitly by a component such as Unify [12] a CTI middleware platform which interfaces with a wide range of telephony and other customer contact service systems [13].","By observing activity on the external system and applying business rules, the addresses to be recorded can be readily identified.","With reference to FIG.","2, once a packet is received and determined to be an IP packet it is examined by the IP Address Filter to determine whether or not it is to be recorded.","It should be appreciated that, according to the network traffic patterns experienced and the proportion to be recorded, the order and/or presence of each of these filtering stages may be changed to deliver optimum performance typically measured by the packet rate that can be processed within a given proportion of the CPU time available.","The IP packet [15] is normally first compared [17] with the IP address of the computer on which the recorder is hosted.","This allows very rapid elimination of all packets that are intended for other processes on this computer and ensures that the hosting of, for example, a search and replay application on the same computer does not adversely impact the performance of the recording sub-system.","The packets which were addressed to other IP addresses are then passed to a circular buffer lookup algorithm [21].","Recently received IP destination addresses including IP port number, are noted in a circular buffer [18].","When a packet is received, its IP address is compared with that of the entry at a Test Point [19] in the circular buffer [18].","If a match is identified, the Test Point [19] is advanced and the entry in the circular buffer is used as a pointer to the Known Destination Object [16] for this IP address.","An entry in the Known Destination object's [16] data structure indicates whether or not the packet is to be recorded [25].","If it is, then it is passed through to the Protocol Filter (next section).","If the packet's IP address does not match the next item in the Circular Buffer [18], it is compared against one or more subsequent entries and if a match is found, the test point [19] is advanced to this point.","The entries that were tested and found not to match will be removed from the buffer as it can be deduced that they are no longer in the correct position or the stream to those addresses has terminated.","If the packet's IP address does not match any of those within the lookahead window described above, it is passed to the following stage of the filter [22] in which the IP address is used to determine if there is an existing Known Destination record [16] for this IP address.","The Known Destination list is typically held in memory in the form of a map with a fast indexing or hash function that allows rapid searching of many such records for the one with a specific address.","However, the nature of IP addresses (e.g.","10.25.34.245) is deliberately hierarchical with the earlier numbers (when represented in text string form as here) representing the larger sub-networks.","It is therefore very common for the vast majority of traffic observed on a single network segment to involve IP addresses from that sub-net i.e.","the first 2 or even 3 of the numbers will be common.","In such cases it is viable to maintain one or more look-up tables of Known Destination Maps [26] which simulate content addressable memory For example, such a table, with 65,536 entries can hold pointers to all the Known Destination records [16] relating to the block of IP addresses 10.10.xxx.yyy where xxx and yyy are any number from 0 to 255.It is therefore very easy and quick to compare the top (leftmost when written as text) two bytes of an IP address against the base address of such a lookup table and, if they match, use the lower (rightmost when written as text) bytes as an index into the table.","A 0 (null) entry in the table infers that the destination does not yet have a Known Destination record associated with it whilst a non-zero entry can contain a pointer to the relevant Known Destination object.","If the Destination is not within the range of any such rapid lookup maps [26], the normal hash table or other indexing method will be used to determine if a Known Destination Record exists.","If it does, it will be inserted into the Circular Buffer [18] at the insertion point normally immediately prior to the test point [19].","If an existing Known Destination record is not found, then a new record will be created and the Record flag will be set to true or false according to the default recording rules [23].","Having found, or created, the appropriate Known Destination Record [16], the packet will at [25] either be discarded or passed to the Protocol Filter according to the Record flag within the Known Destination Record.","It should be noted that the Known Destination Records can also be created, deleted and their recording flag set/reset by the Unify interface [12].","Also in order, to avoid long term build-up of redundant Known Destination Records [16], a background process can serve to periodically review the last time that a packet was observed being sent to that destination and will destroy the Known Destination Record should this exceed a pre-determined threshold.","To enable this function, a data field within the Known Destination Record structure is updated to reflect the current system time whenever it is used to determine whether or not to record a packet.","As illustrated in FIG.","3, the Protocol filter stage can be embodied very simply.","It first examines the contents of the Known Destination Record [16] that is by now associated with the packet being processed.","This record may contain information that allows the protocol filter to determine which protocol the packet should contain.","This is particularly advantageous in the case of RTP data streams as there is no guaranteed method for determining that a packet is indeed an RTP packet.","Such packets are identified as UDP protocol but there is no indication within the UDP packet that the payload is indeed RTP.","Unless an external influence such as Unify [12] has deduced that RTP is to be expected on a given destination IP address, the only alternative is to determine if the first few bytes of the UDP packet's payload represent valid entries for an RTP packet header.","This is time consuming and prone to occasional false detection.","Hence, if the protocol being sent to a specific port is known already, this can be used to direct the packet at either the Packet Data Buffer [30] or the RTP/RTCP Buffer [31].","If the Known Destination Record [16] does not specify the protocol expected, the filter next examines the protocol type specified within the IP packet.","A typical filter will simply check for TCP [28] and pass these packets to the Packet Data Buffer [30] then check for UDP packets [29] and pass these to the RTP/RTCP Packet Buffer [31].","According to pre-configured default behaviour [32], other protocol types may be either discarded [33] or sent to one or other of the two described buffering modules or to a new buffering module specifically optimized for storage of that protocol type such as ARP, ICMP or SNMP.","Turning now to FIG.","4 there is illustrated the packet data buffering of the present invention.","This can prove to be relatively straightforward with each packet simply being preceded by any or all of: A well-known synchronization value which will ensure eventual re-synchronization of a partially corrupt file; A precise time stamp; Offset of local time from Universal Co-ordinated Time (UTC); Daylight Savings Time offset; or Length of packet that follows.","If this is the first packet being recorded for a given Known Destination [16] then the Known Destination record will not yet have had a buffer storage element [37] assigned.","A start recording job is posted onto the queue [35] for the Record Thread Manager and a buffer [37] is assigned.","These buffers are typically several Kbytes in length and can hence hold many individual packets.","The packet to be stored and its associated data as listed above is appended to the buffer.","When subsequent packets are received, these are appended to the buffer [37].","At the point where the buffer fills, it is moved to the FIFO queue [35] to await processing by the Record Thread Manager [39].","When the job reaches the head of the queue, it will be appended to the appropriate file on disk [34].","Meanwhile, a new buffer [37] will be allocated to the Known Destination Record [16] and any remnants of the packet which filled the previous buffer are appended to it.","This process repeats until the recording is terminated at which point the partially filled buffer [37], if any, is appended to the job queue [35] and a Stop Recording job is then added to the job queue.","When the Record Thread Manager reaches a Stop Recording job, it closes the file in question.","Additional details about the call may be included within the Start and Stop Recording messages and these, along with other derived information, such as the total data volume stored are ultimately written to a call detail file, for example in .XML format or direct to a database.","It should be appreciated that a lower priority thread high priority still being lower than Real-time priority—is used for writing data to disk than for capturing, filtering and buffering packets into memory.","This allows RAM buffering to absorb short term peak load conditions which would otherwise cause bottlenecks and limit the peak throughput capacity of the recorder.","FIG.","5 illustrates the RTP/RTCP Packet Buffering.","This component can operate in the same way as the Packet Data Buffering component described above except for the way in which it handles the content of the data packet itself.","Since RTP packets typically form a continuous stream of data, there is no need to store the RTP packet header information for all packets as it is only the payload of actual audio/video data that is required.","When audio is compressed for transmission, the payload can be as little as 20 bytes (G.729A, 20 ms per packet) yet the total packet length, including Ethernet, IP, UDP and RTP headers, will typically exceed 100 bytes.","Hence storing the whole packet is incredibly inefficient.","In the simplest case, the payload of each RTP packet is appended to the buffer [37] and, when full, this is placed on the Record Job Queue [35].","Recording start and stop are exactly as per the Packet Data Buffering component and these two components share a common queue ensuring that data is written to disk in a FIFO manner regardless of which type of recording i.e.","packet or stream, is being made.","In the case of the RTP data, the files that are written by the Record Thread Manager are typically in Microsoft .WAV format and hence readable by any player device that supports this file format and the compression standard used within the file.","The process is complicated somewhat by the need to allow for packet loss.","This is achieved by maintaining in memory, details of the most recently received packet sequence number and timestamp.","It is then possible to compare the currently available packet's sequence number and timestamp with that of the previous packet.","A gap in sequence numbers can be noted and logged to a performance log file or similar [40A].","To avoid gradual buildup of error in the recorded file, any lost packet(s) can be compensated for by the automatic appending of the appropriate number of bytes of silence (or other pre-defined padding sound).","Where a small number of bytes are missing (e.g.","1 or 2 packets) it is more efficient to simply add these to the buffer [37] as if they had been received.","However, in the case of large gaps, it is more efficient to place the current buffer [37] on the Record Job Queue [35] for processing and then place a Padding job specifying the number of bytes of silence to be inserted.","The Record Thread Manager can then append the appropriate number of bytes to the file without using excessive memory space on the Record Job Queue as would be the case if several buffers all full of silence were queued.","This latter technique is particularly advantageous in systems that utilize silence suppression.","In such cases, the RTP stream will stop until the audio level reaches a threshold.","This can save significant bandwidth in voice communications as typically only one party is speaking at a time.","In such cases, the packet sequence numbers in the RTP packet are contiguous but the timestamps are far apart.","Again, this can be detected and the appropriate amount of silence indicated through the placement of a Padding job on the record job queue [35].","This then allows the Record Thread Manager to either pad the call with the appropriate amount of silence or to utilize a scheme such as the Wave List feature within the Microsoft .WAV format.","This allows for the efficient storage of such audio as a sequence of sound, silence, sound, silence etc.","A further refinement includes the processing of RTCP packets associated with the RTP streams being recorded.","By examining the contents of these packets, one can determine the fraction and cumulative number of packets lost by each of the participants in the conversation.","These details can be included with the loss rates experienced at the recorder and included in the Stop Recording job and/or entries to the log file [40A].","This allows subsequent analysis of the recorder's performance in comparison to that experienced by the participants on the call.","To assist with the above description, reference is now made to the general structures of the packets arising in the aspects of the present invention.","Turning now to FIG.","6A, a header 42 is shown as a plurality of boxes, each of which represents a portion or “field” of the header.","The number of bytes occupied by each portion is also shown, it being understood that each layer consists of 32 bits.","The first portion of the header, a “VERS” portion 44, is the protocol version number.","Next, an “H.LEN” portion 46 indicates the number of 32-bit quantities in the header.","A “SERVICE TYPE” portion 48 indicates whether the sender prefers the datagram to travel over a route with minimal delay or a route with maximal through-put.","A “TOTAL LENGTH” portion 50 indicates the total number of octets in both the header and the data.","In the next layer, an “IDENTIFICATION portion 52 identifies the packet itself.","A “FLAGS” portion 54 indicates whether the datagram is a fragment or a complete datagram.","A “FRAGMENT OFFSET” portion 56 specifies the location of this fragment in the original datagram, if the datagram is fragmented.","In the next layer, a “TIME TO LIVE” portion 58 contains a positive integer between 1 and 255, which is progressively decremented at each route travelled.","When the value becomes 0, the packet will no longer be passed and is returned to the sender.","A “TYPE” portion 60 indicates the type of data being passed.","A “HEADER CHECKSUM” portion 62 enables the integrity of the packet to be checked by comparing the actual checksum to the value recorded in portion 62.The next layer of header 42 contains the source IP address 64, after which the following layer contains the destination IP address 66.An optional IP OPTIONS portion 68 is present, after which there is padding (if necessary) and a data portion 70 of the packet containing the data begins.","As shown in FIG.","6B, an RTP packet header 92 features several important fields: a timestamp field 94, a synchronization source (SSRC) identifiers field 96 and a contributing source (CSRC) identifiers field 98.SSRC field 96 is used to determine the source of the RTP packets (the sender), which has a unique identifying address (the SSRC identifier).","The CSRC identifer in CSRC field 98 is used in a conference with multiple parties, and indicates the SSRC identifier of all parties.","Timestamp field 94 is used by an RTP software module to determine the relative time at which the data in each packet should be displayed.","Finally FIG.","6C shows the general structure of an RTCP packet."]],"string":"[\n [\n \"Packet data recording method and system\",\n \"The present invention provides for a data recording system including data recording means having a plurality of network interface cards in which a plurality of network interface cards can be employed within a single recording mean and each card can be provided with a plurality of network connecting ports then, for example, for each RTP packet stream being recorded, the system can advantageously note the sequence number for the most recently accepted packet and any packet received with a lower sequence number can be readily discarded.\"\n ],\n [\n \"1.A data recording system including data recording means having a plurality of network interface cards.\",\n \"2.A system as claimed in claim 1 and including a plurality of network interface cards within a single recording means.\",\n \"3.A system as claimed in claim 2, wherein each Network interface card has multiple network connection ports.\",\n \"4.A system as claimed in claim 1, 2 or 3, wherein each individual recording means is arranged for mutual communication so as to advise each other of the presence of particular packet streams.\",\n \"5.A system as claimed in any one of claims 1 to 4, and arranged such that each recorder is advised of the addresses to be monitored; when a recorder identifies a packet with the required addresses it begins recording it and advises all other recorders to stand down; and other recorders are arranged to note these addresses as being recorded elsewhere.\",\n \"6.A system as claimed in claim 5 and further arranged such that the notification sent to other recorders include within it the sequence number of an RTP packet that was first received and the IP address of the receiving recorder; that in the event that more than one recorder receives packets prior to hearing notification from another recorder that it has commenced recording, the recorder which received the earliest packet can maintain responsibility for recording and the other recorder shall cease recording; that should more than one recorder receive the same packet and send notification of this, then the recorder with highest IP address can maintain its recording and the other(s) will stand down; and that when recording begins, a recorder will not create a record in the database of recordings until a pre-defined time has elapsed in case it receives, during this period, a notification from another recorder that it too has started recording.\",\n \"Should it receive such notification and, according to the algorithms defined in (d) to (f) above, it is to stand down, it will abandon its recording without having altered the database of recordings.\",\n \"7.A system as claimed in claim 6 and further arranged such that should a packet be lost an indication of the sequence number(s) of the lost packets can be maintained as part of the recording control structure for that IP address; and that when packets are received that are determined to be earlier than the most recently accepted packet for that address, their sequence number can subsequently be compared against the list of recently missed packets and, if found, can be stored at the appropriate offset within the recording buffer overwriting the padding that was inserted when the later packet was received and loss of packet(s) identified.\",\n \"8.A method of recording a data employing a plurality of network interface cards and including the steps carried out by the system of any one or more of claims 2 to 7.9.A data packet recording system arranged for determining an IP address of an application and including means for comparing at least a sample of received or transmitted packets with at least a set of pre-programmed signature packets.\",\n \"10.A system as claimed in claim 9 and arranged for comparing all packets received from or sent to each destination against a set of pre-programmed signature packets.\",\n \"11.A system as claimed in claim 9 and arranged such that the sustained absence of such packets is employed as an indicator of an error condition with the device that was previously active on that address.\",\n \"12.A method of recording a data packet for determining an IP address of an application and including the step of comparing at least a sample of received or transmitted packets with at least a set of pre-programmed signature packets.\",\n \"13.A data recording system arranged such that the data packets are arranged to be transmitted via a proxy server and further arranged such that the IP address of at least one party to the conversation is altered.\",\n \"14.A system as claimed in claim 13, and arranged to determine the presence and IP address of the proxy servers, to allow for recorders passing their knowledge of current mappings to other recorders allowing them to refine their filtering algorithms; and to pass examples of specific packets identified as having been mapped by the proxy server to other recorders.\",\n \"15.A data packet recording system including packet filtering means arranged for filtering on the basis of an IP address.\",\n \"16.A data packet recording system as claimed in claim 15, and arranged for filtering on the basis of an IP port number of both source and/or destination address(es).\",\n \"17.A system as claimed in claim 15 or 16, and arranged to analyse the sequence number and timestamp to determine the next packet in the expected sequence based on whether one or more packets have been missed such that performance counters can be incremented appropriately; inserting additional data in the recorded stream to pad-out the space that should have been taken up by the missing packet(s) and whether the payload of the RTP packet can be appended to a buffer and the remainder of the packet discarded.\",\n \"18.A system as claimed in claim 15 or 16 and arranged such that a signature data pattern is appended to a data buffer collecting packets to/from the specific destination; a timestamp indicating the time at which the packet was received to the buffer; a length indicator specifying the number of bytes in the recorded packet is appended to the buffer; and that the contents of the packet are appended to the buffer.\",\n \"19.A data recording system for IP telephones controlled by an IP-PBX application and wherein the controlling application is arranged to request that each phone advise it of the port number for which it can receive incoming audio data.\",\n \"20.A system as claimed in claim 19, and arranged for the identification and storage of only the payloads of successive RTP messages.\",\n \"21.A system as claimed in any one of the preceding claims, and arranged to compare the sequence number of the received packet with that of the previously received packet and to identify if a difference exists.\",\n \"22.A data recording system as claimed in any one of the preceding claims and arranged such that recordings stored independently for each direction of transmission.\"\n ],\n [\n \"The present invention relates to a packet data recording method and related system.\",\n \"The integration of computers into office communication systems has enabled many functions previously performed by separate devices to be combined into a single management system.\",\n \"For example, computer-based voice logging systems enable a computer to: receive voice communication through a hardware connection to the regular telephony network; to record either a conversation, in which at least two parties converse, or a message from at least one party to one or more parties; and to replay such recorded conversations or messages upon request.\",\n \"It is therefore appreciated that commercial entities perform substantial amounts of business via telephone or Internet contact with their customers.\",\n \"The analysis of such contact can help businesses improve the quality and efficiency of their services, and assist with customer retention and, ultimately, profitability.\",\n \"Attempts have been made previously to achieve such analysis in a satisfactory manner and to a satisfactory degree.\",\n \"For example, many businesses have, for some time, recorded some of their staff/customer interactions.\",\n \"Traditionally this was done to satisfy regulatory requirements or to help resolve disputes.\",\n \"More recently, the emphasis has moved towards the reviewing of these interactions from a quality perspective.\",\n \"This is intended to identify good and bad aspects of particular interaction calls with a view to improving the level of customer service.\",\n \"Recently, for example, recording the activity on a PC screen has been undertaken to improve the completeness of the review procedure with the reviewer able to see how accurately staff are entering information received via the telephone.\",\n \"Also, it has been known to employ Call Detail Recording (CDR) systems to prevent perceived abuse of telephone systems and to apportion costs to the department or individual making the calls.\",\n \"Originally such records were printed out directly from the Private Automatic Branch Exchange (PABX) onto a line printer.\",\n \"Later, systems were designed to store this information in a database allowing more sophisticated reporting and searching fro calls on the basis of one or more of the stored call details.\",\n \"More recently, Computer Telephony Integration (CTI) interfaces have been provided that give this information in real-time, during the call.\",\n \"Further, several systems currently exist that use call recording in combination with CDR or CTI and a database application in order to perform routine monitoring of calls with the intention of identifying weaknesses in individual Customer Service Representatives (CSR).\",\n \"Typically a small percentage of the CSR calls are reviewed and “scored” against a set of predetermined criteria to give an indication of the quality of that particular member of staff.\",\n \"Within a call-centre environment, it should also be noted that rather than simply using standard PC office automation applications when dealing with customers, staff in most call centres use increasingly sophisticated applications that help them to handle the calls more efficiently and effectively.\",\n \"Help desk applications and telemarketing call scripting applications are examples of such applications.\",\n \"U.S. Pat.\",\n \"No.\",\n \"6,122,665 discloses a method and a system for: the management of communication sessions for computer network-based telephone communication, and in particular for the identification of packets containing audio and/or video data; the storage of these packets; and for the reconstruction of selected communication sessions for audio and/or video display as needed.\",\n \"In particular, this document teaches the provision of a system and a method arranged to: record communication sessions performed over a computer network; to provide such a system and method for analysing data transmitted over the computer network in order to detect audio and video data transmitted over the computer network in order to detect audio and video data for recording; to provide such as system and method for displaying recorded video and audio data upon request; and to provide such a system and method for analysing, recording and displaying communication session conducted with a LAN-based telephone system.\",\n \"As will be appreciated, U.S. Pat.\",\n \"No.\",\n \"6,122,665 serves to illustrate a general move towards Voice over IP (VoIP) Communications as bringing together voice and data environments.\",\n \"However, there remains a need to record interactions over data networks irrespective of whether these are voice communication sessions, video or data interaction between people and systems, systems and systems or people and people.\",\n \"While it is noted that systems produced by, e.g.\",\n \"Hewlett Packard, have long been able to monitor data networks and store details and contents of the packets they observe, the primary function of such devices has normally been for network performance analysis and diagnostics.\",\n \"Disadvantageously they do not provide for the management of large volumes of recordings, and related archival and indexing.\",\n \"Thus, such systems, and related methods, do exhibit limitations and disadvantages.\",\n \"As a further illustration U.S. Pat.\",\n \"No.\",\n \"6,122,665 merely serves to address the recording of a single network segment.\",\n \"However, with the prevalence of Ethernet switches and routers, it is rare for a system of any size to be implemented as a single network segment.\",\n \"Whenever multiple segments are introduced, the packets are routed between these segments as required in order to reach their destination.\",\n \"There is therefore no single point at which all packets can be observed.\",\n \"It would therefore prove advantageous to provide a system which can be extended to tap into the network at however many points are needed to achieve the required coverage.\",\n \"Unfortunately, merely extending a simple system designed for a single tap-point will result in highly redundant recordings as many of the packets to be recorded will arise at more than one tap point.\",\n \"Further, known systems require knowledge of network addresses to operate properly.\",\n \"To determine what, and indeed how to record, some knowledge of the identity of specific IP addresses is often required.\",\n \"For example, knowing which IP address hosts an IP-PBX will allow better filtering of information and the analysis of call control packets involving that node.\",\n \"Unfortunately, many IP addresses are dynamically assigned, e.g.\",\n \"via DHCP.\",\n \"Also network configurations are regularly changed and updated as networks expand and evolve.\",\n \"Neither do known systems address the use of proxy addresses.\",\n \"In many IP networks, packets are readdressed en route by Proxy Servers.\",\n \"This makes it difficult for the controlling software to identify which packets should be recorded as a single IP address is often only appropriate for packets transmitted over the leg of the journey from which they emanate, or which they finally reach having regard to the network node having that IP address.\",\n \"The Known systems also do not support the recording of packet data streams along with voice/video.\",\n \"The system disclosed in U.S. Pat.\",\n \"No.\",\n \"6,122,665 filters packets out that are not part of a voice or video stream.\",\n \"This prohibits its use in cases where the recording of a combination of data streams is required.\",\n \"With regard to packet filtering, known systems are far from optimal.\",\n \"It is important that a recording system is able to process all the packets it receives and eliminate those that are not required as efficiently as possible.\",\n \"Often only a small proportion of received traffic is to be recorded.\",\n \"The described system performs its filtering initially by determining the type of traffic contained within the packet, for example voice or video versus other.\",\n \"In a large voice/video system with many concurrent sessions occurring, there will be many thousands of packets per second being received at the network tap point.\",\n \"In many cases only a small proportion of these will be required.\",\n \"Known systems will not filter out any of these at the first level and will pass all packets on to the following stage.\",\n \"Such manner of operation proves to be sub-optimal.\",\n \"Disadvantageously, the integration of such a system with the systems controlling call placement is not generally achievable.\",\n \"Also, the determination of which IP ports to record on the basis of H323 signalling information also received via the network tap point is often superfluous.\",\n \"Rather, better information regarding the contacts and the requirement to record them can be obtained by interfacing to real-time event outputs, i.e.\",\n \"CTI ports, of the systems that are controlling the placement of calls.\",\n \"These ACDs or PABXs will often include additional information regarding the calls that cannot be deduced by observing the session control messages.\",\n \"For example, the linkage of one call to the next as calls are transferred between people is not always available from the session control protocol.\",\n \"Also, tolerance issues such as the loss of critical packets is not addressed in known systems.\",\n \"However, in situations where session control information can be gleaned from analysis of session control packets, it can be disadvantageously adversely affected by the loss of a single packet.\",\n \"This can readily lead to a call not being recorded.\",\n \"In many systems, there can prove to be multiple ways to deduce such information, thereby making the system tolerant to the loss of certain packets.\",\n \"As with filtering, known systems prove to be sub-optimal with regard to the storage of received packet data.\",\n \"The described system does not disclose in detail how the packets are stored and it appears that these are simply kept as raw, independent packets such that a significant potential for storage overhead has been found to arise.\",\n \"Importantly, the known systems do not integrate well with traditional (non VoIP) storage and replay mechanisms.\",\n \"Again, the described system appears to store the recorded packets as packets and replays these by retransmitting them to a device similar to that which would have received them originally.\",\n \"In many systems, however, only part of the voice system is packet-based and certainly the replay mechanisms are often not packet based.\",\n \"Hence there is a need to store, retrieve and replay conversations in a way that is both efficient and compatible with existing circuit-switched voice systems.\",\n \"Still further, such known systems do not address the specific requirements of testing and monitoring, nor do they attempt performance measure.\",\n \"In any packet system, there is the chance of the loss of a particular packet and the system described makes no reference to a determination of how well it is performing and whether any packets not recorded were lost by the network or at the recording device's network interface.\",\n \"It is now appreciated that with simple extensions, such recording systems can be used to record data steams in a form suitable for use in simulated loading tests.\",\n \"The capability for advanced recording options such as stereo and multi-party (conference call) recording are also disadvantageously absent from known systems.\",\n \"In particular, the system described does not address the added value that can be obtained by recording conversations so that they may be replayed in “stereo” with multiple tracks allowing the replayer to separate the audio travelling in each direction.\",\n \"Nor does it describe the extension of this technique into the recording of conference calls where the separation and identification of individual speakers is of great benefit in aiding the listener to follow the flow of conversation.\",\n \"The present invention seeks to provide for a data recording system and method and exhibiting an advantage over such known systems.\",\n \"According to one aspect of the present invention, there is provided a data recording system including data recording means having a plurality of network interface cards.\",\n \"Of course, alternatively, where more than one segment must be tapped in order to receive all required packets, this can be done either by providing multiple systems, each tapping a single segment but this can result in the same recordings being made by multiple recorders.\",\n \"A preferred embodiment is to use multiple network interface cards (NICs) within a single recorder.\",\n \"Advantageously, each NIC may have multiple network connection ports, such as typically are used to provide fault tolerant connection to multiple Ethernet switches.\",\n \"In this case, the filtering mechanism is enhanced to allow rapid elimination of duplicate packets.\",\n \"For each RTP packet stream being recorded, the system can advantageously note the sequence number of the most recently accepted packet.\",\n \"Any packet received with a lower sequence number (account being taken of sequence numbers wrapping as they overflow their assigned binary range) will immediately be discarded.\",\n \"In this way, the system advantageously need not actively keep track of network topology and will operate regardless of how many copies of each packet it receives.\",\n \"In larger or physically separated systems which require multiple recorders, it can prove impractical to bring all packets into a single recorder.\",\n \"In such cases, the individual recorders can be arranged to communicate with each other so as to advise each other of the presence of particular packet streams.\",\n \"In a prepared example, it is desired to record packets travelling between a specific pair of addresses: a) each recorder is advised of the addresses to be monitored; b) when a recorder identifies a packet with the required addresses it begins recording it and advises all other recorders to stand down; and c) other recorders are arranged to note these addresses as being recorded elsewhere.\",\n \"Further advantageous refinements to such a scheme can seek to ensure that only one recorder records each call or session in that: d) the notification sent to other recorders call include within it the sequence number (or timestamp) of the RTP packet that was first received and the IP address (or other identifier) of the receiving recorder; e) in the event that more than one recorder receives packets prior to hearing notification from another recorder that it has commenced recording, the recorder which received the earliest packet can maintain responsibility for recording and the other recorder shall cease recording; f) should more than one recorder receive the same packet and send notification of this, then the recorder with highest IP address (or other algorithm allowing unique determination of priority) can maintain its recording and the other(s) will stand down; g) when recording begins, a recorder will not create a record in the database of recordings until a pre-defined time has elapsed in case it receives, during this period, a notification from another recorder that it too has started recording.\",\n \"Should it receive such notification and, according to the algorithms defined in (d) to (f) above, it is to stand down, it will abandon its recording without having altered the database of recordings.\",\n \"The following further refinements can be employed to ensure that packet loss is minimized: h) should a packet be lost (as can be determined from a gap in the sequence number of received packets) an indication of the sequence number(s) of the lost packets can be maintained as part of the recording control structure for that IP address; and i) when packets are received that are determined to be earlier than the most recently accepted packet for that address, their sequence number can subsequently be compared against the list of recently missed packets and, if found, can be stored at the appropriate offset within the recording buffer overwriting the padding that was inserted when the later packet was received and loss of packet(s) identified.\",\n \"It should be appreciated that the invention also provides for a data recording method employing a plurality of network interface cards and as described above.\",\n \"According to another aspect of the present invention, there is provided a data packet recording system arranged for determining an IP address of an application and including means for comparing at least a sample of received or transmitted packets with at least a set of pre-programmed signature packets.\",\n \"The system can then advantageously determine the IP addresses of significant applications by comparing all packets received from or sent to each destination against a set of pre-programmed signature packets.\",\n \"These packets are preferably chosen to be examples of the traffic expected to involve the node being sought.\",\n \"For example, in many IP-PBX systems, each IP-phone regularly heartbeats by sending a simple packet to the IP-PBX control application every minute or so.\",\n \"By monitoring for these packets it is possible to determine the location of the IP-PBXes in the system being monitored.\",\n \"Preferably, the sustained absence of such packets can also be used as an indicator of an error condition with the device that was previously active on that address and can trigger an alarm condition and/or fallback mode of operation.\",\n \"It should be appreciated that the invention also provides for a data packet recording method including a comparison step as described above.\",\n \"According to yet another aspect of the present invention, a data recording system is provided wherein data packets are arranged to be transmitted via a Proxy server and further arranged such that the IP address of at least one party to the conversation is altered.\",\n \"This advantageously allows for security and re-use of address space although difficulties can be experienced in determining which packets should be recorded.\",\n \"The invention can preferably address this by being arranged to: a) determine the presence and IP address of such Proxy servers either by explicit configuration information or, preferably, by analysing the pattern of packets entering and leaving the IP node.\",\n \"This can be achieved for example by comparing the contents of packets transmitted by each node with the contents of those received by that node.\",\n \"Should it find packets that are identical bar the IP address, it can both deduce that this node is performing a Proxy function and determine the current mapping of IP addresses that it is performing; b) allow for recorders passing their knowledge of current mappings to other recorders allowing them to refine their filtering algorithms; and c) pass examples of specific packets identified as having been mapped by the Proxy server to other recorders.\",\n \"This advantageously allows recorders to differentiate between overlapping address ranges.\",\n \"For example, two different Proxy servers may both be mapping addresses in a given range.\",\n \"By passing occasional packets to each other, along with their pre-mapping addresses, recorders can determine whether the stream of data they are observing is indeed that for this address or a completely different stream mapped by a different Proxy server.\",\n \"As before, the invention also provides for a corresponding method including IP address alteration.\",\n \"According to still another aspect of the present invention, there is provided a data packet recording system including packet filtering means arranged to perform on the basis of an IP address and preferably, an IP port number of both source and/or destination address(es).\",\n \"Having determined that the packet is one that is to be recorded, the system can preferably then store the packet in the most appropriate way.\",\n \"For example, for RTP streaming data: a) The system is arranged to analyse the sequence number and timestamp to determine whether this is the next packet in the expected sequence; b) if one or more packets have been missed then performance counters can be incremented appropriately; c) additional data can preferably be inserted in the recorded stream to pad out the space that should have been taken up by the missing packet(s).\",\n \"This advantageously avoids gradual drift between the recording and real-time which would otherwise build up and result in the call being shorter when replayed than it had been originally; and d) the payload of the RTP packet can be appended to a buffer and the remainder of the packet discarded; and e) optionally, information can be flagged from the packet header so as to be retained where this is not the default header, i.e.\",\n \"it cannot be fully deduced from knowledge of the previous header.\",\n \"As an example for other packet oriented data: a) optionally, a signature data pattern (e.g.\",\n \"well known particular 4 byte value) can be appended to the data buffer collecting packets to/from the specific destination.\",\n \"This advantageously ensures that any application reading the recording subsequently can re-synch with these inter-packet boundaries should the recording become corrupted or the start of the recording cannot be accessed; b) a timestamp indicating the time at which the packet was received is appended to said buffer.\",\n \"This will typically be precise to at least millisecond and can often achieve micro-nanosecond precision; c) a length indicator specifying the number of bytes in the recorded packet is appended to said buffer; and d) finally, the contents of the packet can be appended to said buffer Again, the invention also provides for a related method.\",\n \"In an alternative data filtering arrangement and method filtering according solely to address data, or at least before looking at packet content type can be provided.\",\n \"In some cases a given IP port might only be transmitting one type of data and so filtering on its address alone can advantageously prove sufficient in whether or not to record the packet.\",\n \"A preferred arrangement can involve looking up the address of the IP destination of each packet, or equally source data, as the primary filter.\",\n \"A “map” of known addresses is maintained and each entry serves to identify whether data going to (or from) that address should be recorded.\",\n \"Advantageously, by utilizing the IP address as the primary key into this map, extremely fast look-up can be achieved.\",\n \"Further preferred refinements to this scheme include: a) the pre-filtering of packets on IP address so as to eliminate packets sent to or from the recorder itself.\",\n \"This allows the same network interface card to be used for normal IP communications (e.g.\",\n \"with clients searching and replaying previously recorded calls) whilst it is also being used a promiscuous mode tap.\",\n \"This simple filtering out on a single (32-bit) integer IP address avoids the need to look up the address map repeatedly for all packets that are directed at other sockets on this node.\",\n \"With the introduction of IP Version 6, this will become a 128-bit integer comparison; b) in preference to hashing or sorted table indexing, the map of required addresses can be implemented as a memory array.\",\n \"By using the fact that most IP addresses required will lie within a narrow range of possible values it is faster to compare the high order bytes of the IP address with the sub net or nets on which all the target nodes are located.\",\n \"The remainder of the address can then be used as an index into a (typically sparsely populated) memory map in which each address to be recorded is represented by a binary 1 and the remainder as 0.For a Class B address as described here, only 64K bits of information are required to achieve this though the use of 65,536×32 bit words allows for each entry in the map to be either null (0x00000000) or a pointer to the structure describing the known address and its recording details e.g.\",\n \"buffer storage location.\",\n \"It should be noted that a similar scheme can be used in IP Version 6 albeit with a much smaller portion of the overall numbering range being covered by such a table; c) in RTP based systems, many of the data streams being transmitted are constant bit rate.\",\n \"In these cases, many sessions will be transmitting packets at a regular rate resulting in a strong correlation between the order in which packets are received.\",\n \"This is especially true in cases where all transmitters are of the same type e.g.\",\n \"an IP-PBX with many identical IP-phones, all configured with the same packet interval.\",\n \"This can be used to advantage in filtering the incoming packets.\",\n \"If a record is maintained of the recently received packet addresses, the following optimized search algorithm can be deployed I. note the IP destination address of the first RTP packet received at the head of a list of such addresses.\",\n \"Look up the known destination for this address and include a pointer to it at the head of a list of such pointers, II.\",\n \"as subsequent packets are received, compare their address against the packet at the head of the list, III.\",\n \"if they are different, append their addresses to the list; look up their known destination descriptor and append it to the list of such pointers, IV.\",\n \"if it matches the address at the head of the list, use the pointer to the known destination at the head of such list.\",\n \"Note that the next packet should be compared to the following entry in the list, V. subsequent packets should be compared to the next entry in the list.\",\n \"If they match, repeat step IV, if not, insert them into the list at this point.\",\n \"d) The algorithm of c above can preferably be further refined to accommodate the cessation of transmission from any address as happens when a call terminates.\",\n \"By comparing the new packets address against the following item in the list, we can determine that the previous address has probably ceased transmitting and it can be removed from the list.\",\n \"e) To allow for the case where two streams cease in one cycle of the list, it is necessary to look two addresses ahead if the new packet s address is not found at the head or subsequent entries in the list.\",\n \"This is a common case as both directions of the call will typically terminate at or about the same time.\",\n \"However, given a typical interval between transmission of 50 ms it is rare that more than one call terminates in any given cycle of the list.\",\n \"As will be appreciated, traditional telephone recording systems are often controlled by or determine call details by interacting with the telephony switch via a Computer Telephony Integration (CTI) interface.\",\n \"Such an interface typically advises the recording system of call setup and teardown as well as associated details such as dialled digits, calling line identifier (CLI) etc.\",\n \"As such systems move to packet transmission of voice, many are doing so incrementally and hence supporting a mix of packet and traditional calls.\",\n \"Where such an interface exists, it is beneficial to retain the existing investment in its design and also to allow support of VoIP and traditional calls with a single interface rather than having to support a further interface.\",\n \"The present system and related method advantageously supports the use of existing interfaces, provided that where such interface previously provided call identification in the form of telecoms circuit and timeslot information allowing the matching of recorded data stream with call details, it is enhanced to provide equivalent information in the form of IP address and, preferably IP port number.\",\n \"Ideally the addresses of both parties to the conversation (be they IP phone and/or gateway) are provided.\",\n \"However, in some cases, the telephony system is only aware of the IP address of one or other party involved in the call.\",\n \"It is a preferred feature of the present invention that the instruction to record a specified address (source or destination) can optionally be extended with the instruction to automatically start recording of the counterpart with which that address starts to exchange data.\",\n \"In the case of an IP destination being recorded, this is achieved by noting the source of the packet that is recorded and creating a corresponding known destination record for that address.\",\n \"In this way, subsequent packets sent to the newly identified address will also be recorded.\",\n \"Obviously the converse applies should packet source be specified originally.\",\n \"Further, where call control packets are analysed to determine which addresses are to be recorded, there is a danger that the failure to receive such a packet could result in the subsequent failure to record the entire conversation e.g.\",\n \"if the packet lost contained the extension number of the IP phone and hence it was not recognized that a call to a recorded extension had started.\",\n \"Fortunately, many such protocols are redundant and the required information can be deduced from several of the packets.\",\n \"According to a further aspect of the present invention, the system can specifically address the case where IP telephones are controlled by an IP-PBX application.\",\n \"In such cases it is typically possible to deduce the IP port address to be used for transmission in multiple ways.\",\n \"In one case (Cisco CallManager) the controlling application requests that each phone advise it of the port number it can receive incoming audio data.\",\n \"It then advises the other phone in the call of that number.\",\n \"Hence the recorder can determine the port number to record should it receive either of these interactions.\",\n \"Should it receive both, the redundant information can easily be recognized as such and ignored.\",\n \"Yet further, when recording RTP streams, the header of these packets can be almost entirely derived from that of the previous packet and hence contains little of value.\",\n \"By advantageously arranging for the identification and storage of only the payloads of successive RTP messages, the storage requirements for recording a session are dramatically reduced.\",\n \"This is particularly so in the case of compressed audio transmission where the audio payload may be as little as twenty bytes in a packet of over one hundred bytes in total.\",\n \"An embodiment of the present invention also addresses the issue of packet loss by being arranged to compare the sequence number of the received packet with that of the previously received packet.\",\n \"Should these differ by more than one, packet loss can be deduced.\",\n \"In addition to noting the loss in system performance counters, the missing packets can be “padded” out in the buffer being used to receive the recording.\",\n \"Aspects of the present invention can also address the use of silence suppression which results in breaks in the transmission stream.\",\n \"In such cases, the timestamps of successive packets indicate the extent of the suppressed silence whilst the successive sequence numbers let us differentiate between deliberate silence suppression and the accidental loss of packets as described above.\",\n \"When packets are suppressed in this way, the present invention provides for either: the insertion of the appropriate amount of “padding” data which may be pure silence or “noise” derived according to a compression scheme; or the insertion of explicit indication of the silent period into the recorded stream.\",\n \"Such as scheme is described by Microsoft in the definition of the “.WAV” file format.\",\n \"This provides more efficient storage of such silence periods but unfortunately, most “.WAV” file players do not support this mechanism.\",\n \"The present invention can advantageously further optimize storage by supporting a range of compression formats and acting on the received data according to user defined rules.\",\n \"These can include: compressing data received in a specified format (e.g.\",\n \"G.711 mu-law) to a specified second format (e.g.\",\n \"G.726 16 kbps); leaving data received in a compressed format in that format (to avoid the inherent quality reduction associated with decompression and further compression; optionally mixing the two halves of a communication session into a single, mono, recording (preferably) prior to compression; or optionally saving to non-volatile storage (e.g.\",\n \"disk) a summary representation of the audio levels on each of the two channels prior to compression and/or mixing.\",\n \"This Energy Envelope representation allows a compact (20 samples per second, 2 byte values) representation that permits the graphical display of the call showing which party was speaking at what level even though the two halves of the call are subsequently mixed and only available as a mono rather than stereo signal.\",\n \"Within aspects of the present invention, by storing the packet payload in a standard .WAV file, the recorded audio can be replayed alongside traditional recordings using any existing replay mechanism.\",\n \"Also, given the ability of the system to record packet data with timestamps for each packet received, the system may be run in a special mode in which all packets recorded are processed and stored as described in above for the aforesaid other packet oriented data.\",\n \"By storing all packets, including RTP streams, in this way, the recordings made can subsequently be used advantageously as known test cases for exercising and testing the invention.\",\n \"By replaying recordings made in this way over the network, recorders under test will receive exactly the same packet stream as the recorder that made the original recording did.\",\n \"Hence known test cases can be run automatically and the output of the recorders compared with that of previous test runs.\",\n \"It is a further feature of the present invention that said recording files can be arranged to be processed offline, so as to multiply the traffic and hence simulate larger systems than were actually recorded.\",\n \"By replicating the packets in the recording file and, for example, incrementing the IP port numbers for each replication, large volumes of traffic can be simulated from a single recording.\",\n \"In another aspect of the invention, it is important that such recorders do not lose data packets as this will reflect in loss of audio and/or loss of control information and hence possibly missed calls.\",\n \"The present invention therefore can be arranged to monitor its performance in the following way: 1.the gaps in the received packet stream can be determined as described above; 2.by analysing the RTCP packets received alongside the RTP packet streams it is possible to see the loss statistics being experienced by the actual participants in the call; 3.by comparing the above two figures an estimate of the packet losses occurring somewhere other than en route between the end points can be determined.\",\n \"Some of this will be due to the recorder and can be used as an indication of the quality of the recording; In yet another aspect and as discussed above, recordings may advantageously be stored independently for each direction of transmission or combined into a single, mixed recording.\",\n \"The former is highly advantageous should analysis of who was talking be required e.g.\",\n \"to identify angry callers from high interruption levels or in performing speech recognition on the files.\",\n \"The latter however is both less accurate and less useful when performed on a mixed recording since a single speaker model cannot be used and it is not clear who said each word.\",\n \"On conference calls it is particularly beneficial to retain a recording of each party involved in the call since with many speakers, it can be difficult to differentiate between speakers with similar voices, and there is often background noise from one or more parties that makes it difficult to hear the principal speaker It is a preferred feature of the present invention that the recordings of a multiparty interaction can be kept as independent streams or mixed together to optimize storage space.\",\n \"As mentioned above, it is possible to retain a summary record of the level of audio from each speaker even though the actual audio signal has been mixed.\",\n \"It is a further preferred feature of the present invention that when such conferences are handled by a conference bridge, which mixes the signals, storage of the conversations can be limited to the minimum set of data streams needed to reconstruct the conference audio.\",\n \"This advantageous aspect is achieved as follows for a conference between parties A, B and C using conference bridge X A, B and C each transmit audio to X X transmits audio from (B+C) to A X transmits audio from (A+C) to B X transmits audio from (A+B) to C The recorder may simply be instructed not to record packet streams coming from the conference bridge X and hence only records the pure audio from each of the contributing parties.\",\n \"The required mixed signals can be reconstructed at replay time if required from these three streams.\",\n \"Of course, it should be appreciated that the invention can comprise any one or more of the aspects and embodiments described above in combination or alone.\",\n \"The invention is described further hereinafter, by way of example only, with reference to the accompanying drawings in which: FIG.\",\n \"1 is a block diagram of a recording sub-system embodying the present invention; FIG.\",\n \"2 is a schematic block diagram showing the operation of an IP address filter embodying an aspect of the present invention; FIG.\",\n \"3 is a schematic block diagram showing the operation of a protocol filter embodying an aspect of the present invention; FIG.\",\n \"4 is an illustration of packet data buffering as arising in an embodiment of the present invention; FIG.\",\n \"5 is an illustration of RTP/PTCP Packet Buffering as arising in an embodiment of the present invention; and FIGS.\",\n \"6A-6C illustrate the structure of IP packets, RTP packets and RTCP packets as arising within the present invention.\",\n \"Turning now to FIG.\",\n \"1, there is illustrated a recording sub-system embodying the present invention and in which a recorder is connected to a network from which calls are to be recorded by connecting into allocated ports on one or network routers, switches or hubs such as the 10/100 Mbps Ethernet hubs [1], [2], [3].\",\n \"In the case of intelligent switches, which only transmit data packets to those ports that need to receive the data, a “SPAN” setting is normally applied to the port used for the recorder.\",\n \"This setting is normally used for network diagnostics and forces the switch to copy packets destined for one or more of its other ports to this port as well.\",\n \"Each router/switch/hub is connected to an appropriate form of network interface card (NIC), for example 10/100 Mbps Ethernet, Token Ring, Gigabit Ethernet [4], [5].\",\n \"The NIC [5] is an example of a multiport NIC which allows connection to more than one network access point.\",\n \"This allows efficient recording of larger networks where calls are spread across several network concentrator devices, without the need for a separate recorder for each of these.\",\n \"Within the operating system on the Recorder, such as Windows 2000 there will be installed one or more drivers that allow applications to communicate with the NIC(s) installed.\",\n \"A common NDIS driver (6) is illustrated.\",\n \"This typically allows other applications running on the same computer to share access to the NICs without having to be aware of each other.\",\n \"Hence this diagram only shows the recording sub-system.\",\n \"Note the archival, search and retrieval functions typical of a bulk voice recorder can still be hosted on the same PC providing of course that there are sufficient memory, processor and disk resources for these to co-exist.\",\n \"The system described communicates with the NDIS driver [6] and establishes a data channel which it places in promiscuous mode.\",\n \"This overrides the normal behaviour in which only data packets destined for this application will be delivered and instead requests that all data packets received via the network access point are passed through to an IP Address Filter mechanism [7].\",\n \"The IP Address Filter mechanism [7] is described in more detail below with reference to FIG.\",\n \"2.In summary, it compares the IP address and port number of each received IP packet against a list of known destinations.\",\n \"This could be equally well be applied to known sources rather than destinations.\",\n \"A rapid decision is made as to whether this packet is to be recorded or not.\",\n \"If the answer is yes, it will pass the packet on to a Protocol Filter [8].\",\n \"This second stage filter [8] can therefore now use both knowledge of the destination to which the packet was sent and information from the IP packet itself to determine whether the packet contains RTP or RTCP data or other packet data.\",\n \"The filter [8] passes the packet on to either a Packet Data Buffering module [9] or a RTP Packet Buffering module [10].\",\n \"Each of these two buffering modules [9], [10] applies schemes appropriate to the data type being recorded before passing on data, in relatively large, contiguous blocks, to the Record Thread Manager [14a] which, running at slightly lower priority than the previous components will write this data to the appropriate files on the hard disk.\",\n \"This results in efficient large data writes to the permanent storage such as a hard disk [11].\",\n \"The end result is typically a pair of files one containing the actual content of the data stream, for example a .WAV audio file for RTP transmitted telephony, and the other containing details of the recording such as a .XML file containing start time, duration, IP address etc.\",\n \"Alternatively the details of the recordings may be inserted directly into a database.\",\n \"In buffering the RTP data streams, the RTP Packet Buffering module analyses the sequence numbers and timestamps of the received packets.\",\n \"It compares the packet loss rates it is experiencing against those reported by the participants in the streaming interaction and can hence deduce performance levels which it logs for example to a file [14].\",\n \"It could equally well pass this information into a network management tool such as via Simple Network Management Protocol (SNMP).\",\n \"The set of known destinations that are to be recorded can be fixed in a variety of ways.\",\n \"For example, it could be achieved by direct, local configuration of the IP Address Filter [7], through blanket rules such as recording all streams; or it can be instructed explicitly by a component such as Unify [12] a CTI middleware platform which interfaces with a wide range of telephony and other customer contact service systems [13].\",\n \"By observing activity on the external system and applying business rules, the addresses to be recorded can be readily identified.\",\n \"With reference to FIG.\",\n \"2, once a packet is received and determined to be an IP packet it is examined by the IP Address Filter to determine whether or not it is to be recorded.\",\n \"It should be appreciated that, according to the network traffic patterns experienced and the proportion to be recorded, the order and/or presence of each of these filtering stages may be changed to deliver optimum performance typically measured by the packet rate that can be processed within a given proportion of the CPU time available.\",\n \"The IP packet [15] is normally first compared [17] with the IP address of the computer on which the recorder is hosted.\",\n \"This allows very rapid elimination of all packets that are intended for other processes on this computer and ensures that the hosting of, for example, a search and replay application on the same computer does not adversely impact the performance of the recording sub-system.\",\n \"The packets which were addressed to other IP addresses are then passed to a circular buffer lookup algorithm [21].\",\n \"Recently received IP destination addresses including IP port number, are noted in a circular buffer [18].\",\n \"When a packet is received, its IP address is compared with that of the entry at a Test Point [19] in the circular buffer [18].\",\n \"If a match is identified, the Test Point [19] is advanced and the entry in the circular buffer is used as a pointer to the Known Destination Object [16] for this IP address.\",\n \"An entry in the Known Destination object's [16] data structure indicates whether or not the packet is to be recorded [25].\",\n \"If it is, then it is passed through to the Protocol Filter (next section).\",\n \"If the packet's IP address does not match the next item in the Circular Buffer [18], it is compared against one or more subsequent entries and if a match is found, the test point [19] is advanced to this point.\",\n \"The entries that were tested and found not to match will be removed from the buffer as it can be deduced that they are no longer in the correct position or the stream to those addresses has terminated.\",\n \"If the packet's IP address does not match any of those within the lookahead window described above, it is passed to the following stage of the filter [22] in which the IP address is used to determine if there is an existing Known Destination record [16] for this IP address.\",\n \"The Known Destination list is typically held in memory in the form of a map with a fast indexing or hash function that allows rapid searching of many such records for the one with a specific address.\",\n \"However, the nature of IP addresses (e.g.\",\n \"10.25.34.245) is deliberately hierarchical with the earlier numbers (when represented in text string form as here) representing the larger sub-networks.\",\n \"It is therefore very common for the vast majority of traffic observed on a single network segment to involve IP addresses from that sub-net i.e.\",\n \"the first 2 or even 3 of the numbers will be common.\",\n \"In such cases it is viable to maintain one or more look-up tables of Known Destination Maps [26] which simulate content addressable memory For example, such a table, with 65,536 entries can hold pointers to all the Known Destination records [16] relating to the block of IP addresses 10.10.xxx.yyy where xxx and yyy are any number from 0 to 255.It is therefore very easy and quick to compare the top (leftmost when written as text) two bytes of an IP address against the base address of such a lookup table and, if they match, use the lower (rightmost when written as text) bytes as an index into the table.\",\n \"A 0 (null) entry in the table infers that the destination does not yet have a Known Destination record associated with it whilst a non-zero entry can contain a pointer to the relevant Known Destination object.\",\n \"If the Destination is not within the range of any such rapid lookup maps [26], the normal hash table or other indexing method will be used to determine if a Known Destination Record exists.\",\n \"If it does, it will be inserted into the Circular Buffer [18] at the insertion point normally immediately prior to the test point [19].\",\n \"If an existing Known Destination record is not found, then a new record will be created and the Record flag will be set to true or false according to the default recording rules [23].\",\n \"Having found, or created, the appropriate Known Destination Record [16], the packet will at [25] either be discarded or passed to the Protocol Filter according to the Record flag within the Known Destination Record.\",\n \"It should be noted that the Known Destination Records can also be created, deleted and their recording flag set/reset by the Unify interface [12].\",\n \"Also in order, to avoid long term build-up of redundant Known Destination Records [16], a background process can serve to periodically review the last time that a packet was observed being sent to that destination and will destroy the Known Destination Record should this exceed a pre-determined threshold.\",\n \"To enable this function, a data field within the Known Destination Record structure is updated to reflect the current system time whenever it is used to determine whether or not to record a packet.\",\n \"As illustrated in FIG.\",\n \"3, the Protocol filter stage can be embodied very simply.\",\n \"It first examines the contents of the Known Destination Record [16] that is by now associated with the packet being processed.\",\n \"This record may contain information that allows the protocol filter to determine which protocol the packet should contain.\",\n \"This is particularly advantageous in the case of RTP data streams as there is no guaranteed method for determining that a packet is indeed an RTP packet.\",\n \"Such packets are identified as UDP protocol but there is no indication within the UDP packet that the payload is indeed RTP.\",\n \"Unless an external influence such as Unify [12] has deduced that RTP is to be expected on a given destination IP address, the only alternative is to determine if the first few bytes of the UDP packet's payload represent valid entries for an RTP packet header.\",\n \"This is time consuming and prone to occasional false detection.\",\n \"Hence, if the protocol being sent to a specific port is known already, this can be used to direct the packet at either the Packet Data Buffer [30] or the RTP/RTCP Buffer [31].\",\n \"If the Known Destination Record [16] does not specify the protocol expected, the filter next examines the protocol type specified within the IP packet.\",\n \"A typical filter will simply check for TCP [28] and pass these packets to the Packet Data Buffer [30] then check for UDP packets [29] and pass these to the RTP/RTCP Packet Buffer [31].\",\n \"According to pre-configured default behaviour [32], other protocol types may be either discarded [33] or sent to one or other of the two described buffering modules or to a new buffering module specifically optimized for storage of that protocol type such as ARP, ICMP or SNMP.\",\n \"Turning now to FIG.\",\n \"4 there is illustrated the packet data buffering of the present invention.\",\n \"This can prove to be relatively straightforward with each packet simply being preceded by any or all of: A well-known synchronization value which will ensure eventual re-synchronization of a partially corrupt file; A precise time stamp; Offset of local time from Universal Co-ordinated Time (UTC); Daylight Savings Time offset; or Length of packet that follows.\",\n \"If this is the first packet being recorded for a given Known Destination [16] then the Known Destination record will not yet have had a buffer storage element [37] assigned.\",\n \"A start recording job is posted onto the queue [35] for the Record Thread Manager and a buffer [37] is assigned.\",\n \"These buffers are typically several Kbytes in length and can hence hold many individual packets.\",\n \"The packet to be stored and its associated data as listed above is appended to the buffer.\",\n \"When subsequent packets are received, these are appended to the buffer [37].\",\n \"At the point where the buffer fills, it is moved to the FIFO queue [35] to await processing by the Record Thread Manager [39].\",\n \"When the job reaches the head of the queue, it will be appended to the appropriate file on disk [34].\",\n \"Meanwhile, a new buffer [37] will be allocated to the Known Destination Record [16] and any remnants of the packet which filled the previous buffer are appended to it.\",\n \"This process repeats until the recording is terminated at which point the partially filled buffer [37], if any, is appended to the job queue [35] and a Stop Recording job is then added to the job queue.\",\n \"When the Record Thread Manager reaches a Stop Recording job, it closes the file in question.\",\n \"Additional details about the call may be included within the Start and Stop Recording messages and these, along with other derived information, such as the total data volume stored are ultimately written to a call detail file, for example in .XML format or direct to a database.\",\n \"It should be appreciated that a lower priority thread high priority still being lower than Real-time priority—is used for writing data to disk than for capturing, filtering and buffering packets into memory.\",\n \"This allows RAM buffering to absorb short term peak load conditions which would otherwise cause bottlenecks and limit the peak throughput capacity of the recorder.\",\n \"FIG.\",\n \"5 illustrates the RTP/RTCP Packet Buffering.\",\n \"This component can operate in the same way as the Packet Data Buffering component described above except for the way in which it handles the content of the data packet itself.\",\n \"Since RTP packets typically form a continuous stream of data, there is no need to store the RTP packet header information for all packets as it is only the payload of actual audio/video data that is required.\",\n \"When audio is compressed for transmission, the payload can be as little as 20 bytes (G.729A, 20 ms per packet) yet the total packet length, including Ethernet, IP, UDP and RTP headers, will typically exceed 100 bytes.\",\n \"Hence storing the whole packet is incredibly inefficient.\",\n \"In the simplest case, the payload of each RTP packet is appended to the buffer [37] and, when full, this is placed on the Record Job Queue [35].\",\n \"Recording start and stop are exactly as per the Packet Data Buffering component and these two components share a common queue ensuring that data is written to disk in a FIFO manner regardless of which type of recording i.e.\",\n \"packet or stream, is being made.\",\n \"In the case of the RTP data, the files that are written by the Record Thread Manager are typically in Microsoft .WAV format and hence readable by any player device that supports this file format and the compression standard used within the file.\",\n \"The process is complicated somewhat by the need to allow for packet loss.\",\n \"This is achieved by maintaining in memory, details of the most recently received packet sequence number and timestamp.\",\n \"It is then possible to compare the currently available packet's sequence number and timestamp with that of the previous packet.\",\n \"A gap in sequence numbers can be noted and logged to a performance log file or similar [40A].\",\n \"To avoid gradual buildup of error in the recorded file, any lost packet(s) can be compensated for by the automatic appending of the appropriate number of bytes of silence (or other pre-defined padding sound).\",\n \"Where a small number of bytes are missing (e.g.\",\n \"1 or 2 packets) it is more efficient to simply add these to the buffer [37] as if they had been received.\",\n \"However, in the case of large gaps, it is more efficient to place the current buffer [37] on the Record Job Queue [35] for processing and then place a Padding job specifying the number of bytes of silence to be inserted.\",\n \"The Record Thread Manager can then append the appropriate number of bytes to the file without using excessive memory space on the Record Job Queue as would be the case if several buffers all full of silence were queued.\",\n \"This latter technique is particularly advantageous in systems that utilize silence suppression.\",\n \"In such cases, the RTP stream will stop until the audio level reaches a threshold.\",\n \"This can save significant bandwidth in voice communications as typically only one party is speaking at a time.\",\n \"In such cases, the packet sequence numbers in the RTP packet are contiguous but the timestamps are far apart.\",\n \"Again, this can be detected and the appropriate amount of silence indicated through the placement of a Padding job on the record job queue [35].\",\n \"This then allows the Record Thread Manager to either pad the call with the appropriate amount of silence or to utilize a scheme such as the Wave List feature within the Microsoft .WAV format.\",\n \"This allows for the efficient storage of such audio as a sequence of sound, silence, sound, silence etc.\",\n \"A further refinement includes the processing of RTCP packets associated with the RTP streams being recorded.\",\n \"By examining the contents of these packets, one can determine the fraction and cumulative number of packets lost by each of the participants in the conversation.\",\n \"These details can be included with the loss rates experienced at the recorder and included in the Stop Recording job and/or entries to the log file [40A].\",\n \"This allows subsequent analysis of the recorder's performance in comparison to that experienced by the participants on the call.\",\n \"To assist with the above description, reference is now made to the general structures of the packets arising in the aspects of the present invention.\",\n \"Turning now to FIG.\",\n \"6A, a header 42 is shown as a plurality of boxes, each of which represents a portion or “field” of the header.\",\n \"The number of bytes occupied by each portion is also shown, it being understood that each layer consists of 32 bits.\",\n \"The first portion of the header, a “VERS” portion 44, is the protocol version number.\",\n \"Next, an “H.LEN” portion 46 indicates the number of 32-bit quantities in the header.\",\n \"A “SERVICE TYPE” portion 48 indicates whether the sender prefers the datagram to travel over a route with minimal delay or a route with maximal through-put.\",\n \"A “TOTAL LENGTH” portion 50 indicates the total number of octets in both the header and the data.\",\n \"In the next layer, an “IDENTIFICATION portion 52 identifies the packet itself.\",\n \"A “FLAGS” portion 54 indicates whether the datagram is a fragment or a complete datagram.\",\n \"A “FRAGMENT OFFSET” portion 56 specifies the location of this fragment in the original datagram, if the datagram is fragmented.\",\n \"In the next layer, a “TIME TO LIVE” portion 58 contains a positive integer between 1 and 255, which is progressively decremented at each route travelled.\",\n \"When the value becomes 0, the packet will no longer be passed and is returned to the sender.\",\n \"A “TYPE” portion 60 indicates the type of data being passed.\",\n \"A “HEADER CHECKSUM” portion 62 enables the integrity of the packet to be checked by comparing the actual checksum to the value recorded in portion 62.The next layer of header 42 contains the source IP address 64, after which the following layer contains the destination IP address 66.An optional IP OPTIONS portion 68 is present, after which there is padding (if necessary) and a data portion 70 of the packet containing the data begins.\",\n \"As shown in FIG.\",\n \"6B, an RTP packet header 92 features several important fields: a timestamp field 94, a synchronization source (SSRC) identifiers field 96 and a contributing source (CSRC) identifiers field 98.SSRC field 96 is used to determine the source of the RTP packets (the sender), which has a unique identifying address (the SSRC identifier).\",\n \"The CSRC identifer in CSRC field 98 is used in a conference with multiple parties, and indicates the SSRC identifier of all parties.\",\n \"Timestamp field 94 is used by an RTP software module to determine the relative time at which the data in each packet should be displayed.\",\n \"Finally FIG.\",\n \"6C shows the general structure of an RTCP packet.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10467899"}}},{"rowIdx":390,"cells":{"text":{"kind":"list like","value":[["Method and system for multiple hosts anycast routing","Conventional anycast networks provide a service which allows a sender to access the nearest of a group of receivers sharing a common anycast address.","In contrast thereto, multicast networks establish data communications between a sender and all receivers confined by a group having the same multicast address.","Thus, the networks providing both anycast and multicast routing only allow for accesses by a sender to one receiver being specified according to the employed anycast protocol or to a plurality of receivers forming a multicast group.","In order to provide accesses by a sender to a specified number of nearest receivers, the present invention provides a method and a system for multiple hosts anycast routing in a network.","An indicator specifying the number of nearest receivers to be set up for data communications with a sender is included in an anycast address or associated thereto as an extension.","On the basis of the indicator, a corresponding number of anycast members of an anycast group identified by the anycast address are contacted/allocated for data communications with the sender."],["1.A method for multiple hosts anycast routing in a network, the method comprising: communicating sender data from a sender addressed to an anycast group including anycast members, said sender data including an anycast address for addressing said anycast group and a first indicator specifying a first number of said anycast members within said anycast group for data communications with said sender, and determining a number of nearest of said anycast members within said anycast group as hosts for data communications with said sender, said number of nearest anycast members being defined by said first indicator.","2.The method according to claim 1, comprising: communicating further sender data from said sender addressed to said anycast group including said anycast members, said further sender data further including a second indicator specifying a second number of said anycast members within said anycast group, said second number being indicative of already determined nearest of said anycast members within said anycast group, and determining said number of nearest of said anycast members within said anycast group, said number of nearest anycast members being defined by said first and said second indicator.","3.A method for multiple hosts anycast routing in an anycast capable network, the method comprising: associating a first indicator with an anycast address identifying an anycast group having anycast members in a network, said first indicator specifying a first number of anycast members within said anycast group, communicating at least said anycast address and said first indicator in said network to said anycast group, and determining a number of nearest of said anycast members within said anycast group as hosts for data communications in said network, said number of nearest anycast members being defined by said first indicator.","4.The method according to claim 3, comprising: associating a second indicator with said anycast address, said indicator specifying a second number of said anycast members within said anycast group, said second number being indicative of already determined nearest of said anycast members within said anycast group, communicating at least said anycast address, said first indicator and said second indicator in said network to said anycast group and determining said number of nearest of said anycast members within said anycast group, said number of nearest anycast members being defined by said first and said second indicator.","5.The method according to claim 2, wherein said number of nearest anycast members to be determined is defined by the difference between said first number and said second number.","6.The method according to claim 1, wherein said first and/or said second indicator is included in said anycast address.","7.The method according to claim 1, wherein said anycast address is extended by said first and/or said second indicator.","8.The method according to claim 1, wherein an anycast address prefix is included in said anycast address, said anycast address prefix identifying a topological region in said network confining said anycast group, said anycast address prefix including said first and/or said second indicator or being extended by said first and/or said second indicator.","9.The method according to claim 1, wherein said anycast address and said first and/or said second indicator are communicated by communicating an Internet Protocol data packet header including said anycast address.","10.The method according to claim 9, wherein said first and/or said second indicator is included in said Internet Protocol data packet header as a part or an extension of said anycast address, or said Internet Protocol data packet header is extended by said first and/or said second indicator by means of an options field for Internet Protocol data packet headers.","11.The method according to claim 1, wherein said number of nearest anycast members is determined by utilizing a measure of distance of a routing protocol for said network and/or determined by utilizing a measure of distance specified by said sender.","12.The method according to claim 1, wherein said anycast address and said first and/or said second indicator are communicated in said network between domains thereof via routers to network domains having at least one of said anycast members.","13.The method according to claim 12, wherein an ordering of said routers is defined such that said anycast address and said first and/or said second indicator are communicated to the nearest network domains, the nearest network domains being specified according to a measure of distance of a routing protocol for said network and/or according to a measure of distance specified by said sender.","14.The method according to claim 1, wherein said number of anycast members is contacted sequentially.","15.","(Cancel) 16.The method according to claim 1, wherein said first indicator is modified according to a number of already determined nearest anycast members such that said modified indicator specifies a number of said anycast members still to be determined as receivers.","17.The method according to claim 16, wherein said first indicator is decreased by the value of said second indicator.","18.The method according to claim 1, wherein said second indicator is decreased to a value corresponding to a number of 0 and said first indicator is decreased correspondingly to the decrease of the second indicator and said determining of said number of nearest of said anycast members within said anycast group is performed in dependence of said decreased first indicator if said second indicator has a value corresponding to a number of 0.19.The method according to claim 1, wherein said anycast address and said first and/or said second indicator are communicated to at least two network domains including at least one of said anycast members by multiplying said anycast address and said first indicator, and communicating said multiplied anycast address and said first and/or said second indicator to anycast members.","20.The method according to claim 1, wherein said first number specified by said first indicator is decreased corresponding to a number of said anycast members in a network domain to which said anycast address and said first indicator are communicated.","21.The method according to claim 1, wherein said communicating is performed according to the strict routing mechanism or the loose routing mechanism as defined in for the Internet Protocol.","22.The method according to claim 1, wherein said first and said second indicator are communicated together.","23.A system for multiple hosts anycast routing, comprising: a network a sender, and a group of receivers being identified by an anycast address, wherein data communications between said sender and a number of nearest of said receivers are established on the basis of a first indicator provided by said sender specifying said number of receivers.","24.The system according to claim 23, wherein data communications between said sender and said number of nearest of said receivers are further established on the basis of a second indicator provided by said sender specifying a number of receivers already determined for data communications with said sender.","25-28.","(Cancel) 29.The method according to claim 4, wherein said number of nearest anycast members to be determined is defined by the difference between said first number and said second number.","30.The method according to claim 3, wherein said first and/or said second indicator is included in said anycast address.","31.The method according to claim 3, wherein said anycast address is extended by said first and/or said second indicator.","32.The method according to claim 3, wherein an anycast address prefix is included in said anycast address, said anycast address prefix identifying a topological region in said network confining said anycast group, said anycast address prefix including said first and/or said second indicator or being extended by said first and/or said second indicator.","33.The method according to claim 3, wherein said number of nearest anycast members is determined by utilizing a measure of distance of a routing protocol for said network and/or determined by utilizing a measure of distance specified by said sender.","34.The method according to claim 3, wherein said first indicator is modified according to a number of already determined nearest anycast members such that said modified indicator specifies a number of said anycast members still to be determined as receivers.","35.The method according to claim 34, wherein said first indicator is decreased by the value of said second indicator."],["<SOH> BACKGROUND OF THE INVENTION <EOH>1.Technical Field The present invention relates to a method and a system for establishing data communications between a sender and receivers, the number thereof being defined by the sender, wherein the receivers are not individually addressed by the sender, but selected from a group of receivers being addressed from the sender by means of a common address.","2.Background of the Invention An anycast network is a network providing a service which allows a sender to access the nearest of a group of receivers having a common address.","The common address, in particular, the same anycast address for the group of receivers enables a sender to identify a plurality of receivers by providing only one address, namely an anycast address, instead of individually addressing each receiver.","In contrast to a multicast system, wherein data communications are performed between a sender and all receivers sharing a common address, i.e.","the same multicast address, data communications in an anycast network are performed between a sender and a particular receiver of a group of receivers sharing the same anycast address.","In the context, data communications include any kind of data, such as alphanumerical, graphic, multimedia, audio, video, voice data, information, signals etc.","which can be exchanged between systems, devices, network components etc.","(e.g.","computers, end user devices, hosts, servers, routers, modes).","In particular, data communications in an anycast network are performed between a sender and the nearest receiver of the group of receivers sharing the same anycast address.","In this respect, the nearest receiver in relation to a sender is defined according to a measure of a distance employed by a routing protocol or routing system used for data communications in a respective network.","Since the routing protocol or routing system of a network which identifies the nearest receiver for accesses by a sender, the sender does not need to care about how to select the closest destination, i.e.","the nearest receiver.","Usually, receivers in an anycast group are so-called replicas being able to support a same network service for requesting sender.","Examples for such replicas are mirrored web servers.","For accessing a desired network service, among a group of receivers sharing the same anycast address, i.e.","anycast receivers, each thereof providing the desired network service, the nearest receiver is determined and respective data communications are performed between the nearest receiver and the requesting sender.","Thus, accessing the nearest receiver enhances the network performance perceived by the requesting sender, saves network capacities such as network's bandwidth, and provides the desired network service.","In FIG.","1 , the basic principle of an anycast network is illustrated.","Both, members M 1 and M 2 are members of the same anycast group and share the same respective anycast address.","In line with the measure of a distance employed for routing of data in the network, the distances between requesting senders S 1 and S 2 and all members of the anycast group are computed.","Since the distance 2 calculated for the sender S 1 in relation to the anycast group members is the smallest distance, data communications will be performed between the sender S 1 and the member M 1 acting as a receiver for the sender S 1 .","In this manner, it is possible for example to direct Internet Protocol (IP) datagrams from the sender S 1 towards the member M 1 .","Accordingly, data communications are established between the sender S 2 and the member M 2 being a nearest receiver with respect to the sender S 2 , for example to direct IP datagrams from the sender S 2 towards the member M 2 .","Anycast Addresses According to the Internet Protocol version 6 (IPv6), special anycast addresses have been specified in addition to unicast addresses for individually addressing single receivers and multicast addresses for addressing all receivers sharing the same multicast address.","Anycast addresses are allocated from the address space employed for unicast addresses, using any of the formats defined for unicast addresses.","As a result, anycast addresses cannot be syntactically distinguished from unicast addresses.","An anycast address identifies a set of interfaces which typically belong to different nodes of a network.","Data, e.g.","a data packet, communicated from a sender to an anycast address is delivered to one of the interfaces identified by the respective anycast address, in particular, to the nearest interface according to the measure of a distance employed for the network routing protocol or system.","In case, a unicast address is assigned to more than one interface, thus turning the unicast address into a respective anycast address, the nodes of the network to which the address is assigned must be explicitly configured to interpret the address as an anycast address.","According to the IPv6, an anycast address must not be assigned to a host, but may be assigned to an IPv6 router, only.","Usually, for any assigned anycast address, an address prefix P is utilized which identifies a topological region in which all interfaces belonging to an anycast address reside.","In the region identified by the address prefix P, each member of the respective anycast group is advertised as separate entry in a network routing protocol or system.","Outside the region identified by the address prefix P, the anycast address may be aggregated into the routing advertisement for the address prefix P. Anycast Host Registration In order to join an anycast group, a host instructs its first hop router, i.e.","the closest router in relation to the host for data communications via a network, to advertise the anycast address of the anycast group on its behalf.","This can be achieved by adding a new message type to either the Internet Group Management Protocol (IGMP) or the Neighbor Discovery Protocol.","Then, the first hop router advertises the anycast address according to an anycast routing protocol employed for the domain including the host.","Anycast Routing Each anycast group is confined to a particular topological region with which it shares a common address prefix.","Within the topological region identified by the shared address prefix, each member of the anycast group is advertised as a separate entry in the employed unicast routing protocol or system.","The principle of anycast routing within a topological region identified by an address prefix shared by an anycast group is illustrated in FIG.","2 .","As can be derived from the table in FIG.","2 , there are multiple paths to the anycast address.","In case, multiple paths exist to a destination address prefix, a routing table look up of the router will return multiple next hops.","The selection of the next hop router to be used for data communications, e.g.","a particular data packet, depends on the implemented routing protocol or system.","Further, the path selected for data communications can also be effected by the type of data communications to be performed.","For example in case of the Internet Protocol standard, the Type Of Service (TOS) field in the IP header of a data packet can be used to define a data communications path.","Thus, a TOS designation of a data packet would help the router to chose an appropriate communication path for the given data packet.","The selection of an appropriate path is accomplished on the basis on a determination of the distances to the destination defined by the address prefix according to a measure of distance of the respective routing protocol or system.","In case of the Internet Protocol standard, for example, the OSPF knows the distance related to the corresponding matrix as identified in the TOS field of a data packet to the destination, i.e.","the hop count.","As a result, for an anycast routing it is possible to select the nearest next hop on the basis of the employed matrix.","In this respect, it is not necessary to analyze the whole IP address of a given data packet.","In FIG.","2 , this in indicated by the destination address Mx wherein only the prefix “M” is analyzed.","For an anycast routing outside the topological region identified by the shared address prefix, the anycast address may be aggregerated into the routing advertisement for the shared address prefix.","This principle is illustrated in FIG.","3 .","The destination address Ax in FIG.","3 denotes that only the shared address prefix is utilized in order to determine that data communications are to be routed to the corresponding domain.","Since the anycast address for the anycast group shares the address prefix with network domain A, network domain B cannot aggregerate the anycast address in its address prefix.","Therefore, network domain B should advertise the anycast address as a separate entry covering both anycast members A 4 and A 5 .","This is indicated in FIG.","3 by the destination address Ax being based on the address prefix for domain A and destination address Ax′ containing the complete anycast address.","The routing for data communications within the network domains A and B is performed as described before with reference to FIG.","2 .","Strict and Loose Routing According to the Internet Protocol version 4 (IPv4) and the Internet Protocol version 6 (IPv6), the following options for routing data communications from a source (e.g.","sender, requesting system) to a destination (e.g.","receiver, accessed system) have been defined as part of the IP data packet header: Strict Routing This option defines a complete data communications path from a source to a destination by means of a sequence of IP addresses.","Data to be communicated between the source and the destination is required to exactly follow the defined path.","Loose Routing This option specifies a number of routers and an order thereof.","Data to be communicated from a source to a destination is required to traverse the specified routers in the specified order, but is allowed to be communicated via other routers on its way from the source to the destination.","Problems Existing solutions for networks being capable of anycast routing are restricted to the selection of a single nearest receiver for data communications with an accessing sender.","In case a sender intends to access more than one receiver, existing anycast networks do no provide such a service.","A situation for which accesses to more than one receiver are desired is, for example, a network user wanting to access a number of nearest network servers in order to negotiate for the best network service conditions.","A further example is a network user wishing to contact a number of nearest restaurants by accessing the network servers thereof in order to check e.g.","meals, prices, available seats, etc.","or wishing to access a number of nearest emergency service providers in order to ensure that at least one is able to assist (note for the latter examples, sufficient information for the accessing user is only provided in case there is a geographical relationship between the restaurants and the emergency service providers and the corresponding servers).","In conventional networks, accesses to more than one receiver can be accomplished by accessing a number of receivers by individually addressing the same on the basis of a unicast routing or by accessing a group of receivers by commonly addressing the same on the basis of a shared multicasting address.","Such a multiple unicast routing requires that an accessing user must know each individual network address of the desired receivers and individually perform accesses thereto.","This is a time consuming procedure which cannot lead to the desired information for a user in a case, for example, he/she is not provided the respective network addresses and/or information concerning distances to the accessed servers.","With respect to the above given example concerning a negotiation for the best network service conditions by accessing a number of nearest servers, such a multiple unicast routing is also not appropriate since no information indicative of distances to receivers is provided and/or available.","Accesses on the basis of a multicast routing can result in a number of contacted receivers being too high in view of the demands and desired information of a requesting sender/user.","Further, with multicast routing it is not possible to access a desired number of receivers and, in particular, a desired number of nearest receivers, since all receivers being a member of the respective multicasting group are addressed together.","Furthermore, a second attempt, e.g.","by a multiple unicast routing, to access/contact receivers, the number thereof being higher compared to the number of receivers specified in a preceding, first attempt, would unnecessarily return the results from the first attempt again.","This repetition of data communications with respect to the result of the first attempt unnecessarily utilizes network resources in case the requesting network user is not interested in being provided the first attempt results again."],["<SOH> BRIEF DESCRIPTION OF THE INVENTION <EOH>The approach underlying the present invention to obtain the above object is to extend principles employed in existing anycast networks for accessing a single nearest client in order to allow for a multiple anycast routing to a specified number of nearest receivers.","In principle, this is achieved by associating data communications from a sender directed to a network by means of an anycast routing to data which indicate the number of nearest receivers.","In particular, such data which will be referred to as a first indicator in the following, specifies the number of nearest receivers being anycast members of an anycast group identified by an anycast address given by the sender (left) to be contacted by and/or allocated for the sender for data communications, such as requests, accesses, data transmissions, etc.","According to the present invention, a method for multiple hosts anycast routing in a network is provided wherein sender data is communicated from a sender to an anycast group including anycast members.","For communicating the sender data to the anycast group, the sender data includes an anycast address for addressing the anycast group.","In order to specify a number of the anycast members for data communications with the sender, the sender data further includes a first indicator being indicative of the desired, first number of anycast members.","On the basis of the sender data, a number of nearest anycast members is determined as hosts with respect to the sender for data communications.","In particular, the number of nearest anycast members being determined is defined by the first indicator.","This solution provides for data communications between a sender and a number of receivers without addressing the same separately, but addressing a plurality of possible receivers by a single address being common for all receivers and selecting a desired number therefrom by means of a data being simply amended to the single common address.","A further benefit is that it can be employed for any network, even for networks not providing known anycast routing services.","After having performed the above described method it may be possible that a further multiple host anycast routing should be performed wherein the number of anycast members to be determined is varied compared to the number of anycast members previously specified.","In order to avoid that anycast members already determined in the previous multiple host anycast routing are contacted again a second indicator can be used.","The second indicator specifies the number of anycast members previously determined in the preceding multiple host anycast routing.","Then, the number of nearest anycast members can be determined on the basis of the first and the second indicator.","In case the first indicator specifies a number of anycast members then the second indicator does, the number of nearest anycast members indicated by the second indicator (in the following: second number of anycast members) are skipped and not contacted, while a number of anycast member is determined for data communications with the sender up to a number obtained by a comparison of the number of anycast members specified by the first indicator (in the following: first number) and the second number.","In particular, the number of nearest anycast members to be determined corresponds to the difference between the first number and the second number.","In case the first number corresponds to the second number, no further anycast members have to be determined.","The same applies for the case wherein the first number is smaller than the second number.","In order to provide for a method according to the invention for multiple hosts anycast routing to be employed in a network providing a known anycast routing, a first indicator specifying a first number of anycast members to be contacted/allocated for data communications is associated to an anycast address.","By means of the anycast address, an anycast group having anycast members is identified in the network.","To establish the desired data communications, at least the anycast address and the first indicator is communicated in the network to the anycast group.","The anycast group is identified by the anycast address, while the number of anycast members to be contacted/allocated for data communications is selected according to the first indicator.","In particular, the first indicator defines the number of nearest anycast members and according to the first indicator, the specified number of nearest anycast members is determined as hosts for data communications in the network.","Comparable to the embodiment of the above described method for multiple hosts anycast routing in a network, this method for multiple hosts anycast routing in an anycast capable network can employ a second indicator specifying a number of anycast members (in the following: second number) already determined for data communications in the network, e.g.","on the basis of a previously performed multiple hosts anycast routing in a (anycast capable) network.","For providing the first and/or the second indicator, it is possible to include the indicator(s) in the anycast address or to extend the same by the first and/or the second indicator.","Preferably, the anycast address is included an anycast address prefix which identifies a topological region in the network, the topological region confining the anycast group.","Here it is possible, that the anycast address prefix includes the first and/or the second indicator or is extended by the same.","In a case of a network being operated according to an Internet Protocol standard (e.g.","IPv4 or IPv6) the anycast address and the indicator can be communicated by a transmission of an Internet Protocol data packet header which includes at least the anycast address.","For communicating the first and/or the second indicator, the Internet Protocol data packet header can be included the first and/or the second indicator as a part of or an extension for the anycast address.","With respect to the options mechanisms defined for the Internet Protocols, the first and/or the second indicator can be provided by extending the Internet Protocol data packet header by an options field and, in particular, a so-called multiple anycasting options field including the first and/or the second indicator.","It is preferred, that the property “nearest” of anycast members is determined according to a measure of distance of the respective routing protocol employed for the network.","In particular, “nearest” may be understood as the shortest distance between a sender and anycast members.","Additionally or as an option, “nearest” may be specified according to a distance metric requested by the sender.","Usually, the distance metric requested by the sender, e.g.","in the TOS field of an IP header, indicates a smaller distance metrics or nearest distance, respectively.","In case the network comprises domains at least some of which including at least of the anycast members, the anycast address and the first and/or the second indicator can be communicated via network routers to and/or between domains having anycast members.","Since it is assumed that a nearest network domain will also include anycast members being nearer compared to anycast members of more distant domains, it is preferred that the ordering of routers for network domain communications is set up such that the anycast address and the first and/or the second indicator are communicated to nearest network domains.","The nearest network domains may specified according to a measure of distance of a routing protocol for the network and/or according to a (e.g.","smallest) distance metric requested by the sender, e.g.","in the TOS field.","In order to provide for a fast multiple anycasting, the number of anycast members can be contacted sequentially, in particular for a small number specified by the first and/or the second indicator.","Within a network domain, multiple anycast routing and, in particular, a communication of the anycast address and the first and/or the second indicator can be accomplished on the basis of data identifying each of the anycast members.","Such an identifying data can be, for example, provided by means of separate entries for anycast members in a routing table according to the employed routing protocol.","With respect to the anycast routing performed for a network domain, the first indicator can be updated by decreasing the same according to a number of anycast members comprised by a network domain being identified by means of the anycast address and receiving the first indicator.","Further, the anycast routing can be enhanced by multiplying the anycast address and the first and/or the second indicator and communicating the multiplied anycast addresses and indicators to network domains and/or anycast members.","Here, it is preferred to communicate the multiplied data simultaneously to the further network domain and/or anycast members.","In order to precisely select the specified number of anycast members, while determining the number of anycast members, the indicator is modified according to a number of already determined/contacted/allocated nearest anycast members such that the modified indicator being communicated in the network specifies a number of anycast members still to be determined as receivers.","This embodiment avoids a utilization of further data beside the indicator to provide information how many receivers are already obtained and how many are still to be selected.","According to an Internet Protocol standard, the communication in the network can be performed by using the strict routing mechanism or the loose routing mechanism.","Preferably, the first and the second indicator are communicated together and, advantageously in the same manner, e.g.","both indicators being included in the anycast address or extending the same.","Further, the present invention provides a system for multiple hosts anycast routing and an anycast routing capable network which are preferred to be operated according to the method according to the invention as set forth above.","Moreover, the present invention provides a computer program product being adapted to carry out the method steps according to the invention and embodiments thereof as set forth above."],["BACKGROUND OF THE INVENTION 1.Technical Field The present invention relates to a method and a system for establishing data communications between a sender and receivers, the number thereof being defined by the sender, wherein the receivers are not individually addressed by the sender, but selected from a group of receivers being addressed from the sender by means of a common address.","2.Background of the Invention An anycast network is a network providing a service which allows a sender to access the nearest of a group of receivers having a common address.","The common address, in particular, the same anycast address for the group of receivers enables a sender to identify a plurality of receivers by providing only one address, namely an anycast address, instead of individually addressing each receiver.","In contrast to a multicast system, wherein data communications are performed between a sender and all receivers sharing a common address, i.e.","the same multicast address, data communications in an anycast network are performed between a sender and a particular receiver of a group of receivers sharing the same anycast address.","In the context, data communications include any kind of data, such as alphanumerical, graphic, multimedia, audio, video, voice data, information, signals etc.","which can be exchanged between systems, devices, network components etc.","(e.g.","computers, end user devices, hosts, servers, routers, modes).","In particular, data communications in an anycast network are performed between a sender and the nearest receiver of the group of receivers sharing the same anycast address.","In this respect, the nearest receiver in relation to a sender is defined according to a measure of a distance employed by a routing protocol or routing system used for data communications in a respective network.","Since the routing protocol or routing system of a network which identifies the nearest receiver for accesses by a sender, the sender does not need to care about how to select the closest destination, i.e.","the nearest receiver.","Usually, receivers in an anycast group are so-called replicas being able to support a same network service for requesting sender.","Examples for such replicas are mirrored web servers.","For accessing a desired network service, among a group of receivers sharing the same anycast address, i.e.","anycast receivers, each thereof providing the desired network service, the nearest receiver is determined and respective data communications are performed between the nearest receiver and the requesting sender.","Thus, accessing the nearest receiver enhances the network performance perceived by the requesting sender, saves network capacities such as network's bandwidth, and provides the desired network service.","In FIG.","1, the basic principle of an anycast network is illustrated.","Both, members M1 and M2 are members of the same anycast group and share the same respective anycast address.","In line with the measure of a distance employed for routing of data in the network, the distances between requesting senders S1 and S2 and all members of the anycast group are computed.","Since the distance 2 calculated for the sender S1 in relation to the anycast group members is the smallest distance, data communications will be performed between the sender S1 and the member M1 acting as a receiver for the sender S1.In this manner, it is possible for example to direct Internet Protocol (IP) datagrams from the sender S1 towards the member M1.Accordingly, data communications are established between the sender S2 and the member M2 being a nearest receiver with respect to the sender S2, for example to direct IP datagrams from the sender S2 towards the member M2.Anycast Addresses According to the Internet Protocol version 6 (IPv6), special anycast addresses have been specified in addition to unicast addresses for individually addressing single receivers and multicast addresses for addressing all receivers sharing the same multicast address.","Anycast addresses are allocated from the address space employed for unicast addresses, using any of the formats defined for unicast addresses.","As a result, anycast addresses cannot be syntactically distinguished from unicast addresses.","An anycast address identifies a set of interfaces which typically belong to different nodes of a network.","Data, e.g.","a data packet, communicated from a sender to an anycast address is delivered to one of the interfaces identified by the respective anycast address, in particular, to the nearest interface according to the measure of a distance employed for the network routing protocol or system.","In case, a unicast address is assigned to more than one interface, thus turning the unicast address into a respective anycast address, the nodes of the network to which the address is assigned must be explicitly configured to interpret the address as an anycast address.","According to the IPv6, an anycast address must not be assigned to a host, but may be assigned to an IPv6 router, only.","Usually, for any assigned anycast address, an address prefix P is utilized which identifies a topological region in which all interfaces belonging to an anycast address reside.","In the region identified by the address prefix P, each member of the respective anycast group is advertised as separate entry in a network routing protocol or system.","Outside the region identified by the address prefix P, the anycast address may be aggregated into the routing advertisement for the address prefix P. Anycast Host Registration In order to join an anycast group, a host instructs its first hop router, i.e.","the closest router in relation to the host for data communications via a network, to advertise the anycast address of the anycast group on its behalf.","This can be achieved by adding a new message type to either the Internet Group Management Protocol (IGMP) or the Neighbor Discovery Protocol.","Then, the first hop router advertises the anycast address according to an anycast routing protocol employed for the domain including the host.","Anycast Routing Each anycast group is confined to a particular topological region with which it shares a common address prefix.","Within the topological region identified by the shared address prefix, each member of the anycast group is advertised as a separate entry in the employed unicast routing protocol or system.","The principle of anycast routing within a topological region identified by an address prefix shared by an anycast group is illustrated in FIG.","2.As can be derived from the table in FIG.","2, there are multiple paths to the anycast address.","In case, multiple paths exist to a destination address prefix, a routing table look up of the router will return multiple next hops.","The selection of the next hop router to be used for data communications, e.g.","a particular data packet, depends on the implemented routing protocol or system.","Further, the path selected for data communications can also be effected by the type of data communications to be performed.","For example in case of the Internet Protocol standard, the Type Of Service (TOS) field in the IP header of a data packet can be used to define a data communications path.","Thus, a TOS designation of a data packet would help the router to chose an appropriate communication path for the given data packet.","The selection of an appropriate path is accomplished on the basis on a determination of the distances to the destination defined by the address prefix according to a measure of distance of the respective routing protocol or system.","In case of the Internet Protocol standard, for example, the OSPF knows the distance related to the corresponding matrix as identified in the TOS field of a data packet to the destination, i.e.","the hop count.","As a result, for an anycast routing it is possible to select the nearest next hop on the basis of the employed matrix.","In this respect, it is not necessary to analyze the whole IP address of a given data packet.","In FIG.","2, this in indicated by the destination address Mx wherein only the prefix “M” is analyzed.","For an anycast routing outside the topological region identified by the shared address prefix, the anycast address may be aggregerated into the routing advertisement for the shared address prefix.","This principle is illustrated in FIG.","3.The destination address Ax in FIG.","3 denotes that only the shared address prefix is utilized in order to determine that data communications are to be routed to the corresponding domain.","Since the anycast address for the anycast group shares the address prefix with network domain A, network domain B cannot aggregerate the anycast address in its address prefix.","Therefore, network domain B should advertise the anycast address as a separate entry covering both anycast members A4 and A5.This is indicated in FIG.","3 by the destination address Ax being based on the address prefix for domain A and destination address Ax′ containing the complete anycast address.","The routing for data communications within the network domains A and B is performed as described before with reference to FIG.","2.Strict and Loose Routing According to the Internet Protocol version 4 (IPv4) and the Internet Protocol version 6 (IPv6), the following options for routing data communications from a source (e.g.","sender, requesting system) to a destination (e.g.","receiver, accessed system) have been defined as part of the IP data packet header: Strict Routing This option defines a complete data communications path from a source to a destination by means of a sequence of IP addresses.","Data to be communicated between the source and the destination is required to exactly follow the defined path.","Loose Routing This option specifies a number of routers and an order thereof.","Data to be communicated from a source to a destination is required to traverse the specified routers in the specified order, but is allowed to be communicated via other routers on its way from the source to the destination.","Problems Existing solutions for networks being capable of anycast routing are restricted to the selection of a single nearest receiver for data communications with an accessing sender.","In case a sender intends to access more than one receiver, existing anycast networks do no provide such a service.","A situation for which accesses to more than one receiver are desired is, for example, a network user wanting to access a number of nearest network servers in order to negotiate for the best network service conditions.","A further example is a network user wishing to contact a number of nearest restaurants by accessing the network servers thereof in order to check e.g.","meals, prices, available seats, etc.","or wishing to access a number of nearest emergency service providers in order to ensure that at least one is able to assist (note for the latter examples, sufficient information for the accessing user is only provided in case there is a geographical relationship between the restaurants and the emergency service providers and the corresponding servers).","In conventional networks, accesses to more than one receiver can be accomplished by accessing a number of receivers by individually addressing the same on the basis of a unicast routing or by accessing a group of receivers by commonly addressing the same on the basis of a shared multicasting address.","Such a multiple unicast routing requires that an accessing user must know each individual network address of the desired receivers and individually perform accesses thereto.","This is a time consuming procedure which cannot lead to the desired information for a user in a case, for example, he/she is not provided the respective network addresses and/or information concerning distances to the accessed servers.","With respect to the above given example concerning a negotiation for the best network service conditions by accessing a number of nearest servers, such a multiple unicast routing is also not appropriate since no information indicative of distances to receivers is provided and/or available.","Accesses on the basis of a multicast routing can result in a number of contacted receivers being too high in view of the demands and desired information of a requesting sender/user.","Further, with multicast routing it is not possible to access a desired number of receivers and, in particular, a desired number of nearest receivers, since all receivers being a member of the respective multicasting group are addressed together.","Furthermore, a second attempt, e.g.","by a multiple unicast routing, to access/contact receivers, the number thereof being higher compared to the number of receivers specified in a preceding, first attempt, would unnecessarily return the results from the first attempt again.","This repetition of data communications with respect to the result of the first attempt unnecessarily utilizes network resources in case the requesting network user is not interested in being provided the first attempt results again.","OBJECT OF THE INVENTION The object of the present invention is to provide for a method and a system allowing for accesses of a sender to a specified number of nearest receivers and/or data communications between a sender and a specified number of nearest receivers.","BRIEF DESCRIPTION OF THE INVENTION The approach underlying the present invention to obtain the above object is to extend principles employed in existing anycast networks for accessing a single nearest client in order to allow for a multiple anycast routing to a specified number of nearest receivers.","In principle, this is achieved by associating data communications from a sender directed to a network by means of an anycast routing to data which indicate the number of nearest receivers.","In particular, such data which will be referred to as a first indicator in the following, specifies the number of nearest receivers being anycast members of an anycast group identified by an anycast address given by the sender (left) to be contacted by and/or allocated for the sender for data communications, such as requests, accesses, data transmissions, etc.","According to the present invention, a method for multiple hosts anycast routing in a network is provided wherein sender data is communicated from a sender to an anycast group including anycast members.","For communicating the sender data to the anycast group, the sender data includes an anycast address for addressing the anycast group.","In order to specify a number of the anycast members for data communications with the sender, the sender data further includes a first indicator being indicative of the desired, first number of anycast members.","On the basis of the sender data, a number of nearest anycast members is determined as hosts with respect to the sender for data communications.","In particular, the number of nearest anycast members being determined is defined by the first indicator.","This solution provides for data communications between a sender and a number of receivers without addressing the same separately, but addressing a plurality of possible receivers by a single address being common for all receivers and selecting a desired number therefrom by means of a data being simply amended to the single common address.","A further benefit is that it can be employed for any network, even for networks not providing known anycast routing services.","After having performed the above described method it may be possible that a further multiple host anycast routing should be performed wherein the number of anycast members to be determined is varied compared to the number of anycast members previously specified.","In order to avoid that anycast members already determined in the previous multiple host anycast routing are contacted again a second indicator can be used.","The second indicator specifies the number of anycast members previously determined in the preceding multiple host anycast routing.","Then, the number of nearest anycast members can be determined on the basis of the first and the second indicator.","In case the first indicator specifies a number of anycast members then the second indicator does, the number of nearest anycast members indicated by the second indicator (in the following: second number of anycast members) are skipped and not contacted, while a number of anycast member is determined for data communications with the sender up to a number obtained by a comparison of the number of anycast members specified by the first indicator (in the following: first number) and the second number.","In particular, the number of nearest anycast members to be determined corresponds to the difference between the first number and the second number.","In case the first number corresponds to the second number, no further anycast members have to be determined.","The same applies for the case wherein the first number is smaller than the second number.","In order to provide for a method according to the invention for multiple hosts anycast routing to be employed in a network providing a known anycast routing, a first indicator specifying a first number of anycast members to be contacted/allocated for data communications is associated to an anycast address.","By means of the anycast address, an anycast group having anycast members is identified in the network.","To establish the desired data communications, at least the anycast address and the first indicator is communicated in the network to the anycast group.","The anycast group is identified by the anycast address, while the number of anycast members to be contacted/allocated for data communications is selected according to the first indicator.","In particular, the first indicator defines the number of nearest anycast members and according to the first indicator, the specified number of nearest anycast members is determined as hosts for data communications in the network.","Comparable to the embodiment of the above described method for multiple hosts anycast routing in a network, this method for multiple hosts anycast routing in an anycast capable network can employ a second indicator specifying a number of anycast members (in the following: second number) already determined for data communications in the network, e.g.","on the basis of a previously performed multiple hosts anycast routing in a (anycast capable) network.","For providing the first and/or the second indicator, it is possible to include the indicator(s) in the anycast address or to extend the same by the first and/or the second indicator.","Preferably, the anycast address is included an anycast address prefix which identifies a topological region in the network, the topological region confining the anycast group.","Here it is possible, that the anycast address prefix includes the first and/or the second indicator or is extended by the same.","In a case of a network being operated according to an Internet Protocol standard (e.g.","IPv4 or IPv6) the anycast address and the indicator can be communicated by a transmission of an Internet Protocol data packet header which includes at least the anycast address.","For communicating the first and/or the second indicator, the Internet Protocol data packet header can be included the first and/or the second indicator as a part of or an extension for the anycast address.","With respect to the options mechanisms defined for the Internet Protocols, the first and/or the second indicator can be provided by extending the Internet Protocol data packet header by an options field and, in particular, a so-called multiple anycasting options field including the first and/or the second indicator.","It is preferred, that the property “nearest” of anycast members is determined according to a measure of distance of the respective routing protocol employed for the network.","In particular, “nearest” may be understood as the shortest distance between a sender and anycast members.","Additionally or as an option, “nearest” may be specified according to a distance metric requested by the sender.","Usually, the distance metric requested by the sender, e.g.","in the TOS field of an IP header, indicates a smaller distance metrics or nearest distance, respectively.","In case the network comprises domains at least some of which including at least of the anycast members, the anycast address and the first and/or the second indicator can be communicated via network routers to and/or between domains having anycast members.","Since it is assumed that a nearest network domain will also include anycast members being nearer compared to anycast members of more distant domains, it is preferred that the ordering of routers for network domain communications is set up such that the anycast address and the first and/or the second indicator are communicated to nearest network domains.","The nearest network domains may specified according to a measure of distance of a routing protocol for the network and/or according to a (e.g.","smallest) distance metric requested by the sender, e.g.","in the TOS field.","In order to provide for a fast multiple anycasting, the number of anycast members can be contacted sequentially, in particular for a small number specified by the first and/or the second indicator.","Within a network domain, multiple anycast routing and, in particular, a communication of the anycast address and the first and/or the second indicator can be accomplished on the basis of data identifying each of the anycast members.","Such an identifying data can be, for example, provided by means of separate entries for anycast members in a routing table according to the employed routing protocol.","With respect to the anycast routing performed for a network domain, the first indicator can be updated by decreasing the same according to a number of anycast members comprised by a network domain being identified by means of the anycast address and receiving the first indicator.","Further, the anycast routing can be enhanced by multiplying the anycast address and the first and/or the second indicator and communicating the multiplied anycast addresses and indicators to network domains and/or anycast members.","Here, it is preferred to communicate the multiplied data simultaneously to the further network domain and/or anycast members.","In order to precisely select the specified number of anycast members, while determining the number of anycast members, the indicator is modified according to a number of already determined/contacted/allocated nearest anycast members such that the modified indicator being communicated in the network specifies a number of anycast members still to be determined as receivers.","This embodiment avoids a utilization of further data beside the indicator to provide information how many receivers are already obtained and how many are still to be selected.","According to an Internet Protocol standard, the communication in the network can be performed by using the strict routing mechanism or the loose routing mechanism.","Preferably, the first and the second indicator are communicated together and, advantageously in the same manner, e.g.","both indicators being included in the anycast address or extending the same.","Further, the present invention provides a system for multiple hosts anycast routing and an anycast routing capable network which are preferred to be operated according to the method according to the invention as set forth above.","Moreover, the present invention provides a computer program product being adapted to carry out the method steps according to the invention and embodiments thereof as set forth above.","BRIEF DESCRIPTION OF THE FIGURES In the following description of preferred embodiments of the present invention it is referred to the accompanying figures wherein: FIG.","1 illustrates the basic principle of anycast routing in a network according to the state of the art, FIG.","2 illustrates an anycast routing within a topological region identified by a common address prefix according to the state of the art, FIG.","3 illustrates an anycast routing outside a topological region identified by a common address prefix according to the state of the art, FIG.","4 illustrates a data structure for an anycast address according to the present invention, and FIG.","5 illustrates a multiple anycast routing according to the present invention.","DESCRIPTION OF PREFERRED EMBODIMENTS In order to promote the understanding of the present invention preferred embodiments are described with respect to an network according to the Internet Protocol standard.","In such a network, data communications are performed by transmitting IP data packets, short packets, each including an IP packet header, short header, between hosts, i.e.","sending and receiving systems within the network.","Each host is associated to a network domain defining a topological network region.","Data communications between network domains are performed via exterior gateway routers, whereas data communications within a network domain are performed via interior gateway routers.","Exterior gateway routers directly receiving packets from a network domain are called first exterior gateway routers and interior gateway routers directly receiving packets from an exterior gateway router are called border routers.","Hops designate network components via which packets are communicated.","In particular, hops include hosts and routers.","For data communications in the network, i.e.","routing of packets from a source to a destination, different routing protocols are used for transmission within a domain (intra-domain routing) and between domains (inter-domain routing).","For example, an intra-domain routing can be based on interior gateway routing protocols, such as OSPF, while inter-domain routing can employ exterior gateway routing protocols such as BGP.","According to the routing protocols, a routing table is defined which specifies a communication path for packets from a source to a destination and, in particular, hops via which packets are to be transmitted.","As explained above, both, loose routing and strict routing are possible, whereas for a multiple hosts anycasting most probably loose routing will be applied.","IP packet headers include information specifying a destination in the network to which packets are to be communicated from a source by means of destination addresses.","In case of unicast routing a destination corresponds to a receiving host, short receiver, whereas in case of multicast routing, conventional anycast routing, and, specifically, multiple host anycast routing a destination comprises a group of hosts each thereof, only one thereof, and a specified number thereof acting as receiver(s) with respect to a sending host, short sender.","In case of a multiple hosts anycast routing, short multiple anycasting, the number of hosts, i.e.","members of an anycast group, to be contacted is specified by extending a packet and, in particular, its header by an indicator.","The indicator providing information of the number of hosts to be contacted/allocated as originally specified by the sender and the number of hosts which still have to be contacted/allocated as receivers indicates the number of hosts that (still) have to be informed by the corresponding branch utilizing the following embodiments.","Indicator as Part of the IP Destination Address Field in the IP Header Since anycast routing is mostly based on resolving the above described address prefix, data (e.g.","some bits) of an IP destination address and, in particular, an address prefix is used for the indication of a sender-specified number of nearest hosts being anycast members of an anycast group indicated by the address prefix.","In the exemplary illustration of FIG.","4, the IP packet header includes an anycast address prefix and an indicator specifying the number of nearest anycast members (left) to be contacted/allocated.","Initially, the indicator specifies the number of nearest anycast members as desired by a sender, while communicating the packet within the network the indicator is decreased corresponding with the number of already selected receivers (already allocated anycast members).","For the examples given above wherein more than one host is to be contacted as receiver, it is assumed that the sender specified number of desired nearest anycast members is rather low, e.g.","less than 10.In such applications of multiple anycasting, it might be sufficient to, for example, use 4 bits of an IP packet header for the indicator to allow for a maximum of 24(16) nearest anycast members to be contacted.","In applications wherein a specification of a higher number of nearest anycast members should be provided, the amount of data (e.g.","number of bits) of an IP packet header is to be set correspondingly.","Indicator as New Options Extension to an IP Extension of an IP Header On the basis of existing IP options mechanism (e.g.","as explained above with respect to strict and loose routing), an IP packet header is extended by a so-called “multiple anycasting” options field containing an indicator as specified above.","In case a packet is destinated for an anycast address, the respective header including anycasting address information, anycast capable routers only analyses the extension, namely the “multiple anycasting” option field.","Modifications of the extension can be performed, for example, to update the “multiple anycasting” option field to be in line with the number of anycast members still to be contacted/allocated in view of already contacted/allocated anycast members.","As set forth above, data communications are differently performed for intra-domain and inter-domain communications.","As a result, multiple anycasting will work different for intra-domain cases employing interior gateway routing protocols such as OSPF and inter-domain cases employing exterior gateway protocols such as BGP.","In the following inter-domain multiple hosts anycasting and intra-domain multiple hosts anycasting will be described with reference to FIG.","5 illustrating an exemplary topology and routing scenario wherein a host H in a domain C communicates an anycast packet within a network comprising domains A, B and C and exterior gateway routers R1, R2, R3 and R4.For this example it is assumed that the anycast group addressed by the host H comprises the anycast members A1, A2 and A3 in domain A and anycast members A4 and A5 in domain B.","In particular, host H communicates an anycast packet including an indicator specifying a number of three anycast members to be contacted as receivers, i.e.","nearest anycast members.","Inter-Domain Multiple Hosts Anycasting Inter-domain multiple hosts anycasting is indicated by steps 1 and 3 shown in FIG.","5.On the basis of an employed exterior gateway routing protocol, the first exterior gateway router with respect to host H in domain C, i.e.","router R4, will check its routing tables in order to determine anycast members of the anycast group as specified by the anycast packet of host H and, in particular, all domains including the respective anycast members.","The corresponding routers for determined domains, i.e.","routers R1 and R3 to domains A and B, will be added to the IP packet header of the anycast packet from host H by utilizing strict or loose routing options.","Depending on the metrics used for the employed exterior gateway routing protocol and/or the smallest distance metric requested by the sender, i.e.","host H, (e.g.","defined in the TOS field), the ordering of the routers will be based on distances between domains including anycast members and anycast members, respectively, and host H. According to FIG.","5, the anycast packet of host H will be first communicated to router R3.In case the number of anycast members contacted/allocated in a domain receiving the anycast packet from router R3 is less than the number of nearest anycast members to be contacted as defined by host H, the anycast packet will be communicated to router R1.The anycast packet and, in particular, the header thereof transmitted to router R1 includes an indicator specifying the number of anycast members which are still to be contacted/allocated.","With reference to FIG.","5, the indicator communicated to router R1 indicates that one anycast member is to be contacted/allocated assuming that anycast members R4 and R5 of domain B are selected as nearest anycast members.","As specified by the strict or loose routing options of the IP packet header, the specified number of anycast members may be contacted sequentially.","Assuming the specified number being rather low, e.g.","2 to 5 anycast members, this procedure will not effect the network service performance received by requesting host, here host H. In case a high number of anycast member should be allowed to be specified by a sender/requesting host, various known data communication methods for enhancement of data transmissions in a network can be utilized for contacting anycast members.","Intra-Domain Multiple Hosts Anycasting Intra-domain multiple hosts anycasting will be explained with reference to steps 2 and 4 of FIG.","5.At reception of an anycast packet in an border router for a domain, the border router will forward the anycast packet to all next hops, in particular, to the next anycast members of the respective domain.","This can be accomplished since, as explained above, interior gateway routing protocols provide a separate entry in the routing table for each anycast member.","For forwarding the anycast packet, the border router may multiply the anycast packet and simultaneously communicate the multiplied anycast packets to multiple destinations, i.e.","next hops/anycast members.","According to the number of anycast members and, in particular, of anycast members contacted/allocated in the corresponding domain, the number specified by the indicator in the header of the anycast packet is decreased.","In case the indicator is 0 after an intra-domain multiple host anycasting, no data communications are required to contact/allocate further anycast members, e.g.","of a different domain, since the number of nearest anycast members as desired by a requesting host is provided.","Otherwise, the border router will forward the anycast packet including the decreased indicator to the next exterior gateway router for inter-domain multiple hosts anycasting as specified above.","According to FIG.","5, a border router of domain B receives the anycast packet from exterior gateway router R3 and will forward the anycast packet to the anycast members A4 and A5.For example, router R3 may serve as border router for domain B by combining exterior and interior gateway functions.","In step 2 the border router of domain B contacts all anycast members in domain B such that they are specified for nearest anycast members for data communications with host H. As a result, still one further anycast member has to be contacted/allocated, whereby the anycast packet communicated from the border router of domain B via the exterior gateway router R3 to the exterior gateway router R1 includes a correspondingly decreased indicator, i.e.","an indicator having the value of 1.A border router of domain A forwards the received anycast packet within domain A as specified above for contacting/allocating an anycast member.","Comparable to router R3, router R1 combining exterior and interior gateway capabilities may be operated as border router for domain A.","The routing within a domain may be performed in a manner comparable as the above described inter-domain routing in case hierarchies are used for the respective domain.","Then, all levels of the same hierarchy in the domain can be contemplated as inter-domain cases, wherein levels of a lower hierarchy can be seen as intra-domain cases.","Skipping the Number of Anycast Members Specified in a First Multiple Hosts Anycasting for Performing a Second Multiple Hosts Anycasting After having performed a first multiple hosts anycasting, it is possible that the respective host performs a second multiple hosts anycasting wherein the number of anycast members specified in the first multiple hosts anycasting should not be contacted/allocated (again).","For that purpose it is contemplated as an ad-on to the above described multiple hosts anycast routing, but also to the existing anycast specification as known in the state of the art, that a sending/requesting host can specify the number of first anycast members (i.e.","anycast members contacted/allocated in a first anycasting) that should be not contacted/allocated (again) in a second (multiple hosts) anycasting.","After a previous attempt did not result in a successful anycast member search there may be no need to contact/allocate the same anycast members once more if the number of anycast members is to be extended in a subsequent attempt.","In that case, the sending/requesting host could specify in a new optional IP header extension the number of first anycast members that should not be contacted/allocated in the subsequent attempt.","Such an indication can be provided by a second indicator (counter) which is additionally checked, e.g.","by routers, while performing the second (multiple hosts) anycast routing.","In case a network domain includes anycast members, the additional indicator (counter) is first checked.","If the second indicator indicates a number greater than 0, the second indicator is to be decreased until it reaches 0.For each decrease of the second indicator, the first multiple hosts indicator (counter), as described above, is decreased as well, but without contacting/allocating an anycast group member.","In this way, it is ensured that the number of anycast members contaced/allocated in the first, preceding (multiple hosts) anycasting is not contacted again in the second attempt.","As set forth above, this optional addition may already be implemented in the single hosts anycasting as known in the state of the art."]],"string":"[\n [\n \"Method and system for multiple hosts anycast routing\",\n \"Conventional anycast networks provide a service which allows a sender to access the nearest of a group of receivers sharing a common anycast address.\",\n \"In contrast thereto, multicast networks establish data communications between a sender and all receivers confined by a group having the same multicast address.\",\n \"Thus, the networks providing both anycast and multicast routing only allow for accesses by a sender to one receiver being specified according to the employed anycast protocol or to a plurality of receivers forming a multicast group.\",\n \"In order to provide accesses by a sender to a specified number of nearest receivers, the present invention provides a method and a system for multiple hosts anycast routing in a network.\",\n \"An indicator specifying the number of nearest receivers to be set up for data communications with a sender is included in an anycast address or associated thereto as an extension.\",\n \"On the basis of the indicator, a corresponding number of anycast members of an anycast group identified by the anycast address are contacted/allocated for data communications with the sender.\"\n ],\n [\n \"1.A method for multiple hosts anycast routing in a network, the method comprising: communicating sender data from a sender addressed to an anycast group including anycast members, said sender data including an anycast address for addressing said anycast group and a first indicator specifying a first number of said anycast members within said anycast group for data communications with said sender, and determining a number of nearest of said anycast members within said anycast group as hosts for data communications with said sender, said number of nearest anycast members being defined by said first indicator.\",\n \"2.The method according to claim 1, comprising: communicating further sender data from said sender addressed to said anycast group including said anycast members, said further sender data further including a second indicator specifying a second number of said anycast members within said anycast group, said second number being indicative of already determined nearest of said anycast members within said anycast group, and determining said number of nearest of said anycast members within said anycast group, said number of nearest anycast members being defined by said first and said second indicator.\",\n \"3.A method for multiple hosts anycast routing in an anycast capable network, the method comprising: associating a first indicator with an anycast address identifying an anycast group having anycast members in a network, said first indicator specifying a first number of anycast members within said anycast group, communicating at least said anycast address and said first indicator in said network to said anycast group, and determining a number of nearest of said anycast members within said anycast group as hosts for data communications in said network, said number of nearest anycast members being defined by said first indicator.\",\n \"4.The method according to claim 3, comprising: associating a second indicator with said anycast address, said indicator specifying a second number of said anycast members within said anycast group, said second number being indicative of already determined nearest of said anycast members within said anycast group, communicating at least said anycast address, said first indicator and said second indicator in said network to said anycast group and determining said number of nearest of said anycast members within said anycast group, said number of nearest anycast members being defined by said first and said second indicator.\",\n \"5.The method according to claim 2, wherein said number of nearest anycast members to be determined is defined by the difference between said first number and said second number.\",\n \"6.The method according to claim 1, wherein said first and/or said second indicator is included in said anycast address.\",\n \"7.The method according to claim 1, wherein said anycast address is extended by said first and/or said second indicator.\",\n \"8.The method according to claim 1, wherein an anycast address prefix is included in said anycast address, said anycast address prefix identifying a topological region in said network confining said anycast group, said anycast address prefix including said first and/or said second indicator or being extended by said first and/or said second indicator.\",\n \"9.The method according to claim 1, wherein said anycast address and said first and/or said second indicator are communicated by communicating an Internet Protocol data packet header including said anycast address.\",\n \"10.The method according to claim 9, wherein said first and/or said second indicator is included in said Internet Protocol data packet header as a part or an extension of said anycast address, or said Internet Protocol data packet header is extended by said first and/or said second indicator by means of an options field for Internet Protocol data packet headers.\",\n \"11.The method according to claim 1, wherein said number of nearest anycast members is determined by utilizing a measure of distance of a routing protocol for said network and/or determined by utilizing a measure of distance specified by said sender.\",\n \"12.The method according to claim 1, wherein said anycast address and said first and/or said second indicator are communicated in said network between domains thereof via routers to network domains having at least one of said anycast members.\",\n \"13.The method according to claim 12, wherein an ordering of said routers is defined such that said anycast address and said first and/or said second indicator are communicated to the nearest network domains, the nearest network domains being specified according to a measure of distance of a routing protocol for said network and/or according to a measure of distance specified by said sender.\",\n \"14.The method according to claim 1, wherein said number of anycast members is contacted sequentially.\",\n \"15.\",\n \"(Cancel) 16.The method according to claim 1, wherein said first indicator is modified according to a number of already determined nearest anycast members such that said modified indicator specifies a number of said anycast members still to be determined as receivers.\",\n \"17.The method according to claim 16, wherein said first indicator is decreased by the value of said second indicator.\",\n \"18.The method according to claim 1, wherein said second indicator is decreased to a value corresponding to a number of 0 and said first indicator is decreased correspondingly to the decrease of the second indicator and said determining of said number of nearest of said anycast members within said anycast group is performed in dependence of said decreased first indicator if said second indicator has a value corresponding to a number of 0.19.The method according to claim 1, wherein said anycast address and said first and/or said second indicator are communicated to at least two network domains including at least one of said anycast members by multiplying said anycast address and said first indicator, and communicating said multiplied anycast address and said first and/or said second indicator to anycast members.\",\n \"20.The method according to claim 1, wherein said first number specified by said first indicator is decreased corresponding to a number of said anycast members in a network domain to which said anycast address and said first indicator are communicated.\",\n \"21.The method according to claim 1, wherein said communicating is performed according to the strict routing mechanism or the loose routing mechanism as defined in for the Internet Protocol.\",\n \"22.The method according to claim 1, wherein said first and said second indicator are communicated together.\",\n \"23.A system for multiple hosts anycast routing, comprising: a network a sender, and a group of receivers being identified by an anycast address, wherein data communications between said sender and a number of nearest of said receivers are established on the basis of a first indicator provided by said sender specifying said number of receivers.\",\n \"24.The system according to claim 23, wherein data communications between said sender and said number of nearest of said receivers are further established on the basis of a second indicator provided by said sender specifying a number of receivers already determined for data communications with said sender.\",\n \"25-28.\",\n \"(Cancel) 29.The method according to claim 4, wherein said number of nearest anycast members to be determined is defined by the difference between said first number and said second number.\",\n \"30.The method according to claim 3, wherein said first and/or said second indicator is included in said anycast address.\",\n \"31.The method according to claim 3, wherein said anycast address is extended by said first and/or said second indicator.\",\n \"32.The method according to claim 3, wherein an anycast address prefix is included in said anycast address, said anycast address prefix identifying a topological region in said network confining said anycast group, said anycast address prefix including said first and/or said second indicator or being extended by said first and/or said second indicator.\",\n \"33.The method according to claim 3, wherein said number of nearest anycast members is determined by utilizing a measure of distance of a routing protocol for said network and/or determined by utilizing a measure of distance specified by said sender.\",\n \"34.The method according to claim 3, wherein said first indicator is modified according to a number of already determined nearest anycast members such that said modified indicator specifies a number of said anycast members still to be determined as receivers.\",\n \"35.The method according to claim 34, wherein said first indicator is decreased by the value of said second indicator.\"\n ],\n [\n \"<SOH> BACKGROUND OF THE INVENTION <EOH>1.Technical Field The present invention relates to a method and a system for establishing data communications between a sender and receivers, the number thereof being defined by the sender, wherein the receivers are not individually addressed by the sender, but selected from a group of receivers being addressed from the sender by means of a common address.\",\n \"2.Background of the Invention An anycast network is a network providing a service which allows a sender to access the nearest of a group of receivers having a common address.\",\n \"The common address, in particular, the same anycast address for the group of receivers enables a sender to identify a plurality of receivers by providing only one address, namely an anycast address, instead of individually addressing each receiver.\",\n \"In contrast to a multicast system, wherein data communications are performed between a sender and all receivers sharing a common address, i.e.\",\n \"the same multicast address, data communications in an anycast network are performed between a sender and a particular receiver of a group of receivers sharing the same anycast address.\",\n \"In the context, data communications include any kind of data, such as alphanumerical, graphic, multimedia, audio, video, voice data, information, signals etc.\",\n \"which can be exchanged between systems, devices, network components etc.\",\n \"(e.g.\",\n \"computers, end user devices, hosts, servers, routers, modes).\",\n \"In particular, data communications in an anycast network are performed between a sender and the nearest receiver of the group of receivers sharing the same anycast address.\",\n \"In this respect, the nearest receiver in relation to a sender is defined according to a measure of a distance employed by a routing protocol or routing system used for data communications in a respective network.\",\n \"Since the routing protocol or routing system of a network which identifies the nearest receiver for accesses by a sender, the sender does not need to care about how to select the closest destination, i.e.\",\n \"the nearest receiver.\",\n \"Usually, receivers in an anycast group are so-called replicas being able to support a same network service for requesting sender.\",\n \"Examples for such replicas are mirrored web servers.\",\n \"For accessing a desired network service, among a group of receivers sharing the same anycast address, i.e.\",\n \"anycast receivers, each thereof providing the desired network service, the nearest receiver is determined and respective data communications are performed between the nearest receiver and the requesting sender.\",\n \"Thus, accessing the nearest receiver enhances the network performance perceived by the requesting sender, saves network capacities such as network's bandwidth, and provides the desired network service.\",\n \"In FIG.\",\n \"1 , the basic principle of an anycast network is illustrated.\",\n \"Both, members M 1 and M 2 are members of the same anycast group and share the same respective anycast address.\",\n \"In line with the measure of a distance employed for routing of data in the network, the distances between requesting senders S 1 and S 2 and all members of the anycast group are computed.\",\n \"Since the distance 2 calculated for the sender S 1 in relation to the anycast group members is the smallest distance, data communications will be performed between the sender S 1 and the member M 1 acting as a receiver for the sender S 1 .\",\n \"In this manner, it is possible for example to direct Internet Protocol (IP) datagrams from the sender S 1 towards the member M 1 .\",\n \"Accordingly, data communications are established between the sender S 2 and the member M 2 being a nearest receiver with respect to the sender S 2 , for example to direct IP datagrams from the sender S 2 towards the member M 2 .\",\n \"Anycast Addresses According to the Internet Protocol version 6 (IPv6), special anycast addresses have been specified in addition to unicast addresses for individually addressing single receivers and multicast addresses for addressing all receivers sharing the same multicast address.\",\n \"Anycast addresses are allocated from the address space employed for unicast addresses, using any of the formats defined for unicast addresses.\",\n \"As a result, anycast addresses cannot be syntactically distinguished from unicast addresses.\",\n \"An anycast address identifies a set of interfaces which typically belong to different nodes of a network.\",\n \"Data, e.g.\",\n \"a data packet, communicated from a sender to an anycast address is delivered to one of the interfaces identified by the respective anycast address, in particular, to the nearest interface according to the measure of a distance employed for the network routing protocol or system.\",\n \"In case, a unicast address is assigned to more than one interface, thus turning the unicast address into a respective anycast address, the nodes of the network to which the address is assigned must be explicitly configured to interpret the address as an anycast address.\",\n \"According to the IPv6, an anycast address must not be assigned to a host, but may be assigned to an IPv6 router, only.\",\n \"Usually, for any assigned anycast address, an address prefix P is utilized which identifies a topological region in which all interfaces belonging to an anycast address reside.\",\n \"In the region identified by the address prefix P, each member of the respective anycast group is advertised as separate entry in a network routing protocol or system.\",\n \"Outside the region identified by the address prefix P, the anycast address may be aggregated into the routing advertisement for the address prefix P. Anycast Host Registration In order to join an anycast group, a host instructs its first hop router, i.e.\",\n \"the closest router in relation to the host for data communications via a network, to advertise the anycast address of the anycast group on its behalf.\",\n \"This can be achieved by adding a new message type to either the Internet Group Management Protocol (IGMP) or the Neighbor Discovery Protocol.\",\n \"Then, the first hop router advertises the anycast address according to an anycast routing protocol employed for the domain including the host.\",\n \"Anycast Routing Each anycast group is confined to a particular topological region with which it shares a common address prefix.\",\n \"Within the topological region identified by the shared address prefix, each member of the anycast group is advertised as a separate entry in the employed unicast routing protocol or system.\",\n \"The principle of anycast routing within a topological region identified by an address prefix shared by an anycast group is illustrated in FIG.\",\n \"2 .\",\n \"As can be derived from the table in FIG.\",\n \"2 , there are multiple paths to the anycast address.\",\n \"In case, multiple paths exist to a destination address prefix, a routing table look up of the router will return multiple next hops.\",\n \"The selection of the next hop router to be used for data communications, e.g.\",\n \"a particular data packet, depends on the implemented routing protocol or system.\",\n \"Further, the path selected for data communications can also be effected by the type of data communications to be performed.\",\n \"For example in case of the Internet Protocol standard, the Type Of Service (TOS) field in the IP header of a data packet can be used to define a data communications path.\",\n \"Thus, a TOS designation of a data packet would help the router to chose an appropriate communication path for the given data packet.\",\n \"The selection of an appropriate path is accomplished on the basis on a determination of the distances to the destination defined by the address prefix according to a measure of distance of the respective routing protocol or system.\",\n \"In case of the Internet Protocol standard, for example, the OSPF knows the distance related to the corresponding matrix as identified in the TOS field of a data packet to the destination, i.e.\",\n \"the hop count.\",\n \"As a result, for an anycast routing it is possible to select the nearest next hop on the basis of the employed matrix.\",\n \"In this respect, it is not necessary to analyze the whole IP address of a given data packet.\",\n \"In FIG.\",\n \"2 , this in indicated by the destination address Mx wherein only the prefix “M” is analyzed.\",\n \"For an anycast routing outside the topological region identified by the shared address prefix, the anycast address may be aggregerated into the routing advertisement for the shared address prefix.\",\n \"This principle is illustrated in FIG.\",\n \"3 .\",\n \"The destination address Ax in FIG.\",\n \"3 denotes that only the shared address prefix is utilized in order to determine that data communications are to be routed to the corresponding domain.\",\n \"Since the anycast address for the anycast group shares the address prefix with network domain A, network domain B cannot aggregerate the anycast address in its address prefix.\",\n \"Therefore, network domain B should advertise the anycast address as a separate entry covering both anycast members A 4 and A 5 .\",\n \"This is indicated in FIG.\",\n \"3 by the destination address Ax being based on the address prefix for domain A and destination address Ax′ containing the complete anycast address.\",\n \"The routing for data communications within the network domains A and B is performed as described before with reference to FIG.\",\n \"2 .\",\n \"Strict and Loose Routing According to the Internet Protocol version 4 (IPv4) and the Internet Protocol version 6 (IPv6), the following options for routing data communications from a source (e.g.\",\n \"sender, requesting system) to a destination (e.g.\",\n \"receiver, accessed system) have been defined as part of the IP data packet header: Strict Routing This option defines a complete data communications path from a source to a destination by means of a sequence of IP addresses.\",\n \"Data to be communicated between the source and the destination is required to exactly follow the defined path.\",\n \"Loose Routing This option specifies a number of routers and an order thereof.\",\n \"Data to be communicated from a source to a destination is required to traverse the specified routers in the specified order, but is allowed to be communicated via other routers on its way from the source to the destination.\",\n \"Problems Existing solutions for networks being capable of anycast routing are restricted to the selection of a single nearest receiver for data communications with an accessing sender.\",\n \"In case a sender intends to access more than one receiver, existing anycast networks do no provide such a service.\",\n \"A situation for which accesses to more than one receiver are desired is, for example, a network user wanting to access a number of nearest network servers in order to negotiate for the best network service conditions.\",\n \"A further example is a network user wishing to contact a number of nearest restaurants by accessing the network servers thereof in order to check e.g.\",\n \"meals, prices, available seats, etc.\",\n \"or wishing to access a number of nearest emergency service providers in order to ensure that at least one is able to assist (note for the latter examples, sufficient information for the accessing user is only provided in case there is a geographical relationship between the restaurants and the emergency service providers and the corresponding servers).\",\n \"In conventional networks, accesses to more than one receiver can be accomplished by accessing a number of receivers by individually addressing the same on the basis of a unicast routing or by accessing a group of receivers by commonly addressing the same on the basis of a shared multicasting address.\",\n \"Such a multiple unicast routing requires that an accessing user must know each individual network address of the desired receivers and individually perform accesses thereto.\",\n \"This is a time consuming procedure which cannot lead to the desired information for a user in a case, for example, he/she is not provided the respective network addresses and/or information concerning distances to the accessed servers.\",\n \"With respect to the above given example concerning a negotiation for the best network service conditions by accessing a number of nearest servers, such a multiple unicast routing is also not appropriate since no information indicative of distances to receivers is provided and/or available.\",\n \"Accesses on the basis of a multicast routing can result in a number of contacted receivers being too high in view of the demands and desired information of a requesting sender/user.\",\n \"Further, with multicast routing it is not possible to access a desired number of receivers and, in particular, a desired number of nearest receivers, since all receivers being a member of the respective multicasting group are addressed together.\",\n \"Furthermore, a second attempt, e.g.\",\n \"by a multiple unicast routing, to access/contact receivers, the number thereof being higher compared to the number of receivers specified in a preceding, first attempt, would unnecessarily return the results from the first attempt again.\",\n \"This repetition of data communications with respect to the result of the first attempt unnecessarily utilizes network resources in case the requesting network user is not interested in being provided the first attempt results again.\"\n ],\n [\n \"<SOH> BRIEF DESCRIPTION OF THE INVENTION <EOH>The approach underlying the present invention to obtain the above object is to extend principles employed in existing anycast networks for accessing a single nearest client in order to allow for a multiple anycast routing to a specified number of nearest receivers.\",\n \"In principle, this is achieved by associating data communications from a sender directed to a network by means of an anycast routing to data which indicate the number of nearest receivers.\",\n \"In particular, such data which will be referred to as a first indicator in the following, specifies the number of nearest receivers being anycast members of an anycast group identified by an anycast address given by the sender (left) to be contacted by and/or allocated for the sender for data communications, such as requests, accesses, data transmissions, etc.\",\n \"According to the present invention, a method for multiple hosts anycast routing in a network is provided wherein sender data is communicated from a sender to an anycast group including anycast members.\",\n \"For communicating the sender data to the anycast group, the sender data includes an anycast address for addressing the anycast group.\",\n \"In order to specify a number of the anycast members for data communications with the sender, the sender data further includes a first indicator being indicative of the desired, first number of anycast members.\",\n \"On the basis of the sender data, a number of nearest anycast members is determined as hosts with respect to the sender for data communications.\",\n \"In particular, the number of nearest anycast members being determined is defined by the first indicator.\",\n \"This solution provides for data communications between a sender and a number of receivers without addressing the same separately, but addressing a plurality of possible receivers by a single address being common for all receivers and selecting a desired number therefrom by means of a data being simply amended to the single common address.\",\n \"A further benefit is that it can be employed for any network, even for networks not providing known anycast routing services.\",\n \"After having performed the above described method it may be possible that a further multiple host anycast routing should be performed wherein the number of anycast members to be determined is varied compared to the number of anycast members previously specified.\",\n \"In order to avoid that anycast members already determined in the previous multiple host anycast routing are contacted again a second indicator can be used.\",\n \"The second indicator specifies the number of anycast members previously determined in the preceding multiple host anycast routing.\",\n \"Then, the number of nearest anycast members can be determined on the basis of the first and the second indicator.\",\n \"In case the first indicator specifies a number of anycast members then the second indicator does, the number of nearest anycast members indicated by the second indicator (in the following: second number of anycast members) are skipped and not contacted, while a number of anycast member is determined for data communications with the sender up to a number obtained by a comparison of the number of anycast members specified by the first indicator (in the following: first number) and the second number.\",\n \"In particular, the number of nearest anycast members to be determined corresponds to the difference between the first number and the second number.\",\n \"In case the first number corresponds to the second number, no further anycast members have to be determined.\",\n \"The same applies for the case wherein the first number is smaller than the second number.\",\n \"In order to provide for a method according to the invention for multiple hosts anycast routing to be employed in a network providing a known anycast routing, a first indicator specifying a first number of anycast members to be contacted/allocated for data communications is associated to an anycast address.\",\n \"By means of the anycast address, an anycast group having anycast members is identified in the network.\",\n \"To establish the desired data communications, at least the anycast address and the first indicator is communicated in the network to the anycast group.\",\n \"The anycast group is identified by the anycast address, while the number of anycast members to be contacted/allocated for data communications is selected according to the first indicator.\",\n \"In particular, the first indicator defines the number of nearest anycast members and according to the first indicator, the specified number of nearest anycast members is determined as hosts for data communications in the network.\",\n \"Comparable to the embodiment of the above described method for multiple hosts anycast routing in a network, this method for multiple hosts anycast routing in an anycast capable network can employ a second indicator specifying a number of anycast members (in the following: second number) already determined for data communications in the network, e.g.\",\n \"on the basis of a previously performed multiple hosts anycast routing in a (anycast capable) network.\",\n \"For providing the first and/or the second indicator, it is possible to include the indicator(s) in the anycast address or to extend the same by the first and/or the second indicator.\",\n \"Preferably, the anycast address is included an anycast address prefix which identifies a topological region in the network, the topological region confining the anycast group.\",\n \"Here it is possible, that the anycast address prefix includes the first and/or the second indicator or is extended by the same.\",\n \"In a case of a network being operated according to an Internet Protocol standard (e.g.\",\n \"IPv4 or IPv6) the anycast address and the indicator can be communicated by a transmission of an Internet Protocol data packet header which includes at least the anycast address.\",\n \"For communicating the first and/or the second indicator, the Internet Protocol data packet header can be included the first and/or the second indicator as a part of or an extension for the anycast address.\",\n \"With respect to the options mechanisms defined for the Internet Protocols, the first and/or the second indicator can be provided by extending the Internet Protocol data packet header by an options field and, in particular, a so-called multiple anycasting options field including the first and/or the second indicator.\",\n \"It is preferred, that the property “nearest” of anycast members is determined according to a measure of distance of the respective routing protocol employed for the network.\",\n \"In particular, “nearest” may be understood as the shortest distance between a sender and anycast members.\",\n \"Additionally or as an option, “nearest” may be specified according to a distance metric requested by the sender.\",\n \"Usually, the distance metric requested by the sender, e.g.\",\n \"in the TOS field of an IP header, indicates a smaller distance metrics or nearest distance, respectively.\",\n \"In case the network comprises domains at least some of which including at least of the anycast members, the anycast address and the first and/or the second indicator can be communicated via network routers to and/or between domains having anycast members.\",\n \"Since it is assumed that a nearest network domain will also include anycast members being nearer compared to anycast members of more distant domains, it is preferred that the ordering of routers for network domain communications is set up such that the anycast address and the first and/or the second indicator are communicated to nearest network domains.\",\n \"The nearest network domains may specified according to a measure of distance of a routing protocol for the network and/or according to a (e.g.\",\n \"smallest) distance metric requested by the sender, e.g.\",\n \"in the TOS field.\",\n \"In order to provide for a fast multiple anycasting, the number of anycast members can be contacted sequentially, in particular for a small number specified by the first and/or the second indicator.\",\n \"Within a network domain, multiple anycast routing and, in particular, a communication of the anycast address and the first and/or the second indicator can be accomplished on the basis of data identifying each of the anycast members.\",\n \"Such an identifying data can be, for example, provided by means of separate entries for anycast members in a routing table according to the employed routing protocol.\",\n \"With respect to the anycast routing performed for a network domain, the first indicator can be updated by decreasing the same according to a number of anycast members comprised by a network domain being identified by means of the anycast address and receiving the first indicator.\",\n \"Further, the anycast routing can be enhanced by multiplying the anycast address and the first and/or the second indicator and communicating the multiplied anycast addresses and indicators to network domains and/or anycast members.\",\n \"Here, it is preferred to communicate the multiplied data simultaneously to the further network domain and/or anycast members.\",\n \"In order to precisely select the specified number of anycast members, while determining the number of anycast members, the indicator is modified according to a number of already determined/contacted/allocated nearest anycast members such that the modified indicator being communicated in the network specifies a number of anycast members still to be determined as receivers.\",\n \"This embodiment avoids a utilization of further data beside the indicator to provide information how many receivers are already obtained and how many are still to be selected.\",\n \"According to an Internet Protocol standard, the communication in the network can be performed by using the strict routing mechanism or the loose routing mechanism.\",\n \"Preferably, the first and the second indicator are communicated together and, advantageously in the same manner, e.g.\",\n \"both indicators being included in the anycast address or extending the same.\",\n \"Further, the present invention provides a system for multiple hosts anycast routing and an anycast routing capable network which are preferred to be operated according to the method according to the invention as set forth above.\",\n \"Moreover, the present invention provides a computer program product being adapted to carry out the method steps according to the invention and embodiments thereof as set forth above.\"\n ],\n [\n \"BACKGROUND OF THE INVENTION 1.Technical Field The present invention relates to a method and a system for establishing data communications between a sender and receivers, the number thereof being defined by the sender, wherein the receivers are not individually addressed by the sender, but selected from a group of receivers being addressed from the sender by means of a common address.\",\n \"2.Background of the Invention An anycast network is a network providing a service which allows a sender to access the nearest of a group of receivers having a common address.\",\n \"The common address, in particular, the same anycast address for the group of receivers enables a sender to identify a plurality of receivers by providing only one address, namely an anycast address, instead of individually addressing each receiver.\",\n \"In contrast to a multicast system, wherein data communications are performed between a sender and all receivers sharing a common address, i.e.\",\n \"the same multicast address, data communications in an anycast network are performed between a sender and a particular receiver of a group of receivers sharing the same anycast address.\",\n \"In the context, data communications include any kind of data, such as alphanumerical, graphic, multimedia, audio, video, voice data, information, signals etc.\",\n \"which can be exchanged between systems, devices, network components etc.\",\n \"(e.g.\",\n \"computers, end user devices, hosts, servers, routers, modes).\",\n \"In particular, data communications in an anycast network are performed between a sender and the nearest receiver of the group of receivers sharing the same anycast address.\",\n \"In this respect, the nearest receiver in relation to a sender is defined according to a measure of a distance employed by a routing protocol or routing system used for data communications in a respective network.\",\n \"Since the routing protocol or routing system of a network which identifies the nearest receiver for accesses by a sender, the sender does not need to care about how to select the closest destination, i.e.\",\n \"the nearest receiver.\",\n \"Usually, receivers in an anycast group are so-called replicas being able to support a same network service for requesting sender.\",\n \"Examples for such replicas are mirrored web servers.\",\n \"For accessing a desired network service, among a group of receivers sharing the same anycast address, i.e.\",\n \"anycast receivers, each thereof providing the desired network service, the nearest receiver is determined and respective data communications are performed between the nearest receiver and the requesting sender.\",\n \"Thus, accessing the nearest receiver enhances the network performance perceived by the requesting sender, saves network capacities such as network's bandwidth, and provides the desired network service.\",\n \"In FIG.\",\n \"1, the basic principle of an anycast network is illustrated.\",\n \"Both, members M1 and M2 are members of the same anycast group and share the same respective anycast address.\",\n \"In line with the measure of a distance employed for routing of data in the network, the distances between requesting senders S1 and S2 and all members of the anycast group are computed.\",\n \"Since the distance 2 calculated for the sender S1 in relation to the anycast group members is the smallest distance, data communications will be performed between the sender S1 and the member M1 acting as a receiver for the sender S1.In this manner, it is possible for example to direct Internet Protocol (IP) datagrams from the sender S1 towards the member M1.Accordingly, data communications are established between the sender S2 and the member M2 being a nearest receiver with respect to the sender S2, for example to direct IP datagrams from the sender S2 towards the member M2.Anycast Addresses According to the Internet Protocol version 6 (IPv6), special anycast addresses have been specified in addition to unicast addresses for individually addressing single receivers and multicast addresses for addressing all receivers sharing the same multicast address.\",\n \"Anycast addresses are allocated from the address space employed for unicast addresses, using any of the formats defined for unicast addresses.\",\n \"As a result, anycast addresses cannot be syntactically distinguished from unicast addresses.\",\n \"An anycast address identifies a set of interfaces which typically belong to different nodes of a network.\",\n \"Data, e.g.\",\n \"a data packet, communicated from a sender to an anycast address is delivered to one of the interfaces identified by the respective anycast address, in particular, to the nearest interface according to the measure of a distance employed for the network routing protocol or system.\",\n \"In case, a unicast address is assigned to more than one interface, thus turning the unicast address into a respective anycast address, the nodes of the network to which the address is assigned must be explicitly configured to interpret the address as an anycast address.\",\n \"According to the IPv6, an anycast address must not be assigned to a host, but may be assigned to an IPv6 router, only.\",\n \"Usually, for any assigned anycast address, an address prefix P is utilized which identifies a topological region in which all interfaces belonging to an anycast address reside.\",\n \"In the region identified by the address prefix P, each member of the respective anycast group is advertised as separate entry in a network routing protocol or system.\",\n \"Outside the region identified by the address prefix P, the anycast address may be aggregated into the routing advertisement for the address prefix P. Anycast Host Registration In order to join an anycast group, a host instructs its first hop router, i.e.\",\n \"the closest router in relation to the host for data communications via a network, to advertise the anycast address of the anycast group on its behalf.\",\n \"This can be achieved by adding a new message type to either the Internet Group Management Protocol (IGMP) or the Neighbor Discovery Protocol.\",\n \"Then, the first hop router advertises the anycast address according to an anycast routing protocol employed for the domain including the host.\",\n \"Anycast Routing Each anycast group is confined to a particular topological region with which it shares a common address prefix.\",\n \"Within the topological region identified by the shared address prefix, each member of the anycast group is advertised as a separate entry in the employed unicast routing protocol or system.\",\n \"The principle of anycast routing within a topological region identified by an address prefix shared by an anycast group is illustrated in FIG.\",\n \"2.As can be derived from the table in FIG.\",\n \"2, there are multiple paths to the anycast address.\",\n \"In case, multiple paths exist to a destination address prefix, a routing table look up of the router will return multiple next hops.\",\n \"The selection of the next hop router to be used for data communications, e.g.\",\n \"a particular data packet, depends on the implemented routing protocol or system.\",\n \"Further, the path selected for data communications can also be effected by the type of data communications to be performed.\",\n \"For example in case of the Internet Protocol standard, the Type Of Service (TOS) field in the IP header of a data packet can be used to define a data communications path.\",\n \"Thus, a TOS designation of a data packet would help the router to chose an appropriate communication path for the given data packet.\",\n \"The selection of an appropriate path is accomplished on the basis on a determination of the distances to the destination defined by the address prefix according to a measure of distance of the respective routing protocol or system.\",\n \"In case of the Internet Protocol standard, for example, the OSPF knows the distance related to the corresponding matrix as identified in the TOS field of a data packet to the destination, i.e.\",\n \"the hop count.\",\n \"As a result, for an anycast routing it is possible to select the nearest next hop on the basis of the employed matrix.\",\n \"In this respect, it is not necessary to analyze the whole IP address of a given data packet.\",\n \"In FIG.\",\n \"2, this in indicated by the destination address Mx wherein only the prefix “M” is analyzed.\",\n \"For an anycast routing outside the topological region identified by the shared address prefix, the anycast address may be aggregerated into the routing advertisement for the shared address prefix.\",\n \"This principle is illustrated in FIG.\",\n \"3.The destination address Ax in FIG.\",\n \"3 denotes that only the shared address prefix is utilized in order to determine that data communications are to be routed to the corresponding domain.\",\n \"Since the anycast address for the anycast group shares the address prefix with network domain A, network domain B cannot aggregerate the anycast address in its address prefix.\",\n \"Therefore, network domain B should advertise the anycast address as a separate entry covering both anycast members A4 and A5.This is indicated in FIG.\",\n \"3 by the destination address Ax being based on the address prefix for domain A and destination address Ax′ containing the complete anycast address.\",\n \"The routing for data communications within the network domains A and B is performed as described before with reference to FIG.\",\n \"2.Strict and Loose Routing According to the Internet Protocol version 4 (IPv4) and the Internet Protocol version 6 (IPv6), the following options for routing data communications from a source (e.g.\",\n \"sender, requesting system) to a destination (e.g.\",\n \"receiver, accessed system) have been defined as part of the IP data packet header: Strict Routing This option defines a complete data communications path from a source to a destination by means of a sequence of IP addresses.\",\n \"Data to be communicated between the source and the destination is required to exactly follow the defined path.\",\n \"Loose Routing This option specifies a number of routers and an order thereof.\",\n \"Data to be communicated from a source to a destination is required to traverse the specified routers in the specified order, but is allowed to be communicated via other routers on its way from the source to the destination.\",\n \"Problems Existing solutions for networks being capable of anycast routing are restricted to the selection of a single nearest receiver for data communications with an accessing sender.\",\n \"In case a sender intends to access more than one receiver, existing anycast networks do no provide such a service.\",\n \"A situation for which accesses to more than one receiver are desired is, for example, a network user wanting to access a number of nearest network servers in order to negotiate for the best network service conditions.\",\n \"A further example is a network user wishing to contact a number of nearest restaurants by accessing the network servers thereof in order to check e.g.\",\n \"meals, prices, available seats, etc.\",\n \"or wishing to access a number of nearest emergency service providers in order to ensure that at least one is able to assist (note for the latter examples, sufficient information for the accessing user is only provided in case there is a geographical relationship between the restaurants and the emergency service providers and the corresponding servers).\",\n \"In conventional networks, accesses to more than one receiver can be accomplished by accessing a number of receivers by individually addressing the same on the basis of a unicast routing or by accessing a group of receivers by commonly addressing the same on the basis of a shared multicasting address.\",\n \"Such a multiple unicast routing requires that an accessing user must know each individual network address of the desired receivers and individually perform accesses thereto.\",\n \"This is a time consuming procedure which cannot lead to the desired information for a user in a case, for example, he/she is not provided the respective network addresses and/or information concerning distances to the accessed servers.\",\n \"With respect to the above given example concerning a negotiation for the best network service conditions by accessing a number of nearest servers, such a multiple unicast routing is also not appropriate since no information indicative of distances to receivers is provided and/or available.\",\n \"Accesses on the basis of a multicast routing can result in a number of contacted receivers being too high in view of the demands and desired information of a requesting sender/user.\",\n \"Further, with multicast routing it is not possible to access a desired number of receivers and, in particular, a desired number of nearest receivers, since all receivers being a member of the respective multicasting group are addressed together.\",\n \"Furthermore, a second attempt, e.g.\",\n \"by a multiple unicast routing, to access/contact receivers, the number thereof being higher compared to the number of receivers specified in a preceding, first attempt, would unnecessarily return the results from the first attempt again.\",\n \"This repetition of data communications with respect to the result of the first attempt unnecessarily utilizes network resources in case the requesting network user is not interested in being provided the first attempt results again.\",\n \"OBJECT OF THE INVENTION The object of the present invention is to provide for a method and a system allowing for accesses of a sender to a specified number of nearest receivers and/or data communications between a sender and a specified number of nearest receivers.\",\n \"BRIEF DESCRIPTION OF THE INVENTION The approach underlying the present invention to obtain the above object is to extend principles employed in existing anycast networks for accessing a single nearest client in order to allow for a multiple anycast routing to a specified number of nearest receivers.\",\n \"In principle, this is achieved by associating data communications from a sender directed to a network by means of an anycast routing to data which indicate the number of nearest receivers.\",\n \"In particular, such data which will be referred to as a first indicator in the following, specifies the number of nearest receivers being anycast members of an anycast group identified by an anycast address given by the sender (left) to be contacted by and/or allocated for the sender for data communications, such as requests, accesses, data transmissions, etc.\",\n \"According to the present invention, a method for multiple hosts anycast routing in a network is provided wherein sender data is communicated from a sender to an anycast group including anycast members.\",\n \"For communicating the sender data to the anycast group, the sender data includes an anycast address for addressing the anycast group.\",\n \"In order to specify a number of the anycast members for data communications with the sender, the sender data further includes a first indicator being indicative of the desired, first number of anycast members.\",\n \"On the basis of the sender data, a number of nearest anycast members is determined as hosts with respect to the sender for data communications.\",\n \"In particular, the number of nearest anycast members being determined is defined by the first indicator.\",\n \"This solution provides for data communications between a sender and a number of receivers without addressing the same separately, but addressing a plurality of possible receivers by a single address being common for all receivers and selecting a desired number therefrom by means of a data being simply amended to the single common address.\",\n \"A further benefit is that it can be employed for any network, even for networks not providing known anycast routing services.\",\n \"After having performed the above described method it may be possible that a further multiple host anycast routing should be performed wherein the number of anycast members to be determined is varied compared to the number of anycast members previously specified.\",\n \"In order to avoid that anycast members already determined in the previous multiple host anycast routing are contacted again a second indicator can be used.\",\n \"The second indicator specifies the number of anycast members previously determined in the preceding multiple host anycast routing.\",\n \"Then, the number of nearest anycast members can be determined on the basis of the first and the second indicator.\",\n \"In case the first indicator specifies a number of anycast members then the second indicator does, the number of nearest anycast members indicated by the second indicator (in the following: second number of anycast members) are skipped and not contacted, while a number of anycast member is determined for data communications with the sender up to a number obtained by a comparison of the number of anycast members specified by the first indicator (in the following: first number) and the second number.\",\n \"In particular, the number of nearest anycast members to be determined corresponds to the difference between the first number and the second number.\",\n \"In case the first number corresponds to the second number, no further anycast members have to be determined.\",\n \"The same applies for the case wherein the first number is smaller than the second number.\",\n \"In order to provide for a method according to the invention for multiple hosts anycast routing to be employed in a network providing a known anycast routing, a first indicator specifying a first number of anycast members to be contacted/allocated for data communications is associated to an anycast address.\",\n \"By means of the anycast address, an anycast group having anycast members is identified in the network.\",\n \"To establish the desired data communications, at least the anycast address and the first indicator is communicated in the network to the anycast group.\",\n \"The anycast group is identified by the anycast address, while the number of anycast members to be contacted/allocated for data communications is selected according to the first indicator.\",\n \"In particular, the first indicator defines the number of nearest anycast members and according to the first indicator, the specified number of nearest anycast members is determined as hosts for data communications in the network.\",\n \"Comparable to the embodiment of the above described method for multiple hosts anycast routing in a network, this method for multiple hosts anycast routing in an anycast capable network can employ a second indicator specifying a number of anycast members (in the following: second number) already determined for data communications in the network, e.g.\",\n \"on the basis of a previously performed multiple hosts anycast routing in a (anycast capable) network.\",\n \"For providing the first and/or the second indicator, it is possible to include the indicator(s) in the anycast address or to extend the same by the first and/or the second indicator.\",\n \"Preferably, the anycast address is included an anycast address prefix which identifies a topological region in the network, the topological region confining the anycast group.\",\n \"Here it is possible, that the anycast address prefix includes the first and/or the second indicator or is extended by the same.\",\n \"In a case of a network being operated according to an Internet Protocol standard (e.g.\",\n \"IPv4 or IPv6) the anycast address and the indicator can be communicated by a transmission of an Internet Protocol data packet header which includes at least the anycast address.\",\n \"For communicating the first and/or the second indicator, the Internet Protocol data packet header can be included the first and/or the second indicator as a part of or an extension for the anycast address.\",\n \"With respect to the options mechanisms defined for the Internet Protocols, the first and/or the second indicator can be provided by extending the Internet Protocol data packet header by an options field and, in particular, a so-called multiple anycasting options field including the first and/or the second indicator.\",\n \"It is preferred, that the property “nearest” of anycast members is determined according to a measure of distance of the respective routing protocol employed for the network.\",\n \"In particular, “nearest” may be understood as the shortest distance between a sender and anycast members.\",\n \"Additionally or as an option, “nearest” may be specified according to a distance metric requested by the sender.\",\n \"Usually, the distance metric requested by the sender, e.g.\",\n \"in the TOS field of an IP header, indicates a smaller distance metrics or nearest distance, respectively.\",\n \"In case the network comprises domains at least some of which including at least of the anycast members, the anycast address and the first and/or the second indicator can be communicated via network routers to and/or between domains having anycast members.\",\n \"Since it is assumed that a nearest network domain will also include anycast members being nearer compared to anycast members of more distant domains, it is preferred that the ordering of routers for network domain communications is set up such that the anycast address and the first and/or the second indicator are communicated to nearest network domains.\",\n \"The nearest network domains may specified according to a measure of distance of a routing protocol for the network and/or according to a (e.g.\",\n \"smallest) distance metric requested by the sender, e.g.\",\n \"in the TOS field.\",\n \"In order to provide for a fast multiple anycasting, the number of anycast members can be contacted sequentially, in particular for a small number specified by the first and/or the second indicator.\",\n \"Within a network domain, multiple anycast routing and, in particular, a communication of the anycast address and the first and/or the second indicator can be accomplished on the basis of data identifying each of the anycast members.\",\n \"Such an identifying data can be, for example, provided by means of separate entries for anycast members in a routing table according to the employed routing protocol.\",\n \"With respect to the anycast routing performed for a network domain, the first indicator can be updated by decreasing the same according to a number of anycast members comprised by a network domain being identified by means of the anycast address and receiving the first indicator.\",\n \"Further, the anycast routing can be enhanced by multiplying the anycast address and the first and/or the second indicator and communicating the multiplied anycast addresses and indicators to network domains and/or anycast members.\",\n \"Here, it is preferred to communicate the multiplied data simultaneously to the further network domain and/or anycast members.\",\n \"In order to precisely select the specified number of anycast members, while determining the number of anycast members, the indicator is modified according to a number of already determined/contacted/allocated nearest anycast members such that the modified indicator being communicated in the network specifies a number of anycast members still to be determined as receivers.\",\n \"This embodiment avoids a utilization of further data beside the indicator to provide information how many receivers are already obtained and how many are still to be selected.\",\n \"According to an Internet Protocol standard, the communication in the network can be performed by using the strict routing mechanism or the loose routing mechanism.\",\n \"Preferably, the first and the second indicator are communicated together and, advantageously in the same manner, e.g.\",\n \"both indicators being included in the anycast address or extending the same.\",\n \"Further, the present invention provides a system for multiple hosts anycast routing and an anycast routing capable network which are preferred to be operated according to the method according to the invention as set forth above.\",\n \"Moreover, the present invention provides a computer program product being adapted to carry out the method steps according to the invention and embodiments thereof as set forth above.\",\n \"BRIEF DESCRIPTION OF THE FIGURES In the following description of preferred embodiments of the present invention it is referred to the accompanying figures wherein: FIG.\",\n \"1 illustrates the basic principle of anycast routing in a network according to the state of the art, FIG.\",\n \"2 illustrates an anycast routing within a topological region identified by a common address prefix according to the state of the art, FIG.\",\n \"3 illustrates an anycast routing outside a topological region identified by a common address prefix according to the state of the art, FIG.\",\n \"4 illustrates a data structure for an anycast address according to the present invention, and FIG.\",\n \"5 illustrates a multiple anycast routing according to the present invention.\",\n \"DESCRIPTION OF PREFERRED EMBODIMENTS In order to promote the understanding of the present invention preferred embodiments are described with respect to an network according to the Internet Protocol standard.\",\n \"In such a network, data communications are performed by transmitting IP data packets, short packets, each including an IP packet header, short header, between hosts, i.e.\",\n \"sending and receiving systems within the network.\",\n \"Each host is associated to a network domain defining a topological network region.\",\n \"Data communications between network domains are performed via exterior gateway routers, whereas data communications within a network domain are performed via interior gateway routers.\",\n \"Exterior gateway routers directly receiving packets from a network domain are called first exterior gateway routers and interior gateway routers directly receiving packets from an exterior gateway router are called border routers.\",\n \"Hops designate network components via which packets are communicated.\",\n \"In particular, hops include hosts and routers.\",\n \"For data communications in the network, i.e.\",\n \"routing of packets from a source to a destination, different routing protocols are used for transmission within a domain (intra-domain routing) and between domains (inter-domain routing).\",\n \"For example, an intra-domain routing can be based on interior gateway routing protocols, such as OSPF, while inter-domain routing can employ exterior gateway routing protocols such as BGP.\",\n \"According to the routing protocols, a routing table is defined which specifies a communication path for packets from a source to a destination and, in particular, hops via which packets are to be transmitted.\",\n \"As explained above, both, loose routing and strict routing are possible, whereas for a multiple hosts anycasting most probably loose routing will be applied.\",\n \"IP packet headers include information specifying a destination in the network to which packets are to be communicated from a source by means of destination addresses.\",\n \"In case of unicast routing a destination corresponds to a receiving host, short receiver, whereas in case of multicast routing, conventional anycast routing, and, specifically, multiple host anycast routing a destination comprises a group of hosts each thereof, only one thereof, and a specified number thereof acting as receiver(s) with respect to a sending host, short sender.\",\n \"In case of a multiple hosts anycast routing, short multiple anycasting, the number of hosts, i.e.\",\n \"members of an anycast group, to be contacted is specified by extending a packet and, in particular, its header by an indicator.\",\n \"The indicator providing information of the number of hosts to be contacted/allocated as originally specified by the sender and the number of hosts which still have to be contacted/allocated as receivers indicates the number of hosts that (still) have to be informed by the corresponding branch utilizing the following embodiments.\",\n \"Indicator as Part of the IP Destination Address Field in the IP Header Since anycast routing is mostly based on resolving the above described address prefix, data (e.g.\",\n \"some bits) of an IP destination address and, in particular, an address prefix is used for the indication of a sender-specified number of nearest hosts being anycast members of an anycast group indicated by the address prefix.\",\n \"In the exemplary illustration of FIG.\",\n \"4, the IP packet header includes an anycast address prefix and an indicator specifying the number of nearest anycast members (left) to be contacted/allocated.\",\n \"Initially, the indicator specifies the number of nearest anycast members as desired by a sender, while communicating the packet within the network the indicator is decreased corresponding with the number of already selected receivers (already allocated anycast members).\",\n \"For the examples given above wherein more than one host is to be contacted as receiver, it is assumed that the sender specified number of desired nearest anycast members is rather low, e.g.\",\n \"less than 10.In such applications of multiple anycasting, it might be sufficient to, for example, use 4 bits of an IP packet header for the indicator to allow for a maximum of 24(16) nearest anycast members to be contacted.\",\n \"In applications wherein a specification of a higher number of nearest anycast members should be provided, the amount of data (e.g.\",\n \"number of bits) of an IP packet header is to be set correspondingly.\",\n \"Indicator as New Options Extension to an IP Extension of an IP Header On the basis of existing IP options mechanism (e.g.\",\n \"as explained above with respect to strict and loose routing), an IP packet header is extended by a so-called “multiple anycasting” options field containing an indicator as specified above.\",\n \"In case a packet is destinated for an anycast address, the respective header including anycasting address information, anycast capable routers only analyses the extension, namely the “multiple anycasting” option field.\",\n \"Modifications of the extension can be performed, for example, to update the “multiple anycasting” option field to be in line with the number of anycast members still to be contacted/allocated in view of already contacted/allocated anycast members.\",\n \"As set forth above, data communications are differently performed for intra-domain and inter-domain communications.\",\n \"As a result, multiple anycasting will work different for intra-domain cases employing interior gateway routing protocols such as OSPF and inter-domain cases employing exterior gateway protocols such as BGP.\",\n \"In the following inter-domain multiple hosts anycasting and intra-domain multiple hosts anycasting will be described with reference to FIG.\",\n \"5 illustrating an exemplary topology and routing scenario wherein a host H in a domain C communicates an anycast packet within a network comprising domains A, B and C and exterior gateway routers R1, R2, R3 and R4.For this example it is assumed that the anycast group addressed by the host H comprises the anycast members A1, A2 and A3 in domain A and anycast members A4 and A5 in domain B.\",\n \"In particular, host H communicates an anycast packet including an indicator specifying a number of three anycast members to be contacted as receivers, i.e.\",\n \"nearest anycast members.\",\n \"Inter-Domain Multiple Hosts Anycasting Inter-domain multiple hosts anycasting is indicated by steps 1 and 3 shown in FIG.\",\n \"5.On the basis of an employed exterior gateway routing protocol, the first exterior gateway router with respect to host H in domain C, i.e.\",\n \"router R4, will check its routing tables in order to determine anycast members of the anycast group as specified by the anycast packet of host H and, in particular, all domains including the respective anycast members.\",\n \"The corresponding routers for determined domains, i.e.\",\n \"routers R1 and R3 to domains A and B, will be added to the IP packet header of the anycast packet from host H by utilizing strict or loose routing options.\",\n \"Depending on the metrics used for the employed exterior gateway routing protocol and/or the smallest distance metric requested by the sender, i.e.\",\n \"host H, (e.g.\",\n \"defined in the TOS field), the ordering of the routers will be based on distances between domains including anycast members and anycast members, respectively, and host H. According to FIG.\",\n \"5, the anycast packet of host H will be first communicated to router R3.In case the number of anycast members contacted/allocated in a domain receiving the anycast packet from router R3 is less than the number of nearest anycast members to be contacted as defined by host H, the anycast packet will be communicated to router R1.The anycast packet and, in particular, the header thereof transmitted to router R1 includes an indicator specifying the number of anycast members which are still to be contacted/allocated.\",\n \"With reference to FIG.\",\n \"5, the indicator communicated to router R1 indicates that one anycast member is to be contacted/allocated assuming that anycast members R4 and R5 of domain B are selected as nearest anycast members.\",\n \"As specified by the strict or loose routing options of the IP packet header, the specified number of anycast members may be contacted sequentially.\",\n \"Assuming the specified number being rather low, e.g.\",\n \"2 to 5 anycast members, this procedure will not effect the network service performance received by requesting host, here host H. In case a high number of anycast member should be allowed to be specified by a sender/requesting host, various known data communication methods for enhancement of data transmissions in a network can be utilized for contacting anycast members.\",\n \"Intra-Domain Multiple Hosts Anycasting Intra-domain multiple hosts anycasting will be explained with reference to steps 2 and 4 of FIG.\",\n \"5.At reception of an anycast packet in an border router for a domain, the border router will forward the anycast packet to all next hops, in particular, to the next anycast members of the respective domain.\",\n \"This can be accomplished since, as explained above, interior gateway routing protocols provide a separate entry in the routing table for each anycast member.\",\n \"For forwarding the anycast packet, the border router may multiply the anycast packet and simultaneously communicate the multiplied anycast packets to multiple destinations, i.e.\",\n \"next hops/anycast members.\",\n \"According to the number of anycast members and, in particular, of anycast members contacted/allocated in the corresponding domain, the number specified by the indicator in the header of the anycast packet is decreased.\",\n \"In case the indicator is 0 after an intra-domain multiple host anycasting, no data communications are required to contact/allocate further anycast members, e.g.\",\n \"of a different domain, since the number of nearest anycast members as desired by a requesting host is provided.\",\n \"Otherwise, the border router will forward the anycast packet including the decreased indicator to the next exterior gateway router for inter-domain multiple hosts anycasting as specified above.\",\n \"According to FIG.\",\n \"5, a border router of domain B receives the anycast packet from exterior gateway router R3 and will forward the anycast packet to the anycast members A4 and A5.For example, router R3 may serve as border router for domain B by combining exterior and interior gateway functions.\",\n \"In step 2 the border router of domain B contacts all anycast members in domain B such that they are specified for nearest anycast members for data communications with host H. As a result, still one further anycast member has to be contacted/allocated, whereby the anycast packet communicated from the border router of domain B via the exterior gateway router R3 to the exterior gateway router R1 includes a correspondingly decreased indicator, i.e.\",\n \"an indicator having the value of 1.A border router of domain A forwards the received anycast packet within domain A as specified above for contacting/allocating an anycast member.\",\n \"Comparable to router R3, router R1 combining exterior and interior gateway capabilities may be operated as border router for domain A.\",\n \"The routing within a domain may be performed in a manner comparable as the above described inter-domain routing in case hierarchies are used for the respective domain.\",\n \"Then, all levels of the same hierarchy in the domain can be contemplated as inter-domain cases, wherein levels of a lower hierarchy can be seen as intra-domain cases.\",\n \"Skipping the Number of Anycast Members Specified in a First Multiple Hosts Anycasting for Performing a Second Multiple Hosts Anycasting After having performed a first multiple hosts anycasting, it is possible that the respective host performs a second multiple hosts anycasting wherein the number of anycast members specified in the first multiple hosts anycasting should not be contacted/allocated (again).\",\n \"For that purpose it is contemplated as an ad-on to the above described multiple hosts anycast routing, but also to the existing anycast specification as known in the state of the art, that a sending/requesting host can specify the number of first anycast members (i.e.\",\n \"anycast members contacted/allocated in a first anycasting) that should be not contacted/allocated (again) in a second (multiple hosts) anycasting.\",\n \"After a previous attempt did not result in a successful anycast member search there may be no need to contact/allocate the same anycast members once more if the number of anycast members is to be extended in a subsequent attempt.\",\n \"In that case, the sending/requesting host could specify in a new optional IP header extension the number of first anycast members that should not be contacted/allocated in the subsequent attempt.\",\n \"Such an indication can be provided by a second indicator (counter) which is additionally checked, e.g.\",\n \"by routers, while performing the second (multiple hosts) anycast routing.\",\n \"In case a network domain includes anycast members, the additional indicator (counter) is first checked.\",\n \"If the second indicator indicates a number greater than 0, the second indicator is to be decreased until it reaches 0.For each decrease of the second indicator, the first multiple hosts indicator (counter), as described above, is decreased as well, but without contacting/allocating an anycast group member.\",\n \"In this way, it is ensured that the number of anycast members contaced/allocated in the first, preceding (multiple hosts) anycasting is not contacted again in the second attempt.\",\n \"As set forth above, this optional addition may already be implemented in the single hosts anycasting as known in the state of the art.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10467910"}}},{"rowIdx":391,"cells":{"text":{"kind":"list like","value":[["Enhanced data storage and retrieval devices and systems and methods for utilizing the same","The invention relates generally to store information on magnetic or optical storage media by using one or more novel approaches alone or in combination.","These novel approaches are capable of using at least one code which may comprise more than two values (FIG.","4d, 43).","A first series of approaches for the storage of information applies generally to optical recording and reproducing system (FIGS.","7-10, optical media 66), while a second series of approaches applies generally to electric or magnetic recording and reproducing systems (FIG.","1-2, magnetic medium 1), Each series of approaches is capable of storing information data in one or more codes (FIG.","4d, 42a, 42b, 42c, 42d) and the use of at least one higher order code which is different from the traditional binary code of “0” and “1”."],["1.A method for storing data comprising: storing at least one magnetic field strength; utilizing said at least one stored magnetic field strength to cause an amount of splitting or shifting of at least one frequency in at least one material; determining the amount of shifting or splitting in said at least one material; and assigning a data value to said amount of splitting or shifting.","2.The method of claim 1, wherein said determining comprises a means for interrogating said at least one material with electromagnetic energy to determine said amount of shifting or splitting.","3.The method of claim 1, wherein said at least one material comprises at least one magnetic material.","4.The method of claim 3, wherein said at least one material comprises at least one material contiguous to at least one magnetic material.","5.The method of claim 1, wherein said method for storing data comprises a base-2 data storage system.","6.The method of claim 1, wherein said method for storing data comprises, comprises greater than a base-2 data storage system.","7.The method of claim 1, wherein said splitting or shifting comprises at least one Zeeman effect.","8.A method for reading data comprising: creating at least one magnetic field strength; utilizing at least one magnetic field strength to cause an amount of splitting or shifting of at least one frequency and at least one material; determining the amount of shifting or splitting in said at least one material; and assigning a data value to said amount of splitting or shifting.","9.A data storage apparatus comprising: means for storing at least one magnetic field strength; means for utilizing said at least one stored magnetic field strength to cause an amount of splitting or shifting of at least one frequency in said at least one material; means for determining the amount of shifting or splitting in said at least one material; and means for assigning a data value to said amount of shifting or splitting.","10.A method for storing information optically comprising: an optical source; a means for achieving different optical intensity; a means for recording said different optical intensity; and a means for reading said different recorded intensities.","11.A method for storing information optically comprising: an optical source; a means for achieving different optical wavelengths; a means for recording said different optical wavelengths; and a means for reading said different recorded optical wavelengths."],["<SOH> BACKGROUND OF THE INVENTION <EOH>Data storage devices have been becoming smaller in size (as well as faster) due to, for example, the continued improvement in the ability to store more information in smaller spaces.","For example, over the past twenty years the ability to place more data on magnetic storage media such as magnetized hard-disk drives and/or floppy disks, as well as on optical media (e.g., compact disks “CD's” and more recently, Digital Versatile Disk “DVD's”) has been increasing at a phenomenal pace.","The impressive increase in the ability to store more data in smaller areas has been one of the main driving forces for rendering computers, and other electronic devices with reasonably sized memories, accessible to a typical home or small business consumer.","Further, the high operating speeds of microprocessors have required stored data in computers to be rapidly accessible (e.g., both the rate at which information is delivered to a microprocessor as well as the amount of time it takes between the time that a microprocessor requests a file and when the first piece of data begins to flow from the file to the microprocessor are important).","Magnetic memory has undergone many recent improvements and has been utilized extensively in data storage/retrieval devices because magnetic memory currently provides the least expensive alternative for the fast writing and reading capabilities for data.","However, the various currently utilized magnetic technologies face a variety of technical challenges and appear to be headed for a stopping point.","Specifically, these various approaches for the storage and/or retrieval of information may no longer be capable of meeting the growing demands of the industry, without experiencing significant technical enhancements.","For applications where speed has historically been less important, various optical technologies have been growing in popularity.","Many technical advances have been made in the recording and/or retrieval of information by optical means.","In systems which use compact disks (“CD's”), compact disk read-only memory (“CD-ROM's”) and/or Direct Video Disks (“DVD's”), the storage and/or retrieval of information has also been of great interest.","For example, the first CD's manufactured were CD-ROM's that are currently capable of storing about 650 MB of data.","This equates to about 74 minutes of play-time for music that has been recorded in a typical recording mode.","In particular, the standard current approach for utilizing CD-ROM's, includes the use of a solid state interrogating laser (or in some cases more than one laser) which has a wavelength of about 780 nanometers (0.78 μm).","The standard CD-ROM is about 12 cm in total diameter and has a thickness of about 1.2 mm.","The standard CD-ROM has information recorded on plateaus (“bumps”) and valleys (“pits”) that spiral from the inside of the disk toward the outside of the disk.","The digital data stored in the tracks that comprise the spirals must be separated by enough space (e.g., tracks are typically separated radially by about 1.6 μm) so that when the data is read with an interrogating solid state laser, crosstalk from adjacent tracks does not occur (i.e., recorded information is received from only one track at a time).","Accordingly, the placement of more information onto a typical CD has been limited by the required size (e.g., length and width) of the plateaus (“bumps”) and valleys (“pits”).","The size of the plateaus and valleys (e.g., typically not less than 830 nanometers in length) has been a function of various factors, including the wavelength of the laser utilized to read the information from the CD.","Various attempts have been made to increase the memory capacity of current CD's, including the stacking of a plurality of disks one on top of the other so as to provide more memory space, as well as other attempts to miniaturize the digitized information stored in the tracks.","The most recent significant improvements in this type of optical storage/retrieval technology have been the creation of the DVD, which is known as the “Direct Video Disk” or the “Digital Versatile Disk”.","The first commercial products of DVD's entered the marketplace in late 1997.The DVD technology is similar to the technology in CD's, except that, for example, the wavelength of lasers utilized to read information from DVD's is smaller than that used for CD's, namely, about 635 nanometers or 650 nanometers, compared to about 780 nanometers for CD's.","The size of the plateaus (“bumps”) that make up the DVD are about 320 nanometers wide, a minimum of about 400 nanometers long and about 120 nanometers thick.","About 740 nanometers separate one track from the next, compared to the about 1600 nanometer separation of tracks in CD's.","A standard DVD is physically about the same size as a CD, but has a memory capacity that is about seven times greater than that of CD's.","In particular, the memory capacity of a one-sided, one-track, DVD is now about 4.7 GB.","However, recent improvements in DVD have allowed digital information to be placed on both sides of a single track, as well as both sides of a double track.","In this regard, a first interrogating laser reads the digitized data on a first side of a first track (i.e., in the form of bits arranged in a spiral track that begin in the innermost portion of the DVD and spirals outward).","Then, information can be retrieved from the opposite side of the first track in a similar manner from a second laser; however, the spiral track of information typically begins on the outermost portion of the disk and spirals inward toward the center.","Accordingly, when dual-track, dual-sided DVD disks are used, the current amount of memory that can be achieved is about 17 GB.","Another optical data storage technique which shows promise is known as holographic memory.","The potential for high storage capacity and high storage retrieval rates using the holographic technique is tremendous.","In particular, for example, recent work in a system known as Holographic Data Storage System (“HDSS”), has shown that data can be accessed in 100 microseconds or less, which is several orders of magnitude faster than the millisecond retrieval of data in typical magnetic-disk drives.","Briefly, in this technology, an optical-interference pattern is created within a desired photosensitive material (e.g., a crystal or a polymer).","The optical interference pattern is imprinted as a result of physical changes in such materials.","The interference pattern is created by the interaction between an object beam and a reference beam, both of which typically originate from a single light source (e.g., a laser), but are split into two different pathways by a beam splitter.","Specifically, the object beam is incident on a target object that contains data, and modifies the object beam, while the reference beam, taking a separate path which leaves it unmodified, interferes with the object beam at the holographic storage medium.","When the two beams meet at the holographic storage means, the interference pattern which results from their interaction is stored on or in the holographic storage means.","The holographic storage means is later interrogated by a reading or reference beam, which upon interacting with the holographically stored interference pattern, can then recreate the holographic image containing the data originally stored in the object beam.","While the holographic optical technique shows great promise for the future, current industrial emphasis is being placed primarily on improving existing digital optical and magnetic storage/retrieval systems so that more information can be stored in smaller spaces, and retrieval times can be decreased without significantly changing the basic methodology and hardware for the storing and retrieving of information.","With regard to placing more data onto magnetic recording media, effects such as the paramagnetic effect (e.g., magnetic crosstalk between magnetic domains comprising bits) are already beginning to cause problems.","Experts have postulated that in coming years, magnetic storage technologies could reach a limit imposed by the superparamagnetic effect (“SPE”).","The physical phenomenon known as SPE can occur in a magnetic data storage system when the magnetic spins of electrons in the domains which comprise a bit (e.g., in current technology typically either a “0” or a “1”) become unstable due to, for example, the environmental thermal energy surrounding the bits.","Instability of bits occurs when smaller and smaller numbers of domains or atoms are used to comprise a bit and the ability for the domains or atoms to maintain their given spin directions (e.g., corresponding to, for example, a “0” or a “1”) becomes difficult.","Non-maintenance of spin directions results in a “flipping” of spins back and forth and thus changing “0's” to “1's” and “1's” to “0's”, etc.","Such changing or flipping of spins will result in a corruption of the data or information that the “0's” and “1's” represent.","Accordingly, in order for smaller numbers of atoms to be used to form a bit and in order for the bit to be viable, techniques and/or materials for preventing the superparamagnetic effect need to be developed to overcome the deficiencies of the prior art.","Another potential problem in recording information magnetically is that the write head which is used to align magnetic domains in the magnetic storage media may be limited in the ability to impart a required field strength over the entire domain area comprising a bit to cause the magnetic domains to behave as though they have been exposed to a substantially uniform field.","In such cases, it is possible that differently oriented domains within a bit (e.g., differently oriented due to being subjected to different magnetic field strengths within a single writing head due to, for example, non-uniformity of the magnetic flux lines emanating from the write head and/or differently oriented because some domains require more energy than other domains to align due to, for example, their location within a bit) may have a higher tendency to become corrupted, as well as adding to signal/noise ratio problems.","One attempt for miniaturizing domains involves the use of so-called “hard” magnetic materials.","The use of hard magnetic materials is desirable when compared to “soft” magnetic materials because hard magnetic materials are more likely to maintain their magnetic spin directions when domains comprising the hard magnetic materials are closely spaced.","In this regard, various rare-earth and transition elements have been found to be magnetically stable and are thus termed “hard”.","Such magnetically stable materials are known to have a higher coercivity, which is typically represented by “H c ”.","Stated in an over simplified manner, the higher the H c value, the greater the resistance of the material to outside influences, such as outside magnetic fields (e.g., the harder it is for material to lose its imprinted spin direction due to an increase in temperature and/or the influence of other magnetic fields, etc.).","However, one drawback for utilizing “hard” magnetic materials is that hard magnetic materials are more difficult to magnetize than soft magnetic materials (e.g., in some cases, depending on the particular materials chosen, much more difficult to magnetize).","This makes it more difficult to record data initially in hard magnetic materials.","There are various known approaches for solving the problem of encoding data into hard magnetic materials which approaches utilize a laser in conjunction with an appropriate magnetic write head.","These various approaches have been referred to generally as the “magneto-optical” approach.","These magneto-optical approaches effectively reduce the H c value of the magnetic domains by causing the domains to be locally heated just prior to being subjected to the magnetic write head.","Thus, the coercivity (H c ) of the domains is effectively reduced and when such effective reduction in H c has occurred, a somewhat standard write head, or in some cases a very different write head, can be used to alter favorably the magnetic domains.","In other words, the use of a laser, which provides thermal energy to the domains of the hard magnetic material, causes the hard magnetic material to behave in a manner which is similar to a somewhat softer and thus easier to magnetize magnetic material.","Each of the known approaches for utilizing the magneto-optic techniques involves the use of at least one laser and/or at least one laser focusing system.","However, one of the common problems facing these magneto-optical approaches is that the lasers that are utilized to soften the hard magnetic material prior to subjecting the hard magnetic material to a magnetic field is that the lasers typically generate excess amounts of heat that can flow to neighboring bits or tracks (e.g., thermal energy is caused to migrate to undesirable neighboring tracks or areas resulting in a potential corruption of stored information).","Accordingly, various laser applications and/or storage solutions have been devised to minimize the transfer of heat from the laser to neighboring tracks, and thus minimize the loss of recorded information.","However, to date this thermal transfer of laser energy still presents problems to varying degrees in all the prior art approaches.","One interesting technique for the reading of data in connection with one optical-magnetic system is the use of an effect known as the “Kerr effect”, and more particularly, the “magneto-optical Kerr effect”.","Briefly, current uses of the magneto-optical Kerr effect involve incident polarized light being reflected from the surface of a magnetic domain in different ways, depending on the orientation of the magnetic domains (e.g., depending on electron spin directions).","The changes in the reflected polarized light are made, by algorithms, to correspond to different digitized data.","Specifically, when the incident polarized light encounters different magnetic field orientations (e.g., north or south domains comprising a bit which are ordered to represent either a “0” or a “1”) the polarization state of the reflected light is changed.","Another approach to the digital storage of information that shows future promise is known as atomic resolution storage (“ARS”).","This approach, similar to holographic techniques, is capable of storing tremendous amounts of information in a small amount of space.","In this technique, generally, very small electron probes, which are used to generate electron beams, are formed into an array.","The electron probes have tips that are roughly the size of atoms.","The electron beams from the electron probes are made to be incident upon a storage medium so that the incident beams cause some sort of physical change in the storage medium (i.e., the medium comprising the computer memory).","One example of such a storage medium is a material that is capable of containing at least two stable phases at ambient operating conditions so that an incident electron beam emitted from an electron probe changes the storage material from one phase to the other.","In this technique, encoded bits of information are represented by the change in phase of the phase-change storage material.","This technique, similar to the magneto-optical technique discussed above, also suffers from the problem of heat flowing between data spots created by the incident electron beams when the spots or bits are initially recorded.","Accordingly, one of the challenges faced by this relatively new technology includes reducing the flow of heat between data spots which are created by the array of electron beam generators.","The retrieval of stored information is also a challenge.","In this regard, as bits become smaller and include lesser numbers of magnetic domains per bit and/or the domains are oriented differently, (e.g., domains are aligned perpendicular to the substrate containing at least one magnetic surface thereon instead of being parallel to the substrate surface) such bits are capable of being packed more tightly together.","Assuming that the bits are not corrupted by their tight packing and/or smaller numbers of magnetic domains and/or non-uniform alignment within a bit, then reading of these tighter packed domains comprising the bits also becomes a challenge which the prior art is still struggling to overcome.","Historically, large numbers of magnetic domains were present in each bit which contained digital data.","Thus, the resulting or induced magnetic fields were relatively large and various forms of known inductive reading techniques were sufficient to ascertain whether the hundreds or thousands of aligned domains in a bit corresponded to a “0” or a “1”.","However, as prior art miniaturization recording techniques progressed, the known inductive reading techniques were typically not sensitive enough to determine accurately the alignment of ever decreasing numbers of domains comprising a bit (e.g., signal to noise problems in the storage and/or retrieval of digital data bits were exacerbated).","The development of magnetoresistive heads, and the subsequent development of giant magnetoresistive heads, has enabled smaller numbers of domains comprising a digital bit to be read accurately.","In both of magnetoresistive and giant magnetoresistive heads, the electrical resistance of one or more materials comprising the read head(s) is examined.","In particular, both magnetoresistive and giant magnetoresistive heads are made from materials that exhibit small changes in their electrical resistance as a function of magnetic fields created by oriented domains to which such heads are exposed.","Giant magnetoresistive heads are two or three times more sensitive than magnetoresistive heads due to their novel structure which includes layering of different materials stacked on top of each other.","One problem that continues in these technologies is the presence of magnetization vortices in the recorded domains.","Such vortices may result from the difficulty in achieving uniform domain alignment within a bit due to, for example, the difficulty in applying a required magnetic field (e.g., uniform field) to the domains during the recording or writing process.","While use of the magneto-optical Kerr effect and new read heads such as magnetoresistive heads and giant magnetoresistive heads have improved digital data retrieval techniques, further miniaturization is pushing the limits of these systems.","Accordingly, additional improvements are necessary in the ability for hardware, combined with suitable programming, to detect even smaller numbers and/or smaller sizes of domains comprising digitized (or analog) data bits.","Thus, additional innovations in the detection of stored data are required.","In addition to the storage of information digitally, the possibility or promise of storing information in an analog manner continues to intrigue various investigators.","While certain improvements have been made in, for example, the processing of analog information (e.g., the advent of VLSI (Very Large Scale Interrogation) chips, the techniques for the storing of analog information need much improvement.","Particularly, while analog computing has been known for several decades, an accurate and fast-paced system for the storage and retrieval of information has continued to elude researchers.","In this regard, digital computing has received the greatest amount of attention because of it's relative dependability for the storing and retrieving of information in a “1” or a “0” digitized format.","Briefly, digital approaches typically include storing a sampling of the analog information that is to be recorded.","The number of samples taken (e.g., such sampling typically occurs in predetermined units of space or time) from the analog source will correspond to the accuracy between the original analog image or sound and the stored digital data (i.e., the more digital samples that are recorded of an original analog image or sound, the more accurate the reproduction of the original analog image or sound will be).","These techniques of predetermined quantum amounts of sampling of analog images have been adequate for most digitized applications, however, as the need for more accurate recording and retrieval of information occurs (e.g., in, for example, the fields of computing or determining various engineering and scientific relationships in the following representative areas: gravitational; electrostatic; magnetic; thermal; stress; fluid flow field analysis; wave propagation; image processing; etc.","), the availability of computing with more accurate data becomes important.","However, the digitization of tighter and and/or smaller sampling amounts from an analog source can result in tremendous amounts of digitized data being stored to represent a small actual amount of analog information.","This is one area where analog computing has a distinct advantage compared to digital computing.","In particular, an analog computer will store only a single piece of information corresponding to a single sampling area, whereas a digital computer will need to store somewhere between, for example, 8 and 16 bits of data to represent the same single piece of information stored in an analog fashion.","The large number of bits required to store digital information corresponds to the requirement to store information in an ASCII or Unicode format.","However, in order to escape from the current ASCII or Unicode format, analog storage requires a very large number of different data bits (e.g., data corresponding to much greater numbers than merely “0s” and “1's”) to be stored and retrieved.","Current prior art techniques have great difficulty in storing and/or reading bits corresponding to anything other than “ 0 's” and “ 1 's”, let alone the attendant problems of storing and/or reading, for example, a near infinite number of possibilities (e.g., 1, 2, 3 .",".",".","n+1).","However, when performing complex calculations, the digital process may result in very slow processing times as well as requiring very large memories.","It is for this, as well as other reasons, that analog computing is still very attractive.","Thus, while advances in the processing of analog information have been made, reliable techniques for the storage and retrieval of analog information are still being sought without any good solutions currently existing.","As stated above, with regard to digital computing, much of the recent technical emphasis in data storage and retrieval for both optical and magnetic memory enhancement has been on shrinking more information into smaller areas.","The ability to place bits, which are the smallest elements of information used by current digital memory systems, into a binary code format, which is a combination of “0's” or “1's”, into a smaller area on, for example, magnetic and/or optical disk storage media (e.g., a disk or a tape) may soon reach a limit.","Thus, current technologies for writing and/or reading binary data onto and/or from current storage media will be pushed to the maximum limit.","Computer designers are already facing a performance gap whereby processors can process information faster than the information can be retrieved from computer memory.","The majority of the currently proposed approaches for the digital storage of information, suffer from the same problem, namely, that the magnetic and/or optical information which is stored uses a binary code (i.e., a base- 2 number system) which is a combination of “0's” and “1's”.","The predominance of the use of a “binary” code has been due to, for example, difficulty in distinguishing signals (e.g., a “0” or a “1”) from background noise.","In this regard, many techniques have been proposed which use various hardware, as well as algorithms, which attempt to read accurately the stored information as either a “0” or a “1”.","However, certain prior art techniques have been postulated for utilizing codes other than binary.","In particular, U.S. Pat.","No.","6,154,432, entitled “Optical Storage System” discloses a holographic system which records information in very small spots and postulates that 10 or more digits may be possible to record on a single bit.","Accordingly, this holographic technique discloses codes higher order than binary.","Further, for example U.S. Pat.","No.","5,450,363, entitled “Gray Coding for a Multi Level Cell Memory System” discloses the use of a so-called “gray code” approach (also known as “gray scale” by others) for data storage.","This approach also uses a system which is higher order than binary.","In particular, reference is made to the storage of multiple bits of information in a single memory cell thus creating a multi-level cell.","Still further, U.S. Pat.","No.","6,061,005, entitled “Encoding of Data”, discloses a method of encoding data on magnetic cards which uses symbology other than binary, such as trinary, quaternary or even higher symbology, for the recording of information.","However, while various techniques have been postulated for systems which use codes higher than binary, none of such codes have been commercially adopted due to, for example, signal to noise ratio problems, etc.","In particular, reading errors can occur due to lack of contrast between data values.","Such lack of contrast can be caused by random noise (e.g., vibrating inaccurate positioning of recording/reading heads, fidelity loss during recording, etc.)","and determinate variations.","Accordingly, a need exists for techniques which can be commercially viable which can use codes reliably that are higher order than traditional binary codes for the storage and/or retrieval of information.","A very particular combination of “0's” and “1's” is known as the American Standard Code for Information Exchange (i.e., the ASCII code) which assigns a unique code number between 0 and 127 to each of the 128, 7-bit, binary number characters.","Table 1 below shows in table form the current ASCII characters arranged by Hexidecimal digits (i.e., 128 different encoding combinations of groups of seven bits).","Reading within the Table from left to right and then down, the first 32 values are codes which correspond to various computer control functions such as line feed, carriage return, etc.","The 33 rd value corresponds to the “space” character.","Values 34-48 correspond to symbols and punctuation marks.","Values 49-58 correspond to digits, and so on, until reaching value 128, which corresponds to “delete”.","In addition, each of these values also has a corresponding hexidecimal character.","For example, the hexidecimal digit corresponding to the “space” character is “20”, while the hexidecimal digit corresponding to the letter “n” is “6E”, and so on.","TABLE 1 00 01 02 03 04 05 06 07 08 09 0A 0B 0C 0D 0E 0F 00 NUL SOH STX ETX EOT ENQ ACK BEL BS TAB LF VT FF CR SO SI 10 DLE DC1 DC2 DC3 DC4 NAK SYN ETB CAN EM SUB ESC FS GS RS US 20 !","“ # $ % & ‘ ( ) * + , − .","/ 30 0 1 2 3 4 5 6 7 8 9 : ; < = > ?","40 @ A B C D E F G H I J K L M N O 50 P Q R S T U V W X Y Z [ \\ ] {circumflex over ( )} — 60 ’ a b c d e f g h x j k l m n o 70 p q r s t u v w x y z { | } ˜ DEL These 128 characters are enough characters for North American English to be stored and/or retrieved, but are not enough for many other languages (Note: In ASCII, the eighth bit is used, and is thus normally set to “0”).","To represent more than 128 characters, 8-bit (rather than 7-bit) binary numbers are utilized.","For each digit in these eight bits there are two choices for the digit, either “0” or “1”.","This results in a possibility of an additional 128 characters or a total of 256 different combinations of “0's” and “1's” (i.e., 2 8 ).","Accordingly, the current data storage approaches utilize these 256 combinations of “0's” and “1's”.","However, these 8-bit numbers still do not provide enough room for all characters that need to be used in the world (e.g., certain Asian languages have thousands of characters each).","In addition, there is no universal agreement in the world on what all the characters should be, whether referring to the 128 characters, or the 256 characters, or any number of characters beyond these.","However, certain approaches for dealing with all the various languages in the world, which collectively contain many thousands of characters, are beginning to utilize something referred to as the “Unicode”, which uses a 16-bit character set.","In order to adopt something similar to the Unicode, even greater strain will be placed on current memory techniques.","Whether using an 8-bit, 16-bit, or some other approach, all such approaches have been limited primarily to using a binary system of data storage.","However, due to: (1) the impending size barrier for the storage of information on, for example, magnetic and/or optical media; (2) the performance gap between how fast a processor can process information versus how fast a processor can access information; and (3) the goal for a universal adoption of a common set of characters throughout the world, a different approach for the storage of information, either by digital or analog approaches, is clearly needed.","The present invention satisfies the current and future data storage needs by using different approaches for the storage and/or retrieval of data; and/or different codes that use a code higher than the current “base- 2 code”."],["<SOH> SUMMARY OF THE INVENTION <EOH>It is an object of the invention to provide a means for storing/retrieving digitized information optically.","It is an object of the invention to provide a means for storing/retrieving digitized information magnetically.","It is an object of the invention to provide a means for storing/retrieving analog information optically.","It is an object of the invention to provide a means for storing/retrieving analog information magnetically.","It is an object of the invention to provide a storage/retrieval means for utilizing codes other than binary.","It is an object of the present invention to provide an increased number of characters that can be stored as a single bit of information.","It is an object of the invention to provide the ability to store the 128 ASCII-type characters in a much smaller space than the space that is used in a standard binary code of “0's” and “1's”.","It is an object of the invention to provide a means for storing/retrieving codes other than binary.","It is an object of the invention to utilize at least a base- 3 optical storing/retrieving system.","It is an object of the invention to utilize a least a base- 4 coding storing/retrieving system.","It is an object of the invention to utilize at least a base- 6 coding storing/retrieving system.","It is an object of the invention to utilize at least a base- 8 coding storing/retrieving system.","It is an object of the invention to utilize at least a base- 10 coding storing/retrieving system.","It is an object of the invention to utilize particular aspects of the Zeeman effect in connection with the storage/retrieval of digitized information.","It is an object of the invention to utilize particular aspects of the Zeeman effect in connection with the storage/retrieval of analog information.","It is an object of the invention to utilize different shades or colors of spots or dots in connection the optical storage of digitized information.","It is an object of the current invention to utilize different shades or color or spots or dots in connection with the optical storage of analog information.","It is an object of the invention to utilize either monochromatic or polychromatic light in connection with the optical storage of digital and analog information.","It is an object of the invention to utilize an analog-like approach to the storage of data.","The current process of storing bits, which is the smallest element of information used by current memory systems, uses a binary format of only “0's” and “1's”.","A bit which is “off” is considered to be “false” or “not set”, while a bit that is “on” is considered to be “true” or “set”.","Historically, because bits could only be one of two values, bits have been combined together into larger units in order to represent more characters or values.","“Nibbles” are groups of four bits and “bytes” are groups of eight bits (i.e., two “nibbles”).","Bytes are typically what are used to store “characters”.","“Characters” typically include letters, numbers and/or symbols.","For example, the ASCII code uses 128 different encoding combinations to represent: (i) The letters “A-Z” in both upper and lower case (i.e., values 65-90 and 97-122, respectively); (ii) Special characters (e.g., “<”, “.”, “%”, “$” “?”, etc) (e.g., values 33-47); (iii) The numbers 0 - 9 (i.e., values 48-57); and (iv) Certain control codes used for device control (i.e., values 0-31).","To represent more than 128 characters, an eighth bit can be utilized.","However, the ability to store a binary code of only “0's” and “1's” limits the ability for information to be miniaturized.","For example, to store a single character from the ASCII code, at least seven bits of memory (typically eight bits are currently required because the last bit is usually set to zero) are needed.","Table 2 sets forth one example of how a typical computer memory system would store the word “Patent”.","TABLE 2 ASCII LET- CHAR- BINARY HEXADECIMEL BITS OF TER ACTER NUMBER DIGIT OR BYTE MEMORY P = 80 = 01010000 50 8 a = 97 = 01100001 61 8 t = 116 = 01110100 74 8 e = 101 = 01100101 65 8 n = 110 = 01101110 6E 8 t = 116 = 01110100 74 8 Thus, the letter “P” corresponds to the ASCII character 80 , which is represented by the binary code “01010000”.","The first “0” in the sequence is often referred to as “the last bit” and is typically set to “0” for all ASCII characters.","The corresponding hexidecimal digit or byte representation is “50”.","In order for the computer memory system to store the letter “P”, eight bits must be sent to memory in the order “0-1-0-1-0-0-0-0”.","Likewise, to store the letter “a”, eight bits must be sent to memory in the order “0-1-1-0-0-0-0-1”.","Accordingly, 48 bits are used to store the six (6) letter word “Patent”.","Similarly, when a microprocessor unit accesses the portion of memory containing the set of bits representing the word “Patent”, the same number of bits (i.e., 48) or six (6) bytes must be processed by the microprocessor.","Clearly, if the word “Patent” could use a smaller amount of memory, less memory capacity would be needed in a computer and/or the rate at which data could be stored and/or retrieved from memory would be enhanced.","In a first aspect of the invention, a coding system that is a higher order system than a binary coding system is utilized for the digitized storage of information.","For example, any digital coding system which uses three or more different code characters (i.e., a coding system which operates in “base-3” or greater) will result in a reduction in the number of bits needed to store the same amount of digital information relative to a binary system.","Thus, by using a higher order memory system, the possibility of storing more digitized information in a smaller memory area exists.","Likewise, the rate of storing and/or retrieving digitized information should also increase due to memory being stored in a physically smaller area.","This permits a data retrieval device to function more efficiently.","The present invention permits the use of a digitized memory system which is base- 3 or greater.","Moreover, as more code characters are used (e.g., higher order systems of more than 10 or more than 20 different characters are utilized) the digitized system begins to behave in a manner which is similar to analog systems and can thus be referred to as “analog-like”.","In other words, as the amount of digital sampling of an analog source increases, the recorded digital signal begins to approach the storage of most features of the analog signal.","However, current data storage/retrieval techniques utilize, for example, 8 or 16 bits to store a single piece of analog information, which limits the amount of sampling of an analog source that can be reasonably performed when creating a digital representation of the analog source.","Accordingly, the digital data storage/retrieval techniques of the present invention can be vastly superior to current digital techniques which utilize only “0's” and “1's” and are limited to sampling much smaller areas of analog sources than would be desirable in many cases (e.g., for complex computer modeling of numerous events, etc.).","A first embodiment of an alternative memory storage system of the present invention may function in a manner somewhat analogous to the manner in which a Compact Disk (CD) or Digital Versatile Disk (DVD) player functions.","Current CD and DVD technologies utilize a rotating disk comprised of a number of layers of different materials.","The layers may include, for example, polycarbonate plastic, aluminum and acrylic.","The aluminum in the disk includes a series of plateaus and valleys sometimes referred to as “bumps” and “pits”, respectively.","Laser light is caused to focus upon the bumps (plateaus) and pits (valleys) in the rotating disk.","The reflection of the laser from the successive plateaus and valleys corresponds to a data stream (e.g., a binary stream) of information.","CD's and DVD's use, for example, 16-bit strings of binary coded data.","Specifically, laser light reflects differently from a valley compared to the reflection from a plateau and these different reflective properties of the laser, when reflected from these valleys and plateaus, correspond to the binary code data values of “0” and “1” for each bit.","In one embodiment of the present invention, a similar concept to the CD and DVD is utilized, however, rather than utilizing valleys and plateaus (i.e., bumps and pits) in an aluminum layer embedded in a polycarbonate plastic CD or DVD, the present invention utilizes at least two data values per bit represented by different intensity, colors or color width (e.g., different wavelength or frequency).","These data values can be optically created, stored in dots or spots, and optically examined or interrogated (e.g., by known transmission and/or reflection techniques).","These optical dots or spots may be of a size which is approximately the same order of magnitude in size as the bumps and pits which comprise current data tracks used on CD's and DVD's, they may be larger, or they may be smaller.","In particular, similar to current CD's and DVD's, these dots or spots can be put together to form strings or tracks of data which begin at an innermost portion of a rotatable CD/DVD disk and extend spirally out therefrom in the form of tracks.","Accordingly, current CD's of approximately 12 centimeters in diameter would be an acceptable size.","However, if the dots or spots of the present invention are smaller than the “bumps” of the current technology, it is possible for CD's to be smaller in size, if desired, or more data can be placed into the same amount of memory space.","Thus, it is possible to put an amount of optical information onto a rotatable disk of approximately 12 centimeters in diameter which exceeds that amount of information which is currently stored on CD's/DVD's.","However, it is possible for the dots or spots to be larger in size than current bumps and pits in CD's/DVD's while still storing more information relative to standard CD's/DVD's.","This is possible due to at least two effects.","First, tracks may be capable of being located closer together due to, for example, lesser amounts of crosstalk between bits (e.g., recording and/or reading operations can take place in a lesser amount of space).","Second, if digital codes higher than binary are adopted, less actual bits need to be stored relative to a binary system (e.g., rather than storing 8 or 16 bits to represent a single piece of information, a lesser number of bits can be used to represent the same piece of information that is being stored).","It should be noted that as the number of characters that can be stored in a single bit become larger, the digital storage system actually approaches behaviors similar to an analog storage system and can be referred to as “analog-like”.","In particular, the present invention is capable of utilizing a coding system other than the binary coding system of “0's” and “1's” now used in CD/DVD systems.","For example, a series or range of dots or spots of different color intensities (e.g., white-gray-black), and/or different colors (e.g., red-yellow-violet), and/or both as in varied intensity of polychromatic light, can be placed upon a surface (or embedded somewhere within a material), such as a suitable photoferroelectric material such as “PLZT” (i.e., lead lanthanum zirconate titanate ceramic), which is shaped in the form of, for example, a disk.","When, for example, such a disk containing these different intensity data value dots or spots is rotated and interrogated by, for example, a known optical system (e.g., a laser system) with a suitable focusing lens and intensity detecting means (e.g., a suitable photosensitive material or photodiode), the different intensities of the dots or spots can be registered and can be coded to correspond to different values (e.g., “0”, “1”, “2”, “3”, etc.).","Thus, rather than using a simple binary or base- 2 system where a black dot corresponds to “0” and a white dot corresponds to “1”, the present invention provides for a larger number of options for digitized data storage.","However, as discussed in more detail herein, depending on the sensitivity of the system, this technique could also be used for actual analog computing.","Accordingly, for example, in a simple digitized quaternary system having a quaternary code consisting of 0's, 1's, 2's and 3's, there would be 4 different choices of basic digital information storage (i.e., a “base-4” system) rather than only two choices in a binary or base- 2 digital system.","Likewise, in a digital system which utilizes 8 different combinations of spots or dots (i.e., a “base-8” system) eight different options exist for encoding a piece of information rather than only two options in a standard binary or base- 2 digital system.","The use of a larger number of possibilities other than “0's” and “1's” (e.g., 0, 1, 2, 3, 4, 5, 6, 7, etc.)","render it possible to store a much greater amount of information in a much smaller physical area due to the elimination of unneeded bit space.","Moreover, as the number of possibilities other than “0's” and “1's” increases, (e.g., systems greater than base- 10 such as base- 20 or greater systems) the closer the digital system comes to being or performing like an analog system, which has certain advantages over digital systems.","An apparatus according to the present invention permits suitable optical data to be stored onto an appropriately constructed and shaped optical recording medium by using, for example, a laser light source.","The optical recording media (e.g., a PLZT ceramic) can be in the form of a tape, disk, or credit card or any other acceptable shape.","Alternatively, a photodiode or photodiode assembly can also be used.","In all such cases, a light source (e.g., laser light source) can be used in combination with, for example, various means for controlling the direction, polarization, intensity and/or final color of the light source.","Various examples of such systems include the following: (1) a rotatable polarizing filter (or set of filters), sometimes referred to as an analyzer, used in combination with a suitable light source which is subjected to a first polarizing filter(s) selectively modifying the polarized light from the light source.","Specifically, once monochromatic or polychromatic light has been polarized into a first direction polarizer the polarized light is directed toward a second polarizer (sometimes referred to as an “analyzer”) which, when rotated, results in, for example, different intensities of light being emitted therefrom.","The different intensities of light can be caused to be incident upon an appropriate recording/detecting means; (2) a light source (e.g., a laser light source), which is used in conjunction with a first polarizing filter is caused to be incident on a suitable light diffractor (e.g., a Bragg modulator) which can cause the polarized light from the light source to communicate with one or more of a series of additional polarizing filters (or analyzers) each of which analyzers are rotated, relative to the polarized source, at different angles, thus resulting an different amounts or intensities of light passing through each of the analyzers.","Thus, by selectively transmitting the polarized light through the different analyzers (e.g., directed by, for example, the Bragg modulator) different intensities of light can be achieved.","Such different intensities can be recorded and/or detected by a suitable means; (3) a light source (e.g., a laser light source) used in combination with an acousto-optic Bragg modulator, whereby different driving signals are input to the Bragg modulator which results in different intensities of light being emitted from the Bragg modulator.","The multiple intensity emissions are then directed to a suitable detecting/recording means and are made to correspond to various data points in base- 2 or higher order digital data storage systems; and (4) rather than utilizing polarizing filters to polarize a monochromatic or polychromatic source of light, liquid crystal cells can also be utilized.","In particular, light can be caused to be incident upon a liquid crystal cell.","Depending upon whether voltage is applied to the liquid crystal cell, the output from the liquid crystal cell is oriented into one direction or another.","Liquid crystals of this type can be made of, for example, lithium niobate.","However, similar to the embodiments (1)-(3) discussed above, intensities can be varied due to relative polarization directions.","The outputs of light from the liquid crystal cell can be directed, by appropriate beam displacers (e.g., a Bragg modulator), toward a suitable recording/detecting means.","In each of the examples (1)-(3) discussed above, it is possible that a polychromatic light source could be utilized.","In this regard, when polychromatic light is made to be incident upon a first polarizer, and thereafter upon one or more additional polarizers (e.g., analyzers), in addition to different intensities of light flowing through the analyzer(s), a spreading of these different intensities over a particular frequency or wavelength range will also occur.","Specifically, when using polychromatic light, a spectrum of frequencies of various intensities results in the formation of a somewhat continuous curve of varying light intensities over a particular frequency or wavelength range.","The use of a polychromatic source can therefore result in secondary indicia for determining the value assigned to stored bits of data (e.g., a “0” or a “1”) and thus enhances data storage.","For example, not only could the highest intensity of light be recorded/detected, but also the starting and/or ending points which correspond to the lowest frequency intensity of light being emitted, and the highest frequency of light being emitted, respectively, or, alternatively, the area under each curve could also be compared.","In other words, more than one piece of information can be checked to verify the accuracy of each data point that is stored and read.","The presence of more than a single piece of information can result in more accurate reading of data and thus result in signal/noise improvements.","In each of the examples (1)-(4) discussed above, the different intensities or colors of light can be made to be incident upon an appropriate receiving/recording means (e.g., an optical storage means, such as a pohtopolymerisable/photocrosslinkable material, a photodiode assembly, etc.","), such output light being suitably focussed onto the appropriate receiving/recording means by known lens or optical systems etc., all of which are discussed in greater detail herein).","Suitable materials for recording various intensities or colors of light include the following materials: inorganic photochromic crystals, photorefractive materials, chalcogenide semiconductors, organic and polymer materials, thermoplastic media, reversible recording in Tellerium compounds, photothermal recording in Antimony compounds, magneto-optic recording and light stimulated recombination luminescence.","For example, in the simplest embodiment (i.e., that embodiment numbered “(1)” above) for understanding the basic concept for recording various intensities of light (even though this simple embodiment is not as commercially significant as the other embodiments discussed herein), each such recorded intensity corresponds to a different value for a digitized data bit.","In this basic case, different intensities can be achieved as follows: a polarized light source, such as a laser and polarized filter or polarizer, when used in combination with at least one rotatable polarizing filter (e.g., an analyzer) can result in light of different intensities being transmitted from the at least one rotatable analyzer.","Specifically, when the laser light source is passed through a first polarizing filter and a second polarizer (e.g., an analyzer) is sequentially rotated (e.g., from 0-90°) a predetermined amount each time relative to the first polarizing filter (i.e., either the first or second filter can be rotated), different intensity dots or spots corresponding to data values having different meanings (e.g., “0”, “1”, “2”, etc.)","can be produced.","The amount of rotation of the analyzer in relation to the polarizing filter can correspond to a different intensity of light being recorded on a suitable optical recording medium (e.g., a photoferroelectric material such as “PLZT”, inorganic photochromic crystals, photorefractive materals, chalcogenide semiconductors, organic and polymer materials, thermoplastic media, reversible recording in Tellerium compounds, photothermal recording in Antimony compounds, magneto-optic recording and light stimulated recombination luminescence).","If a photoferroelectric material is utilized, the different intensities of light can be caused to be incident upon the photoferroelectric material by use of, for example, a suitable optic lens system or a fiberoptic cable (or a series of cables) that is capable of accurately directing the optical signal to the photoferroelectric material for the proper recording of the various optical signals.","Thus, a large number of different intensity spots or dots corresponding to data values for a single bit can be created.","This basic system and apparatus permits higher order coding systems (e.g., base-3 or greater) to be utilized.","The manner in which such optical data is stored, on, for example, a rotatable disk, as well as interrogated, can be by techniques which are substantially parallel, and/or use hardware which is similar, to those techniques and/or hardware currently used in CD and DVD systems.","For example, a series of bits of an appropriate binary or higher order digital code system can be recorded onto a rotatable disk by starting to record the different intensity dots or spots near a central potion of the disk.","The recording continues to record data in a spiral manner to form tracks of data, such data being suitably separated, physically, to avoid corruption and crosstalk, until an outermost radial portion of the disk is encountered.","Likewise, such a disk can be read or interrogated by, for example, a laser system which focuses light onto the dots or spots and suitably measures the intensities (e.g., reflectance or transmittance) of the spots of data.","The different intensities can be interpreted by suitable algorithms and programs to correspond to different values in the desired coding system.","Moreover, by utilizing a digital optical coding technique other than binary, the present invention permits a much more rapid adoption of the desired “Unicode”, which takes into account, for example, all English language characters, Chinese characters, numbers and symbols, etc., in various digitized forms.","In another embodiment of the invention, a system which utilizes magnetic memory in combination with a suitable retrieval/reading means is utilized.","These memory/retrieval systems can be caused to function, generally, in a manner that is somewhat similar to current magnetic memory systems (e.g., magnetic tapes, cards, disks, hard drives, etc).","In addition, these memory systems can also use digital coding/reading systems higher than a base- 2 system, if desired.","Still further, these memory systems can also store/retrieve information in an analog or analog-like format.","Specifically, it is another object of the invention to utilize certain aspects of the phenomena known as the Zeeman effects to result in a means for the storage and retrieval of information onto/from suitable magnetic media.","The term “Zeeman effects”, as used herein, denotes the effects of external magnetic fields on frequencies of atoms, molecules and components thereof, in the broadest sense.","Thus, as used herein, the term “Zeeman effect” includes not only what may be considered to be Zeeman effects in the more narrow, classical sense but also other magnetic effects such as magnetic quadropole effects, etc.","Digital data may be stored as either a binary code or some higher order code that is greater than the binary or “base-2” code.","Moreover, due to the apparent sensitivities of these data storage techniques, data may be stored/retrieved in an analog or analog-like manner due to the inherent operation of the Zeeman effects.","Specifically, without wishing to be bound by any particular theory or explanation, it appears that use of the Zeeman effects can result in a somewhat continuous set of differing data values that can be stored/retrieved.","The present invention utilizes one aspect of the Zeeman effects in the following general manner.","A magnetic field is applied to a suitable media that is capable of receiving and storing at least a portion of the applied magnetic field.","One example of such a suitable magnetic material is a ferromagnetic material that is capable of exhibiting different amounts of magnetic domain alignment as a function of increasing magnetic field strength up to its natural magnetic saturation point (i.e., whereby any further increases in applied magnetic field do not result in any measurable further alignment of magnetic domains).","Various known materials exist that meet this basic criteria, including many materials currently utilized in the magnetic storage industry (e.g., gamma Fe 2 O 3 , gamma Fe 2 O 3 modified by Co, CrO 2 , etc.).","The various materials can be held together by a variety of organic polymers including, for example, vinyl chloride, polyvinylchloride, methylacrylate, polymethylmethyacrylate, polyurethane, epoxy, polyamides, etc.","Suitable substrates include typical polyesters for flexible materials and aluminum for rigid materials.","The differing amounts of stored magnetic field can result in a corresponding splitting or shifting of the electromagnetic energy frequencies or spectral frequencies (e.g., fine spectrum splitting, hyperfine spectrum splitting, vibrational spectrum splitting, etc.)","of one or more atoms or molecules of the magnetic material itself, or similar splitting or shifting in one or more materials that are located contiguous (e.g., in the magnetic material or in a read/write head) to the material containing the stored magnetic fields.","In the Zeeman effect, the amount of splitting or shifting of one or more frequency spectrum(s) is a function of the strength of the applied/stored magnetic fields.","Typically, the higher the magnetic field strength that is stored/applied (e.g., stored in a first material containing magnetic domains and thereafter applied to (e.g., influencing) a second contiguous material that exhibits Zeeman splitting at the stored magnetic field strengths), the further apart the splitting or shifting will be of one or more of the electromagnetic energy frequencies of a chosen spectrum of the targeted atom(s) or molecule(s).","In this Zeeman effect, when a relatively wide splitting of electromagnetic energy frequencies occurs due to the applied magnetic field, the amount of splitting may correspond to frequencies in the microwave region; whereas a narrow splitting will typically correspond to lower frequencies (e.g., radio).","The amount of splitting that occurs is important because in order to determine the amount of splitting that has occurred, a suitable interrogating electromagnetic energy is caused to be incident upon, for example, the magnetic material itself or the aforementioned contiguous material.","The contiguous material that exhibits Zeeman splitting at the stored magnetic field can be present as, for example, one or more separate sheets of material that are located adjacent to, and/or contiguous with, the material that contains the stored magnetic field.","When multiple contiguous materials are utilized, the present invention contemplates the use of a comparative analysis of the differences in Zeeman splitting between two or more such contiguous materials.","Alternatively, a material comprising, for example, one or more material(s) that are mixed with the magnetic material (e.g., a binder which holds together at least a portion of the material that stores the magnetic field) could also be used.","More specifically, a particular interrogating frequency, or set or sweep of frequencies, needs to be capable of favorably interacting with one or more of the split, shifted or splitting frequencies which result in the magnetic material, or in an alternative embodiment in one or more of the contiguous material(s), due to the different magnetic fields stored therein.","In other words, a first magnetic material is desirably chosen so that an applied magnetic field can be reasonably stored therein.","However, it is desirable for the magnetic material to be capable of storing reliably several magnetic fields of different strength (e.g., capable of having varying numbers of magnetic domains align with an ever changing magnetic field and such domains remain frozen or pinned so that they do not change with time) and, for example, in one embodiment of the invention, the contiguous material needs to produce split, shifted and/or splitting frequencies in response to the stored magnetic fields.","In this regard, if one or more contiguous material(s) are utilized in connection with the magnetic material(s), the contiguous material(s) needs to exhibit a splitting and/or shifting of one or more frequencies, for example, of its rotational, vibrational, etc., bands (e.g., at the atomic level) and the resultant splitting or shifting that occurs in the contiguous material needs to be detectable by an appropriate interrogating electromagnetic source (discussed in greater detail later herein).","Alternatively, a contiguous material does not need to be utilized.","In this case, the magnetic material per se, or at least a portion thereof, needs to exhibit desirable and detectable splitting and/or shifting of one or more frequencies at varying stored magnetic strengths.","Still further, it should be noted that the contiguous material could actually be located in a suitable read head which, similar to magnetoresistive and giant magnetoresistive heads, is influenced by the magnetic field strengths stored in the magnetic media.","However, rather than measuring changes in resistivity in materials comprising the read heads, the splitting or shifting of frequencies will be detected by a suitable technique, discussed elsewhere herein.","In this embodiment of the invention, the contiguous material may be a solid, a liquid, a gas or plasma (e.g., any material that shows desirable Zeeman splitting effects).","With specific regard to interrogating with an applied electromagnetic source to determine the amount of Zeeman splitting and/or shifting that has occurred at a spot on the material that corresponds to a stored bit of data, at least one suitable frequency can be applied as an interrogating frequency, or, alternatively a sweep or combination of frequencies can be applied as interrogating frequencies.","In this regard, if a single suitable frequency is chosen as the interrogating frequency, then heterodyne interactions of the various split, shifted and/or splitting frequencies with the single interrogating frequency can be measured and determined.","Alternatively, resonance interactions of the various split, shifted and/or splitting frequencies can also be measured and determined.","Each such determined heterodyned and/or resonant frequency can be made to correspond, by suitable programming or algorithmic techniques, to various data values.","Alternatively, a series or sweep of frequencies which may resonate with or heterodyne with various split, shifted and/or splitting frequencies achieved in the contiguous material can also be utilized.","These particular affects will be discussed in much greater detail later herein, however, it should be understood that these data acquisition techniques could be performed using hardware which is somewhat similar to that hardware currently used for magnetoresistive and giant magnetoresistive heads.","For example, once an interrogating frequency has been input to a material exhibiting Zeeman splitting and/or shifting, the output (e.g., a heterodyned frequency) can be used as a driving signal or input (e.g., depending on strength or frequency either amplified or unamplified) for various other devices.","One example of such other device is an acousto-optic Bragg modulator.","In this detection scheme, an appropriate light source is caused to be incident upon an appropriate Bragg modulator.","Suitable materials for a Bragg modulator include, for example, lithium niobate crystals and gallium arsenide semiconductor materials.","The incident light beam can be caused to be deflected in differing amounts due to different driving signal inputs (e.g., depending on strength or frequency either amplified or unamplified) being directed into the Bragg modulator.","In particular, for example, the different heterodyne outputs that correspond to different Zeeman splitting amounts (and thus different data points such as a “0” or a “1”) can be input into a Bragg modulator and cause light which is incident on the Bragg modulator to be deflected in different amounts.","Thus, the various Zeeman splitting and/or shifting frequencies that are produced can eventually result in different amounts of light deflection due to the use of a Bragg modulator.","The amount of deflection of light can be observed by the use of, for example, the light detection devices discussed earlier herein.","Similarly, the different heterodyne outputs could be directed into a suitable filter which, due to the different inputs, would regulate the intensity of light passing therethrough.","Accordingly, the different intensities of light could then be detected/recorded by a suitable means discussed elsewhere herein.","This particular embodiment of the invention which utilizes differing magnetic fields to result in differing amounts of an appropriate frequency splitting and/or shifting is very suitable to be used for the analog storage/retrieval of information.","In particular, a substantial continuum of splitting and shifting frequencies results in most materials that are subjected to a corresponding continuum of changing magnetic field strengths.","If a continuum of such differing split, shifted and/or splitting frequencies can be reliably detected, then analog or analog-like (e.g., higher order than binary systems) computing with this technique is facilitated.","Additionally, many of the aforementioned embodiments of the invention can also be used in combination with various holographic and/or three dimensional memory approaches.","To achieve all the foregoing objects and advantages, and to overcome the looming problem in the art of an impending memory barrier, the present invention discloses various means for storing and retrieving information in a digital and/or analog format.","With regard to the digital storage of information, a code other than a binary code for the storage of information can be utilized.","The result of using a higher order code for digital information storage (i.e., 3 or more characters) is the ability to store more information in the same amount of area or the same amount of information in a smaller area.","Accordingly, the present invention provides significant advancement in the art of data storage and/or data retrieval which will assist in preventing the memory barrier from being realized."],["TECHNICAL FIELD The present invention relates generally to the storage and/or retrieval of information on magnetic and/or optical storage media by using one or more novel approaches alone or in combination.","These novel approaches are capable of using at least one code which may comprise more than two values (i.e., more than a “0” and a “1”).","A first series of approaches for the storage of information applies generally to existing optical storage/retrieval systems (e.g., CD's, DVD's, etc.)","as well as novel optical systems; while a second series of approaches applies generally to existing electric and/or magnetic storage/retrieval systems (e.g., magnetic, magneto-optic, etc.)","as well as other novel electrical/magnetic systems.","Each series of approaches is capable of storing information in one or more codes, wherein such approaches permit, if desired, the use of at least one higher order code which is different from the traditional binary code of “0's” and “1's” currently utilized for the storage of digital information.","Said at least one higher order code may comprise three or more optical and/or magnetic values or bits that are used to represent, for example, ASCII or Unicode characters that are currently represented predominately by the traditional binary code.","This higher order code may also be an analog or analog-like code.","BACKGROUND OF THE INVENTION Data storage devices have been becoming smaller in size (as well as faster) due to, for example, the continued improvement in the ability to store more information in smaller spaces.","For example, over the past twenty years the ability to place more data on magnetic storage media such as magnetized hard-disk drives and/or floppy disks, as well as on optical media (e.g., compact disks “CD's” and more recently, Digital Versatile Disk “DVD's”) has been increasing at a phenomenal pace.","The impressive increase in the ability to store more data in smaller areas has been one of the main driving forces for rendering computers, and other electronic devices with reasonably sized memories, accessible to a typical home or small business consumer.","Further, the high operating speeds of microprocessors have required stored data in computers to be rapidly accessible (e.g., both the rate at which information is delivered to a microprocessor as well as the amount of time it takes between the time that a microprocessor requests a file and when the first piece of data begins to flow from the file to the microprocessor are important).","Magnetic memory has undergone many recent improvements and has been utilized extensively in data storage/retrieval devices because magnetic memory currently provides the least expensive alternative for the fast writing and reading capabilities for data.","However, the various currently utilized magnetic technologies face a variety of technical challenges and appear to be headed for a stopping point.","Specifically, these various approaches for the storage and/or retrieval of information may no longer be capable of meeting the growing demands of the industry, without experiencing significant technical enhancements.","For applications where speed has historically been less important, various optical technologies have been growing in popularity.","Many technical advances have been made in the recording and/or retrieval of information by optical means.","In systems which use compact disks (“CD's”), compact disk read-only memory (“CD-ROM's”) and/or Direct Video Disks (“DVD's”), the storage and/or retrieval of information has also been of great interest.","For example, the first CD's manufactured were CD-ROM's that are currently capable of storing about 650 MB of data.","This equates to about 74 minutes of play-time for music that has been recorded in a typical recording mode.","In particular, the standard current approach for utilizing CD-ROM's, includes the use of a solid state interrogating laser (or in some cases more than one laser) which has a wavelength of about 780 nanometers (0.78 μm).","The standard CD-ROM is about 12 cm in total diameter and has a thickness of about 1.2 mm.","The standard CD-ROM has information recorded on plateaus (“bumps”) and valleys (“pits”) that spiral from the inside of the disk toward the outside of the disk.","The digital data stored in the tracks that comprise the spirals must be separated by enough space (e.g., tracks are typically separated radially by about 1.6 μm) so that when the data is read with an interrogating solid state laser, crosstalk from adjacent tracks does not occur (i.e., recorded information is received from only one track at a time).","Accordingly, the placement of more information onto a typical CD has been limited by the required size (e.g., length and width) of the plateaus (“bumps”) and valleys (“pits”).","The size of the plateaus and valleys (e.g., typically not less than 830 nanometers in length) has been a function of various factors, including the wavelength of the laser utilized to read the information from the CD.","Various attempts have been made to increase the memory capacity of current CD's, including the stacking of a plurality of disks one on top of the other so as to provide more memory space, as well as other attempts to miniaturize the digitized information stored in the tracks.","The most recent significant improvements in this type of optical storage/retrieval technology have been the creation of the DVD, which is known as the “Direct Video Disk” or the “Digital Versatile Disk”.","The first commercial products of DVD's entered the marketplace in late 1997.The DVD technology is similar to the technology in CD's, except that, for example, the wavelength of lasers utilized to read information from DVD's is smaller than that used for CD's, namely, about 635 nanometers or 650 nanometers, compared to about 780 nanometers for CD's.","The size of the plateaus (“bumps”) that make up the DVD are about 320 nanometers wide, a minimum of about 400 nanometers long and about 120 nanometers thick.","About 740 nanometers separate one track from the next, compared to the about 1600 nanometer separation of tracks in CD's.","A standard DVD is physically about the same size as a CD, but has a memory capacity that is about seven times greater than that of CD's.","In particular, the memory capacity of a one-sided, one-track, DVD is now about 4.7 GB.","However, recent improvements in DVD have allowed digital information to be placed on both sides of a single track, as well as both sides of a double track.","In this regard, a first interrogating laser reads the digitized data on a first side of a first track (i.e., in the form of bits arranged in a spiral track that begin in the innermost portion of the DVD and spirals outward).","Then, information can be retrieved from the opposite side of the first track in a similar manner from a second laser; however, the spiral track of information typically begins on the outermost portion of the disk and spirals inward toward the center.","Accordingly, when dual-track, dual-sided DVD disks are used, the current amount of memory that can be achieved is about 17 GB.","Another optical data storage technique which shows promise is known as holographic memory.","The potential for high storage capacity and high storage retrieval rates using the holographic technique is tremendous.","In particular, for example, recent work in a system known as Holographic Data Storage System (“HDSS”), has shown that data can be accessed in 100 microseconds or less, which is several orders of magnitude faster than the millisecond retrieval of data in typical magnetic-disk drives.","Briefly, in this technology, an optical-interference pattern is created within a desired photosensitive material (e.g., a crystal or a polymer).","The optical interference pattern is imprinted as a result of physical changes in such materials.","The interference pattern is created by the interaction between an object beam and a reference beam, both of which typically originate from a single light source (e.g., a laser), but are split into two different pathways by a beam splitter.","Specifically, the object beam is incident on a target object that contains data, and modifies the object beam, while the reference beam, taking a separate path which leaves it unmodified, interferes with the object beam at the holographic storage medium.","When the two beams meet at the holographic storage means, the interference pattern which results from their interaction is stored on or in the holographic storage means.","The holographic storage means is later interrogated by a reading or reference beam, which upon interacting with the holographically stored interference pattern, can then recreate the holographic image containing the data originally stored in the object beam.","While the holographic optical technique shows great promise for the future, current industrial emphasis is being placed primarily on improving existing digital optical and magnetic storage/retrieval systems so that more information can be stored in smaller spaces, and retrieval times can be decreased without significantly changing the basic methodology and hardware for the storing and retrieving of information.","With regard to placing more data onto magnetic recording media, effects such as the paramagnetic effect (e.g., magnetic crosstalk between magnetic domains comprising bits) are already beginning to cause problems.","Experts have postulated that in coming years, magnetic storage technologies could reach a limit imposed by the superparamagnetic effect (“SPE”).","The physical phenomenon known as SPE can occur in a magnetic data storage system when the magnetic spins of electrons in the domains which comprise a bit (e.g., in current technology typically either a “0” or a “1”) become unstable due to, for example, the environmental thermal energy surrounding the bits.","Instability of bits occurs when smaller and smaller numbers of domains or atoms are used to comprise a bit and the ability for the domains or atoms to maintain their given spin directions (e.g., corresponding to, for example, a “0” or a “1”) becomes difficult.","Non-maintenance of spin directions results in a “flipping” of spins back and forth and thus changing “0's” to “1's” and “1's” to “0's”, etc.","Such changing or flipping of spins will result in a corruption of the data or information that the “0's” and “1's” represent.","Accordingly, in order for smaller numbers of atoms to be used to form a bit and in order for the bit to be viable, techniques and/or materials for preventing the superparamagnetic effect need to be developed to overcome the deficiencies of the prior art.","Another potential problem in recording information magnetically is that the write head which is used to align magnetic domains in the magnetic storage media may be limited in the ability to impart a required field strength over the entire domain area comprising a bit to cause the magnetic domains to behave as though they have been exposed to a substantially uniform field.","In such cases, it is possible that differently oriented domains within a bit (e.g., differently oriented due to being subjected to different magnetic field strengths within a single writing head due to, for example, non-uniformity of the magnetic flux lines emanating from the write head and/or differently oriented because some domains require more energy than other domains to align due to, for example, their location within a bit) may have a higher tendency to become corrupted, as well as adding to signal/noise ratio problems.","One attempt for miniaturizing domains involves the use of so-called “hard” magnetic materials.","The use of hard magnetic materials is desirable when compared to “soft” magnetic materials because hard magnetic materials are more likely to maintain their magnetic spin directions when domains comprising the hard magnetic materials are closely spaced.","In this regard, various rare-earth and transition elements have been found to be magnetically stable and are thus termed “hard”.","Such magnetically stable materials are known to have a higher coercivity, which is typically represented by “Hc”.","Stated in an over simplified manner, the higher the Hc value, the greater the resistance of the material to outside influences, such as outside magnetic fields (e.g., the harder it is for material to lose its imprinted spin direction due to an increase in temperature and/or the influence of other magnetic fields, etc.).","However, one drawback for utilizing “hard” magnetic materials is that hard magnetic materials are more difficult to magnetize than soft magnetic materials (e.g., in some cases, depending on the particular materials chosen, much more difficult to magnetize).","This makes it more difficult to record data initially in hard magnetic materials.","There are various known approaches for solving the problem of encoding data into hard magnetic materials which approaches utilize a laser in conjunction with an appropriate magnetic write head.","These various approaches have been referred to generally as the “magneto-optical” approach.","These magneto-optical approaches effectively reduce the Hc value of the magnetic domains by causing the domains to be locally heated just prior to being subjected to the magnetic write head.","Thus, the coercivity (Hc) of the domains is effectively reduced and when such effective reduction in Hc has occurred, a somewhat standard write head, or in some cases a very different write head, can be used to alter favorably the magnetic domains.","In other words, the use of a laser, which provides thermal energy to the domains of the hard magnetic material, causes the hard magnetic material to behave in a manner which is similar to a somewhat softer and thus easier to magnetize magnetic material.","Each of the known approaches for utilizing the magneto-optic techniques involves the use of at least one laser and/or at least one laser focusing system.","However, one of the common problems facing these magneto-optical approaches is that the lasers that are utilized to soften the hard magnetic material prior to subjecting the hard magnetic material to a magnetic field is that the lasers typically generate excess amounts of heat that can flow to neighboring bits or tracks (e.g., thermal energy is caused to migrate to undesirable neighboring tracks or areas resulting in a potential corruption of stored information).","Accordingly, various laser applications and/or storage solutions have been devised to minimize the transfer of heat from the laser to neighboring tracks, and thus minimize the loss of recorded information.","However, to date this thermal transfer of laser energy still presents problems to varying degrees in all the prior art approaches.","One interesting technique for the reading of data in connection with one optical-magnetic system is the use of an effect known as the “Kerr effect”, and more particularly, the “magneto-optical Kerr effect”.","Briefly, current uses of the magneto-optical Kerr effect involve incident polarized light being reflected from the surface of a magnetic domain in different ways, depending on the orientation of the magnetic domains (e.g., depending on electron spin directions).","The changes in the reflected polarized light are made, by algorithms, to correspond to different digitized data.","Specifically, when the incident polarized light encounters different magnetic field orientations (e.g., north or south domains comprising a bit which are ordered to represent either a “0” or a “1”) the polarization state of the reflected light is changed.","Another approach to the digital storage of information that shows future promise is known as atomic resolution storage (“ARS”).","This approach, similar to holographic techniques, is capable of storing tremendous amounts of information in a small amount of space.","In this technique, generally, very small electron probes, which are used to generate electron beams, are formed into an array.","The electron probes have tips that are roughly the size of atoms.","The electron beams from the electron probes are made to be incident upon a storage medium so that the incident beams cause some sort of physical change in the storage medium (i.e., the medium comprising the computer memory).","One example of such a storage medium is a material that is capable of containing at least two stable phases at ambient operating conditions so that an incident electron beam emitted from an electron probe changes the storage material from one phase to the other.","In this technique, encoded bits of information are represented by the change in phase of the phase-change storage material.","This technique, similar to the magneto-optical technique discussed above, also suffers from the problem of heat flowing between data spots created by the incident electron beams when the spots or bits are initially recorded.","Accordingly, one of the challenges faced by this relatively new technology includes reducing the flow of heat between data spots which are created by the array of electron beam generators.","The retrieval of stored information is also a challenge.","In this regard, as bits become smaller and include lesser numbers of magnetic domains per bit and/or the domains are oriented differently, (e.g., domains are aligned perpendicular to the substrate containing at least one magnetic surface thereon instead of being parallel to the substrate surface) such bits are capable of being packed more tightly together.","Assuming that the bits are not corrupted by their tight packing and/or smaller numbers of magnetic domains and/or non-uniform alignment within a bit, then reading of these tighter packed domains comprising the bits also becomes a challenge which the prior art is still struggling to overcome.","Historically, large numbers of magnetic domains were present in each bit which contained digital data.","Thus, the resulting or induced magnetic fields were relatively large and various forms of known inductive reading techniques were sufficient to ascertain whether the hundreds or thousands of aligned domains in a bit corresponded to a “0” or a “1”.","However, as prior art miniaturization recording techniques progressed, the known inductive reading techniques were typically not sensitive enough to determine accurately the alignment of ever decreasing numbers of domains comprising a bit (e.g., signal to noise problems in the storage and/or retrieval of digital data bits were exacerbated).","The development of magnetoresistive heads, and the subsequent development of giant magnetoresistive heads, has enabled smaller numbers of domains comprising a digital bit to be read accurately.","In both of magnetoresistive and giant magnetoresistive heads, the electrical resistance of one or more materials comprising the read head(s) is examined.","In particular, both magnetoresistive and giant magnetoresistive heads are made from materials that exhibit small changes in their electrical resistance as a function of magnetic fields created by oriented domains to which such heads are exposed.","Giant magnetoresistive heads are two or three times more sensitive than magnetoresistive heads due to their novel structure which includes layering of different materials stacked on top of each other.","One problem that continues in these technologies is the presence of magnetization vortices in the recorded domains.","Such vortices may result from the difficulty in achieving uniform domain alignment within a bit due to, for example, the difficulty in applying a required magnetic field (e.g., uniform field) to the domains during the recording or writing process.","While use of the magneto-optical Kerr effect and new read heads such as magnetoresistive heads and giant magnetoresistive heads have improved digital data retrieval techniques, further miniaturization is pushing the limits of these systems.","Accordingly, additional improvements are necessary in the ability for hardware, combined with suitable programming, to detect even smaller numbers and/or smaller sizes of domains comprising digitized (or analog) data bits.","Thus, additional innovations in the detection of stored data are required.","In addition to the storage of information digitally, the possibility or promise of storing information in an analog manner continues to intrigue various investigators.","While certain improvements have been made in, for example, the processing of analog information (e.g., the advent of VLSI (Very Large Scale Interrogation) chips, the techniques for the storing of analog information need much improvement.","Particularly, while analog computing has been known for several decades, an accurate and fast-paced system for the storage and retrieval of information has continued to elude researchers.","In this regard, digital computing has received the greatest amount of attention because of it's relative dependability for the storing and retrieving of information in a “1” or a “0” digitized format.","Briefly, digital approaches typically include storing a sampling of the analog information that is to be recorded.","The number of samples taken (e.g., such sampling typically occurs in predetermined units of space or time) from the analog source will correspond to the accuracy between the original analog image or sound and the stored digital data (i.e., the more digital samples that are recorded of an original analog image or sound, the more accurate the reproduction of the original analog image or sound will be).","These techniques of predetermined quantum amounts of sampling of analog images have been adequate for most digitized applications, however, as the need for more accurate recording and retrieval of information occurs (e.g., in, for example, the fields of computing or determining various engineering and scientific relationships in the following representative areas: gravitational; electrostatic; magnetic; thermal; stress; fluid flow field analysis; wave propagation; image processing; etc.","), the availability of computing with more accurate data becomes important.","However, the digitization of tighter and and/or smaller sampling amounts from an analog source can result in tremendous amounts of digitized data being stored to represent a small actual amount of analog information.","This is one area where analog computing has a distinct advantage compared to digital computing.","In particular, an analog computer will store only a single piece of information corresponding to a single sampling area, whereas a digital computer will need to store somewhere between, for example, 8 and 16 bits of data to represent the same single piece of information stored in an analog fashion.","The large number of bits required to store digital information corresponds to the requirement to store information in an ASCII or Unicode format.","However, in order to escape from the current ASCII or Unicode format, analog storage requires a very large number of different data bits (e.g., data corresponding to much greater numbers than merely “0s” and “1's”) to be stored and retrieved.","Current prior art techniques have great difficulty in storing and/or reading bits corresponding to anything other than “0's” and “1's”, let alone the attendant problems of storing and/or reading, for example, a near infinite number of possibilities (e.g., 1, 2, 3 .",".",".","n+1).","However, when performing complex calculations, the digital process may result in very slow processing times as well as requiring very large memories.","It is for this, as well as other reasons, that analog computing is still very attractive.","Thus, while advances in the processing of analog information have been made, reliable techniques for the storage and retrieval of analog information are still being sought without any good solutions currently existing.","As stated above, with regard to digital computing, much of the recent technical emphasis in data storage and retrieval for both optical and magnetic memory enhancement has been on shrinking more information into smaller areas.","The ability to place bits, which are the smallest elements of information used by current digital memory systems, into a binary code format, which is a combination of “0's” or “1's”, into a smaller area on, for example, magnetic and/or optical disk storage media (e.g., a disk or a tape) may soon reach a limit.","Thus, current technologies for writing and/or reading binary data onto and/or from current storage media will be pushed to the maximum limit.","Computer designers are already facing a performance gap whereby processors can process information faster than the information can be retrieved from computer memory.","The majority of the currently proposed approaches for the digital storage of information, suffer from the same problem, namely, that the magnetic and/or optical information which is stored uses a binary code (i.e., a base-2 number system) which is a combination of “0's” and “1's”.","The predominance of the use of a “binary” code has been due to, for example, difficulty in distinguishing signals (e.g., a “0” or a “1”) from background noise.","In this regard, many techniques have been proposed which use various hardware, as well as algorithms, which attempt to read accurately the stored information as either a “0” or a “1”.","However, certain prior art techniques have been postulated for utilizing codes other than binary.","In particular, U.S. Pat.","No.","6,154,432, entitled “Optical Storage System” discloses a holographic system which records information in very small spots and postulates that 10 or more digits may be possible to record on a single bit.","Accordingly, this holographic technique discloses codes higher order than binary.","Further, for example U.S. Pat.","No.","5,450,363, entitled “Gray Coding for a Multi Level Cell Memory System” discloses the use of a so-called “gray code” approach (also known as “gray scale” by others) for data storage.","This approach also uses a system which is higher order than binary.","In particular, reference is made to the storage of multiple bits of information in a single memory cell thus creating a multi-level cell.","Still further, U.S. Pat.","No.","6,061,005, entitled “Encoding of Data”, discloses a method of encoding data on magnetic cards which uses symbology other than binary, such as trinary, quaternary or even higher symbology, for the recording of information.","However, while various techniques have been postulated for systems which use codes higher than binary, none of such codes have been commercially adopted due to, for example, signal to noise ratio problems, etc.","In particular, reading errors can occur due to lack of contrast between data values.","Such lack of contrast can be caused by random noise (e.g., vibrating inaccurate positioning of recording/reading heads, fidelity loss during recording, etc.)","and determinate variations.","Accordingly, a need exists for techniques which can be commercially viable which can use codes reliably that are higher order than traditional binary codes for the storage and/or retrieval of information.","A very particular combination of “0's” and “1's” is known as the American Standard Code for Information Exchange (i.e., the ASCII code) which assigns a unique code number between 0 and 127 to each of the 128, 7-bit, binary number characters.","Table 1 below shows in table form the current ASCII characters arranged by Hexidecimal digits (i.e., 128 different encoding combinations of groups of seven bits).","Reading within the Table from left to right and then down, the first 32 values are codes which correspond to various computer control functions such as line feed, carriage return, etc.","The 33rd value corresponds to the “space” character.","Values 34-48 correspond to symbols and punctuation marks.","Values 49-58 correspond to digits, and so on, until reaching value 128, which corresponds to “delete”.","In addition, each of these values also has a corresponding hexidecimal character.","For example, the hexidecimal digit corresponding to the “space” character is “20”, while the hexidecimal digit corresponding to the letter “n” is “6E”, and so on.","TABLE 1 00 01 02 03 04 05 06 07 08 09 0A 0B 0C 0D 0E 0F 00 NUL SOH STX ETX EOT ENQ ACK BEL BS TAB LF VT FF CR SO SI 10 DLE DC1 DC2 DC3 DC4 NAK SYN ETB CAN EM SUB ESC FS GS RS US 20 !","“ # $ % & ‘ ( ) * + , − .","/ 30 0 1 2 3 4 5 6 7 8 9 : ; < = > ?","40 @ A B C D E F G H I J K L M N O 50 P Q R S T U V W X Y Z [ \\ ] {circumflex over ( )} — 60 ’ a b c d e f g h x j k l m n o 70 p q r s t u v w x y z { | } ˜ DEL These 128 characters are enough characters for North American English to be stored and/or retrieved, but are not enough for many other languages (Note: In ASCII, the eighth bit is used, and is thus normally set to “0”).","To represent more than 128 characters, 8-bit (rather than 7-bit) binary numbers are utilized.","For each digit in these eight bits there are two choices for the digit, either “0” or “1”.","This results in a possibility of an additional 128 characters or a total of 256 different combinations of “0's” and “1's” (i.e., 28).","Accordingly, the current data storage approaches utilize these 256 combinations of “0's” and “1's”.","However, these 8-bit numbers still do not provide enough room for all characters that need to be used in the world (e.g., certain Asian languages have thousands of characters each).","In addition, there is no universal agreement in the world on what all the characters should be, whether referring to the 128 characters, or the 256 characters, or any number of characters beyond these.","However, certain approaches for dealing with all the various languages in the world, which collectively contain many thousands of characters, are beginning to utilize something referred to as the “Unicode”, which uses a 16-bit character set.","In order to adopt something similar to the Unicode, even greater strain will be placed on current memory techniques.","Whether using an 8-bit, 16-bit, or some other approach, all such approaches have been limited primarily to using a binary system of data storage.","However, due to: (1) the impending size barrier for the storage of information on, for example, magnetic and/or optical media; (2) the performance gap between how fast a processor can process information versus how fast a processor can access information; and (3) the goal for a universal adoption of a common set of characters throughout the world, a different approach for the storage of information, either by digital or analog approaches, is clearly needed.","The present invention satisfies the current and future data storage needs by using different approaches for the storage and/or retrieval of data; and/or different codes that use a code higher than the current “base-2 code”.","SUMMARY OF THE INVENTION It is an object of the invention to provide a means for storing/retrieving digitized information optically.","It is an object of the invention to provide a means for storing/retrieving digitized information magnetically.","It is an object of the invention to provide a means for storing/retrieving analog information optically.","It is an object of the invention to provide a means for storing/retrieving analog information magnetically.","It is an object of the invention to provide a storage/retrieval means for utilizing codes other than binary.","It is an object of the present invention to provide an increased number of characters that can be stored as a single bit of information.","It is an object of the invention to provide the ability to store the 128 ASCII-type characters in a much smaller space than the space that is used in a standard binary code of “0's” and “1's”.","It is an object of the invention to provide a means for storing/retrieving codes other than binary.","It is an object of the invention to utilize at least a base-3 optical storing/retrieving system.","It is an object of the invention to utilize a least a base-4 coding storing/retrieving system.","It is an object of the invention to utilize at least a base-6 coding storing/retrieving system.","It is an object of the invention to utilize at least a base-8 coding storing/retrieving system.","It is an object of the invention to utilize at least a base-10 coding storing/retrieving system.","It is an object of the invention to utilize particular aspects of the Zeeman effect in connection with the storage/retrieval of digitized information.","It is an object of the invention to utilize particular aspects of the Zeeman effect in connection with the storage/retrieval of analog information.","It is an object of the invention to utilize different shades or colors of spots or dots in connection the optical storage of digitized information.","It is an object of the current invention to utilize different shades or color or spots or dots in connection with the optical storage of analog information.","It is an object of the invention to utilize either monochromatic or polychromatic light in connection with the optical storage of digital and analog information.","It is an object of the invention to utilize an analog-like approach to the storage of data.","The current process of storing bits, which is the smallest element of information used by current memory systems, uses a binary format of only “0's” and “1's”.","A bit which is “off” is considered to be “false” or “not set”, while a bit that is “on” is considered to be “true” or “set”.","Historically, because bits could only be one of two values, bits have been combined together into larger units in order to represent more characters or values.","“Nibbles” are groups of four bits and “bytes” are groups of eight bits (i.e., two “nibbles”).","Bytes are typically what are used to store “characters”.","“Characters” typically include letters, numbers and/or symbols.","For example, the ASCII code uses 128 different encoding combinations to represent: (i) The letters “A-Z” in both upper and lower case (i.e., values 65-90 and 97-122, respectively); (ii) Special characters (e.g., “<”, “.”, “%”, “$” “?”, etc) (e.g., values 33-47); (iii) The numbers 0 - 9 (i.e., values 48-57); and (iv) Certain control codes used for device control (i.e., values 0-31).","To represent more than 128 characters, an eighth bit can be utilized.","However, the ability to store a binary code of only “0's” and “1's” limits the ability for information to be miniaturized.","For example, to store a single character from the ASCII code, at least seven bits of memory (typically eight bits are currently required because the last bit is usually set to zero) are needed.","Table 2 sets forth one example of how a typical computer memory system would store the word “Patent”.","TABLE 2 ASCII LET- CHAR- BINARY HEXADECIMEL BITS OF TER ACTER NUMBER DIGIT OR BYTE MEMORY P = 80 = 01010000 50 8 a = 97 = 01100001 61 8 t = 116 = 01110100 74 8 e = 101 = 01100101 65 8 n = 110 = 01101110 6E 8 t = 116 = 01110100 74 8 Thus, the letter “P” corresponds to the ASCII character 80, which is represented by the binary code “01010000”.","The first “0” in the sequence is often referred to as “the last bit” and is typically set to “0” for all ASCII characters.","The corresponding hexidecimal digit or byte representation is “50”.","In order for the computer memory system to store the letter “P”, eight bits must be sent to memory in the order “0-1-0-1-0-0-0-0”.","Likewise, to store the letter “a”, eight bits must be sent to memory in the order “0-1-1-0-0-0-0-1”.","Accordingly, 48 bits are used to store the six (6) letter word “Patent”.","Similarly, when a microprocessor unit accesses the portion of memory containing the set of bits representing the word “Patent”, the same number of bits (i.e., 48) or six (6) bytes must be processed by the microprocessor.","Clearly, if the word “Patent” could use a smaller amount of memory, less memory capacity would be needed in a computer and/or the rate at which data could be stored and/or retrieved from memory would be enhanced.","In a first aspect of the invention, a coding system that is a higher order system than a binary coding system is utilized for the digitized storage of information.","For example, any digital coding system which uses three or more different code characters (i.e., a coding system which operates in “base-3” or greater) will result in a reduction in the number of bits needed to store the same amount of digital information relative to a binary system.","Thus, by using a higher order memory system, the possibility of storing more digitized information in a smaller memory area exists.","Likewise, the rate of storing and/or retrieving digitized information should also increase due to memory being stored in a physically smaller area.","This permits a data retrieval device to function more efficiently.","The present invention permits the use of a digitized memory system which is base-3 or greater.","Moreover, as more code characters are used (e.g., higher order systems of more than 10 or more than 20 different characters are utilized) the digitized system begins to behave in a manner which is similar to analog systems and can thus be referred to as “analog-like”.","In other words, as the amount of digital sampling of an analog source increases, the recorded digital signal begins to approach the storage of most features of the analog signal.","However, current data storage/retrieval techniques utilize, for example, 8 or 16 bits to store a single piece of analog information, which limits the amount of sampling of an analog source that can be reasonably performed when creating a digital representation of the analog source.","Accordingly, the digital data storage/retrieval techniques of the present invention can be vastly superior to current digital techniques which utilize only “0's” and “1's” and are limited to sampling much smaller areas of analog sources than would be desirable in many cases (e.g., for complex computer modeling of numerous events, etc.).","A first embodiment of an alternative memory storage system of the present invention may function in a manner somewhat analogous to the manner in which a Compact Disk (CD) or Digital Versatile Disk (DVD) player functions.","Current CD and DVD technologies utilize a rotating disk comprised of a number of layers of different materials.","The layers may include, for example, polycarbonate plastic, aluminum and acrylic.","The aluminum in the disk includes a series of plateaus and valleys sometimes referred to as “bumps” and “pits”, respectively.","Laser light is caused to focus upon the bumps (plateaus) and pits (valleys) in the rotating disk.","The reflection of the laser from the successive plateaus and valleys corresponds to a data stream (e.g., a binary stream) of information.","CD's and DVD's use, for example, 16-bit strings of binary coded data.","Specifically, laser light reflects differently from a valley compared to the reflection from a plateau and these different reflective properties of the laser, when reflected from these valleys and plateaus, correspond to the binary code data values of “0” and “1” for each bit.","In one embodiment of the present invention, a similar concept to the CD and DVD is utilized, however, rather than utilizing valleys and plateaus (i.e., bumps and pits) in an aluminum layer embedded in a polycarbonate plastic CD or DVD, the present invention utilizes at least two data values per bit represented by different intensity, colors or color width (e.g., different wavelength or frequency).","These data values can be optically created, stored in dots or spots, and optically examined or interrogated (e.g., by known transmission and/or reflection techniques).","These optical dots or spots may be of a size which is approximately the same order of magnitude in size as the bumps and pits which comprise current data tracks used on CD's and DVD's, they may be larger, or they may be smaller.","In particular, similar to current CD's and DVD's, these dots or spots can be put together to form strings or tracks of data which begin at an innermost portion of a rotatable CD/DVD disk and extend spirally out therefrom in the form of tracks.","Accordingly, current CD's of approximately 12 centimeters in diameter would be an acceptable size.","However, if the dots or spots of the present invention are smaller than the “bumps” of the current technology, it is possible for CD's to be smaller in size, if desired, or more data can be placed into the same amount of memory space.","Thus, it is possible to put an amount of optical information onto a rotatable disk of approximately 12 centimeters in diameter which exceeds that amount of information which is currently stored on CD's/DVD's.","However, it is possible for the dots or spots to be larger in size than current bumps and pits in CD's/DVD's while still storing more information relative to standard CD's/DVD's.","This is possible due to at least two effects.","First, tracks may be capable of being located closer together due to, for example, lesser amounts of crosstalk between bits (e.g., recording and/or reading operations can take place in a lesser amount of space).","Second, if digital codes higher than binary are adopted, less actual bits need to be stored relative to a binary system (e.g., rather than storing 8 or 16 bits to represent a single piece of information, a lesser number of bits can be used to represent the same piece of information that is being stored).","It should be noted that as the number of characters that can be stored in a single bit become larger, the digital storage system actually approaches behaviors similar to an analog storage system and can be referred to as “analog-like”.","In particular, the present invention is capable of utilizing a coding system other than the binary coding system of “0's” and “1's” now used in CD/DVD systems.","For example, a series or range of dots or spots of different color intensities (e.g., white-gray-black), and/or different colors (e.g., red-yellow-violet), and/or both as in varied intensity of polychromatic light, can be placed upon a surface (or embedded somewhere within a material), such as a suitable photoferroelectric material such as “PLZT” (i.e., lead lanthanum zirconate titanate ceramic), which is shaped in the form of, for example, a disk.","When, for example, such a disk containing these different intensity data value dots or spots is rotated and interrogated by, for example, a known optical system (e.g., a laser system) with a suitable focusing lens and intensity detecting means (e.g., a suitable photosensitive material or photodiode), the different intensities of the dots or spots can be registered and can be coded to correspond to different values (e.g., “0”, “1”, “2”, “3”, etc.).","Thus, rather than using a simple binary or base-2 system where a black dot corresponds to “0” and a white dot corresponds to “1”, the present invention provides for a larger number of options for digitized data storage.","However, as discussed in more detail herein, depending on the sensitivity of the system, this technique could also be used for actual analog computing.","Accordingly, for example, in a simple digitized quaternary system having a quaternary code consisting of 0's, 1's, 2's and 3's, there would be 4 different choices of basic digital information storage (i.e., a “base-4” system) rather than only two choices in a binary or base-2 digital system.","Likewise, in a digital system which utilizes 8 different combinations of spots or dots (i.e., a “base-8” system) eight different options exist for encoding a piece of information rather than only two options in a standard binary or base-2 digital system.","The use of a larger number of possibilities other than “0's” and “1's” (e.g., 0, 1, 2, 3, 4, 5, 6, 7, etc.)","render it possible to store a much greater amount of information in a much smaller physical area due to the elimination of unneeded bit space.","Moreover, as the number of possibilities other than “0's” and “1's” increases, (e.g., systems greater than base-10 such as base-20 or greater systems) the closer the digital system comes to being or performing like an analog system, which has certain advantages over digital systems.","An apparatus according to the present invention permits suitable optical data to be stored onto an appropriately constructed and shaped optical recording medium by using, for example, a laser light source.","The optical recording media (e.g., a PLZT ceramic) can be in the form of a tape, disk, or credit card or any other acceptable shape.","Alternatively, a photodiode or photodiode assembly can also be used.","In all such cases, a light source (e.g., laser light source) can be used in combination with, for example, various means for controlling the direction, polarization, intensity and/or final color of the light source.","Various examples of such systems include the following: (1) a rotatable polarizing filter (or set of filters), sometimes referred to as an analyzer, used in combination with a suitable light source which is subjected to a first polarizing filter(s) selectively modifying the polarized light from the light source.","Specifically, once monochromatic or polychromatic light has been polarized into a first direction polarizer the polarized light is directed toward a second polarizer (sometimes referred to as an “analyzer”) which, when rotated, results in, for example, different intensities of light being emitted therefrom.","The different intensities of light can be caused to be incident upon an appropriate recording/detecting means; (2) a light source (e.g., a laser light source), which is used in conjunction with a first polarizing filter is caused to be incident on a suitable light diffractor (e.g., a Bragg modulator) which can cause the polarized light from the light source to communicate with one or more of a series of additional polarizing filters (or analyzers) each of which analyzers are rotated, relative to the polarized source, at different angles, thus resulting an different amounts or intensities of light passing through each of the analyzers.","Thus, by selectively transmitting the polarized light through the different analyzers (e.g., directed by, for example, the Bragg modulator) different intensities of light can be achieved.","Such different intensities can be recorded and/or detected by a suitable means; (3) a light source (e.g., a laser light source) used in combination with an acousto-optic Bragg modulator, whereby different driving signals are input to the Bragg modulator which results in different intensities of light being emitted from the Bragg modulator.","The multiple intensity emissions are then directed to a suitable detecting/recording means and are made to correspond to various data points in base-2 or higher order digital data storage systems; and (4) rather than utilizing polarizing filters to polarize a monochromatic or polychromatic source of light, liquid crystal cells can also be utilized.","In particular, light can be caused to be incident upon a liquid crystal cell.","Depending upon whether voltage is applied to the liquid crystal cell, the output from the liquid crystal cell is oriented into one direction or another.","Liquid crystals of this type can be made of, for example, lithium niobate.","However, similar to the embodiments (1)-(3) discussed above, intensities can be varied due to relative polarization directions.","The outputs of light from the liquid crystal cell can be directed, by appropriate beam displacers (e.g., a Bragg modulator), toward a suitable recording/detecting means.","In each of the examples (1)-(3) discussed above, it is possible that a polychromatic light source could be utilized.","In this regard, when polychromatic light is made to be incident upon a first polarizer, and thereafter upon one or more additional polarizers (e.g., analyzers), in addition to different intensities of light flowing through the analyzer(s), a spreading of these different intensities over a particular frequency or wavelength range will also occur.","Specifically, when using polychromatic light, a spectrum of frequencies of various intensities results in the formation of a somewhat continuous curve of varying light intensities over a particular frequency or wavelength range.","The use of a polychromatic source can therefore result in secondary indicia for determining the value assigned to stored bits of data (e.g., a “0” or a “1”) and thus enhances data storage.","For example, not only could the highest intensity of light be recorded/detected, but also the starting and/or ending points which correspond to the lowest frequency intensity of light being emitted, and the highest frequency of light being emitted, respectively, or, alternatively, the area under each curve could also be compared.","In other words, more than one piece of information can be checked to verify the accuracy of each data point that is stored and read.","The presence of more than a single piece of information can result in more accurate reading of data and thus result in signal/noise improvements.","In each of the examples (1)-(4) discussed above, the different intensities or colors of light can be made to be incident upon an appropriate receiving/recording means (e.g., an optical storage means, such as a pohtopolymerisable/photocrosslinkable material, a photodiode assembly, etc.","), such output light being suitably focussed onto the appropriate receiving/recording means by known lens or optical systems etc., all of which are discussed in greater detail herein).","Suitable materials for recording various intensities or colors of light include the following materials: inorganic photochromic crystals, photorefractive materials, chalcogenide semiconductors, organic and polymer materials, thermoplastic media, reversible recording in Tellerium compounds, photothermal recording in Antimony compounds, magneto-optic recording and light stimulated recombination luminescence.","For example, in the simplest embodiment (i.e., that embodiment numbered “(1)” above) for understanding the basic concept for recording various intensities of light (even though this simple embodiment is not as commercially significant as the other embodiments discussed herein), each such recorded intensity corresponds to a different value for a digitized data bit.","In this basic case, different intensities can be achieved as follows: a polarized light source, such as a laser and polarized filter or polarizer, when used in combination with at least one rotatable polarizing filter (e.g., an analyzer) can result in light of different intensities being transmitted from the at least one rotatable analyzer.","Specifically, when the laser light source is passed through a first polarizing filter and a second polarizer (e.g., an analyzer) is sequentially rotated (e.g., from 0-90°) a predetermined amount each time relative to the first polarizing filter (i.e., either the first or second filter can be rotated), different intensity dots or spots corresponding to data values having different meanings (e.g., “0”, “1”, “2”, etc.)","can be produced.","The amount of rotation of the analyzer in relation to the polarizing filter can correspond to a different intensity of light being recorded on a suitable optical recording medium (e.g., a photoferroelectric material such as “PLZT”, inorganic photochromic crystals, photorefractive materals, chalcogenide semiconductors, organic and polymer materials, thermoplastic media, reversible recording in Tellerium compounds, photothermal recording in Antimony compounds, magneto-optic recording and light stimulated recombination luminescence).","If a photoferroelectric material is utilized, the different intensities of light can be caused to be incident upon the photoferroelectric material by use of, for example, a suitable optic lens system or a fiberoptic cable (or a series of cables) that is capable of accurately directing the optical signal to the photoferroelectric material for the proper recording of the various optical signals.","Thus, a large number of different intensity spots or dots corresponding to data values for a single bit can be created.","This basic system and apparatus permits higher order coding systems (e.g., base-3 or greater) to be utilized.","The manner in which such optical data is stored, on, for example, a rotatable disk, as well as interrogated, can be by techniques which are substantially parallel, and/or use hardware which is similar, to those techniques and/or hardware currently used in CD and DVD systems.","For example, a series of bits of an appropriate binary or higher order digital code system can be recorded onto a rotatable disk by starting to record the different intensity dots or spots near a central potion of the disk.","The recording continues to record data in a spiral manner to form tracks of data, such data being suitably separated, physically, to avoid corruption and crosstalk, until an outermost radial portion of the disk is encountered.","Likewise, such a disk can be read or interrogated by, for example, a laser system which focuses light onto the dots or spots and suitably measures the intensities (e.g., reflectance or transmittance) of the spots of data.","The different intensities can be interpreted by suitable algorithms and programs to correspond to different values in the desired coding system.","Moreover, by utilizing a digital optical coding technique other than binary, the present invention permits a much more rapid adoption of the desired “Unicode”, which takes into account, for example, all English language characters, Chinese characters, numbers and symbols, etc., in various digitized forms.","In another embodiment of the invention, a system which utilizes magnetic memory in combination with a suitable retrieval/reading means is utilized.","These memory/retrieval systems can be caused to function, generally, in a manner that is somewhat similar to current magnetic memory systems (e.g., magnetic tapes, cards, disks, hard drives, etc).","In addition, these memory systems can also use digital coding/reading systems higher than a base-2 system, if desired.","Still further, these memory systems can also store/retrieve information in an analog or analog-like format.","Specifically, it is another object of the invention to utilize certain aspects of the phenomena known as the Zeeman effects to result in a means for the storage and retrieval of information onto/from suitable magnetic media.","The term “Zeeman effects”, as used herein, denotes the effects of external magnetic fields on frequencies of atoms, molecules and components thereof, in the broadest sense.","Thus, as used herein, the term “Zeeman effect” includes not only what may be considered to be Zeeman effects in the more narrow, classical sense but also other magnetic effects such as magnetic quadropole effects, etc.","Digital data may be stored as either a binary code or some higher order code that is greater than the binary or “base-2” code.","Moreover, due to the apparent sensitivities of these data storage techniques, data may be stored/retrieved in an analog or analog-like manner due to the inherent operation of the Zeeman effects.","Specifically, without wishing to be bound by any particular theory or explanation, it appears that use of the Zeeman effects can result in a somewhat continuous set of differing data values that can be stored/retrieved.","The present invention utilizes one aspect of the Zeeman effects in the following general manner.","A magnetic field is applied to a suitable media that is capable of receiving and storing at least a portion of the applied magnetic field.","One example of such a suitable magnetic material is a ferromagnetic material that is capable of exhibiting different amounts of magnetic domain alignment as a function of increasing magnetic field strength up to its natural magnetic saturation point (i.e., whereby any further increases in applied magnetic field do not result in any measurable further alignment of magnetic domains).","Various known materials exist that meet this basic criteria, including many materials currently utilized in the magnetic storage industry (e.g., gamma Fe2O3, gamma Fe2O3 modified by Co, CrO2, etc.).","The various materials can be held together by a variety of organic polymers including, for example, vinyl chloride, polyvinylchloride, methylacrylate, polymethylmethyacrylate, polyurethane, epoxy, polyamides, etc.","Suitable substrates include typical polyesters for flexible materials and aluminum for rigid materials.","The differing amounts of stored magnetic field can result in a corresponding splitting or shifting of the electromagnetic energy frequencies or spectral frequencies (e.g., fine spectrum splitting, hyperfine spectrum splitting, vibrational spectrum splitting, etc.)","of one or more atoms or molecules of the magnetic material itself, or similar splitting or shifting in one or more materials that are located contiguous (e.g., in the magnetic material or in a read/write head) to the material containing the stored magnetic fields.","In the Zeeman effect, the amount of splitting or shifting of one or more frequency spectrum(s) is a function of the strength of the applied/stored magnetic fields.","Typically, the higher the magnetic field strength that is stored/applied (e.g., stored in a first material containing magnetic domains and thereafter applied to (e.g., influencing) a second contiguous material that exhibits Zeeman splitting at the stored magnetic field strengths), the further apart the splitting or shifting will be of one or more of the electromagnetic energy frequencies of a chosen spectrum of the targeted atom(s) or molecule(s).","In this Zeeman effect, when a relatively wide splitting of electromagnetic energy frequencies occurs due to the applied magnetic field, the amount of splitting may correspond to frequencies in the microwave region; whereas a narrow splitting will typically correspond to lower frequencies (e.g., radio).","The amount of splitting that occurs is important because in order to determine the amount of splitting that has occurred, a suitable interrogating electromagnetic energy is caused to be incident upon, for example, the magnetic material itself or the aforementioned contiguous material.","The contiguous material that exhibits Zeeman splitting at the stored magnetic field can be present as, for example, one or more separate sheets of material that are located adjacent to, and/or contiguous with, the material that contains the stored magnetic field.","When multiple contiguous materials are utilized, the present invention contemplates the use of a comparative analysis of the differences in Zeeman splitting between two or more such contiguous materials.","Alternatively, a material comprising, for example, one or more material(s) that are mixed with the magnetic material (e.g., a binder which holds together at least a portion of the material that stores the magnetic field) could also be used.","More specifically, a particular interrogating frequency, or set or sweep of frequencies, needs to be capable of favorably interacting with one or more of the split, shifted or splitting frequencies which result in the magnetic material, or in an alternative embodiment in one or more of the contiguous material(s), due to the different magnetic fields stored therein.","In other words, a first magnetic material is desirably chosen so that an applied magnetic field can be reasonably stored therein.","However, it is desirable for the magnetic material to be capable of storing reliably several magnetic fields of different strength (e.g., capable of having varying numbers of magnetic domains align with an ever changing magnetic field and such domains remain frozen or pinned so that they do not change with time) and, for example, in one embodiment of the invention, the contiguous material needs to produce split, shifted and/or splitting frequencies in response to the stored magnetic fields.","In this regard, if one or more contiguous material(s) are utilized in connection with the magnetic material(s), the contiguous material(s) needs to exhibit a splitting and/or shifting of one or more frequencies, for example, of its rotational, vibrational, etc., bands (e.g., at the atomic level) and the resultant splitting or shifting that occurs in the contiguous material needs to be detectable by an appropriate interrogating electromagnetic source (discussed in greater detail later herein).","Alternatively, a contiguous material does not need to be utilized.","In this case, the magnetic material per se, or at least a portion thereof, needs to exhibit desirable and detectable splitting and/or shifting of one or more frequencies at varying stored magnetic strengths.","Still further, it should be noted that the contiguous material could actually be located in a suitable read head which, similar to magnetoresistive and giant magnetoresistive heads, is influenced by the magnetic field strengths stored in the magnetic media.","However, rather than measuring changes in resistivity in materials comprising the read heads, the splitting or shifting of frequencies will be detected by a suitable technique, discussed elsewhere herein.","In this embodiment of the invention, the contiguous material may be a solid, a liquid, a gas or plasma (e.g., any material that shows desirable Zeeman splitting effects).","With specific regard to interrogating with an applied electromagnetic source to determine the amount of Zeeman splitting and/or shifting that has occurred at a spot on the material that corresponds to a stored bit of data, at least one suitable frequency can be applied as an interrogating frequency, or, alternatively a sweep or combination of frequencies can be applied as interrogating frequencies.","In this regard, if a single suitable frequency is chosen as the interrogating frequency, then heterodyne interactions of the various split, shifted and/or splitting frequencies with the single interrogating frequency can be measured and determined.","Alternatively, resonance interactions of the various split, shifted and/or splitting frequencies can also be measured and determined.","Each such determined heterodyned and/or resonant frequency can be made to correspond, by suitable programming or algorithmic techniques, to various data values.","Alternatively, a series or sweep of frequencies which may resonate with or heterodyne with various split, shifted and/or splitting frequencies achieved in the contiguous material can also be utilized.","These particular affects will be discussed in much greater detail later herein, however, it should be understood that these data acquisition techniques could be performed using hardware which is somewhat similar to that hardware currently used for magnetoresistive and giant magnetoresistive heads.","For example, once an interrogating frequency has been input to a material exhibiting Zeeman splitting and/or shifting, the output (e.g., a heterodyned frequency) can be used as a driving signal or input (e.g., depending on strength or frequency either amplified or unamplified) for various other devices.","One example of such other device is an acousto-optic Bragg modulator.","In this detection scheme, an appropriate light source is caused to be incident upon an appropriate Bragg modulator.","Suitable materials for a Bragg modulator include, for example, lithium niobate crystals and gallium arsenide semiconductor materials.","The incident light beam can be caused to be deflected in differing amounts due to different driving signal inputs (e.g., depending on strength or frequency either amplified or unamplified) being directed into the Bragg modulator.","In particular, for example, the different heterodyne outputs that correspond to different Zeeman splitting amounts (and thus different data points such as a “0” or a “1”) can be input into a Bragg modulator and cause light which is incident on the Bragg modulator to be deflected in different amounts.","Thus, the various Zeeman splitting and/or shifting frequencies that are produced can eventually result in different amounts of light deflection due to the use of a Bragg modulator.","The amount of deflection of light can be observed by the use of, for example, the light detection devices discussed earlier herein.","Similarly, the different heterodyne outputs could be directed into a suitable filter which, due to the different inputs, would regulate the intensity of light passing therethrough.","Accordingly, the different intensities of light could then be detected/recorded by a suitable means discussed elsewhere herein.","This particular embodiment of the invention which utilizes differing magnetic fields to result in differing amounts of an appropriate frequency splitting and/or shifting is very suitable to be used for the analog storage/retrieval of information.","In particular, a substantial continuum of splitting and shifting frequencies results in most materials that are subjected to a corresponding continuum of changing magnetic field strengths.","If a continuum of such differing split, shifted and/or splitting frequencies can be reliably detected, then analog or analog-like (e.g., higher order than binary systems) computing with this technique is facilitated.","Additionally, many of the aforementioned embodiments of the invention can also be used in combination with various holographic and/or three dimensional memory approaches.","To achieve all the foregoing objects and advantages, and to overcome the looming problem in the art of an impending memory barrier, the present invention discloses various means for storing and retrieving information in a digital and/or analog format.","With regard to the digital storage of information, a code other than a binary code for the storage of information can be utilized.","The result of using a higher order code for digital information storage (i.e., 3 or more characters) is the ability to store more information in the same amount of area or the same amount of information in a smaller area.","Accordingly, the present invention provides significant advancement in the art of data storage and/or data retrieval which will assist in preventing the memory barrier from being realized.","BRIEF DESCRIPTION OF THE DRAWINGS FIG.","1 is a view of a typical hard-disk drive that can be used in accordance with the techniques of the present invention.","FIG.","2 shows a read-write head for arranging magnetic domains in a magnetic disk.","FIG.","3 is a graph which shows an electromagnetic frequency as a function of magnetic field strength which results in different electromagnetic splitting frequencies which can correspond to stored bits of information on magnetic storage media.","FIGS.","4a-4d show a polarized light source and a polarized filter utilized to create dots other than white and black in an optical system (e.g., optical CD's).","FIG.","4e shows a photo detector/reader which is utilized to read the optical images stored in a disk 43.FIG.","5 shows a representative hysteresis curve for a ferromagnetic material that is used as a magnetic memory material in the present invention.","FIG.","6 is a schematic of a means for recording optical information in accordance with the teachings of the present invention.","FIG.","7 is a schematic of a means for recording optical information in accordance with the teachings of the present invention.","FIG.","8 is a schematic of a means for recording optical information in accordance with the teachings of the present invention.","FIG.","9 is a schematic of a means for recording optical information in accordance with the teachings of the present invention.","FIGS.","10a and 10b show a curve of polychromatic light that has been incident upon a polarizing filter and an analyzer.","FIG.","11 shows a fine structure spectrum for SF6 from 0-300 being magnified.","FIGS.","12a and 12b show the magnification of two curves from the fine structure of SF6 showing hyperfine structure frequencies.","Note the regular spacing of the hyperfine structure curves.","FIG.","12a shows magnification of the curve marked with a single asterisk (*) in FIG.","11 and FIG.","12b shows the magnification of the curve marked with a double asterisk (**) in FIG.","11.FIG.","13 shows an energy level diagram corresponding to the hyperfine splitting for the hyperfine structure in the n=2 to n=3 transition for hydrogen.","FIG.","14a shows the Zeeman effect for sodium “D” lines; and FIG.","14b shows the energy level diagram for transitions in the Zeeman effect for sodium “D” lines.","FIG.","15 is a graph which shows the splitting of the ground term of the oxygen atom as a function of magnetic field.","FIG.","16 is a graphic which shows the dependence of the Zeeman effect on magnetic field strength for the “3P” state of silicon.","FIG.","17a is a pictorial which shows a normal Zeeman effect and FIG.","17b is a pictorial which shows an anomalous Zeeman effect.","FIG.","18 shows anomalous Zeeman effect for zinc 3P→3S.","FIG.","19a shows a graphic representation of four Zeeman splitting frequencies and FIG.","19b shows a graphic representation of four new heterodyned differences.","FIG.","20 is a schematic of a means for recording optical information in accordance with one example of the present invention.","FIG.","21 is a schematic of a means for reading recorded optical information in accordance with an example of the present invention.","FIGS.","22a, 22b and 22c show in graphic form plots of power spectral density (i.e., intensity) for six (6) different polarizing filter settings.","FIG.","23 shows a graph of upper and lower intensity limit deviations corresponding to the six (6) different filter settings of FIGS.","22a-22c.","FIGS.","24a, 24b and 24c show in graphic form plots of full width half max (FWHM) data points versus six (6) different polarizing filter settings.","FIG.","25 shows in graphic form the deviation in upper and lower limits for the data shown in FIGS.","24a-24c.","FIG.","26 shows in schematic form the apparatus used to create and determine shifting/splitting frequencies according to the present invention.","FIG.","27 shows an optical layout of a typical holographic memory system that can be used according to the present invention.","DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention utilizes new and revolutionary techniques for the storage of data digitially, or in some cases, by analog or, at least a method approaching analog, such storage of data occurring optically, magnetically and/or electrically, magneto-optically and/or combinations thereof.","The storage/retrieval methods of the present invention are capable of utilizing traditional binary or base-2 code system approaches as well as non-traditional base-3 and higher order approaches.","The use of base-3 and higher order approaches may permit more data to be stored in approximately the same memory area relative to conventional binary techniques and/or substantially the same amount of information is capable of being stored in a smaller area relative to conventional binary techniques.","Certain novel magnetic storage/retrieval techniques will be discussed first, and then followed by discussions of certain novel optical techniques, according to the present invention.","FIG.","1 shows a typical prior art hard-disk drive in a computer.","This prior art hard-disk drive can be used substantially as shown in accordance with certain embodiments and novel techniques of the present invention.","The prior art hard-disk drive typically comprises one or more disks 1 (in FIG.","1, three disks are shown) that are in communication with read-write heads 2.The read-write heads are capable of communicating with each disk in one of several predetermined manners.","Specifically, as shown generally in FIG.","2, the heads 2 are capable of influencing magnetic domains 30 present in the disk 1.These influenced magnetic domains are represented by the number 31, while non-influenced domains are represented by the number 30, as shown in FIG.","2.The read-write heads 2 can first influence the magnetic domains 30 on the disks 1 by applying, for example, small currents through the heads 2 to create a magnetic field in the heads 2, which heads 2 in turn implant a desired effect into said domains (e.g., the magnetic field created by heads 2 can cause the unaligned magnetic domains 30 to become oriented domains 31, such orientation occurring in a controlled manner).","Once the domains 31 are appropriately oriented, they can then be read, if desired, by the same read-write heads 2.The domains 31 can be oriented parallel to the plane of rotation of the disk 1, or the domains 31 can be oriented perpendicular to the plane of rotation of the disk 1.When the domains 31 are oriented substantially parallel to the rotation plane of the disk 1, various electrical/magnetic induction techniques for the reading of the domains 31 are appropriate.","However, when the domains 31 are oriented substantially perpendicular to the rotation plane of the disk 1, standard induction reading techniques do not function as well (e.g., crosstalk between closely located bits can occur and unacceptable signal to noise ratios result).","Accordingly, development of magnetoresistive and giant magnetoresistive heads has occurred.","Briefly, these techniques involve the detection of very small changes in the electrical resistance of one or more materials comprising the read head 2.A primary difference between magnetoresistive heads and giant magnetoresistive heads is that giant magnetoresistive heads are two or three times more sensitive than magnetoresistive heads due to their novel structure which includes the layering of different materials on top of each other.","Somewhat conventional data storage techniques can be used in combination with the techniques of the present invention without the requirement for significant changes in current hardware being utilized.","For example, the magnetic storage media of the prior art can be used to store data associated with Zeeman splitting and/or shifting effects.","Moreover, the digital information that is stored on such media can be similar to or different from prior art digital data storage techniques.","Specifically, rather than storing only a combination of sequences of “0”s and “1”s, the present invention can use more than two stored magnetic field strengths to result in different field strengths that can be applied elsewhere and which can thus result in Zeeman splitting or shifting in the magnetic media, or Zeeman splitting or shifting occurring in a contiguous media (e.g., either with one or more materials comprising the magnetic storage media or either one or more materials, such as a solid, a liquid, a gas and/or a plasma, comprising the read head).","In other words, the different field strengths stored in a suitable magnetic media can result in differing amounts of Zeeman splitting or shifting in an appropriately chosen material, which detected Zeeman splitting or shifting corresponds to the different digital (or analog) values within a coded system.","For example, in a base-4 system, a “0” stored on the magnetic media could correspond to a bit with no magnetic field; a “1” could correspond to a stored field strength of “x” microTesla to the bit; a “2” could correspond to a field strength of “5x” microTesla stored to the bit; and a “3” could correspond to a field strength of “10x” microTesla stored to the bit.","The particular field strengths chosen here are exemplary, and it should be understood that differences in field strengths are simply a matter of design choice (e.g., convenient differences in magnetic field strengths should be a function of the magnetic media utilized to store information as well as the hardware utilized to store and retrieve such information and the media in which Zeeman splitting and/or shifting is desired).","The sensitivity of such media and/or hardware may dictate how much the difference in stored field strength needs to be between each bit so that the likelihood of misreading the information stored on any one bit, or combination of bits, due to, for example, poor signal/noise ratio or crosstalk, is minimized.","Moreover, any Zeeman splitting and/or shifting outputs can be appropriately modified or amplified by conventional techniques.","One example of a suitable magnetic material which can be used to store a magnetic field is a ferromagnetic material.","In particular, a ferromagnetic material that is capable of exhibiting different amounts of magnetic domain alignment as a function of increasing applied magnetic field, up to its natural magnetic saturation point.","Examples of suitable ferromagnetic materials that can be used in connection with the present invention include gamma-Fe2O3, gamma—Fe2O3 modified by Co, CrO2, etc.","FIG.","5 shows a typical hysteresis curve or hysteresis loop for a typical ferromagnetic material that can be used in the present invention.","Initially, at a point “D” no field “H” is applied and no magnetization is present in the virgin ferromagnetic material.","However, as a field H is applied, the magnetization of domains begins at the point “D” and increases along the curve “M” up to the point of saturization (Ms).","It should be noted that the initial magnetization or alignment of domains along the curve “M” is up to about the point “E”, in this FIG.","4, and is reversible.","However, when enough field H is applied to achieve a position on the curve M which corresponds to a magnetic field which is beyond the point “E”, the domains are frozen into place (i.e., will not move substantially unless influenced by, for example, another external magnetic field of greater strength or of a reverse polarity).","This curve is known as a “hysteresis curve”.","A suitable ferromagnetic material for use in the present invention exhibits a substantially infinite number of possible induced magnetizations along the curve M. It is these varying induced magnetizations that will be utilized to create different amounts of Zeeman splitting or shifting (discussed later herein) in the ferromagnetic materials per se, or in one or more materials which are placed contiguous to the ferromagnetic materials.","The magnetization induced in a ferromagnetic material can be increased up to the point Ms, whereby any higher magnetic fields will not result in any additional domain alignment.","As the applied magnetic field H is reduced to zero, the induced magnetization in the ferromagnetic material does not reduce to zero, but rather, has a remnant magnetization or resonance noted as Br.","It should be noted that ferromagnetic materials do not typically exhibit or hold the same magnetic field that is applied to them.","For example, if a field of 10 micro Tesla was applied, the magnetization induced in the ferromagnetic material, if measured as a field, would be less than 10 micro Tesla, for example, 7 micro Tesla.","This factor needs to be accounted for in the present invention, but is not a substantial issue and an artisan of ordinary skill in this field will understand how to proceed in accordance with the other teachings contained herein.","In order to understand the influence of magnetic fields on various Zeeman splitting or shifting frequencies, the following discussions of Fine Structure Frequencies, Hyperfine Structure Frequencies and Magnetic Fields are provided.","These discussions are taken, in part, from PCT Application No.","PCT/US01/28,392, entitled “Spectral Chemistry”, which was filed on Sep. 11, 2001, the subject matter of which is expressly incorporated herein by reference.","Fine Structure Frequencies The field of science concerned generally with measuring the frequencies of energy and matter, known as spectroscopy is well known.","Specifically, there are three broad classes of atomic and molecular spectra: (1) electronic spectra, which are due to electron transitions, have frequencies which occur primarily in the ultraviolet (UV), visible, and infrared (IR) regions, and are present in both atoms and molecules; (2) vibrational spectra, which are due to, for example, bond motion between individual atoms within molecules, occur primarily in the IR regions, and are present in molecules and (3) rotational spectra which are due primarily to the rotation of molecules in space and have microwave or radiowave frequencies, and also occur in molecules.","The three broad classes of spectra discussed above have been oversimplified.","There are actually at least three additional sets of spectra, namely, the fine structure spectra, the hyperfine structure spectra and the superfine structure spectra.","These additional spectra occur in atoms and molecules, and extend in frequencies, for example, from the ultraviolet down to the low radio regions.","These additional spectra are often mentioned in prior art chemistry and spectroscopy books typically as an aside, because prior art chemists usually focus more on the traditional types of spectroscopy, namely, electronic, vibrational, and rotational.","The fine and hyperfine spectra are quite prevalent in the areas of physics and radio astronomy.","For example, cosmologists map the locations of interstellar clouds of hydrogen, and collect data regarding the origins of the universe by detecting signals from outerspace, for example, at 1.420 GHz, a microwave frequency which is one of the hyperfine splitting frequencies for hydrogen.","Most of the large databases concerning the microwave and radio frequencies of molecules and atoms have been developed by astronomers and physicists, rather than by chemists.","This apparent inattention by chemists, physicists and materials scientists of the fine, hyperfine, and superfine spectra, has apparently resulted in little attention being given, if any, to these potentially useful spectra.","In addition to the fine splitting frequencies for atoms (i.e., heterodynes), molecules also have similar fine structure frequencies.","The origin and derivation for molecular fine structure and splitting or shifting is different from that for atoms, however, the graphical and practical results are quite similar.","In atoms, the fine structure frequencies are said to result from the interaction of the spinning electron with its' own magnetic field.","Basically, this means the electron cloud of a single atomic sphere, rotating and interacting with its' own magnetic field, produces the atomic fine structure frequencies.","The prior art refers to this phenomena as “spin-orbit coupling”.","For molecules, the fine structure frequencies correspond to the actual rotational frequencies of the electronic or vibrational frequencies.","So the fine structure frequencies for atoms and molecules both result from rotation.","In the case of atoms, it is the atom spinning and rotating around itself, much the way the earth rotates around its axis.","In the case of molecules, it is the molecule spinning and rotating through space.","Hyperfine Structure Frequencies Hyperfine structure frequencies are similar to the fine structure frequencies.","Fine structure frequencies can be seen by magnifying a portion of a standard frequency spectrum.","Hyperfine frequencies can be seen by magnifying a portion of a fine structure spectrum.","Fine structure splitting frequencies occur at lower frequencies than the electronic spectra, and are located primarily in the infrared and microwave regions of the electromagnetic spectrum.","Hyperfine splitting frequencies occur at even lower frequencies than the fine structure spectra, primarily in the microwave and radio wave regions of the electromagnetic spectrum.","Fine structure frequencies are generally caused by at least the electron interacting with its' own magnetic field.","Hyperfine frequencies are generally caused by at least the electron interacting with the magnetic field of the nucleus.","An example of hyperfine splitting can be seen in FIGS.","11 and 12.Specifically, what appears to be a single crisp curve in FIG.","11, turns out to be a series of several curves spaced very close together.","These are the hyperfine frequency curves.","Accordingly, the fine structure spectra is comprised of several more curves spaced very close together.","These other curves spaced even closer together correspond to the hyperfine frequencies.","FIGS.","12a and 12b show that the spacing of the hyperfine frequency curves are very close together and at somewhat regular intervals.","The small amount that the hyperfine curves are split apart is called the hyperfine splitting frequency.","The hyperfine splitting frequency is also a heterodyne.","This concept is substantially similar to the concept of the fine splitting frequency.","The difference between two curves that are split apart is called a splitting frequency.","As before, the difference between two curves is referred to as a heterodyne frequency.","So, hyperfine splitting frequencies are all heterodynes of hyperfine frequencies.","Because the hyperfine frequency curves result from a magnification of the fine structure curves, the hyperfine splitting frequencies occur at only a fraction of the fine structure splitting frequencies.","The fine structure splitting frequencies are really just several curves, spaced very close together around the regular spectrum frequency.","Magnification of fine structure splitting frequencies results in hyperfine splitting frequencies.","The hyperfine splitting frequencies are really just several more curves, spaced very close together.","The closer together the curves are, the smaller the distance or frequency separating them.","Now the distance separating any two curves is a heterodyne frequency.","So, the closer together any two curves are, the smaller (lower) is the heterodyne frequency between them.","The distance between hyperfine splitting frequencies (i.e., the amount that hyperfine frequencies are split apart) is the hyperfine splitting frequency.","It can also be called a constant or interval.","As a general example, the electronic spectrum frequency of hydrogen is 2,466 THz.","The 2,466 THz frequency is made up of fine structure curves spaced 10.87 GHz (0.01087 THz) apart.","Thus, the fine splitting frequency is 10.87 GHz.","Now the fine structure curves are made up of hyperfine curves.","These hyperfine curves are spaced just 23.68 and 59.21 MHz apart.","Thus, 23 and 59 MHz are both hyperfine splitting frequencies for hydrogen.","Other hyperfine splitting frequencies for hydrogen include 2.71, 4.21, 7.02, 17.55, 52.63, 177.64, and 1,420.0 MHz.","The hyperfine structure frequencies are spaced even closer together than the fine structure frequencies, so the hyperfine splitting frequencies are smaller and lower than the fine splitting frequencies.","Thus, the hyperfine splitting frequencies are lower than the fine splitting frequencies.","This means that rather than being in the infrared and microwave regions, as the fine splitting frequencies can be, the hyperfine splitting frequencies are in the microwave and radiowave regions.","These lower frequencies are in the GHz (109 hertz), MHz (106 hertz) and KHz (103 hertz) regions of the electromagnetic spectrum.","Several of the hyperfine splitting frequencies for hydrogen are shown in FIG.","13.(FIG.","13 shows hyperfine structure in the n=2 to n=3 transition of hydrogen).","Magnetic Fields In spectral terms, magnetic fields can behave in a similar manner to electric fields.","Specifically, the spectral frequency lines of, for example, atoms and molecules, can be split and/or shifted by a magnetic field.","In this case, the external magnetic field from outside the atom or molecule, interacts with the electric and magnetic fields already inside the atom or molecule.","This action of an external magnetic field on spectral lines is called the “Zeeman Effect”, in honor of its' discoverer, Dutch physicist Pieter Zeeman.","In 1896, Zeeman discovered that the yellow flame spectroscopy “D” lines of sodium were broadened when the flame was held between strong magnetic poles.","It was later discovered that the apparent broadening of the sodium spectral lines was actually due to their splitting and shifting.","Zeeman's original observation has evolved into a separate branch of spectroscopy relating to the study of atoms and molecules by measuring the changes of the spectral lines in atoms and molecules caused by a magnetic field.","This in turn has evolved into the nuclear magnetic resonance spectroscopy and magnetic resonance imaging used in medicine, as well as the laser magnetic resonance and electron spin resonance spectroscopy used in physics and chemistry.","The Zeeman effect for the famous “D” lines of sodium is shown in FIGS.","14a and 14b.","FIG.","14a shows the Zeeman effect for sodium “D” lines; whereas FIG.","14b shows the energy level diagram for the transitions in the Zeeman effect for the sodium “D” lines.","The “D” lines are traditionally said to result from transition between the 3p2p and 3s2S electron orbitals.","As is shown, each of the single spectral frequencies is split into two or more slightly different frequencies, which center around the original unsplit frequency.","In this Zeeman effect, the amount that the spectral frequencies are split apart depends on the strength of the applied magnetic field.","FIG.","15 shows Zeeman splitting effects for the oxygen atom as a function of magnetic field.","When there is no magnetic field, there are two single frequencies at zero and 4.8.When the magnetic field is at low strength (e.g., 0.2 Tesla) there is just slight splitting and shifting of the original two frequencies.","However, as the magnetic field is increased, the frequencies are split and shifted farther and farther apart.","The degree of splitting and shifting in the Zeeman effect, depending on magnetic field strength, is shown in FIG.","16 for the 3P state of silicon.","This Zeeman effect, due to an externally applied magnetic field, is slightly different from the Zeeman effect from an internal magnetic field depending on whether an atom or molecule is subjected to the magnetic field.","The Zeeman effect on atoms can be divided into three different magnetic field strengths: weak; moderate; and strong.","If the magnetic field strength is weak, the amount that the spectral frequencies will be shifted and/or split apart will be very small.","The shifting away from the original spectral frequency will still stimulate the shifted frequencies.","This is because they will be so close to the original spectral frequency that they will still be well within its resonance curve.","As for the splitting, it is so small, that it is even less than the hyperfine splitting that normally occurs.","This means that in a weak magnetic field, there will be only very slight splitting of spectral frequencies, translating into very low splitting frequencies in the lower regions of the radio spectrum and down into the very low frequency region.","For example, the Zeeman splitting frequency for the hydrogen atom, which is caused by the earth's magnetic field, is around 30 KHz.","Larger atoms have even lower frequencies in the lower kilohertz and even hertz regions of the electromagnetic spectrum.","At the other end of magnetic field strength, is the very strong magnetic field.","In this case, the splitting apart and shifting of the spectral frequencies will be very wide.","With this wide shifting of frequencies, the difference between the split and/or shifting frequencies will be much larger than the difference between the hyperfine splitting frequencies.","This translates to Zeeman effect splitting and/or splitting frequencies at higher frequencies than the hyperfine splitting frequencies.","This splitting occurs somewhere around the microwave region.","The moderate magnetic field strength case is more complicated.","The shifting and splitting caused by the Zeeman effect from a moderate magnetic field will be approximately equal to the hyperfine splitting and/or shifting.","Although not widely discussed in the prior art, it is possible to apply a moderate magnetic field to an atom, to produce Zeeman splitting which is substantially equivalent to its' hyperfine splitting.","The moderate magnetic field causes low frequency Zeeman splitting that matches and hence energizes the low frequency hyperfine splitting frequency in the atom.","However, the low hyperfine splitting frequencies actually correspond to the heterodyned difference between two vibrational or fine structure frequencies.","When the hyperfine splitting frequency is stimulated, the vibrational or fine structure frequencies will be stimulated, and electronic frequencies will eventually be stimulated.","This in turn causes the atom to be, for example, stimulated.","There is also a difference between the “normal” Zeeman effect and the “anomalous” Zeeman effect.","With the “normal” Zeeman effect, a spectral frequency is split by a magnetic field into three frequencies, with expected even spacing between them (see FIG.","17a which shows the “normal” Zeeman effects and FIG.","17b which shows the “anomalous” Zeeman effects).","One of the new split frequencies is above the original frequency, and the other new split frequency is below the original frequency.","Both new frequencies are split the same distance away from the original frequency.","Thus, the difference between the upper and original and the lower and original frequencies is about the same.","This means that in terms of heterodyne differences, there are at most, two new heterodyned differences with the normal Zeeman effect.","The first heterodyne or splitting difference is the difference between one of the new split frequencies and the original frequency.","The other splitting difference is between the upper and lower new split frequencies.","It is, of course, twice the frequency difference between either of the upper or lower frequencies and the original frequency.","In many instances the Zeeman splitting produced by a magnetic field results in more than three frequencies, or in splitting that is spaced differently than expected.","This is called the “anomalous” Zeeman effect (see FIGS.","17 and 18; wherein FIG.","18 shows an anomalous Zeeman effect for zinc 3p→3s.","If there are still just three frequencies, and the Zeeman effect is anomalous because the spacing is different than expected, the situation is similar to the normal effect.","However, there are at most, two new splitting frequencies that can be used.","If, however, the effect is anomalous because more than three frequencies are produced, then there will be a much more richly varied situation.","Assume an easy case where there are four Zeeman splitting frequencies (see FIGS.","19a and FIG.","19b).","FIG.","19a shows four Zeeman splitting frequencies and FIG.","19b shows four new heterodyned differences.","In this example of anomalous Zeeman splitting, there are a total of four frequencies, where once existed only one frequency.","For simplicity's sake, the new Zeeman frequencies will be labeled 1, 2, 3, and 4.Frequencies 3 and 4 are also split apart by the same difference “w”.","Thus, “w” is a heterodyned splitting frequency.","Frequencies 2 and 3 are also split apart by a different amount “x”.","So far there are two heterodyned splitting frequencies, as in the normal Zeeman effect.","However, frequencies 1 and 3 are split apart by a third amount “y”, where “y” is the sum of “w” and “x”.","And, frequencies 2 and 4 are also split apart by the same third amount “y”.","Finally, frequencies 1 and 4 are split even farther apart by an amount “z”.","Once again, “z” is a summation amount from adding “w+x+w”.","Thus, the result is four heterodyned frequencies: w, x, y, and z in the anomalous Zeeman effect.","If there were six frequencies present from the anomalous Zeeman effect, there would be even more heterodyned differences.","Thus, the anomalous Zeeman effect results in far greater flexibility in the choice of frequencies when compared to the normal Zeeman effect.","In the normal Zeeman effect the original frequency is split into three evenly spaced frequencies, with a total of just two heterodyned frequencies.","In the anomalous Zeeman effect the original frequency is split into four or more unevenly spaced frequencies, with at least four or more heterodyned frequencies.","The following discussion will focus on the Zeeman effect in molecules.","Molecules come in three basic varieties: ferromagnetic; paramagnetic; and diamagnetic.","Ferromagnetic molecules are what comprises typical magnetic storage media and are thus capable of being used in the present invention.","These materials typically hold a strong magnetic field due to domain alignment induced by the application of an external magnetic material and are composed of typical magnetic elements such as iron, cobalt, and/or nickel.","Paramagnetic molecules hold only a weak magnetic field and are thus not of primary interest in the present invention as magnetic strength storage materials.","A strong magnetic field will produce splitting greater than the hyperfine frequencies, in the microwave and infrared regions of the EM spectrum in atoms and paramagnetic molecules.","A moderate magnetic field will produce Zeeman splitting in atoms and paramagnetic molecules at frequencies on par with the hyperfine and rotational splitting frequencies.","Finally, consider the direction of the magnetic field in relation to the orientation of the molecule.","When the magnetic field is parallel to an exciting electromagnetic field, π frequencies are produced.","When the magnetic field is perpendicular to an exciting electromagnetic field, a frequencies are found.","It is important to remember that field orientation can result in different frequencies.","Accordingly, in a first embodiment of the present invention, in order to determine any field strength data stored on appropriate magnetic media, the magnetic media per se (or in an alternative embodiment, a material (e.g., a solid, a liquid, a gas and/or a plasma) contiguous to the magnetic media either forming a part of the magnetic media or part of an interrogating (i.e., read head) needs to be appropriately interrogated for the presence of differing amounts of Zeeman splitting and/or shifting.","A suitable form of interrogation of such stored information could be, for example, a spectroscopic interrogation.","For example, in reference to FIG.","3 the magnetic media utilized to store the different induced field strengths of “0”, “x”, 5x” and “10x” microTesla, could themselves exhibit or show particular Zeeman splitting frequencies for each of the different magnetic field strengths.","Alternatively, the different stored field strengths could result in Zeeman splitting in a contiguous material (or multiple contiguous materials).","The contiguous material could form part of the magnetic media, or could form part of an interrogation assembly such as read/write head.","The Zeeman splitting and/or shifting frequencies which arise are a function of the actual material which comprises the magnetic media, as well as the field strength applied thereto.","In this regard, for example, as shown in FIG.","3, assuming for illustrative purposes that the exemplary magnetic media exhibited a Zeeman splitting frequency of 10 KHz for a magnetic field strength of “x” microTesla, 25 KHz for a magnetic field strength of “5x” microTesla and 80 KHz for a magnetic field strength of “10x” microTesla.","Thus, in order to read any of the bits of information stored on the exemplary magnetic media, each bit of stored information would need to be interrogated directly by applying one or more of the frequencies mentioned above (alternatively, a material contiguous (as discussed elsewhere herein) to the magnetic media could be similarly interrogated to determine Zeeman splitting and/or shifting).","Specifically, in a first interrogation approach, a system could be designed such that each bit of information stored in a magnetic media could be interrogated with each known potential splitting frequency that could be present on each bit; or a system could be designed such that once a known splitting frequency had been found to be located at the bit, the interrogation process could stop.","The particular approach is a matter of preference and reliability.","For example, one interrogation process according to the present invention proceeds as follows.","If the magnetic media per se exhibits Zeeman splitting and/or shifting, then the first bit (i.e., the bit with no magnetic field), could be interrogated with all three frequencies (i.e., 10, 25 and 80 KHz) or a sweep of frequencies (i.e., 10 KHz to 80 KHz).","The interrogation system would recognize that none of these frequencies were present, and thus there is no stored magnetic field strength and the bit is a “0”.","The next bit could then be interrogated with the same frequencies and it would be discovered by the interrogation that a resonant amplitude spike returned from the bit or was absorbed by the bit at a frequency of 10 KHz.","Further interrogation of the bit would show no other resonant amplitude spikes at the interrogation frequencies of 25 and 80 KHz.","Thus, this bit would be “1”.","The next bit could then be interrogated with the same frequencies and it would be discovered by the interrogation process that a resonant amplitude spike returned from the bit at a frequency of 25 KHz.","Further interrogation of the bit would show no other resonant amplitude spikes at the frequencies of 10 KHz and 80 KHz.","Thus, this bit would be “2”.","The final bit could then be interrogated with the same frequencies and it would be discovered that a resonant amplitude spike returned from the bit at a frequency of 80 KHz.","Further interrogation of the bit would show no other resonant amplitude spikes at the frequencies of 10 KHz and 25 KHz.","Thus, the bit would be a “3”.","Accordingly, a reliable base-4 system can be achieved.","Alternatively, another interrogation process according to the present invention could proceed as follows: if the magnetic media has a plurality of induced field strengths recorded and each of the recorded magnetic field strengths per se exhibits different amounts of Zeeman splitting in either the magnetic media or in one or more contiguous material(s), then each of the bits providing different magnetic field strengths could be interrogated with, for example, a single interrogating electromagnetic energy of a suitable frequency.","In this regard, if a frequency of, for example, 50 KHz was used as the interrogating frequency, then heterodynes (side bands) sum and difference frequencies, etc., could be expected to occur with those Zeeman splitting frequencies which occur in the magnetic media or in the contiguous material.","Specifically, heterodyning can occur with each of the stored frequencies, 10, 25 and 80 KHz.","In particular, some, but not all of the possible heterodyned frequencies would be as follows: 60 KHz (i.e., 50+10); 75 KHz (i.e., 50+25); and 130 KHz (i.e., 50+80).","In other words, the application of a 50 KHz interrogating frequency would result in at least the aforementioned heterodyned frequencies, each of which could be caused to correspond to, for example, either a “1”, a “2”, or a “3”, with no heterodynes present indicating the value of “0”.","It should be noted that of each of the aforementioned processes, namely applying a series of frequencies or a single frequency when interrogating for heterodynes, can be used for interrogating either the magnetic material per se, or a contiguous material, said contiguous material being present in the magnetic media per se, adjacent to the magnetic media or as part of an interrogating system, such as a read/write head.","The following brief description of heterodyning is provided to assist in understanding certain aspects of the invention.","However, for a more complete discussion of this important area, the reader is directed toward the aforementioned PCT Application entitled “Spectral Chemistry”, which has been expressly incorporated herein by reference.","Electromagnetic energy consists of waves.","The number of waves in a period of time can be counted.","Usually, time is placed on the horizontal X-axis.","The vertical Y-axis shows the strength or intensity of the wave.","This is also called the amplitude.","A weak wave will be of weak intensity and will have low amplitude.","A strong wave will have high amplitude.","Traditionally, the number of waves per second is counted, to obtain the frequency.","Another name for “waves per second”, is “hertz” (abbreviated “Hz”).","Frequency=Number of waves/time=Waves/second=Hz.","Energy waves and frequency have some interesting properties, and may interact in some interesting ways.","The manner in which wave energies interact, depends largely on the frequency.","For example, when two waves of energy interact, each having the same amplitude, but one at a frequency of 400 Hz and the other at 100 Hz, the waves will add their frequencies, to produce a new frequency of 500 Hz (i.e., the “sum” frequency).","The frequency of the waves will also subtract to produce a frequency of 300 HZ (i.e., the “difference” frequency).","All wave energies typically add and subtract in this manner, and such adding and subtracting is referred to as heterodyning.","Common results of heterodyning are familiar to most as harmonics in music.","There is a mathematical, as well as musical basis, to the harmonics produced by heterodyning.","Consider, for example, a continuous progression of heterodyned frequencies.","As discussed above, beginning with 400 Hz and 100 Hz, the sum frequency is 500 Hz and the difference frequency is 300 Hz.","If these frequencies are further heterodyned (added and subtracted) then new frequencies of 800 (i.e., 500+300) and 200 (i.e., 500−300) are obtained.","The further heterodyning of 800 and 200 results in 1,000 and 600 Hz as shown in FIG.","4.A mathematical pattern begins to emerge.","Both the sum and the difference columns contain alternating series of numbers that double with each set of heterodynes.","In the sum column, 400 Hz, 800 Hz, and 1,600 Hz, alternates with 500 Hz, 1000 Hz, and 2000 Hz.","The same sort of doubling phenomenon occurs in the difference column.","Heterodyning of frequencies is the natural process that occurs whenever waveform energies interact.","Heterodyning results in patterns of increasing numbers that are mathematically derived.","The number patterns are integer multiples of the original frequencies.","These multiples are called harmonics.","For example, 800 Hz and 1600 Hz are harmonics of 400 Hz.","In musical terms, 800 Hz is one octave above 400 Hz, and 1600 Hz is two octaves higher.","It is important to understand the mathematical heterodyne basis for harmonics, which occurs in all waveform energies, and thus in all of nature.","A heterodyne is a mathematical function, governed by mathematical equations, just like a fractal.","A heterodyne also produces self-similar patterns of numbers, like a fractal.","Heterodynes are fractals; the conclusion is inescapable.","Heterodynes and fractals are both mathematical functions which produce a series of self-similar patterns or numbers.","Wave energies interact in heterodyne patterns.","Thus, all wave energies interact as fractal patterns.","Accordingly, since energy interacts by heterodyning, matter should also be capable of interacting by a heterodyning process.","All matter whether in large or small forms, has what is called a natural oscillatory frequency.","The natural oscillatory frequency (“NOF”) of an object, is the frequency at which the object prefers to vibrate, once set in motion.","The NOF of an object is related to many factors including size, shape, dimension, and composition.","The smaller an object is, the smaller the distance it has to cover when it oscillates back and forth.","The smaller the distance, the faster it can oscillate, and the higher its NOF.","For example, consider a wire composed of metal atoms.","The wire has a natural oscillatory frequency.","The individual metal atoms also have unique natural oscillatory frequencies.","The NOF of the atoms and the NOF of the wire heterodyne by adding and subtracting, just the way energy heterodynes.","NOFatom+NOFwire=Sum Frequencyatom+wire and NOFatom−NOFwire=Difference Frequencyatom-wire If the wire is stimulated with the Difference Frequencyatom-wire, the difference frequency will heterodyne (add) with the NOFwire to produce NOFatom, (natural oscillatory frequency of the atom) and the atom will absorb with the energy, thereby becoming stimulated to a higher energy level.","Cirac and Zoeller reported this phenomenon in 1995, and they used a laser to generate the Difference Frequency.","Difference Frequencyatom-wire+NOFwire=NOFatom Matter heterodynes with matter in a manner similar to the way in which wave energies heterodyne with other wave energies.","This means that matter in its various states may also interact in fractal processes.","This interaction of matter by fractal processes assists in explaining why so many creatures and systems in nature exhibit fractal processes and patterns.","Matter, as well as energy, interacts by the mathematical equations of heterodynes, to produce harmonics and fractal patterns.","That is why there are fractals everywhere around us.","Thus, energy heterodynes with energy, and matter heterodynes with matter.","However, perhaps even more important is that matter can heterodyne with energy (and visa versa).","In the metal wire discussion above, the Difference Frequencyatom-wire in the experiment by Cirac and Zoeller was provided by a laser which used electromagnetic wave energy at a frequency equal to the Difference Frequencyatom-wire.","The matter in the wire, via its natural oscillatory frequency, heterodyned with the electromagnetic wave energy frequency of the laser to produce the frequency of an individual atom of matter.","This shows that energy and matter do heterodyne with each other.","In general, when energy encounters matter, one of three possibilities occur.","The energy either bounces off the matter (i.e., is reflected energy), passes through the matter (i.e., is transmitted energy), or interacts and/or combines with the matter (e.g., is absorbed or heterodynes with the matter).","If the energy heterodynes with the matter, new frequencies of energy and/or matter will be produced by mathematical processes of sums and differences.","If the frequency thus produced matches an NOF of the matter, the energy will be, at least partially, absorbed, and the matter will be stimulated to, for example, a higher energy level, (i.e., it possesses more energy).","A crucial factor which determines which of these three possibilities will happen is the frequency of the energy compared to the frequency of the matter.","If the frequencies do not match, the energy will either be reflected, or will pass on through as transmitted energy.","If the frequencies of the energy and the matter match either directly (e.g., are close to each other, as discussed in greater detail later herein), or match indirectly (e.g., heterodynes), then the energy is capable of interacting and/or combining with the matter.","Another term often used for describing the matching of frequencies is resonance.","In this invention, use of the term resonance will typically mean that frequencies of matter and/or energy match.","For example, if the frequency of energy and the frequency of matter match, the energy and matter are in resonance and the energy is capable of combining with the matter.","Resonance, or frequency matching, is merely an aspect of heterodyning that permits the coherent transfer and combination of energy with matter.","In the example above with the wire and atoms, resonance could have been created with the atom, by stimulating the atom with a laser frequency exactly matching the NOF of the atom.","In this case, the atom would be energized with its own resonant frequency and the energy would be transferred to the atom directly.","Alternatively, as was performed in the actual wire/laser experiment, resonance could also have been created with the atom by using the heterodyning that naturally occurs between differing frequencies.","Thus, the resonant frequency of the atom (NOFatom) can be produced indirectly, as an additive (or subtractive) heterodyned frequency, between the resonant frequency of the wire (NOFwire) and the applied frequency of the laser.","Either direct resonance, or indirect resonance through heterodyned frequency matching, produces resonance and thus permits the combining of matter and energy.","When frequencies match, energy transfers.","Heterodyning produces indirect resonance.","Heterodyning also produces harmonics, (i.e., frequencies that are integer multiples of the resonant (NOF) frequency.","For example, the music note “A” is approximately 440 Hz.","If that frequency is doubled to about 880 Hz, the note “A” is heard an octave higher.","This first octave is called the first harmonic.","Doubling the note or frequency again, from 880 Hz to 1,760 Hz (i.e., four times the frequency of the original note) results in another “A”, two octaves above the original note.","This is called the third harmonic.","Every time the frequency is doubled another octave is achieved, so these are the even integer multiples of the resonant frequency.","In between the first and third harmonic is the second harmonic, which is three times the original note.","Musically, this is not an octave like the first and third harmonics.","It is an octave and a fifth, equal to the second “E” above the original “A”.","All of the odd integer multiples are fifths, rather than octaves.","Because harmonics are simply multiples of the fundamental natural oscillatory frequency, harmonics stimulate the NOF or resonant frequency indirectly.","Thus by playing the high “A” at 880 Hz on a piano, the string for middle “A” at 440 Hz should also begin to vibrate due to the phenomenon of harmonics.","Matter and energy in chemical reactions respond to harmonics of resonant frequencies much the way musical instruments do.","Thus, the resonant frequency of the atom (NOFatom) can be stimulated indirectly, using one or more of its' harmonic frequencies.","This is because the harmonic frequency heterodynes with the resonant frequency of the atom itself (NOFatom).","For example, in the wire/atom example above, if the laser is tuned to 800 THz and the atom resonates at 400 THz, heterodyning the two frequencies results in: 800 THz−400 THz=400 THz The 800 THz (the atom's first harmonic), heterodynes with the resonant frequency of the atom, to produce the atom's own resonant frequency.","Thus the first harmonic indirectly resonates with the atom's NOF, and stimulates the atom's resonant frequency as a first generation heterodyne.","Of course, the two frequencies will also heterodyne in the other direction, producing: 800 THz+400 THz=1,200 THz The 1,200 THz frequency is not the resonant frequency of the atom.","Thus, part of the energy of the laser will heterodyne to produce the resonant frequency of the atom.","The other part of the energy of the laser heterodynes to a different frequency, that does not itself stimulate the resonant frequency of the atom.","That is why the stimulation of an object by a harmonic frequency of particular strength of amplitude, is typically less than the stimulation by its' own resonant (NOF) frequency at the same particular strength.","Although it appears that half the energy of a harmonic is wasted, that is not necessarily the case.","Referring again to the exemplary atom vibrating at 400 THz, exposing the atom to electromagnetic energy vibrating at 800 THz will result in frequencies subtracting and adding as follows: 800 THz−400 THz=400 THz and 800 THz+400 THz=1,200 THz The 1,200 THz heterodyne, for which about 50% of the energy appear to be wasted, will heterodyne with other frequencies also, such as 800 THz.","Thus, 1,200 THz−800 THz=400 THz Also, the 1,200 THz will heterodyne with 400 THz: 1,200 THz−400 THz=800 THz, thus producing 800 THz, and the 800 THz will heterodyne with 400 THz: 800 THz−400 THz=400 THz, thus producing 400 THz frequency again.","When other generations of heterodynes of the seemingly wasted energy are taken into consideration, the amount of energy transferred by a first harmonic frequency is much greater than the previously suggested 50% transfer of energy.","There is not as much energy transferred by this approach when compared to direct resonance, but this energy transfer is sufficient to produce a desired effect.","While FIG.","3 (discussed above in greater detail) shows, in graphic form, three specifically selected magnetic field strengths (i.e., 1×, 5× and 10×) and their corresponding (resultant) Zeeman splitting frequencies, it should be obvious to one of ordinary skill in this art that a virtually limitless series of possibilities exists for storage of higher order systems (i.e., higher than base-2 or a binary system), such systems being a function of the magnetic materials comprising the magnetic media, the Zeeman splitting/shifting frequencies generated in the magnetic media, or in a contiguous material, the accuracy of whatever transmitter(s) (e.g., a magnetic inductor, which generates a magnetic field) is used to induce a magnetic field in a magnetic media and/or the accuracy of the reading system which is used to detect Zeeman splitting and/or shifting effects in the magnetic media (or in a material contiguous to the magnetic material) once such magnetic media (or contiguous material) has been interrogated.","Thus, if large numbers of stored fields can result in correspondingly large numbers of Zeeman splitting or shifting frequencies, the use of Zeeman splitting and/or shifting effects can result in an analog or analog-like storage/retrieval of information.","For example, again referring to FIGS.","1 and 2, the Zeeman effect can be used to read information from a series of bits on the disks 1, but such information is retrieved differently from prior art approaches.","In this regard, by utilizing the Zeeman splitting and/or shifting effects, for example, the present invention uses the magnetic domains 31 in a manner different (e.g., the magnetic domains may comprise more than two values and/or the magnetic domains may be interrogated, directly, or indirectly through the use of one or more contiguous material(s), in a different manner) from prior art approaches.","Specifically, the magnetic domains 31 are present on a disk 1.Current techniques allow these magnetic domains to assume “0” or “1” mode (i.e., a base-2 or binary code) and these are aligned in one of two possible combinations at pre-selected magnetic strengths.","The present invention permits the magnetic storage media to be utilized in several unique ways.","Specifically, for example, rather than storing only a combination or sequence of “0”s and “1”s, the present invention stores more than two different field strengths to the magnetic disk 1, by influencing the magnetic media, for example, in a somewhat standard prior art approach.","These different field strengths are stored by utilizing a read-write head 2 that is similar to conventional heads currently being utilized.","The read-write head 2 is capable of imparting, and thus causing storage of different field strengths of different microTesla values upon the disk 1.Any desired combination of different field strengths can be stored upon the disk 1, and can be selected as a function of convenience and/or accuracy.","In this regard, with regard to a base-6 system (i.e., “0, 1, 2, 3, 4, and 5”), the first bit of data stored can be no magnetic field, and can be made to correspond to “0”.","Each of the remaining five different possible values can be selected so that they correspond to different points along the curve “M” shown in FIG.","5.The differences between the stored field strengths are a matter of material design and system designer preferences.","For this system, the choices of magnetic materials and/or materials which exhibit desirable Zeeman splitting or shifting may assist in dictating the difference in magnetic field strengths between different values in a coded system.","Stated more particularly, materials engineers may desire certain types of magnetic materials to be utilized as magnetic storage media and such materials may have preferential field strengths which are unique to those materials.","Moreover, such stored field strengths may result in desirable and detectable Zeeman splitting and/or shifting in the storage media per se, or such stored field strengths may be selected to result in desirable field splitting and/or shifting in one or more materials (e.g., a solid, a liquid, a gas and/or a plasma) contiguous to the magnetic media.","Such preferential field strengths could then be maximized as both inputs and outputs in designing systems to be efficient, accurate and inexpensive.","Such combinations of materials and/or systems should be apparent to those of ordinary skill of the art in view of the teachings herein.","In this Zeeman effect, when a relatively wide splitting of electromagnetic energy frequencies occurs due to the applied magnetic field, the amount of, for example, splitting may correspond to higher frequencies (e.g., microwave); whereas a narrow splitting will typically correspond to low frequencies (e.g., radio).","The amount of splitting that occurs is important because in order to determine the amount of splitting that has occurred, a suitable interrogating electromagnetic energy is caused to be incident upon, for example, the magnetic material itself or the aforementioned contiguous material.","The contiguous material that exhibits Zeeman splitting at the stored magnetic field can be present as, for example, one or more separate sheets of material that are located adjacent to, and/or contiguous with, the material that contains the stored magnetic field.","When multiple contiguous materials are utilized, the present invention contemplates the use of a comparative analysis of the differences in Zeeman splitting between two or more such contiguous materials.","Alternatively, a material comprising, for example, one or more material(s) that are mixed with the magnetic material (e.g., a binder which holds together at least a portion of the material that stores the magnetic field) could also be used.","More specifically, a particular interrogating frequency, or set of frequencies, needs to be capable of favorably interacting with one-or more of the splitting/shifting frequencies which result in the magnetic material, or in an alternative embodiment in one or more of the contiguous materials (e.g., a solid, a liquid, a gas and/or a plasma), due to the different magnetic fields stored in the magnetic material.","In other words, a first material is desirably chosen so that an applied magnetic field can be reasonably stored in a magnetic material.","However, it is desirable for the magnetic material to be capable of storing reliably several magnetic fields of different strength (e.g., capable of having varying numbers of magnetic domains align with an ever increasing magnetic field and such domains remain frozen or pinned so that they do not change with time) and, for example, in one embodiment of the invention, the contiguous material needs to be sensitive to the stored magnetic fields.","In this regard, if one or more contiguous material(s) are utilized in connection with the magnetic material(s), the contiguous material(s) needs to exhibit a splitting of one or more of its rotational, vibrational, etc., bands (e.g., at the atomic or molecular level) and the resultant splitting that occurs in the contiguous material needs to be detectable by an appropriate interrogating electromagnetic source (discussed in greater detail elsewhere herein).","Alternatively, a contiguous material does not need to be utilized.","In this case, the magnetic material per se, or at least a portion thereof, needs to exhibit desirable and detectable splitting or shifting of one or more frequencies.","Accordingly, it should be clear that the Zeeman effect techniques of the present invention could be utilized in conjunction with current hardware systems.","In this regard, so long as an appropriate field can cause to be stored in a magnetic media and the resultant Zeeman effect which occurs in the magnetic material per se, or various material(s) contiguous to the stored magnetic field, can be appropriately interrogated.","Thus, the present invention is a significant improvement over the prior art because current read-write heads use the ASCII code which is comprised of 0's and 1's in 8 different bits resulting in 256 different combinations of 0's and 1's.","The present invention can harvest the advantage of codes that are higher order than binary.","The present invention also provides significant improvements over various optical memory techniques.","Specifically, in another embodiment of an alternative memory system of the present invention, a system somewhat analogous to current optical systems utilized in compact disk (CD) players is utilized.","Current CD technology utilizes a rotating disk comprised of a number of layers of different materials.","The layers include polycarbonate plastic, aluminum and acrylic.","The aluminum in the disk includes a series of plateaus and valleys sometimes referred to as “bumps”.","Laser light is caused to focus upon the plateaus and valleys in the rotating disk.","The reflection of the laser off of the plateaus and valleys corresponds to a binary stream of information.","CD's use, for example, 16-bit strings of binary coded data.","Specifically, laser light reflects differently from a valley compared to the reflection off of a plateau and these different reflective properties of the laser when shined upon these valleys and plateaus correspond to the binary code of “0's” and “1's”.","The present invention utilizes a similar concept to the CD disk, however, rather than utilizing valleys and plateaus (i.e., bumps) in an aluminum layer embedded in a polycarbonate plastic CD, the present invention utilizes at least two dots of different intensity color, or color width.","These dots or spots are optically created and/or optically examined or interrogated.","These optical dots or spots may be of a size which is approximately the same order of magnitude in size as the current data tracks used on CD's, they may be larger, or they may be smaller.","Similar to the current CD's, these dots or spots are put together in strings which begin at an innermost portion of a rotatable CD disk and extend spirally out therefrom.","Accordingly, current CD's of approximately 12 centimeters would be an acceptable size.","However, if the dots or spots of the present invention are smaller than the “bumps” of the current technology, it is possible for CD's to be smaller in size while maintaining the same amount of memory space.","Thus, it is possible to put an amount of optical information onto a rotatable disk of approximately 12 centimeters in diameter which exceeds that amount of information which is currently stored on CD's.","A first example of the invention utilizes a quaternary system which is depicted schematically in FIG.","4.In this regard, with reference to FIG.","4A, a polarized light source 40 (e.g., a light source from a laser that has been subjected to a polarizing filter) is projected through a polarizing filter 41 (sometimes referred to herein as an analyzer).","When the polarized light source 40 and polarizing filter 41 are in alignment, then a white dot 42a is caused to appear on a recording media 43.This white dot may correspond to “0” in a base-4 code.","With regard to FIG.","4B, the polarized light source 40 is projected upon the polarizing filter 41, however, in this Example, the filter 41 has been rotated about 30° with regard to the polarized light source 40.The disk 43 has also been rotated an appropriate amount of “x” so as to permit a separate recording of the dots 42a and 42b.","This 30° rotation of the filter 41 results in the placement or recording of a dot 42b, that is somewhat gray upon the disk 43.FIG.","4C shows the same polarized light source 40, however, the polarizing filter 41 is now arranged at an angle of about 60° relative to the source 40 so that a somewhat darker spot 42c is created.","Again, the disk 43 has been rotated an amount “x” so as to permit the separate recording of the spot 42c upon the disk 43.Likewise, with regard to FIG.","4D, the polarized light source 40 and the polarizing filter 41 are now at an angle of about 90° relative to the starting position of the polarizing filter 41 in FIG.","4a, resulting in a completely dark spot 42d being recorded upon the disk 43.FIG.","4e shows a traditional photo detector 50 projecting a light source onto the disk 43 such that the dots 42a, 42b, 42c, and 42d on the disk 43 can be illuminated.","Light is then reflected back to, for example, the detector portion of the photo detector 50.Specifically, the detector 50 is capable of distinguishing the white dot 42a from the black dot 42d, as well as the shades of gray dots 42b and 42c.","It should be apparent that the example depicted in FIG.","4 has been greatly exaggerated for clarity purposes, and, for example, the amount “x” that the disk 43 needs to move to accommodate the sequential dots 42a-42d, is minute.","Additionally, as a result of recording the four different dots 42a-42d upon the disk 43, a base-4 or quaternary system has been utilized with the result recorded being “0”-“1”-“2”-“3”.","Still further, any appropriate media such as, for example, a PLZT ceramic, could be utilized to store different photo intensity data or spots.","It should be apparent that the dots 42a-42d are the beginning of a track of data which can be recorded spirally outward from a center portion of a disk 43.Another example of the invention comprises a base-8 code utilizing 8 different shades of dots.","While this particular example is not shown in the Figures, it is clear that 8 different shades of dots would require the turning of the polarizing filter 41 shown in FIG.","4 at approximately 12.85° each time.","Another example, similarly, could utilize a 16 shade code system, which would require turning the polarizing filter 41 of FIG.","4 a total of 6° each time to result in 16 different shades of dots.","It should be clear to an artisan of ordinary skill that the amount of data storage becomes a function of how finely the polarized filter 42 is rotated relative to the polarizing light source 41 as well as how accurately an optical detector 50 can distinguish between the different light intensities that are reflected or transmitted by the different shades of gray dots.","It is certainly possible that an entire 256 character ASCII-type code can be written into a single bit.","In such a case, the amount of data storage is significantly improved over the current techniques.","Still further, it should be apparent that different colors (e.g., red-yellow-violet), corresponding to different wavelengths of light can be placed upon a surface of a disk 43.When, for example, such a disk containing these different color dots or spots is rotated and interrogated by, for example, a laser lens system, the different colors (wavelengths or frequencies) of the dots or spots can be registered and could be coded to correspond to different values (e.g., “0”, “1”, “2”, “3”, etc.).","Thus, rather than using a simple binary or base-2 system where a black dot corresponds to “0” and white dot corresponds to “1”, the present invention provides for a larger number of options for data storage by providing methods to store data based on color, intensity or both.","Accordingly, for example, in a simple quaternary system having a quaternary code consisting of 0's, 1's, 2's and 3's, there would be 4 different choices of basic information storage (i.e., a “base-4” system) rather than only two choices in a binary or base-2 system.","Moreover, rather than utilizing only a monochromatic light source, it is possible that a polychromatic light source could also be utilized.","In this regard, when polychromatic light is made to be incident upon a first polarizer, and thereafter upon one or more additional polarizers (e.g., analyzers), in addition to different intensities of light flowing through the analyzer(s), a spreading of different colors over a particular frequency or wavelength range will also occur.","Specifically, as shown in FIGS.","10a and 10b, when using polychromatic light, a curve 90 representing a spectrum of frequencies ranging from f1 to f2 of various intensities results in the formation of a somewhat continuous curve of varying light intensities over a particular frequency or wavelength range.","The use of a polychromatic source can therefore result in secondary indicia for determining the value assigned to stored bits of data (e.g., a “0” or a “1”) and thus enhances data storage.","For example, not only could the highest intensity of light I1 be recorded/detected, but also the starting and/or ending points f1 and/or f2 which correspond to the lowest frequency of light being detected, and the highest frequency of light being detected, respectively.","Alternatively, the area “A” under each curve could also be compared as, for example, with integral area denoted as power spectral density.","Further, the FWHM (i.e., full width half max) can be used as an indicia, representing the frequency ranger, f1 to f2 at half the maximal intensity.","A range of full widths at varying intensities (i.e., 100% maximal, 1% minimal, 50% half maximal, 75% three-fourths maximal, etc.)","can also be used.","In other words, more than one piece of information can be checked to verify the accuracy of each data point that is stored and read.","The presence of more than a single piece of information can result in more accurate reading of data and thus result in signal/noise improvements.","FIG.","6 shows a light source 60 which emits unpolarized light 61 into a polarizing filter 62 which creates a polarized light beam 63.The polarized light 63 is incident upon a suitable light modulator 64 which causes the light beam 63 from the polarizing filter 62 to be directed to one of a plurality of different polarizing (or analyzing) filters 81, 82, 83, 84 and 85.The modulator 64 is known in the art and can function at frequencies of about 1 GHz and is thus compatible with most computer processing systems.","While only five analyzing filters 81-85 are shown, the number of filters that can be used in accordance with this embodiment is based on the data coding system in which it is desired to work.","This particular system which utilizes five filters 81-85 is a base-5 system.","When the modulator 64 directs the polarized light beam 63 from the polarizing filter 62 to the different analyzing filters 81-85, the result will be different intensities of light 65 being transmitted through each of the different analyzing filters 81-85 onto (or into) a suitable optical recording medium 66.Specifically, each of the polarizing filters 81-85 are fixedly rotated a different amount relative to the polarizing direction of the light beam 63 emanating from the polarizing filter 62.Once the light 65 has been transmitted through one of the different polarizing filters 81-85, the light can then be suitably focussed and/or redirected by appropriate optical and/or magneto-optical or acousto-optical (not shown in this FIG.","6) means onto or into the recording medium 66.Examples of suitable optical recording mediums include: inorganic photochromic crystals, photorefractive materials, chalcogenide semiconductors, organic and polymer materials, thermoplastic media, reversible recording in Tellerium compounds, photothermal recording in Antimony compounds, magneto-optic recording and light stimulated recombination luminescence.","With regard to FIG.","7, an alternative to the embodiment of FIG.","6 is shown.","In this embodiment, the plurality of polarizing filters 81-85 are replaced with a polarization modulator 67.In particular, the polarization modulator 67 can be made of a suitable material such as a silica glass or an X-cut lithium niobate crystal (see, for example, the disclosure in U.S. Pat.","No.","6,154,432 for suitable materials).","Voltages are applied to electrodes on the modulator 67 to produce an electric field that can orient the polarization of the propagating wave in any suitable direction.","This technique provides a very robust and mechanically stable method of polarization control.","The polarization of the light wave 70 (as well as the light wave 65) will be maintained through appropriate transmission of the light wave through other non-polarizing optics.","The modulator 67 has been shown to have transmission rates of over 500 Mbits/second.","A polarizing filter 62, oriented in a fixed direction, then provides an output 65 of varying intensities to a light modulator 64.The light modulator 64 then appropriately directs the output 65′ to a similar suitable recording medium 66.FIG.","8 shows another embodiment of the invention wherein a light source 60 emits unpolarized light 61 into an acousto-optic Bragg modulator 68.The acousto-optic Bragg modulator 68 has inputted into it different electrical driving signals which result in different intensities of light 65″ being emitted therefrom.","The different intensities of light can then be suitably focused and/or redirected by appropriate optical and/or magneto-optical and/or acousto-optical means into or onto an appropriate recording medium 66.FIG.","9 shows another embodiment similar to that embodiment shown in FIG.","7, except that the polarization modulator 67, has been replaced with at least one liquid crystal cell.","In this regard, depending upon what voltages are applied to the liquid crystal cell 69 (the output which is directed into a polarizing filter 62) will result in a variable intensity output 65′″.","The output 65′″ can thereafter be suitably directed to an appropriate optical storage medium 66 by any of the means for directing (not sown in FIG.","9) the output 65′″, as discussed above herein.","It should be also be clear to an artisan of ordinary skill that the same general technique disclosed herein can be utilized for the optical layout that is shown in FIG.","27.In this regard, FIG.","27 shows a holographic memory system of how a crystal can be imprinted with pages of data.","In this regard, similar to the techniques shown in FIGS.","4a-e, the system would work just as well for a holographic memory system.","OPTICAL EXAMPLES The following experiments were performed to show that various light intensities can be recorded by rotating at least two polarizable filters (e.g., a polarizer and an analyzer) relative to each other and recording the different resultant intensities on ordinary photographic film (i.e., color slide film).","The recorded intensities were thereafter interrogated with an optical spectrometer by passing a light source through the recorded intensities on the slides and into a probe connected to the optical spectrometer.","All optical recording and interrogation steps were performed on a Newport optics table having one (1) inch grids thereon.","In particular, mounting holes were provided at each corner of one inch squares for rigidly securing all optical equipment thereto to minimize data distortion due to vibrations.","Different optical data values were obtained and recorded by using varying amounts of light which were caused to be incident on a Canon EOS Rebel 4 camera with zoom lens, which was rigidly mounted to the optics table.","In particular, the camera was operated in a manual mode and the manual focus and was set to 50 mm.","The film used in the camera was ordinary color slide film known as Fujichrome Color Slide film RH-400 speed.","The source of light for all optic experiments was a Stocker and Yale, Model 20 Imagelite, polychromatic light source.","The fiberoptic bundle end of the light source was also rigidly mounted to the optics table, with its end being located about 3 cm from a rigidly mounted polarizer/analyzer filter set (Orion Variable Polarizing Filter Set #5558).","In particular, as shown in FIGS.","20 and 21, the polychromatic light source 60 was caused to communicate with a polarizing filter set 62 and 81 through a fiber optic bundle 101 (i.e., a Stocker-Yale SF 38036P single S/O; ⅜″ bundle, 36″ long, MTL-PVC over metal hose).","The function of the fiber optic bundle 101 was to transport polychromatic light from the Imagelite light source 60 (which was operating at a setting of “4”) to the polarizing filter set 62 and 81.The polarizing filter set 62 and 81 was located at a distance of about 3.3 cm from the lens of the Canon camera 100.Light from the light source 60 was directed through the fiber optic bundle 101 and caused to be made incident upon the polarizing filter set 62 and 81.The polarizing filter set 62 and 81 was set at six (6) different relative settings which permitted six (6) different intensities of light to be transmitted therethrough.","In particular, the polarizing directions of each filter were set at the following relative rotations to each other: 0° relative rotation, 18° relative rotation, 36° relative rotation, 54° relative rotation, 72° relative rotation, and 90° relative rotation.","These six (6) different relative rotation filter settings resulted in six (6) different intensities of light being transmitted therethrough.","Specifically, the different intensities of polychromatic light were caused to be incident upon the lens of the camera 100 with care being taken so that the fiber optic bundle 101 transmitted light roughly perpendicular into and through the polarizing filter set 62 and 81 and into the lens of the camera 100.The result was a different intensity spot being recorded on each of the six (6) different slides 102 corresponding to each of the six (6) different relative rotation amounts.","The differences in each of the slides 102 was readily observable by eye.","Once the different images were recorded onto the photographic film comprising the slides 102, the images or spots were then interrogated by a transmission technique that utilized an optic spectrometer.","Specifically, as shown in FIG.","21, data values were retrieved from each of the six (6) different slides 102 by using a StellarNet EPP 2000 spectrometer 104 which utilized SpectraWiz software.","Specifically, the same light source 60 used to create the images on the slides 102 was again connected with the same fiber optic bundle 101, still rigidly mounted on the optics table.","Each of the data slides 102 was rigidly mounted in a holder such that the output from the fiber optics bundle 101 was reproducibly and directly incident upon that portion of the slide 102 containing the data or spot, from a distance of about 7.2 cm.","Care was taken to assure that the fiber optic bundle 101 was mounted perpendicular to the slides 102 in the slide holder.","A fiber optic probe 103 of the StellarNet EPP 2000 spectrometer 104 was rigidly mounted on the other side of each slide 102 about 7.0 cm away, so that transmission information could be obtained.","Again, care was taken to assure proper alignment so that transmission data would not be skewed.","The relevant settings on the StellarNet EPP 2000 fiber optic spectrometer were as follows: Mode Scope Detector integration time 4 ms Number of scans to average 5 Pixel resolution via smoothing 0 level Temperature compensation On Spectrometer channels 1 X axis Wavelength (nm) Y axis Counts The image light source was then set at a standard setting of “7”, in order to obtain information.","The EPP 2000 provided information regarding the maximum integral area under the curve (power spectral density, i.e., Counts2) and full width half max (“FWHM”) of the peak (in nm) for each slide via the automated Area-PSD function.","It is noted that each slide corresponded to the six (6) different rotatable filter settings.","Measurements were reliably reproducible.","Measurements of the six (6) slides, corresponding to the six (6) filter positions ranged, in power spectral densities, from 347,690 at 0° to 36,674 at 90°.","FWHM ranged from 466 nm at 0° to 160 nm at 90°.","Using either power spectral density alone, FWRM alone, or both in combination, six (6) different and distinct data values were obtained.","Specifically, as shown in FIGS.","22a, 22b and 22c, three sets of measurements were taken at different times from the same aforementioned six (6) slides.","FIGS.","22a-22c are a plot of Power Spectral Density (i.e., intensity) versus the six (6) different filter settings.","The deviation in the upper and/lower limits of the data in FIGS.","22a-22c is superimposed on FIG.","23.It is clear from FIG.","23 that the upper and lower intensity limit deviations corresponding to each of the six (6) different filter settings do not overlap with each other.","Thus, this technique shows that intensity alone can provide a basis for a base-6 data storage system.","Further, optical transmission data from the six (6) slides was analyzed by a second approach.","Specifically, FIGS.","24a, 24b and 24c show plots of full width half max (FWHM) data points versus the six (6) different filter settings.","Similar to the data in FIGS.","22a-22c, these data were obtained at three (3) different times to assure the accuracy of the measurements.","FIG.","25 shows the deviation in upper and lower limits for the data shown in FIGS.","24a-24c.","It is clear from FIG.","25 that the upper limit deviation values for FWHM corresponding to each filter setting do not overlap.","Thus, this technique provides another method for data recognition in, for example, a higher order system.","MAGNETIC SPLITTING/SHIFTING EXAMPLES The following magnetic experiments were performed to show that different amounts of Zeeman splitting/shifting can be achieved in a magnetic material and/or a contiguous material.","Specifically, varying magnetic field strengths were applied to a contiguous material comprising aluminum foil to simulate one potential configuration of magnetic storage media exhibiting desirable Zeeman splitting/shifting effects.","The source of the magnetic fields utilized were three (3) permanent magnets of different field strengths.","In particular, the magnets were obtained from DuraMag, Inc., and were characterized as follows: Magnet A: (Low Field Strength) NS-10010050, Neodymium-Iron-Boron Magnet, 1.000″×1.000″×0.500” thick with a field strength near the center thereof of about 5,090 G, increasing to about 8,380 G at the edge; Magnet B: (Intermediate Field Strength) ND-4514 Neodymium Magnet, 1.0″×1.0″×1.0″ thick with a field strength near the center thereof of about 8,730 G, increasing to about 10,300 G at the edge; and Magnet C: (High Field Strength) ND-4514 Neodymium Magnet, 1″×1″×1.9″ thick with a field strength near the center thereof of about 11,500 G, increasing to about 11,900 G at the edge.","All spectroscopy measurements were performed using a Hewlett-Packard microwave spectroscopy system (30-minute warm-up).","The following are the salient features of the spectroscopy system used in these examples.","(1) HP Display Processor—Ref 0.0 dB, S11 mode, log MAG display, 10.0 dB/and marker pips with frequency and dB readout.","(2) HP 8510B Network Analyzer-Ch 1.","(3) HP 8513A Reflection/Transmission Test Set—45 MHz-26.5 GHz, Port 1.","(4) HP 8350B Sweep Oscillator—Instrument State REM, Sweep trigger—INT, Sweep—Time.","(5) HP 83595A RF Plug-in—0.1-26.5 GHz, Power 10.0, mW RF—On, CS Filter—On, ALC—INT.","(6) Cables—W.","L. Gore & Associates DAS ATE (55 AT) Test cables, 24 inches long.","(7) Waveguides (Maury Microwave, Inc., and Narda, Inc.).","It is noted that all waveguides produced flat baselines at 0.0 dB within the various frequency ranges noted below.","a. Narda 616: Range 1.12 to 1.7 GHz b. Maury R229A1: Range 1.7 to 2.6 GHz c. Maury S209C2: Range 2.60 to 3.96 GHz d. Maury G229A1: Range 3.95 to 5.85 GHz e. Maury C209D2: Range 6.85 to 8.2 GHz.","FIG.","26 shows a schematic of the basic experimental set-up that was used to determine Zeeman shifting amounts in an aluminum foil contiguous material that was subjected to different magnetic field strengths.","A microwave spectrometer 204 was comprised of Hewlett Packard items (1)-(5) described above.","The microwave spectrometer 204 was connected by the cable 203 described in item (6) above.","Different waveguides 202, described in item (7) above, were placed over each magnet 201 described as Magnets A, B and C above.","Each of the magnets 201 (i.e., A, B and C) were sequentially placed into contact with a thin sheet of commonly available aluminum foil 200 (Tamco Distr.","), the magnets 201 being oriented with their magnetization direction pointing parallel with the electromagnetic beam propagation in the waveguide 202.The following Table 3 shows the different amounts of Zeeman shifting that were obtained with three (3) different magnetic field strengths generated by the three (3) magnets A, B and C, applied to the contiguous aluminum foil 200.The initial frequency of 1.885 GHz was obtained from the aluminum foil 200 without the application of any magnetic field.","The other values correspond to the shifted aluminum foil 200 frequency determined by microwave spectroscopy, as well as the power frequencies in dB.","It is clear from this simple Example that meaningful Zeeman shifting can occur in a contiguous material to which a “permanent” magnetic field has been applied.","Accordingly, this Example demonstrates that storage of varying magnetic field strengths can function as a memory storage system and that Zeeman shifting/splitting can function as a memory retrieval system.","More particularly, both systems can function in higher order memory systems.","TABLE 3 Magnet A Magnet B Magnet C Waveguide No Magnet (Low) (Intermediate) (High) 1.12-1.7 GHz 1.885 GHz, 1.855 GHz, 1.645 GHz, 1.115 −4.2 dB −4.6 dB −7.2 dB GHz, −3.4 dB 1.7-2.6 GHz - 0 - - 0 - - 0 - - 0 - 2.60-3.95 GHz - 0 - - 0 - - 0 - - 0 - 3.95-5.85 GHz - 0 - - 0 - - 0 - - 0 - While there has been illustrated and described what is at present considered to be the preferred embodiments of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made, and equivalents may be substituted for elements thereof without department from the true scope of the invention.","In addition, many modifications may be made to adapt the teachings of the invention to a particular situation without departing from the central scope of the invention.","Therefore, it is intended that this invention not be limited to the particular embodiments disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims."]],"string":"[\n [\n \"Enhanced data storage and retrieval devices and systems and methods for utilizing the same\",\n \"The invention relates generally to store information on magnetic or optical storage media by using one or more novel approaches alone or in combination.\",\n \"These novel approaches are capable of using at least one code which may comprise more than two values (FIG.\",\n \"4d, 43).\",\n \"A first series of approaches for the storage of information applies generally to optical recording and reproducing system (FIGS.\",\n \"7-10, optical media 66), while a second series of approaches applies generally to electric or magnetic recording and reproducing systems (FIG.\",\n \"1-2, magnetic medium 1), Each series of approaches is capable of storing information data in one or more codes (FIG.\",\n \"4d, 42a, 42b, 42c, 42d) and the use of at least one higher order code which is different from the traditional binary code of “0” and “1”.\"\n ],\n [\n \"1.A method for storing data comprising: storing at least one magnetic field strength; utilizing said at least one stored magnetic field strength to cause an amount of splitting or shifting of at least one frequency in at least one material; determining the amount of shifting or splitting in said at least one material; and assigning a data value to said amount of splitting or shifting.\",\n \"2.The method of claim 1, wherein said determining comprises a means for interrogating said at least one material with electromagnetic energy to determine said amount of shifting or splitting.\",\n \"3.The method of claim 1, wherein said at least one material comprises at least one magnetic material.\",\n \"4.The method of claim 3, wherein said at least one material comprises at least one material contiguous to at least one magnetic material.\",\n \"5.The method of claim 1, wherein said method for storing data comprises a base-2 data storage system.\",\n \"6.The method of claim 1, wherein said method for storing data comprises, comprises greater than a base-2 data storage system.\",\n \"7.The method of claim 1, wherein said splitting or shifting comprises at least one Zeeman effect.\",\n \"8.A method for reading data comprising: creating at least one magnetic field strength; utilizing at least one magnetic field strength to cause an amount of splitting or shifting of at least one frequency and at least one material; determining the amount of shifting or splitting in said at least one material; and assigning a data value to said amount of splitting or shifting.\",\n \"9.A data storage apparatus comprising: means for storing at least one magnetic field strength; means for utilizing said at least one stored magnetic field strength to cause an amount of splitting or shifting of at least one frequency in said at least one material; means for determining the amount of shifting or splitting in said at least one material; and means for assigning a data value to said amount of shifting or splitting.\",\n \"10.A method for storing information optically comprising: an optical source; a means for achieving different optical intensity; a means for recording said different optical intensity; and a means for reading said different recorded intensities.\",\n \"11.A method for storing information optically comprising: an optical source; a means for achieving different optical wavelengths; a means for recording said different optical wavelengths; and a means for reading said different recorded optical wavelengths.\"\n ],\n [\n \"<SOH> BACKGROUND OF THE INVENTION <EOH>Data storage devices have been becoming smaller in size (as well as faster) due to, for example, the continued improvement in the ability to store more information in smaller spaces.\",\n \"For example, over the past twenty years the ability to place more data on magnetic storage media such as magnetized hard-disk drives and/or floppy disks, as well as on optical media (e.g., compact disks “CD's” and more recently, Digital Versatile Disk “DVD's”) has been increasing at a phenomenal pace.\",\n \"The impressive increase in the ability to store more data in smaller areas has been one of the main driving forces for rendering computers, and other electronic devices with reasonably sized memories, accessible to a typical home or small business consumer.\",\n \"Further, the high operating speeds of microprocessors have required stored data in computers to be rapidly accessible (e.g., both the rate at which information is delivered to a microprocessor as well as the amount of time it takes between the time that a microprocessor requests a file and when the first piece of data begins to flow from the file to the microprocessor are important).\",\n \"Magnetic memory has undergone many recent improvements and has been utilized extensively in data storage/retrieval devices because magnetic memory currently provides the least expensive alternative for the fast writing and reading capabilities for data.\",\n \"However, the various currently utilized magnetic technologies face a variety of technical challenges and appear to be headed for a stopping point.\",\n \"Specifically, these various approaches for the storage and/or retrieval of information may no longer be capable of meeting the growing demands of the industry, without experiencing significant technical enhancements.\",\n \"For applications where speed has historically been less important, various optical technologies have been growing in popularity.\",\n \"Many technical advances have been made in the recording and/or retrieval of information by optical means.\",\n \"In systems which use compact disks (“CD's”), compact disk read-only memory (“CD-ROM's”) and/or Direct Video Disks (“DVD's”), the storage and/or retrieval of information has also been of great interest.\",\n \"For example, the first CD's manufactured were CD-ROM's that are currently capable of storing about 650 MB of data.\",\n \"This equates to about 74 minutes of play-time for music that has been recorded in a typical recording mode.\",\n \"In particular, the standard current approach for utilizing CD-ROM's, includes the use of a solid state interrogating laser (or in some cases more than one laser) which has a wavelength of about 780 nanometers (0.78 μm).\",\n \"The standard CD-ROM is about 12 cm in total diameter and has a thickness of about 1.2 mm.\",\n \"The standard CD-ROM has information recorded on plateaus (“bumps”) and valleys (“pits”) that spiral from the inside of the disk toward the outside of the disk.\",\n \"The digital data stored in the tracks that comprise the spirals must be separated by enough space (e.g., tracks are typically separated radially by about 1.6 μm) so that when the data is read with an interrogating solid state laser, crosstalk from adjacent tracks does not occur (i.e., recorded information is received from only one track at a time).\",\n \"Accordingly, the placement of more information onto a typical CD has been limited by the required size (e.g., length and width) of the plateaus (“bumps”) and valleys (“pits”).\",\n \"The size of the plateaus and valleys (e.g., typically not less than 830 nanometers in length) has been a function of various factors, including the wavelength of the laser utilized to read the information from the CD.\",\n \"Various attempts have been made to increase the memory capacity of current CD's, including the stacking of a plurality of disks one on top of the other so as to provide more memory space, as well as other attempts to miniaturize the digitized information stored in the tracks.\",\n \"The most recent significant improvements in this type of optical storage/retrieval technology have been the creation of the DVD, which is known as the “Direct Video Disk” or the “Digital Versatile Disk”.\",\n \"The first commercial products of DVD's entered the marketplace in late 1997.The DVD technology is similar to the technology in CD's, except that, for example, the wavelength of lasers utilized to read information from DVD's is smaller than that used for CD's, namely, about 635 nanometers or 650 nanometers, compared to about 780 nanometers for CD's.\",\n \"The size of the plateaus (“bumps”) that make up the DVD are about 320 nanometers wide, a minimum of about 400 nanometers long and about 120 nanometers thick.\",\n \"About 740 nanometers separate one track from the next, compared to the about 1600 nanometer separation of tracks in CD's.\",\n \"A standard DVD is physically about the same size as a CD, but has a memory capacity that is about seven times greater than that of CD's.\",\n \"In particular, the memory capacity of a one-sided, one-track, DVD is now about 4.7 GB.\",\n \"However, recent improvements in DVD have allowed digital information to be placed on both sides of a single track, as well as both sides of a double track.\",\n \"In this regard, a first interrogating laser reads the digitized data on a first side of a first track (i.e., in the form of bits arranged in a spiral track that begin in the innermost portion of the DVD and spirals outward).\",\n \"Then, information can be retrieved from the opposite side of the first track in a similar manner from a second laser; however, the spiral track of information typically begins on the outermost portion of the disk and spirals inward toward the center.\",\n \"Accordingly, when dual-track, dual-sided DVD disks are used, the current amount of memory that can be achieved is about 17 GB.\",\n \"Another optical data storage technique which shows promise is known as holographic memory.\",\n \"The potential for high storage capacity and high storage retrieval rates using the holographic technique is tremendous.\",\n \"In particular, for example, recent work in a system known as Holographic Data Storage System (“HDSS”), has shown that data can be accessed in 100 microseconds or less, which is several orders of magnitude faster than the millisecond retrieval of data in typical magnetic-disk drives.\",\n \"Briefly, in this technology, an optical-interference pattern is created within a desired photosensitive material (e.g., a crystal or a polymer).\",\n \"The optical interference pattern is imprinted as a result of physical changes in such materials.\",\n \"The interference pattern is created by the interaction between an object beam and a reference beam, both of which typically originate from a single light source (e.g., a laser), but are split into two different pathways by a beam splitter.\",\n \"Specifically, the object beam is incident on a target object that contains data, and modifies the object beam, while the reference beam, taking a separate path which leaves it unmodified, interferes with the object beam at the holographic storage medium.\",\n \"When the two beams meet at the holographic storage means, the interference pattern which results from their interaction is stored on or in the holographic storage means.\",\n \"The holographic storage means is later interrogated by a reading or reference beam, which upon interacting with the holographically stored interference pattern, can then recreate the holographic image containing the data originally stored in the object beam.\",\n \"While the holographic optical technique shows great promise for the future, current industrial emphasis is being placed primarily on improving existing digital optical and magnetic storage/retrieval systems so that more information can be stored in smaller spaces, and retrieval times can be decreased without significantly changing the basic methodology and hardware for the storing and retrieving of information.\",\n \"With regard to placing more data onto magnetic recording media, effects such as the paramagnetic effect (e.g., magnetic crosstalk between magnetic domains comprising bits) are already beginning to cause problems.\",\n \"Experts have postulated that in coming years, magnetic storage technologies could reach a limit imposed by the superparamagnetic effect (“SPE”).\",\n \"The physical phenomenon known as SPE can occur in a magnetic data storage system when the magnetic spins of electrons in the domains which comprise a bit (e.g., in current technology typically either a “0” or a “1”) become unstable due to, for example, the environmental thermal energy surrounding the bits.\",\n \"Instability of bits occurs when smaller and smaller numbers of domains or atoms are used to comprise a bit and the ability for the domains or atoms to maintain their given spin directions (e.g., corresponding to, for example, a “0” or a “1”) becomes difficult.\",\n \"Non-maintenance of spin directions results in a “flipping” of spins back and forth and thus changing “0's” to “1's” and “1's” to “0's”, etc.\",\n \"Such changing or flipping of spins will result in a corruption of the data or information that the “0's” and “1's” represent.\",\n \"Accordingly, in order for smaller numbers of atoms to be used to form a bit and in order for the bit to be viable, techniques and/or materials for preventing the superparamagnetic effect need to be developed to overcome the deficiencies of the prior art.\",\n \"Another potential problem in recording information magnetically is that the write head which is used to align magnetic domains in the magnetic storage media may be limited in the ability to impart a required field strength over the entire domain area comprising a bit to cause the magnetic domains to behave as though they have been exposed to a substantially uniform field.\",\n \"In such cases, it is possible that differently oriented domains within a bit (e.g., differently oriented due to being subjected to different magnetic field strengths within a single writing head due to, for example, non-uniformity of the magnetic flux lines emanating from the write head and/or differently oriented because some domains require more energy than other domains to align due to, for example, their location within a bit) may have a higher tendency to become corrupted, as well as adding to signal/noise ratio problems.\",\n \"One attempt for miniaturizing domains involves the use of so-called “hard” magnetic materials.\",\n \"The use of hard magnetic materials is desirable when compared to “soft” magnetic materials because hard magnetic materials are more likely to maintain their magnetic spin directions when domains comprising the hard magnetic materials are closely spaced.\",\n \"In this regard, various rare-earth and transition elements have been found to be magnetically stable and are thus termed “hard”.\",\n \"Such magnetically stable materials are known to have a higher coercivity, which is typically represented by “H c ”.\",\n \"Stated in an over simplified manner, the higher the H c value, the greater the resistance of the material to outside influences, such as outside magnetic fields (e.g., the harder it is for material to lose its imprinted spin direction due to an increase in temperature and/or the influence of other magnetic fields, etc.).\",\n \"However, one drawback for utilizing “hard” magnetic materials is that hard magnetic materials are more difficult to magnetize than soft magnetic materials (e.g., in some cases, depending on the particular materials chosen, much more difficult to magnetize).\",\n \"This makes it more difficult to record data initially in hard magnetic materials.\",\n \"There are various known approaches for solving the problem of encoding data into hard magnetic materials which approaches utilize a laser in conjunction with an appropriate magnetic write head.\",\n \"These various approaches have been referred to generally as the “magneto-optical” approach.\",\n \"These magneto-optical approaches effectively reduce the H c value of the magnetic domains by causing the domains to be locally heated just prior to being subjected to the magnetic write head.\",\n \"Thus, the coercivity (H c ) of the domains is effectively reduced and when such effective reduction in H c has occurred, a somewhat standard write head, or in some cases a very different write head, can be used to alter favorably the magnetic domains.\",\n \"In other words, the use of a laser, which provides thermal energy to the domains of the hard magnetic material, causes the hard magnetic material to behave in a manner which is similar to a somewhat softer and thus easier to magnetize magnetic material.\",\n \"Each of the known approaches for utilizing the magneto-optic techniques involves the use of at least one laser and/or at least one laser focusing system.\",\n \"However, one of the common problems facing these magneto-optical approaches is that the lasers that are utilized to soften the hard magnetic material prior to subjecting the hard magnetic material to a magnetic field is that the lasers typically generate excess amounts of heat that can flow to neighboring bits or tracks (e.g., thermal energy is caused to migrate to undesirable neighboring tracks or areas resulting in a potential corruption of stored information).\",\n \"Accordingly, various laser applications and/or storage solutions have been devised to minimize the transfer of heat from the laser to neighboring tracks, and thus minimize the loss of recorded information.\",\n \"However, to date this thermal transfer of laser energy still presents problems to varying degrees in all the prior art approaches.\",\n \"One interesting technique for the reading of data in connection with one optical-magnetic system is the use of an effect known as the “Kerr effect”, and more particularly, the “magneto-optical Kerr effect”.\",\n \"Briefly, current uses of the magneto-optical Kerr effect involve incident polarized light being reflected from the surface of a magnetic domain in different ways, depending on the orientation of the magnetic domains (e.g., depending on electron spin directions).\",\n \"The changes in the reflected polarized light are made, by algorithms, to correspond to different digitized data.\",\n \"Specifically, when the incident polarized light encounters different magnetic field orientations (e.g., north or south domains comprising a bit which are ordered to represent either a “0” or a “1”) the polarization state of the reflected light is changed.\",\n \"Another approach to the digital storage of information that shows future promise is known as atomic resolution storage (“ARS”).\",\n \"This approach, similar to holographic techniques, is capable of storing tremendous amounts of information in a small amount of space.\",\n \"In this technique, generally, very small electron probes, which are used to generate electron beams, are formed into an array.\",\n \"The electron probes have tips that are roughly the size of atoms.\",\n \"The electron beams from the electron probes are made to be incident upon a storage medium so that the incident beams cause some sort of physical change in the storage medium (i.e., the medium comprising the computer memory).\",\n \"One example of such a storage medium is a material that is capable of containing at least two stable phases at ambient operating conditions so that an incident electron beam emitted from an electron probe changes the storage material from one phase to the other.\",\n \"In this technique, encoded bits of information are represented by the change in phase of the phase-change storage material.\",\n \"This technique, similar to the magneto-optical technique discussed above, also suffers from the problem of heat flowing between data spots created by the incident electron beams when the spots or bits are initially recorded.\",\n \"Accordingly, one of the challenges faced by this relatively new technology includes reducing the flow of heat between data spots which are created by the array of electron beam generators.\",\n \"The retrieval of stored information is also a challenge.\",\n \"In this regard, as bits become smaller and include lesser numbers of magnetic domains per bit and/or the domains are oriented differently, (e.g., domains are aligned perpendicular to the substrate containing at least one magnetic surface thereon instead of being parallel to the substrate surface) such bits are capable of being packed more tightly together.\",\n \"Assuming that the bits are not corrupted by their tight packing and/or smaller numbers of magnetic domains and/or non-uniform alignment within a bit, then reading of these tighter packed domains comprising the bits also becomes a challenge which the prior art is still struggling to overcome.\",\n \"Historically, large numbers of magnetic domains were present in each bit which contained digital data.\",\n \"Thus, the resulting or induced magnetic fields were relatively large and various forms of known inductive reading techniques were sufficient to ascertain whether the hundreds or thousands of aligned domains in a bit corresponded to a “0” or a “1”.\",\n \"However, as prior art miniaturization recording techniques progressed, the known inductive reading techniques were typically not sensitive enough to determine accurately the alignment of ever decreasing numbers of domains comprising a bit (e.g., signal to noise problems in the storage and/or retrieval of digital data bits were exacerbated).\",\n \"The development of magnetoresistive heads, and the subsequent development of giant magnetoresistive heads, has enabled smaller numbers of domains comprising a digital bit to be read accurately.\",\n \"In both of magnetoresistive and giant magnetoresistive heads, the electrical resistance of one or more materials comprising the read head(s) is examined.\",\n \"In particular, both magnetoresistive and giant magnetoresistive heads are made from materials that exhibit small changes in their electrical resistance as a function of magnetic fields created by oriented domains to which such heads are exposed.\",\n \"Giant magnetoresistive heads are two or three times more sensitive than magnetoresistive heads due to their novel structure which includes layering of different materials stacked on top of each other.\",\n \"One problem that continues in these technologies is the presence of magnetization vortices in the recorded domains.\",\n \"Such vortices may result from the difficulty in achieving uniform domain alignment within a bit due to, for example, the difficulty in applying a required magnetic field (e.g., uniform field) to the domains during the recording or writing process.\",\n \"While use of the magneto-optical Kerr effect and new read heads such as magnetoresistive heads and giant magnetoresistive heads have improved digital data retrieval techniques, further miniaturization is pushing the limits of these systems.\",\n \"Accordingly, additional improvements are necessary in the ability for hardware, combined with suitable programming, to detect even smaller numbers and/or smaller sizes of domains comprising digitized (or analog) data bits.\",\n \"Thus, additional innovations in the detection of stored data are required.\",\n \"In addition to the storage of information digitally, the possibility or promise of storing information in an analog manner continues to intrigue various investigators.\",\n \"While certain improvements have been made in, for example, the processing of analog information (e.g., the advent of VLSI (Very Large Scale Interrogation) chips, the techniques for the storing of analog information need much improvement.\",\n \"Particularly, while analog computing has been known for several decades, an accurate and fast-paced system for the storage and retrieval of information has continued to elude researchers.\",\n \"In this regard, digital computing has received the greatest amount of attention because of it's relative dependability for the storing and retrieving of information in a “1” or a “0” digitized format.\",\n \"Briefly, digital approaches typically include storing a sampling of the analog information that is to be recorded.\",\n \"The number of samples taken (e.g., such sampling typically occurs in predetermined units of space or time) from the analog source will correspond to the accuracy between the original analog image or sound and the stored digital data (i.e., the more digital samples that are recorded of an original analog image or sound, the more accurate the reproduction of the original analog image or sound will be).\",\n \"These techniques of predetermined quantum amounts of sampling of analog images have been adequate for most digitized applications, however, as the need for more accurate recording and retrieval of information occurs (e.g., in, for example, the fields of computing or determining various engineering and scientific relationships in the following representative areas: gravitational; electrostatic; magnetic; thermal; stress; fluid flow field analysis; wave propagation; image processing; etc.\",\n \"), the availability of computing with more accurate data becomes important.\",\n \"However, the digitization of tighter and and/or smaller sampling amounts from an analog source can result in tremendous amounts of digitized data being stored to represent a small actual amount of analog information.\",\n \"This is one area where analog computing has a distinct advantage compared to digital computing.\",\n \"In particular, an analog computer will store only a single piece of information corresponding to a single sampling area, whereas a digital computer will need to store somewhere between, for example, 8 and 16 bits of data to represent the same single piece of information stored in an analog fashion.\",\n \"The large number of bits required to store digital information corresponds to the requirement to store information in an ASCII or Unicode format.\",\n \"However, in order to escape from the current ASCII or Unicode format, analog storage requires a very large number of different data bits (e.g., data corresponding to much greater numbers than merely “0s” and “1's”) to be stored and retrieved.\",\n \"Current prior art techniques have great difficulty in storing and/or reading bits corresponding to anything other than “ 0 's” and “ 1 's”, let alone the attendant problems of storing and/or reading, for example, a near infinite number of possibilities (e.g., 1, 2, 3 .\",\n \".\",\n \".\",\n \"n+1).\",\n \"However, when performing complex calculations, the digital process may result in very slow processing times as well as requiring very large memories.\",\n \"It is for this, as well as other reasons, that analog computing is still very attractive.\",\n \"Thus, while advances in the processing of analog information have been made, reliable techniques for the storage and retrieval of analog information are still being sought without any good solutions currently existing.\",\n \"As stated above, with regard to digital computing, much of the recent technical emphasis in data storage and retrieval for both optical and magnetic memory enhancement has been on shrinking more information into smaller areas.\",\n \"The ability to place bits, which are the smallest elements of information used by current digital memory systems, into a binary code format, which is a combination of “0's” or “1's”, into a smaller area on, for example, magnetic and/or optical disk storage media (e.g., a disk or a tape) may soon reach a limit.\",\n \"Thus, current technologies for writing and/or reading binary data onto and/or from current storage media will be pushed to the maximum limit.\",\n \"Computer designers are already facing a performance gap whereby processors can process information faster than the information can be retrieved from computer memory.\",\n \"The majority of the currently proposed approaches for the digital storage of information, suffer from the same problem, namely, that the magnetic and/or optical information which is stored uses a binary code (i.e., a base- 2 number system) which is a combination of “0's” and “1's”.\",\n \"The predominance of the use of a “binary” code has been due to, for example, difficulty in distinguishing signals (e.g., a “0” or a “1”) from background noise.\",\n \"In this regard, many techniques have been proposed which use various hardware, as well as algorithms, which attempt to read accurately the stored information as either a “0” or a “1”.\",\n \"However, certain prior art techniques have been postulated for utilizing codes other than binary.\",\n \"In particular, U.S. Pat.\",\n \"No.\",\n \"6,154,432, entitled “Optical Storage System” discloses a holographic system which records information in very small spots and postulates that 10 or more digits may be possible to record on a single bit.\",\n \"Accordingly, this holographic technique discloses codes higher order than binary.\",\n \"Further, for example U.S. Pat.\",\n \"No.\",\n \"5,450,363, entitled “Gray Coding for a Multi Level Cell Memory System” discloses the use of a so-called “gray code” approach (also known as “gray scale” by others) for data storage.\",\n \"This approach also uses a system which is higher order than binary.\",\n \"In particular, reference is made to the storage of multiple bits of information in a single memory cell thus creating a multi-level cell.\",\n \"Still further, U.S. Pat.\",\n \"No.\",\n \"6,061,005, entitled “Encoding of Data”, discloses a method of encoding data on magnetic cards which uses symbology other than binary, such as trinary, quaternary or even higher symbology, for the recording of information.\",\n \"However, while various techniques have been postulated for systems which use codes higher than binary, none of such codes have been commercially adopted due to, for example, signal to noise ratio problems, etc.\",\n \"In particular, reading errors can occur due to lack of contrast between data values.\",\n \"Such lack of contrast can be caused by random noise (e.g., vibrating inaccurate positioning of recording/reading heads, fidelity loss during recording, etc.)\",\n \"and determinate variations.\",\n \"Accordingly, a need exists for techniques which can be commercially viable which can use codes reliably that are higher order than traditional binary codes for the storage and/or retrieval of information.\",\n \"A very particular combination of “0's” and “1's” is known as the American Standard Code for Information Exchange (i.e., the ASCII code) which assigns a unique code number between 0 and 127 to each of the 128, 7-bit, binary number characters.\",\n \"Table 1 below shows in table form the current ASCII characters arranged by Hexidecimal digits (i.e., 128 different encoding combinations of groups of seven bits).\",\n \"Reading within the Table from left to right and then down, the first 32 values are codes which correspond to various computer control functions such as line feed, carriage return, etc.\",\n \"The 33 rd value corresponds to the “space” character.\",\n \"Values 34-48 correspond to symbols and punctuation marks.\",\n \"Values 49-58 correspond to digits, and so on, until reaching value 128, which corresponds to “delete”.\",\n \"In addition, each of these values also has a corresponding hexidecimal character.\",\n \"For example, the hexidecimal digit corresponding to the “space” character is “20”, while the hexidecimal digit corresponding to the letter “n” is “6E”, and so on.\",\n \"TABLE 1 00 01 02 03 04 05 06 07 08 09 0A 0B 0C 0D 0E 0F 00 NUL SOH STX ETX EOT ENQ ACK BEL BS TAB LF VT FF CR SO SI 10 DLE DC1 DC2 DC3 DC4 NAK SYN ETB CAN EM SUB ESC FS GS RS US 20 !\",\n \"“ # $ % & ‘ ( ) * + , − .\",\n \"/ 30 0 1 2 3 4 5 6 7 8 9 : ; < = > ?\",\n \"40 @ A B C D E F G H I J K L M N O 50 P Q R S T U V W X Y Z [ \\\\ ] {circumflex over ( )} — 60 ’ a b c d e f g h x j k l m n o 70 p q r s t u v w x y z { | } ˜ DEL These 128 characters are enough characters for North American English to be stored and/or retrieved, but are not enough for many other languages (Note: In ASCII, the eighth bit is used, and is thus normally set to “0”).\",\n \"To represent more than 128 characters, 8-bit (rather than 7-bit) binary numbers are utilized.\",\n \"For each digit in these eight bits there are two choices for the digit, either “0” or “1”.\",\n \"This results in a possibility of an additional 128 characters or a total of 256 different combinations of “0's” and “1's” (i.e., 2 8 ).\",\n \"Accordingly, the current data storage approaches utilize these 256 combinations of “0's” and “1's”.\",\n \"However, these 8-bit numbers still do not provide enough room for all characters that need to be used in the world (e.g., certain Asian languages have thousands of characters each).\",\n \"In addition, there is no universal agreement in the world on what all the characters should be, whether referring to the 128 characters, or the 256 characters, or any number of characters beyond these.\",\n \"However, certain approaches for dealing with all the various languages in the world, which collectively contain many thousands of characters, are beginning to utilize something referred to as the “Unicode”, which uses a 16-bit character set.\",\n \"In order to adopt something similar to the Unicode, even greater strain will be placed on current memory techniques.\",\n \"Whether using an 8-bit, 16-bit, or some other approach, all such approaches have been limited primarily to using a binary system of data storage.\",\n \"However, due to: (1) the impending size barrier for the storage of information on, for example, magnetic and/or optical media; (2) the performance gap between how fast a processor can process information versus how fast a processor can access information; and (3) the goal for a universal adoption of a common set of characters throughout the world, a different approach for the storage of information, either by digital or analog approaches, is clearly needed.\",\n \"The present invention satisfies the current and future data storage needs by using different approaches for the storage and/or retrieval of data; and/or different codes that use a code higher than the current “base- 2 code”.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>It is an object of the invention to provide a means for storing/retrieving digitized information optically.\",\n \"It is an object of the invention to provide a means for storing/retrieving digitized information magnetically.\",\n \"It is an object of the invention to provide a means for storing/retrieving analog information optically.\",\n \"It is an object of the invention to provide a means for storing/retrieving analog information magnetically.\",\n \"It is an object of the invention to provide a storage/retrieval means for utilizing codes other than binary.\",\n \"It is an object of the present invention to provide an increased number of characters that can be stored as a single bit of information.\",\n \"It is an object of the invention to provide the ability to store the 128 ASCII-type characters in a much smaller space than the space that is used in a standard binary code of “0's” and “1's”.\",\n \"It is an object of the invention to provide a means for storing/retrieving codes other than binary.\",\n \"It is an object of the invention to utilize at least a base- 3 optical storing/retrieving system.\",\n \"It is an object of the invention to utilize a least a base- 4 coding storing/retrieving system.\",\n \"It is an object of the invention to utilize at least a base- 6 coding storing/retrieving system.\",\n \"It is an object of the invention to utilize at least a base- 8 coding storing/retrieving system.\",\n \"It is an object of the invention to utilize at least a base- 10 coding storing/retrieving system.\",\n \"It is an object of the invention to utilize particular aspects of the Zeeman effect in connection with the storage/retrieval of digitized information.\",\n \"It is an object of the invention to utilize particular aspects of the Zeeman effect in connection with the storage/retrieval of analog information.\",\n \"It is an object of the invention to utilize different shades or colors of spots or dots in connection the optical storage of digitized information.\",\n \"It is an object of the current invention to utilize different shades or color or spots or dots in connection with the optical storage of analog information.\",\n \"It is an object of the invention to utilize either monochromatic or polychromatic light in connection with the optical storage of digital and analog information.\",\n \"It is an object of the invention to utilize an analog-like approach to the storage of data.\",\n \"The current process of storing bits, which is the smallest element of information used by current memory systems, uses a binary format of only “0's” and “1's”.\",\n \"A bit which is “off” is considered to be “false” or “not set”, while a bit that is “on” is considered to be “true” or “set”.\",\n \"Historically, because bits could only be one of two values, bits have been combined together into larger units in order to represent more characters or values.\",\n \"“Nibbles” are groups of four bits and “bytes” are groups of eight bits (i.e., two “nibbles”).\",\n \"Bytes are typically what are used to store “characters”.\",\n \"“Characters” typically include letters, numbers and/or symbols.\",\n \"For example, the ASCII code uses 128 different encoding combinations to represent: (i) The letters “A-Z” in both upper and lower case (i.e., values 65-90 and 97-122, respectively); (ii) Special characters (e.g., “<”, “.”, “%”, “$” “?”, etc) (e.g., values 33-47); (iii) The numbers 0 - 9 (i.e., values 48-57); and (iv) Certain control codes used for device control (i.e., values 0-31).\",\n \"To represent more than 128 characters, an eighth bit can be utilized.\",\n \"However, the ability to store a binary code of only “0's” and “1's” limits the ability for information to be miniaturized.\",\n \"For example, to store a single character from the ASCII code, at least seven bits of memory (typically eight bits are currently required because the last bit is usually set to zero) are needed.\",\n \"Table 2 sets forth one example of how a typical computer memory system would store the word “Patent”.\",\n \"TABLE 2 ASCII LET- CHAR- BINARY HEXADECIMEL BITS OF TER ACTER NUMBER DIGIT OR BYTE MEMORY P = 80 = 01010000 50 8 a = 97 = 01100001 61 8 t = 116 = 01110100 74 8 e = 101 = 01100101 65 8 n = 110 = 01101110 6E 8 t = 116 = 01110100 74 8 Thus, the letter “P” corresponds to the ASCII character 80 , which is represented by the binary code “01010000”.\",\n \"The first “0” in the sequence is often referred to as “the last bit” and is typically set to “0” for all ASCII characters.\",\n \"The corresponding hexidecimal digit or byte representation is “50”.\",\n \"In order for the computer memory system to store the letter “P”, eight bits must be sent to memory in the order “0-1-0-1-0-0-0-0”.\",\n \"Likewise, to store the letter “a”, eight bits must be sent to memory in the order “0-1-1-0-0-0-0-1”.\",\n \"Accordingly, 48 bits are used to store the six (6) letter word “Patent”.\",\n \"Similarly, when a microprocessor unit accesses the portion of memory containing the set of bits representing the word “Patent”, the same number of bits (i.e., 48) or six (6) bytes must be processed by the microprocessor.\",\n \"Clearly, if the word “Patent” could use a smaller amount of memory, less memory capacity would be needed in a computer and/or the rate at which data could be stored and/or retrieved from memory would be enhanced.\",\n \"In a first aspect of the invention, a coding system that is a higher order system than a binary coding system is utilized for the digitized storage of information.\",\n \"For example, any digital coding system which uses three or more different code characters (i.e., a coding system which operates in “base-3” or greater) will result in a reduction in the number of bits needed to store the same amount of digital information relative to a binary system.\",\n \"Thus, by using a higher order memory system, the possibility of storing more digitized information in a smaller memory area exists.\",\n \"Likewise, the rate of storing and/or retrieving digitized information should also increase due to memory being stored in a physically smaller area.\",\n \"This permits a data retrieval device to function more efficiently.\",\n \"The present invention permits the use of a digitized memory system which is base- 3 or greater.\",\n \"Moreover, as more code characters are used (e.g., higher order systems of more than 10 or more than 20 different characters are utilized) the digitized system begins to behave in a manner which is similar to analog systems and can thus be referred to as “analog-like”.\",\n \"In other words, as the amount of digital sampling of an analog source increases, the recorded digital signal begins to approach the storage of most features of the analog signal.\",\n \"However, current data storage/retrieval techniques utilize, for example, 8 or 16 bits to store a single piece of analog information, which limits the amount of sampling of an analog source that can be reasonably performed when creating a digital representation of the analog source.\",\n \"Accordingly, the digital data storage/retrieval techniques of the present invention can be vastly superior to current digital techniques which utilize only “0's” and “1's” and are limited to sampling much smaller areas of analog sources than would be desirable in many cases (e.g., for complex computer modeling of numerous events, etc.).\",\n \"A first embodiment of an alternative memory storage system of the present invention may function in a manner somewhat analogous to the manner in which a Compact Disk (CD) or Digital Versatile Disk (DVD) player functions.\",\n \"Current CD and DVD technologies utilize a rotating disk comprised of a number of layers of different materials.\",\n \"The layers may include, for example, polycarbonate plastic, aluminum and acrylic.\",\n \"The aluminum in the disk includes a series of plateaus and valleys sometimes referred to as “bumps” and “pits”, respectively.\",\n \"Laser light is caused to focus upon the bumps (plateaus) and pits (valleys) in the rotating disk.\",\n \"The reflection of the laser from the successive plateaus and valleys corresponds to a data stream (e.g., a binary stream) of information.\",\n \"CD's and DVD's use, for example, 16-bit strings of binary coded data.\",\n \"Specifically, laser light reflects differently from a valley compared to the reflection from a plateau and these different reflective properties of the laser, when reflected from these valleys and plateaus, correspond to the binary code data values of “0” and “1” for each bit.\",\n \"In one embodiment of the present invention, a similar concept to the CD and DVD is utilized, however, rather than utilizing valleys and plateaus (i.e., bumps and pits) in an aluminum layer embedded in a polycarbonate plastic CD or DVD, the present invention utilizes at least two data values per bit represented by different intensity, colors or color width (e.g., different wavelength or frequency).\",\n \"These data values can be optically created, stored in dots or spots, and optically examined or interrogated (e.g., by known transmission and/or reflection techniques).\",\n \"These optical dots or spots may be of a size which is approximately the same order of magnitude in size as the bumps and pits which comprise current data tracks used on CD's and DVD's, they may be larger, or they may be smaller.\",\n \"In particular, similar to current CD's and DVD's, these dots or spots can be put together to form strings or tracks of data which begin at an innermost portion of a rotatable CD/DVD disk and extend spirally out therefrom in the form of tracks.\",\n \"Accordingly, current CD's of approximately 12 centimeters in diameter would be an acceptable size.\",\n \"However, if the dots or spots of the present invention are smaller than the “bumps” of the current technology, it is possible for CD's to be smaller in size, if desired, or more data can be placed into the same amount of memory space.\",\n \"Thus, it is possible to put an amount of optical information onto a rotatable disk of approximately 12 centimeters in diameter which exceeds that amount of information which is currently stored on CD's/DVD's.\",\n \"However, it is possible for the dots or spots to be larger in size than current bumps and pits in CD's/DVD's while still storing more information relative to standard CD's/DVD's.\",\n \"This is possible due to at least two effects.\",\n \"First, tracks may be capable of being located closer together due to, for example, lesser amounts of crosstalk between bits (e.g., recording and/or reading operations can take place in a lesser amount of space).\",\n \"Second, if digital codes higher than binary are adopted, less actual bits need to be stored relative to a binary system (e.g., rather than storing 8 or 16 bits to represent a single piece of information, a lesser number of bits can be used to represent the same piece of information that is being stored).\",\n \"It should be noted that as the number of characters that can be stored in a single bit become larger, the digital storage system actually approaches behaviors similar to an analog storage system and can be referred to as “analog-like”.\",\n \"In particular, the present invention is capable of utilizing a coding system other than the binary coding system of “0's” and “1's” now used in CD/DVD systems.\",\n \"For example, a series or range of dots or spots of different color intensities (e.g., white-gray-black), and/or different colors (e.g., red-yellow-violet), and/or both as in varied intensity of polychromatic light, can be placed upon a surface (or embedded somewhere within a material), such as a suitable photoferroelectric material such as “PLZT” (i.e., lead lanthanum zirconate titanate ceramic), which is shaped in the form of, for example, a disk.\",\n \"When, for example, such a disk containing these different intensity data value dots or spots is rotated and interrogated by, for example, a known optical system (e.g., a laser system) with a suitable focusing lens and intensity detecting means (e.g., a suitable photosensitive material or photodiode), the different intensities of the dots or spots can be registered and can be coded to correspond to different values (e.g., “0”, “1”, “2”, “3”, etc.).\",\n \"Thus, rather than using a simple binary or base- 2 system where a black dot corresponds to “0” and a white dot corresponds to “1”, the present invention provides for a larger number of options for digitized data storage.\",\n \"However, as discussed in more detail herein, depending on the sensitivity of the system, this technique could also be used for actual analog computing.\",\n \"Accordingly, for example, in a simple digitized quaternary system having a quaternary code consisting of 0's, 1's, 2's and 3's, there would be 4 different choices of basic digital information storage (i.e., a “base-4” system) rather than only two choices in a binary or base- 2 digital system.\",\n \"Likewise, in a digital system which utilizes 8 different combinations of spots or dots (i.e., a “base-8” system) eight different options exist for encoding a piece of information rather than only two options in a standard binary or base- 2 digital system.\",\n \"The use of a larger number of possibilities other than “0's” and “1's” (e.g., 0, 1, 2, 3, 4, 5, 6, 7, etc.)\",\n \"render it possible to store a much greater amount of information in a much smaller physical area due to the elimination of unneeded bit space.\",\n \"Moreover, as the number of possibilities other than “0's” and “1's” increases, (e.g., systems greater than base- 10 such as base- 20 or greater systems) the closer the digital system comes to being or performing like an analog system, which has certain advantages over digital systems.\",\n \"An apparatus according to the present invention permits suitable optical data to be stored onto an appropriately constructed and shaped optical recording medium by using, for example, a laser light source.\",\n \"The optical recording media (e.g., a PLZT ceramic) can be in the form of a tape, disk, or credit card or any other acceptable shape.\",\n \"Alternatively, a photodiode or photodiode assembly can also be used.\",\n \"In all such cases, a light source (e.g., laser light source) can be used in combination with, for example, various means for controlling the direction, polarization, intensity and/or final color of the light source.\",\n \"Various examples of such systems include the following: (1) a rotatable polarizing filter (or set of filters), sometimes referred to as an analyzer, used in combination with a suitable light source which is subjected to a first polarizing filter(s) selectively modifying the polarized light from the light source.\",\n \"Specifically, once monochromatic or polychromatic light has been polarized into a first direction polarizer the polarized light is directed toward a second polarizer (sometimes referred to as an “analyzer”) which, when rotated, results in, for example, different intensities of light being emitted therefrom.\",\n \"The different intensities of light can be caused to be incident upon an appropriate recording/detecting means; (2) a light source (e.g., a laser light source), which is used in conjunction with a first polarizing filter is caused to be incident on a suitable light diffractor (e.g., a Bragg modulator) which can cause the polarized light from the light source to communicate with one or more of a series of additional polarizing filters (or analyzers) each of which analyzers are rotated, relative to the polarized source, at different angles, thus resulting an different amounts or intensities of light passing through each of the analyzers.\",\n \"Thus, by selectively transmitting the polarized light through the different analyzers (e.g., directed by, for example, the Bragg modulator) different intensities of light can be achieved.\",\n \"Such different intensities can be recorded and/or detected by a suitable means; (3) a light source (e.g., a laser light source) used in combination with an acousto-optic Bragg modulator, whereby different driving signals are input to the Bragg modulator which results in different intensities of light being emitted from the Bragg modulator.\",\n \"The multiple intensity emissions are then directed to a suitable detecting/recording means and are made to correspond to various data points in base- 2 or higher order digital data storage systems; and (4) rather than utilizing polarizing filters to polarize a monochromatic or polychromatic source of light, liquid crystal cells can also be utilized.\",\n \"In particular, light can be caused to be incident upon a liquid crystal cell.\",\n \"Depending upon whether voltage is applied to the liquid crystal cell, the output from the liquid crystal cell is oriented into one direction or another.\",\n \"Liquid crystals of this type can be made of, for example, lithium niobate.\",\n \"However, similar to the embodiments (1)-(3) discussed above, intensities can be varied due to relative polarization directions.\",\n \"The outputs of light from the liquid crystal cell can be directed, by appropriate beam displacers (e.g., a Bragg modulator), toward a suitable recording/detecting means.\",\n \"In each of the examples (1)-(3) discussed above, it is possible that a polychromatic light source could be utilized.\",\n \"In this regard, when polychromatic light is made to be incident upon a first polarizer, and thereafter upon one or more additional polarizers (e.g., analyzers), in addition to different intensities of light flowing through the analyzer(s), a spreading of these different intensities over a particular frequency or wavelength range will also occur.\",\n \"Specifically, when using polychromatic light, a spectrum of frequencies of various intensities results in the formation of a somewhat continuous curve of varying light intensities over a particular frequency or wavelength range.\",\n \"The use of a polychromatic source can therefore result in secondary indicia for determining the value assigned to stored bits of data (e.g., a “0” or a “1”) and thus enhances data storage.\",\n \"For example, not only could the highest intensity of light be recorded/detected, but also the starting and/or ending points which correspond to the lowest frequency intensity of light being emitted, and the highest frequency of light being emitted, respectively, or, alternatively, the area under each curve could also be compared.\",\n \"In other words, more than one piece of information can be checked to verify the accuracy of each data point that is stored and read.\",\n \"The presence of more than a single piece of information can result in more accurate reading of data and thus result in signal/noise improvements.\",\n \"In each of the examples (1)-(4) discussed above, the different intensities or colors of light can be made to be incident upon an appropriate receiving/recording means (e.g., an optical storage means, such as a pohtopolymerisable/photocrosslinkable material, a photodiode assembly, etc.\",\n \"), such output light being suitably focussed onto the appropriate receiving/recording means by known lens or optical systems etc., all of which are discussed in greater detail herein).\",\n \"Suitable materials for recording various intensities or colors of light include the following materials: inorganic photochromic crystals, photorefractive materials, chalcogenide semiconductors, organic and polymer materials, thermoplastic media, reversible recording in Tellerium compounds, photothermal recording in Antimony compounds, magneto-optic recording and light stimulated recombination luminescence.\",\n \"For example, in the simplest embodiment (i.e., that embodiment numbered “(1)” above) for understanding the basic concept for recording various intensities of light (even though this simple embodiment is not as commercially significant as the other embodiments discussed herein), each such recorded intensity corresponds to a different value for a digitized data bit.\",\n \"In this basic case, different intensities can be achieved as follows: a polarized light source, such as a laser and polarized filter or polarizer, when used in combination with at least one rotatable polarizing filter (e.g., an analyzer) can result in light of different intensities being transmitted from the at least one rotatable analyzer.\",\n \"Specifically, when the laser light source is passed through a first polarizing filter and a second polarizer (e.g., an analyzer) is sequentially rotated (e.g., from 0-90°) a predetermined amount each time relative to the first polarizing filter (i.e., either the first or second filter can be rotated), different intensity dots or spots corresponding to data values having different meanings (e.g., “0”, “1”, “2”, etc.)\",\n \"can be produced.\",\n \"The amount of rotation of the analyzer in relation to the polarizing filter can correspond to a different intensity of light being recorded on a suitable optical recording medium (e.g., a photoferroelectric material such as “PLZT”, inorganic photochromic crystals, photorefractive materals, chalcogenide semiconductors, organic and polymer materials, thermoplastic media, reversible recording in Tellerium compounds, photothermal recording in Antimony compounds, magneto-optic recording and light stimulated recombination luminescence).\",\n \"If a photoferroelectric material is utilized, the different intensities of light can be caused to be incident upon the photoferroelectric material by use of, for example, a suitable optic lens system or a fiberoptic cable (or a series of cables) that is capable of accurately directing the optical signal to the photoferroelectric material for the proper recording of the various optical signals.\",\n \"Thus, a large number of different intensity spots or dots corresponding to data values for a single bit can be created.\",\n \"This basic system and apparatus permits higher order coding systems (e.g., base-3 or greater) to be utilized.\",\n \"The manner in which such optical data is stored, on, for example, a rotatable disk, as well as interrogated, can be by techniques which are substantially parallel, and/or use hardware which is similar, to those techniques and/or hardware currently used in CD and DVD systems.\",\n \"For example, a series of bits of an appropriate binary or higher order digital code system can be recorded onto a rotatable disk by starting to record the different intensity dots or spots near a central potion of the disk.\",\n \"The recording continues to record data in a spiral manner to form tracks of data, such data being suitably separated, physically, to avoid corruption and crosstalk, until an outermost radial portion of the disk is encountered.\",\n \"Likewise, such a disk can be read or interrogated by, for example, a laser system which focuses light onto the dots or spots and suitably measures the intensities (e.g., reflectance or transmittance) of the spots of data.\",\n \"The different intensities can be interpreted by suitable algorithms and programs to correspond to different values in the desired coding system.\",\n \"Moreover, by utilizing a digital optical coding technique other than binary, the present invention permits a much more rapid adoption of the desired “Unicode”, which takes into account, for example, all English language characters, Chinese characters, numbers and symbols, etc., in various digitized forms.\",\n \"In another embodiment of the invention, a system which utilizes magnetic memory in combination with a suitable retrieval/reading means is utilized.\",\n \"These memory/retrieval systems can be caused to function, generally, in a manner that is somewhat similar to current magnetic memory systems (e.g., magnetic tapes, cards, disks, hard drives, etc).\",\n \"In addition, these memory systems can also use digital coding/reading systems higher than a base- 2 system, if desired.\",\n \"Still further, these memory systems can also store/retrieve information in an analog or analog-like format.\",\n \"Specifically, it is another object of the invention to utilize certain aspects of the phenomena known as the Zeeman effects to result in a means for the storage and retrieval of information onto/from suitable magnetic media.\",\n \"The term “Zeeman effects”, as used herein, denotes the effects of external magnetic fields on frequencies of atoms, molecules and components thereof, in the broadest sense.\",\n \"Thus, as used herein, the term “Zeeman effect” includes not only what may be considered to be Zeeman effects in the more narrow, classical sense but also other magnetic effects such as magnetic quadropole effects, etc.\",\n \"Digital data may be stored as either a binary code or some higher order code that is greater than the binary or “base-2” code.\",\n \"Moreover, due to the apparent sensitivities of these data storage techniques, data may be stored/retrieved in an analog or analog-like manner due to the inherent operation of the Zeeman effects.\",\n \"Specifically, without wishing to be bound by any particular theory or explanation, it appears that use of the Zeeman effects can result in a somewhat continuous set of differing data values that can be stored/retrieved.\",\n \"The present invention utilizes one aspect of the Zeeman effects in the following general manner.\",\n \"A magnetic field is applied to a suitable media that is capable of receiving and storing at least a portion of the applied magnetic field.\",\n \"One example of such a suitable magnetic material is a ferromagnetic material that is capable of exhibiting different amounts of magnetic domain alignment as a function of increasing magnetic field strength up to its natural magnetic saturation point (i.e., whereby any further increases in applied magnetic field do not result in any measurable further alignment of magnetic domains).\",\n \"Various known materials exist that meet this basic criteria, including many materials currently utilized in the magnetic storage industry (e.g., gamma Fe 2 O 3 , gamma Fe 2 O 3 modified by Co, CrO 2 , etc.).\",\n \"The various materials can be held together by a variety of organic polymers including, for example, vinyl chloride, polyvinylchloride, methylacrylate, polymethylmethyacrylate, polyurethane, epoxy, polyamides, etc.\",\n \"Suitable substrates include typical polyesters for flexible materials and aluminum for rigid materials.\",\n \"The differing amounts of stored magnetic field can result in a corresponding splitting or shifting of the electromagnetic energy frequencies or spectral frequencies (e.g., fine spectrum splitting, hyperfine spectrum splitting, vibrational spectrum splitting, etc.)\",\n \"of one or more atoms or molecules of the magnetic material itself, or similar splitting or shifting in one or more materials that are located contiguous (e.g., in the magnetic material or in a read/write head) to the material containing the stored magnetic fields.\",\n \"In the Zeeman effect, the amount of splitting or shifting of one or more frequency spectrum(s) is a function of the strength of the applied/stored magnetic fields.\",\n \"Typically, the higher the magnetic field strength that is stored/applied (e.g., stored in a first material containing magnetic domains and thereafter applied to (e.g., influencing) a second contiguous material that exhibits Zeeman splitting at the stored magnetic field strengths), the further apart the splitting or shifting will be of one or more of the electromagnetic energy frequencies of a chosen spectrum of the targeted atom(s) or molecule(s).\",\n \"In this Zeeman effect, when a relatively wide splitting of electromagnetic energy frequencies occurs due to the applied magnetic field, the amount of splitting may correspond to frequencies in the microwave region; whereas a narrow splitting will typically correspond to lower frequencies (e.g., radio).\",\n \"The amount of splitting that occurs is important because in order to determine the amount of splitting that has occurred, a suitable interrogating electromagnetic energy is caused to be incident upon, for example, the magnetic material itself or the aforementioned contiguous material.\",\n \"The contiguous material that exhibits Zeeman splitting at the stored magnetic field can be present as, for example, one or more separate sheets of material that are located adjacent to, and/or contiguous with, the material that contains the stored magnetic field.\",\n \"When multiple contiguous materials are utilized, the present invention contemplates the use of a comparative analysis of the differences in Zeeman splitting between two or more such contiguous materials.\",\n \"Alternatively, a material comprising, for example, one or more material(s) that are mixed with the magnetic material (e.g., a binder which holds together at least a portion of the material that stores the magnetic field) could also be used.\",\n \"More specifically, a particular interrogating frequency, or set or sweep of frequencies, needs to be capable of favorably interacting with one or more of the split, shifted or splitting frequencies which result in the magnetic material, or in an alternative embodiment in one or more of the contiguous material(s), due to the different magnetic fields stored therein.\",\n \"In other words, a first magnetic material is desirably chosen so that an applied magnetic field can be reasonably stored therein.\",\n \"However, it is desirable for the magnetic material to be capable of storing reliably several magnetic fields of different strength (e.g., capable of having varying numbers of magnetic domains align with an ever changing magnetic field and such domains remain frozen or pinned so that they do not change with time) and, for example, in one embodiment of the invention, the contiguous material needs to produce split, shifted and/or splitting frequencies in response to the stored magnetic fields.\",\n \"In this regard, if one or more contiguous material(s) are utilized in connection with the magnetic material(s), the contiguous material(s) needs to exhibit a splitting and/or shifting of one or more frequencies, for example, of its rotational, vibrational, etc., bands (e.g., at the atomic level) and the resultant splitting or shifting that occurs in the contiguous material needs to be detectable by an appropriate interrogating electromagnetic source (discussed in greater detail later herein).\",\n \"Alternatively, a contiguous material does not need to be utilized.\",\n \"In this case, the magnetic material per se, or at least a portion thereof, needs to exhibit desirable and detectable splitting and/or shifting of one or more frequencies at varying stored magnetic strengths.\",\n \"Still further, it should be noted that the contiguous material could actually be located in a suitable read head which, similar to magnetoresistive and giant magnetoresistive heads, is influenced by the magnetic field strengths stored in the magnetic media.\",\n \"However, rather than measuring changes in resistivity in materials comprising the read heads, the splitting or shifting of frequencies will be detected by a suitable technique, discussed elsewhere herein.\",\n \"In this embodiment of the invention, the contiguous material may be a solid, a liquid, a gas or plasma (e.g., any material that shows desirable Zeeman splitting effects).\",\n \"With specific regard to interrogating with an applied electromagnetic source to determine the amount of Zeeman splitting and/or shifting that has occurred at a spot on the material that corresponds to a stored bit of data, at least one suitable frequency can be applied as an interrogating frequency, or, alternatively a sweep or combination of frequencies can be applied as interrogating frequencies.\",\n \"In this regard, if a single suitable frequency is chosen as the interrogating frequency, then heterodyne interactions of the various split, shifted and/or splitting frequencies with the single interrogating frequency can be measured and determined.\",\n \"Alternatively, resonance interactions of the various split, shifted and/or splitting frequencies can also be measured and determined.\",\n \"Each such determined heterodyned and/or resonant frequency can be made to correspond, by suitable programming or algorithmic techniques, to various data values.\",\n \"Alternatively, a series or sweep of frequencies which may resonate with or heterodyne with various split, shifted and/or splitting frequencies achieved in the contiguous material can also be utilized.\",\n \"These particular affects will be discussed in much greater detail later herein, however, it should be understood that these data acquisition techniques could be performed using hardware which is somewhat similar to that hardware currently used for magnetoresistive and giant magnetoresistive heads.\",\n \"For example, once an interrogating frequency has been input to a material exhibiting Zeeman splitting and/or shifting, the output (e.g., a heterodyned frequency) can be used as a driving signal or input (e.g., depending on strength or frequency either amplified or unamplified) for various other devices.\",\n \"One example of such other device is an acousto-optic Bragg modulator.\",\n \"In this detection scheme, an appropriate light source is caused to be incident upon an appropriate Bragg modulator.\",\n \"Suitable materials for a Bragg modulator include, for example, lithium niobate crystals and gallium arsenide semiconductor materials.\",\n \"The incident light beam can be caused to be deflected in differing amounts due to different driving signal inputs (e.g., depending on strength or frequency either amplified or unamplified) being directed into the Bragg modulator.\",\n \"In particular, for example, the different heterodyne outputs that correspond to different Zeeman splitting amounts (and thus different data points such as a “0” or a “1”) can be input into a Bragg modulator and cause light which is incident on the Bragg modulator to be deflected in different amounts.\",\n \"Thus, the various Zeeman splitting and/or shifting frequencies that are produced can eventually result in different amounts of light deflection due to the use of a Bragg modulator.\",\n \"The amount of deflection of light can be observed by the use of, for example, the light detection devices discussed earlier herein.\",\n \"Similarly, the different heterodyne outputs could be directed into a suitable filter which, due to the different inputs, would regulate the intensity of light passing therethrough.\",\n \"Accordingly, the different intensities of light could then be detected/recorded by a suitable means discussed elsewhere herein.\",\n \"This particular embodiment of the invention which utilizes differing magnetic fields to result in differing amounts of an appropriate frequency splitting and/or shifting is very suitable to be used for the analog storage/retrieval of information.\",\n \"In particular, a substantial continuum of splitting and shifting frequencies results in most materials that are subjected to a corresponding continuum of changing magnetic field strengths.\",\n \"If a continuum of such differing split, shifted and/or splitting frequencies can be reliably detected, then analog or analog-like (e.g., higher order than binary systems) computing with this technique is facilitated.\",\n \"Additionally, many of the aforementioned embodiments of the invention can also be used in combination with various holographic and/or three dimensional memory approaches.\",\n \"To achieve all the foregoing objects and advantages, and to overcome the looming problem in the art of an impending memory barrier, the present invention discloses various means for storing and retrieving information in a digital and/or analog format.\",\n \"With regard to the digital storage of information, a code other than a binary code for the storage of information can be utilized.\",\n \"The result of using a higher order code for digital information storage (i.e., 3 or more characters) is the ability to store more information in the same amount of area or the same amount of information in a smaller area.\",\n \"Accordingly, the present invention provides significant advancement in the art of data storage and/or data retrieval which will assist in preventing the memory barrier from being realized.\"\n ],\n [\n \"TECHNICAL FIELD The present invention relates generally to the storage and/or retrieval of information on magnetic and/or optical storage media by using one or more novel approaches alone or in combination.\",\n \"These novel approaches are capable of using at least one code which may comprise more than two values (i.e., more than a “0” and a “1”).\",\n \"A first series of approaches for the storage of information applies generally to existing optical storage/retrieval systems (e.g., CD's, DVD's, etc.)\",\n \"as well as novel optical systems; while a second series of approaches applies generally to existing electric and/or magnetic storage/retrieval systems (e.g., magnetic, magneto-optic, etc.)\",\n \"as well as other novel electrical/magnetic systems.\",\n \"Each series of approaches is capable of storing information in one or more codes, wherein such approaches permit, if desired, the use of at least one higher order code which is different from the traditional binary code of “0's” and “1's” currently utilized for the storage of digital information.\",\n \"Said at least one higher order code may comprise three or more optical and/or magnetic values or bits that are used to represent, for example, ASCII or Unicode characters that are currently represented predominately by the traditional binary code.\",\n \"This higher order code may also be an analog or analog-like code.\",\n \"BACKGROUND OF THE INVENTION Data storage devices have been becoming smaller in size (as well as faster) due to, for example, the continued improvement in the ability to store more information in smaller spaces.\",\n \"For example, over the past twenty years the ability to place more data on magnetic storage media such as magnetized hard-disk drives and/or floppy disks, as well as on optical media (e.g., compact disks “CD's” and more recently, Digital Versatile Disk “DVD's”) has been increasing at a phenomenal pace.\",\n \"The impressive increase in the ability to store more data in smaller areas has been one of the main driving forces for rendering computers, and other electronic devices with reasonably sized memories, accessible to a typical home or small business consumer.\",\n \"Further, the high operating speeds of microprocessors have required stored data in computers to be rapidly accessible (e.g., both the rate at which information is delivered to a microprocessor as well as the amount of time it takes between the time that a microprocessor requests a file and when the first piece of data begins to flow from the file to the microprocessor are important).\",\n \"Magnetic memory has undergone many recent improvements and has been utilized extensively in data storage/retrieval devices because magnetic memory currently provides the least expensive alternative for the fast writing and reading capabilities for data.\",\n \"However, the various currently utilized magnetic technologies face a variety of technical challenges and appear to be headed for a stopping point.\",\n \"Specifically, these various approaches for the storage and/or retrieval of information may no longer be capable of meeting the growing demands of the industry, without experiencing significant technical enhancements.\",\n \"For applications where speed has historically been less important, various optical technologies have been growing in popularity.\",\n \"Many technical advances have been made in the recording and/or retrieval of information by optical means.\",\n \"In systems which use compact disks (“CD's”), compact disk read-only memory (“CD-ROM's”) and/or Direct Video Disks (“DVD's”), the storage and/or retrieval of information has also been of great interest.\",\n \"For example, the first CD's manufactured were CD-ROM's that are currently capable of storing about 650 MB of data.\",\n \"This equates to about 74 minutes of play-time for music that has been recorded in a typical recording mode.\",\n \"In particular, the standard current approach for utilizing CD-ROM's, includes the use of a solid state interrogating laser (or in some cases more than one laser) which has a wavelength of about 780 nanometers (0.78 μm).\",\n \"The standard CD-ROM is about 12 cm in total diameter and has a thickness of about 1.2 mm.\",\n \"The standard CD-ROM has information recorded on plateaus (“bumps”) and valleys (“pits”) that spiral from the inside of the disk toward the outside of the disk.\",\n \"The digital data stored in the tracks that comprise the spirals must be separated by enough space (e.g., tracks are typically separated radially by about 1.6 μm) so that when the data is read with an interrogating solid state laser, crosstalk from adjacent tracks does not occur (i.e., recorded information is received from only one track at a time).\",\n \"Accordingly, the placement of more information onto a typical CD has been limited by the required size (e.g., length and width) of the plateaus (“bumps”) and valleys (“pits”).\",\n \"The size of the plateaus and valleys (e.g., typically not less than 830 nanometers in length) has been a function of various factors, including the wavelength of the laser utilized to read the information from the CD.\",\n \"Various attempts have been made to increase the memory capacity of current CD's, including the stacking of a plurality of disks one on top of the other so as to provide more memory space, as well as other attempts to miniaturize the digitized information stored in the tracks.\",\n \"The most recent significant improvements in this type of optical storage/retrieval technology have been the creation of the DVD, which is known as the “Direct Video Disk” or the “Digital Versatile Disk”.\",\n \"The first commercial products of DVD's entered the marketplace in late 1997.The DVD technology is similar to the technology in CD's, except that, for example, the wavelength of lasers utilized to read information from DVD's is smaller than that used for CD's, namely, about 635 nanometers or 650 nanometers, compared to about 780 nanometers for CD's.\",\n \"The size of the plateaus (“bumps”) that make up the DVD are about 320 nanometers wide, a minimum of about 400 nanometers long and about 120 nanometers thick.\",\n \"About 740 nanometers separate one track from the next, compared to the about 1600 nanometer separation of tracks in CD's.\",\n \"A standard DVD is physically about the same size as a CD, but has a memory capacity that is about seven times greater than that of CD's.\",\n \"In particular, the memory capacity of a one-sided, one-track, DVD is now about 4.7 GB.\",\n \"However, recent improvements in DVD have allowed digital information to be placed on both sides of a single track, as well as both sides of a double track.\",\n \"In this regard, a first interrogating laser reads the digitized data on a first side of a first track (i.e., in the form of bits arranged in a spiral track that begin in the innermost portion of the DVD and spirals outward).\",\n \"Then, information can be retrieved from the opposite side of the first track in a similar manner from a second laser; however, the spiral track of information typically begins on the outermost portion of the disk and spirals inward toward the center.\",\n \"Accordingly, when dual-track, dual-sided DVD disks are used, the current amount of memory that can be achieved is about 17 GB.\",\n \"Another optical data storage technique which shows promise is known as holographic memory.\",\n \"The potential for high storage capacity and high storage retrieval rates using the holographic technique is tremendous.\",\n \"In particular, for example, recent work in a system known as Holographic Data Storage System (“HDSS”), has shown that data can be accessed in 100 microseconds or less, which is several orders of magnitude faster than the millisecond retrieval of data in typical magnetic-disk drives.\",\n \"Briefly, in this technology, an optical-interference pattern is created within a desired photosensitive material (e.g., a crystal or a polymer).\",\n \"The optical interference pattern is imprinted as a result of physical changes in such materials.\",\n \"The interference pattern is created by the interaction between an object beam and a reference beam, both of which typically originate from a single light source (e.g., a laser), but are split into two different pathways by a beam splitter.\",\n \"Specifically, the object beam is incident on a target object that contains data, and modifies the object beam, while the reference beam, taking a separate path which leaves it unmodified, interferes with the object beam at the holographic storage medium.\",\n \"When the two beams meet at the holographic storage means, the interference pattern which results from their interaction is stored on or in the holographic storage means.\",\n \"The holographic storage means is later interrogated by a reading or reference beam, which upon interacting with the holographically stored interference pattern, can then recreate the holographic image containing the data originally stored in the object beam.\",\n \"While the holographic optical technique shows great promise for the future, current industrial emphasis is being placed primarily on improving existing digital optical and magnetic storage/retrieval systems so that more information can be stored in smaller spaces, and retrieval times can be decreased without significantly changing the basic methodology and hardware for the storing and retrieving of information.\",\n \"With regard to placing more data onto magnetic recording media, effects such as the paramagnetic effect (e.g., magnetic crosstalk between magnetic domains comprising bits) are already beginning to cause problems.\",\n \"Experts have postulated that in coming years, magnetic storage technologies could reach a limit imposed by the superparamagnetic effect (“SPE”).\",\n \"The physical phenomenon known as SPE can occur in a magnetic data storage system when the magnetic spins of electrons in the domains which comprise a bit (e.g., in current technology typically either a “0” or a “1”) become unstable due to, for example, the environmental thermal energy surrounding the bits.\",\n \"Instability of bits occurs when smaller and smaller numbers of domains or atoms are used to comprise a bit and the ability for the domains or atoms to maintain their given spin directions (e.g., corresponding to, for example, a “0” or a “1”) becomes difficult.\",\n \"Non-maintenance of spin directions results in a “flipping” of spins back and forth and thus changing “0's” to “1's” and “1's” to “0's”, etc.\",\n \"Such changing or flipping of spins will result in a corruption of the data or information that the “0's” and “1's” represent.\",\n \"Accordingly, in order for smaller numbers of atoms to be used to form a bit and in order for the bit to be viable, techniques and/or materials for preventing the superparamagnetic effect need to be developed to overcome the deficiencies of the prior art.\",\n \"Another potential problem in recording information magnetically is that the write head which is used to align magnetic domains in the magnetic storage media may be limited in the ability to impart a required field strength over the entire domain area comprising a bit to cause the magnetic domains to behave as though they have been exposed to a substantially uniform field.\",\n \"In such cases, it is possible that differently oriented domains within a bit (e.g., differently oriented due to being subjected to different magnetic field strengths within a single writing head due to, for example, non-uniformity of the magnetic flux lines emanating from the write head and/or differently oriented because some domains require more energy than other domains to align due to, for example, their location within a bit) may have a higher tendency to become corrupted, as well as adding to signal/noise ratio problems.\",\n \"One attempt for miniaturizing domains involves the use of so-called “hard” magnetic materials.\",\n \"The use of hard magnetic materials is desirable when compared to “soft” magnetic materials because hard magnetic materials are more likely to maintain their magnetic spin directions when domains comprising the hard magnetic materials are closely spaced.\",\n \"In this regard, various rare-earth and transition elements have been found to be magnetically stable and are thus termed “hard”.\",\n \"Such magnetically stable materials are known to have a higher coercivity, which is typically represented by “Hc”.\",\n \"Stated in an over simplified manner, the higher the Hc value, the greater the resistance of the material to outside influences, such as outside magnetic fields (e.g., the harder it is for material to lose its imprinted spin direction due to an increase in temperature and/or the influence of other magnetic fields, etc.).\",\n \"However, one drawback for utilizing “hard” magnetic materials is that hard magnetic materials are more difficult to magnetize than soft magnetic materials (e.g., in some cases, depending on the particular materials chosen, much more difficult to magnetize).\",\n \"This makes it more difficult to record data initially in hard magnetic materials.\",\n \"There are various known approaches for solving the problem of encoding data into hard magnetic materials which approaches utilize a laser in conjunction with an appropriate magnetic write head.\",\n \"These various approaches have been referred to generally as the “magneto-optical” approach.\",\n \"These magneto-optical approaches effectively reduce the Hc value of the magnetic domains by causing the domains to be locally heated just prior to being subjected to the magnetic write head.\",\n \"Thus, the coercivity (Hc) of the domains is effectively reduced and when such effective reduction in Hc has occurred, a somewhat standard write head, or in some cases a very different write head, can be used to alter favorably the magnetic domains.\",\n \"In other words, the use of a laser, which provides thermal energy to the domains of the hard magnetic material, causes the hard magnetic material to behave in a manner which is similar to a somewhat softer and thus easier to magnetize magnetic material.\",\n \"Each of the known approaches for utilizing the magneto-optic techniques involves the use of at least one laser and/or at least one laser focusing system.\",\n \"However, one of the common problems facing these magneto-optical approaches is that the lasers that are utilized to soften the hard magnetic material prior to subjecting the hard magnetic material to a magnetic field is that the lasers typically generate excess amounts of heat that can flow to neighboring bits or tracks (e.g., thermal energy is caused to migrate to undesirable neighboring tracks or areas resulting in a potential corruption of stored information).\",\n \"Accordingly, various laser applications and/or storage solutions have been devised to minimize the transfer of heat from the laser to neighboring tracks, and thus minimize the loss of recorded information.\",\n \"However, to date this thermal transfer of laser energy still presents problems to varying degrees in all the prior art approaches.\",\n \"One interesting technique for the reading of data in connection with one optical-magnetic system is the use of an effect known as the “Kerr effect”, and more particularly, the “magneto-optical Kerr effect”.\",\n \"Briefly, current uses of the magneto-optical Kerr effect involve incident polarized light being reflected from the surface of a magnetic domain in different ways, depending on the orientation of the magnetic domains (e.g., depending on electron spin directions).\",\n \"The changes in the reflected polarized light are made, by algorithms, to correspond to different digitized data.\",\n \"Specifically, when the incident polarized light encounters different magnetic field orientations (e.g., north or south domains comprising a bit which are ordered to represent either a “0” or a “1”) the polarization state of the reflected light is changed.\",\n \"Another approach to the digital storage of information that shows future promise is known as atomic resolution storage (“ARS”).\",\n \"This approach, similar to holographic techniques, is capable of storing tremendous amounts of information in a small amount of space.\",\n \"In this technique, generally, very small electron probes, which are used to generate electron beams, are formed into an array.\",\n \"The electron probes have tips that are roughly the size of atoms.\",\n \"The electron beams from the electron probes are made to be incident upon a storage medium so that the incident beams cause some sort of physical change in the storage medium (i.e., the medium comprising the computer memory).\",\n \"One example of such a storage medium is a material that is capable of containing at least two stable phases at ambient operating conditions so that an incident electron beam emitted from an electron probe changes the storage material from one phase to the other.\",\n \"In this technique, encoded bits of information are represented by the change in phase of the phase-change storage material.\",\n \"This technique, similar to the magneto-optical technique discussed above, also suffers from the problem of heat flowing between data spots created by the incident electron beams when the spots or bits are initially recorded.\",\n \"Accordingly, one of the challenges faced by this relatively new technology includes reducing the flow of heat between data spots which are created by the array of electron beam generators.\",\n \"The retrieval of stored information is also a challenge.\",\n \"In this regard, as bits become smaller and include lesser numbers of magnetic domains per bit and/or the domains are oriented differently, (e.g., domains are aligned perpendicular to the substrate containing at least one magnetic surface thereon instead of being parallel to the substrate surface) such bits are capable of being packed more tightly together.\",\n \"Assuming that the bits are not corrupted by their tight packing and/or smaller numbers of magnetic domains and/or non-uniform alignment within a bit, then reading of these tighter packed domains comprising the bits also becomes a challenge which the prior art is still struggling to overcome.\",\n \"Historically, large numbers of magnetic domains were present in each bit which contained digital data.\",\n \"Thus, the resulting or induced magnetic fields were relatively large and various forms of known inductive reading techniques were sufficient to ascertain whether the hundreds or thousands of aligned domains in a bit corresponded to a “0” or a “1”.\",\n \"However, as prior art miniaturization recording techniques progressed, the known inductive reading techniques were typically not sensitive enough to determine accurately the alignment of ever decreasing numbers of domains comprising a bit (e.g., signal to noise problems in the storage and/or retrieval of digital data bits were exacerbated).\",\n \"The development of magnetoresistive heads, and the subsequent development of giant magnetoresistive heads, has enabled smaller numbers of domains comprising a digital bit to be read accurately.\",\n \"In both of magnetoresistive and giant magnetoresistive heads, the electrical resistance of one or more materials comprising the read head(s) is examined.\",\n \"In particular, both magnetoresistive and giant magnetoresistive heads are made from materials that exhibit small changes in their electrical resistance as a function of magnetic fields created by oriented domains to which such heads are exposed.\",\n \"Giant magnetoresistive heads are two or three times more sensitive than magnetoresistive heads due to their novel structure which includes layering of different materials stacked on top of each other.\",\n \"One problem that continues in these technologies is the presence of magnetization vortices in the recorded domains.\",\n \"Such vortices may result from the difficulty in achieving uniform domain alignment within a bit due to, for example, the difficulty in applying a required magnetic field (e.g., uniform field) to the domains during the recording or writing process.\",\n \"While use of the magneto-optical Kerr effect and new read heads such as magnetoresistive heads and giant magnetoresistive heads have improved digital data retrieval techniques, further miniaturization is pushing the limits of these systems.\",\n \"Accordingly, additional improvements are necessary in the ability for hardware, combined with suitable programming, to detect even smaller numbers and/or smaller sizes of domains comprising digitized (or analog) data bits.\",\n \"Thus, additional innovations in the detection of stored data are required.\",\n \"In addition to the storage of information digitally, the possibility or promise of storing information in an analog manner continues to intrigue various investigators.\",\n \"While certain improvements have been made in, for example, the processing of analog information (e.g., the advent of VLSI (Very Large Scale Interrogation) chips, the techniques for the storing of analog information need much improvement.\",\n \"Particularly, while analog computing has been known for several decades, an accurate and fast-paced system for the storage and retrieval of information has continued to elude researchers.\",\n \"In this regard, digital computing has received the greatest amount of attention because of it's relative dependability for the storing and retrieving of information in a “1” or a “0” digitized format.\",\n \"Briefly, digital approaches typically include storing a sampling of the analog information that is to be recorded.\",\n \"The number of samples taken (e.g., such sampling typically occurs in predetermined units of space or time) from the analog source will correspond to the accuracy between the original analog image or sound and the stored digital data (i.e., the more digital samples that are recorded of an original analog image or sound, the more accurate the reproduction of the original analog image or sound will be).\",\n \"These techniques of predetermined quantum amounts of sampling of analog images have been adequate for most digitized applications, however, as the need for more accurate recording and retrieval of information occurs (e.g., in, for example, the fields of computing or determining various engineering and scientific relationships in the following representative areas: gravitational; electrostatic; magnetic; thermal; stress; fluid flow field analysis; wave propagation; image processing; etc.\",\n \"), the availability of computing with more accurate data becomes important.\",\n \"However, the digitization of tighter and and/or smaller sampling amounts from an analog source can result in tremendous amounts of digitized data being stored to represent a small actual amount of analog information.\",\n \"This is one area where analog computing has a distinct advantage compared to digital computing.\",\n \"In particular, an analog computer will store only a single piece of information corresponding to a single sampling area, whereas a digital computer will need to store somewhere between, for example, 8 and 16 bits of data to represent the same single piece of information stored in an analog fashion.\",\n \"The large number of bits required to store digital information corresponds to the requirement to store information in an ASCII or Unicode format.\",\n \"However, in order to escape from the current ASCII or Unicode format, analog storage requires a very large number of different data bits (e.g., data corresponding to much greater numbers than merely “0s” and “1's”) to be stored and retrieved.\",\n \"Current prior art techniques have great difficulty in storing and/or reading bits corresponding to anything other than “0's” and “1's”, let alone the attendant problems of storing and/or reading, for example, a near infinite number of possibilities (e.g., 1, 2, 3 .\",\n \".\",\n \".\",\n \"n+1).\",\n \"However, when performing complex calculations, the digital process may result in very slow processing times as well as requiring very large memories.\",\n \"It is for this, as well as other reasons, that analog computing is still very attractive.\",\n \"Thus, while advances in the processing of analog information have been made, reliable techniques for the storage and retrieval of analog information are still being sought without any good solutions currently existing.\",\n \"As stated above, with regard to digital computing, much of the recent technical emphasis in data storage and retrieval for both optical and magnetic memory enhancement has been on shrinking more information into smaller areas.\",\n \"The ability to place bits, which are the smallest elements of information used by current digital memory systems, into a binary code format, which is a combination of “0's” or “1's”, into a smaller area on, for example, magnetic and/or optical disk storage media (e.g., a disk or a tape) may soon reach a limit.\",\n \"Thus, current technologies for writing and/or reading binary data onto and/or from current storage media will be pushed to the maximum limit.\",\n \"Computer designers are already facing a performance gap whereby processors can process information faster than the information can be retrieved from computer memory.\",\n \"The majority of the currently proposed approaches for the digital storage of information, suffer from the same problem, namely, that the magnetic and/or optical information which is stored uses a binary code (i.e., a base-2 number system) which is a combination of “0's” and “1's”.\",\n \"The predominance of the use of a “binary” code has been due to, for example, difficulty in distinguishing signals (e.g., a “0” or a “1”) from background noise.\",\n \"In this regard, many techniques have been proposed which use various hardware, as well as algorithms, which attempt to read accurately the stored information as either a “0” or a “1”.\",\n \"However, certain prior art techniques have been postulated for utilizing codes other than binary.\",\n \"In particular, U.S. Pat.\",\n \"No.\",\n \"6,154,432, entitled “Optical Storage System” discloses a holographic system which records information in very small spots and postulates that 10 or more digits may be possible to record on a single bit.\",\n \"Accordingly, this holographic technique discloses codes higher order than binary.\",\n \"Further, for example U.S. Pat.\",\n \"No.\",\n \"5,450,363, entitled “Gray Coding for a Multi Level Cell Memory System” discloses the use of a so-called “gray code” approach (also known as “gray scale” by others) for data storage.\",\n \"This approach also uses a system which is higher order than binary.\",\n \"In particular, reference is made to the storage of multiple bits of information in a single memory cell thus creating a multi-level cell.\",\n \"Still further, U.S. Pat.\",\n \"No.\",\n \"6,061,005, entitled “Encoding of Data”, discloses a method of encoding data on magnetic cards which uses symbology other than binary, such as trinary, quaternary or even higher symbology, for the recording of information.\",\n \"However, while various techniques have been postulated for systems which use codes higher than binary, none of such codes have been commercially adopted due to, for example, signal to noise ratio problems, etc.\",\n \"In particular, reading errors can occur due to lack of contrast between data values.\",\n \"Such lack of contrast can be caused by random noise (e.g., vibrating inaccurate positioning of recording/reading heads, fidelity loss during recording, etc.)\",\n \"and determinate variations.\",\n \"Accordingly, a need exists for techniques which can be commercially viable which can use codes reliably that are higher order than traditional binary codes for the storage and/or retrieval of information.\",\n \"A very particular combination of “0's” and “1's” is known as the American Standard Code for Information Exchange (i.e., the ASCII code) which assigns a unique code number between 0 and 127 to each of the 128, 7-bit, binary number characters.\",\n \"Table 1 below shows in table form the current ASCII characters arranged by Hexidecimal digits (i.e., 128 different encoding combinations of groups of seven bits).\",\n \"Reading within the Table from left to right and then down, the first 32 values are codes which correspond to various computer control functions such as line feed, carriage return, etc.\",\n \"The 33rd value corresponds to the “space” character.\",\n \"Values 34-48 correspond to symbols and punctuation marks.\",\n \"Values 49-58 correspond to digits, and so on, until reaching value 128, which corresponds to “delete”.\",\n \"In addition, each of these values also has a corresponding hexidecimal character.\",\n \"For example, the hexidecimal digit corresponding to the “space” character is “20”, while the hexidecimal digit corresponding to the letter “n” is “6E”, and so on.\",\n \"TABLE 1 00 01 02 03 04 05 06 07 08 09 0A 0B 0C 0D 0E 0F 00 NUL SOH STX ETX EOT ENQ ACK BEL BS TAB LF VT FF CR SO SI 10 DLE DC1 DC2 DC3 DC4 NAK SYN ETB CAN EM SUB ESC FS GS RS US 20 !\",\n \"“ # $ % & ‘ ( ) * + , − .\",\n \"/ 30 0 1 2 3 4 5 6 7 8 9 : ; < = > ?\",\n \"40 @ A B C D E F G H I J K L M N O 50 P Q R S T U V W X Y Z [ \\\\ ] {circumflex over ( )} — 60 ’ a b c d e f g h x j k l m n o 70 p q r s t u v w x y z { | } ˜ DEL These 128 characters are enough characters for North American English to be stored and/or retrieved, but are not enough for many other languages (Note: In ASCII, the eighth bit is used, and is thus normally set to “0”).\",\n \"To represent more than 128 characters, 8-bit (rather than 7-bit) binary numbers are utilized.\",\n \"For each digit in these eight bits there are two choices for the digit, either “0” or “1”.\",\n \"This results in a possibility of an additional 128 characters or a total of 256 different combinations of “0's” and “1's” (i.e., 28).\",\n \"Accordingly, the current data storage approaches utilize these 256 combinations of “0's” and “1's”.\",\n \"However, these 8-bit numbers still do not provide enough room for all characters that need to be used in the world (e.g., certain Asian languages have thousands of characters each).\",\n \"In addition, there is no universal agreement in the world on what all the characters should be, whether referring to the 128 characters, or the 256 characters, or any number of characters beyond these.\",\n \"However, certain approaches for dealing with all the various languages in the world, which collectively contain many thousands of characters, are beginning to utilize something referred to as the “Unicode”, which uses a 16-bit character set.\",\n \"In order to adopt something similar to the Unicode, even greater strain will be placed on current memory techniques.\",\n \"Whether using an 8-bit, 16-bit, or some other approach, all such approaches have been limited primarily to using a binary system of data storage.\",\n \"However, due to: (1) the impending size barrier for the storage of information on, for example, magnetic and/or optical media; (2) the performance gap between how fast a processor can process information versus how fast a processor can access information; and (3) the goal for a universal adoption of a common set of characters throughout the world, a different approach for the storage of information, either by digital or analog approaches, is clearly needed.\",\n \"The present invention satisfies the current and future data storage needs by using different approaches for the storage and/or retrieval of data; and/or different codes that use a code higher than the current “base-2 code”.\",\n \"SUMMARY OF THE INVENTION It is an object of the invention to provide a means for storing/retrieving digitized information optically.\",\n \"It is an object of the invention to provide a means for storing/retrieving digitized information magnetically.\",\n \"It is an object of the invention to provide a means for storing/retrieving analog information optically.\",\n \"It is an object of the invention to provide a means for storing/retrieving analog information magnetically.\",\n \"It is an object of the invention to provide a storage/retrieval means for utilizing codes other than binary.\",\n \"It is an object of the present invention to provide an increased number of characters that can be stored as a single bit of information.\",\n \"It is an object of the invention to provide the ability to store the 128 ASCII-type characters in a much smaller space than the space that is used in a standard binary code of “0's” and “1's”.\",\n \"It is an object of the invention to provide a means for storing/retrieving codes other than binary.\",\n \"It is an object of the invention to utilize at least a base-3 optical storing/retrieving system.\",\n \"It is an object of the invention to utilize a least a base-4 coding storing/retrieving system.\",\n \"It is an object of the invention to utilize at least a base-6 coding storing/retrieving system.\",\n \"It is an object of the invention to utilize at least a base-8 coding storing/retrieving system.\",\n \"It is an object of the invention to utilize at least a base-10 coding storing/retrieving system.\",\n \"It is an object of the invention to utilize particular aspects of the Zeeman effect in connection with the storage/retrieval of digitized information.\",\n \"It is an object of the invention to utilize particular aspects of the Zeeman effect in connection with the storage/retrieval of analog information.\",\n \"It is an object of the invention to utilize different shades or colors of spots or dots in connection the optical storage of digitized information.\",\n \"It is an object of the current invention to utilize different shades or color or spots or dots in connection with the optical storage of analog information.\",\n \"It is an object of the invention to utilize either monochromatic or polychromatic light in connection with the optical storage of digital and analog information.\",\n \"It is an object of the invention to utilize an analog-like approach to the storage of data.\",\n \"The current process of storing bits, which is the smallest element of information used by current memory systems, uses a binary format of only “0's” and “1's”.\",\n \"A bit which is “off” is considered to be “false” or “not set”, while a bit that is “on” is considered to be “true” or “set”.\",\n \"Historically, because bits could only be one of two values, bits have been combined together into larger units in order to represent more characters or values.\",\n \"“Nibbles” are groups of four bits and “bytes” are groups of eight bits (i.e., two “nibbles”).\",\n \"Bytes are typically what are used to store “characters”.\",\n \"“Characters” typically include letters, numbers and/or symbols.\",\n \"For example, the ASCII code uses 128 different encoding combinations to represent: (i) The letters “A-Z” in both upper and lower case (i.e., values 65-90 and 97-122, respectively); (ii) Special characters (e.g., “<”, “.”, “%”, “$” “?”, etc) (e.g., values 33-47); (iii) The numbers 0 - 9 (i.e., values 48-57); and (iv) Certain control codes used for device control (i.e., values 0-31).\",\n \"To represent more than 128 characters, an eighth bit can be utilized.\",\n \"However, the ability to store a binary code of only “0's” and “1's” limits the ability for information to be miniaturized.\",\n \"For example, to store a single character from the ASCII code, at least seven bits of memory (typically eight bits are currently required because the last bit is usually set to zero) are needed.\",\n \"Table 2 sets forth one example of how a typical computer memory system would store the word “Patent”.\",\n \"TABLE 2 ASCII LET- CHAR- BINARY HEXADECIMEL BITS OF TER ACTER NUMBER DIGIT OR BYTE MEMORY P = 80 = 01010000 50 8 a = 97 = 01100001 61 8 t = 116 = 01110100 74 8 e = 101 = 01100101 65 8 n = 110 = 01101110 6E 8 t = 116 = 01110100 74 8 Thus, the letter “P” corresponds to the ASCII character 80, which is represented by the binary code “01010000”.\",\n \"The first “0” in the sequence is often referred to as “the last bit” and is typically set to “0” for all ASCII characters.\",\n \"The corresponding hexidecimal digit or byte representation is “50”.\",\n \"In order for the computer memory system to store the letter “P”, eight bits must be sent to memory in the order “0-1-0-1-0-0-0-0”.\",\n \"Likewise, to store the letter “a”, eight bits must be sent to memory in the order “0-1-1-0-0-0-0-1”.\",\n \"Accordingly, 48 bits are used to store the six (6) letter word “Patent”.\",\n \"Similarly, when a microprocessor unit accesses the portion of memory containing the set of bits representing the word “Patent”, the same number of bits (i.e., 48) or six (6) bytes must be processed by the microprocessor.\",\n \"Clearly, if the word “Patent” could use a smaller amount of memory, less memory capacity would be needed in a computer and/or the rate at which data could be stored and/or retrieved from memory would be enhanced.\",\n \"In a first aspect of the invention, a coding system that is a higher order system than a binary coding system is utilized for the digitized storage of information.\",\n \"For example, any digital coding system which uses three or more different code characters (i.e., a coding system which operates in “base-3” or greater) will result in a reduction in the number of bits needed to store the same amount of digital information relative to a binary system.\",\n \"Thus, by using a higher order memory system, the possibility of storing more digitized information in a smaller memory area exists.\",\n \"Likewise, the rate of storing and/or retrieving digitized information should also increase due to memory being stored in a physically smaller area.\",\n \"This permits a data retrieval device to function more efficiently.\",\n \"The present invention permits the use of a digitized memory system which is base-3 or greater.\",\n \"Moreover, as more code characters are used (e.g., higher order systems of more than 10 or more than 20 different characters are utilized) the digitized system begins to behave in a manner which is similar to analog systems and can thus be referred to as “analog-like”.\",\n \"In other words, as the amount of digital sampling of an analog source increases, the recorded digital signal begins to approach the storage of most features of the analog signal.\",\n \"However, current data storage/retrieval techniques utilize, for example, 8 or 16 bits to store a single piece of analog information, which limits the amount of sampling of an analog source that can be reasonably performed when creating a digital representation of the analog source.\",\n \"Accordingly, the digital data storage/retrieval techniques of the present invention can be vastly superior to current digital techniques which utilize only “0's” and “1's” and are limited to sampling much smaller areas of analog sources than would be desirable in many cases (e.g., for complex computer modeling of numerous events, etc.).\",\n \"A first embodiment of an alternative memory storage system of the present invention may function in a manner somewhat analogous to the manner in which a Compact Disk (CD) or Digital Versatile Disk (DVD) player functions.\",\n \"Current CD and DVD technologies utilize a rotating disk comprised of a number of layers of different materials.\",\n \"The layers may include, for example, polycarbonate plastic, aluminum and acrylic.\",\n \"The aluminum in the disk includes a series of plateaus and valleys sometimes referred to as “bumps” and “pits”, respectively.\",\n \"Laser light is caused to focus upon the bumps (plateaus) and pits (valleys) in the rotating disk.\",\n \"The reflection of the laser from the successive plateaus and valleys corresponds to a data stream (e.g., a binary stream) of information.\",\n \"CD's and DVD's use, for example, 16-bit strings of binary coded data.\",\n \"Specifically, laser light reflects differently from a valley compared to the reflection from a plateau and these different reflective properties of the laser, when reflected from these valleys and plateaus, correspond to the binary code data values of “0” and “1” for each bit.\",\n \"In one embodiment of the present invention, a similar concept to the CD and DVD is utilized, however, rather than utilizing valleys and plateaus (i.e., bumps and pits) in an aluminum layer embedded in a polycarbonate plastic CD or DVD, the present invention utilizes at least two data values per bit represented by different intensity, colors or color width (e.g., different wavelength or frequency).\",\n \"These data values can be optically created, stored in dots or spots, and optically examined or interrogated (e.g., by known transmission and/or reflection techniques).\",\n \"These optical dots or spots may be of a size which is approximately the same order of magnitude in size as the bumps and pits which comprise current data tracks used on CD's and DVD's, they may be larger, or they may be smaller.\",\n \"In particular, similar to current CD's and DVD's, these dots or spots can be put together to form strings or tracks of data which begin at an innermost portion of a rotatable CD/DVD disk and extend spirally out therefrom in the form of tracks.\",\n \"Accordingly, current CD's of approximately 12 centimeters in diameter would be an acceptable size.\",\n \"However, if the dots or spots of the present invention are smaller than the “bumps” of the current technology, it is possible for CD's to be smaller in size, if desired, or more data can be placed into the same amount of memory space.\",\n \"Thus, it is possible to put an amount of optical information onto a rotatable disk of approximately 12 centimeters in diameter which exceeds that amount of information which is currently stored on CD's/DVD's.\",\n \"However, it is possible for the dots or spots to be larger in size than current bumps and pits in CD's/DVD's while still storing more information relative to standard CD's/DVD's.\",\n \"This is possible due to at least two effects.\",\n \"First, tracks may be capable of being located closer together due to, for example, lesser amounts of crosstalk between bits (e.g., recording and/or reading operations can take place in a lesser amount of space).\",\n \"Second, if digital codes higher than binary are adopted, less actual bits need to be stored relative to a binary system (e.g., rather than storing 8 or 16 bits to represent a single piece of information, a lesser number of bits can be used to represent the same piece of information that is being stored).\",\n \"It should be noted that as the number of characters that can be stored in a single bit become larger, the digital storage system actually approaches behaviors similar to an analog storage system and can be referred to as “analog-like”.\",\n \"In particular, the present invention is capable of utilizing a coding system other than the binary coding system of “0's” and “1's” now used in CD/DVD systems.\",\n \"For example, a series or range of dots or spots of different color intensities (e.g., white-gray-black), and/or different colors (e.g., red-yellow-violet), and/or both as in varied intensity of polychromatic light, can be placed upon a surface (or embedded somewhere within a material), such as a suitable photoferroelectric material such as “PLZT” (i.e., lead lanthanum zirconate titanate ceramic), which is shaped in the form of, for example, a disk.\",\n \"When, for example, such a disk containing these different intensity data value dots or spots is rotated and interrogated by, for example, a known optical system (e.g., a laser system) with a suitable focusing lens and intensity detecting means (e.g., a suitable photosensitive material or photodiode), the different intensities of the dots or spots can be registered and can be coded to correspond to different values (e.g., “0”, “1”, “2”, “3”, etc.).\",\n \"Thus, rather than using a simple binary or base-2 system where a black dot corresponds to “0” and a white dot corresponds to “1”, the present invention provides for a larger number of options for digitized data storage.\",\n \"However, as discussed in more detail herein, depending on the sensitivity of the system, this technique could also be used for actual analog computing.\",\n \"Accordingly, for example, in a simple digitized quaternary system having a quaternary code consisting of 0's, 1's, 2's and 3's, there would be 4 different choices of basic digital information storage (i.e., a “base-4” system) rather than only two choices in a binary or base-2 digital system.\",\n \"Likewise, in a digital system which utilizes 8 different combinations of spots or dots (i.e., a “base-8” system) eight different options exist for encoding a piece of information rather than only two options in a standard binary or base-2 digital system.\",\n \"The use of a larger number of possibilities other than “0's” and “1's” (e.g., 0, 1, 2, 3, 4, 5, 6, 7, etc.)\",\n \"render it possible to store a much greater amount of information in a much smaller physical area due to the elimination of unneeded bit space.\",\n \"Moreover, as the number of possibilities other than “0's” and “1's” increases, (e.g., systems greater than base-10 such as base-20 or greater systems) the closer the digital system comes to being or performing like an analog system, which has certain advantages over digital systems.\",\n \"An apparatus according to the present invention permits suitable optical data to be stored onto an appropriately constructed and shaped optical recording medium by using, for example, a laser light source.\",\n \"The optical recording media (e.g., a PLZT ceramic) can be in the form of a tape, disk, or credit card or any other acceptable shape.\",\n \"Alternatively, a photodiode or photodiode assembly can also be used.\",\n \"In all such cases, a light source (e.g., laser light source) can be used in combination with, for example, various means for controlling the direction, polarization, intensity and/or final color of the light source.\",\n \"Various examples of such systems include the following: (1) a rotatable polarizing filter (or set of filters), sometimes referred to as an analyzer, used in combination with a suitable light source which is subjected to a first polarizing filter(s) selectively modifying the polarized light from the light source.\",\n \"Specifically, once monochromatic or polychromatic light has been polarized into a first direction polarizer the polarized light is directed toward a second polarizer (sometimes referred to as an “analyzer”) which, when rotated, results in, for example, different intensities of light being emitted therefrom.\",\n \"The different intensities of light can be caused to be incident upon an appropriate recording/detecting means; (2) a light source (e.g., a laser light source), which is used in conjunction with a first polarizing filter is caused to be incident on a suitable light diffractor (e.g., a Bragg modulator) which can cause the polarized light from the light source to communicate with one or more of a series of additional polarizing filters (or analyzers) each of which analyzers are rotated, relative to the polarized source, at different angles, thus resulting an different amounts or intensities of light passing through each of the analyzers.\",\n \"Thus, by selectively transmitting the polarized light through the different analyzers (e.g., directed by, for example, the Bragg modulator) different intensities of light can be achieved.\",\n \"Such different intensities can be recorded and/or detected by a suitable means; (3) a light source (e.g., a laser light source) used in combination with an acousto-optic Bragg modulator, whereby different driving signals are input to the Bragg modulator which results in different intensities of light being emitted from the Bragg modulator.\",\n \"The multiple intensity emissions are then directed to a suitable detecting/recording means and are made to correspond to various data points in base-2 or higher order digital data storage systems; and (4) rather than utilizing polarizing filters to polarize a monochromatic or polychromatic source of light, liquid crystal cells can also be utilized.\",\n \"In particular, light can be caused to be incident upon a liquid crystal cell.\",\n \"Depending upon whether voltage is applied to the liquid crystal cell, the output from the liquid crystal cell is oriented into one direction or another.\",\n \"Liquid crystals of this type can be made of, for example, lithium niobate.\",\n \"However, similar to the embodiments (1)-(3) discussed above, intensities can be varied due to relative polarization directions.\",\n \"The outputs of light from the liquid crystal cell can be directed, by appropriate beam displacers (e.g., a Bragg modulator), toward a suitable recording/detecting means.\",\n \"In each of the examples (1)-(3) discussed above, it is possible that a polychromatic light source could be utilized.\",\n \"In this regard, when polychromatic light is made to be incident upon a first polarizer, and thereafter upon one or more additional polarizers (e.g., analyzers), in addition to different intensities of light flowing through the analyzer(s), a spreading of these different intensities over a particular frequency or wavelength range will also occur.\",\n \"Specifically, when using polychromatic light, a spectrum of frequencies of various intensities results in the formation of a somewhat continuous curve of varying light intensities over a particular frequency or wavelength range.\",\n \"The use of a polychromatic source can therefore result in secondary indicia for determining the value assigned to stored bits of data (e.g., a “0” or a “1”) and thus enhances data storage.\",\n \"For example, not only could the highest intensity of light be recorded/detected, but also the starting and/or ending points which correspond to the lowest frequency intensity of light being emitted, and the highest frequency of light being emitted, respectively, or, alternatively, the area under each curve could also be compared.\",\n \"In other words, more than one piece of information can be checked to verify the accuracy of each data point that is stored and read.\",\n \"The presence of more than a single piece of information can result in more accurate reading of data and thus result in signal/noise improvements.\",\n \"In each of the examples (1)-(4) discussed above, the different intensities or colors of light can be made to be incident upon an appropriate receiving/recording means (e.g., an optical storage means, such as a pohtopolymerisable/photocrosslinkable material, a photodiode assembly, etc.\",\n \"), such output light being suitably focussed onto the appropriate receiving/recording means by known lens or optical systems etc., all of which are discussed in greater detail herein).\",\n \"Suitable materials for recording various intensities or colors of light include the following materials: inorganic photochromic crystals, photorefractive materials, chalcogenide semiconductors, organic and polymer materials, thermoplastic media, reversible recording in Tellerium compounds, photothermal recording in Antimony compounds, magneto-optic recording and light stimulated recombination luminescence.\",\n \"For example, in the simplest embodiment (i.e., that embodiment numbered “(1)” above) for understanding the basic concept for recording various intensities of light (even though this simple embodiment is not as commercially significant as the other embodiments discussed herein), each such recorded intensity corresponds to a different value for a digitized data bit.\",\n \"In this basic case, different intensities can be achieved as follows: a polarized light source, such as a laser and polarized filter or polarizer, when used in combination with at least one rotatable polarizing filter (e.g., an analyzer) can result in light of different intensities being transmitted from the at least one rotatable analyzer.\",\n \"Specifically, when the laser light source is passed through a first polarizing filter and a second polarizer (e.g., an analyzer) is sequentially rotated (e.g., from 0-90°) a predetermined amount each time relative to the first polarizing filter (i.e., either the first or second filter can be rotated), different intensity dots or spots corresponding to data values having different meanings (e.g., “0”, “1”, “2”, etc.)\",\n \"can be produced.\",\n \"The amount of rotation of the analyzer in relation to the polarizing filter can correspond to a different intensity of light being recorded on a suitable optical recording medium (e.g., a photoferroelectric material such as “PLZT”, inorganic photochromic crystals, photorefractive materals, chalcogenide semiconductors, organic and polymer materials, thermoplastic media, reversible recording in Tellerium compounds, photothermal recording in Antimony compounds, magneto-optic recording and light stimulated recombination luminescence).\",\n \"If a photoferroelectric material is utilized, the different intensities of light can be caused to be incident upon the photoferroelectric material by use of, for example, a suitable optic lens system or a fiberoptic cable (or a series of cables) that is capable of accurately directing the optical signal to the photoferroelectric material for the proper recording of the various optical signals.\",\n \"Thus, a large number of different intensity spots or dots corresponding to data values for a single bit can be created.\",\n \"This basic system and apparatus permits higher order coding systems (e.g., base-3 or greater) to be utilized.\",\n \"The manner in which such optical data is stored, on, for example, a rotatable disk, as well as interrogated, can be by techniques which are substantially parallel, and/or use hardware which is similar, to those techniques and/or hardware currently used in CD and DVD systems.\",\n \"For example, a series of bits of an appropriate binary or higher order digital code system can be recorded onto a rotatable disk by starting to record the different intensity dots or spots near a central potion of the disk.\",\n \"The recording continues to record data in a spiral manner to form tracks of data, such data being suitably separated, physically, to avoid corruption and crosstalk, until an outermost radial portion of the disk is encountered.\",\n \"Likewise, such a disk can be read or interrogated by, for example, a laser system which focuses light onto the dots or spots and suitably measures the intensities (e.g., reflectance or transmittance) of the spots of data.\",\n \"The different intensities can be interpreted by suitable algorithms and programs to correspond to different values in the desired coding system.\",\n \"Moreover, by utilizing a digital optical coding technique other than binary, the present invention permits a much more rapid adoption of the desired “Unicode”, which takes into account, for example, all English language characters, Chinese characters, numbers and symbols, etc., in various digitized forms.\",\n \"In another embodiment of the invention, a system which utilizes magnetic memory in combination with a suitable retrieval/reading means is utilized.\",\n \"These memory/retrieval systems can be caused to function, generally, in a manner that is somewhat similar to current magnetic memory systems (e.g., magnetic tapes, cards, disks, hard drives, etc).\",\n \"In addition, these memory systems can also use digital coding/reading systems higher than a base-2 system, if desired.\",\n \"Still further, these memory systems can also store/retrieve information in an analog or analog-like format.\",\n \"Specifically, it is another object of the invention to utilize certain aspects of the phenomena known as the Zeeman effects to result in a means for the storage and retrieval of information onto/from suitable magnetic media.\",\n \"The term “Zeeman effects”, as used herein, denotes the effects of external magnetic fields on frequencies of atoms, molecules and components thereof, in the broadest sense.\",\n \"Thus, as used herein, the term “Zeeman effect” includes not only what may be considered to be Zeeman effects in the more narrow, classical sense but also other magnetic effects such as magnetic quadropole effects, etc.\",\n \"Digital data may be stored as either a binary code or some higher order code that is greater than the binary or “base-2” code.\",\n \"Moreover, due to the apparent sensitivities of these data storage techniques, data may be stored/retrieved in an analog or analog-like manner due to the inherent operation of the Zeeman effects.\",\n \"Specifically, without wishing to be bound by any particular theory or explanation, it appears that use of the Zeeman effects can result in a somewhat continuous set of differing data values that can be stored/retrieved.\",\n \"The present invention utilizes one aspect of the Zeeman effects in the following general manner.\",\n \"A magnetic field is applied to a suitable media that is capable of receiving and storing at least a portion of the applied magnetic field.\",\n \"One example of such a suitable magnetic material is a ferromagnetic material that is capable of exhibiting different amounts of magnetic domain alignment as a function of increasing magnetic field strength up to its natural magnetic saturation point (i.e., whereby any further increases in applied magnetic field do not result in any measurable further alignment of magnetic domains).\",\n \"Various known materials exist that meet this basic criteria, including many materials currently utilized in the magnetic storage industry (e.g., gamma Fe2O3, gamma Fe2O3 modified by Co, CrO2, etc.).\",\n \"The various materials can be held together by a variety of organic polymers including, for example, vinyl chloride, polyvinylchloride, methylacrylate, polymethylmethyacrylate, polyurethane, epoxy, polyamides, etc.\",\n \"Suitable substrates include typical polyesters for flexible materials and aluminum for rigid materials.\",\n \"The differing amounts of stored magnetic field can result in a corresponding splitting or shifting of the electromagnetic energy frequencies or spectral frequencies (e.g., fine spectrum splitting, hyperfine spectrum splitting, vibrational spectrum splitting, etc.)\",\n \"of one or more atoms or molecules of the magnetic material itself, or similar splitting or shifting in one or more materials that are located contiguous (e.g., in the magnetic material or in a read/write head) to the material containing the stored magnetic fields.\",\n \"In the Zeeman effect, the amount of splitting or shifting of one or more frequency spectrum(s) is a function of the strength of the applied/stored magnetic fields.\",\n \"Typically, the higher the magnetic field strength that is stored/applied (e.g., stored in a first material containing magnetic domains and thereafter applied to (e.g., influencing) a second contiguous material that exhibits Zeeman splitting at the stored magnetic field strengths), the further apart the splitting or shifting will be of one or more of the electromagnetic energy frequencies of a chosen spectrum of the targeted atom(s) or molecule(s).\",\n \"In this Zeeman effect, when a relatively wide splitting of electromagnetic energy frequencies occurs due to the applied magnetic field, the amount of splitting may correspond to frequencies in the microwave region; whereas a narrow splitting will typically correspond to lower frequencies (e.g., radio).\",\n \"The amount of splitting that occurs is important because in order to determine the amount of splitting that has occurred, a suitable interrogating electromagnetic energy is caused to be incident upon, for example, the magnetic material itself or the aforementioned contiguous material.\",\n \"The contiguous material that exhibits Zeeman splitting at the stored magnetic field can be present as, for example, one or more separate sheets of material that are located adjacent to, and/or contiguous with, the material that contains the stored magnetic field.\",\n \"When multiple contiguous materials are utilized, the present invention contemplates the use of a comparative analysis of the differences in Zeeman splitting between two or more such contiguous materials.\",\n \"Alternatively, a material comprising, for example, one or more material(s) that are mixed with the magnetic material (e.g., a binder which holds together at least a portion of the material that stores the magnetic field) could also be used.\",\n \"More specifically, a particular interrogating frequency, or set or sweep of frequencies, needs to be capable of favorably interacting with one or more of the split, shifted or splitting frequencies which result in the magnetic material, or in an alternative embodiment in one or more of the contiguous material(s), due to the different magnetic fields stored therein.\",\n \"In other words, a first magnetic material is desirably chosen so that an applied magnetic field can be reasonably stored therein.\",\n \"However, it is desirable for the magnetic material to be capable of storing reliably several magnetic fields of different strength (e.g., capable of having varying numbers of magnetic domains align with an ever changing magnetic field and such domains remain frozen or pinned so that they do not change with time) and, for example, in one embodiment of the invention, the contiguous material needs to produce split, shifted and/or splitting frequencies in response to the stored magnetic fields.\",\n \"In this regard, if one or more contiguous material(s) are utilized in connection with the magnetic material(s), the contiguous material(s) needs to exhibit a splitting and/or shifting of one or more frequencies, for example, of its rotational, vibrational, etc., bands (e.g., at the atomic level) and the resultant splitting or shifting that occurs in the contiguous material needs to be detectable by an appropriate interrogating electromagnetic source (discussed in greater detail later herein).\",\n \"Alternatively, a contiguous material does not need to be utilized.\",\n \"In this case, the magnetic material per se, or at least a portion thereof, needs to exhibit desirable and detectable splitting and/or shifting of one or more frequencies at varying stored magnetic strengths.\",\n \"Still further, it should be noted that the contiguous material could actually be located in a suitable read head which, similar to magnetoresistive and giant magnetoresistive heads, is influenced by the magnetic field strengths stored in the magnetic media.\",\n \"However, rather than measuring changes in resistivity in materials comprising the read heads, the splitting or shifting of frequencies will be detected by a suitable technique, discussed elsewhere herein.\",\n \"In this embodiment of the invention, the contiguous material may be a solid, a liquid, a gas or plasma (e.g., any material that shows desirable Zeeman splitting effects).\",\n \"With specific regard to interrogating with an applied electromagnetic source to determine the amount of Zeeman splitting and/or shifting that has occurred at a spot on the material that corresponds to a stored bit of data, at least one suitable frequency can be applied as an interrogating frequency, or, alternatively a sweep or combination of frequencies can be applied as interrogating frequencies.\",\n \"In this regard, if a single suitable frequency is chosen as the interrogating frequency, then heterodyne interactions of the various split, shifted and/or splitting frequencies with the single interrogating frequency can be measured and determined.\",\n \"Alternatively, resonance interactions of the various split, shifted and/or splitting frequencies can also be measured and determined.\",\n \"Each such determined heterodyned and/or resonant frequency can be made to correspond, by suitable programming or algorithmic techniques, to various data values.\",\n \"Alternatively, a series or sweep of frequencies which may resonate with or heterodyne with various split, shifted and/or splitting frequencies achieved in the contiguous material can also be utilized.\",\n \"These particular affects will be discussed in much greater detail later herein, however, it should be understood that these data acquisition techniques could be performed using hardware which is somewhat similar to that hardware currently used for magnetoresistive and giant magnetoresistive heads.\",\n \"For example, once an interrogating frequency has been input to a material exhibiting Zeeman splitting and/or shifting, the output (e.g., a heterodyned frequency) can be used as a driving signal or input (e.g., depending on strength or frequency either amplified or unamplified) for various other devices.\",\n \"One example of such other device is an acousto-optic Bragg modulator.\",\n \"In this detection scheme, an appropriate light source is caused to be incident upon an appropriate Bragg modulator.\",\n \"Suitable materials for a Bragg modulator include, for example, lithium niobate crystals and gallium arsenide semiconductor materials.\",\n \"The incident light beam can be caused to be deflected in differing amounts due to different driving signal inputs (e.g., depending on strength or frequency either amplified or unamplified) being directed into the Bragg modulator.\",\n \"In particular, for example, the different heterodyne outputs that correspond to different Zeeman splitting amounts (and thus different data points such as a “0” or a “1”) can be input into a Bragg modulator and cause light which is incident on the Bragg modulator to be deflected in different amounts.\",\n \"Thus, the various Zeeman splitting and/or shifting frequencies that are produced can eventually result in different amounts of light deflection due to the use of a Bragg modulator.\",\n \"The amount of deflection of light can be observed by the use of, for example, the light detection devices discussed earlier herein.\",\n \"Similarly, the different heterodyne outputs could be directed into a suitable filter which, due to the different inputs, would regulate the intensity of light passing therethrough.\",\n \"Accordingly, the different intensities of light could then be detected/recorded by a suitable means discussed elsewhere herein.\",\n \"This particular embodiment of the invention which utilizes differing magnetic fields to result in differing amounts of an appropriate frequency splitting and/or shifting is very suitable to be used for the analog storage/retrieval of information.\",\n \"In particular, a substantial continuum of splitting and shifting frequencies results in most materials that are subjected to a corresponding continuum of changing magnetic field strengths.\",\n \"If a continuum of such differing split, shifted and/or splitting frequencies can be reliably detected, then analog or analog-like (e.g., higher order than binary systems) computing with this technique is facilitated.\",\n \"Additionally, many of the aforementioned embodiments of the invention can also be used in combination with various holographic and/or three dimensional memory approaches.\",\n \"To achieve all the foregoing objects and advantages, and to overcome the looming problem in the art of an impending memory barrier, the present invention discloses various means for storing and retrieving information in a digital and/or analog format.\",\n \"With regard to the digital storage of information, a code other than a binary code for the storage of information can be utilized.\",\n \"The result of using a higher order code for digital information storage (i.e., 3 or more characters) is the ability to store more information in the same amount of area or the same amount of information in a smaller area.\",\n \"Accordingly, the present invention provides significant advancement in the art of data storage and/or data retrieval which will assist in preventing the memory barrier from being realized.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS FIG.\",\n \"1 is a view of a typical hard-disk drive that can be used in accordance with the techniques of the present invention.\",\n \"FIG.\",\n \"2 shows a read-write head for arranging magnetic domains in a magnetic disk.\",\n \"FIG.\",\n \"3 is a graph which shows an electromagnetic frequency as a function of magnetic field strength which results in different electromagnetic splitting frequencies which can correspond to stored bits of information on magnetic storage media.\",\n \"FIGS.\",\n \"4a-4d show a polarized light source and a polarized filter utilized to create dots other than white and black in an optical system (e.g., optical CD's).\",\n \"FIG.\",\n \"4e shows a photo detector/reader which is utilized to read the optical images stored in a disk 43.FIG.\",\n \"5 shows a representative hysteresis curve for a ferromagnetic material that is used as a magnetic memory material in the present invention.\",\n \"FIG.\",\n \"6 is a schematic of a means for recording optical information in accordance with the teachings of the present invention.\",\n \"FIG.\",\n \"7 is a schematic of a means for recording optical information in accordance with the teachings of the present invention.\",\n \"FIG.\",\n \"8 is a schematic of a means for recording optical information in accordance with the teachings of the present invention.\",\n \"FIG.\",\n \"9 is a schematic of a means for recording optical information in accordance with the teachings of the present invention.\",\n \"FIGS.\",\n \"10a and 10b show a curve of polychromatic light that has been incident upon a polarizing filter and an analyzer.\",\n \"FIG.\",\n \"11 shows a fine structure spectrum for SF6 from 0-300 being magnified.\",\n \"FIGS.\",\n \"12a and 12b show the magnification of two curves from the fine structure of SF6 showing hyperfine structure frequencies.\",\n \"Note the regular spacing of the hyperfine structure curves.\",\n \"FIG.\",\n \"12a shows magnification of the curve marked with a single asterisk (*) in FIG.\",\n \"11 and FIG.\",\n \"12b shows the magnification of the curve marked with a double asterisk (**) in FIG.\",\n \"11.FIG.\",\n \"13 shows an energy level diagram corresponding to the hyperfine splitting for the hyperfine structure in the n=2 to n=3 transition for hydrogen.\",\n \"FIG.\",\n \"14a shows the Zeeman effect for sodium “D” lines; and FIG.\",\n \"14b shows the energy level diagram for transitions in the Zeeman effect for sodium “D” lines.\",\n \"FIG.\",\n \"15 is a graph which shows the splitting of the ground term of the oxygen atom as a function of magnetic field.\",\n \"FIG.\",\n \"16 is a graphic which shows the dependence of the Zeeman effect on magnetic field strength for the “3P” state of silicon.\",\n \"FIG.\",\n \"17a is a pictorial which shows a normal Zeeman effect and FIG.\",\n \"17b is a pictorial which shows an anomalous Zeeman effect.\",\n \"FIG.\",\n \"18 shows anomalous Zeeman effect for zinc 3P→3S.\",\n \"FIG.\",\n \"19a shows a graphic representation of four Zeeman splitting frequencies and FIG.\",\n \"19b shows a graphic representation of four new heterodyned differences.\",\n \"FIG.\",\n \"20 is a schematic of a means for recording optical information in accordance with one example of the present invention.\",\n \"FIG.\",\n \"21 is a schematic of a means for reading recorded optical information in accordance with an example of the present invention.\",\n \"FIGS.\",\n \"22a, 22b and 22c show in graphic form plots of power spectral density (i.e., intensity) for six (6) different polarizing filter settings.\",\n \"FIG.\",\n \"23 shows a graph of upper and lower intensity limit deviations corresponding to the six (6) different filter settings of FIGS.\",\n \"22a-22c.\",\n \"FIGS.\",\n \"24a, 24b and 24c show in graphic form plots of full width half max (FWHM) data points versus six (6) different polarizing filter settings.\",\n \"FIG.\",\n \"25 shows in graphic form the deviation in upper and lower limits for the data shown in FIGS.\",\n \"24a-24c.\",\n \"FIG.\",\n \"26 shows in schematic form the apparatus used to create and determine shifting/splitting frequencies according to the present invention.\",\n \"FIG.\",\n \"27 shows an optical layout of a typical holographic memory system that can be used according to the present invention.\",\n \"DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention utilizes new and revolutionary techniques for the storage of data digitially, or in some cases, by analog or, at least a method approaching analog, such storage of data occurring optically, magnetically and/or electrically, magneto-optically and/or combinations thereof.\",\n \"The storage/retrieval methods of the present invention are capable of utilizing traditional binary or base-2 code system approaches as well as non-traditional base-3 and higher order approaches.\",\n \"The use of base-3 and higher order approaches may permit more data to be stored in approximately the same memory area relative to conventional binary techniques and/or substantially the same amount of information is capable of being stored in a smaller area relative to conventional binary techniques.\",\n \"Certain novel magnetic storage/retrieval techniques will be discussed first, and then followed by discussions of certain novel optical techniques, according to the present invention.\",\n \"FIG.\",\n \"1 shows a typical prior art hard-disk drive in a computer.\",\n \"This prior art hard-disk drive can be used substantially as shown in accordance with certain embodiments and novel techniques of the present invention.\",\n \"The prior art hard-disk drive typically comprises one or more disks 1 (in FIG.\",\n \"1, three disks are shown) that are in communication with read-write heads 2.The read-write heads are capable of communicating with each disk in one of several predetermined manners.\",\n \"Specifically, as shown generally in FIG.\",\n \"2, the heads 2 are capable of influencing magnetic domains 30 present in the disk 1.These influenced magnetic domains are represented by the number 31, while non-influenced domains are represented by the number 30, as shown in FIG.\",\n \"2.The read-write heads 2 can first influence the magnetic domains 30 on the disks 1 by applying, for example, small currents through the heads 2 to create a magnetic field in the heads 2, which heads 2 in turn implant a desired effect into said domains (e.g., the magnetic field created by heads 2 can cause the unaligned magnetic domains 30 to become oriented domains 31, such orientation occurring in a controlled manner).\",\n \"Once the domains 31 are appropriately oriented, they can then be read, if desired, by the same read-write heads 2.The domains 31 can be oriented parallel to the plane of rotation of the disk 1, or the domains 31 can be oriented perpendicular to the plane of rotation of the disk 1.When the domains 31 are oriented substantially parallel to the rotation plane of the disk 1, various electrical/magnetic induction techniques for the reading of the domains 31 are appropriate.\",\n \"However, when the domains 31 are oriented substantially perpendicular to the rotation plane of the disk 1, standard induction reading techniques do not function as well (e.g., crosstalk between closely located bits can occur and unacceptable signal to noise ratios result).\",\n \"Accordingly, development of magnetoresistive and giant magnetoresistive heads has occurred.\",\n \"Briefly, these techniques involve the detection of very small changes in the electrical resistance of one or more materials comprising the read head 2.A primary difference between magnetoresistive heads and giant magnetoresistive heads is that giant magnetoresistive heads are two or three times more sensitive than magnetoresistive heads due to their novel structure which includes the layering of different materials on top of each other.\",\n \"Somewhat conventional data storage techniques can be used in combination with the techniques of the present invention without the requirement for significant changes in current hardware being utilized.\",\n \"For example, the magnetic storage media of the prior art can be used to store data associated with Zeeman splitting and/or shifting effects.\",\n \"Moreover, the digital information that is stored on such media can be similar to or different from prior art digital data storage techniques.\",\n \"Specifically, rather than storing only a combination of sequences of “0”s and “1”s, the present invention can use more than two stored magnetic field strengths to result in different field strengths that can be applied elsewhere and which can thus result in Zeeman splitting or shifting in the magnetic media, or Zeeman splitting or shifting occurring in a contiguous media (e.g., either with one or more materials comprising the magnetic storage media or either one or more materials, such as a solid, a liquid, a gas and/or a plasma, comprising the read head).\",\n \"In other words, the different field strengths stored in a suitable magnetic media can result in differing amounts of Zeeman splitting or shifting in an appropriately chosen material, which detected Zeeman splitting or shifting corresponds to the different digital (or analog) values within a coded system.\",\n \"For example, in a base-4 system, a “0” stored on the magnetic media could correspond to a bit with no magnetic field; a “1” could correspond to a stored field strength of “x” microTesla to the bit; a “2” could correspond to a field strength of “5x” microTesla stored to the bit; and a “3” could correspond to a field strength of “10x” microTesla stored to the bit.\",\n \"The particular field strengths chosen here are exemplary, and it should be understood that differences in field strengths are simply a matter of design choice (e.g., convenient differences in magnetic field strengths should be a function of the magnetic media utilized to store information as well as the hardware utilized to store and retrieve such information and the media in which Zeeman splitting and/or shifting is desired).\",\n \"The sensitivity of such media and/or hardware may dictate how much the difference in stored field strength needs to be between each bit so that the likelihood of misreading the information stored on any one bit, or combination of bits, due to, for example, poor signal/noise ratio or crosstalk, is minimized.\",\n \"Moreover, any Zeeman splitting and/or shifting outputs can be appropriately modified or amplified by conventional techniques.\",\n \"One example of a suitable magnetic material which can be used to store a magnetic field is a ferromagnetic material.\",\n \"In particular, a ferromagnetic material that is capable of exhibiting different amounts of magnetic domain alignment as a function of increasing applied magnetic field, up to its natural magnetic saturation point.\",\n \"Examples of suitable ferromagnetic materials that can be used in connection with the present invention include gamma-Fe2O3, gamma—Fe2O3 modified by Co, CrO2, etc.\",\n \"FIG.\",\n \"5 shows a typical hysteresis curve or hysteresis loop for a typical ferromagnetic material that can be used in the present invention.\",\n \"Initially, at a point “D” no field “H” is applied and no magnetization is present in the virgin ferromagnetic material.\",\n \"However, as a field H is applied, the magnetization of domains begins at the point “D” and increases along the curve “M” up to the point of saturization (Ms).\",\n \"It should be noted that the initial magnetization or alignment of domains along the curve “M” is up to about the point “E”, in this FIG.\",\n \"4, and is reversible.\",\n \"However, when enough field H is applied to achieve a position on the curve M which corresponds to a magnetic field which is beyond the point “E”, the domains are frozen into place (i.e., will not move substantially unless influenced by, for example, another external magnetic field of greater strength or of a reverse polarity).\",\n \"This curve is known as a “hysteresis curve”.\",\n \"A suitable ferromagnetic material for use in the present invention exhibits a substantially infinite number of possible induced magnetizations along the curve M. It is these varying induced magnetizations that will be utilized to create different amounts of Zeeman splitting or shifting (discussed later herein) in the ferromagnetic materials per se, or in one or more materials which are placed contiguous to the ferromagnetic materials.\",\n \"The magnetization induced in a ferromagnetic material can be increased up to the point Ms, whereby any higher magnetic fields will not result in any additional domain alignment.\",\n \"As the applied magnetic field H is reduced to zero, the induced magnetization in the ferromagnetic material does not reduce to zero, but rather, has a remnant magnetization or resonance noted as Br.\",\n \"It should be noted that ferromagnetic materials do not typically exhibit or hold the same magnetic field that is applied to them.\",\n \"For example, if a field of 10 micro Tesla was applied, the magnetization induced in the ferromagnetic material, if measured as a field, would be less than 10 micro Tesla, for example, 7 micro Tesla.\",\n \"This factor needs to be accounted for in the present invention, but is not a substantial issue and an artisan of ordinary skill in this field will understand how to proceed in accordance with the other teachings contained herein.\",\n \"In order to understand the influence of magnetic fields on various Zeeman splitting or shifting frequencies, the following discussions of Fine Structure Frequencies, Hyperfine Structure Frequencies and Magnetic Fields are provided.\",\n \"These discussions are taken, in part, from PCT Application No.\",\n \"PCT/US01/28,392, entitled “Spectral Chemistry”, which was filed on Sep. 11, 2001, the subject matter of which is expressly incorporated herein by reference.\",\n \"Fine Structure Frequencies The field of science concerned generally with measuring the frequencies of energy and matter, known as spectroscopy is well known.\",\n \"Specifically, there are three broad classes of atomic and molecular spectra: (1) electronic spectra, which are due to electron transitions, have frequencies which occur primarily in the ultraviolet (UV), visible, and infrared (IR) regions, and are present in both atoms and molecules; (2) vibrational spectra, which are due to, for example, bond motion between individual atoms within molecules, occur primarily in the IR regions, and are present in molecules and (3) rotational spectra which are due primarily to the rotation of molecules in space and have microwave or radiowave frequencies, and also occur in molecules.\",\n \"The three broad classes of spectra discussed above have been oversimplified.\",\n \"There are actually at least three additional sets of spectra, namely, the fine structure spectra, the hyperfine structure spectra and the superfine structure spectra.\",\n \"These additional spectra occur in atoms and molecules, and extend in frequencies, for example, from the ultraviolet down to the low radio regions.\",\n \"These additional spectra are often mentioned in prior art chemistry and spectroscopy books typically as an aside, because prior art chemists usually focus more on the traditional types of spectroscopy, namely, electronic, vibrational, and rotational.\",\n \"The fine and hyperfine spectra are quite prevalent in the areas of physics and radio astronomy.\",\n \"For example, cosmologists map the locations of interstellar clouds of hydrogen, and collect data regarding the origins of the universe by detecting signals from outerspace, for example, at 1.420 GHz, a microwave frequency which is one of the hyperfine splitting frequencies for hydrogen.\",\n \"Most of the large databases concerning the microwave and radio frequencies of molecules and atoms have been developed by astronomers and physicists, rather than by chemists.\",\n \"This apparent inattention by chemists, physicists and materials scientists of the fine, hyperfine, and superfine spectra, has apparently resulted in little attention being given, if any, to these potentially useful spectra.\",\n \"In addition to the fine splitting frequencies for atoms (i.e., heterodynes), molecules also have similar fine structure frequencies.\",\n \"The origin and derivation for molecular fine structure and splitting or shifting is different from that for atoms, however, the graphical and practical results are quite similar.\",\n \"In atoms, the fine structure frequencies are said to result from the interaction of the spinning electron with its' own magnetic field.\",\n \"Basically, this means the electron cloud of a single atomic sphere, rotating and interacting with its' own magnetic field, produces the atomic fine structure frequencies.\",\n \"The prior art refers to this phenomena as “spin-orbit coupling”.\",\n \"For molecules, the fine structure frequencies correspond to the actual rotational frequencies of the electronic or vibrational frequencies.\",\n \"So the fine structure frequencies for atoms and molecules both result from rotation.\",\n \"In the case of atoms, it is the atom spinning and rotating around itself, much the way the earth rotates around its axis.\",\n \"In the case of molecules, it is the molecule spinning and rotating through space.\",\n \"Hyperfine Structure Frequencies Hyperfine structure frequencies are similar to the fine structure frequencies.\",\n \"Fine structure frequencies can be seen by magnifying a portion of a standard frequency spectrum.\",\n \"Hyperfine frequencies can be seen by magnifying a portion of a fine structure spectrum.\",\n \"Fine structure splitting frequencies occur at lower frequencies than the electronic spectra, and are located primarily in the infrared and microwave regions of the electromagnetic spectrum.\",\n \"Hyperfine splitting frequencies occur at even lower frequencies than the fine structure spectra, primarily in the microwave and radio wave regions of the electromagnetic spectrum.\",\n \"Fine structure frequencies are generally caused by at least the electron interacting with its' own magnetic field.\",\n \"Hyperfine frequencies are generally caused by at least the electron interacting with the magnetic field of the nucleus.\",\n \"An example of hyperfine splitting can be seen in FIGS.\",\n \"11 and 12.Specifically, what appears to be a single crisp curve in FIG.\",\n \"11, turns out to be a series of several curves spaced very close together.\",\n \"These are the hyperfine frequency curves.\",\n \"Accordingly, the fine structure spectra is comprised of several more curves spaced very close together.\",\n \"These other curves spaced even closer together correspond to the hyperfine frequencies.\",\n \"FIGS.\",\n \"12a and 12b show that the spacing of the hyperfine frequency curves are very close together and at somewhat regular intervals.\",\n \"The small amount that the hyperfine curves are split apart is called the hyperfine splitting frequency.\",\n \"The hyperfine splitting frequency is also a heterodyne.\",\n \"This concept is substantially similar to the concept of the fine splitting frequency.\",\n \"The difference between two curves that are split apart is called a splitting frequency.\",\n \"As before, the difference between two curves is referred to as a heterodyne frequency.\",\n \"So, hyperfine splitting frequencies are all heterodynes of hyperfine frequencies.\",\n \"Because the hyperfine frequency curves result from a magnification of the fine structure curves, the hyperfine splitting frequencies occur at only a fraction of the fine structure splitting frequencies.\",\n \"The fine structure splitting frequencies are really just several curves, spaced very close together around the regular spectrum frequency.\",\n \"Magnification of fine structure splitting frequencies results in hyperfine splitting frequencies.\",\n \"The hyperfine splitting frequencies are really just several more curves, spaced very close together.\",\n \"The closer together the curves are, the smaller the distance or frequency separating them.\",\n \"Now the distance separating any two curves is a heterodyne frequency.\",\n \"So, the closer together any two curves are, the smaller (lower) is the heterodyne frequency between them.\",\n \"The distance between hyperfine splitting frequencies (i.e., the amount that hyperfine frequencies are split apart) is the hyperfine splitting frequency.\",\n \"It can also be called a constant or interval.\",\n \"As a general example, the electronic spectrum frequency of hydrogen is 2,466 THz.\",\n \"The 2,466 THz frequency is made up of fine structure curves spaced 10.87 GHz (0.01087 THz) apart.\",\n \"Thus, the fine splitting frequency is 10.87 GHz.\",\n \"Now the fine structure curves are made up of hyperfine curves.\",\n \"These hyperfine curves are spaced just 23.68 and 59.21 MHz apart.\",\n \"Thus, 23 and 59 MHz are both hyperfine splitting frequencies for hydrogen.\",\n \"Other hyperfine splitting frequencies for hydrogen include 2.71, 4.21, 7.02, 17.55, 52.63, 177.64, and 1,420.0 MHz.\",\n \"The hyperfine structure frequencies are spaced even closer together than the fine structure frequencies, so the hyperfine splitting frequencies are smaller and lower than the fine splitting frequencies.\",\n \"Thus, the hyperfine splitting frequencies are lower than the fine splitting frequencies.\",\n \"This means that rather than being in the infrared and microwave regions, as the fine splitting frequencies can be, the hyperfine splitting frequencies are in the microwave and radiowave regions.\",\n \"These lower frequencies are in the GHz (109 hertz), MHz (106 hertz) and KHz (103 hertz) regions of the electromagnetic spectrum.\",\n \"Several of the hyperfine splitting frequencies for hydrogen are shown in FIG.\",\n \"13.(FIG.\",\n \"13 shows hyperfine structure in the n=2 to n=3 transition of hydrogen).\",\n \"Magnetic Fields In spectral terms, magnetic fields can behave in a similar manner to electric fields.\",\n \"Specifically, the spectral frequency lines of, for example, atoms and molecules, can be split and/or shifted by a magnetic field.\",\n \"In this case, the external magnetic field from outside the atom or molecule, interacts with the electric and magnetic fields already inside the atom or molecule.\",\n \"This action of an external magnetic field on spectral lines is called the “Zeeman Effect”, in honor of its' discoverer, Dutch physicist Pieter Zeeman.\",\n \"In 1896, Zeeman discovered that the yellow flame spectroscopy “D” lines of sodium were broadened when the flame was held between strong magnetic poles.\",\n \"It was later discovered that the apparent broadening of the sodium spectral lines was actually due to their splitting and shifting.\",\n \"Zeeman's original observation has evolved into a separate branch of spectroscopy relating to the study of atoms and molecules by measuring the changes of the spectral lines in atoms and molecules caused by a magnetic field.\",\n \"This in turn has evolved into the nuclear magnetic resonance spectroscopy and magnetic resonance imaging used in medicine, as well as the laser magnetic resonance and electron spin resonance spectroscopy used in physics and chemistry.\",\n \"The Zeeman effect for the famous “D” lines of sodium is shown in FIGS.\",\n \"14a and 14b.\",\n \"FIG.\",\n \"14a shows the Zeeman effect for sodium “D” lines; whereas FIG.\",\n \"14b shows the energy level diagram for the transitions in the Zeeman effect for the sodium “D” lines.\",\n \"The “D” lines are traditionally said to result from transition between the 3p2p and 3s2S electron orbitals.\",\n \"As is shown, each of the single spectral frequencies is split into two or more slightly different frequencies, which center around the original unsplit frequency.\",\n \"In this Zeeman effect, the amount that the spectral frequencies are split apart depends on the strength of the applied magnetic field.\",\n \"FIG.\",\n \"15 shows Zeeman splitting effects for the oxygen atom as a function of magnetic field.\",\n \"When there is no magnetic field, there are two single frequencies at zero and 4.8.When the magnetic field is at low strength (e.g., 0.2 Tesla) there is just slight splitting and shifting of the original two frequencies.\",\n \"However, as the magnetic field is increased, the frequencies are split and shifted farther and farther apart.\",\n \"The degree of splitting and shifting in the Zeeman effect, depending on magnetic field strength, is shown in FIG.\",\n \"16 for the 3P state of silicon.\",\n \"This Zeeman effect, due to an externally applied magnetic field, is slightly different from the Zeeman effect from an internal magnetic field depending on whether an atom or molecule is subjected to the magnetic field.\",\n \"The Zeeman effect on atoms can be divided into three different magnetic field strengths: weak; moderate; and strong.\",\n \"If the magnetic field strength is weak, the amount that the spectral frequencies will be shifted and/or split apart will be very small.\",\n \"The shifting away from the original spectral frequency will still stimulate the shifted frequencies.\",\n \"This is because they will be so close to the original spectral frequency that they will still be well within its resonance curve.\",\n \"As for the splitting, it is so small, that it is even less than the hyperfine splitting that normally occurs.\",\n \"This means that in a weak magnetic field, there will be only very slight splitting of spectral frequencies, translating into very low splitting frequencies in the lower regions of the radio spectrum and down into the very low frequency region.\",\n \"For example, the Zeeman splitting frequency for the hydrogen atom, which is caused by the earth's magnetic field, is around 30 KHz.\",\n \"Larger atoms have even lower frequencies in the lower kilohertz and even hertz regions of the electromagnetic spectrum.\",\n \"At the other end of magnetic field strength, is the very strong magnetic field.\",\n \"In this case, the splitting apart and shifting of the spectral frequencies will be very wide.\",\n \"With this wide shifting of frequencies, the difference between the split and/or shifting frequencies will be much larger than the difference between the hyperfine splitting frequencies.\",\n \"This translates to Zeeman effect splitting and/or splitting frequencies at higher frequencies than the hyperfine splitting frequencies.\",\n \"This splitting occurs somewhere around the microwave region.\",\n \"The moderate magnetic field strength case is more complicated.\",\n \"The shifting and splitting caused by the Zeeman effect from a moderate magnetic field will be approximately equal to the hyperfine splitting and/or shifting.\",\n \"Although not widely discussed in the prior art, it is possible to apply a moderate magnetic field to an atom, to produce Zeeman splitting which is substantially equivalent to its' hyperfine splitting.\",\n \"The moderate magnetic field causes low frequency Zeeman splitting that matches and hence energizes the low frequency hyperfine splitting frequency in the atom.\",\n \"However, the low hyperfine splitting frequencies actually correspond to the heterodyned difference between two vibrational or fine structure frequencies.\",\n \"When the hyperfine splitting frequency is stimulated, the vibrational or fine structure frequencies will be stimulated, and electronic frequencies will eventually be stimulated.\",\n \"This in turn causes the atom to be, for example, stimulated.\",\n \"There is also a difference between the “normal” Zeeman effect and the “anomalous” Zeeman effect.\",\n \"With the “normal” Zeeman effect, a spectral frequency is split by a magnetic field into three frequencies, with expected even spacing between them (see FIG.\",\n \"17a which shows the “normal” Zeeman effects and FIG.\",\n \"17b which shows the “anomalous” Zeeman effects).\",\n \"One of the new split frequencies is above the original frequency, and the other new split frequency is below the original frequency.\",\n \"Both new frequencies are split the same distance away from the original frequency.\",\n \"Thus, the difference between the upper and original and the lower and original frequencies is about the same.\",\n \"This means that in terms of heterodyne differences, there are at most, two new heterodyned differences with the normal Zeeman effect.\",\n \"The first heterodyne or splitting difference is the difference between one of the new split frequencies and the original frequency.\",\n \"The other splitting difference is between the upper and lower new split frequencies.\",\n \"It is, of course, twice the frequency difference between either of the upper or lower frequencies and the original frequency.\",\n \"In many instances the Zeeman splitting produced by a magnetic field results in more than three frequencies, or in splitting that is spaced differently than expected.\",\n \"This is called the “anomalous” Zeeman effect (see FIGS.\",\n \"17 and 18; wherein FIG.\",\n \"18 shows an anomalous Zeeman effect for zinc 3p→3s.\",\n \"If there are still just three frequencies, and the Zeeman effect is anomalous because the spacing is different than expected, the situation is similar to the normal effect.\",\n \"However, there are at most, two new splitting frequencies that can be used.\",\n \"If, however, the effect is anomalous because more than three frequencies are produced, then there will be a much more richly varied situation.\",\n \"Assume an easy case where there are four Zeeman splitting frequencies (see FIGS.\",\n \"19a and FIG.\",\n \"19b).\",\n \"FIG.\",\n \"19a shows four Zeeman splitting frequencies and FIG.\",\n \"19b shows four new heterodyned differences.\",\n \"In this example of anomalous Zeeman splitting, there are a total of four frequencies, where once existed only one frequency.\",\n \"For simplicity's sake, the new Zeeman frequencies will be labeled 1, 2, 3, and 4.Frequencies 3 and 4 are also split apart by the same difference “w”.\",\n \"Thus, “w” is a heterodyned splitting frequency.\",\n \"Frequencies 2 and 3 are also split apart by a different amount “x”.\",\n \"So far there are two heterodyned splitting frequencies, as in the normal Zeeman effect.\",\n \"However, frequencies 1 and 3 are split apart by a third amount “y”, where “y” is the sum of “w” and “x”.\",\n \"And, frequencies 2 and 4 are also split apart by the same third amount “y”.\",\n \"Finally, frequencies 1 and 4 are split even farther apart by an amount “z”.\",\n \"Once again, “z” is a summation amount from adding “w+x+w”.\",\n \"Thus, the result is four heterodyned frequencies: w, x, y, and z in the anomalous Zeeman effect.\",\n \"If there were six frequencies present from the anomalous Zeeman effect, there would be even more heterodyned differences.\",\n \"Thus, the anomalous Zeeman effect results in far greater flexibility in the choice of frequencies when compared to the normal Zeeman effect.\",\n \"In the normal Zeeman effect the original frequency is split into three evenly spaced frequencies, with a total of just two heterodyned frequencies.\",\n \"In the anomalous Zeeman effect the original frequency is split into four or more unevenly spaced frequencies, with at least four or more heterodyned frequencies.\",\n \"The following discussion will focus on the Zeeman effect in molecules.\",\n \"Molecules come in three basic varieties: ferromagnetic; paramagnetic; and diamagnetic.\",\n \"Ferromagnetic molecules are what comprises typical magnetic storage media and are thus capable of being used in the present invention.\",\n \"These materials typically hold a strong magnetic field due to domain alignment induced by the application of an external magnetic material and are composed of typical magnetic elements such as iron, cobalt, and/or nickel.\",\n \"Paramagnetic molecules hold only a weak magnetic field and are thus not of primary interest in the present invention as magnetic strength storage materials.\",\n \"A strong magnetic field will produce splitting greater than the hyperfine frequencies, in the microwave and infrared regions of the EM spectrum in atoms and paramagnetic molecules.\",\n \"A moderate magnetic field will produce Zeeman splitting in atoms and paramagnetic molecules at frequencies on par with the hyperfine and rotational splitting frequencies.\",\n \"Finally, consider the direction of the magnetic field in relation to the orientation of the molecule.\",\n \"When the magnetic field is parallel to an exciting electromagnetic field, π frequencies are produced.\",\n \"When the magnetic field is perpendicular to an exciting electromagnetic field, a frequencies are found.\",\n \"It is important to remember that field orientation can result in different frequencies.\",\n \"Accordingly, in a first embodiment of the present invention, in order to determine any field strength data stored on appropriate magnetic media, the magnetic media per se (or in an alternative embodiment, a material (e.g., a solid, a liquid, a gas and/or a plasma) contiguous to the magnetic media either forming a part of the magnetic media or part of an interrogating (i.e., read head) needs to be appropriately interrogated for the presence of differing amounts of Zeeman splitting and/or shifting.\",\n \"A suitable form of interrogation of such stored information could be, for example, a spectroscopic interrogation.\",\n \"For example, in reference to FIG.\",\n \"3 the magnetic media utilized to store the different induced field strengths of “0”, “x”, 5x” and “10x” microTesla, could themselves exhibit or show particular Zeeman splitting frequencies for each of the different magnetic field strengths.\",\n \"Alternatively, the different stored field strengths could result in Zeeman splitting in a contiguous material (or multiple contiguous materials).\",\n \"The contiguous material could form part of the magnetic media, or could form part of an interrogation assembly such as read/write head.\",\n \"The Zeeman splitting and/or shifting frequencies which arise are a function of the actual material which comprises the magnetic media, as well as the field strength applied thereto.\",\n \"In this regard, for example, as shown in FIG.\",\n \"3, assuming for illustrative purposes that the exemplary magnetic media exhibited a Zeeman splitting frequency of 10 KHz for a magnetic field strength of “x” microTesla, 25 KHz for a magnetic field strength of “5x” microTesla and 80 KHz for a magnetic field strength of “10x” microTesla.\",\n \"Thus, in order to read any of the bits of information stored on the exemplary magnetic media, each bit of stored information would need to be interrogated directly by applying one or more of the frequencies mentioned above (alternatively, a material contiguous (as discussed elsewhere herein) to the magnetic media could be similarly interrogated to determine Zeeman splitting and/or shifting).\",\n \"Specifically, in a first interrogation approach, a system could be designed such that each bit of information stored in a magnetic media could be interrogated with each known potential splitting frequency that could be present on each bit; or a system could be designed such that once a known splitting frequency had been found to be located at the bit, the interrogation process could stop.\",\n \"The particular approach is a matter of preference and reliability.\",\n \"For example, one interrogation process according to the present invention proceeds as follows.\",\n \"If the magnetic media per se exhibits Zeeman splitting and/or shifting, then the first bit (i.e., the bit with no magnetic field), could be interrogated with all three frequencies (i.e., 10, 25 and 80 KHz) or a sweep of frequencies (i.e., 10 KHz to 80 KHz).\",\n \"The interrogation system would recognize that none of these frequencies were present, and thus there is no stored magnetic field strength and the bit is a “0”.\",\n \"The next bit could then be interrogated with the same frequencies and it would be discovered by the interrogation that a resonant amplitude spike returned from the bit or was absorbed by the bit at a frequency of 10 KHz.\",\n \"Further interrogation of the bit would show no other resonant amplitude spikes at the interrogation frequencies of 25 and 80 KHz.\",\n \"Thus, this bit would be “1”.\",\n \"The next bit could then be interrogated with the same frequencies and it would be discovered by the interrogation process that a resonant amplitude spike returned from the bit at a frequency of 25 KHz.\",\n \"Further interrogation of the bit would show no other resonant amplitude spikes at the frequencies of 10 KHz and 80 KHz.\",\n \"Thus, this bit would be “2”.\",\n \"The final bit could then be interrogated with the same frequencies and it would be discovered that a resonant amplitude spike returned from the bit at a frequency of 80 KHz.\",\n \"Further interrogation of the bit would show no other resonant amplitude spikes at the frequencies of 10 KHz and 25 KHz.\",\n \"Thus, the bit would be a “3”.\",\n \"Accordingly, a reliable base-4 system can be achieved.\",\n \"Alternatively, another interrogation process according to the present invention could proceed as follows: if the magnetic media has a plurality of induced field strengths recorded and each of the recorded magnetic field strengths per se exhibits different amounts of Zeeman splitting in either the magnetic media or in one or more contiguous material(s), then each of the bits providing different magnetic field strengths could be interrogated with, for example, a single interrogating electromagnetic energy of a suitable frequency.\",\n \"In this regard, if a frequency of, for example, 50 KHz was used as the interrogating frequency, then heterodynes (side bands) sum and difference frequencies, etc., could be expected to occur with those Zeeman splitting frequencies which occur in the magnetic media or in the contiguous material.\",\n \"Specifically, heterodyning can occur with each of the stored frequencies, 10, 25 and 80 KHz.\",\n \"In particular, some, but not all of the possible heterodyned frequencies would be as follows: 60 KHz (i.e., 50+10); 75 KHz (i.e., 50+25); and 130 KHz (i.e., 50+80).\",\n \"In other words, the application of a 50 KHz interrogating frequency would result in at least the aforementioned heterodyned frequencies, each of which could be caused to correspond to, for example, either a “1”, a “2”, or a “3”, with no heterodynes present indicating the value of “0”.\",\n \"It should be noted that of each of the aforementioned processes, namely applying a series of frequencies or a single frequency when interrogating for heterodynes, can be used for interrogating either the magnetic material per se, or a contiguous material, said contiguous material being present in the magnetic media per se, adjacent to the magnetic media or as part of an interrogating system, such as a read/write head.\",\n \"The following brief description of heterodyning is provided to assist in understanding certain aspects of the invention.\",\n \"However, for a more complete discussion of this important area, the reader is directed toward the aforementioned PCT Application entitled “Spectral Chemistry”, which has been expressly incorporated herein by reference.\",\n \"Electromagnetic energy consists of waves.\",\n \"The number of waves in a period of time can be counted.\",\n \"Usually, time is placed on the horizontal X-axis.\",\n \"The vertical Y-axis shows the strength or intensity of the wave.\",\n \"This is also called the amplitude.\",\n \"A weak wave will be of weak intensity and will have low amplitude.\",\n \"A strong wave will have high amplitude.\",\n \"Traditionally, the number of waves per second is counted, to obtain the frequency.\",\n \"Another name for “waves per second”, is “hertz” (abbreviated “Hz”).\",\n \"Frequency=Number of waves/time=Waves/second=Hz.\",\n \"Energy waves and frequency have some interesting properties, and may interact in some interesting ways.\",\n \"The manner in which wave energies interact, depends largely on the frequency.\",\n \"For example, when two waves of energy interact, each having the same amplitude, but one at a frequency of 400 Hz and the other at 100 Hz, the waves will add their frequencies, to produce a new frequency of 500 Hz (i.e., the “sum” frequency).\",\n \"The frequency of the waves will also subtract to produce a frequency of 300 HZ (i.e., the “difference” frequency).\",\n \"All wave energies typically add and subtract in this manner, and such adding and subtracting is referred to as heterodyning.\",\n \"Common results of heterodyning are familiar to most as harmonics in music.\",\n \"There is a mathematical, as well as musical basis, to the harmonics produced by heterodyning.\",\n \"Consider, for example, a continuous progression of heterodyned frequencies.\",\n \"As discussed above, beginning with 400 Hz and 100 Hz, the sum frequency is 500 Hz and the difference frequency is 300 Hz.\",\n \"If these frequencies are further heterodyned (added and subtracted) then new frequencies of 800 (i.e., 500+300) and 200 (i.e., 500−300) are obtained.\",\n \"The further heterodyning of 800 and 200 results in 1,000 and 600 Hz as shown in FIG.\",\n \"4.A mathematical pattern begins to emerge.\",\n \"Both the sum and the difference columns contain alternating series of numbers that double with each set of heterodynes.\",\n \"In the sum column, 400 Hz, 800 Hz, and 1,600 Hz, alternates with 500 Hz, 1000 Hz, and 2000 Hz.\",\n \"The same sort of doubling phenomenon occurs in the difference column.\",\n \"Heterodyning of frequencies is the natural process that occurs whenever waveform energies interact.\",\n \"Heterodyning results in patterns of increasing numbers that are mathematically derived.\",\n \"The number patterns are integer multiples of the original frequencies.\",\n \"These multiples are called harmonics.\",\n \"For example, 800 Hz and 1600 Hz are harmonics of 400 Hz.\",\n \"In musical terms, 800 Hz is one octave above 400 Hz, and 1600 Hz is two octaves higher.\",\n \"It is important to understand the mathematical heterodyne basis for harmonics, which occurs in all waveform energies, and thus in all of nature.\",\n \"A heterodyne is a mathematical function, governed by mathematical equations, just like a fractal.\",\n \"A heterodyne also produces self-similar patterns of numbers, like a fractal.\",\n \"Heterodynes are fractals; the conclusion is inescapable.\",\n \"Heterodynes and fractals are both mathematical functions which produce a series of self-similar patterns or numbers.\",\n \"Wave energies interact in heterodyne patterns.\",\n \"Thus, all wave energies interact as fractal patterns.\",\n \"Accordingly, since energy interacts by heterodyning, matter should also be capable of interacting by a heterodyning process.\",\n \"All matter whether in large or small forms, has what is called a natural oscillatory frequency.\",\n \"The natural oscillatory frequency (“NOF”) of an object, is the frequency at which the object prefers to vibrate, once set in motion.\",\n \"The NOF of an object is related to many factors including size, shape, dimension, and composition.\",\n \"The smaller an object is, the smaller the distance it has to cover when it oscillates back and forth.\",\n \"The smaller the distance, the faster it can oscillate, and the higher its NOF.\",\n \"For example, consider a wire composed of metal atoms.\",\n \"The wire has a natural oscillatory frequency.\",\n \"The individual metal atoms also have unique natural oscillatory frequencies.\",\n \"The NOF of the atoms and the NOF of the wire heterodyne by adding and subtracting, just the way energy heterodynes.\",\n \"NOFatom+NOFwire=Sum Frequencyatom+wire and NOFatom−NOFwire=Difference Frequencyatom-wire If the wire is stimulated with the Difference Frequencyatom-wire, the difference frequency will heterodyne (add) with the NOFwire to produce NOFatom, (natural oscillatory frequency of the atom) and the atom will absorb with the energy, thereby becoming stimulated to a higher energy level.\",\n \"Cirac and Zoeller reported this phenomenon in 1995, and they used a laser to generate the Difference Frequency.\",\n \"Difference Frequencyatom-wire+NOFwire=NOFatom Matter heterodynes with matter in a manner similar to the way in which wave energies heterodyne with other wave energies.\",\n \"This means that matter in its various states may also interact in fractal processes.\",\n \"This interaction of matter by fractal processes assists in explaining why so many creatures and systems in nature exhibit fractal processes and patterns.\",\n \"Matter, as well as energy, interacts by the mathematical equations of heterodynes, to produce harmonics and fractal patterns.\",\n \"That is why there are fractals everywhere around us.\",\n \"Thus, energy heterodynes with energy, and matter heterodynes with matter.\",\n \"However, perhaps even more important is that matter can heterodyne with energy (and visa versa).\",\n \"In the metal wire discussion above, the Difference Frequencyatom-wire in the experiment by Cirac and Zoeller was provided by a laser which used electromagnetic wave energy at a frequency equal to the Difference Frequencyatom-wire.\",\n \"The matter in the wire, via its natural oscillatory frequency, heterodyned with the electromagnetic wave energy frequency of the laser to produce the frequency of an individual atom of matter.\",\n \"This shows that energy and matter do heterodyne with each other.\",\n \"In general, when energy encounters matter, one of three possibilities occur.\",\n \"The energy either bounces off the matter (i.e., is reflected energy), passes through the matter (i.e., is transmitted energy), or interacts and/or combines with the matter (e.g., is absorbed or heterodynes with the matter).\",\n \"If the energy heterodynes with the matter, new frequencies of energy and/or matter will be produced by mathematical processes of sums and differences.\",\n \"If the frequency thus produced matches an NOF of the matter, the energy will be, at least partially, absorbed, and the matter will be stimulated to, for example, a higher energy level, (i.e., it possesses more energy).\",\n \"A crucial factor which determines which of these three possibilities will happen is the frequency of the energy compared to the frequency of the matter.\",\n \"If the frequencies do not match, the energy will either be reflected, or will pass on through as transmitted energy.\",\n \"If the frequencies of the energy and the matter match either directly (e.g., are close to each other, as discussed in greater detail later herein), or match indirectly (e.g., heterodynes), then the energy is capable of interacting and/or combining with the matter.\",\n \"Another term often used for describing the matching of frequencies is resonance.\",\n \"In this invention, use of the term resonance will typically mean that frequencies of matter and/or energy match.\",\n \"For example, if the frequency of energy and the frequency of matter match, the energy and matter are in resonance and the energy is capable of combining with the matter.\",\n \"Resonance, or frequency matching, is merely an aspect of heterodyning that permits the coherent transfer and combination of energy with matter.\",\n \"In the example above with the wire and atoms, resonance could have been created with the atom, by stimulating the atom with a laser frequency exactly matching the NOF of the atom.\",\n \"In this case, the atom would be energized with its own resonant frequency and the energy would be transferred to the atom directly.\",\n \"Alternatively, as was performed in the actual wire/laser experiment, resonance could also have been created with the atom by using the heterodyning that naturally occurs between differing frequencies.\",\n \"Thus, the resonant frequency of the atom (NOFatom) can be produced indirectly, as an additive (or subtractive) heterodyned frequency, between the resonant frequency of the wire (NOFwire) and the applied frequency of the laser.\",\n \"Either direct resonance, or indirect resonance through heterodyned frequency matching, produces resonance and thus permits the combining of matter and energy.\",\n \"When frequencies match, energy transfers.\",\n \"Heterodyning produces indirect resonance.\",\n \"Heterodyning also produces harmonics, (i.e., frequencies that are integer multiples of the resonant (NOF) frequency.\",\n \"For example, the music note “A” is approximately 440 Hz.\",\n \"If that frequency is doubled to about 880 Hz, the note “A” is heard an octave higher.\",\n \"This first octave is called the first harmonic.\",\n \"Doubling the note or frequency again, from 880 Hz to 1,760 Hz (i.e., four times the frequency of the original note) results in another “A”, two octaves above the original note.\",\n \"This is called the third harmonic.\",\n \"Every time the frequency is doubled another octave is achieved, so these are the even integer multiples of the resonant frequency.\",\n \"In between the first and third harmonic is the second harmonic, which is three times the original note.\",\n \"Musically, this is not an octave like the first and third harmonics.\",\n \"It is an octave and a fifth, equal to the second “E” above the original “A”.\",\n \"All of the odd integer multiples are fifths, rather than octaves.\",\n \"Because harmonics are simply multiples of the fundamental natural oscillatory frequency, harmonics stimulate the NOF or resonant frequency indirectly.\",\n \"Thus by playing the high “A” at 880 Hz on a piano, the string for middle “A” at 440 Hz should also begin to vibrate due to the phenomenon of harmonics.\",\n \"Matter and energy in chemical reactions respond to harmonics of resonant frequencies much the way musical instruments do.\",\n \"Thus, the resonant frequency of the atom (NOFatom) can be stimulated indirectly, using one or more of its' harmonic frequencies.\",\n \"This is because the harmonic frequency heterodynes with the resonant frequency of the atom itself (NOFatom).\",\n \"For example, in the wire/atom example above, if the laser is tuned to 800 THz and the atom resonates at 400 THz, heterodyning the two frequencies results in: 800 THz−400 THz=400 THz The 800 THz (the atom's first harmonic), heterodynes with the resonant frequency of the atom, to produce the atom's own resonant frequency.\",\n \"Thus the first harmonic indirectly resonates with the atom's NOF, and stimulates the atom's resonant frequency as a first generation heterodyne.\",\n \"Of course, the two frequencies will also heterodyne in the other direction, producing: 800 THz+400 THz=1,200 THz The 1,200 THz frequency is not the resonant frequency of the atom.\",\n \"Thus, part of the energy of the laser will heterodyne to produce the resonant frequency of the atom.\",\n \"The other part of the energy of the laser heterodynes to a different frequency, that does not itself stimulate the resonant frequency of the atom.\",\n \"That is why the stimulation of an object by a harmonic frequency of particular strength of amplitude, is typically less than the stimulation by its' own resonant (NOF) frequency at the same particular strength.\",\n \"Although it appears that half the energy of a harmonic is wasted, that is not necessarily the case.\",\n \"Referring again to the exemplary atom vibrating at 400 THz, exposing the atom to electromagnetic energy vibrating at 800 THz will result in frequencies subtracting and adding as follows: 800 THz−400 THz=400 THz and 800 THz+400 THz=1,200 THz The 1,200 THz heterodyne, for which about 50% of the energy appear to be wasted, will heterodyne with other frequencies also, such as 800 THz.\",\n \"Thus, 1,200 THz−800 THz=400 THz Also, the 1,200 THz will heterodyne with 400 THz: 1,200 THz−400 THz=800 THz, thus producing 800 THz, and the 800 THz will heterodyne with 400 THz: 800 THz−400 THz=400 THz, thus producing 400 THz frequency again.\",\n \"When other generations of heterodynes of the seemingly wasted energy are taken into consideration, the amount of energy transferred by a first harmonic frequency is much greater than the previously suggested 50% transfer of energy.\",\n \"There is not as much energy transferred by this approach when compared to direct resonance, but this energy transfer is sufficient to produce a desired effect.\",\n \"While FIG.\",\n \"3 (discussed above in greater detail) shows, in graphic form, three specifically selected magnetic field strengths (i.e., 1×, 5× and 10×) and their corresponding (resultant) Zeeman splitting frequencies, it should be obvious to one of ordinary skill in this art that a virtually limitless series of possibilities exists for storage of higher order systems (i.e., higher than base-2 or a binary system), such systems being a function of the magnetic materials comprising the magnetic media, the Zeeman splitting/shifting frequencies generated in the magnetic media, or in a contiguous material, the accuracy of whatever transmitter(s) (e.g., a magnetic inductor, which generates a magnetic field) is used to induce a magnetic field in a magnetic media and/or the accuracy of the reading system which is used to detect Zeeman splitting and/or shifting effects in the magnetic media (or in a material contiguous to the magnetic material) once such magnetic media (or contiguous material) has been interrogated.\",\n \"Thus, if large numbers of stored fields can result in correspondingly large numbers of Zeeman splitting or shifting frequencies, the use of Zeeman splitting and/or shifting effects can result in an analog or analog-like storage/retrieval of information.\",\n \"For example, again referring to FIGS.\",\n \"1 and 2, the Zeeman effect can be used to read information from a series of bits on the disks 1, but such information is retrieved differently from prior art approaches.\",\n \"In this regard, by utilizing the Zeeman splitting and/or shifting effects, for example, the present invention uses the magnetic domains 31 in a manner different (e.g., the magnetic domains may comprise more than two values and/or the magnetic domains may be interrogated, directly, or indirectly through the use of one or more contiguous material(s), in a different manner) from prior art approaches.\",\n \"Specifically, the magnetic domains 31 are present on a disk 1.Current techniques allow these magnetic domains to assume “0” or “1” mode (i.e., a base-2 or binary code) and these are aligned in one of two possible combinations at pre-selected magnetic strengths.\",\n \"The present invention permits the magnetic storage media to be utilized in several unique ways.\",\n \"Specifically, for example, rather than storing only a combination or sequence of “0”s and “1”s, the present invention stores more than two different field strengths to the magnetic disk 1, by influencing the magnetic media, for example, in a somewhat standard prior art approach.\",\n \"These different field strengths are stored by utilizing a read-write head 2 that is similar to conventional heads currently being utilized.\",\n \"The read-write head 2 is capable of imparting, and thus causing storage of different field strengths of different microTesla values upon the disk 1.Any desired combination of different field strengths can be stored upon the disk 1, and can be selected as a function of convenience and/or accuracy.\",\n \"In this regard, with regard to a base-6 system (i.e., “0, 1, 2, 3, 4, and 5”), the first bit of data stored can be no magnetic field, and can be made to correspond to “0”.\",\n \"Each of the remaining five different possible values can be selected so that they correspond to different points along the curve “M” shown in FIG.\",\n \"5.The differences between the stored field strengths are a matter of material design and system designer preferences.\",\n \"For this system, the choices of magnetic materials and/or materials which exhibit desirable Zeeman splitting or shifting may assist in dictating the difference in magnetic field strengths between different values in a coded system.\",\n \"Stated more particularly, materials engineers may desire certain types of magnetic materials to be utilized as magnetic storage media and such materials may have preferential field strengths which are unique to those materials.\",\n \"Moreover, such stored field strengths may result in desirable and detectable Zeeman splitting and/or shifting in the storage media per se, or such stored field strengths may be selected to result in desirable field splitting and/or shifting in one or more materials (e.g., a solid, a liquid, a gas and/or a plasma) contiguous to the magnetic media.\",\n \"Such preferential field strengths could then be maximized as both inputs and outputs in designing systems to be efficient, accurate and inexpensive.\",\n \"Such combinations of materials and/or systems should be apparent to those of ordinary skill of the art in view of the teachings herein.\",\n \"In this Zeeman effect, when a relatively wide splitting of electromagnetic energy frequencies occurs due to the applied magnetic field, the amount of, for example, splitting may correspond to higher frequencies (e.g., microwave); whereas a narrow splitting will typically correspond to low frequencies (e.g., radio).\",\n \"The amount of splitting that occurs is important because in order to determine the amount of splitting that has occurred, a suitable interrogating electromagnetic energy is caused to be incident upon, for example, the magnetic material itself or the aforementioned contiguous material.\",\n \"The contiguous material that exhibits Zeeman splitting at the stored magnetic field can be present as, for example, one or more separate sheets of material that are located adjacent to, and/or contiguous with, the material that contains the stored magnetic field.\",\n \"When multiple contiguous materials are utilized, the present invention contemplates the use of a comparative analysis of the differences in Zeeman splitting between two or more such contiguous materials.\",\n \"Alternatively, a material comprising, for example, one or more material(s) that are mixed with the magnetic material (e.g., a binder which holds together at least a portion of the material that stores the magnetic field) could also be used.\",\n \"More specifically, a particular interrogating frequency, or set of frequencies, needs to be capable of favorably interacting with one-or more of the splitting/shifting frequencies which result in the magnetic material, or in an alternative embodiment in one or more of the contiguous materials (e.g., a solid, a liquid, a gas and/or a plasma), due to the different magnetic fields stored in the magnetic material.\",\n \"In other words, a first material is desirably chosen so that an applied magnetic field can be reasonably stored in a magnetic material.\",\n \"However, it is desirable for the magnetic material to be capable of storing reliably several magnetic fields of different strength (e.g., capable of having varying numbers of magnetic domains align with an ever increasing magnetic field and such domains remain frozen or pinned so that they do not change with time) and, for example, in one embodiment of the invention, the contiguous material needs to be sensitive to the stored magnetic fields.\",\n \"In this regard, if one or more contiguous material(s) are utilized in connection with the magnetic material(s), the contiguous material(s) needs to exhibit a splitting of one or more of its rotational, vibrational, etc., bands (e.g., at the atomic or molecular level) and the resultant splitting that occurs in the contiguous material needs to be detectable by an appropriate interrogating electromagnetic source (discussed in greater detail elsewhere herein).\",\n \"Alternatively, a contiguous material does not need to be utilized.\",\n \"In this case, the magnetic material per se, or at least a portion thereof, needs to exhibit desirable and detectable splitting or shifting of one or more frequencies.\",\n \"Accordingly, it should be clear that the Zeeman effect techniques of the present invention could be utilized in conjunction with current hardware systems.\",\n \"In this regard, so long as an appropriate field can cause to be stored in a magnetic media and the resultant Zeeman effect which occurs in the magnetic material per se, or various material(s) contiguous to the stored magnetic field, can be appropriately interrogated.\",\n \"Thus, the present invention is a significant improvement over the prior art because current read-write heads use the ASCII code which is comprised of 0's and 1's in 8 different bits resulting in 256 different combinations of 0's and 1's.\",\n \"The present invention can harvest the advantage of codes that are higher order than binary.\",\n \"The present invention also provides significant improvements over various optical memory techniques.\",\n \"Specifically, in another embodiment of an alternative memory system of the present invention, a system somewhat analogous to current optical systems utilized in compact disk (CD) players is utilized.\",\n \"Current CD technology utilizes a rotating disk comprised of a number of layers of different materials.\",\n \"The layers include polycarbonate plastic, aluminum and acrylic.\",\n \"The aluminum in the disk includes a series of plateaus and valleys sometimes referred to as “bumps”.\",\n \"Laser light is caused to focus upon the plateaus and valleys in the rotating disk.\",\n \"The reflection of the laser off of the plateaus and valleys corresponds to a binary stream of information.\",\n \"CD's use, for example, 16-bit strings of binary coded data.\",\n \"Specifically, laser light reflects differently from a valley compared to the reflection off of a plateau and these different reflective properties of the laser when shined upon these valleys and plateaus correspond to the binary code of “0's” and “1's”.\",\n \"The present invention utilizes a similar concept to the CD disk, however, rather than utilizing valleys and plateaus (i.e., bumps) in an aluminum layer embedded in a polycarbonate plastic CD, the present invention utilizes at least two dots of different intensity color, or color width.\",\n \"These dots or spots are optically created and/or optically examined or interrogated.\",\n \"These optical dots or spots may be of a size which is approximately the same order of magnitude in size as the current data tracks used on CD's, they may be larger, or they may be smaller.\",\n \"Similar to the current CD's, these dots or spots are put together in strings which begin at an innermost portion of a rotatable CD disk and extend spirally out therefrom.\",\n \"Accordingly, current CD's of approximately 12 centimeters would be an acceptable size.\",\n \"However, if the dots or spots of the present invention are smaller than the “bumps” of the current technology, it is possible for CD's to be smaller in size while maintaining the same amount of memory space.\",\n \"Thus, it is possible to put an amount of optical information onto a rotatable disk of approximately 12 centimeters in diameter which exceeds that amount of information which is currently stored on CD's.\",\n \"A first example of the invention utilizes a quaternary system which is depicted schematically in FIG.\",\n \"4.In this regard, with reference to FIG.\",\n \"4A, a polarized light source 40 (e.g., a light source from a laser that has been subjected to a polarizing filter) is projected through a polarizing filter 41 (sometimes referred to herein as an analyzer).\",\n \"When the polarized light source 40 and polarizing filter 41 are in alignment, then a white dot 42a is caused to appear on a recording media 43.This white dot may correspond to “0” in a base-4 code.\",\n \"With regard to FIG.\",\n \"4B, the polarized light source 40 is projected upon the polarizing filter 41, however, in this Example, the filter 41 has been rotated about 30° with regard to the polarized light source 40.The disk 43 has also been rotated an appropriate amount of “x” so as to permit a separate recording of the dots 42a and 42b.\",\n \"This 30° rotation of the filter 41 results in the placement or recording of a dot 42b, that is somewhat gray upon the disk 43.FIG.\",\n \"4C shows the same polarized light source 40, however, the polarizing filter 41 is now arranged at an angle of about 60° relative to the source 40 so that a somewhat darker spot 42c is created.\",\n \"Again, the disk 43 has been rotated an amount “x” so as to permit the separate recording of the spot 42c upon the disk 43.Likewise, with regard to FIG.\",\n \"4D, the polarized light source 40 and the polarizing filter 41 are now at an angle of about 90° relative to the starting position of the polarizing filter 41 in FIG.\",\n \"4a, resulting in a completely dark spot 42d being recorded upon the disk 43.FIG.\",\n \"4e shows a traditional photo detector 50 projecting a light source onto the disk 43 such that the dots 42a, 42b, 42c, and 42d on the disk 43 can be illuminated.\",\n \"Light is then reflected back to, for example, the detector portion of the photo detector 50.Specifically, the detector 50 is capable of distinguishing the white dot 42a from the black dot 42d, as well as the shades of gray dots 42b and 42c.\",\n \"It should be apparent that the example depicted in FIG.\",\n \"4 has been greatly exaggerated for clarity purposes, and, for example, the amount “x” that the disk 43 needs to move to accommodate the sequential dots 42a-42d, is minute.\",\n \"Additionally, as a result of recording the four different dots 42a-42d upon the disk 43, a base-4 or quaternary system has been utilized with the result recorded being “0”-“1”-“2”-“3”.\",\n \"Still further, any appropriate media such as, for example, a PLZT ceramic, could be utilized to store different photo intensity data or spots.\",\n \"It should be apparent that the dots 42a-42d are the beginning of a track of data which can be recorded spirally outward from a center portion of a disk 43.Another example of the invention comprises a base-8 code utilizing 8 different shades of dots.\",\n \"While this particular example is not shown in the Figures, it is clear that 8 different shades of dots would require the turning of the polarizing filter 41 shown in FIG.\",\n \"4 at approximately 12.85° each time.\",\n \"Another example, similarly, could utilize a 16 shade code system, which would require turning the polarizing filter 41 of FIG.\",\n \"4 a total of 6° each time to result in 16 different shades of dots.\",\n \"It should be clear to an artisan of ordinary skill that the amount of data storage becomes a function of how finely the polarized filter 42 is rotated relative to the polarizing light source 41 as well as how accurately an optical detector 50 can distinguish between the different light intensities that are reflected or transmitted by the different shades of gray dots.\",\n \"It is certainly possible that an entire 256 character ASCII-type code can be written into a single bit.\",\n \"In such a case, the amount of data storage is significantly improved over the current techniques.\",\n \"Still further, it should be apparent that different colors (e.g., red-yellow-violet), corresponding to different wavelengths of light can be placed upon a surface of a disk 43.When, for example, such a disk containing these different color dots or spots is rotated and interrogated by, for example, a laser lens system, the different colors (wavelengths or frequencies) of the dots or spots can be registered and could be coded to correspond to different values (e.g., “0”, “1”, “2”, “3”, etc.).\",\n \"Thus, rather than using a simple binary or base-2 system where a black dot corresponds to “0” and white dot corresponds to “1”, the present invention provides for a larger number of options for data storage by providing methods to store data based on color, intensity or both.\",\n \"Accordingly, for example, in a simple quaternary system having a quaternary code consisting of 0's, 1's, 2's and 3's, there would be 4 different choices of basic information storage (i.e., a “base-4” system) rather than only two choices in a binary or base-2 system.\",\n \"Moreover, rather than utilizing only a monochromatic light source, it is possible that a polychromatic light source could also be utilized.\",\n \"In this regard, when polychromatic light is made to be incident upon a first polarizer, and thereafter upon one or more additional polarizers (e.g., analyzers), in addition to different intensities of light flowing through the analyzer(s), a spreading of different colors over a particular frequency or wavelength range will also occur.\",\n \"Specifically, as shown in FIGS.\",\n \"10a and 10b, when using polychromatic light, a curve 90 representing a spectrum of frequencies ranging from f1 to f2 of various intensities results in the formation of a somewhat continuous curve of varying light intensities over a particular frequency or wavelength range.\",\n \"The use of a polychromatic source can therefore result in secondary indicia for determining the value assigned to stored bits of data (e.g., a “0” or a “1”) and thus enhances data storage.\",\n \"For example, not only could the highest intensity of light I1 be recorded/detected, but also the starting and/or ending points f1 and/or f2 which correspond to the lowest frequency of light being detected, and the highest frequency of light being detected, respectively.\",\n \"Alternatively, the area “A” under each curve could also be compared as, for example, with integral area denoted as power spectral density.\",\n \"Further, the FWHM (i.e., full width half max) can be used as an indicia, representing the frequency ranger, f1 to f2 at half the maximal intensity.\",\n \"A range of full widths at varying intensities (i.e., 100% maximal, 1% minimal, 50% half maximal, 75% three-fourths maximal, etc.)\",\n \"can also be used.\",\n \"In other words, more than one piece of information can be checked to verify the accuracy of each data point that is stored and read.\",\n \"The presence of more than a single piece of information can result in more accurate reading of data and thus result in signal/noise improvements.\",\n \"FIG.\",\n \"6 shows a light source 60 which emits unpolarized light 61 into a polarizing filter 62 which creates a polarized light beam 63.The polarized light 63 is incident upon a suitable light modulator 64 which causes the light beam 63 from the polarizing filter 62 to be directed to one of a plurality of different polarizing (or analyzing) filters 81, 82, 83, 84 and 85.The modulator 64 is known in the art and can function at frequencies of about 1 GHz and is thus compatible with most computer processing systems.\",\n \"While only five analyzing filters 81-85 are shown, the number of filters that can be used in accordance with this embodiment is based on the data coding system in which it is desired to work.\",\n \"This particular system which utilizes five filters 81-85 is a base-5 system.\",\n \"When the modulator 64 directs the polarized light beam 63 from the polarizing filter 62 to the different analyzing filters 81-85, the result will be different intensities of light 65 being transmitted through each of the different analyzing filters 81-85 onto (or into) a suitable optical recording medium 66.Specifically, each of the polarizing filters 81-85 are fixedly rotated a different amount relative to the polarizing direction of the light beam 63 emanating from the polarizing filter 62.Once the light 65 has been transmitted through one of the different polarizing filters 81-85, the light can then be suitably focussed and/or redirected by appropriate optical and/or magneto-optical or acousto-optical (not shown in this FIG.\",\n \"6) means onto or into the recording medium 66.Examples of suitable optical recording mediums include: inorganic photochromic crystals, photorefractive materials, chalcogenide semiconductors, organic and polymer materials, thermoplastic media, reversible recording in Tellerium compounds, photothermal recording in Antimony compounds, magneto-optic recording and light stimulated recombination luminescence.\",\n \"With regard to FIG.\",\n \"7, an alternative to the embodiment of FIG.\",\n \"6 is shown.\",\n \"In this embodiment, the plurality of polarizing filters 81-85 are replaced with a polarization modulator 67.In particular, the polarization modulator 67 can be made of a suitable material such as a silica glass or an X-cut lithium niobate crystal (see, for example, the disclosure in U.S. Pat.\",\n \"No.\",\n \"6,154,432 for suitable materials).\",\n \"Voltages are applied to electrodes on the modulator 67 to produce an electric field that can orient the polarization of the propagating wave in any suitable direction.\",\n \"This technique provides a very robust and mechanically stable method of polarization control.\",\n \"The polarization of the light wave 70 (as well as the light wave 65) will be maintained through appropriate transmission of the light wave through other non-polarizing optics.\",\n \"The modulator 67 has been shown to have transmission rates of over 500 Mbits/second.\",\n \"A polarizing filter 62, oriented in a fixed direction, then provides an output 65 of varying intensities to a light modulator 64.The light modulator 64 then appropriately directs the output 65′ to a similar suitable recording medium 66.FIG.\",\n \"8 shows another embodiment of the invention wherein a light source 60 emits unpolarized light 61 into an acousto-optic Bragg modulator 68.The acousto-optic Bragg modulator 68 has inputted into it different electrical driving signals which result in different intensities of light 65″ being emitted therefrom.\",\n \"The different intensities of light can then be suitably focused and/or redirected by appropriate optical and/or magneto-optical and/or acousto-optical means into or onto an appropriate recording medium 66.FIG.\",\n \"9 shows another embodiment similar to that embodiment shown in FIG.\",\n \"7, except that the polarization modulator 67, has been replaced with at least one liquid crystal cell.\",\n \"In this regard, depending upon what voltages are applied to the liquid crystal cell 69 (the output which is directed into a polarizing filter 62) will result in a variable intensity output 65′″.\",\n \"The output 65′″ can thereafter be suitably directed to an appropriate optical storage medium 66 by any of the means for directing (not sown in FIG.\",\n \"9) the output 65′″, as discussed above herein.\",\n \"It should be also be clear to an artisan of ordinary skill that the same general technique disclosed herein can be utilized for the optical layout that is shown in FIG.\",\n \"27.In this regard, FIG.\",\n \"27 shows a holographic memory system of how a crystal can be imprinted with pages of data.\",\n \"In this regard, similar to the techniques shown in FIGS.\",\n \"4a-e, the system would work just as well for a holographic memory system.\",\n \"OPTICAL EXAMPLES The following experiments were performed to show that various light intensities can be recorded by rotating at least two polarizable filters (e.g., a polarizer and an analyzer) relative to each other and recording the different resultant intensities on ordinary photographic film (i.e., color slide film).\",\n \"The recorded intensities were thereafter interrogated with an optical spectrometer by passing a light source through the recorded intensities on the slides and into a probe connected to the optical spectrometer.\",\n \"All optical recording and interrogation steps were performed on a Newport optics table having one (1) inch grids thereon.\",\n \"In particular, mounting holes were provided at each corner of one inch squares for rigidly securing all optical equipment thereto to minimize data distortion due to vibrations.\",\n \"Different optical data values were obtained and recorded by using varying amounts of light which were caused to be incident on a Canon EOS Rebel 4 camera with zoom lens, which was rigidly mounted to the optics table.\",\n \"In particular, the camera was operated in a manual mode and the manual focus and was set to 50 mm.\",\n \"The film used in the camera was ordinary color slide film known as Fujichrome Color Slide film RH-400 speed.\",\n \"The source of light for all optic experiments was a Stocker and Yale, Model 20 Imagelite, polychromatic light source.\",\n \"The fiberoptic bundle end of the light source was also rigidly mounted to the optics table, with its end being located about 3 cm from a rigidly mounted polarizer/analyzer filter set (Orion Variable Polarizing Filter Set #5558).\",\n \"In particular, as shown in FIGS.\",\n \"20 and 21, the polychromatic light source 60 was caused to communicate with a polarizing filter set 62 and 81 through a fiber optic bundle 101 (i.e., a Stocker-Yale SF 38036P single S/O; ⅜″ bundle, 36″ long, MTL-PVC over metal hose).\",\n \"The function of the fiber optic bundle 101 was to transport polychromatic light from the Imagelite light source 60 (which was operating at a setting of “4”) to the polarizing filter set 62 and 81.The polarizing filter set 62 and 81 was located at a distance of about 3.3 cm from the lens of the Canon camera 100.Light from the light source 60 was directed through the fiber optic bundle 101 and caused to be made incident upon the polarizing filter set 62 and 81.The polarizing filter set 62 and 81 was set at six (6) different relative settings which permitted six (6) different intensities of light to be transmitted therethrough.\",\n \"In particular, the polarizing directions of each filter were set at the following relative rotations to each other: 0° relative rotation, 18° relative rotation, 36° relative rotation, 54° relative rotation, 72° relative rotation, and 90° relative rotation.\",\n \"These six (6) different relative rotation filter settings resulted in six (6) different intensities of light being transmitted therethrough.\",\n \"Specifically, the different intensities of polychromatic light were caused to be incident upon the lens of the camera 100 with care being taken so that the fiber optic bundle 101 transmitted light roughly perpendicular into and through the polarizing filter set 62 and 81 and into the lens of the camera 100.The result was a different intensity spot being recorded on each of the six (6) different slides 102 corresponding to each of the six (6) different relative rotation amounts.\",\n \"The differences in each of the slides 102 was readily observable by eye.\",\n \"Once the different images were recorded onto the photographic film comprising the slides 102, the images or spots were then interrogated by a transmission technique that utilized an optic spectrometer.\",\n \"Specifically, as shown in FIG.\",\n \"21, data values were retrieved from each of the six (6) different slides 102 by using a StellarNet EPP 2000 spectrometer 104 which utilized SpectraWiz software.\",\n \"Specifically, the same light source 60 used to create the images on the slides 102 was again connected with the same fiber optic bundle 101, still rigidly mounted on the optics table.\",\n \"Each of the data slides 102 was rigidly mounted in a holder such that the output from the fiber optics bundle 101 was reproducibly and directly incident upon that portion of the slide 102 containing the data or spot, from a distance of about 7.2 cm.\",\n \"Care was taken to assure that the fiber optic bundle 101 was mounted perpendicular to the slides 102 in the slide holder.\",\n \"A fiber optic probe 103 of the StellarNet EPP 2000 spectrometer 104 was rigidly mounted on the other side of each slide 102 about 7.0 cm away, so that transmission information could be obtained.\",\n \"Again, care was taken to assure proper alignment so that transmission data would not be skewed.\",\n \"The relevant settings on the StellarNet EPP 2000 fiber optic spectrometer were as follows: Mode Scope Detector integration time 4 ms Number of scans to average 5 Pixel resolution via smoothing 0 level Temperature compensation On Spectrometer channels 1 X axis Wavelength (nm) Y axis Counts The image light source was then set at a standard setting of “7”, in order to obtain information.\",\n \"The EPP 2000 provided information regarding the maximum integral area under the curve (power spectral density, i.e., Counts2) and full width half max (“FWHM”) of the peak (in nm) for each slide via the automated Area-PSD function.\",\n \"It is noted that each slide corresponded to the six (6) different rotatable filter settings.\",\n \"Measurements were reliably reproducible.\",\n \"Measurements of the six (6) slides, corresponding to the six (6) filter positions ranged, in power spectral densities, from 347,690 at 0° to 36,674 at 90°.\",\n \"FWHM ranged from 466 nm at 0° to 160 nm at 90°.\",\n \"Using either power spectral density alone, FWRM alone, or both in combination, six (6) different and distinct data values were obtained.\",\n \"Specifically, as shown in FIGS.\",\n \"22a, 22b and 22c, three sets of measurements were taken at different times from the same aforementioned six (6) slides.\",\n \"FIGS.\",\n \"22a-22c are a plot of Power Spectral Density (i.e., intensity) versus the six (6) different filter settings.\",\n \"The deviation in the upper and/lower limits of the data in FIGS.\",\n \"22a-22c is superimposed on FIG.\",\n \"23.It is clear from FIG.\",\n \"23 that the upper and lower intensity limit deviations corresponding to each of the six (6) different filter settings do not overlap with each other.\",\n \"Thus, this technique shows that intensity alone can provide a basis for a base-6 data storage system.\",\n \"Further, optical transmission data from the six (6) slides was analyzed by a second approach.\",\n \"Specifically, FIGS.\",\n \"24a, 24b and 24c show plots of full width half max (FWHM) data points versus the six (6) different filter settings.\",\n \"Similar to the data in FIGS.\",\n \"22a-22c, these data were obtained at three (3) different times to assure the accuracy of the measurements.\",\n \"FIG.\",\n \"25 shows the deviation in upper and lower limits for the data shown in FIGS.\",\n \"24a-24c.\",\n \"It is clear from FIG.\",\n \"25 that the upper limit deviation values for FWHM corresponding to each filter setting do not overlap.\",\n \"Thus, this technique provides another method for data recognition in, for example, a higher order system.\",\n \"MAGNETIC SPLITTING/SHIFTING EXAMPLES The following magnetic experiments were performed to show that different amounts of Zeeman splitting/shifting can be achieved in a magnetic material and/or a contiguous material.\",\n \"Specifically, varying magnetic field strengths were applied to a contiguous material comprising aluminum foil to simulate one potential configuration of magnetic storage media exhibiting desirable Zeeman splitting/shifting effects.\",\n \"The source of the magnetic fields utilized were three (3) permanent magnets of different field strengths.\",\n \"In particular, the magnets were obtained from DuraMag, Inc., and were characterized as follows: Magnet A: (Low Field Strength) NS-10010050, Neodymium-Iron-Boron Magnet, 1.000″×1.000″×0.500” thick with a field strength near the center thereof of about 5,090 G, increasing to about 8,380 G at the edge; Magnet B: (Intermediate Field Strength) ND-4514 Neodymium Magnet, 1.0″×1.0″×1.0″ thick with a field strength near the center thereof of about 8,730 G, increasing to about 10,300 G at the edge; and Magnet C: (High Field Strength) ND-4514 Neodymium Magnet, 1″×1″×1.9″ thick with a field strength near the center thereof of about 11,500 G, increasing to about 11,900 G at the edge.\",\n \"All spectroscopy measurements were performed using a Hewlett-Packard microwave spectroscopy system (30-minute warm-up).\",\n \"The following are the salient features of the spectroscopy system used in these examples.\",\n \"(1) HP Display Processor—Ref 0.0 dB, S11 mode, log MAG display, 10.0 dB/and marker pips with frequency and dB readout.\",\n \"(2) HP 8510B Network Analyzer-Ch 1.\",\n \"(3) HP 8513A Reflection/Transmission Test Set—45 MHz-26.5 GHz, Port 1.\",\n \"(4) HP 8350B Sweep Oscillator—Instrument State REM, Sweep trigger—INT, Sweep—Time.\",\n \"(5) HP 83595A RF Plug-in—0.1-26.5 GHz, Power 10.0, mW RF—On, CS Filter—On, ALC—INT.\",\n \"(6) Cables—W.\",\n \"L. Gore & Associates DAS ATE (55 AT) Test cables, 24 inches long.\",\n \"(7) Waveguides (Maury Microwave, Inc., and Narda, Inc.).\",\n \"It is noted that all waveguides produced flat baselines at 0.0 dB within the various frequency ranges noted below.\",\n \"a. Narda 616: Range 1.12 to 1.7 GHz b. Maury R229A1: Range 1.7 to 2.6 GHz c. Maury S209C2: Range 2.60 to 3.96 GHz d. Maury G229A1: Range 3.95 to 5.85 GHz e. Maury C209D2: Range 6.85 to 8.2 GHz.\",\n \"FIG.\",\n \"26 shows a schematic of the basic experimental set-up that was used to determine Zeeman shifting amounts in an aluminum foil contiguous material that was subjected to different magnetic field strengths.\",\n \"A microwave spectrometer 204 was comprised of Hewlett Packard items (1)-(5) described above.\",\n \"The microwave spectrometer 204 was connected by the cable 203 described in item (6) above.\",\n \"Different waveguides 202, described in item (7) above, were placed over each magnet 201 described as Magnets A, B and C above.\",\n \"Each of the magnets 201 (i.e., A, B and C) were sequentially placed into contact with a thin sheet of commonly available aluminum foil 200 (Tamco Distr.\",\n \"), the magnets 201 being oriented with their magnetization direction pointing parallel with the electromagnetic beam propagation in the waveguide 202.The following Table 3 shows the different amounts of Zeeman shifting that were obtained with three (3) different magnetic field strengths generated by the three (3) magnets A, B and C, applied to the contiguous aluminum foil 200.The initial frequency of 1.885 GHz was obtained from the aluminum foil 200 without the application of any magnetic field.\",\n \"The other values correspond to the shifted aluminum foil 200 frequency determined by microwave spectroscopy, as well as the power frequencies in dB.\",\n \"It is clear from this simple Example that meaningful Zeeman shifting can occur in a contiguous material to which a “permanent” magnetic field has been applied.\",\n \"Accordingly, this Example demonstrates that storage of varying magnetic field strengths can function as a memory storage system and that Zeeman shifting/splitting can function as a memory retrieval system.\",\n \"More particularly, both systems can function in higher order memory systems.\",\n \"TABLE 3 Magnet A Magnet B Magnet C Waveguide No Magnet (Low) (Intermediate) (High) 1.12-1.7 GHz 1.885 GHz, 1.855 GHz, 1.645 GHz, 1.115 −4.2 dB −4.6 dB −7.2 dB GHz, −3.4 dB 1.7-2.6 GHz - 0 - - 0 - - 0 - - 0 - 2.60-3.95 GHz - 0 - - 0 - - 0 - - 0 - 3.95-5.85 GHz - 0 - - 0 - - 0 - - 0 - While there has been illustrated and described what is at present considered to be the preferred embodiments of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made, and equivalents may be substituted for elements thereof without department from the true scope of the invention.\",\n \"In addition, many modifications may be made to adapt the teachings of the invention to a particular situation without departing from the central scope of the invention.\",\n \"Therefore, it is intended that this invention not be limited to the particular embodiments disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10467912"}}},{"rowIdx":392,"cells":{"text":{"kind":"list like","value":[["Apparatus and method for splicing sliver of yarn during yarn formation and processing","An apparatus (20) and methods are provided for splicing selected portions of sliver (S) such as during yarn formation and processing.","The apparatus (20) preferably includes a needle carrying member (35) having a plurality of needles (32) to engage silver (S) when positioned adjacent thereto and a needle engaging member (45) positioned to underlie the needle carrying member (35) and to receive the plurality of needles (32) from the first needle carrying member (35).","The apparatus (20) also preferably includes a hand-activated needle actuation device (30, 40) connected to the needle carrying member (35) and the needle engaging member (45) to position the needle carrying member (35) in an open position to allow sliver (S) to be spliced to be readily positioned therebetween and responsive to closing at least portions of the hand (H) of a user for actuating the engaging of the needle carrying member (35) with the sliver (S) and the needle engaging member (45) so that the engaging of plurality of needles (32) of the needle carrying member (35) with the needle engaging member (45) thereby defines a closed position."],["1-18.","(canceled) 19.An apparatus for splicing selected portions of sliver, the apparatus comprising: a needle carrying member having a plurality of needles to engage sliver when positioned adjacent thereto; a needle engaging member positioned to receive the plurality of needles from the needle carrying member when the plurality of needles engage the sliver in a closed position; and a hand-activated needle actuation device connected to the needle carrying member and the needle engaging member to position the needle carrying member in an open position so that the plurality of needles is spaced-apart from the needle engaging member to allow sliver to be spliced to be readily positioned therebetween, the hand-activated needle actuation device being responsive to grippingly closing at least portions of a hand of a user to actuate the engaging of the needle carrying member with the sliver and the needle engaging member when the sliver is positioned between the needle carrying member and the needle engaging member so that the engaging of the plurality of needles of the needle carrying member with the needle engaging member thereby defines a closed position.","20.An apparatus as defined in claim 19, wherein the hand-activated needle actuation device includes a first handle portion connected to the needle carrying member and positioned to be gripped by a hand of a user, a second handle portion connected to the needle engaging member and positioned to be gripped by a hand of a user, a pivot member connected to the first and second handle portions to allow either the first or second handle portions to pivot about the pivot member between the respective open and closed positions, and a biasing member connected to the pivot member and positioned to bias the needle carrying member in the open position.","21.An apparatus as defined in claim 19, wherein the hand-activated needle actuation device includes a first handle portion connected to the needle carrying member and positioned to be gripped by a hand of a user, a second handle portion connected to the needle engaging member and positioned to be gripped by a hand of a user, a pivot member connected to the first and second handle portions to allow either the first or second handle portions to pivot about the pivot member between the respective open and closed positions, and a biasing member associated with the first and second handle portions and positioned to bias the needle carrying member in the closed position.","22.An apparatus as defined in claim 19, wherein the needle engaging member includes a body member and a plurality of openings formed in the body member and each extending to a depth in the body member so that none of the plurality of needles extend outwardly from the needle engaging member when in the closed position, the number of the plurality of openings being equal to the number of the plurality of needles, each of the plurality of openings being positioned to underlie a corresponding one of the plurality of needles of the needles carrying member so that the plurality of openings matingly receive the plurality of needles as the plurality of needles pass into and out of the plurality of openings to thereby engage the needles and sliver being carried therewith.","23.An apparatus as defined in claim 22, wherein the body member of the needle engaging member includes a substantially closed bottom, and wherein the bottom underlies each of the plurality of openings so that portions of the bottom define a floor for each of the plurality of openings.","24.An apparatus as defined in claim 19, further comprising a lock associated with the hand activated needle actuation device to lock the hand activated needle actuation device in the closed position so that none of the plurality of needles extend outwardly from the needle engaging member when locked in the closed position.","25.An apparatus as defined in claims 19, wherein each of the plurality of needles includes a needle body and a recessed portion formed in the needle body and positioned to assist in the engaging of and interconnecting of the sliver when each needle engages sliver during movement to the closed position.","26.An apparatus as defined in claims 20, wherein the needle carrying member is detachably connected to at least one of the first handle portion and the second handle portion and defines a needle cartridge to readily remove from the respective first handle portion and second handle portion.","27.An apparatus as defined in claims 26, further comprising at least one replacement auxiliary needle cartridge adapted to be readily positioned in the at least one of the first handle portion and the second handle portion.","28.An apparatus for splicing sliver, the apparatus comprising a base and a needle receiving member associated with the base and positioned to receive a plurality of needles, the apparatus being characterized by having: a first cartridge member detachably connected to the base to be readily removed therefrom, the first cartridge member having a combination of a body portion and a plurality of needles each connected to the body portion, each of the plurality of needles having a recessed portion to engage and intertwine first portions of sliver with adjacent second portions of sliver; and a second auxiliary replacement cartridge member adapted to be detachably connected to the base and to be placed in substantially the same position as the first cartridge member, the second auxiliary replacement cartridge member also having a combination of a body portion and a plurality of needles each connected to the body portion and adapted to be positioned to be received by the needle receiving member, each of the plurality of needles having a recessed portion to engage and intertwine first portions of sliver with adjacent second portions of sliver.","29.An apparatus as defined in claim 28, further comprising a pivot member associated with the first cartridge member and the needle receiving member to allow either the first cartridge member or needle receiving member to pivot about the pivot member between respective open and closed positions, the open position being defined by portions of the first cartridge member being spaced-apart from portions of the needle receiving member to receive the first cartridge member and the closed position being defined by the first cartridge member being received by portions of the needle receiving member when positioned closely adjacent thereto.","30.An apparatus as defined in claim 29, further comprising a biasing member associated with the pivot member and positioned to bias either the first cartridge member or the needle receiving member in a preselected biased position.","31.An apparatus as defined in claim 28, wherein the recessed portion of each of the plurality of needles of the first and second cartridge members includes at least one of the following: a barb, a groove, and a channel.","32.An apparatus as defined in claims 28, a lock associated with the first cartridge member and the needle receiving member to lock the first cartridge member and the needle receiving member in a closed position.","33.A method of splicing sliver, the method being characterized by the steps of: grippingly closing a first handle portion and a second handle portion of a sliver splicer having at least one sliver engaging member by a hand of a user so that the at least one sliver engaging member engages and splices sliver positioned adjacent thereto when in a closed position, the at least one sliver engaging member having a plurality of needles each including a recessed portion to engage and intertwine first portions of the sliver with second portions of the sliver to thereby join the first portions to the second portions of the sliver; and releasingly opening the first handle portion and the second handle portion by the hand of the user from the closed position to an open position to thereby release the spliced portion of sliver from the at least one sliver engaging member.","34.A method as defined in claim 33, further comprising the steps of removing the spliced portion from adjacent the at least one sliver engaging member, grippingly closing the first handle portion and the second handle portion to the closed position without sliver positioned therebetween, and locking the first handle portion and the second handle portion in the closed position.","35.A method as defined in claim 34, further comprising removing the spliced portion from adjacent the at least one sliver engaging member and biasingly closing the first handle portion and the second handle portion to the closed position without sliver positioned therebetween.","36.A method as defined in claim 35, wherein none of the recessed portion of each of the plurality of needles is exposed when in the closed position.","37.A method of splicing sliver comprising joining first portions of sliver with a plurality of needles having a recessed portion to engage and intertwine with adjacent second portions of sliver, the method characterized by having: the plurality of needles being connected to a body portion so that the body portion and the plurality of needles in the combination define a needle cartridge member; and replacing the needle cartridge member with an auxiliary cartridge member also having a body portion and a plurality of needles connected to the body portion.","38.A method as defined in claims 37, wherein each of the plurality of needles includes a needle body, and wherein the recessed portion is formed in the needle body and positioned to assist in the engaging of and interconnecting of the sliver when each needle engages sliver."],["<SOH> BACKGROUND OF THE INVENTION <EOH>In the textile industry, yarn is formed of a plurality of slivers.","To form the yarn, however, various steps are required to obtain the type and texture of the yarn desired.","During sliver processing, sliver is often provided for shipment and use in cans or other containers which allow a first end of the sliver to be drawn or pulled from the can.","A second end of the sliver is often left available for splicing to the first end of another can.","This splicing is conventionally accomplished by a hand-type braid, twist, or other connection between the second end of the first can and the first end of the second can to join these respective ends of sliver together so that when the first can of sliver empties, the second can is ready to go for additional sliver processing.","This hand-type braiding or twisting of the sliver has also become somewhat of a specialty of different textile personnel in sliver handling and processing.","This manual procedure, however, can be time consuming, labor intensive, costly, inconsistent from person to person forming the braid or twist, and often is not strong or secure enough when the sliver is further handled or processed.","Other types of automatic splicing systems have been developed over the years.","Examples can be seen in U.S. Pat.","No.","5,544,389 by Onoue et al.","titled “Sliver Piecing In Spinning Machines,” U.S. Pat.","No.","5,140,722 by Akiyama titled “Sliver Piecing Device Having Fiber Entangling Needles And Air Jets,” U.S. Pat.","No.","5,058,241 by Haigh et al.","titled “Method And Apparatus For Combining Fibres Formed Into Slivers For Supply To Textile Machinery,” U.S. Pat.","No.","4,445,318 by Becker et al.","titled “Method And Device For Making A Knot-Free Thread Connection By Splicing,” U.S. Pat.","Nos.","4,969,323 and 4,982,563 each by Stahlecker and each titled “Sliver Splicing Arrangement For A Spinning Machine,” U.S. Pat.","No.","2,608,725 by Strew titled “Sliver Piecing Device,” U.S. Pat.","No.","3 , 308 , 520 by Gagnon titled “Process Of Splicing Tow,” U.S. Pat.","No.","5,359,758 by Stahlecker et al.","titled “Process And An Arrangement For The Piercing Of A Sliver,” Japanese Patent Document Application No.","05105652 by Takashi titled “Sliver Joining Apparatus In Spinning Machine,” and German Patent Document No.","90-210593/28 titled “Automatic Splicer For Roving On Ring-Spinning Frame—Has Needle Arrangement To Felt Fibres.” These automated systems, however, can be quite expensive to install, can be costly to operate, can have various complex mechanical and/or electrical problems, can take up additional floor space in manufacturing environments, can be bulky and awkward to use, and can often require extensive special training for personnel or the hiring of special personnel to oversee this automated equipment."],["<SOH> SUMMARY OF THE INVENTION <EOH>With the foregoing in mind, the present invention advantageously provides an apparatus and methods for splicing slivers of yarn during formation and processing which is compact, relatively simple to use, and readily portable.","The present invention also advantageously provides an apparatus and methods for splicing various types of textile slivers which is relatively inexpensive and does not require extensive personnel training to understand and operate.","The present invention additionally advantageously provides an apparatus and methods for splicing various types of textile sliver which allows the user to easily handle, carry, and tote and which is of such a size as to be easily inserted into a pocket of a garment or a carrying case worn by the user.","The present invention still also advantageously provides an apparatus and methods for splicing textile sliver which forms a substantially secure connection between ends of sliver being spliced or joined for later handling and/or processing.","The present invention further advantageously provides an apparatus and methods for splicing textile sliver which is less complex, easy to repair or replace parts, and is readily adaptable to various splicing needs and situations.","More particularly, the present invention provides an apparatus for splicing selected portions of sliver which preferably includes a needle carrying member having a plurality of needles to engage sliver when positioned adjacent thereto, a needle engaging member positioned to receive the plurality of needles from the first needle carrying member when the plurality of needles engage the sliver in a closed position, and a hand-activated needle actuation device connected to the needle carrying member and the needle engaging member to position the needle carrying member in an open position so that the plurality of needles is spaced-apart from the needle engaging member to allow sliver to be spliced to be readily positioned therebetween and responsive to grippingly closing at least portions of the hand of a user to actuate the engaging of the needle carrying member with the sliver and the needle engaging member when the sliver is positioned between the needle carrying member and the needle engaging member so that the engaging of plurality of needles of the needle carrying member with the needle engaging member thereby defines a closed position.","The present invention also advantageously provides an apparatus for splicing sliver which preferably includes a first handle portion having at least one sliver engaging member, a second handle portion positioned to receive the at least one sliver engaging member in a closed position, and a pivot member associated with the first and second handle portions to allow either the first or second handle portions to pivot about the pivot member between respective open and closed positions.","The open position preferably is defined by portions of the first handle member having the at least one sliver engaging member being spaced-apart from portions of the second handle portion positioned to receive the at least one sliver engaging member, and the closed position preferably is defined by the at least one sliver engaging member of the first handle portion being received by the portions of the second handle member when positioned closely adjacent thereto.","The apparatus preferably further includes a biasing member associated with the pivot member and positioned to bias either the first or the second handle portions in a preselected biased position.","The present invention additionally provides an apparatus for splicing sliver which preferably includes a first handle portion having a first handle body and at least one sliver engaging member detachably connected to the first handle body to thereby define a cartridge member, a second handle portion pivotally connected to and positioned to receive the at least one sliver engaging member and pivot between open and closed positions.","The open position is preferably defined by the at least one sliver engaging member being spaced-apart from portions of the second handle portion positioned to receive the at least one sliver engaging member, and the closed position preferably is defined by the at least one sliver engaging member of the first handle portion being received by the portions of the second handle member when positioned closely adjacent thereto.","The apparatus preferably also includes a biasing member positioned to bias either the first or the second handle portions in a preselected biased position.","The present invention further provides methods of splicing sliver.","A first method preferably includes grippingly closing a handle portion of a sliver splicer having at least one sliver engaging member by the hand of a user so that the at least one sliver engaging member engages and splices sliver positioned adjacent thereto and releasingly opening the handle portion by the hand of the user to thereby release the spliced portion of sliver from the at least one sliver engaging member.","Another method of splicing sliver, according to the present invention, preferably includes closing a handle portion of a needle engaging member having a plurality of needles so that the plurality of needles engages and splices sliver positioned adjacent thereto and opening the handle portion of the needle engaging member so that the plurality of needles release the spliced portions of sliver therefrom.","Yet another method of splicing sliver according to the present invention preferably includes joining first portions of sliver with a plurality of needles each having a recessed portion to engage and intertwine with adjacent second portions of sliver, the plurality of needles being connected to a body portion so that the body portion and the plurality of needles in combination define a needle cartridge member and replacing the needle cartridge member with an auxiliary cartridge member also having a body portion and a plurality of needles connected to the body portion.","The apparatus and methods of the present invention provide additional manufacturing, handling, processing, and formation flexibility in the use of the splicers for sliver.","For example, manufacturing personnel can walk around a facility with an apparatus of the present invention positioned in a pocket, holster, or harness when the splicing apparatus or splicer is preferably in a locked closed position so that the manufacturing personnel can readily remove the splicer, unlock the splicer, accomplish the splicing function, relock the splicer, and return the splicer to the pocket, holster, or harness.","Additionally, the splicing apparatus of the present invention can be strapped to a chain or belt which can enhance carrying and portability.","Further, when one or more needles or other sliver engaging members are damaged, according to one embodiment of the present invention, a cartridge member can readily be removed which carries the needles and replaced with an auxiliary cartridge member.","This cartridge replacement, for example, prevents the need to replace the entire splicing apparatus and saves money and reordering time.","Also, because the splicing apparatus is portable, compact, and relatively of simple construction and low cost, many different types of manufacturing personnel can use the splicing apparatus and can readily order additional or readily replace the entire splicing apparatus if desired without incurring extensive costs."],["FIELD OF THE INVENTION The present invention relates to the textile industry and, more particularly, to the field of textile splicing and methods.","BACKGROUND OF THE INVENTION In the textile industry, yarn is formed of a plurality of slivers.","To form the yarn, however, various steps are required to obtain the type and texture of the yarn desired.","During sliver processing, sliver is often provided for shipment and use in cans or other containers which allow a first end of the sliver to be drawn or pulled from the can.","A second end of the sliver is often left available for splicing to the first end of another can.","This splicing is conventionally accomplished by a hand-type braid, twist, or other connection between the second end of the first can and the first end of the second can to join these respective ends of sliver together so that when the first can of sliver empties, the second can is ready to go for additional sliver processing.","This hand-type braiding or twisting of the sliver has also become somewhat of a specialty of different textile personnel in sliver handling and processing.","This manual procedure, however, can be time consuming, labor intensive, costly, inconsistent from person to person forming the braid or twist, and often is not strong or secure enough when the sliver is further handled or processed.","Other types of automatic splicing systems have been developed over the years.","Examples can be seen in U.S. Pat.","No.","5,544,389 by Onoue et al.","titled “Sliver Piecing In Spinning Machines,” U.S. Pat.","No.","5,140,722 by Akiyama titled “Sliver Piecing Device Having Fiber Entangling Needles And Air Jets,” U.S. Pat.","No.","5,058,241 by Haigh et al.","titled “Method And Apparatus For Combining Fibres Formed Into Slivers For Supply To Textile Machinery,” U.S. Pat.","No.","4,445,318 by Becker et al.","titled “Method And Device For Making A Knot-Free Thread Connection By Splicing,” U.S. Pat.","Nos.","4,969,323 and 4,982,563 each by Stahlecker and each titled “Sliver Splicing Arrangement For A Spinning Machine,” U.S. Pat.","No.","2,608,725 by Strew titled “Sliver Piecing Device,” U.S. Pat.","No.","3,308,520 by Gagnon titled “Process Of Splicing Tow,” U.S. Pat.","No.","5,359,758 by Stahlecker et al.","titled “Process And An Arrangement For The Piercing Of A Sliver,” Japanese Patent Document Application No.","05105652 by Takashi titled “Sliver Joining Apparatus In Spinning Machine,” and German Patent Document No.","90-210593/28 titled “Automatic Splicer For Roving On Ring-Spinning Frame—Has Needle Arrangement To Felt Fibres.” These automated systems, however, can be quite expensive to install, can be costly to operate, can have various complex mechanical and/or electrical problems, can take up additional floor space in manufacturing environments, can be bulky and awkward to use, and can often require extensive special training for personnel or the hiring of special personnel to oversee this automated equipment.","SUMMARY OF THE INVENTION With the foregoing in mind, the present invention advantageously provides an apparatus and methods for splicing slivers of yarn during formation and processing which is compact, relatively simple to use, and readily portable.","The present invention also advantageously provides an apparatus and methods for splicing various types of textile slivers which is relatively inexpensive and does not require extensive personnel training to understand and operate.","The present invention additionally advantageously provides an apparatus and methods for splicing various types of textile sliver which allows the user to easily handle, carry, and tote and which is of such a size as to be easily inserted into a pocket of a garment or a carrying case worn by the user.","The present invention still also advantageously provides an apparatus and methods for splicing textile sliver which forms a substantially secure connection between ends of sliver being spliced or joined for later handling and/or processing.","The present invention further advantageously provides an apparatus and methods for splicing textile sliver which is less complex, easy to repair or replace parts, and is readily adaptable to various splicing needs and situations.","More particularly, the present invention provides an apparatus for splicing selected portions of sliver which preferably includes a needle carrying member having a plurality of needles to engage sliver when positioned adjacent thereto, a needle engaging member positioned to receive the plurality of needles from the first needle carrying member when the plurality of needles engage the sliver in a closed position, and a hand-activated needle actuation device connected to the needle carrying member and the needle engaging member to position the needle carrying member in an open position so that the plurality of needles is spaced-apart from the needle engaging member to allow sliver to be spliced to be readily positioned therebetween and responsive to grippingly closing at least portions of the hand of a user to actuate the engaging of the needle carrying member with the sliver and the needle engaging member when the sliver is positioned between the needle carrying member and the needle engaging member so that the engaging of plurality of needles of the needle carrying member with the needle engaging member thereby defines a closed position.","The present invention also advantageously provides an apparatus for splicing sliver which preferably includes a first handle portion having at least one sliver engaging member, a second handle portion positioned to receive the at least one sliver engaging member in a closed position, and a pivot member associated with the first and second handle portions to allow either the first or second handle portions to pivot about the pivot member between respective open and closed positions.","The open position preferably is defined by portions of the first handle member having the at least one sliver engaging member being spaced-apart from portions of the second handle portion positioned to receive the at least one sliver engaging member, and the closed position preferably is defined by the at least one sliver engaging member of the first handle portion being received by the portions of the second handle member when positioned closely adjacent thereto.","The apparatus preferably further includes a biasing member associated with the pivot member and positioned to bias either the first or the second handle portions in a preselected biased position.","The present invention additionally provides an apparatus for splicing sliver which preferably includes a first handle portion having a first handle body and at least one sliver engaging member detachably connected to the first handle body to thereby define a cartridge member, a second handle portion pivotally connected to and positioned to receive the at least one sliver engaging member and pivot between open and closed positions.","The open position is preferably defined by the at least one sliver engaging member being spaced-apart from portions of the second handle portion positioned to receive the at least one sliver engaging member, and the closed position preferably is defined by the at least one sliver engaging member of the first handle portion being received by the portions of the second handle member when positioned closely adjacent thereto.","The apparatus preferably also includes a biasing member positioned to bias either the first or the second handle portions in a preselected biased position.","The present invention further provides methods of splicing sliver.","A first method preferably includes grippingly closing a handle portion of a sliver splicer having at least one sliver engaging member by the hand of a user so that the at least one sliver engaging member engages and splices sliver positioned adjacent thereto and releasingly opening the handle portion by the hand of the user to thereby release the spliced portion of sliver from the at least one sliver engaging member.","Another method of splicing sliver, according to the present invention, preferably includes closing a handle portion of a needle engaging member having a plurality of needles so that the plurality of needles engages and splices sliver positioned adjacent thereto and opening the handle portion of the needle engaging member so that the plurality of needles release the spliced portions of sliver therefrom.","Yet another method of splicing sliver according to the present invention preferably includes joining first portions of sliver with a plurality of needles each having a recessed portion to engage and intertwine with adjacent second portions of sliver, the plurality of needles being connected to a body portion so that the body portion and the plurality of needles in combination define a needle cartridge member and replacing the needle cartridge member with an auxiliary cartridge member also having a body portion and a plurality of needles connected to the body portion.","The apparatus and methods of the present invention provide additional manufacturing, handling, processing, and formation flexibility in the use of the splicers for sliver.","For example, manufacturing personnel can walk around a facility with an apparatus of the present invention positioned in a pocket, holster, or harness when the splicing apparatus or splicer is preferably in a locked closed position so that the manufacturing personnel can readily remove the splicer, unlock the splicer, accomplish the splicing function, relock the splicer, and return the splicer to the pocket, holster, or harness.","Additionally, the splicing apparatus of the present invention can be strapped to a chain or belt which can enhance carrying and portability.","Further, when one or more needles or other sliver engaging members are damaged, according to one embodiment of the present invention, a cartridge member can readily be removed which carries the needles and replaced with an auxiliary cartridge member.","This cartridge replacement, for example, prevents the need to replace the entire splicing apparatus and saves money and reordering time.","Also, because the splicing apparatus is portable, compact, and relatively of simple construction and low cost, many different types of manufacturing personnel can use the splicing apparatus and can readily order additional or readily replace the entire splicing apparatus if desired without incurring extensive costs.","BRIEF DESCRIPTION OF THE DRAWINGS Some of the features, advantages, and benefits of the present invention having been stated, others will become apparent as the description proceeds when taken in conjunction with the accompanying drawings in which: FIG.","1 is a perspective view of an apparatus for splicing sliver according to a first embodiment of the present invention; FIG.","2 is a side elevational view of an apparatus for splicing sliver being actuated by the hand of a user according to a first embodiment of the present invention; FIG.","3 is an enlarged fragmentary elevational view of a type of needle of an apparatus for splicing sliver according to the present invention; FIG.","4 is a sectional view of a needle of an apparatus for splicing sliver taken along line 4-4 of FIG.","3 according to the present invention; FIG.","5 is an enlarged fragmentary elevational view of a needle of an apparatus for splicing sliver according to the present invention; FIG.","6 is a sectional view of a needle of an apparatus for splicing sliver taken along line 6-6 of FIG.","5 according to the present invention; FIGS.","7A-7D are schematic perspective views of the operation of an apparatus for splicing sliver according to a first embodiment of the present invention; FIG.","8A is an enlarged fragmentary perspective view of an apparatus for splicing sliver prior to engagement with sliver in an open position according to the present invention; FIG.","8B is an enlarged fragmentary perspective view of an apparatus for splicing sliver after engagement with the sliver in a closed position according to the present invention; FIG.","8C is an enlarged fragmentary perspective view of an apparatus for splicing sliver after engagement with the sliver in an open position according to the present invention; FIG.","9 is an enlarged fragmentary view of sliver after being spliced with an apparatus for splicing sliver according to the present invention; FIG.","10 is a perspective view of an apparatus for splicing sliver according to a second embodiment of the present invention; FIG.","11 is an exploded fragmentary perspective, view of a needle cartridge for positioning in an apparatus for splicing sliver according to a second embodiment of the present invention; FIG.","12 is a side elevational view of an apparatus for splicing sliver having a needle cartridge illustrated by broken lines for clarity according to a second embodiment of the present invention; FIG.","13 is an exploded side elevational view of an apparatus for splicing sliver having a needle cartridge member and a plurality of replacement auxiliary needle cartridge members according to a second embodiment of the present invention; FIG.","14 is a fragmentary perspective view a portion of an apparatus for splicing sliver illustrating a connection between first and second handle portions along a pivot member and the position of a biasing member to bias the apparatus in an open position according to the present invention; and FIG.","15 is an apparatus for splicing sliver according to a third embodiment of the present invention.","DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS The present invention will now be described more fully hereinafter with reference to the accompanying drawings which illustrate preferred embodiments of the invention.","This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.","Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.","Like numbers refer to like elements throughout, the prime notation, if used, indicates similar elements in alternative embodiments.","FIGS.","1-9 illustrate a first embodiment of an apparatus 20 for splicing sliver S. The apparatus 20 preferably includes a first handle portion 30 having at least one sliver engaging member.","The at least one sliver engaging member is preferably one or more needles 32, and more preferably a plurality of needles 32, connected to the first handle portion 30.A second handle portion 40 preferably is positioned to receive the at least one sliver engaging member, e.g., the plurality of needles 32, in a closed position such as through or within one or more openings or bores 42, 42′ as shown (see also FIG.","10).. As perhaps be shown in FIGS.","1-2 and 14, a pivot member 25 is associated with, and preferably connected to or mounted as illustrated, to the first and second handle portions 30, 40 to allow either the first or second handle portions 30, 40 to pivot about the pivot member 25 between respective open and closed positions.","The open position preferably is defined by distal portions 31 of the first handle portion 30 having the at least one sliver engaging member being spaced-apart from distal portions 31 of the second handle portion 40 positioned to receive the at least one sliver engaging member, and the closed position preferably is defined by the at least one sliver engaging member of the first handle portion 30 being received by the distal portions of the second handle portion 40 when positioned closely adjacent thereto.","The first and second handle portions 30, 40 can advantageously defined a base which is advantageously hand operated, but other base structures as understood by those skilled in the art which may or may not be hand operated can be used as well according to the present invention.","The apparatus 20 preferably includes a biasing member 28 associated with the pivot member 25 and positioned to bias either the first or the second handle portions 30, 40 in a preselected biased position.","In the embodiment of the splicing apparatus 20 as illustrated, the first handle portion 30 is biased by the biasing member 28 to the open position.","As shown in FIG.","14, the biasing member 28 is preferably a spring as understood by those skilled in the art and preferably is connected to the pivot member 25 and positioned to bias the first handle portion 30 in the open position.","Other types of biasing members, including various types of springs, can be used as well.","Although the illustrated embodiment of biasing the first handle portion 30 in the open position is particularly advantageous due to the movement desired in the splicing process and the ease of use by a user's hand, for example, it will also be understood by those skilled in the art that the present invention would include biasing the splicing apparatus 2011 in the closed position (see also FIG.","15).","Further, the apparatus 20 preferably also includes a lock 50 associated with the first and second handle portions 30, 40 to lock the first and second handle portions 30, 40 in a closed position.","In the embodiment shown, the lock 50 is primarily connected to the first handle portion 30 and slidably engages or locks, e.g., a latch, with the second handle portion 40, e.g., into a slot or channel by connecting to the needle receiving portion or body 45 of the second handle portion 40 (see FIG.","2).","As perhaps best illustrated in FIG.","2, the first and second handle portions 30, 40 define a hand-activated needle actuation device which advantageously allows a ready grip by a user's hand H to apply pressure from the user's hand H to actuate the movement of the plurality of needles 32.As described above, and as illustrated in FIGS.","7A-7D and 8A-8C, the at least one sliver engaging member preferably includes a plurality of needles 32, and the hand-activated needle actuation device is responsive to grippingly closing at least portions of the hand H of a user to actuate the engaging of the plurality of needles 32 with the sliver S to be spliced and the distal portions 45, e.g., through the openings or bores 42, 42′ of the second handle portion 40 when the sliver S is positioned between the plurality of needles 32 and the distal portions 45 of the second handle portion 40 so that the engaging of plurality of needles 32 with the distal portions 45 of the second handle portion 40 thereby further defines the closed position (see also FIG.","10).","Also, as perhaps best shown in FIGS.","3-6 and 8A-8C, each of the plurality of needles 32 preferably includes a needle body 33, 33′ and a recessed portion 34, 34′ formed in the needle body 33, 33′ and positioned to assist in the engaging of and interconnecting of the sliver S when each needle 32, 32′ engages sliver S during movement to the closed position.","The recessed portion 34, 34′ preferably includes at least one of the following: a barb, a groove, and a channel.","The recessed portion 34, 34′ preferably has an upward slope with respect to the downward movement of the needle 32 so that the recessed portion 34, 34′ readily catches, engages, or otherwise contacts the sliver S during the downward motion of the needles and responsively releases the sliver S during upward motion.","This process allows the intertwining or interconnecting of the sliver S to join the portions of sliver S desired to be spliced together.","As shown in FIGS.","10-13 and 15, according to a second embodiment of a splicing apparatus 20′ of the present invention, the at least one sliver engaging member preferably is detachably connected to the first handle portion 30′ and defines a cartridge member 35, 35′ positioned in distal portions 31′ of the first handle portion 30′ to readily remove from the first handle portion 30′.","At least one replacement auxiliary cartridge member 38, and preferably a plurality of replacement auxiliary cartridge members 38, can have the same construction as the cartridge member 35, 35′ and can be adapted to be readily positioned in the first handle portion 30′.","Accordingly, a kit can also be provided which has a portable splicing apparatus 20′ positioned in a container, e.g., box, bag, package, with one or more auxiliary cartridge members 38, so that when a cartridge member 35, 35′ being used is damaged, dulled, or otherwise desired to be replaced, another cartridge member 38 can be readily inserted into an opening 39 in the first handle portion 30′ of the splicing apparatus 20′ after removal of the damaged cartridge member 35, 35′ so that splicing operations proceed with substantially reduced interruptions.","The distal portion 45′ of the second handle portion 40′ preferably has additional springs or other types of biasing members 36 which allow the needles 32′ to retract and extend from openings 37 in the distal portion 31′ as shown.","Further still, as shown in FIG.","15, scissor-type handles 30″, 40″, substantially closed loops, and various other types of handle or finger grips can also be used.","In this embodiment of a splicing apparatus 20″, the pivot member 25″ is also moved forward toward a more medial portion of the handle members 30″, 40″ and the biasing member 28″ is another type of spring, as understood by those skilled in the art, which biases the scissor-type handle embodiment to a closed position so that the distal portions 31″, 45″ of the handles are positioned closely adjacent each other and the plurality of needles 32″ can engage receiving portions of the handle 40″.","As illustrated in FIGS.","1-15, the present invention further provides methods of splicing sliver S. A first method preferably includes grippingly closing a handle portion 30 of a sliver splicer 20 having at least one sliver engaging member 32 by the hand H of a user so that the at least one sliver engaging member 32 engages and splices sliver S positioned adjacent thereto and releasingly opening the handle portion 30 by the hand H of the user to thereby release the spliced portion of sliver S from the at least one sliver engaging member 32.Another method of splicing sliver S according to the present invention preferably includes closing a handle portion 30 of a needle engaging member having a plurality of needles 32 so that the plurality of needles 32 engages and splices sliver S positioned adjacent thereto and opening the handle portion 30 of the needle engaging member so that the plurality of needles 32 release the spliced portions of sliver S therefrom.","Yet another method of splicing sliver S according to the present invention preferably includes joining first portions of sliver S with a plurality of needles 32 each having a recessed portion 34 to engage and intertwine with adjacent second portions of sliver S. The plurality of needles preferably is connected to a body portion so that the body portion and the plurality of needles 32 in combination define a needle cartridge member 35 and replacing the needle cartridge member 35 with an auxiliary cartridge member 38 also having a body portion and a plurality of needles 32 connected to the body portion.","The apparatus 20, 20′, 20″ and methods of the present invention provide additional manufacturing, handling, processing, and formation flexibility in the use of the splicers for sliver.","For example, manufacturing personnel can walk around a facility with an apparatus 20 of the present invention positioned in a pocket, holster, or harness when the splicing apparatus 20 or splicer is preferably in a locked closed position so that the manufacturing personnel can readily remove the splicer 20, unlock the splicer 20, accomplish the splicing function, relock the splicer 20, and return the splicer 20 to the pocket, holster, or harness.","Additionally, the splicing apparatus 20 of the present invention can be strapped to a chain or belt which can enhance carrying and portability.","Further, when one or more needles 32 or other sliver engaging members are damaged, according to one embodiment of the present invention, a cartridge member 35 can readily be removed which carries the needles 32′ and replaced with an auxiliary cartridge member 38.This cartridge replacement, for example, prevents the need to replace the entire splicing apparatus 20′ and saves money and reordering time.","Also, because the splicing apparatus 20 is portable, compact, and relatively of simple construction and low cost, many different types of manufacturing personnel can use the splicing apparatus 20, 20′, 20″ and can readily order additional or readily replace the entire splicing apparatus 20, 20′, 20″ if desired without incurring extensive costs.","In the drawings and specification, there have been disclosed a typical preferred embodiment of the invention, and although specific terms are employed, the terms are used in a descriptive sense only and not for purposes of limitation.","The invention has been described in considerable detail with specific reference to these illustrated embodiments.","It will be apparent, however, that various modifications and changes can be made within the spirit and scope of the invention as described in the foregoing specification and as defined in the appended claims."]],"string":"[\n [\n \"Apparatus and method for splicing sliver of yarn during yarn formation and processing\",\n \"An apparatus (20) and methods are provided for splicing selected portions of sliver (S) such as during yarn formation and processing.\",\n \"The apparatus (20) preferably includes a needle carrying member (35) having a plurality of needles (32) to engage silver (S) when positioned adjacent thereto and a needle engaging member (45) positioned to underlie the needle carrying member (35) and to receive the plurality of needles (32) from the first needle carrying member (35).\",\n \"The apparatus (20) also preferably includes a hand-activated needle actuation device (30, 40) connected to the needle carrying member (35) and the needle engaging member (45) to position the needle carrying member (35) in an open position to allow sliver (S) to be spliced to be readily positioned therebetween and responsive to closing at least portions of the hand (H) of a user for actuating the engaging of the needle carrying member (35) with the sliver (S) and the needle engaging member (45) so that the engaging of plurality of needles (32) of the needle carrying member (35) with the needle engaging member (45) thereby defines a closed position.\"\n ],\n [\n \"1-18.\",\n \"(canceled) 19.An apparatus for splicing selected portions of sliver, the apparatus comprising: a needle carrying member having a plurality of needles to engage sliver when positioned adjacent thereto; a needle engaging member positioned to receive the plurality of needles from the needle carrying member when the plurality of needles engage the sliver in a closed position; and a hand-activated needle actuation device connected to the needle carrying member and the needle engaging member to position the needle carrying member in an open position so that the plurality of needles is spaced-apart from the needle engaging member to allow sliver to be spliced to be readily positioned therebetween, the hand-activated needle actuation device being responsive to grippingly closing at least portions of a hand of a user to actuate the engaging of the needle carrying member with the sliver and the needle engaging member when the sliver is positioned between the needle carrying member and the needle engaging member so that the engaging of the plurality of needles of the needle carrying member with the needle engaging member thereby defines a closed position.\",\n \"20.An apparatus as defined in claim 19, wherein the hand-activated needle actuation device includes a first handle portion connected to the needle carrying member and positioned to be gripped by a hand of a user, a second handle portion connected to the needle engaging member and positioned to be gripped by a hand of a user, a pivot member connected to the first and second handle portions to allow either the first or second handle portions to pivot about the pivot member between the respective open and closed positions, and a biasing member connected to the pivot member and positioned to bias the needle carrying member in the open position.\",\n \"21.An apparatus as defined in claim 19, wherein the hand-activated needle actuation device includes a first handle portion connected to the needle carrying member and positioned to be gripped by a hand of a user, a second handle portion connected to the needle engaging member and positioned to be gripped by a hand of a user, a pivot member connected to the first and second handle portions to allow either the first or second handle portions to pivot about the pivot member between the respective open and closed positions, and a biasing member associated with the first and second handle portions and positioned to bias the needle carrying member in the closed position.\",\n \"22.An apparatus as defined in claim 19, wherein the needle engaging member includes a body member and a plurality of openings formed in the body member and each extending to a depth in the body member so that none of the plurality of needles extend outwardly from the needle engaging member when in the closed position, the number of the plurality of openings being equal to the number of the plurality of needles, each of the plurality of openings being positioned to underlie a corresponding one of the plurality of needles of the needles carrying member so that the plurality of openings matingly receive the plurality of needles as the plurality of needles pass into and out of the plurality of openings to thereby engage the needles and sliver being carried therewith.\",\n \"23.An apparatus as defined in claim 22, wherein the body member of the needle engaging member includes a substantially closed bottom, and wherein the bottom underlies each of the plurality of openings so that portions of the bottom define a floor for each of the plurality of openings.\",\n \"24.An apparatus as defined in claim 19, further comprising a lock associated with the hand activated needle actuation device to lock the hand activated needle actuation device in the closed position so that none of the plurality of needles extend outwardly from the needle engaging member when locked in the closed position.\",\n \"25.An apparatus as defined in claims 19, wherein each of the plurality of needles includes a needle body and a recessed portion formed in the needle body and positioned to assist in the engaging of and interconnecting of the sliver when each needle engages sliver during movement to the closed position.\",\n \"26.An apparatus as defined in claims 20, wherein the needle carrying member is detachably connected to at least one of the first handle portion and the second handle portion and defines a needle cartridge to readily remove from the respective first handle portion and second handle portion.\",\n \"27.An apparatus as defined in claims 26, further comprising at least one replacement auxiliary needle cartridge adapted to be readily positioned in the at least one of the first handle portion and the second handle portion.\",\n \"28.An apparatus for splicing sliver, the apparatus comprising a base and a needle receiving member associated with the base and positioned to receive a plurality of needles, the apparatus being characterized by having: a first cartridge member detachably connected to the base to be readily removed therefrom, the first cartridge member having a combination of a body portion and a plurality of needles each connected to the body portion, each of the plurality of needles having a recessed portion to engage and intertwine first portions of sliver with adjacent second portions of sliver; and a second auxiliary replacement cartridge member adapted to be detachably connected to the base and to be placed in substantially the same position as the first cartridge member, the second auxiliary replacement cartridge member also having a combination of a body portion and a plurality of needles each connected to the body portion and adapted to be positioned to be received by the needle receiving member, each of the plurality of needles having a recessed portion to engage and intertwine first portions of sliver with adjacent second portions of sliver.\",\n \"29.An apparatus as defined in claim 28, further comprising a pivot member associated with the first cartridge member and the needle receiving member to allow either the first cartridge member or needle receiving member to pivot about the pivot member between respective open and closed positions, the open position being defined by portions of the first cartridge member being spaced-apart from portions of the needle receiving member to receive the first cartridge member and the closed position being defined by the first cartridge member being received by portions of the needle receiving member when positioned closely adjacent thereto.\",\n \"30.An apparatus as defined in claim 29, further comprising a biasing member associated with the pivot member and positioned to bias either the first cartridge member or the needle receiving member in a preselected biased position.\",\n \"31.An apparatus as defined in claim 28, wherein the recessed portion of each of the plurality of needles of the first and second cartridge members includes at least one of the following: a barb, a groove, and a channel.\",\n \"32.An apparatus as defined in claims 28, a lock associated with the first cartridge member and the needle receiving member to lock the first cartridge member and the needle receiving member in a closed position.\",\n \"33.A method of splicing sliver, the method being characterized by the steps of: grippingly closing a first handle portion and a second handle portion of a sliver splicer having at least one sliver engaging member by a hand of a user so that the at least one sliver engaging member engages and splices sliver positioned adjacent thereto when in a closed position, the at least one sliver engaging member having a plurality of needles each including a recessed portion to engage and intertwine first portions of the sliver with second portions of the sliver to thereby join the first portions to the second portions of the sliver; and releasingly opening the first handle portion and the second handle portion by the hand of the user from the closed position to an open position to thereby release the spliced portion of sliver from the at least one sliver engaging member.\",\n \"34.A method as defined in claim 33, further comprising the steps of removing the spliced portion from adjacent the at least one sliver engaging member, grippingly closing the first handle portion and the second handle portion to the closed position without sliver positioned therebetween, and locking the first handle portion and the second handle portion in the closed position.\",\n \"35.A method as defined in claim 34, further comprising removing the spliced portion from adjacent the at least one sliver engaging member and biasingly closing the first handle portion and the second handle portion to the closed position without sliver positioned therebetween.\",\n \"36.A method as defined in claim 35, wherein none of the recessed portion of each of the plurality of needles is exposed when in the closed position.\",\n \"37.A method of splicing sliver comprising joining first portions of sliver with a plurality of needles having a recessed portion to engage and intertwine with adjacent second portions of sliver, the method characterized by having: the plurality of needles being connected to a body portion so that the body portion and the plurality of needles in the combination define a needle cartridge member; and replacing the needle cartridge member with an auxiliary cartridge member also having a body portion and a plurality of needles connected to the body portion.\",\n \"38.A method as defined in claims 37, wherein each of the plurality of needles includes a needle body, and wherein the recessed portion is formed in the needle body and positioned to assist in the engaging of and interconnecting of the sliver when each needle engages sliver.\"\n ],\n [\n \"<SOH> BACKGROUND OF THE INVENTION <EOH>In the textile industry, yarn is formed of a plurality of slivers.\",\n \"To form the yarn, however, various steps are required to obtain the type and texture of the yarn desired.\",\n \"During sliver processing, sliver is often provided for shipment and use in cans or other containers which allow a first end of the sliver to be drawn or pulled from the can.\",\n \"A second end of the sliver is often left available for splicing to the first end of another can.\",\n \"This splicing is conventionally accomplished by a hand-type braid, twist, or other connection between the second end of the first can and the first end of the second can to join these respective ends of sliver together so that when the first can of sliver empties, the second can is ready to go for additional sliver processing.\",\n \"This hand-type braiding or twisting of the sliver has also become somewhat of a specialty of different textile personnel in sliver handling and processing.\",\n \"This manual procedure, however, can be time consuming, labor intensive, costly, inconsistent from person to person forming the braid or twist, and often is not strong or secure enough when the sliver is further handled or processed.\",\n \"Other types of automatic splicing systems have been developed over the years.\",\n \"Examples can be seen in U.S. Pat.\",\n \"No.\",\n \"5,544,389 by Onoue et al.\",\n \"titled “Sliver Piecing In Spinning Machines,” U.S. Pat.\",\n \"No.\",\n \"5,140,722 by Akiyama titled “Sliver Piecing Device Having Fiber Entangling Needles And Air Jets,” U.S. Pat.\",\n \"No.\",\n \"5,058,241 by Haigh et al.\",\n \"titled “Method And Apparatus For Combining Fibres Formed Into Slivers For Supply To Textile Machinery,” U.S. Pat.\",\n \"No.\",\n \"4,445,318 by Becker et al.\",\n \"titled “Method And Device For Making A Knot-Free Thread Connection By Splicing,” U.S. Pat.\",\n \"Nos.\",\n \"4,969,323 and 4,982,563 each by Stahlecker and each titled “Sliver Splicing Arrangement For A Spinning Machine,” U.S. Pat.\",\n \"No.\",\n \"2,608,725 by Strew titled “Sliver Piecing Device,” U.S. Pat.\",\n \"No.\",\n \"3 , 308 , 520 by Gagnon titled “Process Of Splicing Tow,” U.S. Pat.\",\n \"No.\",\n \"5,359,758 by Stahlecker et al.\",\n \"titled “Process And An Arrangement For The Piercing Of A Sliver,” Japanese Patent Document Application No.\",\n \"05105652 by Takashi titled “Sliver Joining Apparatus In Spinning Machine,” and German Patent Document No.\",\n \"90-210593/28 titled “Automatic Splicer For Roving On Ring-Spinning Frame—Has Needle Arrangement To Felt Fibres.” These automated systems, however, can be quite expensive to install, can be costly to operate, can have various complex mechanical and/or electrical problems, can take up additional floor space in manufacturing environments, can be bulky and awkward to use, and can often require extensive special training for personnel or the hiring of special personnel to oversee this automated equipment.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>With the foregoing in mind, the present invention advantageously provides an apparatus and methods for splicing slivers of yarn during formation and processing which is compact, relatively simple to use, and readily portable.\",\n \"The present invention also advantageously provides an apparatus and methods for splicing various types of textile slivers which is relatively inexpensive and does not require extensive personnel training to understand and operate.\",\n \"The present invention additionally advantageously provides an apparatus and methods for splicing various types of textile sliver which allows the user to easily handle, carry, and tote and which is of such a size as to be easily inserted into a pocket of a garment or a carrying case worn by the user.\",\n \"The present invention still also advantageously provides an apparatus and methods for splicing textile sliver which forms a substantially secure connection between ends of sliver being spliced or joined for later handling and/or processing.\",\n \"The present invention further advantageously provides an apparatus and methods for splicing textile sliver which is less complex, easy to repair or replace parts, and is readily adaptable to various splicing needs and situations.\",\n \"More particularly, the present invention provides an apparatus for splicing selected portions of sliver which preferably includes a needle carrying member having a plurality of needles to engage sliver when positioned adjacent thereto, a needle engaging member positioned to receive the plurality of needles from the first needle carrying member when the plurality of needles engage the sliver in a closed position, and a hand-activated needle actuation device connected to the needle carrying member and the needle engaging member to position the needle carrying member in an open position so that the plurality of needles is spaced-apart from the needle engaging member to allow sliver to be spliced to be readily positioned therebetween and responsive to grippingly closing at least portions of the hand of a user to actuate the engaging of the needle carrying member with the sliver and the needle engaging member when the sliver is positioned between the needle carrying member and the needle engaging member so that the engaging of plurality of needles of the needle carrying member with the needle engaging member thereby defines a closed position.\",\n \"The present invention also advantageously provides an apparatus for splicing sliver which preferably includes a first handle portion having at least one sliver engaging member, a second handle portion positioned to receive the at least one sliver engaging member in a closed position, and a pivot member associated with the first and second handle portions to allow either the first or second handle portions to pivot about the pivot member between respective open and closed positions.\",\n \"The open position preferably is defined by portions of the first handle member having the at least one sliver engaging member being spaced-apart from portions of the second handle portion positioned to receive the at least one sliver engaging member, and the closed position preferably is defined by the at least one sliver engaging member of the first handle portion being received by the portions of the second handle member when positioned closely adjacent thereto.\",\n \"The apparatus preferably further includes a biasing member associated with the pivot member and positioned to bias either the first or the second handle portions in a preselected biased position.\",\n \"The present invention additionally provides an apparatus for splicing sliver which preferably includes a first handle portion having a first handle body and at least one sliver engaging member detachably connected to the first handle body to thereby define a cartridge member, a second handle portion pivotally connected to and positioned to receive the at least one sliver engaging member and pivot between open and closed positions.\",\n \"The open position is preferably defined by the at least one sliver engaging member being spaced-apart from portions of the second handle portion positioned to receive the at least one sliver engaging member, and the closed position preferably is defined by the at least one sliver engaging member of the first handle portion being received by the portions of the second handle member when positioned closely adjacent thereto.\",\n \"The apparatus preferably also includes a biasing member positioned to bias either the first or the second handle portions in a preselected biased position.\",\n \"The present invention further provides methods of splicing sliver.\",\n \"A first method preferably includes grippingly closing a handle portion of a sliver splicer having at least one sliver engaging member by the hand of a user so that the at least one sliver engaging member engages and splices sliver positioned adjacent thereto and releasingly opening the handle portion by the hand of the user to thereby release the spliced portion of sliver from the at least one sliver engaging member.\",\n \"Another method of splicing sliver, according to the present invention, preferably includes closing a handle portion of a needle engaging member having a plurality of needles so that the plurality of needles engages and splices sliver positioned adjacent thereto and opening the handle portion of the needle engaging member so that the plurality of needles release the spliced portions of sliver therefrom.\",\n \"Yet another method of splicing sliver according to the present invention preferably includes joining first portions of sliver with a plurality of needles each having a recessed portion to engage and intertwine with adjacent second portions of sliver, the plurality of needles being connected to a body portion so that the body portion and the plurality of needles in combination define a needle cartridge member and replacing the needle cartridge member with an auxiliary cartridge member also having a body portion and a plurality of needles connected to the body portion.\",\n \"The apparatus and methods of the present invention provide additional manufacturing, handling, processing, and formation flexibility in the use of the splicers for sliver.\",\n \"For example, manufacturing personnel can walk around a facility with an apparatus of the present invention positioned in a pocket, holster, or harness when the splicing apparatus or splicer is preferably in a locked closed position so that the manufacturing personnel can readily remove the splicer, unlock the splicer, accomplish the splicing function, relock the splicer, and return the splicer to the pocket, holster, or harness.\",\n \"Additionally, the splicing apparatus of the present invention can be strapped to a chain or belt which can enhance carrying and portability.\",\n \"Further, when one or more needles or other sliver engaging members are damaged, according to one embodiment of the present invention, a cartridge member can readily be removed which carries the needles and replaced with an auxiliary cartridge member.\",\n \"This cartridge replacement, for example, prevents the need to replace the entire splicing apparatus and saves money and reordering time.\",\n \"Also, because the splicing apparatus is portable, compact, and relatively of simple construction and low cost, many different types of manufacturing personnel can use the splicing apparatus and can readily order additional or readily replace the entire splicing apparatus if desired without incurring extensive costs.\"\n ],\n [\n \"FIELD OF THE INVENTION The present invention relates to the textile industry and, more particularly, to the field of textile splicing and methods.\",\n \"BACKGROUND OF THE INVENTION In the textile industry, yarn is formed of a plurality of slivers.\",\n \"To form the yarn, however, various steps are required to obtain the type and texture of the yarn desired.\",\n \"During sliver processing, sliver is often provided for shipment and use in cans or other containers which allow a first end of the sliver to be drawn or pulled from the can.\",\n \"A second end of the sliver is often left available for splicing to the first end of another can.\",\n \"This splicing is conventionally accomplished by a hand-type braid, twist, or other connection between the second end of the first can and the first end of the second can to join these respective ends of sliver together so that when the first can of sliver empties, the second can is ready to go for additional sliver processing.\",\n \"This hand-type braiding or twisting of the sliver has also become somewhat of a specialty of different textile personnel in sliver handling and processing.\",\n \"This manual procedure, however, can be time consuming, labor intensive, costly, inconsistent from person to person forming the braid or twist, and often is not strong or secure enough when the sliver is further handled or processed.\",\n \"Other types of automatic splicing systems have been developed over the years.\",\n \"Examples can be seen in U.S. Pat.\",\n \"No.\",\n \"5,544,389 by Onoue et al.\",\n \"titled “Sliver Piecing In Spinning Machines,” U.S. Pat.\",\n \"No.\",\n \"5,140,722 by Akiyama titled “Sliver Piecing Device Having Fiber Entangling Needles And Air Jets,” U.S. Pat.\",\n \"No.\",\n \"5,058,241 by Haigh et al.\",\n \"titled “Method And Apparatus For Combining Fibres Formed Into Slivers For Supply To Textile Machinery,” U.S. Pat.\",\n \"No.\",\n \"4,445,318 by Becker et al.\",\n \"titled “Method And Device For Making A Knot-Free Thread Connection By Splicing,” U.S. Pat.\",\n \"Nos.\",\n \"4,969,323 and 4,982,563 each by Stahlecker and each titled “Sliver Splicing Arrangement For A Spinning Machine,” U.S. Pat.\",\n \"No.\",\n \"2,608,725 by Strew titled “Sliver Piecing Device,” U.S. Pat.\",\n \"No.\",\n \"3,308,520 by Gagnon titled “Process Of Splicing Tow,” U.S. Pat.\",\n \"No.\",\n \"5,359,758 by Stahlecker et al.\",\n \"titled “Process And An Arrangement For The Piercing Of A Sliver,” Japanese Patent Document Application No.\",\n \"05105652 by Takashi titled “Sliver Joining Apparatus In Spinning Machine,” and German Patent Document No.\",\n \"90-210593/28 titled “Automatic Splicer For Roving On Ring-Spinning Frame—Has Needle Arrangement To Felt Fibres.” These automated systems, however, can be quite expensive to install, can be costly to operate, can have various complex mechanical and/or electrical problems, can take up additional floor space in manufacturing environments, can be bulky and awkward to use, and can often require extensive special training for personnel or the hiring of special personnel to oversee this automated equipment.\",\n \"SUMMARY OF THE INVENTION With the foregoing in mind, the present invention advantageously provides an apparatus and methods for splicing slivers of yarn during formation and processing which is compact, relatively simple to use, and readily portable.\",\n \"The present invention also advantageously provides an apparatus and methods for splicing various types of textile slivers which is relatively inexpensive and does not require extensive personnel training to understand and operate.\",\n \"The present invention additionally advantageously provides an apparatus and methods for splicing various types of textile sliver which allows the user to easily handle, carry, and tote and which is of such a size as to be easily inserted into a pocket of a garment or a carrying case worn by the user.\",\n \"The present invention still also advantageously provides an apparatus and methods for splicing textile sliver which forms a substantially secure connection between ends of sliver being spliced or joined for later handling and/or processing.\",\n \"The present invention further advantageously provides an apparatus and methods for splicing textile sliver which is less complex, easy to repair or replace parts, and is readily adaptable to various splicing needs and situations.\",\n \"More particularly, the present invention provides an apparatus for splicing selected portions of sliver which preferably includes a needle carrying member having a plurality of needles to engage sliver when positioned adjacent thereto, a needle engaging member positioned to receive the plurality of needles from the first needle carrying member when the plurality of needles engage the sliver in a closed position, and a hand-activated needle actuation device connected to the needle carrying member and the needle engaging member to position the needle carrying member in an open position so that the plurality of needles is spaced-apart from the needle engaging member to allow sliver to be spliced to be readily positioned therebetween and responsive to grippingly closing at least portions of the hand of a user to actuate the engaging of the needle carrying member with the sliver and the needle engaging member when the sliver is positioned between the needle carrying member and the needle engaging member so that the engaging of plurality of needles of the needle carrying member with the needle engaging member thereby defines a closed position.\",\n \"The present invention also advantageously provides an apparatus for splicing sliver which preferably includes a first handle portion having at least one sliver engaging member, a second handle portion positioned to receive the at least one sliver engaging member in a closed position, and a pivot member associated with the first and second handle portions to allow either the first or second handle portions to pivot about the pivot member between respective open and closed positions.\",\n \"The open position preferably is defined by portions of the first handle member having the at least one sliver engaging member being spaced-apart from portions of the second handle portion positioned to receive the at least one sliver engaging member, and the closed position preferably is defined by the at least one sliver engaging member of the first handle portion being received by the portions of the second handle member when positioned closely adjacent thereto.\",\n \"The apparatus preferably further includes a biasing member associated with the pivot member and positioned to bias either the first or the second handle portions in a preselected biased position.\",\n \"The present invention additionally provides an apparatus for splicing sliver which preferably includes a first handle portion having a first handle body and at least one sliver engaging member detachably connected to the first handle body to thereby define a cartridge member, a second handle portion pivotally connected to and positioned to receive the at least one sliver engaging member and pivot between open and closed positions.\",\n \"The open position is preferably defined by the at least one sliver engaging member being spaced-apart from portions of the second handle portion positioned to receive the at least one sliver engaging member, and the closed position preferably is defined by the at least one sliver engaging member of the first handle portion being received by the portions of the second handle member when positioned closely adjacent thereto.\",\n \"The apparatus preferably also includes a biasing member positioned to bias either the first or the second handle portions in a preselected biased position.\",\n \"The present invention further provides methods of splicing sliver.\",\n \"A first method preferably includes grippingly closing a handle portion of a sliver splicer having at least one sliver engaging member by the hand of a user so that the at least one sliver engaging member engages and splices sliver positioned adjacent thereto and releasingly opening the handle portion by the hand of the user to thereby release the spliced portion of sliver from the at least one sliver engaging member.\",\n \"Another method of splicing sliver, according to the present invention, preferably includes closing a handle portion of a needle engaging member having a plurality of needles so that the plurality of needles engages and splices sliver positioned adjacent thereto and opening the handle portion of the needle engaging member so that the plurality of needles release the spliced portions of sliver therefrom.\",\n \"Yet another method of splicing sliver according to the present invention preferably includes joining first portions of sliver with a plurality of needles each having a recessed portion to engage and intertwine with adjacent second portions of sliver, the plurality of needles being connected to a body portion so that the body portion and the plurality of needles in combination define a needle cartridge member and replacing the needle cartridge member with an auxiliary cartridge member also having a body portion and a plurality of needles connected to the body portion.\",\n \"The apparatus and methods of the present invention provide additional manufacturing, handling, processing, and formation flexibility in the use of the splicers for sliver.\",\n \"For example, manufacturing personnel can walk around a facility with an apparatus of the present invention positioned in a pocket, holster, or harness when the splicing apparatus or splicer is preferably in a locked closed position so that the manufacturing personnel can readily remove the splicer, unlock the splicer, accomplish the splicing function, relock the splicer, and return the splicer to the pocket, holster, or harness.\",\n \"Additionally, the splicing apparatus of the present invention can be strapped to a chain or belt which can enhance carrying and portability.\",\n \"Further, when one or more needles or other sliver engaging members are damaged, according to one embodiment of the present invention, a cartridge member can readily be removed which carries the needles and replaced with an auxiliary cartridge member.\",\n \"This cartridge replacement, for example, prevents the need to replace the entire splicing apparatus and saves money and reordering time.\",\n \"Also, because the splicing apparatus is portable, compact, and relatively of simple construction and low cost, many different types of manufacturing personnel can use the splicing apparatus and can readily order additional or readily replace the entire splicing apparatus if desired without incurring extensive costs.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS Some of the features, advantages, and benefits of the present invention having been stated, others will become apparent as the description proceeds when taken in conjunction with the accompanying drawings in which: FIG.\",\n \"1 is a perspective view of an apparatus for splicing sliver according to a first embodiment of the present invention; FIG.\",\n \"2 is a side elevational view of an apparatus for splicing sliver being actuated by the hand of a user according to a first embodiment of the present invention; FIG.\",\n \"3 is an enlarged fragmentary elevational view of a type of needle of an apparatus for splicing sliver according to the present invention; FIG.\",\n \"4 is a sectional view of a needle of an apparatus for splicing sliver taken along line 4-4 of FIG.\",\n \"3 according to the present invention; FIG.\",\n \"5 is an enlarged fragmentary elevational view of a needle of an apparatus for splicing sliver according to the present invention; FIG.\",\n \"6 is a sectional view of a needle of an apparatus for splicing sliver taken along line 6-6 of FIG.\",\n \"5 according to the present invention; FIGS.\",\n \"7A-7D are schematic perspective views of the operation of an apparatus for splicing sliver according to a first embodiment of the present invention; FIG.\",\n \"8A is an enlarged fragmentary perspective view of an apparatus for splicing sliver prior to engagement with sliver in an open position according to the present invention; FIG.\",\n \"8B is an enlarged fragmentary perspective view of an apparatus for splicing sliver after engagement with the sliver in a closed position according to the present invention; FIG.\",\n \"8C is an enlarged fragmentary perspective view of an apparatus for splicing sliver after engagement with the sliver in an open position according to the present invention; FIG.\",\n \"9 is an enlarged fragmentary view of sliver after being spliced with an apparatus for splicing sliver according to the present invention; FIG.\",\n \"10 is a perspective view of an apparatus for splicing sliver according to a second embodiment of the present invention; FIG.\",\n \"11 is an exploded fragmentary perspective, view of a needle cartridge for positioning in an apparatus for splicing sliver according to a second embodiment of the present invention; FIG.\",\n \"12 is a side elevational view of an apparatus for splicing sliver having a needle cartridge illustrated by broken lines for clarity according to a second embodiment of the present invention; FIG.\",\n \"13 is an exploded side elevational view of an apparatus for splicing sliver having a needle cartridge member and a plurality of replacement auxiliary needle cartridge members according to a second embodiment of the present invention; FIG.\",\n \"14 is a fragmentary perspective view a portion of an apparatus for splicing sliver illustrating a connection between first and second handle portions along a pivot member and the position of a biasing member to bias the apparatus in an open position according to the present invention; and FIG.\",\n \"15 is an apparatus for splicing sliver according to a third embodiment of the present invention.\",\n \"DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS The present invention will now be described more fully hereinafter with reference to the accompanying drawings which illustrate preferred embodiments of the invention.\",\n \"This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.\",\n \"Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.\",\n \"Like numbers refer to like elements throughout, the prime notation, if used, indicates similar elements in alternative embodiments.\",\n \"FIGS.\",\n \"1-9 illustrate a first embodiment of an apparatus 20 for splicing sliver S. The apparatus 20 preferably includes a first handle portion 30 having at least one sliver engaging member.\",\n \"The at least one sliver engaging member is preferably one or more needles 32, and more preferably a plurality of needles 32, connected to the first handle portion 30.A second handle portion 40 preferably is positioned to receive the at least one sliver engaging member, e.g., the plurality of needles 32, in a closed position such as through or within one or more openings or bores 42, 42′ as shown (see also FIG.\",\n \"10).. As perhaps be shown in FIGS.\",\n \"1-2 and 14, a pivot member 25 is associated with, and preferably connected to or mounted as illustrated, to the first and second handle portions 30, 40 to allow either the first or second handle portions 30, 40 to pivot about the pivot member 25 between respective open and closed positions.\",\n \"The open position preferably is defined by distal portions 31 of the first handle portion 30 having the at least one sliver engaging member being spaced-apart from distal portions 31 of the second handle portion 40 positioned to receive the at least one sliver engaging member, and the closed position preferably is defined by the at least one sliver engaging member of the first handle portion 30 being received by the distal portions of the second handle portion 40 when positioned closely adjacent thereto.\",\n \"The first and second handle portions 30, 40 can advantageously defined a base which is advantageously hand operated, but other base structures as understood by those skilled in the art which may or may not be hand operated can be used as well according to the present invention.\",\n \"The apparatus 20 preferably includes a biasing member 28 associated with the pivot member 25 and positioned to bias either the first or the second handle portions 30, 40 in a preselected biased position.\",\n \"In the embodiment of the splicing apparatus 20 as illustrated, the first handle portion 30 is biased by the biasing member 28 to the open position.\",\n \"As shown in FIG.\",\n \"14, the biasing member 28 is preferably a spring as understood by those skilled in the art and preferably is connected to the pivot member 25 and positioned to bias the first handle portion 30 in the open position.\",\n \"Other types of biasing members, including various types of springs, can be used as well.\",\n \"Although the illustrated embodiment of biasing the first handle portion 30 in the open position is particularly advantageous due to the movement desired in the splicing process and the ease of use by a user's hand, for example, it will also be understood by those skilled in the art that the present invention would include biasing the splicing apparatus 2011 in the closed position (see also FIG.\",\n \"15).\",\n \"Further, the apparatus 20 preferably also includes a lock 50 associated with the first and second handle portions 30, 40 to lock the first and second handle portions 30, 40 in a closed position.\",\n \"In the embodiment shown, the lock 50 is primarily connected to the first handle portion 30 and slidably engages or locks, e.g., a latch, with the second handle portion 40, e.g., into a slot or channel by connecting to the needle receiving portion or body 45 of the second handle portion 40 (see FIG.\",\n \"2).\",\n \"As perhaps best illustrated in FIG.\",\n \"2, the first and second handle portions 30, 40 define a hand-activated needle actuation device which advantageously allows a ready grip by a user's hand H to apply pressure from the user's hand H to actuate the movement of the plurality of needles 32.As described above, and as illustrated in FIGS.\",\n \"7A-7D and 8A-8C, the at least one sliver engaging member preferably includes a plurality of needles 32, and the hand-activated needle actuation device is responsive to grippingly closing at least portions of the hand H of a user to actuate the engaging of the plurality of needles 32 with the sliver S to be spliced and the distal portions 45, e.g., through the openings or bores 42, 42′ of the second handle portion 40 when the sliver S is positioned between the plurality of needles 32 and the distal portions 45 of the second handle portion 40 so that the engaging of plurality of needles 32 with the distal portions 45 of the second handle portion 40 thereby further defines the closed position (see also FIG.\",\n \"10).\",\n \"Also, as perhaps best shown in FIGS.\",\n \"3-6 and 8A-8C, each of the plurality of needles 32 preferably includes a needle body 33, 33′ and a recessed portion 34, 34′ formed in the needle body 33, 33′ and positioned to assist in the engaging of and interconnecting of the sliver S when each needle 32, 32′ engages sliver S during movement to the closed position.\",\n \"The recessed portion 34, 34′ preferably includes at least one of the following: a barb, a groove, and a channel.\",\n \"The recessed portion 34, 34′ preferably has an upward slope with respect to the downward movement of the needle 32 so that the recessed portion 34, 34′ readily catches, engages, or otherwise contacts the sliver S during the downward motion of the needles and responsively releases the sliver S during upward motion.\",\n \"This process allows the intertwining or interconnecting of the sliver S to join the portions of sliver S desired to be spliced together.\",\n \"As shown in FIGS.\",\n \"10-13 and 15, according to a second embodiment of a splicing apparatus 20′ of the present invention, the at least one sliver engaging member preferably is detachably connected to the first handle portion 30′ and defines a cartridge member 35, 35′ positioned in distal portions 31′ of the first handle portion 30′ to readily remove from the first handle portion 30′.\",\n \"At least one replacement auxiliary cartridge member 38, and preferably a plurality of replacement auxiliary cartridge members 38, can have the same construction as the cartridge member 35, 35′ and can be adapted to be readily positioned in the first handle portion 30′.\",\n \"Accordingly, a kit can also be provided which has a portable splicing apparatus 20′ positioned in a container, e.g., box, bag, package, with one or more auxiliary cartridge members 38, so that when a cartridge member 35, 35′ being used is damaged, dulled, or otherwise desired to be replaced, another cartridge member 38 can be readily inserted into an opening 39 in the first handle portion 30′ of the splicing apparatus 20′ after removal of the damaged cartridge member 35, 35′ so that splicing operations proceed with substantially reduced interruptions.\",\n \"The distal portion 45′ of the second handle portion 40′ preferably has additional springs or other types of biasing members 36 which allow the needles 32′ to retract and extend from openings 37 in the distal portion 31′ as shown.\",\n \"Further still, as shown in FIG.\",\n \"15, scissor-type handles 30″, 40″, substantially closed loops, and various other types of handle or finger grips can also be used.\",\n \"In this embodiment of a splicing apparatus 20″, the pivot member 25″ is also moved forward toward a more medial portion of the handle members 30″, 40″ and the biasing member 28″ is another type of spring, as understood by those skilled in the art, which biases the scissor-type handle embodiment to a closed position so that the distal portions 31″, 45″ of the handles are positioned closely adjacent each other and the plurality of needles 32″ can engage receiving portions of the handle 40″.\",\n \"As illustrated in FIGS.\",\n \"1-15, the present invention further provides methods of splicing sliver S. A first method preferably includes grippingly closing a handle portion 30 of a sliver splicer 20 having at least one sliver engaging member 32 by the hand H of a user so that the at least one sliver engaging member 32 engages and splices sliver S positioned adjacent thereto and releasingly opening the handle portion 30 by the hand H of the user to thereby release the spliced portion of sliver S from the at least one sliver engaging member 32.Another method of splicing sliver S according to the present invention preferably includes closing a handle portion 30 of a needle engaging member having a plurality of needles 32 so that the plurality of needles 32 engages and splices sliver S positioned adjacent thereto and opening the handle portion 30 of the needle engaging member so that the plurality of needles 32 release the spliced portions of sliver S therefrom.\",\n \"Yet another method of splicing sliver S according to the present invention preferably includes joining first portions of sliver S with a plurality of needles 32 each having a recessed portion 34 to engage and intertwine with adjacent second portions of sliver S. The plurality of needles preferably is connected to a body portion so that the body portion and the plurality of needles 32 in combination define a needle cartridge member 35 and replacing the needle cartridge member 35 with an auxiliary cartridge member 38 also having a body portion and a plurality of needles 32 connected to the body portion.\",\n \"The apparatus 20, 20′, 20″ and methods of the present invention provide additional manufacturing, handling, processing, and formation flexibility in the use of the splicers for sliver.\",\n \"For example, manufacturing personnel can walk around a facility with an apparatus 20 of the present invention positioned in a pocket, holster, or harness when the splicing apparatus 20 or splicer is preferably in a locked closed position so that the manufacturing personnel can readily remove the splicer 20, unlock the splicer 20, accomplish the splicing function, relock the splicer 20, and return the splicer 20 to the pocket, holster, or harness.\",\n \"Additionally, the splicing apparatus 20 of the present invention can be strapped to a chain or belt which can enhance carrying and portability.\",\n \"Further, when one or more needles 32 or other sliver engaging members are damaged, according to one embodiment of the present invention, a cartridge member 35 can readily be removed which carries the needles 32′ and replaced with an auxiliary cartridge member 38.This cartridge replacement, for example, prevents the need to replace the entire splicing apparatus 20′ and saves money and reordering time.\",\n \"Also, because the splicing apparatus 20 is portable, compact, and relatively of simple construction and low cost, many different types of manufacturing personnel can use the splicing apparatus 20, 20′, 20″ and can readily order additional or readily replace the entire splicing apparatus 20, 20′, 20″ if desired without incurring extensive costs.\",\n \"In the drawings and specification, there have been disclosed a typical preferred embodiment of the invention, and although specific terms are employed, the terms are used in a descriptive sense only and not for purposes of limitation.\",\n \"The invention has been described in considerable detail with specific reference to these illustrated embodiments.\",\n \"It will be apparent, however, that various modifications and changes can be made within the spirit and scope of the invention as described in the foregoing specification and as defined in the appended claims.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10467924"}}},{"rowIdx":393,"cells":{"text":{"kind":"list like","value":[["Production of nanoparticles from methyl vinyl ether and maleic anhydride for the administration of hydrophilic pharmaceuticals, more particularly of puric and pyrimidinic bases","Manufacture of nanoparticles on the basis of methyl vinyl ether and maleic acid for the administration of pharmaceuticals of an hydrophilic nature, in particular analogs of puric and pyrimidinic bases.","The nanoparticles are obtained by desolvation with an hydroalcoholic phase of a methyl vinyl ether and maleic acid copolymer solution in acetone.","The particles obtained are next treated with cross-linking agents (diamines or proteins) for the purpose of prolonging their useful life and are, possibly, incubated with a pharmaceutical which will be transported on the surface.","The nanoparticles can carry the pharmaceutical likewise encapsulated which would then be added during the desolvation.","In the case of the nanoparticle-ligand conjugates, the nanoparticles previously obtained and containing inside the pharmaceutical to be transported are incubated with the ligand or molecule which will contribute the property of specifically recognising a particular receptor of the organism.","These pharmaceutical forms have as objective to improve the transport of the pharmaceutical or biologically active molecule to its site of action and/or absorption.","This property improves the specificity and effectiveness of said pharmaceuticals."],["1.a process for the manufacture of nanoparticles and ligand-nanoparticle conjugates with site specific delivery or targeting properties, able to carry drugs or biologically active molecules on their surface or in their interior characterized by the desolvation of poly(methyl vinyl ether-co-maleic anhydride)copolymer, dissolved in an organic solvent, and subsequent cross-linkage reaction with polyfunctional chemical compounds, including polyamines and polyhydroxyls: 2.A process according to claim 1, that comprises: a) desolvation of the poly(methyl vinyl ether-co-maleic anhydride)polymer dissolved in a polar organic phase at a concentration between 0.01 and 10% w/v, which may optionally contain a drug or biologically active molecule, with a hydroalcoholic solution in an organic phase/hydroalcoholic solution ratio of 1/1 to 1/10; b) elimination of the organic solvents by conventional methods such as filtration, centrifugation or evaporation, including the use of vacuum, among others; c) stabilization of the resulting nanoparticles with cross-linking agents; d) optionally, incubation of the nanoparticles with either the drug or the biologically active molecule or, alternatively, the ligand with targeting properties; e) purification of either the obtained nanoparticles or conjugates by conventional techniques such as ultracentrifugation, centrifugation, tangential filtration, among others; f) optional freeze-drying of the obtained nanoparticles or conjugates.","3.A process according to claim 1, characterized in that nanoparticles or conjugates are produced in a way which permits encapsulation of the drug or biologically active molecule in their interior, that comprises: i) the addition of said drug or biologically active molecule in step a) and, ii) optionally, the addition of a second drug or a different biologically active molecule or, alternatively, a ligand with targeting properties in step d).","4.A process according to claim 1, characterized in that nanoparticles are produced with the drug or biologically active molecule on outer layer, that comprises: i) the addition of the aforesaid drug or biologically active molecule in step d) and, ii) optionally, the addition of another drug or biologically active molecule in step a).","5.A process according to claim 1, characterized in that unloaded nanoparticles are produced consisting of carrying out step a) and step d) without adding any drug or biologically active molecule or ligand.","6.A process according to claim 1, characterized in that step c) is carried out by using polyamine or polyhydroxyl type polyfunctional reagents, including among them proteins and polymeric macromolecules such as non-ionic surfactants or polyvinylpyrrolidone.","7.A process according to claim 1, characterized in that step c) is carried out using the cross-linking agent 1,3-diaminopropane (DP) at a concentration ranging between 0 and 1 mg DP/mg poly(methyl vinyl ether co maleic anhydride).","8.A process according to claim 1, characterized in that step c) is carried out using albumin, such as human serum albumin or bovine serum albumin, at a concentration ranging between 0 and 10 mg albumin/mg poly(methyl vinyl ether co maleic anhydride).","9.A process according to claim 1, characterized in that step f) is carried out by adding mannitol or sacarose as a cryoprotector agent at a concentration ranging between 0.1 and 10% in weight.","10.A process according to claim 1, characterized in that the ligands used in the production of the nanoparticle conjugates have the characteristics of being able to recognize specific structures, or cellular or tissular receptors located on the surface or inside specific cell types in the organism.","11.A process according to claim 10, characterized in that the ligands used are lectins, carbohydrates, monoclonal antibodies, vitamins, amino acids, lipids or molecules of a peptidic nature.","12.A process according to claim 11, characterized in that the ligand is the Sambucus nigra lectin which is bound at a concentration ranging between 1 and 100 mg lectin/mg nanoparticles.","13.A process according to claim 1, characterized in that the drugs or the biologically active molecules incorporated inside the nanoparticles and conjugates possess a hydrophilic character and are soluble in organic polar solvents.","14.A process according to claim 13, characterized in that which the drug incorporated is the anti-tumor drug 5-fluorouridine.","15.A process according to claim 14, characterized in that 5-fluorouridine is dissolved in acetone at a concentration ranging between 0.1 and 3.33 mg/mL and then added to the poly(methyl vinyl ether co maleic anhydride)aceton solution, in order to obtain a drug/polymer ratio ranging between 0.01 and 0.4 mg/mg.","16.A process according to claim 1, characterized in that the drug or biologically active substance is carried on the surface of the nanoparticles and in which step d) of incubation is produced in an aqueous solution.","17.A process according to claim 16, characterized in that the drugs or biologically active molecules incorporated into the surface of the nanoparticles are of hydrophilic character or are soluble in aqueous solutions.","18.A process according to claim 17, characterized in that the drugs or biologically active molecules are either analogs of puric or pyrimidinic bases, or they are compounds of protein nature such as peptides, proteins, glycoproteins, lipoproteins, or they are carbohydrates.","19.A process according to claim 18, characterized in that the drug incorporated in the surface of the nanoparticles is the anti-tumor agent 5-fluorouridine.","20.A process according to claim 19, characterized in that 5-fluorouridine is dissolved in water and, subsequently, added to the suspension of nanoparticles at a concentration ranging between 10 and 1000 μg drug/mg polymer, obtaining concentrations greater than 200 μg of drug bounded to the surface per mg nanoparticles.","21.A process according to claim 17, characterized in that the incorporated drug is the antiviral agent ganciclovir.","22.A process according to claim 21, characterized in that the ganciclovir is dissolved in water and then added to a suspension of nanoparticles, at a concentration ranging between 0.1 and 20 mg drug/mg polymer, obtaining entrapment efficiencies greater than 20% of the initially added ganciclovir.","23.A process according to claim 17, characterized in that the incorporated drug is an oligonucleotide.","24.A process according to claim 23, characterized in that the incorporated drug is an antisense oligonucleotide.","25.A process according to claim 24, characterized in that the incorporated drug is the antisense oligonucleotide ISIS 2922.26.A process according to claim 25, characterized in that ISIS 2922 is dissolved in water and later added to the suspension of nanoparticles at a concentration ranging between 0.1 and 200 μg drug/mg polymer, producing nanoparticles whose superficially-bounded drug concentration is greater than 2 μg per mg nanoparticle.","27.Nanoparticles or conjugates obtainable by a manufacturing process according to claim 1, characterized by having an approximate average size of less than 500 nm.","28.Nanoparticles or conjugates according to claim 27, characterized by showing a biphasic release profile, with a first phase of immediate release of up to 60% of the loaded drug or biologically active molecule, followed by a second phase in which the drug or biologically active molecule is released slowly and in a sustained release manner.","29.Nanoparticles or conjugates characterized in that they have the following composition: 13-99% w/w poly(methyl vinyl ether co maleic anhydride), 0.001-15% w/w cross-linking agent, optionally, 0.001-15% w/w drug or biologically active molecule, optionally, 0.01-4.5% w/w ligand with specific targeting properties, optionally, 70-82% w/w cryoprotector.","30.Use of A method comprising using the nanoparticles and conjugates according to claim 27 for the administration of drugs or biologically active molecules to a patient.","31.A method comprising using the nanoparticles and conjugates according to claim 27 in the administration of third generation colloidal pharmaceutical forms to a patient.","32.A method comprising using the nanoparticles and conjugates of claim 27 for the pellicular coating of macroscopic pharmaceutical forms such as tablets, granules, granulates and pellets.","33.A method comprising using the nanoparticles and conjugates of claim 27 loading 5-fluorouridine in the preparation of compositions that are useful in the treatment of certain diseases such as colon cancer, cancers of the gastrointestinal tract, breast cancer, cancers of the cervix and endometrium, as well as cancers of the head and neck, liver, ovary, pancreas, prostrate and skin.","34.A method comprising using the nanoparticles and conjugates of claim 27 loading ganciclovir in the preparation of compositions which are useful for the treatment of infections induced by human cytomegalovirus.","35.A method comprising using the nanoparticles and conjugates of claim 27, loading ganciclovir, as adjuvant, in the preparation of gene therapy compositions, which incorporate suicide genes and, in particular, the thymidine quinase gene.","36.A method comprising using the nanoparticles and conjugates of claim 27 loading the antisense oligonucleotide ISIS 2922 in the preparation of compositions useful for the treatment of infections induced by human cytomegalovirus."],["<SOH> TECHNICAL FIELD OF THE INVENTION <EOH>The present invention is within the scope of the procedures for the production of non-biological vectors, nanoparticles and nanoparticle-ligand conjugates, for the transport and administration of pharmaceuticals or active biological molecules of hydrophilic nature through the use of the methyl vinyl ether and maleic anhydride copolymer, poly(methyl vinyl ether-co-maleic anhydride), as priority element."],["TECHNICAL FIELD OF THE INVENTION The present invention is within the scope of the procedures for the production of non-biological vectors, nanoparticles and nanoparticle-ligand conjugates, for the transport and administration of pharmaceuticals or active biological molecules of hydrophilic nature through the use of the methyl vinyl ether and maleic anhydride copolymer, poly(methyl vinyl ether-co-maleic anhydride), as priority element.","STATE OF THE ART The behavior of a drug after its administration in the organism is determined by a combination of different processes: distribution and elimination when the drug is administered by intravenous route; absorption, distribution, and elimination when an extravascular route is used.","Each one of these processes mainly depends on the physico-chemical properties of the drug substance which are determined by its chemical structure.","After administering the drug by conventional means, it is distributed throughout the organism according to its molecular structure, which determines its physicochemical properties (molecular mass, lipophilia, pKa, crystalline structure, solubility), biochemical properties (affinity with plasmatic or tissue proteins, affinity with the membrane or intracellular receptors, sensibility to the enzymes responsible for its biotransformation, etc.)","and pharmacological properties (ability to clear biological membranes, intensity of the action, etc.).","Numerous studies have shown that the classical pharmaceutical forms (tablets, capsules, suppositories, injections, etc.)","only permit the modulation of the intensity and/or speed of release of the biologically active molecule into the blood circulation (absorption), but they have no effect on the subsequent stages of in vivo behavior of the drug (distribution and elimination).","During the distribution stage, the drug spreads to a large number or to every anatomical area of the organism.","This implies: (i) the administration of doses higher to those which would theoretically be needed to obtain the required effect or (ii) the repeated administration of the drug.","The therapeutic effect is attained in spite of the loss of an important fraction of the drug dose in different tissues other than those where it should act, resulting in unwanted side effects.","This nonspecific distribution of the drug, tolerable for those with high therapeutic indexes (quotient between the dose that provokes the toxic effects and the therapeutic dose), could become the limiting factor for the clinical use of certain biologically active molecules (example: the 5-fluorouridine).","So as to obtain a more rational and better adapted therapeutic use, one of the most promising possibilities is that which uses the concept of vectorization for the increase in the efficiency and specificity of action of the drugs.","This concept consists in associating a drug or a biologically active molecule to an appropriate vector or carrier.","Although there are different types of vectors, in this study only the characteristics and properties of the non-biological vectors (liposomes, nanoparticles and conjugates) will be studied.","The use of these new pharmaceutical forms permits the association between the drug and a system whose mission is to carry the drug to its pharmacological target instead of remaining in a free state.","In fact, the use of non-biological vectors permits the masking of the physicochemical properties of the drug and, logically, the characteristics of the carrier are what permit control over the distribution stage.","In the best of cases, the vector-drug pair should be inactive and remain stable in the different biological media between the site of administration and the place of action.","The most important potentialities that these vectors or non-biological carriers provide are the following (Couvreur & Puisieux.","Adv.","Drug Del.","Rev., 10 (1993) 141-162): protection against the chemical, enzymatic or immunological inactivation of the drug between the administration site and the place of action; improvement in the transport of the biologically active molecule to places of difficult access and its penetration into the cell (in the case of infections located in the intracellular territories which are inaccessible by simple diffusion); increase in the specificity of action by a selective, effective, and regular concentration of drug in the cellular and/or molecular target.","In this way, with smaller doses, the obtained therapeutic activity is, at least, identical and the side effects are milder; decrease in the toxicity for certain organs by means of modifying the tissular distribution of the biologically active molecule that is carried; in some cases, a lengthening of the time of residence of the drug in the organism (interesting for those molecules with high clearance and a low biological half-life) and, control of its release.","All of this implies a decrease of the frequency of drug intake and indirectly, an increase in the observance of the treatment on the part of the patient.","Within the group of non-biological vectors, colloidal systems of solid particle type with a size smaller than a micrometer stand out; they are also called nanoparticles.","These are subdivided into matrix nanospheres and vesicular nanocapsules (Orecchioni e Irache, Formes pharmaceutiques pour application locale.","Lavoisier Tech & Doc., Paris, 1996, 441-457).","The nanocapsules are vesicular systems formed by an internal cavity which is surrounded by a membrane or polymeric wall.","The nanospheres are matrix forms, formed by a tridimensional polymeric net.","In both cases, the molecules of the biologically active substance can be dissolved, entrapped or adhered to the macromolecular structure (in the nanospheres) or encapsulated by the polymeric membrane (in the nanocapsules).","Also, the biologically active substance may remain adsorbed on the surface of the nanoparticles.","Depending on the used material, the nanospheres can be prepared from: reactions of monomer polymerization; macromolecules of natural origin; preformed synthetic polymers.","The nanoparticles produced from preformed polymers are generally obtained by two different procedures: (i) application of techniques based on the emulsion of an organic solution of the polymer (Vanderhoff et al, U.S. Pat.","No.","4,177,177 (1979)) and (ii) by processes derived from the desolvation of the polymer (Fessi et al, FRENCH PAT, (1986) 2 608 988).","On the contrary, nanoparticles of natural macromolecules are generally obtained by means of (i) gelification techniques (Rajaonari-vony et al, J. Pharm.Sci, 82 (1993) 912-917), (ii) emulsion (Vanderhoff et al, U.S. Pat.","No.","4,177,177 (1979) and (iii) controlled desolvation (Marty et al, Pharm Acta Helv., 53 (1978) 79-82).","Generally, a supplementary stage is needed to estabilize the aforesaid systems.","In the organism, the distribution of the nanoparticles generally depends on their physicochemical characteristics (mainly their size and their surface properties).","By intravenous route, the submicronic vectors (for example, the nanoparticles) are captured by the endothelial reticular system (ERS), also called mononuclear phagocyte system, principally located in the liver, spleen, lungs and marrow.","This property that nanoparticles possess converts them into very interesting pharmaceutical forms for the administration of drugs destined to the treatment of diseases located in the ERS or in the treatment of diseases in which the cells of ERS possess a function (infections, cancerous processes, etc.).","On the other hand, administration of nanoparticles through the mucous membranes (oral, nasal, ocular, etc.","routes) enables the development of adhesive interactions which increase the time of residence of the pharmaceutical form in contact with the site of absorption or action of the drug (Ponchel & Irache, Adv.","Drug Del.","Rev., 34 (1998) 191-219).","For the design of pharmaceutical forms (based on nanoparticles) capable of vectorizing cells or tissues that are different from those which the nanoparticles normally distribute, active vectors (also called third generation vectors or nanoparticle-ligand conjugates can be used) are used.","These systems are obtained by means of the binding of molecules capable of interacting specifically with cellular or molecular receptors to the surface of the nanoparticles.","Among these molecules or ligands, polyethylenglycoles (“stealth” vectors; Allen et al., Cancer Res., 52 (1992) 2431-2439), certain monoclonal antibodies (Weinstein et al, Pharm.","Therap., 24 (1984) 207-233), lectins (Irache et al, Biomaterials, 15 (1994), carbohydrates (Maruyama et al, Biomaterials, 15 (1994) 1035-1042), amines (Roser & Kissel, Eur.","J. Pharm.","Biopharm., 39 (1993) 8-12), vitamins (Russell-Jones & Westwood, U.S. Pat.","No.","07/956.003; (1992) WO 92/17167), etc.","can be used.","Nowadays, the preparation of nanoparticle pharmaceutical forms containing hydrophilic drugs is limited by several aspects of their formulation.","In general, hydrophilic substances or drugs do not show any affinity for matrix lipophylic systems (for example, nanospheres).","Therefore, production yields are very low and, logically, can not be applied to drug administration.","Analogs of puric and pirimidinic bases such as oligonucleotides, 5-fluouridine or ganciclovir are included in this category of hydrophilic drugs.","Poly(methyl vinyl ether-co-maleic anhydride)synthetic copolymers have a wide range of applications in the agricultural field (commercial name: Agrimer® VEMA, ISP) as adyuvants and estabilizers in veterinary solutions, in seed, granules and tablet coatings, etc.","These copolymers are also used in chemical and pharmaceutical fields (commercial name: Gantrez® Copolymers, ISP), mainly due to their capacity to form coating films and their high bioadhesive capacity (Esposito er al., Biomaterials, 15 (1994) 177-182).","They have also been described as thickeners, complexing agents and hydrophilic colloids.","Likewise, their use in sustained release systems such as transdermic patches or ocular matrixes has been proposed (Finne et al., Int.","J.","Pharm., 78 (1992) 237-241).","Their main advantages are their relatively low toxicity (LD50=8-9 g/Kg by oral route) and their easy production, regarding both quantity and price.","Poly(methyl vinyl ether-co-maleic anhydride)copolymers have also been used to prepare microparticle vectorial systems by a process of complex coacervation (Mortada et al., J.","Microencas., 4 (1987) 11-21).","Poly(methyl vinyl ether-co-maleic anhydride)copolymer nanospheres are new matrix systems of vectors of submicronic size.","These are able to retain the biologically active substance thanks to (i) solution or entrapping inside the macromolecular structure or matrix, (ii) covalent bonds result of the interaction of the drug and the polymer anhydride groups and (iii) processes of adsorption by means of weak bonds.","The extremely high reactivity of this copolymer due to its cyclic anhydride groups makes it perfect to retain hydrophilic substances in its structure.","They are also very interesting systems for the fixation of ligands to the nanoparticle surface, without having to use highly toxic organic reactants (glutaraldehyde and carbodiimides) .","A variety of current drugs (for example, 5-fluouridine, ganciclovir) and biotechnological products (for example, antisense oligonucleotides) can be mentioned as hydrophilic active ingredients.","All of them are analogs of puric and pirimidinic bases.","Other kind of molecules such as proteins, peptides, lectins and, in general, all those substances which have alcohol groups and/or primary amines (for example: lectins) can also be fixed.","The majority of the vectorial systems that have been described in the literature and are used to administrate hydrophilic substances require laborious manufacturing processes.","Thus, for example, quite complex bonds with toxic reactions (Rimoli et al., J.","Controlled Rel., 58 (1999) 61-68), multiple emulsions (Jeffery et al., Pharm.","Res., 10 (1993) 362-367) or working with very high drug concentrations in conditions not applicable to the industrial field have been proposed.","The conditions of applicability that allow to ascertain of the industrial interest of poly(methyl vinyl ether-co-maleic anhydride)nanoparticle vectors, could be the following: 1.The obtaining of homogeneous populations with submicronic particle size.","This favors the distribution, improves the transport of active substances to its place of action and increases the penetration in the target cells.","2.The use in the chemical-pharmaceutical field of abundant preformed copolymers with a low expense, for the administration of drugs.","3.The obtaining of particles in which part of the drug will be entrapped and other part will be bound to the matrix by a covalent bond.","This favors the bi-phasic release of the drug.","The entrapped fraction enables a very quick initial release, while the bound fraction is released in a controlled way over a long period of time.","This period will depend on particle degradability and on the breakage of the bounds between the polymer and the biologically active molecule.","4.The obtaining of particles in which the drug is bound by a covalent bond to the surface.","Like in the previous case, the drug will be released in a controlled way and depending on the velocity of the breakage of the bonds between the groups located on the nanoparticle surface and the biologically active molecule.","5.The use of the aforementioned nanoparticles, as a base to produce third generation vectors or nanoparticle-ligand conjugates capable of carrying the active principle to certain cells or tissues by a specific recognition system.","6.Working conditions at room temperature that avoid the possible degradation of thermolabile active substances of hydrophilic nature, and of chemical and/or biotechnological origin.","7.The obtaining of particles capable of protection against premature inactivation of the encapsulated active principles.","DESCRIPTION OF THE INVENTION Poly(methyl vinyl ether-co-maleic anhydride)nanoparticles are obtained by a desolvation method consisting in the addition of a polar solvent (miscible with the copolymer solution) to an organic solution of the copolymer.","Subsequently, a second non-solvent liquid is added, in this case an hydroalcoholic solution.","In order to do this, the copolymer is dissolved in a predetermined volume of acetone and then an ethanol solution is added.","Then a similar volume of water is added to this solution.","The particles are instantly formed in the medium, which takes on the appearance of a milky suspension.","The organic solvents (ethanol and acetone) are then eliminated by evaporation under reduced pressure.","The particles remain in a stable aqueous suspension.","The poly(methyl vinyl ether-co-maleic anhydride)copolymer is insoluble in water, but after some time it hydrolyzes to an acid form which is soluble in water.","Although this hydration process starts on contacting with water, it lasts approximately 10 hours.","This is the reason why a supplementary stage is required.","This stage consists in the stabilization of the particles by the cross-linkage of the particles with the purpose of increasing their stability in aqueous media.","This also allows the modulation and control of the release of the encapsulated drug.","As cross-linking agents, polyfunctional chemical compounds, including a number of polyamines and polyhydroxyl molecules, can be used.","The main cross-linking agents used are human serum albumin (HSA) and 1,3-diaminopropane (DP).","Other macromolecules, such as bovine serum albumin (BSA), povidone and gelatin, have also shown to be good stabilizing agents.","The next manufacturing step consists in purifying the resultant nanoparticles in order to eliminate all the remains of the process, mainly polymer and cross-linker residues.","To do this, different techniques such as ultracentrifugation or tangential filtration may be used.","Finally, the purified nanoparticles may be lyophilized or freeze-dried for their long term storage and preservation.","Depending on the aqueous solubility of the drug, two different procedures may be used to associate a drug to the nanoparticles.","The first one consists in binding the drug (hydrophilic or with a low aqueous solubility) to the polymer in an organic solution.","In order to do this, poly(methyl vinyl ether-co-maleic anhydride) and the drug are dissolved in a given volume of acetone.","This solution is incubated and stirred at a predetermined temperature and for a fixed time.","Then a given volume of ethanol is added, followed by the addition of a similar volume of water.","The particles are immediately formed in the medium, and this suspension takes on a milky appearance.","The organic solvents (ethanol and acetone) are removed by means of evaporation under reduced pressure and the particles remain in a stable aqueous suspension.","The particles are cross-linked with one of the aforementioned products (mainly HSA and DP) and purified by ultracentrifugation or tangential filtration.","Finally, the particles can be lyophilized.","The second one is a process for the loading of molecules with a high polarity, in which the drug is bound to the surface of freshly prepared nanoparticles.","In this case, the poly(methyl vinyl ether-co-maleic anhydride)copolymer is dissolved in a given volume of acetone.","Then, a given amount of ethanol is added followed by the addition of a similar amount of water.","The particles are immediately formed in the medium, and this suspension takes on a milky appearance.","The organic solvents (ethanol and acetone) are removed by means of evaporation under reduced pressure, and the particles remain in a stable, aqueous medium.","The freshly prepared nanoparticles are then incubated at a fixed temperature for a predetermined time with an aqueous solution of the drug and the solution is incubated at a predetermined temperature and for a fixed time.","Afterwards, particles are stabilized by cross-linkage using one of the aforementioned agents (mainly HSA and DP) and purified by ultracentrifugation or tangential filtration.","Finally, nanoparticles can be lyophilized.","Finally, to prepare ligand-nanoparticle conjugates, the manufacturing process is quite simple and is based on the incubation between the nanoparticles and the ligand to be bound to the surface of the carriers.","Then, a supplementary purifying step is needed to eliminate the unbound ligand remains.","As in the previous cases, the obtained conjugates can be lyophilized.","The following examples illustrate the present invention, but in no way do they pretend to limit the possible uses of the invention.","In these examples, three different pharmaceuticals, either derivatives from puric or pyrimidinic bases, have been used.","These drugs are the anti-cancer agent 5-fluorouridine, the antiviral ganciclovir and the antisense oligonucleotide ISIS 2922.5-fluorouracile (2,4-dioxo-5-fluropyrimidine) and, its derivative, 5-fluorouridine (2,4-dioxo-5-fluoropyrimidine riboside) are analogs of the pyrimidines, and their molecules contain a fluoride atom.","These drugs are usually used in the treatment of colon cancer, either alone or in combination with other drugs.","They are also used in the treatment of breast, cervix, endometrium, gastrointestinal, head and neck, liver, ovary, pancreas, prostrate and skin tumors.","The 5-fluorouridine has an antitumoral activity which is 100-times more potent than 5-fluorouracile and other derivatives of this drug (Jampel et al.","Arch.","Ophtal., 108 (1990) 430-435).","However, its clinical use is limited due to the severe side effects (leukopenia, thrombocytopenia and gastrointestinal toxicity) when administered in a traditionally pharmaceutical form (Brusa et al., II Farmaco, 52 (1997) 71-81).","Administration of this drug in the form of nanoparticles would allow an increase of the therapeutic index of this drug.","Ganciclovir, (9-[(1,3-dihydroxy-2-propoxy)methyl]guanine, is a synthetic nucleoside analogue.","This molecule exhibits antiviral activity against some herpes viruses (Martin et al., J. Med.","Chem., 26 (1983) 759-761).","Furthermore, it is the most frequently used antiviral drug in the treatment of infections caused by human cytomegalovirus (CMV) in immunocompromised patients, mainly in those with the acquired immunodeficiency syndrome (AIDS), congenital immunodeficiency or in individuals who have undergone organ transplantation.","The principal clinical manifestation is retinitis, which if left untreated, results in irreversible blindness (Markham & Faulde, Drugs, 48 (1994) 455-460).","The use of nanoparticles would permit the obtaining of pharmaceutical dosage forms showing sustained release properties and controlled drug release.","Besides, and given its small size, they would minimally affect vision when administered by intravitreous route.","In addition, during the last few years, ganciclovir has been used as an adjuvant for gene therapy involving suicide genes.","This new therapeutical technique consists of administrating retrovirus, adenovirus or herpes simplex incorporating the thymidine quinase (tk) gene, followed by the adminisration of ganciclovir to the patient.","The viral vectors permit the incorporation of tk into the dividing cells and, after expression, the ganciclovir is metabolized by the expressed enzyme in its phosphate derivative.","This derivative is then transformed into ganciclovir-diphosphate and finally in ganciclovir-triphosphate, which is toxic for the cell and induces its apoptosis.","This is being successfully used for the treatment of pancreas tumors (Carrio et al., Gene Ther., 6 (1999) 547-553), prostate adenocarcinomas (Herman et al., Hum.","Gene Ther., 10 (1999) 1239-1249) and hepatocarcinomas (Qian et al., Hum.","Gene.","Ther., 10 (1997) 349-358).","Antisense oligonucleotides are new therapeutic agents which are able to regulate the genetic expression of living organisms.","This technology consists in the use of synthetic fragments of DNA or RNA, called oligonucleotides, which allow the stoppage of the production of illnesses related to the synthesized proteins.","Antisense compounds block the transmission of genetic information between the nucleus and the place where the protein is produced inside the cell.","There is a variety of applications that include the treatment of certain types of cancer, inflammatory pathologies and viral illnesses.","ISIS 2922 is an antisense oligonucleotide with a sequence complementary to messenger RNA in the human citomegalovirus region IE1 (Anderson et al., Antimicrob.","Agents Chemother., 40 (1996), 2004-2011).","This oligonucleotide blocks the expression of a protein essential for viral replication, thereby inhibiting the replication of the virus (Leeds et al., Drug Metabol.","Dispos., 25 (1997) 921-926) with an in vitro efficiency of about 30-times greater than traditional therapies.","However, physico-chemical oligonucleotide characteristics (high mass, charged, hydrophilic molecules) do not allow its free access into the cell where they have to act.","The use of nanoparticles permits an increase of the penetration capacity of those substances and a protection against exo- and endonucleases.","The size and zeta potential of the nanoparticles were determined in a Zetamaster apparatus (Malvern Instruments/Optilas, Spain).","The drugs, 5-fluoroudine (provided by Sigma; Alcobendas, Spain) and ganciclovir (Cymevene®, Roche; Madrid, Spain), were analyzed by High Performance Liquid Chromatography (HPLC), using a Hewlett Packard series 1050 (Germany).","The antisense oligonucleotide, ISIS 2922, was obtained from Pharmacia Biotech (Cambridge, United Kingdom) and analyzed by zone capillary electrophoresis.","The Sambucus nigra lectin was obtained from Vector Laboratories (USA) and determined by gel permeability chromatography.","The samples of nanoparticles were lyophilized using a Virtis Genesis 12EL apparatus (USA).","EXAMPLE 1 Preparation of Nanoparticles from poly(methyl vinyl ether-co-maleic anhydride)copolumer The process described below is valid for the preparation of colloidal pharmaceutical forms, nanoparticle type, which can be used for the administration of drugs or biologically active molecules.","In addition, these nanoparticles can also be used for the film coating of macroscopic pharmaceutical forms, such as tablets, granules, granulates, and minigranules.","1.1.Cross-Linkage with 1,3-diaminopropane (DP) 100 mg of poly(methyl vinyl ether-co-maleic anhydride)copolymer are dissolved in 5 mL organic solvent (acetone).","Then, under stirring, 10 mL of a miscible organic solvent (ethanol) and 10 mL distilled water are added.","The resulting mixture is stirred for homogenization during 5 minutes and the suspension of nanoparticles is evaporated under reduced pressure for organic solvents elimination.","The final volume is adjusted with water (or an aqueous solution) to 10 mL.","The next step is to cross-link the resulting nanoparticles with 1,3-diaminopropane (DP).","In order to do this, a DP solution is prepared in water (1% v/v), and a predetermined amount of cross-linking agent is added to the suspension of nanoparticles for incubation for 5 minutes.","The suspension is then purified by ultracentrifugation (12 minutes at 10,000 rpm) or tangential filtration.","The supernatants are eliminated and the residues are resuspended in water or in an aqueous solution of mannitol (5% w/v).","Finally, the resulting suspension of nanoparticles is lyophilized maintaining all its properties.","The resulting formulation of the nanoparticles suspension before liophilization is: Poly(methyl vinyl ether-co-maleic anhydride) 1.0% w/v Mannitol 5.0% w/v 1,3-diaminopropane c.s.","Purified water c.s.p.","10 mL The mean size of the obtained nanoparticles is lower than 300 nm and the carriers display a negative superficial charge.","Table 1 shows the influence of the amount of DP (used to cross-link the nanoparticles) on the size and zeta potential of the resulting nanoparticles.","From these results, it is clear that an increase in the amount of cross-linking agent used to harden the nanoparticles increases the particle size and decreases negative zeta potential of these colloidal carriers.","This last fact proves the reaction between the anhydride groups from the surface of the particles and the cross-linking agent.","TABLE 1 Effect of the quantity of the cross-linking agent (DP) on the size and zeta potential of the resulting nanoparticles.","DP Quantity (mL) Size (nm) Zeta potential (mV) 0 206.7 ± 1.13 −51.7 ± 2.45 2 239.8 ± 5.16 −37.8 ± 1.13 3 270.9 ± 4.13 −27.7 ± 2.26 5 270.1 ± 10.53 −23.7 ± 0.85 10 275.0 ± 11.33 −23.7 ± 0.78 The yield of the nanoparticles manufacturing process was calculated as the quotient between the weight of the freeze-dried samples and the initial amount of polymer used to prepare the nanoparticles, either with mannitol or without cryoprotector.","From a previously known amount of the polymer (100 mg) the amount that had been transformed in nanoparticles at the end of the process was determined by gravirnetry.","In any case, the yield of the manufacturing process (expressed in % with respect to the initial mass of polymer) was high; although higher efficiencies were obtained when the manufacturing process was carried out at room temperature rather than at 40° C. (see FIG.","1).","1.2.Cross-Linkage with Human Serum Albumin (HSA) 100 mg of poly(methyl vinyl ether-co-maleic anhydride)copolymer are dissolved in 5 mL of an organic solvent (acetone).","Then, under magnetic stirring, 10 mL of a miscible organic solvent (ethanol) and 10 mL distilled water are added.","The resulting mixture is stirred for 5 minutes, and the suspension of nanoparticles is evaporated under reduced pressure for organic solvent elimination.","The final volume is adjusted with water (or an aqueous solution) to 10 mL.","The next step is to harden the resulting nanoparticles with human serum albumin (HSA).","In order to do this, a HSA solution is prepared in water (30 mg/mL), and a predetermined amount of cross-linking agent is added to the suspension of nanoparticles for incubation for 120 minutes.","The suspension is then purified by ultracentrifugation (12 minutes at 10,000 rpm) or tangential filtration.","The supernatants are eliminated and the residues resuspended in water or in an aqueous solution of mannitol (5% w/v).","Finally, the resulting suspension of nanoparticles is freeze-dried maintaining all its properties.","The resulting formulation of the nanoparticles solution before liophilization is: Poly(methyl vinyl ether-co-maleic anhydride) 1.0% w/v Mannitol 5.0% w/v Human serum albumin c.s.","Purified water c.s.p.","10 mL The mean size of the obtained nanoparticles is lower than 300 nm and the carriers display a negative superficial charge.","Table 2 shows the influence of the amount of HSA (used to cross-link the nanoparticles) on the size and zeta potential of the resulting nanoparticles.","It was observed that an increase in the amount of cross-linking agent used to harden the nanoparticles slightly increases the size of the resulting nanoparticles and decreases their negative zeta potential.","These results prove that the anhydride groups on the surface of the particles react with the cross-linking agent.","TABLE 2 Effect of the quantity of the cross-linking agent HSA on the size and zeta potential of the resulting nanoparticles.","Amount HSA Zeta (mg/mg polymer) Size (nm) potential (mV) 0 206.7 ± 1.13 −51.7 ± 2.45 10 239.8 ± 5.16 −42.7 ± 1.13 15 240.9 ± 15.05 −42.7 ± 1.00 The yield of the nanoparticles manufacturing process was calculated as the quotient between the weight of the freeze-dried samples and the initial amount of polymer used to prepare the nanoparticles, either with mannitol or without cryoprotector.","The amount of poly(methyl vinyl ether-co-maleic anhydride)copolymer that had been transformed in nanoparticles at the end of the process was determined by gravimetry from a previously known amount of the polymer (100 mg).","In any case, the yield of the manufacturing process (expressed in % with respect to the initial mass of polymer) was high; although higher efficiencies were obtained when the manufacturing process was carried out at room temperature rather than at 40° C. (see FIG.","2).","EXAMPLE 2 Manufacturing Process of Nanoparticle Conjugates Based on the Binding of Lectin to the Surface of poly(methyl vinyl ether-co-maleic anhydride)nanoparticles The process described below permits the preparation of third generation pharmaceutical colloidal forms for the site specific delivery or targeting of drugs.","These forms are formed by the binding of a ligand to the surface of copolymer nanoparticles.","The ligand offers the possibility of driving the pharmaceutical dosage form to a specific site in the organism.","In fact, the ligand should specifically recognize certain receptors located on the surface or inside a certain type of cells.","These forms are known as conjugates.","In the next example, the model ligand used was the lectin of Sambucus nigra agglutinin (SNA).","First of all, 100 mg of poly(methyl vinyl ether-co-maleic anhydride) are dissolved in 5 mL organic solvent (acetone).","Then, under magnetic stirring, 10 mL of a miscible organic solvent (ethanol) and 10 mL distilled water are added.","The resulting mixture is stirred for 5 minutes, and the suspension of nanoparticles is concentrated under reduced pressure for organic solvent elimination.","The final volume is adjusted with water (or an aqueous solution) to 10 mL.","The next step is to harden the resulting nanoparticles with 1,3-diaminopropane (DP).","In order to do this, a solution of DP is prepared in water (1% v/v), and a predetermined amount of cross-linking agent is added to the suspension of nanoparticles for incubation for 5 minutes.","The suspension is then purified by ultracentrifugation (12 minutes at 10,000 rpm) or tangential filtration.","The supematants are eliminated and the residues resuspended in water.","Then the purified nanoparticles are incubated with the lectin.","In order to do this, a variable amount of SNA is added to the nanoparticle suspension and this mixture is magnetically stirred at room temperature for 1 hour.","The conjugates are centrifuged twice at 10,000 rpm for 12 minutes, in order to remove the unbound lectin (5% w/v).","Finally, the resulting suspension of conjugates is freeze-dried after the addition of sacarose or mannitol as cryoprotectors.","FIG.","3 shows the influence of the initial amount of cross-linking agent on the lectin bound to the poly(methyl vinyl ether-co-maleic anhydride)nanoparticles.","The lectin was determined by HPLC (High performance Liquid Chromatography).","It was evident that as the amount of cross-linking agent increases, the amount of lectin being loaded by the nanoparticles decreases.","Likewise, the amount of lectin bound to the nanoparticles is higher when the initial amount of lectin increases.","The quantity of active lectin bound to the nanoparticles was determined by means of an erythrocyte agglutination test.","It was observed that by increasing the initial quantity of lectin, a greater amount of lectin bound to the nanoparticles remained active.","When analyzing the influence of the amount of cross-linking agent, it was observed that there are certain ideal conditions that allow the optimal binding of lectins to the nanoparticle surfaces, around 30 μg/mg nanoparticle (FIG.","4).","Similar results were obtained when using human serum albumin (HSA) or bovine serum albumin (BSA) as cross-linking agents.","EXAMPLE 3 Manufacturing Process for the Preparation of 5-fluorouridine-loading poly(methyl vinyl ether-co-maleic anhydride)nanoparticles The process described below permits the preparation of colloidal pharmaceutical forms (i.e., nanoparticles and ligand-nanoparticle conjugates) carrying 5-fluorouridine or any other active molecule with a chemical structure similar to or derived from said drug.","3.1.Cross-Linkage with 1,3-diaminopropane (DP) 100 mg of poly(methyl vinyl ether-co-maleic anhydride) are dissolved in 2 mL organic solvent (acetone).","A predetermined amount of 5-fluorouridine is dissolved in 3 mL acetone, until a theoretical maximum of 10 mg/mL is reached.","The two aforementioned solutions are mixed and then incubated at room temperature for 2 hours.","Then, under magnetic stirring, 10 mL of a miscible organic solvent (ethanol) and 10 mL distilled water are added to the acetone solution.","The resulting mixture is stirred for 5 more minutes, and the suspension of nanoparticles is concentrated under reduced pressure for organic solvents elimination.","The final volume is adjusted with water (or an aqueous solution) to 10 mL.","The next step is to harden the resulting nanoparticles with 1,3-diaminopropane (DP).","In order to do this, a solution of DP is prepared in water (1% v/v), and a predetermined amount of cross-linking agent is added to the suspension of nanoparticles for incubation for 5 minutes.","The suspension is then purified by ultracentrifugation (12 minutes at 10,000 rpm) or tangential filtration.","Optionally, the resulting nanoparticles (loaded with 5-fluorouridine) are incubated with the Sambucus nigra lectin in order to obtain the nanoparticle-lectin conjugates, as described in example 2.The supernatants are analyzed by HPLC.","The quantity of entrapped drug in the nanoparticles is determined as the difference with respect to the initial quantity added.","The residue or pellet is resuspended in water or in an aqueous solution of mannitol (5% w/v).","Finally, the resulting suspension of nanoparticles is freeze-dried.","FIG.","5 shows the influence of the incubation time between 5-fluorouridine and the poly(methyl vinyl ether-co-maleic anhydride) on the drug loading.","In the example of the SNA-nanoparticle conjugates containing 5-fluorouridine (experimental conditions: 200 μg initial lectin), the amount of encapsulated fluorouridine does not change and the amount of lectin bound to the surface of nanoparticles was calculated to be around 40 μg/mg polymer.","3.2.Cross-Linkage with Human Serum Albumin (HSA) 100 mg of poly(methyl vinyl ether-co-maleic anhydride) are dissolved in 2 mL organic solvent (acetone).","A predetermined amount of 5-fluorouridine is dissolved in 3 mL acetone, until a theoretical maximum of 10 mg/mL is reached.","The two aforementioned solutions are mixed and then incubated at room temperature for 2 hours.","Then, under magnetic stirring, 10 mL of a miscible organic solvent (ethanol) and 10 mL distilled water are added to the acetone solution.","The resulting mixture is stirred for 5 more minutes, and the suspension of nanoparticles is concentrated under reduced pressure for organic solvent elimination.","The final volume is adjusted with water (or an aqueous solution) to 10 mL.","The next step is to harden the resulting nanoparticles with human serum albumin (HSA).","In order to do this, a solution of HSA is prepared in water (30 mg/mL), and a predetermined amount of cross-linking agent is added to the suspension of nanoparticles for incubation for 120 minutes.","The suspension is then purified by ultracentrifugation (12 minutes at 10,000 rpm) or tangential filtration.","The supernatants are analyzed by HPLC.","The quantity of entrapped drug in the nanoparticles is determined as the difference with respect to the initial quantity added.","The residue or pellet is resuspended in water or in an aqueous solution of mannitol (5% w/v).","Finally, the resulting suspension of nanoparticles is freeze-dried maintaining all its properties.","FIG.","6 shows the influence of the incubation time between 5-fluorouridine and the copolymer on the drug loading of nanoparticles when the carriers where cross-linked with HSA.","EXAMPLE 4 Manufacturing Process for the Preparation of poly(methyl vinyl ether-co-maleic anhydride)nanoparticles Coated with 5-fluorouridine The process described below permits the preparation of colloidal pharmaceuticai forms (i.e., nanoparticles and ligand-nanoparticle conjugates) containing 5-fluorouridine or any other active molecule with a chemical structure similar to or derived from said drug.","4.1.Cross-Linkage with 1,3-diaminopropane (DP) 100 mg of poly(methyl vinyl ether-co-maleic anhydride) are dissolved in 5 mL organic solvent (acetone).","A predetermined amount of 5-fluorouridine is dissolved in 2 mL water.","Then, under magnetic stirring, 10 mL of a miscible organic solvent (ethanol) and 10 mL distilled water are added to the acetone solution of the copolymer.","The resulting mixture is stirred for 5 more minutes, and the suspension of nanoparticles is evaporated under reduced pressure for organic solvents elimination.","The final volume is adjusted with water (or an aqueous solution) to 10 mL, and the suspension is then incubated with the 5-fluorouridine aqueous solution.","The next step is to harden the resulting nanoparticles with 1,3-diaminopropane (DP).","In order to do this, a solution of DP in water (1% DP v/v water) is prepared and a predetermined amount of cross-linking agent is added to the suspension of nanoparticles for incubation for 5 minutes.","The suspension is then purified by ultracentrifugation (12 minutes at 10,000 rpm) or tangential filtration.","The supernatants are analyzed by HPLC.","The quantity of entrapped drug in the nanoparticles is determined as the difference with respect to the initial quantity added.","The residue or pellet is resuspended in water or in an aqueous solution of mannitol (5% w/v).","Finally, the resulting suspension of nanoparticles is freeze-dried maintaining all its properties.","FIG.","7 shows the influence of the initial amount of 5-fluorouridine (FU) that is adsorbed or interacts with the anhydride groups on the nanoparticle (NP) surface.","It is clear that when the initial amount of the drug increases, the amount of FU carried by the nanoparticles also increases.","Likewise, the higher the initial amount of FU loaded in the nanoparticles, the higher the amount that is released quickly.","In the case of the administration of these colloidal pharmaceutical forms, the drug fraction that is released immediately would act as burst dose.","4.2.Cross-Linkage with Human Serum Albumin (HSA) 100 mg of poly(methyl vinyl ether-co-maleic anhydride) are dissolved in 5 mL organic solvent (acetone).","A predetermined amount of 5-fluorouridine is dissolved in 2 mL water.","Then, under magnetic stirring, 10 mL of a miscible organic solvent (ethanol) and 10 mL distilled water are added to the acetone solution of the copolymer.","The resulting mixture is stirred during 5 minutes more and the suspension of nanoparticles is concentrated under reduced pressure for organic solvents elimination.","The final volume is adjusted with water (or an aqueous solution) to 10 mL and the suspension is, then, incubated with the 5-fluorouridine aqueous solution.","The next step is to harden the resulting nanoparticles with human serum albumin (HSA).","In order to do this, a solution of HSA is prepared in water (30 mg/mL), and a predetermined amount of cross-linking agent is added to the suspension of nanoparticles for incubation for 120 minutes.","The suspension is then purified by ultracentrifugation (12 minutes at 10,000 rpm) or tangential filtration.","The supernatants are analyzed by HPLC.","The quantity of entrapped drug in the nanoparticles is determined as the difference with respect to the initial quantity added.","The residue or pellet is resuspended in water or in an aqueous solution of mannitol (5% w/v).","Finally, the resulting suspension of nanoparticles is freeze-dried maintaining all its properties.","FIG.","8 shows the influence of the initial concentration of 5-fluorouridine that is adsorbed and interacts with the reactive groups on the nanoparticle surface.","It is clear that when the initial amount of drug increases, the amount of FU carried by nanoparticles cross-linked with HSA also increases.","As expected, the immediately released fraction increased by increasing the amount of drug incubated with the freshly prepared nanoparticles.","This burst effect would be acting as an initial dose.","4.3.In Vitro Release Studies For these studies, the nanoparticles were prepared following the conditions described in 4.1.The in vitro release assay was carried out in a temperature-controlled bath, with constant stirring and a constant temperature of 37±1° C. FIG.","9 shows the results obtained for two different batches of nanoparticles prepared from 1 and 2 mg FU per mL suspension.","It can be observed that a fraction of the drug (approximately 30-50% of the drug) is adsorbed, in a labile way, onto the surface of the nanoparticles and is immediately released.","In addition, there is another fraction, which is strongly bound to the nanoparticles.","This second fraction was not released during the time in which the release study was performed.","However, it can be expected that in vivo conditions, the bond between the drug and the copolymer would break given its lability.","This would permit the here described nanoparticles to act as a biphasic releasing drug.","The great potential of this system is based on this property, together with their capacity to target certain organs and inflamed regions of the organism.","EXAMPLE 5 Manufacturing Process for the Preparation poly(methyl vinyl ether-co-maleic anhydride)nanoparticles Coated with Ganciclovir The process described below permits the preparation of colloidal pharmaceutical forms (i.e., nanoparticles and ligand-nanoparticle conjugates) containing ganciclovir or any other active molecule with a chemical structure similar to or derived from said drug.","5.1.Cross-Linkage with Human Serum Albumin (HSA) 100 mg of poly(methyl vinyl ether-co-maleic anhydride) are dissolved in 5 mL organic solvent (acetone).","A predetermined amount of ganciclovir is dissolved in water.","Then, under magnetic stirring, 10 mL of a miscible organic solvent (ethanol) and 10 mL distilled water are added to the acetone solution of the copolymer.","The resulting mixture is stirred during 5 more minutes, and the suspension of nanoparticles is concentrated under reduced pressure for organic solvents elimination.","The final volume is adjusted with water (or an aqueous solution) to 10 mL, and the suspension is then incubated with the ganciclovir aqueous solution.","The next step is to harden the resulting nanoparticles with human serum albumin (HSA).","In order to do this, a solution of HSA is prepared in water (30 mg HSA/mL water) and a predetermined amount of cross-linking agent is added to the suspension of nanoparticles for incubation for 120 minutes.","The suspension is then purified by ultracentrifugation (12 minutes at 10,000 rpm) or tangential filtration.","The supernatants are analyzed by HPLC.","The quantity of entrapped drug in the nanoparticles is determined as the difference with respect to the initial quantity added.","The residue or pellet is resuspended in water or in an aqueous solution of mannitol (5% w/v).","Finally, the resulting suspension of nanoparticles is freeze-dried maintaining its properties.","This manufacturing process gives similar results when nanoparticles are cross-linked with 1,3-diaminopropane (DP) in the conditions indicated in the previous examples.","FIG.","10 shows the influence of the bulk concentration of ganciclovir incubated with the nanoparticles in the percentage of drug bound to them.","It can be observed that an initial ganciclovir amount greater, than 4-5 mg, does not have much effect on the percentage of entrapped drug.","5.2.In Vitro Release Studies These in vitro release studies were carried out with nanoparticulate batches prepared as described in 5.1.In order to do this, 100 μL of different formulation (with similar drug entrapped efficiency but increasing amounts of drug loaded) were dispersed in 1 mL phosphate buffer solution containing tripsin.","The in vitro release assay was carried out in a temperature-controlled bath, with constant stirring and a constant temperature of 37±10° C. taking samples at different predetermined times.","A biphasic release profile was observed for all of the formulations tested.","This profile was characterized by a first phase of rapid (almost immediate) release (also know as a “burst effect”), followed by a second slow and continuous release.","During the first release stage (in the first eight hours), the percentages of drug released ranged between 40 and 50% of the loaded drug.","The second part of the curve was adjusted to a zero-order kinetic (see FIG.","11) that continued for more than 7 days.","FIG.","11 shows the ganciclovir release kinetic from the poly(methyl vinyl ether-co-maleic anhydride)nanoparticles, for a formulation containing 90 μg ganciclovir bound to the nanoparticles per niL suspension.","EXAMPLE 6 Manufacturing Process for the Preparation poly(methyl vinyl ether-co-maleic anhydride)nanoparticles Coated with the antisense oligonucleotide ISIS 2922 The process described below permits the preparation of colloidal pharmaceutical forms (i.e., nanoparticles and ligand-nanoparticle conjugates) containing antisense oligonucleotides or any other active molecule of DNA or RNA.","The example described below was carried out with the oligonucleotide ISIS 2922.ISIS 2922 is an oligonucleotide of 21 bases which possesses antiviral activity against cytomegalovirus.","6.1.Cross-Linkage with 1,3-diaminopropane (DP) 100 mg of poly(methyl vinyl ether-co-maleic anhydride) are dissolved in 5 mL organic solvent (acetone).","A predetermined amount of ISIS 2922 is dissolved in water.","Then, under magnetic stirring, 10 mL of a miscible organic solvent (ethanol) and 10 mL distilled water are added to the acetone solution of the copolymer.","The resulting mixture is stirred during 5 more minutes, and the suspension of nanoparticles is concentrated under reduced pressure for organic solvents elimination.","The final volume is adjusted with water (or an aqueous solution) to 10 mL, and the suspension is then incubated with the ISIS 2922 aqueous solution for 20 minutes.","The next step is to harden the resulting nanoparticles with 1,3-diaminopropane (DP).","For this purpose, a solution of DP is prepared in water (1% DP v/v water), and a predetermined amount of cross-linking agent is added to the suspension of nanoparticles for incubation for 5 minutes.","The suspension is then purified by ultracentrifugation (12 minutes at 10,000 rpm) or tangential filtration.","The supernatants are analyzed by capilar electrophoresis.","The quantity of entrapped drug in the nanoparticles is determined as the difference with respect to the initial quantity added.","The residue or pellet is resuspended in water or in an aqueous solution of mannitol (5% w/v).","Finally, the resulting suspension of nanoparticles is freeze-dried maintaining its properties.","FIG.","12 shows the influence of the initial amount of ISIS 2922 incubated with the nanoparticles on the amount of oligonucleotide loaded.","This procedure gives similar results when cross-linkage is carried out with (DP), in the conditions described in the previous examples.","DESCRIPTION OF THE FIGURES FIG.","1.Influence of the incubation time (abscissas) and temperature on the yield of the manufacturing process (ordinates) of poly(methyl vinyl ether-co-maleic anhydride)nanoparticles cross-linked with DP.","FIG.","2.Influence of the incubation time (abscissas) and temperature on the yield of the manufacturing process (ordinates) of poly(methyl vinyl ether-co-maleic anhydride)nanoparticles cross-linked with HSA.","FIG.","3.Influence of the initial amount of the cross-linking agent (1,3-diaminopropane in μLsol/mg polymer; abscissas) on the amount of lectin bound (μg/mg polymer; ordinates) to the poly(methyl vinyl ether-co-maleic anhydride)nanoparticles.","FIG.","4.Influence of the cross-linking agent (1,3-diaminopropane in μL sol 1% v/v/mg polymer; abscissas) on the amount of active Sambucus nigra lectin (μg/mg polymer; ordinates) bound to the poly(methyl vinyl ether-co-maleic anhydride)nanoparticles.","FIG.","5.Influence of the incubation time (abscissas) on the anti-cancer 5-fluorouridine loading in poly(methyl vinyl ether-co-maleic anhydride)nanoparticles cross-linked with DP (drug loading in μg FU/mg nanoparticles; ordinates).","Experimental conditions: initially 10 mg drug.","FIG.","6.Influence of the incubation time (abscissas) on the 5-fluorouridine loading in poly(methyl vinyl ether-co-maleic anhydride)nanoparticles cross-linked with HSA (Drug loading in μg FU/mg nanoparticles; ordinates).","Experimental conditions: initially 10 mg drug.","FIG.","7.Influence of the initial amount of 5-fluorouridine (FU) (μ/mg polymer; abscissas) on the drug loading in poly(methyl vinyl ether-co-maleic anhydride)nanoparticles cross-linked with 1,3-diaminopropane (μg FU/mg NP; ordinates).","The squares represent the total drug loading and the circles represent the drug immediately released by dilution.","Experimental conditions: Initially 10 mg drug.","FIG.","8.Influence of the initial amount of 5-fluorouridine (μg FU/mg polymer; abscissas) on the drug loading in poly(methyl vinyl ether-co-maleic anhydride)nanoparticles cross-linked with HSA (μg FU/mg NP; ordinates).","The squares represent the total drug loading and the circles represent the drug immediately released by dilution.","Experimental conditions: Initially 10 mg of drug.","FIG.","9.Study of the release (abscissas) of two formulations cross-linked with DP and prepared from two different concentrations of 5-fluouridine (FU 5 ordintaes).","FIG.","10.Influence of the initial amount of ganciclovir (Bulk GCV in mg; abscissas) on the drug encapsulation efficiency (in %, ordinates) in poly(methyl vinyl ether-co-maleic anhydride)nanoparticles cross-linked with HSA.","FIG.","11.In vitro release kinetic (hours in abscissas) in an aqueous solution of tripsin, for a formulation of poly(methyl vinyl ether-co-maleic anhydride)nanoparticles with ganciclovir (GCV-μg ordinates).","FIG.","12.Influence of the initial amount of ISIS 2922 (μg in abscissas) on its loading in poly(methyl vinyl ether-co-maleic anhydride)nanoparticles (μg ISIS/mg NP; ordinates).","Initial amount of nanoparticles (NP)=100 mg."]],"string":"[\n [\n \"Production of nanoparticles from methyl vinyl ether and maleic anhydride for the administration of hydrophilic pharmaceuticals, more particularly of puric and pyrimidinic bases\",\n \"Manufacture of nanoparticles on the basis of methyl vinyl ether and maleic acid for the administration of pharmaceuticals of an hydrophilic nature, in particular analogs of puric and pyrimidinic bases.\",\n \"The nanoparticles are obtained by desolvation with an hydroalcoholic phase of a methyl vinyl ether and maleic acid copolymer solution in acetone.\",\n \"The particles obtained are next treated with cross-linking agents (diamines or proteins) for the purpose of prolonging their useful life and are, possibly, incubated with a pharmaceutical which will be transported on the surface.\",\n \"The nanoparticles can carry the pharmaceutical likewise encapsulated which would then be added during the desolvation.\",\n \"In the case of the nanoparticle-ligand conjugates, the nanoparticles previously obtained and containing inside the pharmaceutical to be transported are incubated with the ligand or molecule which will contribute the property of specifically recognising a particular receptor of the organism.\",\n \"These pharmaceutical forms have as objective to improve the transport of the pharmaceutical or biologically active molecule to its site of action and/or absorption.\",\n \"This property improves the specificity and effectiveness of said pharmaceuticals.\"\n ],\n [\n \"1.a process for the manufacture of nanoparticles and ligand-nanoparticle conjugates with site specific delivery or targeting properties, able to carry drugs or biologically active molecules on their surface or in their interior characterized by the desolvation of poly(methyl vinyl ether-co-maleic anhydride)copolymer, dissolved in an organic solvent, and subsequent cross-linkage reaction with polyfunctional chemical compounds, including polyamines and polyhydroxyls: 2.A process according to claim 1, that comprises: a) desolvation of the poly(methyl vinyl ether-co-maleic anhydride)polymer dissolved in a polar organic phase at a concentration between 0.01 and 10% w/v, which may optionally contain a drug or biologically active molecule, with a hydroalcoholic solution in an organic phase/hydroalcoholic solution ratio of 1/1 to 1/10; b) elimination of the organic solvents by conventional methods such as filtration, centrifugation or evaporation, including the use of vacuum, among others; c) stabilization of the resulting nanoparticles with cross-linking agents; d) optionally, incubation of the nanoparticles with either the drug or the biologically active molecule or, alternatively, the ligand with targeting properties; e) purification of either the obtained nanoparticles or conjugates by conventional techniques such as ultracentrifugation, centrifugation, tangential filtration, among others; f) optional freeze-drying of the obtained nanoparticles or conjugates.\",\n \"3.A process according to claim 1, characterized in that nanoparticles or conjugates are produced in a way which permits encapsulation of the drug or biologically active molecule in their interior, that comprises: i) the addition of said drug or biologically active molecule in step a) and, ii) optionally, the addition of a second drug or a different biologically active molecule or, alternatively, a ligand with targeting properties in step d).\",\n \"4.A process according to claim 1, characterized in that nanoparticles are produced with the drug or biologically active molecule on outer layer, that comprises: i) the addition of the aforesaid drug or biologically active molecule in step d) and, ii) optionally, the addition of another drug or biologically active molecule in step a).\",\n \"5.A process according to claim 1, characterized in that unloaded nanoparticles are produced consisting of carrying out step a) and step d) without adding any drug or biologically active molecule or ligand.\",\n \"6.A process according to claim 1, characterized in that step c) is carried out by using polyamine or polyhydroxyl type polyfunctional reagents, including among them proteins and polymeric macromolecules such as non-ionic surfactants or polyvinylpyrrolidone.\",\n \"7.A process according to claim 1, characterized in that step c) is carried out using the cross-linking agent 1,3-diaminopropane (DP) at a concentration ranging between 0 and 1 mg DP/mg poly(methyl vinyl ether co maleic anhydride).\",\n \"8.A process according to claim 1, characterized in that step c) is carried out using albumin, such as human serum albumin or bovine serum albumin, at a concentration ranging between 0 and 10 mg albumin/mg poly(methyl vinyl ether co maleic anhydride).\",\n \"9.A process according to claim 1, characterized in that step f) is carried out by adding mannitol or sacarose as a cryoprotector agent at a concentration ranging between 0.1 and 10% in weight.\",\n \"10.A process according to claim 1, characterized in that the ligands used in the production of the nanoparticle conjugates have the characteristics of being able to recognize specific structures, or cellular or tissular receptors located on the surface or inside specific cell types in the organism.\",\n \"11.A process according to claim 10, characterized in that the ligands used are lectins, carbohydrates, monoclonal antibodies, vitamins, amino acids, lipids or molecules of a peptidic nature.\",\n \"12.A process according to claim 11, characterized in that the ligand is the Sambucus nigra lectin which is bound at a concentration ranging between 1 and 100 mg lectin/mg nanoparticles.\",\n \"13.A process according to claim 1, characterized in that the drugs or the biologically active molecules incorporated inside the nanoparticles and conjugates possess a hydrophilic character and are soluble in organic polar solvents.\",\n \"14.A process according to claim 13, characterized in that which the drug incorporated is the anti-tumor drug 5-fluorouridine.\",\n \"15.A process according to claim 14, characterized in that 5-fluorouridine is dissolved in acetone at a concentration ranging between 0.1 and 3.33 mg/mL and then added to the poly(methyl vinyl ether co maleic anhydride)aceton solution, in order to obtain a drug/polymer ratio ranging between 0.01 and 0.4 mg/mg.\",\n \"16.A process according to claim 1, characterized in that the drug or biologically active substance is carried on the surface of the nanoparticles and in which step d) of incubation is produced in an aqueous solution.\",\n \"17.A process according to claim 16, characterized in that the drugs or biologically active molecules incorporated into the surface of the nanoparticles are of hydrophilic character or are soluble in aqueous solutions.\",\n \"18.A process according to claim 17, characterized in that the drugs or biologically active molecules are either analogs of puric or pyrimidinic bases, or they are compounds of protein nature such as peptides, proteins, glycoproteins, lipoproteins, or they are carbohydrates.\",\n \"19.A process according to claim 18, characterized in that the drug incorporated in the surface of the nanoparticles is the anti-tumor agent 5-fluorouridine.\",\n \"20.A process according to claim 19, characterized in that 5-fluorouridine is dissolved in water and, subsequently, added to the suspension of nanoparticles at a concentration ranging between 10 and 1000 μg drug/mg polymer, obtaining concentrations greater than 200 μg of drug bounded to the surface per mg nanoparticles.\",\n \"21.A process according to claim 17, characterized in that the incorporated drug is the antiviral agent ganciclovir.\",\n \"22.A process according to claim 21, characterized in that the ganciclovir is dissolved in water and then added to a suspension of nanoparticles, at a concentration ranging between 0.1 and 20 mg drug/mg polymer, obtaining entrapment efficiencies greater than 20% of the initially added ganciclovir.\",\n \"23.A process according to claim 17, characterized in that the incorporated drug is an oligonucleotide.\",\n \"24.A process according to claim 23, characterized in that the incorporated drug is an antisense oligonucleotide.\",\n \"25.A process according to claim 24, characterized in that the incorporated drug is the antisense oligonucleotide ISIS 2922.26.A process according to claim 25, characterized in that ISIS 2922 is dissolved in water and later added to the suspension of nanoparticles at a concentration ranging between 0.1 and 200 μg drug/mg polymer, producing nanoparticles whose superficially-bounded drug concentration is greater than 2 μg per mg nanoparticle.\",\n \"27.Nanoparticles or conjugates obtainable by a manufacturing process according to claim 1, characterized by having an approximate average size of less than 500 nm.\",\n \"28.Nanoparticles or conjugates according to claim 27, characterized by showing a biphasic release profile, with a first phase of immediate release of up to 60% of the loaded drug or biologically active molecule, followed by a second phase in which the drug or biologically active molecule is released slowly and in a sustained release manner.\",\n \"29.Nanoparticles or conjugates characterized in that they have the following composition: 13-99% w/w poly(methyl vinyl ether co maleic anhydride), 0.001-15% w/w cross-linking agent, optionally, 0.001-15% w/w drug or biologically active molecule, optionally, 0.01-4.5% w/w ligand with specific targeting properties, optionally, 70-82% w/w cryoprotector.\",\n \"30.Use of A method comprising using the nanoparticles and conjugates according to claim 27 for the administration of drugs or biologically active molecules to a patient.\",\n \"31.A method comprising using the nanoparticles and conjugates according to claim 27 in the administration of third generation colloidal pharmaceutical forms to a patient.\",\n \"32.A method comprising using the nanoparticles and conjugates of claim 27 for the pellicular coating of macroscopic pharmaceutical forms such as tablets, granules, granulates and pellets.\",\n \"33.A method comprising using the nanoparticles and conjugates of claim 27 loading 5-fluorouridine in the preparation of compositions that are useful in the treatment of certain diseases such as colon cancer, cancers of the gastrointestinal tract, breast cancer, cancers of the cervix and endometrium, as well as cancers of the head and neck, liver, ovary, pancreas, prostrate and skin.\",\n \"34.A method comprising using the nanoparticles and conjugates of claim 27 loading ganciclovir in the preparation of compositions which are useful for the treatment of infections induced by human cytomegalovirus.\",\n \"35.A method comprising using the nanoparticles and conjugates of claim 27, loading ganciclovir, as adjuvant, in the preparation of gene therapy compositions, which incorporate suicide genes and, in particular, the thymidine quinase gene.\",\n \"36.A method comprising using the nanoparticles and conjugates of claim 27 loading the antisense oligonucleotide ISIS 2922 in the preparation of compositions useful for the treatment of infections induced by human cytomegalovirus.\"\n ],\n [\n \"<SOH> TECHNICAL FIELD OF THE INVENTION <EOH>The present invention is within the scope of the procedures for the production of non-biological vectors, nanoparticles and nanoparticle-ligand conjugates, for the transport and administration of pharmaceuticals or active biological molecules of hydrophilic nature through the use of the methyl vinyl ether and maleic anhydride copolymer, poly(methyl vinyl ether-co-maleic anhydride), as priority element.\"\n ],\n [\n \"TECHNICAL FIELD OF THE INVENTION The present invention is within the scope of the procedures for the production of non-biological vectors, nanoparticles and nanoparticle-ligand conjugates, for the transport and administration of pharmaceuticals or active biological molecules of hydrophilic nature through the use of the methyl vinyl ether and maleic anhydride copolymer, poly(methyl vinyl ether-co-maleic anhydride), as priority element.\",\n \"STATE OF THE ART The behavior of a drug after its administration in the organism is determined by a combination of different processes: distribution and elimination when the drug is administered by intravenous route; absorption, distribution, and elimination when an extravascular route is used.\",\n \"Each one of these processes mainly depends on the physico-chemical properties of the drug substance which are determined by its chemical structure.\",\n \"After administering the drug by conventional means, it is distributed throughout the organism according to its molecular structure, which determines its physicochemical properties (molecular mass, lipophilia, pKa, crystalline structure, solubility), biochemical properties (affinity with plasmatic or tissue proteins, affinity with the membrane or intracellular receptors, sensibility to the enzymes responsible for its biotransformation, etc.)\",\n \"and pharmacological properties (ability to clear biological membranes, intensity of the action, etc.).\",\n \"Numerous studies have shown that the classical pharmaceutical forms (tablets, capsules, suppositories, injections, etc.)\",\n \"only permit the modulation of the intensity and/or speed of release of the biologically active molecule into the blood circulation (absorption), but they have no effect on the subsequent stages of in vivo behavior of the drug (distribution and elimination).\",\n \"During the distribution stage, the drug spreads to a large number or to every anatomical area of the organism.\",\n \"This implies: (i) the administration of doses higher to those which would theoretically be needed to obtain the required effect or (ii) the repeated administration of the drug.\",\n \"The therapeutic effect is attained in spite of the loss of an important fraction of the drug dose in different tissues other than those where it should act, resulting in unwanted side effects.\",\n \"This nonspecific distribution of the drug, tolerable for those with high therapeutic indexes (quotient between the dose that provokes the toxic effects and the therapeutic dose), could become the limiting factor for the clinical use of certain biologically active molecules (example: the 5-fluorouridine).\",\n \"So as to obtain a more rational and better adapted therapeutic use, one of the most promising possibilities is that which uses the concept of vectorization for the increase in the efficiency and specificity of action of the drugs.\",\n \"This concept consists in associating a drug or a biologically active molecule to an appropriate vector or carrier.\",\n \"Although there are different types of vectors, in this study only the characteristics and properties of the non-biological vectors (liposomes, nanoparticles and conjugates) will be studied.\",\n \"The use of these new pharmaceutical forms permits the association between the drug and a system whose mission is to carry the drug to its pharmacological target instead of remaining in a free state.\",\n \"In fact, the use of non-biological vectors permits the masking of the physicochemical properties of the drug and, logically, the characteristics of the carrier are what permit control over the distribution stage.\",\n \"In the best of cases, the vector-drug pair should be inactive and remain stable in the different biological media between the site of administration and the place of action.\",\n \"The most important potentialities that these vectors or non-biological carriers provide are the following (Couvreur & Puisieux.\",\n \"Adv.\",\n \"Drug Del.\",\n \"Rev., 10 (1993) 141-162): protection against the chemical, enzymatic or immunological inactivation of the drug between the administration site and the place of action; improvement in the transport of the biologically active molecule to places of difficult access and its penetration into the cell (in the case of infections located in the intracellular territories which are inaccessible by simple diffusion); increase in the specificity of action by a selective, effective, and regular concentration of drug in the cellular and/or molecular target.\",\n \"In this way, with smaller doses, the obtained therapeutic activity is, at least, identical and the side effects are milder; decrease in the toxicity for certain organs by means of modifying the tissular distribution of the biologically active molecule that is carried; in some cases, a lengthening of the time of residence of the drug in the organism (interesting for those molecules with high clearance and a low biological half-life) and, control of its release.\",\n \"All of this implies a decrease of the frequency of drug intake and indirectly, an increase in the observance of the treatment on the part of the patient.\",\n \"Within the group of non-biological vectors, colloidal systems of solid particle type with a size smaller than a micrometer stand out; they are also called nanoparticles.\",\n \"These are subdivided into matrix nanospheres and vesicular nanocapsules (Orecchioni e Irache, Formes pharmaceutiques pour application locale.\",\n \"Lavoisier Tech & Doc., Paris, 1996, 441-457).\",\n \"The nanocapsules are vesicular systems formed by an internal cavity which is surrounded by a membrane or polymeric wall.\",\n \"The nanospheres are matrix forms, formed by a tridimensional polymeric net.\",\n \"In both cases, the molecules of the biologically active substance can be dissolved, entrapped or adhered to the macromolecular structure (in the nanospheres) or encapsulated by the polymeric membrane (in the nanocapsules).\",\n \"Also, the biologically active substance may remain adsorbed on the surface of the nanoparticles.\",\n \"Depending on the used material, the nanospheres can be prepared from: reactions of monomer polymerization; macromolecules of natural origin; preformed synthetic polymers.\",\n \"The nanoparticles produced from preformed polymers are generally obtained by two different procedures: (i) application of techniques based on the emulsion of an organic solution of the polymer (Vanderhoff et al, U.S. Pat.\",\n \"No.\",\n \"4,177,177 (1979)) and (ii) by processes derived from the desolvation of the polymer (Fessi et al, FRENCH PAT, (1986) 2 608 988).\",\n \"On the contrary, nanoparticles of natural macromolecules are generally obtained by means of (i) gelification techniques (Rajaonari-vony et al, J. Pharm.Sci, 82 (1993) 912-917), (ii) emulsion (Vanderhoff et al, U.S. Pat.\",\n \"No.\",\n \"4,177,177 (1979) and (iii) controlled desolvation (Marty et al, Pharm Acta Helv., 53 (1978) 79-82).\",\n \"Generally, a supplementary stage is needed to estabilize the aforesaid systems.\",\n \"In the organism, the distribution of the nanoparticles generally depends on their physicochemical characteristics (mainly their size and their surface properties).\",\n \"By intravenous route, the submicronic vectors (for example, the nanoparticles) are captured by the endothelial reticular system (ERS), also called mononuclear phagocyte system, principally located in the liver, spleen, lungs and marrow.\",\n \"This property that nanoparticles possess converts them into very interesting pharmaceutical forms for the administration of drugs destined to the treatment of diseases located in the ERS or in the treatment of diseases in which the cells of ERS possess a function (infections, cancerous processes, etc.).\",\n \"On the other hand, administration of nanoparticles through the mucous membranes (oral, nasal, ocular, etc.\",\n \"routes) enables the development of adhesive interactions which increase the time of residence of the pharmaceutical form in contact with the site of absorption or action of the drug (Ponchel & Irache, Adv.\",\n \"Drug Del.\",\n \"Rev., 34 (1998) 191-219).\",\n \"For the design of pharmaceutical forms (based on nanoparticles) capable of vectorizing cells or tissues that are different from those which the nanoparticles normally distribute, active vectors (also called third generation vectors or nanoparticle-ligand conjugates can be used) are used.\",\n \"These systems are obtained by means of the binding of molecules capable of interacting specifically with cellular or molecular receptors to the surface of the nanoparticles.\",\n \"Among these molecules or ligands, polyethylenglycoles (“stealth” vectors; Allen et al., Cancer Res., 52 (1992) 2431-2439), certain monoclonal antibodies (Weinstein et al, Pharm.\",\n \"Therap., 24 (1984) 207-233), lectins (Irache et al, Biomaterials, 15 (1994), carbohydrates (Maruyama et al, Biomaterials, 15 (1994) 1035-1042), amines (Roser & Kissel, Eur.\",\n \"J. Pharm.\",\n \"Biopharm., 39 (1993) 8-12), vitamins (Russell-Jones & Westwood, U.S. Pat.\",\n \"No.\",\n \"07/956.003; (1992) WO 92/17167), etc.\",\n \"can be used.\",\n \"Nowadays, the preparation of nanoparticle pharmaceutical forms containing hydrophilic drugs is limited by several aspects of their formulation.\",\n \"In general, hydrophilic substances or drugs do not show any affinity for matrix lipophylic systems (for example, nanospheres).\",\n \"Therefore, production yields are very low and, logically, can not be applied to drug administration.\",\n \"Analogs of puric and pirimidinic bases such as oligonucleotides, 5-fluouridine or ganciclovir are included in this category of hydrophilic drugs.\",\n \"Poly(methyl vinyl ether-co-maleic anhydride)synthetic copolymers have a wide range of applications in the agricultural field (commercial name: Agrimer® VEMA, ISP) as adyuvants and estabilizers in veterinary solutions, in seed, granules and tablet coatings, etc.\",\n \"These copolymers are also used in chemical and pharmaceutical fields (commercial name: Gantrez® Copolymers, ISP), mainly due to their capacity to form coating films and their high bioadhesive capacity (Esposito er al., Biomaterials, 15 (1994) 177-182).\",\n \"They have also been described as thickeners, complexing agents and hydrophilic colloids.\",\n \"Likewise, their use in sustained release systems such as transdermic patches or ocular matrixes has been proposed (Finne et al., Int.\",\n \"J.\",\n \"Pharm., 78 (1992) 237-241).\",\n \"Their main advantages are their relatively low toxicity (LD50=8-9 g/Kg by oral route) and their easy production, regarding both quantity and price.\",\n \"Poly(methyl vinyl ether-co-maleic anhydride)copolymers have also been used to prepare microparticle vectorial systems by a process of complex coacervation (Mortada et al., J.\",\n \"Microencas., 4 (1987) 11-21).\",\n \"Poly(methyl vinyl ether-co-maleic anhydride)copolymer nanospheres are new matrix systems of vectors of submicronic size.\",\n \"These are able to retain the biologically active substance thanks to (i) solution or entrapping inside the macromolecular structure or matrix, (ii) covalent bonds result of the interaction of the drug and the polymer anhydride groups and (iii) processes of adsorption by means of weak bonds.\",\n \"The extremely high reactivity of this copolymer due to its cyclic anhydride groups makes it perfect to retain hydrophilic substances in its structure.\",\n \"They are also very interesting systems for the fixation of ligands to the nanoparticle surface, without having to use highly toxic organic reactants (glutaraldehyde and carbodiimides) .\",\n \"A variety of current drugs (for example, 5-fluouridine, ganciclovir) and biotechnological products (for example, antisense oligonucleotides) can be mentioned as hydrophilic active ingredients.\",\n \"All of them are analogs of puric and pirimidinic bases.\",\n \"Other kind of molecules such as proteins, peptides, lectins and, in general, all those substances which have alcohol groups and/or primary amines (for example: lectins) can also be fixed.\",\n \"The majority of the vectorial systems that have been described in the literature and are used to administrate hydrophilic substances require laborious manufacturing processes.\",\n \"Thus, for example, quite complex bonds with toxic reactions (Rimoli et al., J.\",\n \"Controlled Rel., 58 (1999) 61-68), multiple emulsions (Jeffery et al., Pharm.\",\n \"Res., 10 (1993) 362-367) or working with very high drug concentrations in conditions not applicable to the industrial field have been proposed.\",\n \"The conditions of applicability that allow to ascertain of the industrial interest of poly(methyl vinyl ether-co-maleic anhydride)nanoparticle vectors, could be the following: 1.The obtaining of homogeneous populations with submicronic particle size.\",\n \"This favors the distribution, improves the transport of active substances to its place of action and increases the penetration in the target cells.\",\n \"2.The use in the chemical-pharmaceutical field of abundant preformed copolymers with a low expense, for the administration of drugs.\",\n \"3.The obtaining of particles in which part of the drug will be entrapped and other part will be bound to the matrix by a covalent bond.\",\n \"This favors the bi-phasic release of the drug.\",\n \"The entrapped fraction enables a very quick initial release, while the bound fraction is released in a controlled way over a long period of time.\",\n \"This period will depend on particle degradability and on the breakage of the bounds between the polymer and the biologically active molecule.\",\n \"4.The obtaining of particles in which the drug is bound by a covalent bond to the surface.\",\n \"Like in the previous case, the drug will be released in a controlled way and depending on the velocity of the breakage of the bonds between the groups located on the nanoparticle surface and the biologically active molecule.\",\n \"5.The use of the aforementioned nanoparticles, as a base to produce third generation vectors or nanoparticle-ligand conjugates capable of carrying the active principle to certain cells or tissues by a specific recognition system.\",\n \"6.Working conditions at room temperature that avoid the possible degradation of thermolabile active substances of hydrophilic nature, and of chemical and/or biotechnological origin.\",\n \"7.The obtaining of particles capable of protection against premature inactivation of the encapsulated active principles.\",\n \"DESCRIPTION OF THE INVENTION Poly(methyl vinyl ether-co-maleic anhydride)nanoparticles are obtained by a desolvation method consisting in the addition of a polar solvent (miscible with the copolymer solution) to an organic solution of the copolymer.\",\n \"Subsequently, a second non-solvent liquid is added, in this case an hydroalcoholic solution.\",\n \"In order to do this, the copolymer is dissolved in a predetermined volume of acetone and then an ethanol solution is added.\",\n \"Then a similar volume of water is added to this solution.\",\n \"The particles are instantly formed in the medium, which takes on the appearance of a milky suspension.\",\n \"The organic solvents (ethanol and acetone) are then eliminated by evaporation under reduced pressure.\",\n \"The particles remain in a stable aqueous suspension.\",\n \"The poly(methyl vinyl ether-co-maleic anhydride)copolymer is insoluble in water, but after some time it hydrolyzes to an acid form which is soluble in water.\",\n \"Although this hydration process starts on contacting with water, it lasts approximately 10 hours.\",\n \"This is the reason why a supplementary stage is required.\",\n \"This stage consists in the stabilization of the particles by the cross-linkage of the particles with the purpose of increasing their stability in aqueous media.\",\n \"This also allows the modulation and control of the release of the encapsulated drug.\",\n \"As cross-linking agents, polyfunctional chemical compounds, including a number of polyamines and polyhydroxyl molecules, can be used.\",\n \"The main cross-linking agents used are human serum albumin (HSA) and 1,3-diaminopropane (DP).\",\n \"Other macromolecules, such as bovine serum albumin (BSA), povidone and gelatin, have also shown to be good stabilizing agents.\",\n \"The next manufacturing step consists in purifying the resultant nanoparticles in order to eliminate all the remains of the process, mainly polymer and cross-linker residues.\",\n \"To do this, different techniques such as ultracentrifugation or tangential filtration may be used.\",\n \"Finally, the purified nanoparticles may be lyophilized or freeze-dried for their long term storage and preservation.\",\n \"Depending on the aqueous solubility of the drug, two different procedures may be used to associate a drug to the nanoparticles.\",\n \"The first one consists in binding the drug (hydrophilic or with a low aqueous solubility) to the polymer in an organic solution.\",\n \"In order to do this, poly(methyl vinyl ether-co-maleic anhydride) and the drug are dissolved in a given volume of acetone.\",\n \"This solution is incubated and stirred at a predetermined temperature and for a fixed time.\",\n \"Then a given volume of ethanol is added, followed by the addition of a similar volume of water.\",\n \"The particles are immediately formed in the medium, and this suspension takes on a milky appearance.\",\n \"The organic solvents (ethanol and acetone) are removed by means of evaporation under reduced pressure and the particles remain in a stable aqueous suspension.\",\n \"The particles are cross-linked with one of the aforementioned products (mainly HSA and DP) and purified by ultracentrifugation or tangential filtration.\",\n \"Finally, the particles can be lyophilized.\",\n \"The second one is a process for the loading of molecules with a high polarity, in which the drug is bound to the surface of freshly prepared nanoparticles.\",\n \"In this case, the poly(methyl vinyl ether-co-maleic anhydride)copolymer is dissolved in a given volume of acetone.\",\n \"Then, a given amount of ethanol is added followed by the addition of a similar amount of water.\",\n \"The particles are immediately formed in the medium, and this suspension takes on a milky appearance.\",\n \"The organic solvents (ethanol and acetone) are removed by means of evaporation under reduced pressure, and the particles remain in a stable, aqueous medium.\",\n \"The freshly prepared nanoparticles are then incubated at a fixed temperature for a predetermined time with an aqueous solution of the drug and the solution is incubated at a predetermined temperature and for a fixed time.\",\n \"Afterwards, particles are stabilized by cross-linkage using one of the aforementioned agents (mainly HSA and DP) and purified by ultracentrifugation or tangential filtration.\",\n \"Finally, nanoparticles can be lyophilized.\",\n \"Finally, to prepare ligand-nanoparticle conjugates, the manufacturing process is quite simple and is based on the incubation between the nanoparticles and the ligand to be bound to the surface of the carriers.\",\n \"Then, a supplementary purifying step is needed to eliminate the unbound ligand remains.\",\n \"As in the previous cases, the obtained conjugates can be lyophilized.\",\n \"The following examples illustrate the present invention, but in no way do they pretend to limit the possible uses of the invention.\",\n \"In these examples, three different pharmaceuticals, either derivatives from puric or pyrimidinic bases, have been used.\",\n \"These drugs are the anti-cancer agent 5-fluorouridine, the antiviral ganciclovir and the antisense oligonucleotide ISIS 2922.5-fluorouracile (2,4-dioxo-5-fluropyrimidine) and, its derivative, 5-fluorouridine (2,4-dioxo-5-fluoropyrimidine riboside) are analogs of the pyrimidines, and their molecules contain a fluoride atom.\",\n \"These drugs are usually used in the treatment of colon cancer, either alone or in combination with other drugs.\",\n \"They are also used in the treatment of breast, cervix, endometrium, gastrointestinal, head and neck, liver, ovary, pancreas, prostrate and skin tumors.\",\n \"The 5-fluorouridine has an antitumoral activity which is 100-times more potent than 5-fluorouracile and other derivatives of this drug (Jampel et al.\",\n \"Arch.\",\n \"Ophtal., 108 (1990) 430-435).\",\n \"However, its clinical use is limited due to the severe side effects (leukopenia, thrombocytopenia and gastrointestinal toxicity) when administered in a traditionally pharmaceutical form (Brusa et al., II Farmaco, 52 (1997) 71-81).\",\n \"Administration of this drug in the form of nanoparticles would allow an increase of the therapeutic index of this drug.\",\n \"Ganciclovir, (9-[(1,3-dihydroxy-2-propoxy)methyl]guanine, is a synthetic nucleoside analogue.\",\n \"This molecule exhibits antiviral activity against some herpes viruses (Martin et al., J. Med.\",\n \"Chem., 26 (1983) 759-761).\",\n \"Furthermore, it is the most frequently used antiviral drug in the treatment of infections caused by human cytomegalovirus (CMV) in immunocompromised patients, mainly in those with the acquired immunodeficiency syndrome (AIDS), congenital immunodeficiency or in individuals who have undergone organ transplantation.\",\n \"The principal clinical manifestation is retinitis, which if left untreated, results in irreversible blindness (Markham & Faulde, Drugs, 48 (1994) 455-460).\",\n \"The use of nanoparticles would permit the obtaining of pharmaceutical dosage forms showing sustained release properties and controlled drug release.\",\n \"Besides, and given its small size, they would minimally affect vision when administered by intravitreous route.\",\n \"In addition, during the last few years, ganciclovir has been used as an adjuvant for gene therapy involving suicide genes.\",\n \"This new therapeutical technique consists of administrating retrovirus, adenovirus or herpes simplex incorporating the thymidine quinase (tk) gene, followed by the adminisration of ganciclovir to the patient.\",\n \"The viral vectors permit the incorporation of tk into the dividing cells and, after expression, the ganciclovir is metabolized by the expressed enzyme in its phosphate derivative.\",\n \"This derivative is then transformed into ganciclovir-diphosphate and finally in ganciclovir-triphosphate, which is toxic for the cell and induces its apoptosis.\",\n \"This is being successfully used for the treatment of pancreas tumors (Carrio et al., Gene Ther., 6 (1999) 547-553), prostate adenocarcinomas (Herman et al., Hum.\",\n \"Gene Ther., 10 (1999) 1239-1249) and hepatocarcinomas (Qian et al., Hum.\",\n \"Gene.\",\n \"Ther., 10 (1997) 349-358).\",\n \"Antisense oligonucleotides are new therapeutic agents which are able to regulate the genetic expression of living organisms.\",\n \"This technology consists in the use of synthetic fragments of DNA or RNA, called oligonucleotides, which allow the stoppage of the production of illnesses related to the synthesized proteins.\",\n \"Antisense compounds block the transmission of genetic information between the nucleus and the place where the protein is produced inside the cell.\",\n \"There is a variety of applications that include the treatment of certain types of cancer, inflammatory pathologies and viral illnesses.\",\n \"ISIS 2922 is an antisense oligonucleotide with a sequence complementary to messenger RNA in the human citomegalovirus region IE1 (Anderson et al., Antimicrob.\",\n \"Agents Chemother., 40 (1996), 2004-2011).\",\n \"This oligonucleotide blocks the expression of a protein essential for viral replication, thereby inhibiting the replication of the virus (Leeds et al., Drug Metabol.\",\n \"Dispos., 25 (1997) 921-926) with an in vitro efficiency of about 30-times greater than traditional therapies.\",\n \"However, physico-chemical oligonucleotide characteristics (high mass, charged, hydrophilic molecules) do not allow its free access into the cell where they have to act.\",\n \"The use of nanoparticles permits an increase of the penetration capacity of those substances and a protection against exo- and endonucleases.\",\n \"The size and zeta potential of the nanoparticles were determined in a Zetamaster apparatus (Malvern Instruments/Optilas, Spain).\",\n \"The drugs, 5-fluoroudine (provided by Sigma; Alcobendas, Spain) and ganciclovir (Cymevene®, Roche; Madrid, Spain), were analyzed by High Performance Liquid Chromatography (HPLC), using a Hewlett Packard series 1050 (Germany).\",\n \"The antisense oligonucleotide, ISIS 2922, was obtained from Pharmacia Biotech (Cambridge, United Kingdom) and analyzed by zone capillary electrophoresis.\",\n \"The Sambucus nigra lectin was obtained from Vector Laboratories (USA) and determined by gel permeability chromatography.\",\n \"The samples of nanoparticles were lyophilized using a Virtis Genesis 12EL apparatus (USA).\",\n \"EXAMPLE 1 Preparation of Nanoparticles from poly(methyl vinyl ether-co-maleic anhydride)copolumer The process described below is valid for the preparation of colloidal pharmaceutical forms, nanoparticle type, which can be used for the administration of drugs or biologically active molecules.\",\n \"In addition, these nanoparticles can also be used for the film coating of macroscopic pharmaceutical forms, such as tablets, granules, granulates, and minigranules.\",\n \"1.1.Cross-Linkage with 1,3-diaminopropane (DP) 100 mg of poly(methyl vinyl ether-co-maleic anhydride)copolymer are dissolved in 5 mL organic solvent (acetone).\",\n \"Then, under stirring, 10 mL of a miscible organic solvent (ethanol) and 10 mL distilled water are added.\",\n \"The resulting mixture is stirred for homogenization during 5 minutes and the suspension of nanoparticles is evaporated under reduced pressure for organic solvents elimination.\",\n \"The final volume is adjusted with water (or an aqueous solution) to 10 mL.\",\n \"The next step is to cross-link the resulting nanoparticles with 1,3-diaminopropane (DP).\",\n \"In order to do this, a DP solution is prepared in water (1% v/v), and a predetermined amount of cross-linking agent is added to the suspension of nanoparticles for incubation for 5 minutes.\",\n \"The suspension is then purified by ultracentrifugation (12 minutes at 10,000 rpm) or tangential filtration.\",\n \"The supernatants are eliminated and the residues are resuspended in water or in an aqueous solution of mannitol (5% w/v).\",\n \"Finally, the resulting suspension of nanoparticles is lyophilized maintaining all its properties.\",\n \"The resulting formulation of the nanoparticles suspension before liophilization is: Poly(methyl vinyl ether-co-maleic anhydride) 1.0% w/v Mannitol 5.0% w/v 1,3-diaminopropane c.s.\",\n \"Purified water c.s.p.\",\n \"10 mL The mean size of the obtained nanoparticles is lower than 300 nm and the carriers display a negative superficial charge.\",\n \"Table 1 shows the influence of the amount of DP (used to cross-link the nanoparticles) on the size and zeta potential of the resulting nanoparticles.\",\n \"From these results, it is clear that an increase in the amount of cross-linking agent used to harden the nanoparticles increases the particle size and decreases negative zeta potential of these colloidal carriers.\",\n \"This last fact proves the reaction between the anhydride groups from the surface of the particles and the cross-linking agent.\",\n \"TABLE 1 Effect of the quantity of the cross-linking agent (DP) on the size and zeta potential of the resulting nanoparticles.\",\n \"DP Quantity (mL) Size (nm) Zeta potential (mV) 0 206.7 ± 1.13 −51.7 ± 2.45 2 239.8 ± 5.16 −37.8 ± 1.13 3 270.9 ± 4.13 −27.7 ± 2.26 5 270.1 ± 10.53 −23.7 ± 0.85 10 275.0 ± 11.33 −23.7 ± 0.78 The yield of the nanoparticles manufacturing process was calculated as the quotient between the weight of the freeze-dried samples and the initial amount of polymer used to prepare the nanoparticles, either with mannitol or without cryoprotector.\",\n \"From a previously known amount of the polymer (100 mg) the amount that had been transformed in nanoparticles at the end of the process was determined by gravirnetry.\",\n \"In any case, the yield of the manufacturing process (expressed in % with respect to the initial mass of polymer) was high; although higher efficiencies were obtained when the manufacturing process was carried out at room temperature rather than at 40° C. (see FIG.\",\n \"1).\",\n \"1.2.Cross-Linkage with Human Serum Albumin (HSA) 100 mg of poly(methyl vinyl ether-co-maleic anhydride)copolymer are dissolved in 5 mL of an organic solvent (acetone).\",\n \"Then, under magnetic stirring, 10 mL of a miscible organic solvent (ethanol) and 10 mL distilled water are added.\",\n \"The resulting mixture is stirred for 5 minutes, and the suspension of nanoparticles is evaporated under reduced pressure for organic solvent elimination.\",\n \"The final volume is adjusted with water (or an aqueous solution) to 10 mL.\",\n \"The next step is to harden the resulting nanoparticles with human serum albumin (HSA).\",\n \"In order to do this, a HSA solution is prepared in water (30 mg/mL), and a predetermined amount of cross-linking agent is added to the suspension of nanoparticles for incubation for 120 minutes.\",\n \"The suspension is then purified by ultracentrifugation (12 minutes at 10,000 rpm) or tangential filtration.\",\n \"The supernatants are eliminated and the residues resuspended in water or in an aqueous solution of mannitol (5% w/v).\",\n \"Finally, the resulting suspension of nanoparticles is freeze-dried maintaining all its properties.\",\n \"The resulting formulation of the nanoparticles solution before liophilization is: Poly(methyl vinyl ether-co-maleic anhydride) 1.0% w/v Mannitol 5.0% w/v Human serum albumin c.s.\",\n \"Purified water c.s.p.\",\n \"10 mL The mean size of the obtained nanoparticles is lower than 300 nm and the carriers display a negative superficial charge.\",\n \"Table 2 shows the influence of the amount of HSA (used to cross-link the nanoparticles) on the size and zeta potential of the resulting nanoparticles.\",\n \"It was observed that an increase in the amount of cross-linking agent used to harden the nanoparticles slightly increases the size of the resulting nanoparticles and decreases their negative zeta potential.\",\n \"These results prove that the anhydride groups on the surface of the particles react with the cross-linking agent.\",\n \"TABLE 2 Effect of the quantity of the cross-linking agent HSA on the size and zeta potential of the resulting nanoparticles.\",\n \"Amount HSA Zeta (mg/mg polymer) Size (nm) potential (mV) 0 206.7 ± 1.13 −51.7 ± 2.45 10 239.8 ± 5.16 −42.7 ± 1.13 15 240.9 ± 15.05 −42.7 ± 1.00 The yield of the nanoparticles manufacturing process was calculated as the quotient between the weight of the freeze-dried samples and the initial amount of polymer used to prepare the nanoparticles, either with mannitol or without cryoprotector.\",\n \"The amount of poly(methyl vinyl ether-co-maleic anhydride)copolymer that had been transformed in nanoparticles at the end of the process was determined by gravimetry from a previously known amount of the polymer (100 mg).\",\n \"In any case, the yield of the manufacturing process (expressed in % with respect to the initial mass of polymer) was high; although higher efficiencies were obtained when the manufacturing process was carried out at room temperature rather than at 40° C. (see FIG.\",\n \"2).\",\n \"EXAMPLE 2 Manufacturing Process of Nanoparticle Conjugates Based on the Binding of Lectin to the Surface of poly(methyl vinyl ether-co-maleic anhydride)nanoparticles The process described below permits the preparation of third generation pharmaceutical colloidal forms for the site specific delivery or targeting of drugs.\",\n \"These forms are formed by the binding of a ligand to the surface of copolymer nanoparticles.\",\n \"The ligand offers the possibility of driving the pharmaceutical dosage form to a specific site in the organism.\",\n \"In fact, the ligand should specifically recognize certain receptors located on the surface or inside a certain type of cells.\",\n \"These forms are known as conjugates.\",\n \"In the next example, the model ligand used was the lectin of Sambucus nigra agglutinin (SNA).\",\n \"First of all, 100 mg of poly(methyl vinyl ether-co-maleic anhydride) are dissolved in 5 mL organic solvent (acetone).\",\n \"Then, under magnetic stirring, 10 mL of a miscible organic solvent (ethanol) and 10 mL distilled water are added.\",\n \"The resulting mixture is stirred for 5 minutes, and the suspension of nanoparticles is concentrated under reduced pressure for organic solvent elimination.\",\n \"The final volume is adjusted with water (or an aqueous solution) to 10 mL.\",\n \"The next step is to harden the resulting nanoparticles with 1,3-diaminopropane (DP).\",\n \"In order to do this, a solution of DP is prepared in water (1% v/v), and a predetermined amount of cross-linking agent is added to the suspension of nanoparticles for incubation for 5 minutes.\",\n \"The suspension is then purified by ultracentrifugation (12 minutes at 10,000 rpm) or tangential filtration.\",\n \"The supematants are eliminated and the residues resuspended in water.\",\n \"Then the purified nanoparticles are incubated with the lectin.\",\n \"In order to do this, a variable amount of SNA is added to the nanoparticle suspension and this mixture is magnetically stirred at room temperature for 1 hour.\",\n \"The conjugates are centrifuged twice at 10,000 rpm for 12 minutes, in order to remove the unbound lectin (5% w/v).\",\n \"Finally, the resulting suspension of conjugates is freeze-dried after the addition of sacarose or mannitol as cryoprotectors.\",\n \"FIG.\",\n \"3 shows the influence of the initial amount of cross-linking agent on the lectin bound to the poly(methyl vinyl ether-co-maleic anhydride)nanoparticles.\",\n \"The lectin was determined by HPLC (High performance Liquid Chromatography).\",\n \"It was evident that as the amount of cross-linking agent increases, the amount of lectin being loaded by the nanoparticles decreases.\",\n \"Likewise, the amount of lectin bound to the nanoparticles is higher when the initial amount of lectin increases.\",\n \"The quantity of active lectin bound to the nanoparticles was determined by means of an erythrocyte agglutination test.\",\n \"It was observed that by increasing the initial quantity of lectin, a greater amount of lectin bound to the nanoparticles remained active.\",\n \"When analyzing the influence of the amount of cross-linking agent, it was observed that there are certain ideal conditions that allow the optimal binding of lectins to the nanoparticle surfaces, around 30 μg/mg nanoparticle (FIG.\",\n \"4).\",\n \"Similar results were obtained when using human serum albumin (HSA) or bovine serum albumin (BSA) as cross-linking agents.\",\n \"EXAMPLE 3 Manufacturing Process for the Preparation of 5-fluorouridine-loading poly(methyl vinyl ether-co-maleic anhydride)nanoparticles The process described below permits the preparation of colloidal pharmaceutical forms (i.e., nanoparticles and ligand-nanoparticle conjugates) carrying 5-fluorouridine or any other active molecule with a chemical structure similar to or derived from said drug.\",\n \"3.1.Cross-Linkage with 1,3-diaminopropane (DP) 100 mg of poly(methyl vinyl ether-co-maleic anhydride) are dissolved in 2 mL organic solvent (acetone).\",\n \"A predetermined amount of 5-fluorouridine is dissolved in 3 mL acetone, until a theoretical maximum of 10 mg/mL is reached.\",\n \"The two aforementioned solutions are mixed and then incubated at room temperature for 2 hours.\",\n \"Then, under magnetic stirring, 10 mL of a miscible organic solvent (ethanol) and 10 mL distilled water are added to the acetone solution.\",\n \"The resulting mixture is stirred for 5 more minutes, and the suspension of nanoparticles is concentrated under reduced pressure for organic solvents elimination.\",\n \"The final volume is adjusted with water (or an aqueous solution) to 10 mL.\",\n \"The next step is to harden the resulting nanoparticles with 1,3-diaminopropane (DP).\",\n \"In order to do this, a solution of DP is prepared in water (1% v/v), and a predetermined amount of cross-linking agent is added to the suspension of nanoparticles for incubation for 5 minutes.\",\n \"The suspension is then purified by ultracentrifugation (12 minutes at 10,000 rpm) or tangential filtration.\",\n \"Optionally, the resulting nanoparticles (loaded with 5-fluorouridine) are incubated with the Sambucus nigra lectin in order to obtain the nanoparticle-lectin conjugates, as described in example 2.The supernatants are analyzed by HPLC.\",\n \"The quantity of entrapped drug in the nanoparticles is determined as the difference with respect to the initial quantity added.\",\n \"The residue or pellet is resuspended in water or in an aqueous solution of mannitol (5% w/v).\",\n \"Finally, the resulting suspension of nanoparticles is freeze-dried.\",\n \"FIG.\",\n \"5 shows the influence of the incubation time between 5-fluorouridine and the poly(methyl vinyl ether-co-maleic anhydride) on the drug loading.\",\n \"In the example of the SNA-nanoparticle conjugates containing 5-fluorouridine (experimental conditions: 200 μg initial lectin), the amount of encapsulated fluorouridine does not change and the amount of lectin bound to the surface of nanoparticles was calculated to be around 40 μg/mg polymer.\",\n \"3.2.Cross-Linkage with Human Serum Albumin (HSA) 100 mg of poly(methyl vinyl ether-co-maleic anhydride) are dissolved in 2 mL organic solvent (acetone).\",\n \"A predetermined amount of 5-fluorouridine is dissolved in 3 mL acetone, until a theoretical maximum of 10 mg/mL is reached.\",\n \"The two aforementioned solutions are mixed and then incubated at room temperature for 2 hours.\",\n \"Then, under magnetic stirring, 10 mL of a miscible organic solvent (ethanol) and 10 mL distilled water are added to the acetone solution.\",\n \"The resulting mixture is stirred for 5 more minutes, and the suspension of nanoparticles is concentrated under reduced pressure for organic solvent elimination.\",\n \"The final volume is adjusted with water (or an aqueous solution) to 10 mL.\",\n \"The next step is to harden the resulting nanoparticles with human serum albumin (HSA).\",\n \"In order to do this, a solution of HSA is prepared in water (30 mg/mL), and a predetermined amount of cross-linking agent is added to the suspension of nanoparticles for incubation for 120 minutes.\",\n \"The suspension is then purified by ultracentrifugation (12 minutes at 10,000 rpm) or tangential filtration.\",\n \"The supernatants are analyzed by HPLC.\",\n \"The quantity of entrapped drug in the nanoparticles is determined as the difference with respect to the initial quantity added.\",\n \"The residue or pellet is resuspended in water or in an aqueous solution of mannitol (5% w/v).\",\n \"Finally, the resulting suspension of nanoparticles is freeze-dried maintaining all its properties.\",\n \"FIG.\",\n \"6 shows the influence of the incubation time between 5-fluorouridine and the copolymer on the drug loading of nanoparticles when the carriers where cross-linked with HSA.\",\n \"EXAMPLE 4 Manufacturing Process for the Preparation of poly(methyl vinyl ether-co-maleic anhydride)nanoparticles Coated with 5-fluorouridine The process described below permits the preparation of colloidal pharmaceuticai forms (i.e., nanoparticles and ligand-nanoparticle conjugates) containing 5-fluorouridine or any other active molecule with a chemical structure similar to or derived from said drug.\",\n \"4.1.Cross-Linkage with 1,3-diaminopropane (DP) 100 mg of poly(methyl vinyl ether-co-maleic anhydride) are dissolved in 5 mL organic solvent (acetone).\",\n \"A predetermined amount of 5-fluorouridine is dissolved in 2 mL water.\",\n \"Then, under magnetic stirring, 10 mL of a miscible organic solvent (ethanol) and 10 mL distilled water are added to the acetone solution of the copolymer.\",\n \"The resulting mixture is stirred for 5 more minutes, and the suspension of nanoparticles is evaporated under reduced pressure for organic solvents elimination.\",\n \"The final volume is adjusted with water (or an aqueous solution) to 10 mL, and the suspension is then incubated with the 5-fluorouridine aqueous solution.\",\n \"The next step is to harden the resulting nanoparticles with 1,3-diaminopropane (DP).\",\n \"In order to do this, a solution of DP in water (1% DP v/v water) is prepared and a predetermined amount of cross-linking agent is added to the suspension of nanoparticles for incubation for 5 minutes.\",\n \"The suspension is then purified by ultracentrifugation (12 minutes at 10,000 rpm) or tangential filtration.\",\n \"The supernatants are analyzed by HPLC.\",\n \"The quantity of entrapped drug in the nanoparticles is determined as the difference with respect to the initial quantity added.\",\n \"The residue or pellet is resuspended in water or in an aqueous solution of mannitol (5% w/v).\",\n \"Finally, the resulting suspension of nanoparticles is freeze-dried maintaining all its properties.\",\n \"FIG.\",\n \"7 shows the influence of the initial amount of 5-fluorouridine (FU) that is adsorbed or interacts with the anhydride groups on the nanoparticle (NP) surface.\",\n \"It is clear that when the initial amount of the drug increases, the amount of FU carried by the nanoparticles also increases.\",\n \"Likewise, the higher the initial amount of FU loaded in the nanoparticles, the higher the amount that is released quickly.\",\n \"In the case of the administration of these colloidal pharmaceutical forms, the drug fraction that is released immediately would act as burst dose.\",\n \"4.2.Cross-Linkage with Human Serum Albumin (HSA) 100 mg of poly(methyl vinyl ether-co-maleic anhydride) are dissolved in 5 mL organic solvent (acetone).\",\n \"A predetermined amount of 5-fluorouridine is dissolved in 2 mL water.\",\n \"Then, under magnetic stirring, 10 mL of a miscible organic solvent (ethanol) and 10 mL distilled water are added to the acetone solution of the copolymer.\",\n \"The resulting mixture is stirred during 5 minutes more and the suspension of nanoparticles is concentrated under reduced pressure for organic solvents elimination.\",\n \"The final volume is adjusted with water (or an aqueous solution) to 10 mL and the suspension is, then, incubated with the 5-fluorouridine aqueous solution.\",\n \"The next step is to harden the resulting nanoparticles with human serum albumin (HSA).\",\n \"In order to do this, a solution of HSA is prepared in water (30 mg/mL), and a predetermined amount of cross-linking agent is added to the suspension of nanoparticles for incubation for 120 minutes.\",\n \"The suspension is then purified by ultracentrifugation (12 minutes at 10,000 rpm) or tangential filtration.\",\n \"The supernatants are analyzed by HPLC.\",\n \"The quantity of entrapped drug in the nanoparticles is determined as the difference with respect to the initial quantity added.\",\n \"The residue or pellet is resuspended in water or in an aqueous solution of mannitol (5% w/v).\",\n \"Finally, the resulting suspension of nanoparticles is freeze-dried maintaining all its properties.\",\n \"FIG.\",\n \"8 shows the influence of the initial concentration of 5-fluorouridine that is adsorbed and interacts with the reactive groups on the nanoparticle surface.\",\n \"It is clear that when the initial amount of drug increases, the amount of FU carried by nanoparticles cross-linked with HSA also increases.\",\n \"As expected, the immediately released fraction increased by increasing the amount of drug incubated with the freshly prepared nanoparticles.\",\n \"This burst effect would be acting as an initial dose.\",\n \"4.3.In Vitro Release Studies For these studies, the nanoparticles were prepared following the conditions described in 4.1.The in vitro release assay was carried out in a temperature-controlled bath, with constant stirring and a constant temperature of 37±1° C. FIG.\",\n \"9 shows the results obtained for two different batches of nanoparticles prepared from 1 and 2 mg FU per mL suspension.\",\n \"It can be observed that a fraction of the drug (approximately 30-50% of the drug) is adsorbed, in a labile way, onto the surface of the nanoparticles and is immediately released.\",\n \"In addition, there is another fraction, which is strongly bound to the nanoparticles.\",\n \"This second fraction was not released during the time in which the release study was performed.\",\n \"However, it can be expected that in vivo conditions, the bond between the drug and the copolymer would break given its lability.\",\n \"This would permit the here described nanoparticles to act as a biphasic releasing drug.\",\n \"The great potential of this system is based on this property, together with their capacity to target certain organs and inflamed regions of the organism.\",\n \"EXAMPLE 5 Manufacturing Process for the Preparation poly(methyl vinyl ether-co-maleic anhydride)nanoparticles Coated with Ganciclovir The process described below permits the preparation of colloidal pharmaceutical forms (i.e., nanoparticles and ligand-nanoparticle conjugates) containing ganciclovir or any other active molecule with a chemical structure similar to or derived from said drug.\",\n \"5.1.Cross-Linkage with Human Serum Albumin (HSA) 100 mg of poly(methyl vinyl ether-co-maleic anhydride) are dissolved in 5 mL organic solvent (acetone).\",\n \"A predetermined amount of ganciclovir is dissolved in water.\",\n \"Then, under magnetic stirring, 10 mL of a miscible organic solvent (ethanol) and 10 mL distilled water are added to the acetone solution of the copolymer.\",\n \"The resulting mixture is stirred during 5 more minutes, and the suspension of nanoparticles is concentrated under reduced pressure for organic solvents elimination.\",\n \"The final volume is adjusted with water (or an aqueous solution) to 10 mL, and the suspension is then incubated with the ganciclovir aqueous solution.\",\n \"The next step is to harden the resulting nanoparticles with human serum albumin (HSA).\",\n \"In order to do this, a solution of HSA is prepared in water (30 mg HSA/mL water) and a predetermined amount of cross-linking agent is added to the suspension of nanoparticles for incubation for 120 minutes.\",\n \"The suspension is then purified by ultracentrifugation (12 minutes at 10,000 rpm) or tangential filtration.\",\n \"The supernatants are analyzed by HPLC.\",\n \"The quantity of entrapped drug in the nanoparticles is determined as the difference with respect to the initial quantity added.\",\n \"The residue or pellet is resuspended in water or in an aqueous solution of mannitol (5% w/v).\",\n \"Finally, the resulting suspension of nanoparticles is freeze-dried maintaining its properties.\",\n \"This manufacturing process gives similar results when nanoparticles are cross-linked with 1,3-diaminopropane (DP) in the conditions indicated in the previous examples.\",\n \"FIG.\",\n \"10 shows the influence of the bulk concentration of ganciclovir incubated with the nanoparticles in the percentage of drug bound to them.\",\n \"It can be observed that an initial ganciclovir amount greater, than 4-5 mg, does not have much effect on the percentage of entrapped drug.\",\n \"5.2.In Vitro Release Studies These in vitro release studies were carried out with nanoparticulate batches prepared as described in 5.1.In order to do this, 100 μL of different formulation (with similar drug entrapped efficiency but increasing amounts of drug loaded) were dispersed in 1 mL phosphate buffer solution containing tripsin.\",\n \"The in vitro release assay was carried out in a temperature-controlled bath, with constant stirring and a constant temperature of 37±10° C. taking samples at different predetermined times.\",\n \"A biphasic release profile was observed for all of the formulations tested.\",\n \"This profile was characterized by a first phase of rapid (almost immediate) release (also know as a “burst effect”), followed by a second slow and continuous release.\",\n \"During the first release stage (in the first eight hours), the percentages of drug released ranged between 40 and 50% of the loaded drug.\",\n \"The second part of the curve was adjusted to a zero-order kinetic (see FIG.\",\n \"11) that continued for more than 7 days.\",\n \"FIG.\",\n \"11 shows the ganciclovir release kinetic from the poly(methyl vinyl ether-co-maleic anhydride)nanoparticles, for a formulation containing 90 μg ganciclovir bound to the nanoparticles per niL suspension.\",\n \"EXAMPLE 6 Manufacturing Process for the Preparation poly(methyl vinyl ether-co-maleic anhydride)nanoparticles Coated with the antisense oligonucleotide ISIS 2922 The process described below permits the preparation of colloidal pharmaceutical forms (i.e., nanoparticles and ligand-nanoparticle conjugates) containing antisense oligonucleotides or any other active molecule of DNA or RNA.\",\n \"The example described below was carried out with the oligonucleotide ISIS 2922.ISIS 2922 is an oligonucleotide of 21 bases which possesses antiviral activity against cytomegalovirus.\",\n \"6.1.Cross-Linkage with 1,3-diaminopropane (DP) 100 mg of poly(methyl vinyl ether-co-maleic anhydride) are dissolved in 5 mL organic solvent (acetone).\",\n \"A predetermined amount of ISIS 2922 is dissolved in water.\",\n \"Then, under magnetic stirring, 10 mL of a miscible organic solvent (ethanol) and 10 mL distilled water are added to the acetone solution of the copolymer.\",\n \"The resulting mixture is stirred during 5 more minutes, and the suspension of nanoparticles is concentrated under reduced pressure for organic solvents elimination.\",\n \"The final volume is adjusted with water (or an aqueous solution) to 10 mL, and the suspension is then incubated with the ISIS 2922 aqueous solution for 20 minutes.\",\n \"The next step is to harden the resulting nanoparticles with 1,3-diaminopropane (DP).\",\n \"For this purpose, a solution of DP is prepared in water (1% DP v/v water), and a predetermined amount of cross-linking agent is added to the suspension of nanoparticles for incubation for 5 minutes.\",\n \"The suspension is then purified by ultracentrifugation (12 minutes at 10,000 rpm) or tangential filtration.\",\n \"The supernatants are analyzed by capilar electrophoresis.\",\n \"The quantity of entrapped drug in the nanoparticles is determined as the difference with respect to the initial quantity added.\",\n \"The residue or pellet is resuspended in water or in an aqueous solution of mannitol (5% w/v).\",\n \"Finally, the resulting suspension of nanoparticles is freeze-dried maintaining its properties.\",\n \"FIG.\",\n \"12 shows the influence of the initial amount of ISIS 2922 incubated with the nanoparticles on the amount of oligonucleotide loaded.\",\n \"This procedure gives similar results when cross-linkage is carried out with (DP), in the conditions described in the previous examples.\",\n \"DESCRIPTION OF THE FIGURES FIG.\",\n \"1.Influence of the incubation time (abscissas) and temperature on the yield of the manufacturing process (ordinates) of poly(methyl vinyl ether-co-maleic anhydride)nanoparticles cross-linked with DP.\",\n \"FIG.\",\n \"2.Influence of the incubation time (abscissas) and temperature on the yield of the manufacturing process (ordinates) of poly(methyl vinyl ether-co-maleic anhydride)nanoparticles cross-linked with HSA.\",\n \"FIG.\",\n \"3.Influence of the initial amount of the cross-linking agent (1,3-diaminopropane in μLsol/mg polymer; abscissas) on the amount of lectin bound (μg/mg polymer; ordinates) to the poly(methyl vinyl ether-co-maleic anhydride)nanoparticles.\",\n \"FIG.\",\n \"4.Influence of the cross-linking agent (1,3-diaminopropane in μL sol 1% v/v/mg polymer; abscissas) on the amount of active Sambucus nigra lectin (μg/mg polymer; ordinates) bound to the poly(methyl vinyl ether-co-maleic anhydride)nanoparticles.\",\n \"FIG.\",\n \"5.Influence of the incubation time (abscissas) on the anti-cancer 5-fluorouridine loading in poly(methyl vinyl ether-co-maleic anhydride)nanoparticles cross-linked with DP (drug loading in μg FU/mg nanoparticles; ordinates).\",\n \"Experimental conditions: initially 10 mg drug.\",\n \"FIG.\",\n \"6.Influence of the incubation time (abscissas) on the 5-fluorouridine loading in poly(methyl vinyl ether-co-maleic anhydride)nanoparticles cross-linked with HSA (Drug loading in μg FU/mg nanoparticles; ordinates).\",\n \"Experimental conditions: initially 10 mg drug.\",\n \"FIG.\",\n \"7.Influence of the initial amount of 5-fluorouridine (FU) (μ/mg polymer; abscissas) on the drug loading in poly(methyl vinyl ether-co-maleic anhydride)nanoparticles cross-linked with 1,3-diaminopropane (μg FU/mg NP; ordinates).\",\n \"The squares represent the total drug loading and the circles represent the drug immediately released by dilution.\",\n \"Experimental conditions: Initially 10 mg drug.\",\n \"FIG.\",\n \"8.Influence of the initial amount of 5-fluorouridine (μg FU/mg polymer; abscissas) on the drug loading in poly(methyl vinyl ether-co-maleic anhydride)nanoparticles cross-linked with HSA (μg FU/mg NP; ordinates).\",\n \"The squares represent the total drug loading and the circles represent the drug immediately released by dilution.\",\n \"Experimental conditions: Initially 10 mg of drug.\",\n \"FIG.\",\n \"9.Study of the release (abscissas) of two formulations cross-linked with DP and prepared from two different concentrations of 5-fluouridine (FU 5 ordintaes).\",\n \"FIG.\",\n \"10.Influence of the initial amount of ganciclovir (Bulk GCV in mg; abscissas) on the drug encapsulation efficiency (in %, ordinates) in poly(methyl vinyl ether-co-maleic anhydride)nanoparticles cross-linked with HSA.\",\n \"FIG.\",\n \"11.In vitro release kinetic (hours in abscissas) in an aqueous solution of tripsin, for a formulation of poly(methyl vinyl ether-co-maleic anhydride)nanoparticles with ganciclovir (GCV-μg ordinates).\",\n \"FIG.\",\n \"12.Influence of the initial amount of ISIS 2922 (μg in abscissas) on its loading in poly(methyl vinyl ether-co-maleic anhydride)nanoparticles (μg ISIS/mg NP; ordinates).\",\n \"Initial amount of nanoparticles (NP)=100 mg.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10467983"}}},{"rowIdx":394,"cells":{"text":{"kind":"list like","value":[["Method for the administration of a subscriber card for mobile telephony equipment of the type with auxiliary reader and embedded system for the implementation of the method","The invention concerns a method for the administration of a subscriber SIM card 3 inserted in the main smartcard reader of a mobile telephone of the type including a second smartcard reader, using an additional administrator smartcard temporarily inserted in this second reader.","The administrator smartcard includes at least one specific loader (Ch) applet and one or more files (fl-fn) corresponding to the applets (A1-An) which can be loaded in the SIM card under the control of the loader (Ch).","The administrator card complies with the Sim Toolkit standard.","The method can also be used to carry out the deletion, modification and various operations on data or applets (A1-An) present in the SIM card: display, determination of the memory space, etc.","The invention also concerns an embedded system, more particularly a smartcard, for the implementation of the method."],["1.Administration method for an embedded system for a device of type including a first embedded system reader, the main reader, designed to take the said embedded system and a second embedded system reader, the auxiliary reader, the said embedded system including means for the computer processing and storage of data, wherein it consists of loading in the said storage means of the said embedded system at least one item of digital data contained in an additional embedded system, the administration system, inserted in the said auxiliary embedded system reader, under the control of a specific software module contained in the said additional embedded system.","2.Method according to claim 1, wherein it consists of performing the addition, deletion, modification and management/display administration operations under the control of the said specific software module.","3.Method according to claim 1, wherein it includes at least the following steps: temporary insertion of the said administration embedded system in the said auxiliary embedded system reader; conversion of the said digital data item(s) in the first format into a series of digital data in a second format under the control of the said specific software module; and the selective transfer of the said digital data item(s) in the said second format to the said embedded system and its loading in the said storage means of this embedded system, via the said auxiliary and main embedded system readers, under the control of the said specific software module.","4.Method according to claim 1, wherein the same administration embedded system becomes “master” on powering up although inserted in the said auxiliary embedded system reader.","5.Method according to claim 1, wherein the said device transmits, on being put into service, a command for automatic activation of the said specific software module and in that, the said device including display means, it initialises the display of an interactive menu on the said display means, enabling a user to select one of the said items of digital data for loading in the said storage means of the said embedded system, guiding the user through the loading operations.","6.Method according to claim 1, wherein the said specific software module and the said digital data item(s) to be loaded in the said storage means of the said embedded system are applets encoded in JAVA (registered trademark) language.","7.Method according to claim 1, wherein the said administration of the said embedded system includes the scanning, under the control of the said specific software module, of its storage means, in order to determine predetermined attributes associated with the said digital data item(s) loaded in these storage means and the display of these attributes on the display means of the said device.","8.Method according to claim 1, wherein it includes an additional step, before the loading, which consists of determining whether the remaining memory space is sufficient to perform the said loading, and, in case of negative result, execution of an additional step which consists of the selective and optional deletion, after the choice by a user of the said device and under the control of the said specific software module, of at least one of the said digital data items, in order to release sufficient memory space to carry out the said loading.","9.Method according to claim 1, wherein the said device is a mobile telephony device, the said embedded system is a subscriber embedded system, the said administration embedded system complies with the so-called SIM Toolkit standard, in that it includes an operating system in compliance with this standard enabling the transmission of at least so-called proactive commands in compliance with standard ETSI 11.14, in that the communication protocol used for the loading complies with standard ISO 7816, including the so-called APDU commands, and in that the said proactive commands can be used to activate and send APDU commands, in order to set up a communication session between the said administration and subscriber embedded systems, and obtain the said selective transfer of at least one digital data item and their loading in the storage means of the said subscriber embedded system.","10.Embedded system including means for the computer processing and storage of digital data, characterised in that it includes, in its storage means, a specific software module, including at least one component consisting of a loader program (Ch) and at least one item of digital data in order to load at least one of the said digital data items in the said storage means of a second embedded system, for a device when the said system is inserted in an auxiliary reader of the said device.","11.Embedded system according to claim 10, wherein the said specific software module stored in the said storage means of the said administration embedded system and the said digital data item(s) to be loaded in the said storage means of the said embedded system are applets encoded in JAVA (registered trademark)."],["The invention concerns a method for the administration of an embedded system with electronic chip, more especially a subscriber card for mobile telephony equipment of the type with auxiliary reader.","It is more especially, although not exclusively, intended for mobile telephony terminals complying with the GSM standard, henceforth referred to simply as “mobile telephone”.","One of the most frequently used standards in Europe is the GSM transmission standard (Global System for Mobile communications, public radiocommunications operating in the 900 MHz band).","It must be clearly understood however that the invention is not limited to this standard alone.","In particular, it may be used in the standards under development such as GPRS or UTMS.","The invention concerns an embedded system, more particularly an electronic smartcard, for the implementation of the method.","Henceforth, for simplification, the name “smartcard” will be used, without limiting in any way whatsoever the scope of the invention.","Some mobile telephones are known as “dual slot” since they include a first smartcard reader, which will henceforth be called the main reader, accepting a card equipped with information processing and storage means, including a functional module known by the abbreviation SIM (Subscriber Identification Module).","This smartcard, which will henceforth be called “subscriber” card, may be replaced by a simple electronic module acting as smartcard.","If the smartcard has the format known as ID-1, it includes a card as such made from plastic material on which the above-mentioned module with electronic chip is positioned.","The assembly respects a certain number of well known standards, whether regarding physical (dimensions, location of the chip, etc.","), electrical and/or electronic aspects, which require no further mention.","Otherwise, the module alone, in the Plug-in SIM format, can be inserted in the main reader.","Generally, the latter simply consists of a chamber fitted with a cover.","A back wall has contact studs used to connect the module with the electronic circuits equipping the telephone terminal.","The smartcard inserted in the main reader, or SIM card, stores a certain number of data items concerning the telephone subscription as such the name(s) of the operator(s) that the subscription(s) have been taken out with, subscription, subscriber identification data, etc.","), as well as so-called embedded applications.","These are, in particular, applets providing access to services present on remote servers, via the telephone network, or just executed locally.","The interactive video games displayed on the screen of the mobile telephone are examples of this last category of applications.","To do this, the SIM cards implement the so-called SIM Toolkit technology which complies with standard ETSI 11.14.These cards are programmable.","Like all programmable cards, they not only receive commands from the host terminal, i.e.","the mobile telephone, but they also transmit commands to this terminal.","These commands are known as “proactive”.","As an illustration, a proactive command called “DISPLAY TEXT”, with as parameter, for example, the following text: “Welcome”, will request the mobile telephone, under the control of the applet contained in the card, to display the above-mentioned text “Welcome” on its display screen.","Amongst the proactive commands described by the above-mentioned standard ETSI 11.14, some of them are used to activate and then send so-called APDU (Application Protocol Data Unit) commands, i.e.","complying with standard ISO 7816, to a second smartcard inserted in a second reader of the mobile equipment, henceforth called the auxiliary reader.","To understand the proactive commands sent by the SIM card, the GSM terminals must of course also comply with the standard ETSI 11.14.In the known state of the art, the second smartcard generally consists of a credit card, for example a standard bank card.","The auxiliary reader generally consists of a traditional smartcard reader which can read a smartcard of this type.","In order to do this, it has a slot into which the smartcard is inserted whilst the transaction is being carried out.","For example, the transaction could consist of debiting the credit card or the bank card by a certain amount to reload the SIM card with telephone communication units.","The second smartcard operates in “slave” mode, since it is under the control of the first smartcard.","In the context of the invention, the term “embedded applications” must be taken in its broadest sense.","It normally concerns applets or similar programs, but also includes all types of digital data.","For example, the entries of a telephone directory stored in the SIM card or any other data file.","Henceforth, the term “digital data” will designate either “applications” (or “programs”) or “data or simple data files” stored in the memory means of the SIM card.","We can easily see that for various reasons, it may be useful, or even necessary, to add, delete and/or modify applications or data stored in the SIM card.","It is known that applets need to be added in a SIM card.","Generally, a computer (e.g.","a traditional microcomputer) is used to load applets.","The applet to be loaded is, for example, stored on a hard disk of the computer.","A resident program in the computer, known as the “Loader” receives in input a file containing the applet and converts it into a set of commands that it sends to the SIM card.","These commands can be sent in two main ways: locally, from a smartcard reader connected to the computer.","remotely, for example by using the technology known as Over The Air (O.T.A.)","in GSM terminology.","These methods present several disadvantages.","Firstly, the SIM card is inserted in the smartcard reader of the computer used for loading.","It can be seen immediately that this methods presents the disadvantage of requiring specific hardware.","At least, the subscriber, holder of the SIM card, must physically go to a place where this type of hardware is available.","As regards the second method, it is worth pointing out that digital data is generally transmitted via the Short Message Service (SMS) technology (GSM-Data Service) in compliance with the two standards ETSI 03.40 and ETSI 03.48.Generally, the maximum length of useful in these messages is 160 septets or 140 octets, depending on the applications.","Although with current technology, smartcards can only store programs requiring relatively a limited amount of memory, as is the case with the applets, they often come in the form of files occupying about 10 kB.","Clearly, it would take about 80 messages to send a file of this length.","It must also be pointed out that with transmissions implementing OTA technology, the security and/or reliability rates are not very high.","In particular, especially when a set of SIM cards must be updated with data, even identical, the process has to be repeated for each mobile telephone, since the links between a remote server and a mobile telephone are “point to point”.","Consequently, the above-mentioned method is, in practice, extremely complicated and possibly even impossible to implement.","Since the mobile telephones concerned by the invention are dual slot type, as pointed out earlier, one possible method could have been to use the auxiliary reader to load applets in the SIM card inserted in the main reader.","An obvious solution would in fact be to implement a loader program in the subscriber's SIM card.","The code of the applet to be loaded could then be stored in another smartcard which could be inserted in the auxiliary reader.","The loader program in the SIM card could then transmit one or more proactive command(s) in order to read the code of the applet to be loaded.","Once the program has read the applet code, it can then load it using a suitable means.","Initially, this method seems to offer advantages: 1) The loader program of the SIM card is compatible with the card itself since stored in it; 2) The auxiliary smartcard containing the applet code does not need to comply with the Sim Toolkit standard.","However, this method presents significant disadvantages: 1) Since the loader program takes up a non-negligible amount of space in the SIM card, there is less space for the applets which have to be stored there.","Even though the capacity of the memories used on smartcards has significantly increased over the last few years, it remains relatively limited for this type of application.","2) This solution cannot be considered using the present technology, since: it is impossible to send loading commands.","The commands to be sent by the application to the operating system in the SIM card are in fact specified in the standard ETSI 03.19.Loading commands cannot be sent under this standard.","in addition, the applet cannot request the mobile telephone to send commands to the card it hosts.","The current standards would therefore have be modified in order to consider this type of operation.","it is known from the european patent application EP 0 869 691 a mobile phone in which the SIM card communicates with an external component.","The SIM card behaves as a gateway between the external component and the GSM network and/or the mobile phone so that the external component controls completely the mobile phone.","The modified functionalities are transferred through applets via the SIM card to the external component.","it is also known from the international patent application WO 00/154530 a system allowing to program a set of selectable optional features into the memory of a cellular telephone independtly of the main program.","The systems is constituted of a programmer and a port.","The programmer located outside the housing of the telephone sends the set of selectable optional features to the port for storage in the memory.","The invention aims to overcome the disadvantages of the methods and devices of the known state of the art, some of which have just been described.","The purpose of the invention, for a mobile device of type including two card readers, is to define a method for the administration of digital data, applications and/or simple data, stored on a first smartcard, called subscriber or SIM smartcard, inserted in a first smartcard reader, called the main reader, via a second smartcard, called administration card, inserted in a second smartcard reader, called auxiliary smartcard reader.","The method according to the invention can be used not only to add one or more applets in the SIM card, i.e.","to load applets, but also provides true management or administration of digital data, applications and/or simple data, stored in it.","Apart from loading digital data in the SIM card, the method according to the invention can be used to delete digital data, display it on the screen of the mobile device and modify it.","This method can also be used to obtain information concerning various parameters and/or attributes about the said digital data and/or the SIM card as such, for example by displaying the names or statuses of the applets already stored in the SIM card, the memory space available in the SIM card, etc.","The method according to the invention does not involve any modification of the hardware implemented (ordinary telephony equipment can be used) and remains fully compatible with current standards whether regarding the transmissions the smartcards (SIM Toolkit technology) or the communications between the smartcards and the circuits of the mobile device, which invoke APDU standards.","To do this, according to a first characteristic, an “administrator” smartcard will be used, which implements a specific software module.","It is this specific program module which is used to administer the SIM card.","If the loading function is available, the software module includes at least a loader program and one or more applet codes which can be loaded selectively into the SIM card.","Preferably, the loader program consists of an applet.","Preferably, this applet and the other applets are programmed in JAVA (registered trademark).","If the subscriber SIM card only stores simple digital data (i.e.","no applications), then it does not necessarily need to comply with the SIM Toolkit technology.","The invention also concerns a mobile telephony device to implement the method.","Apart from the numerous possibilities offered by the method and its high flexibility, it must also be pointed out that the administrator card is not a “proprietary” type card.","It does not in fact have to be provided by a telephone network operator.","In all cases, it can be obtained, for example purchased, from various sources: supermarkets, post offices, etc., or sent through the post.","Once the user has simply inserted it in the auxiliary reader; the entire loading process, or more generally the administration process, is carried out automatically (as will be fully detailed below) and/or according to the instructions displayed on the mobile telephone screen.","Once again preferably, the instructions for using the administrator card can be printed on it or displayed on screen when it is inserted in the auxiliary reader, after switching on the mobile telephone.","Still preferably, the administrator card is in ID-1 format, which means that it can be inserted instead of a credit card or a standard bank card.","The main purpose of the invention is therefore to define an administration method for an embedded subscriber system for a mobile telephone device of type including a first embedded system reader, the main reader, designed to take the said subscriber embedded system and a second embedded system reader, the auxiliary reader, the said subscriber embedded system including computer processing and data storage means, the said data possibly including software applications, characterised in that it includes at least the following steps: initial loading, in the digital data storage means of an additional system, the administration system, of a specific software module, including at least one component consisting of a loader program and at least one digital data file in a first format; temporary insertion of the said administration embedded system in the said auxiliary embedded system reader; conversion of the said digital data in the first format into a series of digital data in a second format under the control of the said specific software module; and the selective transfer of at least one series of digital data in the said second format to the said subscriber embedded system and its loading in the said storage means of this embedded system, via the said auxiliary and main embedded system readers, under the control of the said specific software module; and in that the said transfer is carried out according to a given communication protocol.","The invention also concerns an embedded system for the implementation of the method.","The invention will now be described in more detail, referring to the attached drawings, amongst which: FIG.","1 is a diagrammatic representation of a configuration example for mobile telephone including two smartcard readers with a subscriber SIM card and a smartcard according to the invention; FIG.","2 is a block diagram explaining the main steps and phases of the method according to the invention and the interactions between the components implemented; FIG.","3 is a block diagram schematising the main administration operations possible through the use of the method according to the invention; FIG.","4 is a block diagram illustrating a practical example of an applet in the subscriber SIM card, according to the method of the invention; and FIG.","5 is a block diagram illustrating a practical example of deleting an applet in the subscriber SIM card, according to the method of the invention.","Henceforth, without limiting in any way whatsoever the scope of the invention, we will consider the preferred application of the invention, unless otherwise specified, i.e.","the context of a dual slot mobile telephone complying with the GSM standard.","FIG.","1 is a diagrammatic representation of a mobile telephone 1 and the two smartcards implemented, 2 and 3.We have assumed that the SIM subscriber card 3 is a module in Plug-in SIM format.","As such, this SIM card corresponds to the known state of the art.","It includes a support 30 in the above-mentioned Plug-in SIM format, on which is positioned an electronic chip 31 including input-output contact studs, with unique reference 310.The assembly is defined by various standards and/or norms, whether regarding the physical (dimensions, location of the chip, etc.","), electrical and/or electronic aspects.","The electronic chip includes in particular data processing means in a stored program (microprocessor or microcontroller) and non volatile (ROM, EPROM) and volatile (RAM and various registers) storage means.","These various computer resources, also well known, are not shown on FIG.","1.Apart from programs and digital data directly associated with the telephone transmissions, carried out for example in compliance with the above-mentioned GSM standard, the storage means of the module 3 can store various items of digital data, and especially applets, for example n applets, A1 to An.","As such, this characteristic also corresponds to the known state of the art.","If module 3 stores applets, it must comply with the Sim Toolkit technology so that it can receive commands and transmit proactive commands.","In the example shown on FIG.","1, the mobile telephone 1 includes a main body 10 and an auxiliary body 11, which can be folded back by rotation around an axis 110 at the bottom of the main body 10.The main smartcard reader 12 consists of an internal chamber 12 housing the SIM module 3 (represented on FIG.","1 enlarged and outside its housing).","The main body 10 is equipped in particular with a display screen 100, generally liquid crystal type.","The auxiliary smartcard reader 13 is located in the auxiliary folding body 11 It communicates with the exterior via a slot 130 in which a smartcard in ID-1 format, and therefore compatible with standard smartcards, bank or credit cards, can be inserted.","The mobile telephone according to the invention therefore remains fully compatible with the norms and standards of the known state of the art.","It requires no modifications.","In a preferred mode of realisation of the invention, the smartcard used as administration card also complies with the above-mentioned standard ID-1.The auxiliary reader 13 can therefore accept either standard smartcards or smartcards 2 complying with the characteristics of the invention and which are detailed below.","Initially, we assume that the main function of the smartcard 2 is to load new applets in the SIM module 3.Preferably, the smartcard 2 complies with the Sim Toolkit technology.","It can send proactive type commands to the host mobile telephone 1 and read and/or write commands to the files in its Sim Toolkit operating system OSST.","According to a first characteristic of the invention, apart from the above-mentioned Sim Toolkit operating system OSST, the electronic chip 20 of the smartcard 2 contains at least one software module known as the loader Ch and a certain number of files, f1 to fn, capable of being transferred into the SIM module 3, in order to load applications there in addition to any applications already resident (not shown).","Preferably, these applications consist of applets, advantageously coded in JAVA (registered trademark) language.","In this case, the files, f1 to fn, are advantageously coded according to the CAP file format defined by the Java Card forum (registered trademark).","The loader program Ch itself is also a Sim Toolkit applet.","According to a characteristic of the method of the invention, the subscriber's SIM module or card 3 becomes the receiving card.","The administrator card 2, according to another characteristic of the method of the invention becomes the main card during the loading, although it is inserted in the auxiliary smartcard reader 13.In a preferred mode of realisation, due to the fact that the administrator card uses Sim Toolkit technology, it can, unlike the known state of the art, become priority when the mobile telephone 1 is switched on, i.e.","it can become “master”.","The card inserted in the auxiliary reader 13, i.e.","the administrator card 2, then takes priority over the SIM module 3: it is the applets of this administrator card 2 which are proposed to the user and not those resident in the SIM module 3 (receiving card).","However, it must be made quite clear that this arrangement is not mandatory.","The user could, for example, be requested to press a key or a key combination 101 on the keyboard of the mobile telephone 1 so that the process starts “manually” after this mobile telephone 1 is switched on.","For example, the instructions for use may be supplied independently from the administrator card 2 or printed or engraved on one side of it.","In the preferred mode of realisation, since the administrator card 2 takes priority, an initial interactive menu can be displayed on the screen 100 showing in particular the loader applet Ch installed on this card.","Once the user has selected this application, it can read the files present, f1 to fn, so that it can then execute APDU commands on the receiving card, i.e.","the SIM module 3, and thereby load the applet(s) corresponding to the files present on the administrator card.","These operations are carried out in co-operation with the Sim Toolkit operating system OSST.","More precisely, the card inserted in the auxiliary reader 13, i.e.","the administrator card 2, can read the files present on its operating system via one or more traditional APDU commands, such as “READ BINARY”.","Reading the system files, f1 to fn, enables the administrator card 2 to collect all the information required concerning the applet to be installed on the subscriber's SIM module or card 3.In addition, the administrator card 2 can open a session in order to access the subscriber's SIM module 3 via the proactive command “POWER ON CARD”.","When this has been done, the administrator card 2 sends APDU loading commands defined by the standards ETSI 03.48 and ISO/IEC 7816-4 in order to install the applet read previously.","The APDU commands are therefore sent to the SIM module via the proactive command “PERFORM CARD APDU”.","Once the applet has been installed, the administrator card 2 of the auxiliary reader 13 closes the session with the command “POWER OFF CARD”.","The user can now withdraw the administrator card 2 from the auxiliary reader 13 in order to reconnect on the SIM module 3.On la FIG.","1, the applets referenced A1 to An, loaded in addition in the SIM module 3 correspond to files f1 to fn of the administrator card 2.All these commands or sessions comply with the usual standards and norms concerning the communication protocol between a card reader and a smartcard.","As pointed out above, APDU commands in particular are used.","The invention therefore involves no modifications.","It should also be pointed out that, once the administrator card 2 according to the invention has been withdrawn, a traditional smartcard, for example a bank card or a credit card, can be inserted in the auxiliary reader 13.In this case, operation corresponds once again to the known state of the art: the smartcard in the auxiliary reader 13 no longer takes priority.","It remains under the control of the SIM module or card 3.FIG.","2 is a block diagram explaining in more detail the main interactions between the various components implemented.","The main steps and phases of the method are also shown.","We also assumed that that the administrator card can be used not only to load one or more applets in the SIM module 3, but also for other operations: deletion, etc., which will be detailed below, especially in reference to FIG.","3.The software module specific to the invention includes several components, under the single reference 40.Preferably, as indicated, it is activated automatically when the mobile telephone 1 is switched on (FIG.","1: 1).","To do this, a command called “TERMINAL PROFILE” is used, generated by the mobile telephone 1 (FIG.","1) precisely when switching on.","The files stored in the administrator card 2 under the single reference 41, are generally associated not only with applets likely to be loaded in the SIM module 3, but also with digital data.","For example, this data could consist of entries in the telephone directory.","The administration card 2 may also be intended to load data associated with a new telephony operator in the SIM module 3.A priori, since the standards used for the communications between smartcard and reader implement APDU commands, the data and/or applications 41 cannot be loaded directly into the SIM module 3.A prior operation 42 must be carried out, which will be called formatting, under the specific program command 40.Note that are of course, as with any program during execution, interactions between the operating system OSST and this specific program 40.Once the formatting has been carried out, still under the control of the specific program 40, communication sessions, which may be bidirectional, are set up between the administrator card 2 and the module 3, via the auxiliary reader 13 and main reader 12, using a series of APDU commands (standardised communication protocol).","Consequently, one or more additional applets may be loaded in the SIM module 3.However, the method according to the invention is not limited to loading applets.","As shown by the block diagram on FIG.","3, the card 2 can perform the following main administration operations 200: “addition” 201 (as shown above), as well as “deletion” 202 and “modification” 203.These operations can be carried out on applications (applets) 410 or various items of digital data 411.Lastly, the administrator card 2 can perform administration operations as such, which may be called “management” operations, especially various displays (for example multiple choice interactive menus) on the screen 100 (FIG.","1), search and display of the memory space occupied by the applets present in the SIM module 3, search and display of the name and logical status of these applets, etc.","These operations are all performed under the control of the specific program 40 and implement standard APDU commands and sessions.","As an illustration, we will now describe two examples of practical implementation, referring to FIGS.","4 and 5.The first concerns more specifically the selective addition of an applet, which in this case is used for local execution of the well known game called “MINEFIELD”.","The second concerns more specifically the selective deletion of an applet present in the SIM module 3, after detecting insufficient memory space, in order to add a new applet (an address manager in this case).","EXAMPLE 1 Adding an Applet (FIG.","4) We assume that a user has already loaded an applet concerning the well known game “HANGMAN” on the SIM module 3 (FIG.","1).","He buys an administrator card according to the invention in order to load an extra game, in this game the game “MINEFIELD”.","When the user inserts the loader card, i.e.","the administrator card 2 (FIG.","1) in the auxiliary reader 13 (FIG.","1) with the “Loader” applet Ch (FIG.","1) and the file corresponding to the “MINEFIELD” applet, according to the preferred mode, an initial interactive menu is automatically displayed on the screen 100 (FIG.","1), without the user having to press any key 101 (FIG.","1) on the mobile telephone 1 (FIG.","1).","This initial menu indicates to the user the name of the applet that can be loaded and requests whether or not to continue the loading: step 50.The text displayed is as follows: “Do you want to install Minefield on your SIM card?","OK CANCEL”.","If the user refuses (right hand branch: “CANCEL validated by user”), a new menu prompts the user to withdraw the loader card, i.e.","the administrator card 2 (FIG.","1): step 55.The text displayed is as follows: “You can withdraw your loader card.","Goodbye”.","The user withdraws the administrator card 2 from the auxiliary reader 13 (FIG.","1).","Following this operation, the mobile telephone 1 (FIG.","1) switches back over to the SIM module 3 (FIG.","1) and requests the user's PIN code (Personal Identifier Number): step 56.After entering a correct PIN code, the user can use the SIM module or card 3 again and select an applet from those already resident, for example the “HANGMAN” applet: step 57.If the user accepts to load the new applet (left hand branch, “OK” validated by user), the administrator card 2 (FIG.","1) loads the “MINEFIELD” applet on the SIM module 33 (FIG.","1), and informs the user that the operation has been carried out successfully: steps 51 and 52.The following messages are displayed: step 51: “Please wait.","Loading .",".",".","”, and step 52: “Minefield has been loaded correctly.","You can withdraw the loader card”.","When the user has withdrawn the administrator card 2 (FIG.","1), he is prompted to enter his PIN code: step 53.After entering a correct PIN code, the user can use the SIM module or card 3 (FIG.","1) again and select an applet from those already resident.","After selection of the applet menu the two applets present on the SIM module are displayed: “HANGMAN”, previously resident, and “MINEFIELD” which has just been loaded: step 54.He can use this new applet, i.e.","execute the game “MINEFIELD” on his mobile telephone 1 (FIG.","1).","EXAMPLE 2 Deleting an Applet (FIG.","5) We now assume that the user has two applets installed on the SIM module 3 (FIG.","1), for example the above-mentioned “HANGMAN” and “MINEFIELD”, and he wants to install an additional applet, for example an “address manager”.","As before, the user inserts an administrator card 2 (FIG.","1) which has a specific program 40 (FIG.","2), especially including a component to load the “address manager” applet (one of the files 41: FIG.","2).","At step 60, a message is displayed: “Do you want to install the address manager on your SIM card?","OK CANCEL”.","If the user accepts the option presented (left hand branch, “OK” validated), the specific program 40 (FIG.","2) scans the memory of the SIM module 3 (FIG.","2): step 61.If there is sufficient memory space left on the SIM module 3 (FIG.","1) for this third applet, the “address manager” applet can be loaded on the SIM module 3 (FIG.","1).","The execution of this operation is similar to that which has been described with respect to FIG.","4.Consequently, the corresponding steps will not be described again.","Otherwise, if there is not sufficient memory space, a message is displayed to the user indicating that at least one of the two applets already present must be deleted.","In the example described, the message is as follows: “Insufficient memory.","You must first delete an applet OK CANCEL”.","If the user accepts this possibility (“OK” validated), he is prompted to select one of the applets, in response to the message displayed: “Select the applet to be deleted: HANGMAN or MINEFIELD-OK CANCEL”: step 62.If the user accepts one of the options presented, for example if he selects “HANGMAN”, the specific program 40 (FIG.","2) starts a session in order to delete the “HANGMAN” applet.","After deleting one of the applets loaded, the user is prompted to continue the procedure, just as in the previous example (FIG.","4): loop back to step 60.The following message is displayed: “The HANGMAN applet has been deleted from memory.","Do you want to continue?","OK CANCEL”: step 63.In steps 60, 61, 62 and 63, the user has the possibility of refusing the choice proposed (“CANCEL” validated).","The process then continues at step 64.The user is prompted to withdraw the administrator card 2 (FIG.","1) from the auxiliary reader 13 (FIG.","1).","The following message is displayed: “You can withdraw your loader card.","Goodbye”.","The user must then enter his PIN code in order to use the SIM module 3 (FIG.","1): step 65.After reading the above, it is easy to see that the invention does in fact reach the objectives set.","With the method according to the invention, it is possible in particular to use a standard mobile telephone since no modifications are required to the equipment.","It complies fully with current norms and standards.","It is therefore fully compatible with the known state of the art.","However, it offers numerous advantages.","Not only can it be used to add (load) but also to delete and modify applications and/or digital data, as well as to perform various management operations: display, scanning the SIM memory, etc.","It therefore provides true management of the subscriber's SIM card or module.","No other equipment is required, as with certain methods of the known art, since the auxiliary reader of the mobile telephone is used as receiver of the administrator card.","Neither does it rely on downloads from remote servers, with the disadvantages inherent to this type of method, which were pointed out in the preamble of this description.","Lastly, the administrator card can be issued by any entity, it is not only a proprietary card issued by a mobile telephone operator.","The applications and/or data stored are a priori independent from those specific to the telephony operators.","However, the method enables an administrator card issued by a particular operator to be used in order to update its own data or to add a subscription to this operator in the SIM card, or on the contrary to delete it.","The administrator card may be available from various types of point of sale or procurement, or even sent through the post.","The operations possible, especially the loading of additional applets, require no special knowledge.","In a preferred mode of realisation, certain operations are fully automatic, in particular the activation of the loader program or, more generally, of the specific program.","The following steps are interactive, the user being guided by menu.","However, the invention is of course not limited to only those examples of realisation explicitly described, especially in relation to FIGS.","1 to.","In particular the text of the messages displayed was only indicated to describe the method of the invention more clearly.","These messages are related to the particular applications (applets) loaded on the administrator card.","A priori, any messages would be possible without leaving the scope of the invention.","Similarly, the method according to the invention does not interfere in any way with the technology used for the transmissions: GSM, GPRS or UTMS, for example, since the operations are all carried out locally through the implementation of two readers: the main reader containing the SIM module or card and the auxiliary reader intended to receive a traditional smartcard, for example a bank card.","Moreover, the invention is not exclusively intended for mobile telephony.","The invention concerns too for example an administration method for a data processing device as a computer, an organizer, an encoder reader or others."]],"string":"[\n [\n \"Method for the administration of a subscriber card for mobile telephony equipment of the type with auxiliary reader and embedded system for the implementation of the method\",\n \"The invention concerns a method for the administration of a subscriber SIM card 3 inserted in the main smartcard reader of a mobile telephone of the type including a second smartcard reader, using an additional administrator smartcard temporarily inserted in this second reader.\",\n \"The administrator smartcard includes at least one specific loader (Ch) applet and one or more files (fl-fn) corresponding to the applets (A1-An) which can be loaded in the SIM card under the control of the loader (Ch).\",\n \"The administrator card complies with the Sim Toolkit standard.\",\n \"The method can also be used to carry out the deletion, modification and various operations on data or applets (A1-An) present in the SIM card: display, determination of the memory space, etc.\",\n \"The invention also concerns an embedded system, more particularly a smartcard, for the implementation of the method.\"\n ],\n [\n \"1.Administration method for an embedded system for a device of type including a first embedded system reader, the main reader, designed to take the said embedded system and a second embedded system reader, the auxiliary reader, the said embedded system including means for the computer processing and storage of data, wherein it consists of loading in the said storage means of the said embedded system at least one item of digital data contained in an additional embedded system, the administration system, inserted in the said auxiliary embedded system reader, under the control of a specific software module contained in the said additional embedded system.\",\n \"2.Method according to claim 1, wherein it consists of performing the addition, deletion, modification and management/display administration operations under the control of the said specific software module.\",\n \"3.Method according to claim 1, wherein it includes at least the following steps: temporary insertion of the said administration embedded system in the said auxiliary embedded system reader; conversion of the said digital data item(s) in the first format into a series of digital data in a second format under the control of the said specific software module; and the selective transfer of the said digital data item(s) in the said second format to the said embedded system and its loading in the said storage means of this embedded system, via the said auxiliary and main embedded system readers, under the control of the said specific software module.\",\n \"4.Method according to claim 1, wherein the same administration embedded system becomes “master” on powering up although inserted in the said auxiliary embedded system reader.\",\n \"5.Method according to claim 1, wherein the said device transmits, on being put into service, a command for automatic activation of the said specific software module and in that, the said device including display means, it initialises the display of an interactive menu on the said display means, enabling a user to select one of the said items of digital data for loading in the said storage means of the said embedded system, guiding the user through the loading operations.\",\n \"6.Method according to claim 1, wherein the said specific software module and the said digital data item(s) to be loaded in the said storage means of the said embedded system are applets encoded in JAVA (registered trademark) language.\",\n \"7.Method according to claim 1, wherein the said administration of the said embedded system includes the scanning, under the control of the said specific software module, of its storage means, in order to determine predetermined attributes associated with the said digital data item(s) loaded in these storage means and the display of these attributes on the display means of the said device.\",\n \"8.Method according to claim 1, wherein it includes an additional step, before the loading, which consists of determining whether the remaining memory space is sufficient to perform the said loading, and, in case of negative result, execution of an additional step which consists of the selective and optional deletion, after the choice by a user of the said device and under the control of the said specific software module, of at least one of the said digital data items, in order to release sufficient memory space to carry out the said loading.\",\n \"9.Method according to claim 1, wherein the said device is a mobile telephony device, the said embedded system is a subscriber embedded system, the said administration embedded system complies with the so-called SIM Toolkit standard, in that it includes an operating system in compliance with this standard enabling the transmission of at least so-called proactive commands in compliance with standard ETSI 11.14, in that the communication protocol used for the loading complies with standard ISO 7816, including the so-called APDU commands, and in that the said proactive commands can be used to activate and send APDU commands, in order to set up a communication session between the said administration and subscriber embedded systems, and obtain the said selective transfer of at least one digital data item and their loading in the storage means of the said subscriber embedded system.\",\n \"10.Embedded system including means for the computer processing and storage of digital data, characterised in that it includes, in its storage means, a specific software module, including at least one component consisting of a loader program (Ch) and at least one item of digital data in order to load at least one of the said digital data items in the said storage means of a second embedded system, for a device when the said system is inserted in an auxiliary reader of the said device.\",\n \"11.Embedded system according to claim 10, wherein the said specific software module stored in the said storage means of the said administration embedded system and the said digital data item(s) to be loaded in the said storage means of the said embedded system are applets encoded in JAVA (registered trademark).\"\n ],\n [\n \"The invention concerns a method for the administration of an embedded system with electronic chip, more especially a subscriber card for mobile telephony equipment of the type with auxiliary reader.\",\n \"It is more especially, although not exclusively, intended for mobile telephony terminals complying with the GSM standard, henceforth referred to simply as “mobile telephone”.\",\n \"One of the most frequently used standards in Europe is the GSM transmission standard (Global System for Mobile communications, public radiocommunications operating in the 900 MHz band).\",\n \"It must be clearly understood however that the invention is not limited to this standard alone.\",\n \"In particular, it may be used in the standards under development such as GPRS or UTMS.\",\n \"The invention concerns an embedded system, more particularly an electronic smartcard, for the implementation of the method.\",\n \"Henceforth, for simplification, the name “smartcard” will be used, without limiting in any way whatsoever the scope of the invention.\",\n \"Some mobile telephones are known as “dual slot” since they include a first smartcard reader, which will henceforth be called the main reader, accepting a card equipped with information processing and storage means, including a functional module known by the abbreviation SIM (Subscriber Identification Module).\",\n \"This smartcard, which will henceforth be called “subscriber” card, may be replaced by a simple electronic module acting as smartcard.\",\n \"If the smartcard has the format known as ID-1, it includes a card as such made from plastic material on which the above-mentioned module with electronic chip is positioned.\",\n \"The assembly respects a certain number of well known standards, whether regarding physical (dimensions, location of the chip, etc.\",\n \"), electrical and/or electronic aspects, which require no further mention.\",\n \"Otherwise, the module alone, in the Plug-in SIM format, can be inserted in the main reader.\",\n \"Generally, the latter simply consists of a chamber fitted with a cover.\",\n \"A back wall has contact studs used to connect the module with the electronic circuits equipping the telephone terminal.\",\n \"The smartcard inserted in the main reader, or SIM card, stores a certain number of data items concerning the telephone subscription as such the name(s) of the operator(s) that the subscription(s) have been taken out with, subscription, subscriber identification data, etc.\",\n \"), as well as so-called embedded applications.\",\n \"These are, in particular, applets providing access to services present on remote servers, via the telephone network, or just executed locally.\",\n \"The interactive video games displayed on the screen of the mobile telephone are examples of this last category of applications.\",\n \"To do this, the SIM cards implement the so-called SIM Toolkit technology which complies with standard ETSI 11.14.These cards are programmable.\",\n \"Like all programmable cards, they not only receive commands from the host terminal, i.e.\",\n \"the mobile telephone, but they also transmit commands to this terminal.\",\n \"These commands are known as “proactive”.\",\n \"As an illustration, a proactive command called “DISPLAY TEXT”, with as parameter, for example, the following text: “Welcome”, will request the mobile telephone, under the control of the applet contained in the card, to display the above-mentioned text “Welcome” on its display screen.\",\n \"Amongst the proactive commands described by the above-mentioned standard ETSI 11.14, some of them are used to activate and then send so-called APDU (Application Protocol Data Unit) commands, i.e.\",\n \"complying with standard ISO 7816, to a second smartcard inserted in a second reader of the mobile equipment, henceforth called the auxiliary reader.\",\n \"To understand the proactive commands sent by the SIM card, the GSM terminals must of course also comply with the standard ETSI 11.14.In the known state of the art, the second smartcard generally consists of a credit card, for example a standard bank card.\",\n \"The auxiliary reader generally consists of a traditional smartcard reader which can read a smartcard of this type.\",\n \"In order to do this, it has a slot into which the smartcard is inserted whilst the transaction is being carried out.\",\n \"For example, the transaction could consist of debiting the credit card or the bank card by a certain amount to reload the SIM card with telephone communication units.\",\n \"The second smartcard operates in “slave” mode, since it is under the control of the first smartcard.\",\n \"In the context of the invention, the term “embedded applications” must be taken in its broadest sense.\",\n \"It normally concerns applets or similar programs, but also includes all types of digital data.\",\n \"For example, the entries of a telephone directory stored in the SIM card or any other data file.\",\n \"Henceforth, the term “digital data” will designate either “applications” (or “programs”) or “data or simple data files” stored in the memory means of the SIM card.\",\n \"We can easily see that for various reasons, it may be useful, or even necessary, to add, delete and/or modify applications or data stored in the SIM card.\",\n \"It is known that applets need to be added in a SIM card.\",\n \"Generally, a computer (e.g.\",\n \"a traditional microcomputer) is used to load applets.\",\n \"The applet to be loaded is, for example, stored on a hard disk of the computer.\",\n \"A resident program in the computer, known as the “Loader” receives in input a file containing the applet and converts it into a set of commands that it sends to the SIM card.\",\n \"These commands can be sent in two main ways: locally, from a smartcard reader connected to the computer.\",\n \"remotely, for example by using the technology known as Over The Air (O.T.A.)\",\n \"in GSM terminology.\",\n \"These methods present several disadvantages.\",\n \"Firstly, the SIM card is inserted in the smartcard reader of the computer used for loading.\",\n \"It can be seen immediately that this methods presents the disadvantage of requiring specific hardware.\",\n \"At least, the subscriber, holder of the SIM card, must physically go to a place where this type of hardware is available.\",\n \"As regards the second method, it is worth pointing out that digital data is generally transmitted via the Short Message Service (SMS) technology (GSM-Data Service) in compliance with the two standards ETSI 03.40 and ETSI 03.48.Generally, the maximum length of useful in these messages is 160 septets or 140 octets, depending on the applications.\",\n \"Although with current technology, smartcards can only store programs requiring relatively a limited amount of memory, as is the case with the applets, they often come in the form of files occupying about 10 kB.\",\n \"Clearly, it would take about 80 messages to send a file of this length.\",\n \"It must also be pointed out that with transmissions implementing OTA technology, the security and/or reliability rates are not very high.\",\n \"In particular, especially when a set of SIM cards must be updated with data, even identical, the process has to be repeated for each mobile telephone, since the links between a remote server and a mobile telephone are “point to point”.\",\n \"Consequently, the above-mentioned method is, in practice, extremely complicated and possibly even impossible to implement.\",\n \"Since the mobile telephones concerned by the invention are dual slot type, as pointed out earlier, one possible method could have been to use the auxiliary reader to load applets in the SIM card inserted in the main reader.\",\n \"An obvious solution would in fact be to implement a loader program in the subscriber's SIM card.\",\n \"The code of the applet to be loaded could then be stored in another smartcard which could be inserted in the auxiliary reader.\",\n \"The loader program in the SIM card could then transmit one or more proactive command(s) in order to read the code of the applet to be loaded.\",\n \"Once the program has read the applet code, it can then load it using a suitable means.\",\n \"Initially, this method seems to offer advantages: 1) The loader program of the SIM card is compatible with the card itself since stored in it; 2) The auxiliary smartcard containing the applet code does not need to comply with the Sim Toolkit standard.\",\n \"However, this method presents significant disadvantages: 1) Since the loader program takes up a non-negligible amount of space in the SIM card, there is less space for the applets which have to be stored there.\",\n \"Even though the capacity of the memories used on smartcards has significantly increased over the last few years, it remains relatively limited for this type of application.\",\n \"2) This solution cannot be considered using the present technology, since: it is impossible to send loading commands.\",\n \"The commands to be sent by the application to the operating system in the SIM card are in fact specified in the standard ETSI 03.19.Loading commands cannot be sent under this standard.\",\n \"in addition, the applet cannot request the mobile telephone to send commands to the card it hosts.\",\n \"The current standards would therefore have be modified in order to consider this type of operation.\",\n \"it is known from the european patent application EP 0 869 691 a mobile phone in which the SIM card communicates with an external component.\",\n \"The SIM card behaves as a gateway between the external component and the GSM network and/or the mobile phone so that the external component controls completely the mobile phone.\",\n \"The modified functionalities are transferred through applets via the SIM card to the external component.\",\n \"it is also known from the international patent application WO 00/154530 a system allowing to program a set of selectable optional features into the memory of a cellular telephone independtly of the main program.\",\n \"The systems is constituted of a programmer and a port.\",\n \"The programmer located outside the housing of the telephone sends the set of selectable optional features to the port for storage in the memory.\",\n \"The invention aims to overcome the disadvantages of the methods and devices of the known state of the art, some of which have just been described.\",\n \"The purpose of the invention, for a mobile device of type including two card readers, is to define a method for the administration of digital data, applications and/or simple data, stored on a first smartcard, called subscriber or SIM smartcard, inserted in a first smartcard reader, called the main reader, via a second smartcard, called administration card, inserted in a second smartcard reader, called auxiliary smartcard reader.\",\n \"The method according to the invention can be used not only to add one or more applets in the SIM card, i.e.\",\n \"to load applets, but also provides true management or administration of digital data, applications and/or simple data, stored in it.\",\n \"Apart from loading digital data in the SIM card, the method according to the invention can be used to delete digital data, display it on the screen of the mobile device and modify it.\",\n \"This method can also be used to obtain information concerning various parameters and/or attributes about the said digital data and/or the SIM card as such, for example by displaying the names or statuses of the applets already stored in the SIM card, the memory space available in the SIM card, etc.\",\n \"The method according to the invention does not involve any modification of the hardware implemented (ordinary telephony equipment can be used) and remains fully compatible with current standards whether regarding the transmissions the smartcards (SIM Toolkit technology) or the communications between the smartcards and the circuits of the mobile device, which invoke APDU standards.\",\n \"To do this, according to a first characteristic, an “administrator” smartcard will be used, which implements a specific software module.\",\n \"It is this specific program module which is used to administer the SIM card.\",\n \"If the loading function is available, the software module includes at least a loader program and one or more applet codes which can be loaded selectively into the SIM card.\",\n \"Preferably, the loader program consists of an applet.\",\n \"Preferably, this applet and the other applets are programmed in JAVA (registered trademark).\",\n \"If the subscriber SIM card only stores simple digital data (i.e.\",\n \"no applications), then it does not necessarily need to comply with the SIM Toolkit technology.\",\n \"The invention also concerns a mobile telephony device to implement the method.\",\n \"Apart from the numerous possibilities offered by the method and its high flexibility, it must also be pointed out that the administrator card is not a “proprietary” type card.\",\n \"It does not in fact have to be provided by a telephone network operator.\",\n \"In all cases, it can be obtained, for example purchased, from various sources: supermarkets, post offices, etc., or sent through the post.\",\n \"Once the user has simply inserted it in the auxiliary reader; the entire loading process, or more generally the administration process, is carried out automatically (as will be fully detailed below) and/or according to the instructions displayed on the mobile telephone screen.\",\n \"Once again preferably, the instructions for using the administrator card can be printed on it or displayed on screen when it is inserted in the auxiliary reader, after switching on the mobile telephone.\",\n \"Still preferably, the administrator card is in ID-1 format, which means that it can be inserted instead of a credit card or a standard bank card.\",\n \"The main purpose of the invention is therefore to define an administration method for an embedded subscriber system for a mobile telephone device of type including a first embedded system reader, the main reader, designed to take the said subscriber embedded system and a second embedded system reader, the auxiliary reader, the said subscriber embedded system including computer processing and data storage means, the said data possibly including software applications, characterised in that it includes at least the following steps: initial loading, in the digital data storage means of an additional system, the administration system, of a specific software module, including at least one component consisting of a loader program and at least one digital data file in a first format; temporary insertion of the said administration embedded system in the said auxiliary embedded system reader; conversion of the said digital data in the first format into a series of digital data in a second format under the control of the said specific software module; and the selective transfer of at least one series of digital data in the said second format to the said subscriber embedded system and its loading in the said storage means of this embedded system, via the said auxiliary and main embedded system readers, under the control of the said specific software module; and in that the said transfer is carried out according to a given communication protocol.\",\n \"The invention also concerns an embedded system for the implementation of the method.\",\n \"The invention will now be described in more detail, referring to the attached drawings, amongst which: FIG.\",\n \"1 is a diagrammatic representation of a configuration example for mobile telephone including two smartcard readers with a subscriber SIM card and a smartcard according to the invention; FIG.\",\n \"2 is a block diagram explaining the main steps and phases of the method according to the invention and the interactions between the components implemented; FIG.\",\n \"3 is a block diagram schematising the main administration operations possible through the use of the method according to the invention; FIG.\",\n \"4 is a block diagram illustrating a practical example of an applet in the subscriber SIM card, according to the method of the invention; and FIG.\",\n \"5 is a block diagram illustrating a practical example of deleting an applet in the subscriber SIM card, according to the method of the invention.\",\n \"Henceforth, without limiting in any way whatsoever the scope of the invention, we will consider the preferred application of the invention, unless otherwise specified, i.e.\",\n \"the context of a dual slot mobile telephone complying with the GSM standard.\",\n \"FIG.\",\n \"1 is a diagrammatic representation of a mobile telephone 1 and the two smartcards implemented, 2 and 3.We have assumed that the SIM subscriber card 3 is a module in Plug-in SIM format.\",\n \"As such, this SIM card corresponds to the known state of the art.\",\n \"It includes a support 30 in the above-mentioned Plug-in SIM format, on which is positioned an electronic chip 31 including input-output contact studs, with unique reference 310.The assembly is defined by various standards and/or norms, whether regarding the physical (dimensions, location of the chip, etc.\",\n \"), electrical and/or electronic aspects.\",\n \"The electronic chip includes in particular data processing means in a stored program (microprocessor or microcontroller) and non volatile (ROM, EPROM) and volatile (RAM and various registers) storage means.\",\n \"These various computer resources, also well known, are not shown on FIG.\",\n \"1.Apart from programs and digital data directly associated with the telephone transmissions, carried out for example in compliance with the above-mentioned GSM standard, the storage means of the module 3 can store various items of digital data, and especially applets, for example n applets, A1 to An.\",\n \"As such, this characteristic also corresponds to the known state of the art.\",\n \"If module 3 stores applets, it must comply with the Sim Toolkit technology so that it can receive commands and transmit proactive commands.\",\n \"In the example shown on FIG.\",\n \"1, the mobile telephone 1 includes a main body 10 and an auxiliary body 11, which can be folded back by rotation around an axis 110 at the bottom of the main body 10.The main smartcard reader 12 consists of an internal chamber 12 housing the SIM module 3 (represented on FIG.\",\n \"1 enlarged and outside its housing).\",\n \"The main body 10 is equipped in particular with a display screen 100, generally liquid crystal type.\",\n \"The auxiliary smartcard reader 13 is located in the auxiliary folding body 11 It communicates with the exterior via a slot 130 in which a smartcard in ID-1 format, and therefore compatible with standard smartcards, bank or credit cards, can be inserted.\",\n \"The mobile telephone according to the invention therefore remains fully compatible with the norms and standards of the known state of the art.\",\n \"It requires no modifications.\",\n \"In a preferred mode of realisation of the invention, the smartcard used as administration card also complies with the above-mentioned standard ID-1.The auxiliary reader 13 can therefore accept either standard smartcards or smartcards 2 complying with the characteristics of the invention and which are detailed below.\",\n \"Initially, we assume that the main function of the smartcard 2 is to load new applets in the SIM module 3.Preferably, the smartcard 2 complies with the Sim Toolkit technology.\",\n \"It can send proactive type commands to the host mobile telephone 1 and read and/or write commands to the files in its Sim Toolkit operating system OSST.\",\n \"According to a first characteristic of the invention, apart from the above-mentioned Sim Toolkit operating system OSST, the electronic chip 20 of the smartcard 2 contains at least one software module known as the loader Ch and a certain number of files, f1 to fn, capable of being transferred into the SIM module 3, in order to load applications there in addition to any applications already resident (not shown).\",\n \"Preferably, these applications consist of applets, advantageously coded in JAVA (registered trademark) language.\",\n \"In this case, the files, f1 to fn, are advantageously coded according to the CAP file format defined by the Java Card forum (registered trademark).\",\n \"The loader program Ch itself is also a Sim Toolkit applet.\",\n \"According to a characteristic of the method of the invention, the subscriber's SIM module or card 3 becomes the receiving card.\",\n \"The administrator card 2, according to another characteristic of the method of the invention becomes the main card during the loading, although it is inserted in the auxiliary smartcard reader 13.In a preferred mode of realisation, due to the fact that the administrator card uses Sim Toolkit technology, it can, unlike the known state of the art, become priority when the mobile telephone 1 is switched on, i.e.\",\n \"it can become “master”.\",\n \"The card inserted in the auxiliary reader 13, i.e.\",\n \"the administrator card 2, then takes priority over the SIM module 3: it is the applets of this administrator card 2 which are proposed to the user and not those resident in the SIM module 3 (receiving card).\",\n \"However, it must be made quite clear that this arrangement is not mandatory.\",\n \"The user could, for example, be requested to press a key or a key combination 101 on the keyboard of the mobile telephone 1 so that the process starts “manually” after this mobile telephone 1 is switched on.\",\n \"For example, the instructions for use may be supplied independently from the administrator card 2 or printed or engraved on one side of it.\",\n \"In the preferred mode of realisation, since the administrator card 2 takes priority, an initial interactive menu can be displayed on the screen 100 showing in particular the loader applet Ch installed on this card.\",\n \"Once the user has selected this application, it can read the files present, f1 to fn, so that it can then execute APDU commands on the receiving card, i.e.\",\n \"the SIM module 3, and thereby load the applet(s) corresponding to the files present on the administrator card.\",\n \"These operations are carried out in co-operation with the Sim Toolkit operating system OSST.\",\n \"More precisely, the card inserted in the auxiliary reader 13, i.e.\",\n \"the administrator card 2, can read the files present on its operating system via one or more traditional APDU commands, such as “READ BINARY”.\",\n \"Reading the system files, f1 to fn, enables the administrator card 2 to collect all the information required concerning the applet to be installed on the subscriber's SIM module or card 3.In addition, the administrator card 2 can open a session in order to access the subscriber's SIM module 3 via the proactive command “POWER ON CARD”.\",\n \"When this has been done, the administrator card 2 sends APDU loading commands defined by the standards ETSI 03.48 and ISO/IEC 7816-4 in order to install the applet read previously.\",\n \"The APDU commands are therefore sent to the SIM module via the proactive command “PERFORM CARD APDU”.\",\n \"Once the applet has been installed, the administrator card 2 of the auxiliary reader 13 closes the session with the command “POWER OFF CARD”.\",\n \"The user can now withdraw the administrator card 2 from the auxiliary reader 13 in order to reconnect on the SIM module 3.On la FIG.\",\n \"1, the applets referenced A1 to An, loaded in addition in the SIM module 3 correspond to files f1 to fn of the administrator card 2.All these commands or sessions comply with the usual standards and norms concerning the communication protocol between a card reader and a smartcard.\",\n \"As pointed out above, APDU commands in particular are used.\",\n \"The invention therefore involves no modifications.\",\n \"It should also be pointed out that, once the administrator card 2 according to the invention has been withdrawn, a traditional smartcard, for example a bank card or a credit card, can be inserted in the auxiliary reader 13.In this case, operation corresponds once again to the known state of the art: the smartcard in the auxiliary reader 13 no longer takes priority.\",\n \"It remains under the control of the SIM module or card 3.FIG.\",\n \"2 is a block diagram explaining in more detail the main interactions between the various components implemented.\",\n \"The main steps and phases of the method are also shown.\",\n \"We also assumed that that the administrator card can be used not only to load one or more applets in the SIM module 3, but also for other operations: deletion, etc., which will be detailed below, especially in reference to FIG.\",\n \"3.The software module specific to the invention includes several components, under the single reference 40.Preferably, as indicated, it is activated automatically when the mobile telephone 1 is switched on (FIG.\",\n \"1: 1).\",\n \"To do this, a command called “TERMINAL PROFILE” is used, generated by the mobile telephone 1 (FIG.\",\n \"1) precisely when switching on.\",\n \"The files stored in the administrator card 2 under the single reference 41, are generally associated not only with applets likely to be loaded in the SIM module 3, but also with digital data.\",\n \"For example, this data could consist of entries in the telephone directory.\",\n \"The administration card 2 may also be intended to load data associated with a new telephony operator in the SIM module 3.A priori, since the standards used for the communications between smartcard and reader implement APDU commands, the data and/or applications 41 cannot be loaded directly into the SIM module 3.A prior operation 42 must be carried out, which will be called formatting, under the specific program command 40.Note that are of course, as with any program during execution, interactions between the operating system OSST and this specific program 40.Once the formatting has been carried out, still under the control of the specific program 40, communication sessions, which may be bidirectional, are set up between the administrator card 2 and the module 3, via the auxiliary reader 13 and main reader 12, using a series of APDU commands (standardised communication protocol).\",\n \"Consequently, one or more additional applets may be loaded in the SIM module 3.However, the method according to the invention is not limited to loading applets.\",\n \"As shown by the block diagram on FIG.\",\n \"3, the card 2 can perform the following main administration operations 200: “addition” 201 (as shown above), as well as “deletion” 202 and “modification” 203.These operations can be carried out on applications (applets) 410 or various items of digital data 411.Lastly, the administrator card 2 can perform administration operations as such, which may be called “management” operations, especially various displays (for example multiple choice interactive menus) on the screen 100 (FIG.\",\n \"1), search and display of the memory space occupied by the applets present in the SIM module 3, search and display of the name and logical status of these applets, etc.\",\n \"These operations are all performed under the control of the specific program 40 and implement standard APDU commands and sessions.\",\n \"As an illustration, we will now describe two examples of practical implementation, referring to FIGS.\",\n \"4 and 5.The first concerns more specifically the selective addition of an applet, which in this case is used for local execution of the well known game called “MINEFIELD”.\",\n \"The second concerns more specifically the selective deletion of an applet present in the SIM module 3, after detecting insufficient memory space, in order to add a new applet (an address manager in this case).\",\n \"EXAMPLE 1 Adding an Applet (FIG.\",\n \"4) We assume that a user has already loaded an applet concerning the well known game “HANGMAN” on the SIM module 3 (FIG.\",\n \"1).\",\n \"He buys an administrator card according to the invention in order to load an extra game, in this game the game “MINEFIELD”.\",\n \"When the user inserts the loader card, i.e.\",\n \"the administrator card 2 (FIG.\",\n \"1) in the auxiliary reader 13 (FIG.\",\n \"1) with the “Loader” applet Ch (FIG.\",\n \"1) and the file corresponding to the “MINEFIELD” applet, according to the preferred mode, an initial interactive menu is automatically displayed on the screen 100 (FIG.\",\n \"1), without the user having to press any key 101 (FIG.\",\n \"1) on the mobile telephone 1 (FIG.\",\n \"1).\",\n \"This initial menu indicates to the user the name of the applet that can be loaded and requests whether or not to continue the loading: step 50.The text displayed is as follows: “Do you want to install Minefield on your SIM card?\",\n \"OK CANCEL”.\",\n \"If the user refuses (right hand branch: “CANCEL validated by user”), a new menu prompts the user to withdraw the loader card, i.e.\",\n \"the administrator card 2 (FIG.\",\n \"1): step 55.The text displayed is as follows: “You can withdraw your loader card.\",\n \"Goodbye”.\",\n \"The user withdraws the administrator card 2 from the auxiliary reader 13 (FIG.\",\n \"1).\",\n \"Following this operation, the mobile telephone 1 (FIG.\",\n \"1) switches back over to the SIM module 3 (FIG.\",\n \"1) and requests the user's PIN code (Personal Identifier Number): step 56.After entering a correct PIN code, the user can use the SIM module or card 3 again and select an applet from those already resident, for example the “HANGMAN” applet: step 57.If the user accepts to load the new applet (left hand branch, “OK” validated by user), the administrator card 2 (FIG.\",\n \"1) loads the “MINEFIELD” applet on the SIM module 33 (FIG.\",\n \"1), and informs the user that the operation has been carried out successfully: steps 51 and 52.The following messages are displayed: step 51: “Please wait.\",\n \"Loading .\",\n \".\",\n \".\",\n \"”, and step 52: “Minefield has been loaded correctly.\",\n \"You can withdraw the loader card”.\",\n \"When the user has withdrawn the administrator card 2 (FIG.\",\n \"1), he is prompted to enter his PIN code: step 53.After entering a correct PIN code, the user can use the SIM module or card 3 (FIG.\",\n \"1) again and select an applet from those already resident.\",\n \"After selection of the applet menu the two applets present on the SIM module are displayed: “HANGMAN”, previously resident, and “MINEFIELD” which has just been loaded: step 54.He can use this new applet, i.e.\",\n \"execute the game “MINEFIELD” on his mobile telephone 1 (FIG.\",\n \"1).\",\n \"EXAMPLE 2 Deleting an Applet (FIG.\",\n \"5) We now assume that the user has two applets installed on the SIM module 3 (FIG.\",\n \"1), for example the above-mentioned “HANGMAN” and “MINEFIELD”, and he wants to install an additional applet, for example an “address manager”.\",\n \"As before, the user inserts an administrator card 2 (FIG.\",\n \"1) which has a specific program 40 (FIG.\",\n \"2), especially including a component to load the “address manager” applet (one of the files 41: FIG.\",\n \"2).\",\n \"At step 60, a message is displayed: “Do you want to install the address manager on your SIM card?\",\n \"OK CANCEL”.\",\n \"If the user accepts the option presented (left hand branch, “OK” validated), the specific program 40 (FIG.\",\n \"2) scans the memory of the SIM module 3 (FIG.\",\n \"2): step 61.If there is sufficient memory space left on the SIM module 3 (FIG.\",\n \"1) for this third applet, the “address manager” applet can be loaded on the SIM module 3 (FIG.\",\n \"1).\",\n \"The execution of this operation is similar to that which has been described with respect to FIG.\",\n \"4.Consequently, the corresponding steps will not be described again.\",\n \"Otherwise, if there is not sufficient memory space, a message is displayed to the user indicating that at least one of the two applets already present must be deleted.\",\n \"In the example described, the message is as follows: “Insufficient memory.\",\n \"You must first delete an applet OK CANCEL”.\",\n \"If the user accepts this possibility (“OK” validated), he is prompted to select one of the applets, in response to the message displayed: “Select the applet to be deleted: HANGMAN or MINEFIELD-OK CANCEL”: step 62.If the user accepts one of the options presented, for example if he selects “HANGMAN”, the specific program 40 (FIG.\",\n \"2) starts a session in order to delete the “HANGMAN” applet.\",\n \"After deleting one of the applets loaded, the user is prompted to continue the procedure, just as in the previous example (FIG.\",\n \"4): loop back to step 60.The following message is displayed: “The HANGMAN applet has been deleted from memory.\",\n \"Do you want to continue?\",\n \"OK CANCEL”: step 63.In steps 60, 61, 62 and 63, the user has the possibility of refusing the choice proposed (“CANCEL” validated).\",\n \"The process then continues at step 64.The user is prompted to withdraw the administrator card 2 (FIG.\",\n \"1) from the auxiliary reader 13 (FIG.\",\n \"1).\",\n \"The following message is displayed: “You can withdraw your loader card.\",\n \"Goodbye”.\",\n \"The user must then enter his PIN code in order to use the SIM module 3 (FIG.\",\n \"1): step 65.After reading the above, it is easy to see that the invention does in fact reach the objectives set.\",\n \"With the method according to the invention, it is possible in particular to use a standard mobile telephone since no modifications are required to the equipment.\",\n \"It complies fully with current norms and standards.\",\n \"It is therefore fully compatible with the known state of the art.\",\n \"However, it offers numerous advantages.\",\n \"Not only can it be used to add (load) but also to delete and modify applications and/or digital data, as well as to perform various management operations: display, scanning the SIM memory, etc.\",\n \"It therefore provides true management of the subscriber's SIM card or module.\",\n \"No other equipment is required, as with certain methods of the known art, since the auxiliary reader of the mobile telephone is used as receiver of the administrator card.\",\n \"Neither does it rely on downloads from remote servers, with the disadvantages inherent to this type of method, which were pointed out in the preamble of this description.\",\n \"Lastly, the administrator card can be issued by any entity, it is not only a proprietary card issued by a mobile telephone operator.\",\n \"The applications and/or data stored are a priori independent from those specific to the telephony operators.\",\n \"However, the method enables an administrator card issued by a particular operator to be used in order to update its own data or to add a subscription to this operator in the SIM card, or on the contrary to delete it.\",\n \"The administrator card may be available from various types of point of sale or procurement, or even sent through the post.\",\n \"The operations possible, especially the loading of additional applets, require no special knowledge.\",\n \"In a preferred mode of realisation, certain operations are fully automatic, in particular the activation of the loader program or, more generally, of the specific program.\",\n \"The following steps are interactive, the user being guided by menu.\",\n \"However, the invention is of course not limited to only those examples of realisation explicitly described, especially in relation to FIGS.\",\n \"1 to.\",\n \"In particular the text of the messages displayed was only indicated to describe the method of the invention more clearly.\",\n \"These messages are related to the particular applications (applets) loaded on the administrator card.\",\n \"A priori, any messages would be possible without leaving the scope of the invention.\",\n \"Similarly, the method according to the invention does not interfere in any way with the technology used for the transmissions: GSM, GPRS or UTMS, for example, since the operations are all carried out locally through the implementation of two readers: the main reader containing the SIM module or card and the auxiliary reader intended to receive a traditional smartcard, for example a bank card.\",\n \"Moreover, the invention is not exclusively intended for mobile telephony.\",\n \"The invention concerns too for example an administration method for a data processing device as a computer, an organizer, an encoder reader or others.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10468033"}}},{"rowIdx":395,"cells":{"text":{"kind":"list like","value":[["Estimating signal strength measurements in a telecommunications system","The number of measurements being performed in a telecommunications system can be reduced by performing a predetermined measurement in Cell A and in co-located Cell B, step 1, for example a received signal code power (RSCP) measurement.","The difference between the two signals is determined, and stored for future reference, step 3.","Once the difference value has been established, the mobile station no longer needs to perform measurements on both Cell A and Cell B.","Instead, if a subsequent measurement is made on Cell A, step 5, the network can estimate the measurement for Cell B based on the difference signal previously determined, step 7."],["1.A method of estimating a signal measurement in a telecommunications system, the method comprising the steps of: performing a first measurement in first and second co-located cells of the telecommunications system; determining a difference value between the measurements; and using the difference value to estimate a second measurement for the second cell, based on a second measurement being made in the first cell.","2.A method as claimed in claim 1, wherein the second measurement is the same type of measurement as the first measurement.","3.A method as claimed in claim 1, wherein the second measurement is a different type of measurement to the first measurement, but having a predetermined relationship to the first measurement.","4.A method as claimed in claim 3, wherein the second measurement is a CPICH Ec/No measurement.","5.A method as claimed in any one of claims 1 to 4, wherein the first measurement is a received signal code power (RSCP) measurement.","6.A method as claimed in any one of the preceding claims, wherein the measurements are performed at a mobile station in the telecommunications system and the difference value stored at a network side of the telecommunications system.","7.A method as claimed in any one of the preceding claims, wherein the method of estimating a measurement is used to evaluate which cells should belong to an active set on a frequency other than the frequency being used.","8.A method of handover in a cellular telecommunications system, the method of handover using a method of estimating a signal strength measurement as defined in any one of claims 1 to 7.9.A telecommunications system in which signal strength measurements are performed in two or more co-located cells, the system comprising: means for measuring a first measurement in first and second co-located cells of the telecommunications system; means for determining a difference value between the measurements; and means for estimating a second measurement for the second cell, based on a second measurement being made in the first cell and the previously determined difference value.","10.A system as claimed in claim 9, wherein the second measurement is the same type of measurement as the first measurement.","11.A system as claimed in claim 9, wherein the second measurement is a different type of measurement to the first measurement, but having a predetermined relationship to the first measurement.","12.A system as claimed in claim 11, wherein the second measurement is a CPICH Ec/No measurement.","13.A system as claimed in any one of claims 9 to 12, wherein the first measurement is a received signal code power (RSCP) measurement.","14.A system as claimed in any one of claims 9 to 13, wherein the measurements are performed at a mobile station in the telecommunications system and the difference value stored at a network side of the telecommunications system.","15.A system as claimed in any one of claims 9 to 14, having means for evaluating which cells should belong to an active set on a frequency other than the frequency being used.","16.A method of estimating a signal measurement substantially as hereinbefore described, with reference to, and as shown in FIG.","1 or 2 of the accompanying drawings.","17.A telecommunications system substantially as hereinbefore described, with reference to, and as shown in FIG.","1 or 2 of the accompanying drawings.","18 A communications node for a telecommunications system in which signal strength measurements are performed in two or more co-located cells, the communications node comprising: means for receiving a first measurement from first and second co-located cells of the telecommunications system; means for determining a difference value between the measurements; and means for estimating a second measurement for the second cell, based on a second measurement being made in the first cell and the previously determined difference value.","19.A communications node as claimed in claim 18, wherein the second measurement is the same type of measurement as the first measurement.","20.A communications node as claimed in claim 18, wherein the second measurement is a different type of measurement to the first measurement, but having a predetermined relationship to the first measurement.","21.A communications node as claimed in claim 20, wherein the second measurement is a CPICH Ec/No measurement.","22.A communications node as claimed in any one of claims 18 to 21, wherein the first measurement is a received signal code power (RSCP) measurement.","23.A communications node substantially as hereinbefore described, with reference to, and as shown in FIG.","1 or 2 of the accompanying drawings.","24.A method of estimating a signal measurement in a communications node of a telecommunications system, the method comprising the steps of: receiving a first measurement from first and second co-located cells of the telecommunications system; determining a difference value between the measurements; and using the difference value to estimate a second measurement for the second cell, based on a second measurement being made in the first cell.","25.A method as claimed in claim 24, wherein the second measurement is the same type of measurement as the first measurement.","26.A method as claimed in claim 24, wherein the second measurement is a different type of measurement to the first measurement, but having a predetermined relationship to the first measurement.","27.A method as claimed in claim 26, wherein the second measurement is a CPICH Ec/No measurement.","28.A method as claimed in any one of claims 24 to 27, wherein the first measurement is a received signal code power (RSCP) measurement.","29.A method of estimating a signal measurement in a communications node of a telecommunications system, the method being substantially as hereinbefore described, with reference to, and as shown in FIG.","1 or 2 of the accompanying drawings.","30.A mobile communications terminal for use in a telecommunications system as described in any one of claims 9 to 15."],["<SOH> BACKGROUND OF THE INVENTION <EOH>In a telecommunications system, for example a wideband CDMA system (WCDMA), there is always a desire to communicate using the least amount of power.","Therefore, all transmissions should ideally be performed at the lowest possible power level, while still maintaining an acceptable quality level.","However, there are certain factors which act against this general desire to reduce power levels.","Certain communication techniques have an inherent need for the power levels to be increased, both in the mobile station and the network.","For example, data compression can be used to transmit a given amount of data in less time, resulting in gaps being created to allow inter-frequency or inter-RAT (Radio Access Technology) measurements to be carried out.","In addition to the peak power being increased during the data compression, the average power also needs to be increased to compensate for the channel estimates that are not being updated during the time gaps, resulting in the receiver not being optimally tuned after the time gaps.","While increased inter-frequency and inter-RAT measurements typically have the disadvantage of increasing the average power consumption when using real time services with requirements of low delays, for example speech, they also have a degrading effect in that they reduce the available channelization-codes.","During data compression a code for a lower spreading factor needs to be used, which typically blocks several codes used for higher spreading factors in the downlink.","The gaps in transmission mentioned above can also occur in other ways.","For example, reducing the number of bits to be transmitted by reducing the number of extra bits which are used for error correction during some frames (i.e.","code puncturing), or scheduling data transmission from a higher layer.","It is known to reduce the power requirement (and in turn improve data throughput) by reducing the number of measurements carried out in the telecommunications system.","One known example of how this may be achieved is based on the use of neighbouring cell lists, and thresholds for starting measurements.","This type of solution suffers from the disadvantage that each neighbouring cell is treated equally as if they were adjacent neighbours using different antennas covering different, but to some extent overlapping, geographical areas.","The reduction in these schemes is based on limiting the number of neighbouring cells included in the list, and on handover statistics and propagation predictions when cell planning is performed.","The aim of the present invention is to provide a method of estimating measurements in a telecommunications system, thereby reducing the number of measurements being performed.","This has the advantage of reducing power consumption and increasing the capacity of the system."],["<SOH> SUMMARY OF THE INVENTION <EOH>According to a first aspect of the present invention, there is provided a method of estimating a signal measurement in a telecommunications system, the method comprising the steps of: performing a first measurement in first and second co-located cells of the telecommunications system; determining a difference value between the measurements; and using the difference value to estimate a second measurement for the second cell, based on a second measurement being made in the first cell.","According to another aspect of the present invention, there is provided a telecommunications system in which signal strength measurements are performed in two or more co-located cells, the system comprising: means for measuring a first measurement in first and second co-located cells of the telecommunications system; means for determining a difference value between the measurements; and means for estimating a second measurement for the second cell, based on a second measurement being made in the first cell and the previously determined difference value.","According to another aspect of the present invention, there is provided a communications node for a telecommunications system in which signal strength measurements are performed in two or more co-located cells, the communications node comprising means for receiving a first measurement from first and second co-located cells of the telecommunications system, means for determining a difference value between the measurements, and means for estimating a second measurement for the second cell, based on a second measurement being made in the first cell and the previously determined difference value."],["FIELD OF THE INVENTION The present invention relates to performing measurements in a telecommunications system, and in particular, to estimating measurements in a telecommunications system, thereby reducing the number of measurements being performed.","BACKGROUND OF THE INVENTION In a telecommunications system, for example a wideband CDMA system (WCDMA), there is always a desire to communicate using the least amount of power.","Therefore, all transmissions should ideally be performed at the lowest possible power level, while still maintaining an acceptable quality level.","However, there are certain factors which act against this general desire to reduce power levels.","Certain communication techniques have an inherent need for the power levels to be increased, both in the mobile station and the network.","For example, data compression can be used to transmit a given amount of data in less time, resulting in gaps being created to allow inter-frequency or inter-RAT (Radio Access Technology) measurements to be carried out.","In addition to the peak power being increased during the data compression, the average power also needs to be increased to compensate for the channel estimates that are not being updated during the time gaps, resulting in the receiver not being optimally tuned after the time gaps.","While increased inter-frequency and inter-RAT measurements typically have the disadvantage of increasing the average power consumption when using real time services with requirements of low delays, for example speech, they also have a degrading effect in that they reduce the available channelization-codes.","During data compression a code for a lower spreading factor needs to be used, which typically blocks several codes used for higher spreading factors in the downlink.","The gaps in transmission mentioned above can also occur in other ways.","For example, reducing the number of bits to be transmitted by reducing the number of extra bits which are used for error correction during some frames (i.e.","code puncturing), or scheduling data transmission from a higher layer.","It is known to reduce the power requirement (and in turn improve data throughput) by reducing the number of measurements carried out in the telecommunications system.","One known example of how this may be achieved is based on the use of neighbouring cell lists, and thresholds for starting measurements.","This type of solution suffers from the disadvantage that each neighbouring cell is treated equally as if they were adjacent neighbours using different antennas covering different, but to some extent overlapping, geographical areas.","The reduction in these schemes is based on limiting the number of neighbouring cells included in the list, and on handover statistics and propagation predictions when cell planning is performed.","The aim of the present invention is to provide a method of estimating measurements in a telecommunications system, thereby reducing the number of measurements being performed.","This has the advantage of reducing power consumption and increasing the capacity of the system.","SUMMARY OF THE INVENTION According to a first aspect of the present invention, there is provided a method of estimating a signal measurement in a telecommunications system, the method comprising the steps of: performing a first measurement in first and second co-located cells of the telecommunications system; determining a difference value between the measurements; and using the difference value to estimate a second measurement for the second cell, based on a second measurement being made in the first cell.","According to another aspect of the present invention, there is provided a telecommunications system in which signal strength measurements are performed in two or more co-located cells, the system comprising: means for measuring a first measurement in first and second co-located cells of the telecommunications system; means for determining a difference value between the measurements; and means for estimating a second measurement for the second cell, based on a second measurement being made in the first cell and the previously determined difference value.","According to another aspect of the present invention, there is provided a communications node for a telecommunications system in which signal strength measurements are performed in two or more co-located cells, the communications node comprising means for receiving a first measurement from first and second co-located cells of the telecommunications system, means for determining a difference value between the measurements, and means for estimating a second measurement for the second cell, based on a second measurement being made in the first cell and the previously determined difference value.","BRIEF DESCRIPTION OF THE DRAWINGS For a better understanding of the present invention, and to show more clearly how it may be carried into effect, reference will now be made, by way of example, to the accompanying drawings, in which:— FIG.","1 shows the steps involved in a method of estimating a measurement according to a first aspect of the present invention; and, FIG.","2 shows the steps involved in a method of estimating a measurement according to a second aspect of the present invention.","DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT OF THE PRESENT INVENTION In a WCDMA based radio access network, several cells may be co-located using different frequencies.","The network will have information that a particular cell, say Cell A, is co-located with one or more other cells, say Cell B.","A mobile station positioned in a given location is able to make signal measurements on common pilot channels (CPICHs) transmitted from a serving cell and any co-located cells.","FIG.","1 shows the steps involved in estimating a measurement according to a first aspect of the present invention.","In step 1, the network instructs a mobile station to perform a predetermined measurement in Cell A and in co-located Cell B, for example a received signal code power (RSCP) measurement, hereinafter referred to as RSCPA and RSCPB, respectively.","These measurements are reported by the mobile station to the network.","The network determines the difference between the two signals (RSCPA−B) which is stored for future reference, step 3.Once the difference value has been established, the mobile station no longer needs to perform measurements on both Cell A and Cell B.","The network can therefore suspend inter-RAT or inter-frequency RSCP measurements.","Instead, if a subsequent measurement is made on Cell A (RSCPA+t), step 5, the network can estimate the measurement for Cell B based on the difference signal previously determined.","For example, if the difference signal was originally determined by subtracting RSCPB from RSCPA, then the estimated value for Cell B is determined by subtracting the difference value RSCPA−B from the new measurement for Cell A, RSCPA+t, step 7.Thus, RSCPB+t=(RSCPA+t−RSCPA−B) Since Cell A is co-located with Cell B, the path loss difference will remain constant for all locations of the mobile station, which means that the RSCP difference RSCPA−B also remains constant.","This difference value can therefore provide an accurate estimate of a measurement for one cell, using the measurement of a co-located cell.","Measurements in Cell B can be restarted at any time, for example if the estimated measurement approaches a critical value.","According to another aspect of the invention, the difference value described above can be used to provide further reductions in measurements.","In addition to RSCP measurements, a mobile station may perform any one, or any combination, of the following measurements, depending upon the processing utilised in the mobile station: 1.UTPA carrier RSSI (this stands for received signal strength indicator, and relates to signal strength plus interference on a particular frequency from any intra frequency cell) 2.CPICH RSCP (this stands for received signal code power, and relates to signal strength from a particular cell's CPICH on a particular frequency) 3.CPICH Ec/No (this is basically the signal-to-noise ratio used for representing the “cell quality” for handover evaluation).","Each of the measurements described above imposes a different demand on the telecommunications system in terms of the processing power required, and/or the time needed to perform a given measurement.","In other words, certain measurements are more “expensive” than others.","For example, the CPICH Ec/No measurement requires typically more processing power that the CPICH RSCP measurement, which is turn requires more processing power than the UTRA carrier RSSI measurement.","The measurements described above have a predetermined relationship whereby: CPICH Ec/No=(CPICH RSCP)/(UTRA carrier RSSI) Using this relationship, the estimation method according to the first aspect of the invention can be used to reduce the number of “expensive” measurements being made, for example by reducing the number of CPICH Ec/No measurements being performed in the telecommunications system.","Referring to FIG.","2, a difference signal is first determined, as mentioned above, by calculating the difference between the RSCPA and RSCPB measurements, (RSCPA−B), step 101.The RSSIA measurement and (Ec/No)A measurement for Cell A are also performed, step 103.Depending upon the particular implementation in a given mobile station, the (Ec/No)A measurement can either be calculated directly, or calculated from the RSCPA and RSSIA values.","Next, when it is determined that an Ec/No measurement is subsequently required for Cell B, step 105, instead of measuring this value directly, the measurement can be estimated as follows.","First, the RSSI value for cell B is measured, RSSIB, as shown in step 107.The Ec/No measurement can then be estimated from the RSSIB measurement, using a RSCPA measurement for cell A and the previously stored difference value RSCPA−B, as follows: (Ec/No)B=(RSCPA−RSCPA−B)/(RSSIB) Thus, the Ec/No measurement for cell B can be estimated without having to perform a relatively costly CPICH RSCP measurement for cell B.","Instead, only the less costly UTRA RSSI measurement needs to be made for cell B.","Also, since the RSCP and Ec/No measurements for cell A are being made in the same cell that the mobile station is currently using, these measurements are also less costly than the corresponding measurements for cell B (i.e.","being the cell which is not currently being used by the mobile station).","The Ec/No measurement for cell A and the estimated Ec/No measurement for cell B may be used to distribute traffic between cells, for example, to trigger handover between cell A and cell B.","As noted above, since the RSSI measurement for cell B is less demanding than the Ec/No measurement, power consumption is thereby reduced.","For example, assume that one 7 slot wide measurement gap is used every third frame (each frame consisting of 15 slots) for inter-frequency compressed mode measurements on CPICH Ec/No, and that the average DL power needs to be increased by 3 dB to maintain the same connection quality.","The width of the measurement gap can be decreased to one slot used every third frame, and, if the power increase required to maintain the same quality can be assumed to be decreased by {fraction (1/7)} because of that, the following difference in power can be calculated.","This corresponds to a 0.6 dB increase (i.e.","1.143 times increase) instead of 3 dB increase (i.e.","2 times increase) when making CPICH Ec/No measurements.","Thus, if it is assumed that all the mobiles in a cell are using compressed mode at all times then, if the nominal amount of mobiles is planned to be limited to 20 per cell using 1 watt each when not using compressed mode, the estimated amount of mobiles when using compressed mode and the same DL power margins would be: 20/2=10 mobiles when the mobiles are performing inter-frequency CPICH Ec/No measurements all of the time, and 20/1.143=17.5 mobiles when using estimation according to the second aspect of the invention, whereby RSSI measurements are carried out instead.","According to the first and second aspects of the invention, rather than having a mobile station perform measurements on each of the co-located cells, the mobile station determines a relationship between the cells, assumes that this relationship remains constant, and then performs measurements on just one cell—the other cell's measurements being estimated from the known relationship with the first cell.","Also, if different measurements within a given cell have different power requirements, the high power measurements can be estimated by measuring the lower power measurements, and calculating the higher power measurements accordingly.","As can be seen from the above, estimating measurements in this way has the effect of reducing the number of measurements being performed.","This has the effect of reducing power consumption and increasing the capacity of the telecommunications system, thereby enabling a greater number of mobile stations to be supported.","Although the preferred embodiment refers to the difference value being calculated with respect to the respective RSCP measurements from each cell, the invention is equally applicable to other signals being used in this manner, for example determining a difference signal using the BCCH frequency RXLEV in a GSM system, or measured despread signal strength on a IS95 system.","Also, although the invention has been described as performing measurements between two co-located cells, the invention may be used with just one cell, or with any number of co-located cells.","Furthermore, the invention can be used in the same way for evaluating cells on a frequency other than the frequency being used, to determine whether they should belong to the active set.","For example, by using knowledge about the active set on the currently used frequency and also the knowledge that the cells on used and unused frequencies are co-located, the measurement requirements on the unused frequency will only be UTRA Carrier RSSI and information on RSCPB+t, allowing the CPICH Ec/No value to be used for evaluating the cells on the unused frequency, to determine whether they should belong to the active set."]],"string":"[\n [\n \"Estimating signal strength measurements in a telecommunications system\",\n \"The number of measurements being performed in a telecommunications system can be reduced by performing a predetermined measurement in Cell A and in co-located Cell B, step 1, for example a received signal code power (RSCP) measurement.\",\n \"The difference between the two signals is determined, and stored for future reference, step 3.\",\n \"Once the difference value has been established, the mobile station no longer needs to perform measurements on both Cell A and Cell B.\",\n \"Instead, if a subsequent measurement is made on Cell A, step 5, the network can estimate the measurement for Cell B based on the difference signal previously determined, step 7.\"\n ],\n [\n \"1.A method of estimating a signal measurement in a telecommunications system, the method comprising the steps of: performing a first measurement in first and second co-located cells of the telecommunications system; determining a difference value between the measurements; and using the difference value to estimate a second measurement for the second cell, based on a second measurement being made in the first cell.\",\n \"2.A method as claimed in claim 1, wherein the second measurement is the same type of measurement as the first measurement.\",\n \"3.A method as claimed in claim 1, wherein the second measurement is a different type of measurement to the first measurement, but having a predetermined relationship to the first measurement.\",\n \"4.A method as claimed in claim 3, wherein the second measurement is a CPICH Ec/No measurement.\",\n \"5.A method as claimed in any one of claims 1 to 4, wherein the first measurement is a received signal code power (RSCP) measurement.\",\n \"6.A method as claimed in any one of the preceding claims, wherein the measurements are performed at a mobile station in the telecommunications system and the difference value stored at a network side of the telecommunications system.\",\n \"7.A method as claimed in any one of the preceding claims, wherein the method of estimating a measurement is used to evaluate which cells should belong to an active set on a frequency other than the frequency being used.\",\n \"8.A method of handover in a cellular telecommunications system, the method of handover using a method of estimating a signal strength measurement as defined in any one of claims 1 to 7.9.A telecommunications system in which signal strength measurements are performed in two or more co-located cells, the system comprising: means for measuring a first measurement in first and second co-located cells of the telecommunications system; means for determining a difference value between the measurements; and means for estimating a second measurement for the second cell, based on a second measurement being made in the first cell and the previously determined difference value.\",\n \"10.A system as claimed in claim 9, wherein the second measurement is the same type of measurement as the first measurement.\",\n \"11.A system as claimed in claim 9, wherein the second measurement is a different type of measurement to the first measurement, but having a predetermined relationship to the first measurement.\",\n \"12.A system as claimed in claim 11, wherein the second measurement is a CPICH Ec/No measurement.\",\n \"13.A system as claimed in any one of claims 9 to 12, wherein the first measurement is a received signal code power (RSCP) measurement.\",\n \"14.A system as claimed in any one of claims 9 to 13, wherein the measurements are performed at a mobile station in the telecommunications system and the difference value stored at a network side of the telecommunications system.\",\n \"15.A system as claimed in any one of claims 9 to 14, having means for evaluating which cells should belong to an active set on a frequency other than the frequency being used.\",\n \"16.A method of estimating a signal measurement substantially as hereinbefore described, with reference to, and as shown in FIG.\",\n \"1 or 2 of the accompanying drawings.\",\n \"17.A telecommunications system substantially as hereinbefore described, with reference to, and as shown in FIG.\",\n \"1 or 2 of the accompanying drawings.\",\n \"18 A communications node for a telecommunications system in which signal strength measurements are performed in two or more co-located cells, the communications node comprising: means for receiving a first measurement from first and second co-located cells of the telecommunications system; means for determining a difference value between the measurements; and means for estimating a second measurement for the second cell, based on a second measurement being made in the first cell and the previously determined difference value.\",\n \"19.A communications node as claimed in claim 18, wherein the second measurement is the same type of measurement as the first measurement.\",\n \"20.A communications node as claimed in claim 18, wherein the second measurement is a different type of measurement to the first measurement, but having a predetermined relationship to the first measurement.\",\n \"21.A communications node as claimed in claim 20, wherein the second measurement is a CPICH Ec/No measurement.\",\n \"22.A communications node as claimed in any one of claims 18 to 21, wherein the first measurement is a received signal code power (RSCP) measurement.\",\n \"23.A communications node substantially as hereinbefore described, with reference to, and as shown in FIG.\",\n \"1 or 2 of the accompanying drawings.\",\n \"24.A method of estimating a signal measurement in a communications node of a telecommunications system, the method comprising the steps of: receiving a first measurement from first and second co-located cells of the telecommunications system; determining a difference value between the measurements; and using the difference value to estimate a second measurement for the second cell, based on a second measurement being made in the first cell.\",\n \"25.A method as claimed in claim 24, wherein the second measurement is the same type of measurement as the first measurement.\",\n \"26.A method as claimed in claim 24, wherein the second measurement is a different type of measurement to the first measurement, but having a predetermined relationship to the first measurement.\",\n \"27.A method as claimed in claim 26, wherein the second measurement is a CPICH Ec/No measurement.\",\n \"28.A method as claimed in any one of claims 24 to 27, wherein the first measurement is a received signal code power (RSCP) measurement.\",\n \"29.A method of estimating a signal measurement in a communications node of a telecommunications system, the method being substantially as hereinbefore described, with reference to, and as shown in FIG.\",\n \"1 or 2 of the accompanying drawings.\",\n \"30.A mobile communications terminal for use in a telecommunications system as described in any one of claims 9 to 15.\"\n ],\n [\n \"<SOH> BACKGROUND OF THE INVENTION <EOH>In a telecommunications system, for example a wideband CDMA system (WCDMA), there is always a desire to communicate using the least amount of power.\",\n \"Therefore, all transmissions should ideally be performed at the lowest possible power level, while still maintaining an acceptable quality level.\",\n \"However, there are certain factors which act against this general desire to reduce power levels.\",\n \"Certain communication techniques have an inherent need for the power levels to be increased, both in the mobile station and the network.\",\n \"For example, data compression can be used to transmit a given amount of data in less time, resulting in gaps being created to allow inter-frequency or inter-RAT (Radio Access Technology) measurements to be carried out.\",\n \"In addition to the peak power being increased during the data compression, the average power also needs to be increased to compensate for the channel estimates that are not being updated during the time gaps, resulting in the receiver not being optimally tuned after the time gaps.\",\n \"While increased inter-frequency and inter-RAT measurements typically have the disadvantage of increasing the average power consumption when using real time services with requirements of low delays, for example speech, they also have a degrading effect in that they reduce the available channelization-codes.\",\n \"During data compression a code for a lower spreading factor needs to be used, which typically blocks several codes used for higher spreading factors in the downlink.\",\n \"The gaps in transmission mentioned above can also occur in other ways.\",\n \"For example, reducing the number of bits to be transmitted by reducing the number of extra bits which are used for error correction during some frames (i.e.\",\n \"code puncturing), or scheduling data transmission from a higher layer.\",\n \"It is known to reduce the power requirement (and in turn improve data throughput) by reducing the number of measurements carried out in the telecommunications system.\",\n \"One known example of how this may be achieved is based on the use of neighbouring cell lists, and thresholds for starting measurements.\",\n \"This type of solution suffers from the disadvantage that each neighbouring cell is treated equally as if they were adjacent neighbours using different antennas covering different, but to some extent overlapping, geographical areas.\",\n \"The reduction in these schemes is based on limiting the number of neighbouring cells included in the list, and on handover statistics and propagation predictions when cell planning is performed.\",\n \"The aim of the present invention is to provide a method of estimating measurements in a telecommunications system, thereby reducing the number of measurements being performed.\",\n \"This has the advantage of reducing power consumption and increasing the capacity of the system.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>According to a first aspect of the present invention, there is provided a method of estimating a signal measurement in a telecommunications system, the method comprising the steps of: performing a first measurement in first and second co-located cells of the telecommunications system; determining a difference value between the measurements; and using the difference value to estimate a second measurement for the second cell, based on a second measurement being made in the first cell.\",\n \"According to another aspect of the present invention, there is provided a telecommunications system in which signal strength measurements are performed in two or more co-located cells, the system comprising: means for measuring a first measurement in first and second co-located cells of the telecommunications system; means for determining a difference value between the measurements; and means for estimating a second measurement for the second cell, based on a second measurement being made in the first cell and the previously determined difference value.\",\n \"According to another aspect of the present invention, there is provided a communications node for a telecommunications system in which signal strength measurements are performed in two or more co-located cells, the communications node comprising means for receiving a first measurement from first and second co-located cells of the telecommunications system, means for determining a difference value between the measurements, and means for estimating a second measurement for the second cell, based on a second measurement being made in the first cell and the previously determined difference value.\"\n ],\n [\n \"FIELD OF THE INVENTION The present invention relates to performing measurements in a telecommunications system, and in particular, to estimating measurements in a telecommunications system, thereby reducing the number of measurements being performed.\",\n \"BACKGROUND OF THE INVENTION In a telecommunications system, for example a wideband CDMA system (WCDMA), there is always a desire to communicate using the least amount of power.\",\n \"Therefore, all transmissions should ideally be performed at the lowest possible power level, while still maintaining an acceptable quality level.\",\n \"However, there are certain factors which act against this general desire to reduce power levels.\",\n \"Certain communication techniques have an inherent need for the power levels to be increased, both in the mobile station and the network.\",\n \"For example, data compression can be used to transmit a given amount of data in less time, resulting in gaps being created to allow inter-frequency or inter-RAT (Radio Access Technology) measurements to be carried out.\",\n \"In addition to the peak power being increased during the data compression, the average power also needs to be increased to compensate for the channel estimates that are not being updated during the time gaps, resulting in the receiver not being optimally tuned after the time gaps.\",\n \"While increased inter-frequency and inter-RAT measurements typically have the disadvantage of increasing the average power consumption when using real time services with requirements of low delays, for example speech, they also have a degrading effect in that they reduce the available channelization-codes.\",\n \"During data compression a code for a lower spreading factor needs to be used, which typically blocks several codes used for higher spreading factors in the downlink.\",\n \"The gaps in transmission mentioned above can also occur in other ways.\",\n \"For example, reducing the number of bits to be transmitted by reducing the number of extra bits which are used for error correction during some frames (i.e.\",\n \"code puncturing), or scheduling data transmission from a higher layer.\",\n \"It is known to reduce the power requirement (and in turn improve data throughput) by reducing the number of measurements carried out in the telecommunications system.\",\n \"One known example of how this may be achieved is based on the use of neighbouring cell lists, and thresholds for starting measurements.\",\n \"This type of solution suffers from the disadvantage that each neighbouring cell is treated equally as if they were adjacent neighbours using different antennas covering different, but to some extent overlapping, geographical areas.\",\n \"The reduction in these schemes is based on limiting the number of neighbouring cells included in the list, and on handover statistics and propagation predictions when cell planning is performed.\",\n \"The aim of the present invention is to provide a method of estimating measurements in a telecommunications system, thereby reducing the number of measurements being performed.\",\n \"This has the advantage of reducing power consumption and increasing the capacity of the system.\",\n \"SUMMARY OF THE INVENTION According to a first aspect of the present invention, there is provided a method of estimating a signal measurement in a telecommunications system, the method comprising the steps of: performing a first measurement in first and second co-located cells of the telecommunications system; determining a difference value between the measurements; and using the difference value to estimate a second measurement for the second cell, based on a second measurement being made in the first cell.\",\n \"According to another aspect of the present invention, there is provided a telecommunications system in which signal strength measurements are performed in two or more co-located cells, the system comprising: means for measuring a first measurement in first and second co-located cells of the telecommunications system; means for determining a difference value between the measurements; and means for estimating a second measurement for the second cell, based on a second measurement being made in the first cell and the previously determined difference value.\",\n \"According to another aspect of the present invention, there is provided a communications node for a telecommunications system in which signal strength measurements are performed in two or more co-located cells, the communications node comprising means for receiving a first measurement from first and second co-located cells of the telecommunications system, means for determining a difference value between the measurements, and means for estimating a second measurement for the second cell, based on a second measurement being made in the first cell and the previously determined difference value.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS For a better understanding of the present invention, and to show more clearly how it may be carried into effect, reference will now be made, by way of example, to the accompanying drawings, in which:— FIG.\",\n \"1 shows the steps involved in a method of estimating a measurement according to a first aspect of the present invention; and, FIG.\",\n \"2 shows the steps involved in a method of estimating a measurement according to a second aspect of the present invention.\",\n \"DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT OF THE PRESENT INVENTION In a WCDMA based radio access network, several cells may be co-located using different frequencies.\",\n \"The network will have information that a particular cell, say Cell A, is co-located with one or more other cells, say Cell B.\",\n \"A mobile station positioned in a given location is able to make signal measurements on common pilot channels (CPICHs) transmitted from a serving cell and any co-located cells.\",\n \"FIG.\",\n \"1 shows the steps involved in estimating a measurement according to a first aspect of the present invention.\",\n \"In step 1, the network instructs a mobile station to perform a predetermined measurement in Cell A and in co-located Cell B, for example a received signal code power (RSCP) measurement, hereinafter referred to as RSCPA and RSCPB, respectively.\",\n \"These measurements are reported by the mobile station to the network.\",\n \"The network determines the difference between the two signals (RSCPA−B) which is stored for future reference, step 3.Once the difference value has been established, the mobile station no longer needs to perform measurements on both Cell A and Cell B.\",\n \"The network can therefore suspend inter-RAT or inter-frequency RSCP measurements.\",\n \"Instead, if a subsequent measurement is made on Cell A (RSCPA+t), step 5, the network can estimate the measurement for Cell B based on the difference signal previously determined.\",\n \"For example, if the difference signal was originally determined by subtracting RSCPB from RSCPA, then the estimated value for Cell B is determined by subtracting the difference value RSCPA−B from the new measurement for Cell A, RSCPA+t, step 7.Thus, RSCPB+t=(RSCPA+t−RSCPA−B) Since Cell A is co-located with Cell B, the path loss difference will remain constant for all locations of the mobile station, which means that the RSCP difference RSCPA−B also remains constant.\",\n \"This difference value can therefore provide an accurate estimate of a measurement for one cell, using the measurement of a co-located cell.\",\n \"Measurements in Cell B can be restarted at any time, for example if the estimated measurement approaches a critical value.\",\n \"According to another aspect of the invention, the difference value described above can be used to provide further reductions in measurements.\",\n \"In addition to RSCP measurements, a mobile station may perform any one, or any combination, of the following measurements, depending upon the processing utilised in the mobile station: 1.UTPA carrier RSSI (this stands for received signal strength indicator, and relates to signal strength plus interference on a particular frequency from any intra frequency cell) 2.CPICH RSCP (this stands for received signal code power, and relates to signal strength from a particular cell's CPICH on a particular frequency) 3.CPICH Ec/No (this is basically the signal-to-noise ratio used for representing the “cell quality” for handover evaluation).\",\n \"Each of the measurements described above imposes a different demand on the telecommunications system in terms of the processing power required, and/or the time needed to perform a given measurement.\",\n \"In other words, certain measurements are more “expensive” than others.\",\n \"For example, the CPICH Ec/No measurement requires typically more processing power that the CPICH RSCP measurement, which is turn requires more processing power than the UTRA carrier RSSI measurement.\",\n \"The measurements described above have a predetermined relationship whereby: CPICH Ec/No=(CPICH RSCP)/(UTRA carrier RSSI) Using this relationship, the estimation method according to the first aspect of the invention can be used to reduce the number of “expensive” measurements being made, for example by reducing the number of CPICH Ec/No measurements being performed in the telecommunications system.\",\n \"Referring to FIG.\",\n \"2, a difference signal is first determined, as mentioned above, by calculating the difference between the RSCPA and RSCPB measurements, (RSCPA−B), step 101.The RSSIA measurement and (Ec/No)A measurement for Cell A are also performed, step 103.Depending upon the particular implementation in a given mobile station, the (Ec/No)A measurement can either be calculated directly, or calculated from the RSCPA and RSSIA values.\",\n \"Next, when it is determined that an Ec/No measurement is subsequently required for Cell B, step 105, instead of measuring this value directly, the measurement can be estimated as follows.\",\n \"First, the RSSI value for cell B is measured, RSSIB, as shown in step 107.The Ec/No measurement can then be estimated from the RSSIB measurement, using a RSCPA measurement for cell A and the previously stored difference value RSCPA−B, as follows: (Ec/No)B=(RSCPA−RSCPA−B)/(RSSIB) Thus, the Ec/No measurement for cell B can be estimated without having to perform a relatively costly CPICH RSCP measurement for cell B.\",\n \"Instead, only the less costly UTRA RSSI measurement needs to be made for cell B.\",\n \"Also, since the RSCP and Ec/No measurements for cell A are being made in the same cell that the mobile station is currently using, these measurements are also less costly than the corresponding measurements for cell B (i.e.\",\n \"being the cell which is not currently being used by the mobile station).\",\n \"The Ec/No measurement for cell A and the estimated Ec/No measurement for cell B may be used to distribute traffic between cells, for example, to trigger handover between cell A and cell B.\",\n \"As noted above, since the RSSI measurement for cell B is less demanding than the Ec/No measurement, power consumption is thereby reduced.\",\n \"For example, assume that one 7 slot wide measurement gap is used every third frame (each frame consisting of 15 slots) for inter-frequency compressed mode measurements on CPICH Ec/No, and that the average DL power needs to be increased by 3 dB to maintain the same connection quality.\",\n \"The width of the measurement gap can be decreased to one slot used every third frame, and, if the power increase required to maintain the same quality can be assumed to be decreased by {fraction (1/7)} because of that, the following difference in power can be calculated.\",\n \"This corresponds to a 0.6 dB increase (i.e.\",\n \"1.143 times increase) instead of 3 dB increase (i.e.\",\n \"2 times increase) when making CPICH Ec/No measurements.\",\n \"Thus, if it is assumed that all the mobiles in a cell are using compressed mode at all times then, if the nominal amount of mobiles is planned to be limited to 20 per cell using 1 watt each when not using compressed mode, the estimated amount of mobiles when using compressed mode and the same DL power margins would be: 20/2=10 mobiles when the mobiles are performing inter-frequency CPICH Ec/No measurements all of the time, and 20/1.143=17.5 mobiles when using estimation according to the second aspect of the invention, whereby RSSI measurements are carried out instead.\",\n \"According to the first and second aspects of the invention, rather than having a mobile station perform measurements on each of the co-located cells, the mobile station determines a relationship between the cells, assumes that this relationship remains constant, and then performs measurements on just one cell—the other cell's measurements being estimated from the known relationship with the first cell.\",\n \"Also, if different measurements within a given cell have different power requirements, the high power measurements can be estimated by measuring the lower power measurements, and calculating the higher power measurements accordingly.\",\n \"As can be seen from the above, estimating measurements in this way has the effect of reducing the number of measurements being performed.\",\n \"This has the effect of reducing power consumption and increasing the capacity of the telecommunications system, thereby enabling a greater number of mobile stations to be supported.\",\n \"Although the preferred embodiment refers to the difference value being calculated with respect to the respective RSCP measurements from each cell, the invention is equally applicable to other signals being used in this manner, for example determining a difference signal using the BCCH frequency RXLEV in a GSM system, or measured despread signal strength on a IS95 system.\",\n \"Also, although the invention has been described as performing measurements between two co-located cells, the invention may be used with just one cell, or with any number of co-located cells.\",\n \"Furthermore, the invention can be used in the same way for evaluating cells on a frequency other than the frequency being used, to determine whether they should belong to the active set.\",\n \"For example, by using knowledge about the active set on the currently used frequency and also the knowledge that the cells on used and unused frequencies are co-located, the measurement requirements on the unused frequency will only be UTRA Carrier RSSI and information on RSCPB+t, allowing the CPICH Ec/No value to be used for evaluating the cells on the unused frequency, to determine whether they should belong to the active set.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10468046"}}},{"rowIdx":396,"cells":{"text":{"kind":"list like","value":[["Device with flexible roller shutter","This invention concerns a shutter system comprising, firstly, drive systems allowing the movement of the shutter (1) along a path between a closed and an open position, said shutter (1) being intended to close an opening (3) or other aperture and, secondly, detection systems (16) allowing determination of deflection of the shutter (1) from its path, for example due to the presence of an obstruction (13) below the shutter (1), during its movement into its closed or open position, the detection systems (16) combining with the drive systems in such a way as to permit stopping or opening of the shutter (1), each of the side edges of the shutter (1) running in a guide track (9).","This system is characterised by the fact that the detection systems (16) are fitted in such a way as to be able to combine them with the guide track (9) in order to detect whether the shutter (1) is deflected from its path in the vicinity of the guide track (9)."],["1.Shutter system consisting of, firstly, drive systems permitting movement of the shutter (1) along a path between a closed and an open position, intended to close an opening (3) or other aperture and, secondly, detection systems (16) allow determination of whether the shutter (1) is being deflected from its path, for example, by the presence of an obstruction (13) below the shutter (1) during its movement into its closed position or open position, said detection systems (16) being linked to the drive systems in such a way as to stop or open the shutter (1), each of the side edges of the shutter (1) running in a guide track (9), characterised by the fact that the detection systems (16) are fitted in a way permitting their combination with the guide track (9) in order to detect whether the shutter (1) is being deflected from its path in the vicinity of the guide track (9).","2.System in accordance with claim 1, characterised by the fact that the detection systems include at least one switch (16) fitted in such a way as to permit the shutter (1) undergoing deflection from its path to operate the switch (16) and interrupt movement of the shutter (1) towards its closed or open position.","3.System in accordance with claims 1 or 2, characterised by the fact that the part of the path alongside the opening (3) or other aperture is demarcated by a guide track (9) extending downwards from the drive systems (2).","4.System in accordance with claim 3, characterised by the fact that the aforementioned detection systems (16) are fitted in such a way as to permit deflection of the shutter (1) from its path at a point located outside the guide track (9).","5.System in accordance with claim 3, characterised by the fact that the detection systems (16) are mounted on the guide track (9) in the vicinity of that part of the path located outside the guide track (9).","6.System in accordance with claim 5, characterised by the fact that the guide track (9) is elastically mounted on a bracket (20) in a flexible way and/or one which will allow it to pivot about an axis approximately parallel to the longitudinal axis of the guide track (9) so that if a force is applied to the plane of the shutter (1) when it is not fully open, the detection systems (16) will move with the guide track (9) to avoid being operated by the movement of the shutter (1) due to said force.","7.System in accordance with claim 3, characterised by the fact that the guide tracks (9) have a recess (23) which permits the shutter (1) to be deflected across said recess (23) when normal opening or closure of the shutter (1) is disturbed, e.g.","due to the presence of an obstruction (13) below the shutter (1), the aforementioned detection systems (16) being fitted in a way to allow such deflection to be detected.","8.System in accordance with claim 7, characterised by the fact that the guide tracks (9) essentially take the form of U-shaped channel, the aforementioned recess (23) being provided in one of the walls (25) of the U.","9.System in accordance with claim 3, characterised by the fact that the guide tracks (9) are flexibly mounted on a bracket (20) so that if the shutter (1) is deflected from its path when running in the guide tracks (9), the latter are deflected from the bracket (20), the detection systems (16) being fitted in such a way as to permit detection of such movement.","10.System in accordance with claim 9, characterised by the fact that the guide tracks (9) linked to a spring (8) apply a force to the guide tracks (9) towards the bracket (20) in order to flatten the shutter (1).","11.System in accordance with claim 1 or claim 10, characterised in that the aforementioned drive systems include a drum (2) which can be rotated about its centre line (12), the aforementioned detection systems (16) being mounted to the side of the drum (2) and at a certain distance from it, so that if the shutter (1) undergoes specific deflection from its path at the level of the drum (2), the detection systems (16) combine with the drive systems by stopping movement of the shutter (1) into its closed position.","12.System in accordance with claim 11, characterised by the fact that the detection systems include at least one switch (16) mounted to the side of the drum (2) at a certain distance from it and fitted in such a way as to permit the shutter (1) undergoing deflection from its path at the level of the drum (2) to operate the switch (16) and interrupt the movement of the shutter (1) towards its closed position.","13.System in accordance with claim 1, characterised by the fact that a flexible stiffened area (17) is provided on the shutter (1), extending in the direction of movement of the shutter (1).","14.System in accordance with claim 1, characterised by the fact that a flexible stiffened area (17) extends into the vicinity of both the side edges (7) of the shutter (1).","15.System in accordance with one of either claims 13 or 14, characterised by the fact that the stiffened area combines with the detection systems in order to detect deflection of the shutter (1) from its path.","16.Flexible shutter for use in a closure system and intended for the closure of an opening (3) or other aperture in which the shutter (1) is unwound from a drum (2) along a path during closure of the opening (3) or other aperture by moving in guide tracks (9) which are arranged along the opening (3) or other aperture, characterised by the fact that a flexible stiffened area (17) extends into the vicinity of at least one side edge (7) of the shutter (1) in order to be able to bend if the shutter (1) is deflected from its path and to be able to combine with detection systems (16) detecting the curvature (26) of the stiffened area (17)."],["This invention concerns a flexible shutter system combined with drive systems in such a way as to permit the shutter to move along a specific path between a closed and an open position.","The said shutter is intended to close an opening or other aperture.","One of the essential objectives of this invention is the provision of a system of the aforementioned type which provides maximum safety when the shutter is moving towards its closed position and its path is obstructed, or if movement of the shutter is prevented for another reason during closure or opening.","For this purpose, inventive detection systems are provided which allow deflection of the shutter from its path to be determined, e.g.","due to the presence of an obstruction below the shutter when it moves towards its closed or open position.","The detection systems combine with the drive systems to stop or open the shutter.","In an advantageous embodiment of the inventive system, the aforementioned drive systems include a drum which may be rotated about its centre line, the aforementioned detection systems being fitted at the side of the drum at a certain distance from it so that if the shutter undergoes specific deflection from its path at the level of the drum, the detection systems will act upon the drive systems, stopping movement of the shutter towards its closed position.","In a particularly advantageous embodiment of the inventive system, a stiffened area is provided on the shutter, extending in the direction of movement of the latter.","Other details and particular features of the invention emerge from the description provided below, in non-exhaustive examples of several embodiments with references to the drawings appended hereto.","FIG.","1 is a diagrammatic vertical section through a first embodiment of the inventive system with the shutter in its closed position.","FIG.","2 is a view analogous to that in FIG.","1, with the shutter in its open position.","FIG.","3 is a view analogous to that in FIGS.","1 and 2 while the shutter is moving into its closed position and an obstruction is present in its path.","FIG.","4 is a partial lateral section through a shutter of which the side edges are held in guide tracks, on a larger scale.","FIG.","5 is a view, analogous to that in FIG.","4, of a second embodiment of the invention in which a switch is mounted on the guide track.","FIG.","6 is a front elevation of part of a shutter in accordance with the second embodiment of the invention, as shown in FIG.","5.FIG.","7 is a view analogous to that in FIG.","5, of the second embodiment of the invention in which a transverse force is exercised on the plane of the shutter.","FIG.","8 is a diagrammatic vertical section through the second embodiment of the inventive system while the shutter is moving into its closed position and an obstruction is present in its path.","FIG.","9 is a diagrammatic vertical section through a third embodiment of the inventive system with the shutter in its closed position.","FIG.","10 is a horizontal section through the guide track along the line X-X in FIG.","9.FIG.","11 is a horizontal section through the guide track along the line XI-XI in FIG.","9.FIG.","12 is a view analogous to that in FIG.","9 while the shutter is moving into its closed position and an obstruction is present in its path.","FIG.","13 is a diagrammatic horizontal section through part of a guide track in a fourth embodiment of the inventive system.","FIG.","14 is a horizontal section through the guide track along the line XIV-XIV in FIG.","13.FIG.","15 is a view analogous to that in FIG.","13 when the shutter is deflected from its normal path.","FIG.","16 is a horizontal section through the guide track along line XVI-XVI in FIG.","15.The same reference numbers refer to the same or analogous components in the various drawings.","In general, this invention concerns a shutter system 1 combined with drive systems such as a drum 2 of which the centre line is connected to the shaft of a motor (not shown).","The shutter 1, which can move between a closed position and an open position, is intended to close an opening 3 in a wall 4 or any aperture.","In FIG.","1, the system is shown in its closed position with the lower edge 5 of the shutter 1 against the floor 6.For the purposes of this invention, the word “shutter” should be understood as any component at least partially pliant, flexible, rigid or semi-rigid, such as a tarpaulin, a strip of plastic material, an assembly of articulated lamellae, a grille, etc.","More specifically, it involves a shutter which may be folded or wound around a spindle at right angles to its direction of movement between its open and closed positions.","However, it should be noted that pronounced preference is given to pliant shutters formed by a tarpaulin.","The drawings thus concern a shutter made up of a tarpaulin 1 of which the side edges 7 have, for example, a bead or a series of blocks 8, as shown in FIG.","6.The shutter 1 is guided by its side edges 7 in guide tracks 9 extending approximately vertically on either side of the opening 3.When the shutter 1 opens, as shown in FIG.","2, its side edges 7 are moved along the guide tracks 9 in the direction indicated by the arrow 10 as a consequence of the shutter 1 being wound on the drum 2, which rotates around its centre line 12 in the direction indicated by the arrow 11.The guide tracks 9 are fixed to the wall 4 by systems not shown.","The shutter 1 is moved along a specific path during normal opening and closure.","In a standard procedure, during normal operation of the shutter system, within the meaning of this description, this path corresponds to that followed by the shutter 1 during its movement between its closed position, as shown in FIG.","1, and its open position, as shown in FIG.","2.Should any interference with normal operation of the shutter take place during movement of the latter in the direction indicated by the arrow 14 into its closed position, e.g.","by an obstruction 13 located below the shutter 1 which consequently prevents movement of said shutter 1, part of the shutter 1 will be deflected from its normal path, by forming a bulge or gatherings located outside its path.","This bulge makes contact with detection systems, such as a switch 16, which acts upon the drive systems of the shutter 1 in such a way as to interrupt its descent.","In the embodiment shown in FIGS.","1 to 8, the guide tracks 9 extend along part of the path of the shutter 1 to a certain distance from the drive systems so that the shutter 1 is not guided over this distance.","A gap 22 is thus formed between the upper end of the guide tracks 9 and the drive systems in which the shutter 1 is not guided laterally and into which the shutter 1 may be deflected if its movement is retarded, e.g.","by the presence of an obstruction below the shutter 1.As shown in FIG.","3, if an obstruction 13, such as a fork-lift truck, is fouled by the lower edge 5 of the shutter 1, the latter will form a bulge 15 above the guide tracks 9.This is caused by the fact that the drive systems will not stop immediately when the lower edge 5 of the shutter 1 encounters the obstruction 13.In particular, the drum 2 will continue to unroll the shutter 1 of which the movement of the lower edge 5 is prevented by the presence of the obstruction 13 in the path of the shutter 1.Inventive detection systems are provided which allow determination of whether the shutter 1 is being deflected from its normal path.","It is advantageous for these detection systems to be fitted so that deflection of the shutter 1 from its path can be detected outside the guide track 9.As shown in FIGS.","1 to 3, the detection systems include a switch 16 mounted to the side of the drum 2 at a certain distance from the latter and fitted against the wall 4 so that if the shutter 1 undergoes specific deflection from its path in the gap 22 at the level of the drum 2 due to the presence of an obstruction 13 below the shutter 1, the latter will encounter and operate the switch 16 by forming a bulge 15.The switch 16 combines with the drive systems to cause movement of the shutter 1 into its closed position to be interrupted.","In order to remedy the interference caused by the obstruction 13 immediately, shutter 1 is preferably returned to its open position automatically by the drive systems .","In some cases, use of a highly pliant shutter 1 to obtain a very light shutter system which can open and close rapidly is indispensable.","In this case, instead of forming a bulge 15, there is a risk that the shutters will gather if the lower edge 5 fouls an obstruction 13 when moving into its closed position.","In order to ensure that the switch 16 will be operated by the shutter 1 when the latter is deflected from its path, it is advantageous to provide a flexible stiffened area 17 on the shutter 1, as shown in diagrammatic form in FIG.","4.Said stiffened area 17 preferably extends into the vicinity of each of the side edges 7 of the shutter 1.The stiffened area 17 may consist of a continuous plastic or metallic strip affixed to the shutter 1.In a specific embodiment of the invention, the stiffened area 17 is formed by a series of rigid elements which are mounted alongside each other on one surface of the shutter 1.Said rigid elements are mounted on the surface of the shutter 1 facing the switch 16.Such a series of rigid elements ensures that the shutter will not gather and render it flexible in only one direction, so that a bulge 15 of a specific shape will be formed which will encounter the switch 16 if there is an obstruction below the shutter 1.A different embodiment of the inventive shutter system is shown in FIGS.","5 to 8.In this embodiment, the detection systems include a switch 16 which is mounted on the guide track 9.The switch 16 is mounted in the vicinity of the path of the shutter I which is located outside the guide track 9, i.e.","in the aforementioned gap 22 located between the drum 2 and the free upper end of the guide tracks 9, more specifically in the vicinity of said free end.","In this way, the switch 16 can detect whether the shutter 1 is being deflected from the section of the path located above the guide track 9.The fact that the detection systems are mounted on the guide track 9 prevents the shutter 1 from encountering the switch 16 if a force is exercised on shutter 1 in a direction approximately transverse to the plane of the latter, e.g.","by wind.","In FIG.","7, the arrows 19 represent the forces exercised on the shutter 1 by wind.","As illustrated, the shutter 1 is bent in the direction indicated by the arrow 19 by these forces and the guide track 9 is pivoted about its longitudinal axis from the position shown in FIG.","5.Due to the fact that the switch 16 is mounted on the guide track 9, it combines with the guide track 9, moving with it.","However, the distance between the shutter 1 and the switch 16 remains approximately constant.","In this embodiment of the inventive shutter system, the guide track 9 is mounted on a bracket 20 which is fixed to the wall 4 in a flexible way and/or one which will allow it to pivot about an axis approximately parallel to the longitudinal axis of said guide track 9.Such a method of mounting guide tracks 9 has already been described in international patent application no.","PCT/BE92/00017.The switch 16 has an arm 21 which extends above the guide track 9, as clearly illustrated in FIGS.","6 and 8.In this way, if a bulge 15 forms above the guide track 9 due to the presence of an obstruction 13 below the shutter 1, it will encounter the arm 21 which is linked to the switch 16.Consequently, movement of the shutter 1 will be stopped and it will preferably be immediately wound on to the drum 2.A third embodiment of the inventive shutter system is shown in FIGS.","9 to 12.In this embodiment, the guide tracks 9 form an essentially U-shaped channel, as shown in FIG.","10.The shutter 1 is thus guided in the guide tracks 9 by a block 24 which is located in the channel and which is provided on the side edges of the shutter 1.In order to permit detection of whether an obstruction is located below the shutter 1 during closure of the latter, each guide track 9 contains a recess 23 over part of its length.","Said recess 23 is, in particular, provided in one of the walls 25 of the guide track 9, as shown in FIGS.","9 and 11.If closure of the shutter 1 is retarded by the presence of an obstruction, as shown in FIG.","12, shutter 1 may be deflected from its normal path by forming a bulge 26 extending across the recess 23.A switch 16 is fitted opposite the recess 23 in order to detect such deflection of the shutter 1.In this way, the shutter 1, which has been deflected from its path, encounters the switch 16 which combines with the drive systems, thus causing the shutter 1 to stop or open.","In a variation of this inventive embodiment of the system, the guide track 9 has a cover at the height of the recess 23, such as a plate, which will become detached or move easily when the lower edge 5 of the shutter 1 encounters an obstruction and a bulge 26 is formed across said recess 23.The cover then encounters the switch 16 and sends a signal to the drive systems.","FIGS.","13 to 16 show a fourth embodiment of the inventive system, in which the guide tracks 9 are elastically mounted on a bracket 20 which is affixed to the wall.","In this way, the guide tracks 9 can move relative to the bracket 20 if the shutter 1 gathers or if a force is applied to the plane of the shutter.","The shutter 1 gathers, for example, if it is moving along the guide tracks 9 into its closed position and the lower edge 5 of the shutter 1 fouls an obstruction.","Due to the gathering of the shutter 1, its side edges 7 move towards each other and the shutter 1 is deflected from its normal path, moving the guide tracks 9 relative to the bracket 20 in the direction indicated by the arrow 27 in FIG.","16.Such movement of the guide tracks 9 relative to the path is detected by the detection systems, particularly by a switch 16 mounted on the bracket 20 and thus linked to the guide tracks 9.The guide tracks 9 are linked to a spring 28 forcing the former towards the bracket 20, to ensure that the shutter 1 is flat.","It is self-evident that the invention is not restricted to the various embodiments described above and that other variations may be envisaged without departing from the scope of this invention, particularly in respect of the position of the switch.","It is possible to install several switches, for example on the two side edges of the shutter 1.In other respects, the detection systems may include any means which allow the presence of a bulge to be detected, such as, for example, optical detection systems, particularly an electronic eye.","In addition, the stiffened area may be affixed to the shutter continuously, or intermittently by discrete fixings.","For example, the stiffened area may consist of a reinforced side edge of the shutter 1."]],"string":"[\n [\n \"Device with flexible roller shutter\",\n \"This invention concerns a shutter system comprising, firstly, drive systems allowing the movement of the shutter (1) along a path between a closed and an open position, said shutter (1) being intended to close an opening (3) or other aperture and, secondly, detection systems (16) allowing determination of deflection of the shutter (1) from its path, for example due to the presence of an obstruction (13) below the shutter (1), during its movement into its closed or open position, the detection systems (16) combining with the drive systems in such a way as to permit stopping or opening of the shutter (1), each of the side edges of the shutter (1) running in a guide track (9).\",\n \"This system is characterised by the fact that the detection systems (16) are fitted in such a way as to be able to combine them with the guide track (9) in order to detect whether the shutter (1) is deflected from its path in the vicinity of the guide track (9).\"\n ],\n [\n \"1.Shutter system consisting of, firstly, drive systems permitting movement of the shutter (1) along a path between a closed and an open position, intended to close an opening (3) or other aperture and, secondly, detection systems (16) allow determination of whether the shutter (1) is being deflected from its path, for example, by the presence of an obstruction (13) below the shutter (1) during its movement into its closed position or open position, said detection systems (16) being linked to the drive systems in such a way as to stop or open the shutter (1), each of the side edges of the shutter (1) running in a guide track (9), characterised by the fact that the detection systems (16) are fitted in a way permitting their combination with the guide track (9) in order to detect whether the shutter (1) is being deflected from its path in the vicinity of the guide track (9).\",\n \"2.System in accordance with claim 1, characterised by the fact that the detection systems include at least one switch (16) fitted in such a way as to permit the shutter (1) undergoing deflection from its path to operate the switch (16) and interrupt movement of the shutter (1) towards its closed or open position.\",\n \"3.System in accordance with claims 1 or 2, characterised by the fact that the part of the path alongside the opening (3) or other aperture is demarcated by a guide track (9) extending downwards from the drive systems (2).\",\n \"4.System in accordance with claim 3, characterised by the fact that the aforementioned detection systems (16) are fitted in such a way as to permit deflection of the shutter (1) from its path at a point located outside the guide track (9).\",\n \"5.System in accordance with claim 3, characterised by the fact that the detection systems (16) are mounted on the guide track (9) in the vicinity of that part of the path located outside the guide track (9).\",\n \"6.System in accordance with claim 5, characterised by the fact that the guide track (9) is elastically mounted on a bracket (20) in a flexible way and/or one which will allow it to pivot about an axis approximately parallel to the longitudinal axis of the guide track (9) so that if a force is applied to the plane of the shutter (1) when it is not fully open, the detection systems (16) will move with the guide track (9) to avoid being operated by the movement of the shutter (1) due to said force.\",\n \"7.System in accordance with claim 3, characterised by the fact that the guide tracks (9) have a recess (23) which permits the shutter (1) to be deflected across said recess (23) when normal opening or closure of the shutter (1) is disturbed, e.g.\",\n \"due to the presence of an obstruction (13) below the shutter (1), the aforementioned detection systems (16) being fitted in a way to allow such deflection to be detected.\",\n \"8.System in accordance with claim 7, characterised by the fact that the guide tracks (9) essentially take the form of U-shaped channel, the aforementioned recess (23) being provided in one of the walls (25) of the U.\",\n \"9.System in accordance with claim 3, characterised by the fact that the guide tracks (9) are flexibly mounted on a bracket (20) so that if the shutter (1) is deflected from its path when running in the guide tracks (9), the latter are deflected from the bracket (20), the detection systems (16) being fitted in such a way as to permit detection of such movement.\",\n \"10.System in accordance with claim 9, characterised by the fact that the guide tracks (9) linked to a spring (8) apply a force to the guide tracks (9) towards the bracket (20) in order to flatten the shutter (1).\",\n \"11.System in accordance with claim 1 or claim 10, characterised in that the aforementioned drive systems include a drum (2) which can be rotated about its centre line (12), the aforementioned detection systems (16) being mounted to the side of the drum (2) and at a certain distance from it, so that if the shutter (1) undergoes specific deflection from its path at the level of the drum (2), the detection systems (16) combine with the drive systems by stopping movement of the shutter (1) into its closed position.\",\n \"12.System in accordance with claim 11, characterised by the fact that the detection systems include at least one switch (16) mounted to the side of the drum (2) at a certain distance from it and fitted in such a way as to permit the shutter (1) undergoing deflection from its path at the level of the drum (2) to operate the switch (16) and interrupt the movement of the shutter (1) towards its closed position.\",\n \"13.System in accordance with claim 1, characterised by the fact that a flexible stiffened area (17) is provided on the shutter (1), extending in the direction of movement of the shutter (1).\",\n \"14.System in accordance with claim 1, characterised by the fact that a flexible stiffened area (17) extends into the vicinity of both the side edges (7) of the shutter (1).\",\n \"15.System in accordance with one of either claims 13 or 14, characterised by the fact that the stiffened area combines with the detection systems in order to detect deflection of the shutter (1) from its path.\",\n \"16.Flexible shutter for use in a closure system and intended for the closure of an opening (3) or other aperture in which the shutter (1) is unwound from a drum (2) along a path during closure of the opening (3) or other aperture by moving in guide tracks (9) which are arranged along the opening (3) or other aperture, characterised by the fact that a flexible stiffened area (17) extends into the vicinity of at least one side edge (7) of the shutter (1) in order to be able to bend if the shutter (1) is deflected from its path and to be able to combine with detection systems (16) detecting the curvature (26) of the stiffened area (17).\"\n ],\n [\n \"This invention concerns a flexible shutter system combined with drive systems in such a way as to permit the shutter to move along a specific path between a closed and an open position.\",\n \"The said shutter is intended to close an opening or other aperture.\",\n \"One of the essential objectives of this invention is the provision of a system of the aforementioned type which provides maximum safety when the shutter is moving towards its closed position and its path is obstructed, or if movement of the shutter is prevented for another reason during closure or opening.\",\n \"For this purpose, inventive detection systems are provided which allow deflection of the shutter from its path to be determined, e.g.\",\n \"due to the presence of an obstruction below the shutter when it moves towards its closed or open position.\",\n \"The detection systems combine with the drive systems to stop or open the shutter.\",\n \"In an advantageous embodiment of the inventive system, the aforementioned drive systems include a drum which may be rotated about its centre line, the aforementioned detection systems being fitted at the side of the drum at a certain distance from it so that if the shutter undergoes specific deflection from its path at the level of the drum, the detection systems will act upon the drive systems, stopping movement of the shutter towards its closed position.\",\n \"In a particularly advantageous embodiment of the inventive system, a stiffened area is provided on the shutter, extending in the direction of movement of the latter.\",\n \"Other details and particular features of the invention emerge from the description provided below, in non-exhaustive examples of several embodiments with references to the drawings appended hereto.\",\n \"FIG.\",\n \"1 is a diagrammatic vertical section through a first embodiment of the inventive system with the shutter in its closed position.\",\n \"FIG.\",\n \"2 is a view analogous to that in FIG.\",\n \"1, with the shutter in its open position.\",\n \"FIG.\",\n \"3 is a view analogous to that in FIGS.\",\n \"1 and 2 while the shutter is moving into its closed position and an obstruction is present in its path.\",\n \"FIG.\",\n \"4 is a partial lateral section through a shutter of which the side edges are held in guide tracks, on a larger scale.\",\n \"FIG.\",\n \"5 is a view, analogous to that in FIG.\",\n \"4, of a second embodiment of the invention in which a switch is mounted on the guide track.\",\n \"FIG.\",\n \"6 is a front elevation of part of a shutter in accordance with the second embodiment of the invention, as shown in FIG.\",\n \"5.FIG.\",\n \"7 is a view analogous to that in FIG.\",\n \"5, of the second embodiment of the invention in which a transverse force is exercised on the plane of the shutter.\",\n \"FIG.\",\n \"8 is a diagrammatic vertical section through the second embodiment of the inventive system while the shutter is moving into its closed position and an obstruction is present in its path.\",\n \"FIG.\",\n \"9 is a diagrammatic vertical section through a third embodiment of the inventive system with the shutter in its closed position.\",\n \"FIG.\",\n \"10 is a horizontal section through the guide track along the line X-X in FIG.\",\n \"9.FIG.\",\n \"11 is a horizontal section through the guide track along the line XI-XI in FIG.\",\n \"9.FIG.\",\n \"12 is a view analogous to that in FIG.\",\n \"9 while the shutter is moving into its closed position and an obstruction is present in its path.\",\n \"FIG.\",\n \"13 is a diagrammatic horizontal section through part of a guide track in a fourth embodiment of the inventive system.\",\n \"FIG.\",\n \"14 is a horizontal section through the guide track along the line XIV-XIV in FIG.\",\n \"13.FIG.\",\n \"15 is a view analogous to that in FIG.\",\n \"13 when the shutter is deflected from its normal path.\",\n \"FIG.\",\n \"16 is a horizontal section through the guide track along line XVI-XVI in FIG.\",\n \"15.The same reference numbers refer to the same or analogous components in the various drawings.\",\n \"In general, this invention concerns a shutter system 1 combined with drive systems such as a drum 2 of which the centre line is connected to the shaft of a motor (not shown).\",\n \"The shutter 1, which can move between a closed position and an open position, is intended to close an opening 3 in a wall 4 or any aperture.\",\n \"In FIG.\",\n \"1, the system is shown in its closed position with the lower edge 5 of the shutter 1 against the floor 6.For the purposes of this invention, the word “shutter” should be understood as any component at least partially pliant, flexible, rigid or semi-rigid, such as a tarpaulin, a strip of plastic material, an assembly of articulated lamellae, a grille, etc.\",\n \"More specifically, it involves a shutter which may be folded or wound around a spindle at right angles to its direction of movement between its open and closed positions.\",\n \"However, it should be noted that pronounced preference is given to pliant shutters formed by a tarpaulin.\",\n \"The drawings thus concern a shutter made up of a tarpaulin 1 of which the side edges 7 have, for example, a bead or a series of blocks 8, as shown in FIG.\",\n \"6.The shutter 1 is guided by its side edges 7 in guide tracks 9 extending approximately vertically on either side of the opening 3.When the shutter 1 opens, as shown in FIG.\",\n \"2, its side edges 7 are moved along the guide tracks 9 in the direction indicated by the arrow 10 as a consequence of the shutter 1 being wound on the drum 2, which rotates around its centre line 12 in the direction indicated by the arrow 11.The guide tracks 9 are fixed to the wall 4 by systems not shown.\",\n \"The shutter 1 is moved along a specific path during normal opening and closure.\",\n \"In a standard procedure, during normal operation of the shutter system, within the meaning of this description, this path corresponds to that followed by the shutter 1 during its movement between its closed position, as shown in FIG.\",\n \"1, and its open position, as shown in FIG.\",\n \"2.Should any interference with normal operation of the shutter take place during movement of the latter in the direction indicated by the arrow 14 into its closed position, e.g.\",\n \"by an obstruction 13 located below the shutter 1 which consequently prevents movement of said shutter 1, part of the shutter 1 will be deflected from its normal path, by forming a bulge or gatherings located outside its path.\",\n \"This bulge makes contact with detection systems, such as a switch 16, which acts upon the drive systems of the shutter 1 in such a way as to interrupt its descent.\",\n \"In the embodiment shown in FIGS.\",\n \"1 to 8, the guide tracks 9 extend along part of the path of the shutter 1 to a certain distance from the drive systems so that the shutter 1 is not guided over this distance.\",\n \"A gap 22 is thus formed between the upper end of the guide tracks 9 and the drive systems in which the shutter 1 is not guided laterally and into which the shutter 1 may be deflected if its movement is retarded, e.g.\",\n \"by the presence of an obstruction below the shutter 1.As shown in FIG.\",\n \"3, if an obstruction 13, such as a fork-lift truck, is fouled by the lower edge 5 of the shutter 1, the latter will form a bulge 15 above the guide tracks 9.This is caused by the fact that the drive systems will not stop immediately when the lower edge 5 of the shutter 1 encounters the obstruction 13.In particular, the drum 2 will continue to unroll the shutter 1 of which the movement of the lower edge 5 is prevented by the presence of the obstruction 13 in the path of the shutter 1.Inventive detection systems are provided which allow determination of whether the shutter 1 is being deflected from its normal path.\",\n \"It is advantageous for these detection systems to be fitted so that deflection of the shutter 1 from its path can be detected outside the guide track 9.As shown in FIGS.\",\n \"1 to 3, the detection systems include a switch 16 mounted to the side of the drum 2 at a certain distance from the latter and fitted against the wall 4 so that if the shutter 1 undergoes specific deflection from its path in the gap 22 at the level of the drum 2 due to the presence of an obstruction 13 below the shutter 1, the latter will encounter and operate the switch 16 by forming a bulge 15.The switch 16 combines with the drive systems to cause movement of the shutter 1 into its closed position to be interrupted.\",\n \"In order to remedy the interference caused by the obstruction 13 immediately, shutter 1 is preferably returned to its open position automatically by the drive systems .\",\n \"In some cases, use of a highly pliant shutter 1 to obtain a very light shutter system which can open and close rapidly is indispensable.\",\n \"In this case, instead of forming a bulge 15, there is a risk that the shutters will gather if the lower edge 5 fouls an obstruction 13 when moving into its closed position.\",\n \"In order to ensure that the switch 16 will be operated by the shutter 1 when the latter is deflected from its path, it is advantageous to provide a flexible stiffened area 17 on the shutter 1, as shown in diagrammatic form in FIG.\",\n \"4.Said stiffened area 17 preferably extends into the vicinity of each of the side edges 7 of the shutter 1.The stiffened area 17 may consist of a continuous plastic or metallic strip affixed to the shutter 1.In a specific embodiment of the invention, the stiffened area 17 is formed by a series of rigid elements which are mounted alongside each other on one surface of the shutter 1.Said rigid elements are mounted on the surface of the shutter 1 facing the switch 16.Such a series of rigid elements ensures that the shutter will not gather and render it flexible in only one direction, so that a bulge 15 of a specific shape will be formed which will encounter the switch 16 if there is an obstruction below the shutter 1.A different embodiment of the inventive shutter system is shown in FIGS.\",\n \"5 to 8.In this embodiment, the detection systems include a switch 16 which is mounted on the guide track 9.The switch 16 is mounted in the vicinity of the path of the shutter I which is located outside the guide track 9, i.e.\",\n \"in the aforementioned gap 22 located between the drum 2 and the free upper end of the guide tracks 9, more specifically in the vicinity of said free end.\",\n \"In this way, the switch 16 can detect whether the shutter 1 is being deflected from the section of the path located above the guide track 9.The fact that the detection systems are mounted on the guide track 9 prevents the shutter 1 from encountering the switch 16 if a force is exercised on shutter 1 in a direction approximately transverse to the plane of the latter, e.g.\",\n \"by wind.\",\n \"In FIG.\",\n \"7, the arrows 19 represent the forces exercised on the shutter 1 by wind.\",\n \"As illustrated, the shutter 1 is bent in the direction indicated by the arrow 19 by these forces and the guide track 9 is pivoted about its longitudinal axis from the position shown in FIG.\",\n \"5.Due to the fact that the switch 16 is mounted on the guide track 9, it combines with the guide track 9, moving with it.\",\n \"However, the distance between the shutter 1 and the switch 16 remains approximately constant.\",\n \"In this embodiment of the inventive shutter system, the guide track 9 is mounted on a bracket 20 which is fixed to the wall 4 in a flexible way and/or one which will allow it to pivot about an axis approximately parallel to the longitudinal axis of said guide track 9.Such a method of mounting guide tracks 9 has already been described in international patent application no.\",\n \"PCT/BE92/00017.The switch 16 has an arm 21 which extends above the guide track 9, as clearly illustrated in FIGS.\",\n \"6 and 8.In this way, if a bulge 15 forms above the guide track 9 due to the presence of an obstruction 13 below the shutter 1, it will encounter the arm 21 which is linked to the switch 16.Consequently, movement of the shutter 1 will be stopped and it will preferably be immediately wound on to the drum 2.A third embodiment of the inventive shutter system is shown in FIGS.\",\n \"9 to 12.In this embodiment, the guide tracks 9 form an essentially U-shaped channel, as shown in FIG.\",\n \"10.The shutter 1 is thus guided in the guide tracks 9 by a block 24 which is located in the channel and which is provided on the side edges of the shutter 1.In order to permit detection of whether an obstruction is located below the shutter 1 during closure of the latter, each guide track 9 contains a recess 23 over part of its length.\",\n \"Said recess 23 is, in particular, provided in one of the walls 25 of the guide track 9, as shown in FIGS.\",\n \"9 and 11.If closure of the shutter 1 is retarded by the presence of an obstruction, as shown in FIG.\",\n \"12, shutter 1 may be deflected from its normal path by forming a bulge 26 extending across the recess 23.A switch 16 is fitted opposite the recess 23 in order to detect such deflection of the shutter 1.In this way, the shutter 1, which has been deflected from its path, encounters the switch 16 which combines with the drive systems, thus causing the shutter 1 to stop or open.\",\n \"In a variation of this inventive embodiment of the system, the guide track 9 has a cover at the height of the recess 23, such as a plate, which will become detached or move easily when the lower edge 5 of the shutter 1 encounters an obstruction and a bulge 26 is formed across said recess 23.The cover then encounters the switch 16 and sends a signal to the drive systems.\",\n \"FIGS.\",\n \"13 to 16 show a fourth embodiment of the inventive system, in which the guide tracks 9 are elastically mounted on a bracket 20 which is affixed to the wall.\",\n \"In this way, the guide tracks 9 can move relative to the bracket 20 if the shutter 1 gathers or if a force is applied to the plane of the shutter.\",\n \"The shutter 1 gathers, for example, if it is moving along the guide tracks 9 into its closed position and the lower edge 5 of the shutter 1 fouls an obstruction.\",\n \"Due to the gathering of the shutter 1, its side edges 7 move towards each other and the shutter 1 is deflected from its normal path, moving the guide tracks 9 relative to the bracket 20 in the direction indicated by the arrow 27 in FIG.\",\n \"16.Such movement of the guide tracks 9 relative to the path is detected by the detection systems, particularly by a switch 16 mounted on the bracket 20 and thus linked to the guide tracks 9.The guide tracks 9 are linked to a spring 28 forcing the former towards the bracket 20, to ensure that the shutter 1 is flat.\",\n \"It is self-evident that the invention is not restricted to the various embodiments described above and that other variations may be envisaged without departing from the scope of this invention, particularly in respect of the position of the switch.\",\n \"It is possible to install several switches, for example on the two side edges of the shutter 1.In other respects, the detection systems may include any means which allow the presence of a bulge to be detected, such as, for example, optical detection systems, particularly an electronic eye.\",\n \"In addition, the stiffened area may be affixed to the shutter continuously, or intermittently by discrete fixings.\",\n \"For example, the stiffened area may consist of a reinforced side edge of the shutter 1.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10468129"}}},{"rowIdx":397,"cells":{"text":{"kind":"list like","value":[["Plasma surface graft process for reducing thrombogenicity","In accordance with the present invention, there is provided a novel process for modifying the surface properties of a material that is suitable for contact with animal tissue so as to enhance its hemocompatibility and make it less thrombogenic when in use.","This process comprises: Exposing the surface of the material to plasma treatment conditions in order to create reactive groups on said surface; activating a molecule with an activator to produce a reactive molecular species capable of forming convalent bonds with the reactive groups created on the surface of the material to form convalent bonds.","The invention further encompasses the materials produced by this process as well as devices, such as vascular prosthesis, that are comprised of these process-modified materials."],["1.A process for modifying the surface properties of a material suitable for contact with a living tissue comprising: Exposing the surface of the material to plasma treatment conditions in order to create reactive groups on said surface; Activating a molecule with an activator to produce a reactive molecular species capable of forming strong bonds with the reactive groups created on the surface of the material; and Contacting the reactive molecular species with the reactive groups created on the surface of the material to form strong bonds.","2.A process as defined in claim 1, wherein said strong bonds are covalent bonds.","3.A process as defined in claim 1 or 2, wherein the resulting surface of the material is compatible with living tissue.","4.A process as defined in claim 3, wherein said tissue is blood tissue.","5.A process as defined in claim 1, wherein: said plasma is ammonia Radio Frequency (RF) plasma and said reactive groups are amine groups; said molecule is selected from the group consisting of choline, heparin, and other molecules known to those skilled in the art for their hemocompatibility, said activator is phosphoryl chloride (POCl3) and said reactive molecular species is the oxyphosphorodichlorinated derivative of said molecule; and said strong bonds are phosphoamide-type covalent bonds.","6.A process as defined in claim 5, wherein said plasma treatment conditions consist of the application of a RF power of between about 5 watts to about 500 watts for a time of about 10 seconds to about 30 minutes at a pressure of about 50 mtorr to about 5 torr.","7.A process as defined in claim 6, wherein said plasma treatment conditions consist of the application of a RF power of about 20 watts for a time of about 250 seconds at a pressure of about 300 mtorr.","8.A process as defined in claim 6, wherein said plasma treatment conditions consist of the application of a RF power of about 15 watts for a time of about 100 seconds at a pressure of about 250 mtorr.","9.A process as defined in claim 7 or 8, wherein the third step is performed within about 2 hours of the first step.","10.A process as defined in any one of claims 1 to 9, wherein said material suitable for contact with a living tissue is material implantable in an animal's body.","11.A process as defined in claim 10, wherein said material implantable in an animal's body is selected from the group consisting of: Microporous expanded polytetrafluoroethylene (ePTFE), polytetrafluoroethylene (PTFE), polyvinylchloride (PVC), polyethylenes, polyesters (such as polyethylene terephtalate—PET), polypropylenes, polyurethanes, polycarbonates, silicones, PVDF and polymer-coated materials, such as metals and ceramics for example.","12.A process as defined in claim 11, wherein said material implantable in an animal's body is ePTFE.","13.A process as defined in claim 12, wherein said ePTFE is that of the internal surface of a vascular prosthesis.","14.A process as defined in claim 13, wherein said vascular prosthesis has an inner diameter of about 1 to 30 mm.","15.A material implantable in an animal's body produced by the process of any one of claims 1 to 14.16.A vascular prosthesis produced by the process of claim 13 or 14.17.A device comprising a material as defined in claim 15.18.A device as defined in claim 17, wherein said device is to be used in contact with an animal's tissue.","19.A device as defined in claim 18, wherein said tissue is blood tissue.","20.A device as defined in claim 18 or 19, wherein said device is selected from the group consisting of: An implant, a prosthesis, an artificial organ, a stent, a cardiac valve, an apparatus contacting blood during an extra-corporal blood circulation, an apparatus contacting blood during a dialysis treatment or any other material with surfaces coming in contact with blood.","21.Use of a device as defined in any one of claims 17 to 20 for preventing thrombosis.","22.A material suitable for contact with a living tissue characterized in having a surface that is resistant to neutrophil adhesion, platelet adhesion and activation but that enhances the development of fibroblasts and endothelial cells.","23.A material as defined in claim 22 which is plasma-treated ePTFE with grafted PRC.","24.A device comprising a material as defined in claim 22 or 23.25.A device as defined in claim 24, wherein said device is selected from the group consisting of: An implant, a prosthesis, an artificial organ, a stent, a cardiac valve, an apparatus contacting blood during an extra-corporal blood circulation, an apparatus contacting blood during a dialysis treatment or any other material with surfaces coming in contact with blood.","26.Use of a device as defined in claim 24 or 25 for preventing thrombosis."],["<SOH> BACKGROUND OF THE INVENTION <EOH>Approximately 350 000 synthetic vascular prostheses are implanted each year as arterial bypasses in Western countries and Japan.","Made of Dacron™ (polyethyleneterephthalate) or microporous Teflon™ (expanded polytetrafluoroethylene, or ePTFE), they perform quite well in large-diameter vessels under high flow conditions but have a low patency rate when used as small-diameter bypasses (i.e., coronaries) or as medium-diameter bypasses in low-flow conditions and high-resistance locations (i.e., leg arteries).","(The patency rate relates to a biomaterial's ability to remain pervious to blood flow when replacing an artery).","A study on 398 ePTFE vascular prostheses implanted as medium-diameter bypasses and retrieved following complications revealed that 65% of them had to be explanted due to thrombosis.","(1 ) Endothelial cell lining is the natural blood-compatible surface that covers the inside surface of blood vessels and heart chambers, and it is recognized to be the best possible hemocompatible surface.","The principal techniques that have been proposed in the past to promote endothelial cell adhesion and spreading onto the luminal surfaces of vascular prostheses are: (1) endothelial cell seeding; (2) graft pre-treatment with endothelial cell mitogens; (3) increasing structural porosity; and (4) coating the surface with a protein matrix.","While several attempts have been made, the complete endothelial cell coverage of the blood-exposed inside prosthetic surfaces has never been observed in humans.","It is therefore evident that increasing the patency rate of arterial prostheses is a high priority in current cardiovascular research.","It has been shown in many instances that surface modification through plasma treatment is one of the most promising techniques that may be used to improve specific hemocompatibility and general biocompatibility of polymeric materials.","(2,3) One advantage of the plasma treatment is that it allows formation of a covalent bond between the modifier and the surface to be treated, unlike other approaches where a coating is deposited inside the prosthesis without formation of a covalent bond (4,5) , where the bond is ionic (6) or where the bioactive substance is incorporated in the biomaterial.","(7) Following to the covalent bonding of the modifier to the surface of the material, made possible by plasma treatment, a molecule chosen for its biocompatibility properties can be covalently attached to the modifier either directly or through a spacer molecule.","Clinically, the increased stability of a surface treatment involving plasma treatment could result in improved limb salvage rates and a possible future use in small-diameter coronary bypass operations.","This, in turn, could minimize the incidence of re-operation for patients and lower social healthcare costs related to surgical interventions of this type.","Plasma treatment of biomaterials is not the only process that can yield covalent bonding of molecules possessing desirable hemocompatibility and/or biocompatibility.","However, the processes that are described in the relevant prior art frequently involve several steps.","(8-14) To date, most of the experiments performed to improve the performance of biomaterial surfaces have been based on flat specimens of material, particularly polymeric sheets.","Unfortunately, few experiments involving the use of plasma to activate the surface have been carried out directly on commercial prostheses with a non-planar geometry.","Experiments done at the University of Washington, (2,15-20) which consisted mainly in treating commercial Dacron™ arterial prostheses with a plasma treatment in a tetrafluoroethylene (TFE) gas environment so as to coat the internal surfaces of the prostheses with potentially more biocompatible CF 3 groups, were a precursor to the development of the new commercial Radio Frequency Glow Discharge (RFGD)-treated vascular grafts.","(17) Yet, despite the existence of many surface treatment systems for biomaterial surfaces, few have resulted in the treatment, as opposed to the coating, of the internal surface of tubular devices.","(21-23) Over the last 30 years, a number of studies have been designed to test the ability of plasma to either treat or coat the surfaces of biomedical devices with a view to enhancing their biocompatibility or to modulate the interactions of polymers and tissue when prostheses are implanted in situ.","(16,24-30) Plasma polymerizable gas has been used to coat various substrates with a thin polymeric layer, (15,17-19,24,31,32) while non-polymerizable gas has been applied to treat substrate surfaces.","(33-38) Despite the fact that many approaches have been developed to coat or treat the surface of biomaterials, the introduction of amino groups through RFGD treatment is particularly interesting for two reasons.","First, amino groups are known to facilitate cellular spreading on a biomaterial surface.","(39-41) Second, amino groups react readily with other chemical functional groups, allowing for the attachment of specific molecules through interactions sufficiently strong to prevent the leaching of these molecules by the blood stream.","(42) For example, amino groups can be used to ionically immobilize heparin, a well-known polysaccharide anticoagulant.","(22) While many efforts have been made in the last 20 years in the field of cardiovascular prosthetic devices to develop surfaces with improved human blood compatibility, an acceptable long-term blood-compatible synthetic material has yet to be achieved.","The complexity of the interactions between blood and synthetic materials constitutes a major interfacial problem in this respect."],["<SOH> SUMMARY OF THE INVENTION <EOH>Surface treatments allow a modulation of the surface properties of a biomaterial to enhance its interfacial reaction with a biological environment.","Low pressure plasma surface treatments are particularly advantageous in the design and development of new biocompatible materials, since they permit surface modifications without altering the bulk of the materials' properties.","(43) In the context of the present invention, they are particularly useful for modulating different tissue/biomaterial interface properties, but their ultimate utility may reside in their significant improvement of the hemocompatibility of vascular prostheses.","Thus, in accordance with the present invention, there is provided a novel process for modifying the surface properties of a material that is to be placed in contact with living tissues.","This process comprises: Exposing the surface of the material to plasma treatment conditions in order to create reactive groups on the surface of the material; Activating a molecule with an activator to produce a reactive molecular species capable of forming strong bonds with the reactive groups created on the surface of the material; and Contacting the reactive molecular species with the reactive groups created on the surface of the material to form strong bonds.","Preferably, the strong bonds formed are covalent bonds.","In a preferred embodiment, the molecule selected for activation is compatible with living tissue, including human tissue.","In a specific embodiment the novel process of the present invention further comprises the following features: the plasma is ammonia Radio Frequency (RF) plasma and the reactive groups are amine groups; the molecule selected for activation is chosen from the group consisting of choline, heparin, and other molecules known to those skilled in the art for their hemocompatibility, the activator is phosphoryl chloride (POCl 3 ) and the reactive molecular species is the oxyphosphorodichlorinated derivative of the molecule; and the strong bonds are phosphoamide-type covalent bonds.","In a preferred embodiment of the specific embodiment above, the RF power is between about 5 watts to about 500 watts and is applied for a time of about 10 seconds to about 30 minutes at a pressure of about 50 mtorr to about 5 torr.","Those skilled in the art will recognize that care has to be taken in order to avoid combinations of time and power that would deliver excessive amounts of energy to the treated surface.","Such conditions lead to a microscopically brittle surface.","Preferably, the RF power is about 20 watts and is applied for a time of about 250 seconds at a pressure of about 300 mtorr.","In yet another preferred embodiment, the RF power is about 15 watts and is applied for a time of about 100 seconds at a pressure of about 250 mtorr.","Ideally, the third step of the process is performed within about 2 hours of the first step.","The novel process of the present invention is particularly suitable for an implantable material selected from the following group: expanded polytetrafluoroethylene (ePTFE), polytetrafluoroethylene (PTFE), polyethylenes, polyesters, polypropylenes and polyurethanes.","In a preferred embodiment, the implantable material is ePTFE, and the treated surface is the internal surface of a vascular prosthesis.","In a more preferred embodiment, the vascular prosthesis has an inner diameter of about 1 to 30 mm.","The present invention further comprises the materials produced by the novel process, as well as devices comprising these materials, including any of the following: an implant, a prosthesis, an artificial organ, a stent, a cardiac valve, an apparatus contacting blood during an extra-corporal blood circulation, an apparatus contacting blood during a dialysis treatment or any other material with surfaces coming in contact with blood.","Additionally, the present invention is meant to cover the use of any such device, particularly to prevent thrombosis in an animal, including a human being.","By controlling the RF power, the ammonia pressure, and the treatment duration, the atomic substitution percentage may be modulated and up to 15% of the surface atoms may be substituted with nitrogen, as probed by X-ray photoelectron spectroscopy (XPS).","On the surface, different chemical species are present after the treatment, such as amine and imine groups.","Storage in air for up to 80 days shows a defluorination of the surface and a slow decrease of the surface nitrogen concentration during this storage.","Ammonia RF Plasma treatment can be successfully used to uniformly treat the internal surface of an ePTFE arterial prosthesis despite the fact that care has to be taken to prevent a significant surface reorganization upon exposure to the atmosphere.","(44,45) The ammonia RF plasma-treated internal surfaces of vascular prostheses may be reacted with a substance whose molecular structure can render the prostheses more hemocompatible and non-thrombogenic when in use.","The molecule is preferably activated phosphorylcholine (PRC) or any other molecule with similar properties, wherein the two hydroxyl groups on the phosphate moiety have been substituted with chlorine to form the dichloro derivative of the molecule.","This dichloro derivative readily reacts with the amino groups resulting from the ammonia RF plasma treatment of the inside surfaces of the vascular prostheses, allowing for the effective anchoring of the molecule.","It is an object of the present invention to provide a novel process for producing a material suitable for contact with a living tissue.","This material is characterized in having a surface that is resistant to neutrophil adhesion and thrombosis but that enhances the development of fibroblasts and endothelial cells.","The process may be used, for example, to treat the inside surfaces of devices, such as vascular prostheses, so as to enhance their hemocompatibility and make them less thrombogenic when in use.","A further object of the invention is therefore to provide devices treated with the novel process of the present invention.","Other objects, advantages and features of the present invention will become more apparent upon reading of the following non restrictive description of preferred embodiments thereof, given by way of example only with reference to the accompanying drawings."],["FIELD OF THE INVENTION The present invention relates to a novel process for modifying the surface properties of a material that is suitable for contact with living tissue comprising: Exposing the surface of the material to plasma treatment conditions in order to create reactive groups on said surface; activating a molecule to produce a reactive molecular species capable of forming strong bonds with the reactive groups created on the surface of the material; and contacting the reactive molecular species with the reactive groups created on the surface of the material to form strong bonds.","The invention further encompasses the materials produced by this process as well as devices that are comprised of these process-modified materials, such as vascular prostheses.","BACKGROUND OF THE INVENTION Approximately 350 000 synthetic vascular prostheses are implanted each year as arterial bypasses in Western countries and Japan.","Made of Dacron™ (polyethyleneterephthalate) or microporous Teflon™ (expanded polytetrafluoroethylene, or ePTFE), they perform quite well in large-diameter vessels under high flow conditions but have a low patency rate when used as small-diameter bypasses (i.e., coronaries) or as medium-diameter bypasses in low-flow conditions and high-resistance locations (i.e., leg arteries).","(The patency rate relates to a biomaterial's ability to remain pervious to blood flow when replacing an artery).","A study on 398 ePTFE vascular prostheses implanted as medium-diameter bypasses and retrieved following complications revealed that 65% of them had to be explanted due to thrombosis.","(1) Endothelial cell lining is the natural blood-compatible surface that covers the inside surface of blood vessels and heart chambers, and it is recognized to be the best possible hemocompatible surface.","The principal techniques that have been proposed in the past to promote endothelial cell adhesion and spreading onto the luminal surfaces of vascular prostheses are: (1) endothelial cell seeding; (2) graft pre-treatment with endothelial cell mitogens; (3) increasing structural porosity; and (4) coating the surface with a protein matrix.","While several attempts have been made, the complete endothelial cell coverage of the blood-exposed inside prosthetic surfaces has never been observed in humans.","It is therefore evident that increasing the patency rate of arterial prostheses is a high priority in current cardiovascular research.","It has been shown in many instances that surface modification through plasma treatment is one of the most promising techniques that may be used to improve specific hemocompatibility and general biocompatibility of polymeric materials.","(2,3) One advantage of the plasma treatment is that it allows formation of a covalent bond between the modifier and the surface to be treated, unlike other approaches where a coating is deposited inside the prosthesis without formation of a covalent bond(4,5), where the bond is ionic(6) or where the bioactive substance is incorporated in the biomaterial.","(7) Following to the covalent bonding of the modifier to the surface of the material, made possible by plasma treatment, a molecule chosen for its biocompatibility properties can be covalently attached to the modifier either directly or through a spacer molecule.","Clinically, the increased stability of a surface treatment involving plasma treatment could result in improved limb salvage rates and a possible future use in small-diameter coronary bypass operations.","This, in turn, could minimize the incidence of re-operation for patients and lower social healthcare costs related to surgical interventions of this type.","Plasma treatment of biomaterials is not the only process that can yield covalent bonding of molecules possessing desirable hemocompatibility and/or biocompatibility.","However, the processes that are described in the relevant prior art frequently involve several steps.","(8-14) To date, most of the experiments performed to improve the performance of biomaterial surfaces have been based on flat specimens of material, particularly polymeric sheets.","Unfortunately, few experiments involving the use of plasma to activate the surface have been carried out directly on commercial prostheses with a non-planar geometry.","Experiments done at the University of Washington,(2,15-20) which consisted mainly in treating commercial Dacron™ arterial prostheses with a plasma treatment in a tetrafluoroethylene (TFE) gas environment so as to coat the internal surfaces of the prostheses with potentially more biocompatible CF3 groups, were a precursor to the development of the new commercial Radio Frequency Glow Discharge (RFGD)-treated vascular grafts.","(17) Yet, despite the existence of many surface treatment systems for biomaterial surfaces, few have resulted in the treatment, as opposed to the coating, of the internal surface of tubular devices.","(21-23) Over the last 30 years, a number of studies have been designed to test the ability of plasma to either treat or coat the surfaces of biomedical devices with a view to enhancing their biocompatibility or to modulate the interactions of polymers and tissue when prostheses are implanted in situ.","(16,24-30) Plasma polymerizable gas has been used to coat various substrates with a thin polymeric layer,(15,17-19,24,31,32) while non-polymerizable gas has been applied to treat substrate surfaces.","(33-38) Despite the fact that many approaches have been developed to coat or treat the surface of biomaterials, the introduction of amino groups through RFGD treatment is particularly interesting for two reasons.","First, amino groups are known to facilitate cellular spreading on a biomaterial surface.","(39-41) Second, amino groups react readily with other chemical functional groups, allowing for the attachment of specific molecules through interactions sufficiently strong to prevent the leaching of these molecules by the blood stream.","(42) For example, amino groups can be used to ionically immobilize heparin, a well-known polysaccharide anticoagulant.","(22) While many efforts have been made in the last 20 years in the field of cardiovascular prosthetic devices to develop surfaces with improved human blood compatibility, an acceptable long-term blood-compatible synthetic material has yet to be achieved.","The complexity of the interactions between blood and synthetic materials constitutes a major interfacial problem in this respect.","SUMMARY OF THE INVENTION Surface treatments allow a modulation of the surface properties of a biomaterial to enhance its interfacial reaction with a biological environment.","Low pressure plasma surface treatments are particularly advantageous in the design and development of new biocompatible materials, since they permit surface modifications without altering the bulk of the materials' properties.","(43) In the context of the present invention, they are particularly useful for modulating different tissue/biomaterial interface properties, but their ultimate utility may reside in their significant improvement of the hemocompatibility of vascular prostheses.","Thus, in accordance with the present invention, there is provided a novel process for modifying the surface properties of a material that is to be placed in contact with living tissues.","This process comprises: Exposing the surface of the material to plasma treatment conditions in order to create reactive groups on the surface of the material; Activating a molecule with an activator to produce a reactive molecular species capable of forming strong bonds with the reactive groups created on the surface of the material; and Contacting the reactive molecular species with the reactive groups created on the surface of the material to form strong bonds.","Preferably, the strong bonds formed are covalent bonds.","In a preferred embodiment, the molecule selected for activation is compatible with living tissue, including human tissue.","In a specific embodiment the novel process of the present invention further comprises the following features: the plasma is ammonia Radio Frequency (RF) plasma and the reactive groups are amine groups; the molecule selected for activation is chosen from the group consisting of choline, heparin, and other molecules known to those skilled in the art for their hemocompatibility, the activator is phosphoryl chloride (POCl3) and the reactive molecular species is the oxyphosphorodichlorinated derivative of the molecule; and the strong bonds are phosphoamide-type covalent bonds.","In a preferred embodiment of the specific embodiment above, the RF power is between about 5 watts to about 500 watts and is applied for a time of about 10 seconds to about 30 minutes at a pressure of about 50 mtorr to about 5 torr.","Those skilled in the art will recognize that care has to be taken in order to avoid combinations of time and power that would deliver excessive amounts of energy to the treated surface.","Such conditions lead to a microscopically brittle surface.","Preferably, the RF power is about 20 watts and is applied for a time of about 250 seconds at a pressure of about 300 mtorr.","In yet another preferred embodiment, the RF power is about 15 watts and is applied for a time of about 100 seconds at a pressure of about 250 mtorr.","Ideally, the third step of the process is performed within about 2 hours of the first step.","The novel process of the present invention is particularly suitable for an implantable material selected from the following group: expanded polytetrafluoroethylene (ePTFE), polytetrafluoroethylene (PTFE), polyethylenes, polyesters, polypropylenes and polyurethanes.","In a preferred embodiment, the implantable material is ePTFE, and the treated surface is the internal surface of a vascular prosthesis.","In a more preferred embodiment, the vascular prosthesis has an inner diameter of about 1 to 30 mm.","The present invention further comprises the materials produced by the novel process, as well as devices comprising these materials, including any of the following: an implant, a prosthesis, an artificial organ, a stent, a cardiac valve, an apparatus contacting blood during an extra-corporal blood circulation, an apparatus contacting blood during a dialysis treatment or any other material with surfaces coming in contact with blood.","Additionally, the present invention is meant to cover the use of any such device, particularly to prevent thrombosis in an animal, including a human being.","By controlling the RF power, the ammonia pressure, and the treatment duration, the atomic substitution percentage may be modulated and up to 15% of the surface atoms may be substituted with nitrogen, as probed by X-ray photoelectron spectroscopy (XPS).","On the surface, different chemical species are present after the treatment, such as amine and imine groups.","Storage in air for up to 80 days shows a defluorination of the surface and a slow decrease of the surface nitrogen concentration during this storage.","Ammonia RF Plasma treatment can be successfully used to uniformly treat the internal surface of an ePTFE arterial prosthesis despite the fact that care has to be taken to prevent a significant surface reorganization upon exposure to the atmosphere.","(44,45) The ammonia RF plasma-treated internal surfaces of vascular prostheses may be reacted with a substance whose molecular structure can render the prostheses more hemocompatible and non-thrombogenic when in use.","The molecule is preferably activated phosphorylcholine (PRC) or any other molecule with similar properties, wherein the two hydroxyl groups on the phosphate moiety have been substituted with chlorine to form the dichloro derivative of the molecule.","This dichloro derivative readily reacts with the amino groups resulting from the ammonia RF plasma treatment of the inside surfaces of the vascular prostheses, allowing for the effective anchoring of the molecule.","It is an object of the present invention to provide a novel process for producing a material suitable for contact with a living tissue.","This material is characterized in having a surface that is resistant to neutrophil adhesion and thrombosis but that enhances the development of fibroblasts and endothelial cells.","The process may be used, for example, to treat the inside surfaces of devices, such as vascular prostheses, so as to enhance their hemocompatibility and make them less thrombogenic when in use.","A further object of the invention is therefore to provide devices treated with the novel process of the present invention.","Other objects, advantages and features of the present invention will become more apparent upon reading of the following non restrictive description of preferred embodiments thereof, given by way of example only with reference to the accompanying drawings.","BRIEF DESCRIPTION OF THE FIGURES FIG.","1 is a schematic representation of the Radio Frequency Glow Discharge treatment system used in the process of the invention.","FIG.","2 shows the XPS spectrum of the inside surface of an untreated ePTFE vascular prosthesis and the XPS spectrum of the inside surface of an ammonia plasma treated ePTFE vascular prosthesis.","FIG.","3 shows the effect on the surface composition of treatments performed at 300 mtorr of ammonia by varying the power (20, 40 and 50 W) and treatment times (10, 120 and 275 seconds).","FIG.","4 shows the relationship between the pressure within the chamber and the modification of the ePTFE prosthetic surface.","FIG.","5 shows the percentage of nitrogen and the N/C and F/C ratios measured on the surface as a function of ammonia plasma treatment time.","FIG.","6 shows the derivatization reaction of a plasma treated PTFE film with chlorobenzaldehyde.","FIG.","7 shows the relationship between the atomic surface concentration of phosphorous grafted onto the surface (% P) and the intensity of the N+ peak at 402 eV.","FIG.","8 shows the cellular growth determined by the fluorescence of DNA strands using specific markers with untreated and treated prostheses as well as with “control” cell cultures.","FIG.","9 reproduces SEM micrographs showing the degree of accumulation of blood platelets within (a) a prosthesis treated in accordance with the process of the invention and (b) an untreated prosthesis.","FIG.","10 shows the quantification of the adherent blood platelets on untreated and treated prostheses.","FIG.","11 shows the quantification of the adherent neutrophils on untreated and treated prostheses.","FIG.","12 shows the influence of a treated and untreated prostheses on the clotting time.","FIG.","13 shows the influence of a treated and untreated prostheses on the thrombogenic index.","FIG.","14 displays microscopic slides of the growth of fibroblasts within a prosthesis treated in accordance with the process of the invention ((a) and (c)) and an untreated prosthesis ((b) and (d)).","FIG.","15 SEM images at 20× and 2000× magnification for ANBIOPA™-treated prostheses and commercially-available untreated prostheses after a one-month implantation in a canine model: a) ANBIOPA™-treated, 20×; b) untreated, 20×; c) ANBIOPA™-treated, 2000×; and d) untreated, 2000×.","DESCRIPTION OF A PREFERRED EMBODIMENT For the present purposes, we give the following definitions of the terms “untreated” and “treated”.","“Untreated” means a commercially available vascular prosthesis, to which the inventors did not apply any surface modification treatment.","“Treated” means a commercially available vascular prosthesis to which the inventors applied the surface modification procedure hereafter described.","The term “animal” as used herein is meant to signify human beings, primates, domestic animals (such as horses, cows, pigs, goats, sheep, cats, dogs, guinea pigs, mice, etc.)","and other mammals.","Generally, this term is used to indicate living creatures having highly-developed vascular systems.","I. Pre-Treatment of the Inside Surface of Vascular Prostheses with Ammonia Radio Frequency (RF) Plasma Experimental Method (The Experimental Method is described with reference to FIG.","1.)","Materials Microporous ePTFE vascular prostheses 6 (6 and 10 mm ID) with a fibril average length of 25 microns were used, and the method is described in relation to a single prosthesis.","A microporous ePTFE arterial prosthesis (length 3 to 10 cm) was first washed with high purity methanol for 10 minutes and vacuum-dried.","This procedure was performed to remove any contaminants which may have been present on the surface of the “as-received” prosthesis.","Following this washing procedure, two small holes were punctured at one end of the prosthesis, facing each other, and a polypropylene monofilament (5-0—Ethicon) 3 was inserted through both holes and a knot tied loosely above the prosthesis.","The other end of the monofilament was rolled around a pulley 5, itself located on the horizontal shaft of an electric motor 36 to allow a vertical displacement of the sample prosthesis inside the Pyrex™ tube 2 of the RFGD treatment system.","Gravity caused the vascular prosthesis to move down inside the Pyrex™ tube.","For vascular grafts longer than 3 cm, it was preferable, in addition to the aforementioned knot and its attachment via the monofilament to the pulley, to attach a polypropylene monofilament 3 at the bottom of the vascular prosthesis and to attach the monofilament to the pulley by first letting the monofilament run down the tube and then up to the pulley.","With this set-up, the motor can pull on the prosthesis when it is going down the Pyrex™ tube, and therefore friction of the prosthesis on the inside wall of the tube ceases to be a problem when moving the prosthesis down the tube.","Prior to closing the system, a container and its cap were placed inside a bell jar 1 to collect and store the prosthesis in an argon atmosphere following the RFGD treatment, thereby minimizing any contact of the active sites on the surface of the prosthesis with air.","Plasma Equipment As shown in FIG.","1, the RFGD treatment system used can be generally represented by the numeral 10.This system is basically comprised of a Pyrex™ glass tube (400 mm long, 12.5 mm i.d.","for 10 mm i.d.","prostheses and 8 mm i.d.","for 6 mm i.d.","prostheses) 2 with an internal diameter 4 slightly greater than the external diameter of the prosthesis 6, a configuration which allows for RFGD generation on the internal side of the prosthesis only.","Indeed, the path length between the external side of the prosthesis 6 and the Pyrex™ tube 4 is not long enough to allow the electron-induced cascade ionization which is necessary for plasma formation.","(11) Therefore, the treatment of prostheses of various diameter requires the selection of a Pyrex™ tube of the appropriate diameter.","As mentioned above, the criteria of selection is that no plasma is ignited on the outside surface of the prosthesis during its treatment.","A space of about 1 mm all around the outside diameter of the prosthesis and the inside wall of the Pyrex™ glass tube 2 has proven to be appropriate.","The Pyrex™ glass tube 2 has an inlet for gas injection 8 located 5 cm below its upper end.","Three capacitively coupled copper electrodes 12a, 12b and 12c, spaced from 3 to 5 cm from each other (depending on the length of the prosthesis to treat), are attached to the middle of the tube 2.The vacuum within the chamber was created by a turbo-molecular pump 14 with a pumping speed of 60 liters/s (Model TCP015, Balzers Pfeiffer) which, in turn, is connected to a mechanical pump 16 (Model 1402, Welch Scientific Company).","This set-up allows a pressure of close to 4×10−5 torr to be reached.","During treatment, pressure is kept constant within the chamber by means of a capacitance pressure gauge/electromechanical valve controller 18 (type 250-1A, MKS Instruments) connected to an electromechanical valve 20 (type 248A-00200SV, MKS Instruments) and a capacitance manometer 22 (Baratron type 626A01TAE, MKS Instruments).","Finally, gaseous molecules are excited through a radio-frequency field made by an RF generator 24 (YAESU HF transceiver FT-840, YAESU) that transmits the power, through a power meter 26, to an automatic matching network (Smartuner SG-230, SGC) 28 which then couples the RF power with an antenna made of three copper electrodes 12a-c. Electrodes 12a and 12c, located at both ends of the antenna, are connected to a ground (not shown) while the RF power is brought through the central electrode 12b.","The treatment can be operated in a RF power range from 10 to 100 W at 13.56 MHz.","Plasma Treatment Initially, all valves on the system are closed.","After the introduction of the prosthesis into the bell jar 1, valve 30 is opened and the mechanical pump 16 is activated to reach a vacuum of approximately 10−3 torr.","As soon as this pressure is attained inside the system, valve 30 is closed, valve 32 is opened and after a few seconds the turbo-molecular pump 14 is activated.","After about one minute, valve 34 is opened and pumping inside the bell jar continued until it reaches a vacuum greater than 5×10−5 torr.","The chamber is then isolated from the turbo pump 14 by closing valve 34 and high purity ammonia is introduced into the bell jar 1 via electromechanical valve 20 and, when the pressure is above 0.1 torr, the bell jar 1 is pumped by the mechanical pump 16 alone, through valve 30.The electromechanical valve controller 18 automatically regulates the amount of gas entering the chamber and the pressure, read by the capacitance gauge 22, settles within 1 minute to the value selected on the capacitance gauge/electromechanical valve controller.","Typically, this configuration allows plasma generation within a pressure range of 10−3 to 1 torr.","With the above parameters established, the plasma is ignited by turning the radio-frequency generator 24 on.","Sufficient time is allowed for the matching network 28 to reach a standing wave ratio (SWR) signal of between 1 and 1.15, as read on the power meter 26 in order to provide optimal matching of the power input to the discharge.","RFGD treatments are then performed using a static treatment: the prosthesis 6 is initially located just under the inlet 8 while the plasma parameters are adjusted, it is then moved from this position in the Pyrex™ glass tube 2 into the middle of the lighted area (between the three electrodes 12a, 12b and 12c) using the electric motor 36, and is maintained within the plasma for predetermined periods of time.","Prostheses longer than 10 cm could be treated by slowly moving the prostheses across the lighted area using electric motor 36, resulting in similar grafting efficiencies.","(45) At the end of the treatment, the Radio-Frequency generator 24 is turned off and the gas flow stopped by closing the electromechanical valve 20 via its controller 18.The mechanical pump 16 is kept in operation to remove residual gaseous ammonia from the bell-jar 1 until a pressure of approximately 50 mtorr is reached.","It is then flushed with argon for 2 minutes by carefully opening valve 38.This is done in order to allow the excited species resulting from the plasma treatment to reach their ground state.","After isolating the mechanical pump 16 from the bell-jar by closing valve 30, argon flow is continued inside the chamber to bring the bell jar back to atmospheric pressure.","The prosthesis 6 is then lowered to the bottom of the Pyrex™ tube 2, the bell jar 1 is quickly opened and the prosthesis 6 is disconnected from the polypropylene microfilament 3 and put inside a capped container.","It is then placed in a glove box purged with nitrogen where a small sample is taken for XPS studies in order to assess the success of the ammonia treatment.","The prosthesis is then prepared for the anchoring process as described below.","II.","Anchoring of Molecules Mimicry of the non-thrombogenic surface of the erythrocyte has been advocated as the starting point for the development of non-thrombogenic biomaterials.","Since phosphorylcholine (PRC) is a major component of the outer surface of the erythrocyte, materials containing it would be expected to be non-thrombogenic.","(46-50) Moreover, it is known that PRC groups attached to polymer surfaces improve hemocompatibility.","(51-57) In order to enhance the hemocompatibility and non-thrombogenicity of the interior surfaces of vascular prostheses, prostheses which had been pre-treated with plasma (as described above) were made to react with activated analogues of molecules such as phosphorylcholine (PRC).","Since the ultimate goal is the implantation of such prostheses into the human body, the molecules must be solidly attached to the inside walls of the prostheses.","Consequently, it is necessary to form strong bonds (preferably, covalent bonds) between the molecules and the amine groups created by the plasma treatment rather than to rely on the formation of ionic bonds or adsorption phenomenon to secure the molecules onto the inside surfaces of the prostheses.","Additionally, it is desirable that any chemical treatment of this type be simple and inexpensive.","The general structure of the molecules is represented below: Direct reaction between PRC and the amino groups present on the surface of the plasma-treated prostheses results in ionic bonds and/or surface adsorption neither of which, as previously stated, is sufficiently strong or stable for the applications contemplated.","It is therefore necessary to enhance the reactivity of these molecules by synthesizing reactive analogues so that they may partake in the creation of phosphoamide-type covalent bonds with the amino groups on the surface of the ammonia plasma pre-treated prostheses.","In this way, the molecules may be anchored effectively to the interior surface of the prostheses.","In order to achieve the desired covalent reaction, the dichlorinated derivative RPO(Cl)2 of the target molecule RPO(OH)2 must first be synthesized.","The synthesis of the dichlorinated derivatives of the molecules suitable for reaction with the amino groups on the inside surface of the pre-treated vascular prostheses may be achieved in at least two ways, as revealed diagrammatically below with PRC.","In the first pathway, PRC, which is available commercially in the form of a calcium salt, is reacted with an acidic resin(58) to eliminate the calcium.","The product is then made to react with phosphorus trichloride or pentachloride (or any halogenation agent such as phosgene, oxalyl chloride, thionyl chloride) to obtain the dichlorinated derivative.","An alternative synthesis is shown in the second pathway.","Here, the dichlorinated derivative of PRC is synthesized directly from phosphoryl chloride and choline in a single step.","In a preferred embodiment, the preferred pathway is the second one described above which has the advantage of requiring a lesser number of steps.","This experimental protocol takes into account such factors as reproducibility and toxicity, as well as feasibility on an industrial scale.","Experimental Method The dichloro derivative of PRC is synthesized by the addition of phosphoryl chloride (POCl3) in chloroform solution to a chloroform solution containing choline with glass beads (3 mm diameter) at 0° C. (ice bath) in a closed glass flask, under a dry nitrogen stream to exclude the presence of water and displace the HCl resulting from the reaction.","The mixture is then stirred at 0° C. for 1 hr, and at room temperature for 12 hours to allow the reaction to proceed, as indicated by dissolution of the choline crystals and the formation of a oily phase.","A slight excess of choline is used to ensure that all the phosphoryl chloride is consumed during the activation reaction.","Immediately following the synthesis of the dichloro derivative of PRC, the 3 cm prostheses are immersed at room temperature in the abovementioned glass flask and agitation is continued for an additional 2 hours.","In the case of longer vascular prostheses (>3 cm) a simple system was designed with a 1-liter commercial glass reactor with an outlet at the bottom.","In this case, following the 12 hours of mechanical agitation, the chloroform layer at the bottom was removed via the bottom outlet of the flask and was replaced by fresh chloroform in order to remove any remaining POCl3.A PTFE diaphragm pump is then used to circulate the activated PRC solution from the bottom of the reactor, through a section of PTFE tubing, then through a vertical glass tube in which the prosthesis is held.","Finally, a section of PTFE tubing brings back the solution in the glass reactor for recirculation.","When the grafting of the activated PRC has occurred, the prostheses are washed many times in water to eliminate all traces of chlorine that may remain on the phosphoamide moiety of the anchored molecule (see Step 2 of the complete reaction, below) as well as all traces of reactants and solvent.","The prostheses are then dried under vacuum overnight before being analyzed by XPS and static SIMS to determine the level of grafting.","Overall Reaction for ePTFE Vascular Prostheses Inner Surface Treatment The process of the present invention may be extended to bind other molecules that are highly compatible with biological tissue, such as blood tissue.","In fact, preliminary results(63) indicate that the internal surface of commercial ePTFE arterial prostheses that were previously treated with the ammonia plasma treatment described herein can be homogeneously coated, using a grafting method different from the one presented here, with polyethylene-glycol (PEG), which is another highly biocompatible molecule.","Characterization of Ammonia RFGD Plasma Treated Surfaces and PRC-Grafted Surfaces X-Ray Photoelectron Spectroscopy (XPS) Analyses X-ray photoelectron spectroscopy (XPS) was used to characterize the modified surfaces.","XPS spectra were recorded using a PHI 5600-ci spectrometer (Physical Electronics).","A monochromatic aluminum X-ray source (1486.6 eV) was used to record the survey and high resolution spectra of the prosthetic surface.","For both type of analyses, the power source was kept at 100 W to minimize X-ray induced modifications of the specimen and the detection was performed at 45° with respect to the surface.","The pressure within the XPS analytical chamber was kept at about 5×10−9 torr.","Ammonia RFGD Plasma Treated ePTFE Surfaces After RFGD ammonia plasma treatments, specimens were longitudinally opened in a chamber under nitrogen positive pressure and their internal surfaces were analyzed by XPS within 30 minutes of the RFGD treatment.","Surface atomic concentration of oxygen was always lower than 5%.","In order to collect data of the surface modifications along the longitudinally-treated length of a specimen, the XPS analysis area was moved along the length of the prosthesis.","To validate that the RFGD system resulted in the treatment of the internal surface only, XPS analyses of the external surface of a prosthetic specimen treated at 20 W, for 250 seconds with an ammonia pressure of 300 mtorr were conducted.","The data (not shown) confirmed that the external surface was not modified by the glow discharge when the internal diameter of the Pyrex™ tube was chosen to be only slightly larger (1 mm) than the external diameter of the prosthesis.","Under the pressure and power conditions used, the Debye length did not allow the formation of a stable plasma between the external surface of the prosthesis and the internal surface of the tube, because the ionization processes were insufficient to compensate for the recombination processes.","(60) All results are discussed by considering the percentage of atomic concentration of nitrogen on the surface, and the N/C, F/C and O/C surface concentration ratios.","It was observed that because of experimental and instrumental uncertainties, atomic concentration values above 5% are reliable to ±1% (absolute) or better.","As gaseous ammonia was used to create the plasma, it is clear that the percentage of nitrogen would be a valid indicator of the extent of the RFGD treatment.","Moreover, the N/C ratio probes the extent of nitrogen atom insertion onto the ePTFE surface, while the F/C ratio monitors the level of defluorination and is indicative of the amount of fluorine atoms substituted by other atoms and/or multiple bond formations within or between the chains.","Relationship Between the Nature of the Nitrogen-Containing Species Grafted onto the Surface of the Vascular Prostheses and Treatment Parameters With a view to understanding the relationship between the nature of the nitrogen-containing species grafted onto the surface of the RFGD treated ePTFE samples, different tests were performed to investigate the effects of variable parameters such as gas pressure, RF power and treatment time.","Prostheses measuring 3 cm in length were treated under conditions where two of the variables were kept constant while the third was varied.","As may be seen in FIG.","2, the RF plasma treatment allowed a surface atomic nitrogen concentration of up to 15% to be reached, with an average N/C ratio of up to 0.3, at 20 W for 250 seconds and with an ammonia pressure of 300 mtorr.","This represents a significant improvement over previously-reported results following treatment aimed at incorporating nitrogen on ePTFE surfaces, where published data indicates a percentage of nitrogen between 1 and 3%(26,42,61) or a N/C ratio between 0.06 and 0.18(28-43,59).","A possible explanation for the present results may reside in the cylindrical configuration of the reactor chosen maximizes the efficiency of the treatment(59).","The three surface characterization parameters (F/C, N/C and % N) indicate that the surface modifications induced on any given prosthesis are fairly homogenous: for example, (13±1)% atomic nitrogen concentration on a 7 cm-segment of a 6 mm ID prosthesis (250 s, 300 mtorr, 15 W).","The degree of homogeneity required for subsequent grafting of the hemocompatible molecule and obtaining of an improved performance of the vascular prostheses can only be properly determined by hemocompatibility tests.","However, the observed relative uniformity of surface modifications following ammonia plasma treatment provides a good starting point for the subsequent grafting.","FIG.","3 shows the effect on the surface composition of treatments performed by keeping the pressure constant (300 mtorr) while varying the power (20, 40 and 50 W) and treatment times (10, 120 and 275 seconds).","It appears that the nitrogen concentration reached its highest value for a RF power of 40 W. The N/C ratio measured for the experiments performed for 275 seconds was significantly higher than that measured following treatment for 120 seconds.","On the other hand, the F/C ratio was identical for these two treatment times for all RF powers tested.","Thus, this data suggests that, despite leading to a higher nitrogen surface concentration, longer treatment times did not promote increased fluorine substitution.","This result could mean that different treatment times lead to the formation of different chemical species on the surface.","As may be seen in FIG.","4, increasing the ammonia pressure from 100 to 600 mtorr (RF power kept at 20 W and treatment time at 10 s) lead to only a slight increase of the nitrogen content: from lower to higher pressures, this content ranged from 5 to 7%, respectively and the N/C ratio which ranged from 0.14 to 0.16.However, it is clear that the defluorination of the surface is highly pressure-dependent; the F/C ratio decreased from 1.45, for a 100 mtorr pressure, to 1.1 at 600 mtorr.","Therefore, it is evident that despite a similar nitrogen insertion, the chemical moieties formed on the surface depend upon the pressure within the chamber.","Further experiments were carried out to determine the time required to reach an approximately constant surface concentration of nitrogen.","The nitrogen concentration reached a plateau after a treatment time of 160 seconds, as shown by the percentage of nitrogen and the N/C ratio measured on the surface (FIG.","5).","Defluorination of the surface, despite showing a more complex behavior, appears to follow a similar pattern.","The above results have shown that the pressure, the duration and the RF power of the treatment all have an effect on the chemical composition as seen from the surface characterization parameters given by the survey spectra.","In particular, these results raise the possibility of presence of different nitrogen species as a function of the above three treatment parameters.","As several nitrogen containing species may be formed upon the ammonia RFGD plasma treatment, we have performed surface derivatization in order to quantify the percentage of the nitrogen present as NH2 species.","The results of the surface derivatization on model surfaces (not shown) demonstrated that chlorobenzaldehyde specifically reacts with the amine moieties as depicted in FIG.","6.Therefore, the chlorine XPS signal of the derivatized surface allows the determination of the surface amine concentration as described by d'Agostino et al.(62).","Using the surface derivatization technique, it was possible to show that up to 45% of all the nitrogen species grafted onto the surface during the plasma treatment are amine moieties (as shown in Table 1 by % of N0 present as NH2).","In addition, the data presented in the Table 1 also indicate that the surface amine concentration may be controlled by changing the plasma treatment duration.","For example, the surface concentration of NH2 on the surface goes from 3.6% for a 50 second treatment duration to 6.0% when the surface is treated for 250 seconds.","However, too long exposures to the plasma environment should be avoided to minimise the occurrence of polytetrafluoroethylene chain scission or formation of double bonds.","(64) TABLE 1 Quantification of amine groups on plasma-treated PTFE film Plasma treatment % of N0 Surface concentration Duration F/C N/C % N0 present as NH2 of NH2 250 s 0.499 0.269 14.3 42 6.0 100 s 0.626 0.229 11.9 42 5.0 50 s 0.865 0.249 11.6 31 3.6 One skilled in the art will appreciate that the surface amine groups can be created on the surface by methods different from the one presented here without departing from the spirit of the present invention.","For example, amine groups can be created on the surface not only by using an ammonia plasma, but also by using plasmas of N2H4 (hydrazine),(65,66) aliphatic amines,(67) coating-forming gases such as allylamine,(68-71) to name a few non-restrictive possibilities.","U.S. Pat.","No.","6,159,531,(72) for example, briefly discusses the relative merits of these gases in view of their use as precursors to surface amine groups.","Various methods of activating the surface can also be used, other than the continuous capacitively coupled RF plasma used here.","Without departing from the spirit of the present invention, these include, but are not limited to, pulsed plasma,(68,70,73) inductively coupled RF plasma,(74-76) corona discharge,(77,78) microwave plasma(79,80) and UV irradiation.","(81,82) As an example, one can read U.S. Pat.","No.","5,922,161,(83) which teaches the use of such treatments for the partial surface oxidation of polymers.","Furthermore, without departing from the spirit of the present invention, one skilled in the art will appreciate that surface reactive groups other than amine can successfully react with the oxyphosphorodichlorinated derivative form of phosphorylcholine or another so-activated molecule.","Hydroxyl and thiol groups(84-87) are two non-restrictive examples of such surface groups.","Conversely, different pathways, in addition to the one proposed here, can be followed to obtain an activated form of phosphorylcholine, as one skilled in the art knows.","These additional activation methods include, but are not limited to, using reagents such as PCl5, PCl3, PBr5, PBr3, PI3, SOCl2, SOBr2, COCl2, COBr2, (COCl)2, (COBr)2 to directly activate phosphorylcholine through the formation of at least a reactive P-halogen bond.","(87,88) Storage in Atmosphere of the Ammonia Plasma Treated Prostheses Surface Atomic Concentrations as a Function of the Storage Time in Air The surface atomic concentration of nitrogen on the surface of a treated prosthesis tends to slowly decrease (from 14% initially to 12% after 80 days of storage in air or a decrease of N/C from 0.3 to 0.2) and oxygen uptake is also observed (O/C increased from 0.05 to 0.3 after 20 days)(45).","Therefore, it is preferable to perform the grafting of the molecule shortly after the plasma treatment, preferably within two hours(45).","PRC Grafted ePTFE Surfaces The atomic surface compositions were also determined by XPS spectroscopy.","First, the survey spectra clearly showed that there is phosphorus and chlorine atom integration in approximately similar concentrations, as expected from the molecular structure of PRC (chlorine is present as the counterion of the N(CH3)3+ moiety of PRC).","The extent of grafting of the PRC was therefore estimated from the percentage of phosphorus detected by XPS.","Moreover, FIG.","7 shows that the N 1 s High Resolution XPS spectra clearly indicates that there is a good correlation between the trimethylammonium peak at 402 eV (a peak which is not present on an ammonia plasma-treated prosthesis) and the percentage of phosphorus grafted onto the prosthesis.","Indeed, there is a linear relation between the extent of PRC grafting (characterized by % P), and the trimethylammonium peak, +N(CH3)3 (FIG.","7).","The C 1 s peak (not shown) for each of the three methyl groups from the trimethylammonium moiety, +N(CH3)3, followed the same trend and provided further indication of the successful grafting of PRC onto the surface.","The PRC grafting on PTFE films was also confirmed by static SIMS (static Secondary Ion Mass Spectroscopy).","This method identifies molecules adsorbed on a surface by recording the mass spectra of atomic or molecular particles which are emitted when a surface is bombarded by energetic primary particles.","The secondary ions emitted from the surface result from the fragmentation of the initial surface molecules and are only detected under their ionized state (positive and negative state).","The peaks are given in Dalton (molecular weight—gmol−1).","Thus, from an analysis of these spectra it is possible to determine the initial structure of the molecules grafted onto the surface.","The spectra of a PRC-grafted PTFE film (not shown) gave the following fragments and confirmed the PRC grafting.","122.1 78.8 PO3− 58.1 +N—(CH3)3 62.8 PO2− 43 +N—(CH3)2 O—CH2—CH+ 29 +N—CH3 15 CH3+ III.","Sterilization of Inner-Surface Modified Vascular Prostheses For the present method of surface modification to be useful, it is required that sterilization not remove the grafted molecules.","A very widely used method for sterilization is autoclaving.","At least two possible methods of autoclaving may be contemplated: 1) sterilization under vapor pressure: 120° C., 15 PSI, 20 min; and 2) dry sterilization: short duration and high temperature (180° C., 1 h) or for a longer duration at a lower temperature (150° C., 2 h).","According to scientific literature, PRC derivatives are stable when subjected to the first sterilization procedure.","In addition, ePTFE prostheses are more frequently sterilized in this manner.","This method is recognized and used by hospitals.","Sample PRC-grafted films and prostheses were placed in a sterilization bag and sealed.","A sterilization marker, a piece of adhesive tape, was placed on the bag; this indicator darkens when sterilization is successful.","Following the sterilization procedure, the sample prostheses were analyzed by XPS to determine whether their compositions and the distribution of the phosphorus atoms had been altered.","The results are summarized in Table 2 below.","TABLE 2 Effect of Sterilization on PRC-Grafted Prostheses Sterilization Before After After 6 months % P 5 ± 1 4 ± 1 3.4 ± 0.6* *This data was obtained from a 8 cm-segment of a 6 mm ID prosthesis (yielding 60 data points) whereas the other values were estimated from a shorter segment (1 cm, yielding 6 data points), hence the difference in the standard deviations.","Despite the slight decrease in the percentage of phosphorus, which is taken as a measure of the amount of PRC on the surface because it is otherwise absent, PRC grafting was found to be stable when subjected to sterilization under vapor pressure and the distribution of the grafting on the surface remained homogeneous.","The surface concentrations were stable after 6 months and the distribution remained homogeneous (the same was observed for intermediate times of 1 and 2 months for different prostheses).","Consequently, the sterilization procedure most practiced by hospitals does not appear to adversely modify the quantity and distribution of the grafted molecules on the PRC-treated vascular prostheses.","IV.","In Vitro Testing of Inner-Surface Modified Vascular Prostheses A. Non-Toxicity of Ammonia RFGD Plasma Treated Prostheses To verify the non-toxicity of ammonia plasma treated prostheses, we used the diffusion test which determines whether cellular growth is inhibited by the biomaterial by using a coloring agent specific to DNA (Hoechst).","The elution test, also known as “extract dilution”, was also used; it determines whether there is inhibition of cellular growth by looking for an extract of biomaterial.","a) Diffusion Test Human endothelial cells derived from an umbilical cord were cultured in a 24-well plate (1×104 cells/well), taking care to pre-treat each well with gelatin before use.","The cells were allowed to grow in Medium 199 containing 10% FBS, heparin (90 g/ml), L-Glutamine (2 mM), penicillin G (100 I.U./ml), streptomycin sulfate (100 μg/ml), amphotericin B (250 ng/ml) and ECGS (20 μg/ml).","Circular prosthetic samples measuring 7 mm in diameter from both treated and untreated prostheses were placed in the 24 wells.","The cultures were incubated for 3 days with 5% CO2 in a humid atmosphere.","Subsequently, the cultures were washed, lysed, treated with Hoechst #33358 and finally transferred to another plate designed for fluorescent studies.","Readings were taken with a Bio-Tek FL600 fluorometer, and the quantity of DNA calculated from a calibration curve.","At the same time, cells were cultured in wells without prosthetic samples (control).","b) Elution Test Two samples, one from a pre-treated prosthesis and the other from an untreated prosthesis, were incubated in M199 for 7 days.","Twenty-four hours before the end, endothelial cells were cultured in a 24-well plate.","At the seventh day, the cells were cultured in the M199 medium used with the prostheses.","(It is important to note here that, in contrast to what is done in the Diffusion Test, only the medium is used; the sample prosthetic devices are set aside following the incubation period.)","The endothelial cells are incubated for 3 days under the same conditions as those for the Diffusion Test and analyzed in the same way (fluorometry).","Table 3 shows the results for the Diffusion and Elution Tests on untreated (virgin) prostheses, on ammonia plasma treated prostheses and on control samples.","(All experiments were done in duplicate, series A and B.)","These results demonstrate that there are no significant variations in the level of cellular proliferation.","It may therefore be concluded that ammonia plasma treated ePTFE vascular prostheses are no more toxic than untreated ePTFE vascular prostheses.","TABLE 3 Results of in vitro Cellular Cultures Analyzed by Fluorometry Ammonia Plasma Untreated Prostheses treated Prostheses Control Test (*104) (*104) (*104) Diffusion Series A 54 ± 1 52 ± 2 Series B 43.7 ± 0.5 43.1 ± 0.5 40 ± 1 Elution Series A 32.9 ± 0.4 33 ± 1 Series B 28 ± 1 32 ± 1 31 ± 2 Cytotoxicity of the PRC-Grafted Vascular Prostheses As discussed above, toxicity tests performed on prostheses following plasma treatment but prior to the anchoring of the molecules revealed that the plasma-treated prostheses were non-toxic, or at least no more toxic than virgin prostheses.","The next logical step was to determine whether prostheses which had been completely treated by the process of the present invention were also non-toxic to cells, and particularly to endothelial cells which naturally line the internal surface of blood vessels.","Endothelial cells are currently used to verify the toxicity of certain products as well as to check the efficiency of sterilization.","In cytotoxicity testing, the prostheses are not in direct contact with endothelial cells and the incubation period is 3 days at 37° C. Cellular growth is determined by the fluorescence of DNA strands using specific markers.","Testing was repeated several times in order to obtain the best statistical average for the cell culture results.","To gain a better appreciation of the effect of PRC grafting on the cells, tests were also performed on untreated prostheses as well as with “control” cell cultures.","The histogram in FIG.","8 clearly shows that treated prostheses have no more effect on cell growth than do untreated prostheses and neither of them have a significant effect on the growth of the cells.","Hence, the prostheses treated with the process of the invention are non-cytotoxic and in fact seem to slightly favor endothelial cell growth.","These findings are promising given that endothelial cells must line the internal surfaces of the prostheses.","B) Blood Platelets Adhesion Adhesion and Aggregation of Blood Platelets The adhesion and aggregation of blood platelets on prosthetic devices has a direct effect on hemocompatibility, since the platelets are involved in the coagulation cascade.","Indeed, the adherence of platelets on the internal surfaces of prostheses may prevent the adherence of endothelial cells (the natural lining of blood vessels), and may result in thrombosis if the accumulation is too important.","In vitro studies were performed several times in the following manner.","Following the centrifugation of human blood, the platelet-rich plasma was extracted and placed on prostheses for one hour at 37° C. The prostheses were then treated with different solutions before analyses by SEM.","FIG.","9 shows SEM photographs of treated and untreated prosthetic devices.","A comparison of the two photographs reveals that the untreated prosthesis has a significantly greater accumulation and activation of platelets than the prosthesis that had been treated with the process of the invention.","Hence, the treated prosthesis appears to prevent much more effectively the accumulation of blood platelets.","Quantification of Blood Platelets After centrifugation of human blood, the platelet-rich plasma (5*104 platelets/μl count by a hemocytometer) was extracted, radiolabelled with 51Cr and placed on prostheses for two hours at 37° C. The prostheses were then washed three times with sterile PBS before analyses and the amount of platelet uptake was evaluated by a gamma scintillation counter adjusted to 270-370 keV.","This test was performed 3 times with human blood from six donors (FIG.","10).","In all cases, no significant difference in the adherent platelets was noticed for untreated and treated prostheses.","C) Neutrophils Neutrophils are characteristic of the inflammatory response of an individual to a foreign material.","After centrifugation of human blood, the leukocyte-rich suspension was extracted, radiolabelled with 111In and placed on prostheses for two hours at 37° C. The prostheses were then analyzed and the adherent neutrophils were evaluated by a gamma scintillation counter adjusted to 260-480 keV.","This test was performed three times with blood from six different donors (FIG.","11).","For every individual, there were fewer adherent neutrophils on the treated prosthesis than on the untreated prosthesis.","Thus, it appears that the inflammatory response was reduced by the PRC grafted on the prosthesis.","D) Clotting Time Platelet adhesion to biomaterials is often used as an indication of blood compatibility, but a more clinically relevant issue is whether the adherent platelets are able to promote clot formation.","Indeed, clotting events require the presence of other blood elements such as fibrinogen to catalyze the clotting cascade.","Longer clotting times are taken as indicative of a better thrombogenic resistance of a prosthesis in contact with blood.","Normal human blood was collected into a citrate anticoagulant tube and a 1-ml sample was deposited onto the surface of the prosthesis.","The clotting time was determined visually: the blood was deemed to have clotted when there was no longer a movement of the blood in response to a tilting motion of the sample prosthesis.","This test was performed three times with blood from six human donors, on untreated and treated (PRC-grafted) prostheses (FIG.","12).","As observed in FIG.","12, clotting times were significantly longer on PRC-grafted prostheses.","Thus, it appears that the treated prosthesis is less thrombogenic when compared to the untreated prosthesis.","E) Thromboelastography Another important measure of the thrombogenic potential of a material is the evolution of the elastic properties of whole blood clots that form on the surface of the material.","For a given material in contact with blood, the thrombogenicity index takes into account the coagulation time as well as the elasticity of the blood clot.","Surfaces with good blood compatibility should exhibit low thrombogenecity indexes, as measured by a thromboelastograph.","This technique uses a steel piston placed in a cuvette having a 1-mm clearance between the piston and the inner surface of the cuvette.","Blood is placed inside the cuvette.","As coagulation proceeds, the elasticity of the forming blood clot varies and these changes are detected by the torsion wire to which the piston is suspended.","This is converted to an electric signal which is sent to a chart recorder.","The resulting trace is called the thromboelastogram (TEG), which records the elasticity of the formed blood clot.","This test was performed using the blood of six human donors, on samples of untreated and treated prostheses (FIG.","13).","Comparison of the index of thrombogenic potential for treated and untreated prostheses showed a lower thrombogenic potential for the PRC-grafted prostheses.","Values ranged from 17 to 45% lower, except for one individual where the value was 30% higher.","Thus, the treated prostheses improved the non-thrombogenic potential of the initial material.","F) Fibroblast Cultures The development of an endothelium, which is the natural hemocompatible surface, on the surface of prostheses would render them more hemocompatible.","Fibroblasts, which are cells capable of differentiation, are commonly used for in vitro hemocompatibility tests.","By testing the adsorption of fibroblasts, one can measure the ability of a material to act as a viable substrate for the adsorption and spreading of cells.","The adsorption and spreading of cells represent conditions that are necessary for the development of the endothelium.","Fibroblasts were placed in direct contact with the prostheses in order to observe the ability of cells to develop on different materials.","The cells were allowed to grow for 7 days at 37° C. The fibroblasts were then observed through a microscope using two coloring agents: the Hoëscht reagent marked the nucleus (allowing the cells to be counted) and rhodamine dyed the actin filaments in the cytoplasm (allowing the shape of the cells to be determined).","As shown in FIG.","14, the development of fibroblasts on the surface of the prostheses treated by the process of the present invention was greatly enhanced in comparison to the untreated prosthesis: the number of nuclei is greater, and the shape of the fibroblasts is more elongated, which shows that the cells adhere to and develop on the surface.","G) Endothelial Cell Cultures Endothelial cells are the cells that naturally line the internal surface of blood vessels; they therefore constitute the ideal hemocompatible surface.","Because endothelial cells are known to be more fragile and sensitive than fibroblasts, this test is more severe.","Endothelial cells were cultured following the same protocol as that used for the fibroblasts.","The incubation period was 3 days at 37° C. The development of endothelial cells was greatly enhanced on the prosthesis treated by the process of this invention than on the untreated one.","FIG.","14 shows the optical microscope image obtained after using the Hoëscht reagent to mark the nucleus of the endothelial cells.","The number of cell nucleus is clearly more important on the treated prosthesis, as is the spreading of the cells, as observed by SEM (FIG.","14).","V. In Vivo Testing of Inner-Surface Modified Vascular Prostheses The in vivo response of the ANBIOPA™-treated prostheses was tested in a canine model.","The implantation period was one month and six dogs received a prosthesis: three received an untreated Gore-Tex™ ePTFE prosthesis while the three others received an ANBIOPA™-treated prosthesis.","Surgery (The following procedure, while described in relation to a single dog, was used on all six dogs that participated in the trial.)","After a 24-hour fast, a conditioned mongrel dog having a weight of between 20 and 25 kg was pre-anaesthetized by intra-veinous administration of atropin sulfate (0.05 mg/kg) and acepromazin maleate (0.1 mg/kg).","The dog's abdomen was shaved and disinfected by an application of Hibitane and Proviodine.","The dog was then placed on a surgical table in decubitus position, anaesthetized by intra-veinous administration of Penthothal (10 mg/kg) and then ventilated with a mix of air and isofuran to maintain anaesthesia.","After abdominal incision, the sub-renal aorta was localized and cleared of surrounding structures before the collateral arteries were tied.","Prior to clamping, heparin (0.5 mg/kg) was injected as an anticoagulant.","The aorta was clamped upstream and downstream of a 5 cm segment in an aorto-aortic position and this segment was replaced with a 6 mm diameter vascular prosthesis (untreated commercially available prothesis or ANBIOPA™-treated prosthesis) of the same length by termino-terminal anastomosis using a Surgilene™ monofilament.","The dog was stitched and butorphanol tartrate (0.2 mg/kg) was subcutaneously injected as an analgesic for three days after surgery.","Following surgery, the dog was fed with a standard diet until explantation of the prosthesis one (1) month later.","Image Analysis SEM observation on the explanted prostheses shows clear differences between the untreated commercially available prostheses and the ANBIOPA™-treated prostheses (FIG.","15).","At low magnification, the surface of an ANBIOPA™-treated prosthesis remains uniform with a smooth surface and is almost deposit free (FIG.","15a).","Further observations at higher magnification reveal a coating consisting of several layers of red blood cells (FIG.","15c).","The lack of activated platelets and fibrin clot on an ANBIOPA™-treated prosthesis surface suggests an anti-thrombogenic property.","On the other hand, an untreated commercially available prosthesis shows important deposits on the surface at low magnification (FIG.","15b) with deposits mainly composed of a network of activated platelets and red blood cells trapped in a fibrin clot (FIG.","15d), thus providing evidence of thrombus formation.","Although the present invention has been described by way of preferred embodiments thereof, it can be modified without departing from the spirit and nature of the subject invention, as defined in the appended claims.","LIST OF REFERENCES 1.R.","Guidoin, N. Chakfé, S. Maurel, T. How, M. Batt, M. Marois and C. Gosselin, Biomaterials 14, 678 (1993).","2.B.","D. Ratner, A. Chilkoti and G. P. Lopez, Plasma deposition and treatment for biomedical applications, in Plasma Deposition, Treatment, and Etching of Polymers, R. D'Agotino, ed., Academic Press, New York, (1990), p. 463.3.B.","D. Ratner, J. Biomed.","Mat.","Res.","27, 837 (1993).","4.B.","Haimovich; A. Freeman; R. Greco, Thromboresistant surface treatment for biomaterials, U.S. Pat.","No.","5,578,073 (1996).","5.P.","D. Drumheller, Materials and method for the immobilization of bioactive species onto polymeric subtrates, U.S. Pat.","No.","5,874,165 (1999).","6.J.","R. Keogh, Ionic attachement of biomolecules with a guanidine moiety to medical devices surfaces, U.S. Pat.","No.","5,928,916 (1999).","7.J.","M. McHaney and C. E. Banas, Carbon containing vascular graft and method of making same, U.S. Pat.","No.","5,827,327 (1998).","8.J.","R. Keogh, Method for a covalent attachment of biomolecules to surfaces of medical devices, U.S. Pat.","No.","6,033,719 (2000).","9.J.","R. Keogh, Periodate oxidative method for attachment and crosslinking of biomolecules to medical device surfaces, U.S. Pat.","No.","6,017,741 (2000).","10.J.","R. Keogh, Method for attachment of biomolecules to medical devices surfaces, U.S. Pat.","No.","5,925,552 (1999).","11.J.","R. Keogh, Oxidative method for attachment of glycoproteins or glycopeptides to surfaces of medical devices, U.S. Pat.","No.","5,891,506 (1999).","12.J.","R. Keogh, Oxidative method for attachment of glycoproteins to surfaces of medical devices, U.S. Pat.","No.","5,728,420 (1998).","13.P.","T. Cahalan, M. Verhoeven, M. Hendricks, L. Cahalan, Biocompatibility of solid surfaces, U.S. Pat.","No.","5,702,818 (1997).","14.P.","E. Guire, Biocompatible device with covalently bonded biocompatible agent, U.S. Pat.","No.","5,263,992 (1993).","15.D.","G. Castner, P. Favia and B. D. Ratner, Deposition of fluorocarbon films by remote RF glow discharges, in Surface Modification of Polymeric Biomaterials, B. D. Ratner and D. G. Castner, eds., Plenum Press, New York (1997), p. 45.16.B.","D. Ratner, D. Leach-Scampavia and D. G. Castner, Biomaterials 14, 148 (1993).","17.A.","S. Hoffman, A. M. Garfinkle, B. D. Ratner and S. R. Hanson, Plasma gas discharge treatment for improving the compatibility of biomaterials, U.S. Pat.","No.","5,034,265 (1991).","18.T.","A. Horbett, B. D. Ratner, J.","A. Chinn and Y. Haque, Radio frequency plasma deposited polymers that enhance cell growth, U.S. Pat.","No.","4,919,659 (1990).","19.A.","S. Hoffman, A. Garfinkle, B. D. Ratner and S. R. Hanson, Plasma gas discharge treatment for improving the biocompatibility of biomaterials, U.S. Pat.","No.","4,656,083 (1987).","20.M.","Garfinkle, A. S. Hoffmann, B. D. Ratner, L. O. Reynolds, S. R. Hanson, Trans.","Am.","Soc.","Artif.","Intern.","Organs 30, 432 (1984).","21.J.","C. Lin and S. L. Cooper, J. Appl.","Polym.","Sci.",": Appl.","Polym.","Symp.","54, 157 (1994).","22.P.","V. Narayanan, J. Biomater.","Sci.","Polymer Edn, 6, 181 (1994).","23.Y.","Matsuzawa and H. Yasuda, J. Appl.","Polym.","Sci.","38, 65 (1984).","24.P.","Favia, V. H. Perez-Luna, T. Boland, D. G. Castner and B. D. Ratner, Plasmas & Polymers 1, 299, (1996).","25.Y.","Babukutty, M. Kodama, H. Nomiyama, M. Kogoma and S. Okazaki, Preparation of acrylamide coated PVC tube by APG treatment and its characterization, in Advanced Biomaterials in Biomedical Engineering and Drug Delivery Systems, N. Ogata, S. W. Kim, J. Feijen and T. Okano, eds., Springer-Verlag, Tokyo (1996), p. 217.26.A.","Dekker, K. Reitsma, T. Beugeling, A. Bantjes, J. Feijen and W. G. van Aken, Biomaterials 12, 130 (1991).","27.T.","A. Giroux and S. L. Cooper, J. Colloid Interface Sci.","146, 179 (1991).","28.N.","Inagaki, S. Tasaka and H. Kawai, J. Adhesion Sci.","Technol.","3, 637 (1989).","29.Y.","Nakayama, T. Takahagi, F. Soeda, S. Nagaoka, J. Suzuki and A. Ishitani, J. Polym.","Sci., Part A: Polym.","Chem.","26, 559 (1988).","30.J.","R. Hollahan, B.","B. Stafford, R. D. Falb and S. T. Payne, J. Appl.","Polymer Sci.","13, 807 (1969).","31.R.","E. Marchant, Anticoagulant plasma polymer-modified substrate, U.S. Pat.","No.","5,455,040 (1995).","32.H.","K. Yasuda, Method of making a biocompatible prosthesis, U.S. Pat.","No.","4,994,298 (1991).","33.T.","Karwoski and Y. Matsuzawa, Glow discharge process for producing implantable devices, U.S. Pat.","No.","4,632,842 (1986).","34.P.","V. Narayanan, Radiofrequency plasma biocompatibility treatment of inside of medical tubing and the like, U.S. Pat.","No.","5,244,654 (1993).","35.P.","V. Trescony, P. Cahalan and K. Keeney, Vascular prosthesis and method, U.S. Pat.","No.","5,246,451 (1993).","36.P.","V. Narayanan, S. M. Rowland and K. D. Stanley, Treatment of metallic surfaces using radiofrequency plasma deposition and chemical attachment of bioactive agents, U.S. Pat.","No.","5,336,518 (1994).","37.P.","V. Narayanan, Radiofrequency plasma biocompatibility treatement of inside surfaces, U.S. Pat.","No.","5,486,357 (1996).","38.F.","Schué, G. Clarotti, J. Sledz, A. Mas, K. E. Geckeler, W. Göpel, A. Orsetti, Makromol.","Chem, Macromol.","Symp.","73, 217 (1993).","39.R.","Sipehia, Biomat., Art.","Cells & Immob.","Biotech.","21, 647 (1993).","40.U.","König, A. Augsburg, F. Simon, D. Lunkwitz, G. Hermel, C. Werner, M. Müller, H. J. Jacobash, ACS Polymer Preprints.","38, 1079 (1997).","41.F.","Simon, G. Hermel, D. Lunkwitz, C. Werner, K. Eichhorn and H. J. Jacobash, Macromol.","Symp.","103, 243 (1996).","42.R.","Sipehia, G. Martucci, M. Barbarosie and C. Wu, Biomat., Art.","Cells & Immob.","Biotech.","21, 455 (1993).","43.D.","Ratner, Biomaterials science: an interdisciplinary endeavor, in Biomaterials science: an introduction to materials in medicine, B. D. Ratner, A. S. Hoffman, F. J. Schoen, J. E. Lemons, eds., Academic Press, San Diego, (1996), p. 1.44.D.","Mantovani, M. Castonguay, J. F. Pageau, M. Fiset and G. Laroche.","Mater.","Res.","Soc.","Preprints, 544, 21 (1998).","45.D.","Mantovani, M. Castonguay, J. F. Pageau, M. Fiset and G. Laroche.","Plasmas and Polymers, 4, 207 (1999).","46.J.","A. Hayward and D. Chapman, Biomaterials, 5, 135 (1984).","47.R.","F. A. Zwall, P. Comfurius, L. M. M. van Deenen, Nature, 268, 358 (1977).","48.R.","F. A. Zwall, H. C. Hember, Haemostasis, 11, 12 (1982).","49.R.","F. A. Zwall and E. M. Bevers, Subcellular Biochemistry, 9, 163 (1983).","50.Y.","P. Yianni, Biocompatible surfaces based upon biomembrane mimicry, in Structural and dynamic properties of lipids and membranes, eds., P J Quinn & R J Cherry, Portland, Press Research Monograph, 1992, p. 187-216.51.A.","A. Durrani, J.","A. Hayward and D. Chapman, Biomaterials, 7, 121 (1986).","52.Y.","P. Yianni, S. R. Hanson, A.","B. Lumsden, Journal of Surgical Research, 77, 119 (1998).","53.K.","Ishihara, R. Aragaki, A. Watenabe, N. Nakabayashi, J. Biomed.","Mater.","Res., 24, 1069 (1990).","54.K.","Ishihara, N. Nakabayashi, K. Fukumoto, J. Aoki, Biomaterials, 13, 145 (1992).","55.K.","Ishishara, H. Oshida, Y. Endo, T. Ueda, A. Watanabe, N. Nakabayashi, J. Biomed.","Mater.","Res., 26, 1543 (1992).","56.K.","Ishihara, H. Hanuyada, N. Nakabayashi, Biomaterials, 16, 873 (1995).","57.Y.","Iwasaki, K. Ishihara, N. Nakabayashi, G. Khang, J. H. Jeon, J. W. Lee, H. B. Lee, J Biomater Sci Polym Edn, 9, 801 (1998).","58.A.","E. Gal, F. J. Fash, Lipids, 12, 314 (1976).","59.J.","C. Lin and S. L. Cooper, J. Appl.","Polymer Sci.:Appl.","Polymer Symp.","54, 157 (1994).","60.F.","Fracassi, Architecture of RF plasma reactors, in Plasma Processing of Polymers, R. D'Agostino, P. Favia, F. Fracassi, eds., NATO-ASI Series E: Applied Science—Vol.","346, Kluwer Academic Publishers (1997), p. 47.61.M.","E. Ryan and J. P. S. Badyal, Macromolecules 28, 1377 (1995).","62.P.","Favia, M. V. Stendardo and R. d'Agostino, Plasmas and Polymers 1, 91 (1996).","63.S.","F. Popescu, M. Castonguay, C. Doillon, R. C. Gaudreault, G. Laroche.","Society for Biomaterials 25th Annual Conference, Proceeding vol.","XXII, 574, Apr.","28-May 2, 1999, Rhode Island, USA.","64.P.","Chevallier, M. Castonguay, S. Turgeon, D. Mantovani, J. C. Wittmann, G. Laroche, submitted for publication at J. Phys.","Chem.","65.A.","J. Martinez, S. Manolache, V. Gonzalez, R. A.","Young, F. Denes, J. Biomater.","Sci., Polym.","Ed., 11, 415 (2000).","66.X.","Zhang, R. A.","Young, Mater.","Res.","Soc.","Symp.","Proc., 586, 157 (2000).","67.J.","G. A. Terlingen, L. M. Brenneisen, H. T. J. Super, A. P. Pijpers, A. S. Hoffman, J. Feijenand, J. Biomater.","Sci., Polym.","Ed., 4, 165 (1993) 68.W.","R. Gombotz, A. S. Hoffman, J. Appl.","Polym.","Sci.",": Appl.","polym.","Symp., 42, 285 (1988).","69.H.","J. Griesser, Mater.","Forum, 14, 192 (1990).","70.C.","L. Rinsch, X. Chen, V. Panchalingam, R. E. Eberhart, J-H. Wang, R. B. Timmons, Langmuir, 12, 2995 (1996).","71.J.","G. Calderon, A. Harsch, G. W. Gross, R. B. Timmons, J. Biomed.","Mater.","Res., 42, 597 (1998).","72.M.","H. Dang; P. Chiu, Coating having biological activity and medical implant having surface carrying the same and method, U.S. Pat.","No.","6,159,531 (2000).","73.J.","G. Calderon, R. B. Timmons, Macromolecules, 31, 3216 (1998).","74.P.","Colpo, R. Ernst, J.-P. Keradec, Plasma Sources Sci.","Technol., 8, 587 (1999).","75.M.","Desilets, B. Davies, G. Soucy, P. Proulx, Can.","J. Chem.","Eng., 76, 707 (1998).","76.J.","T. Gudmundsson, A. M. Marakhtanov, K. K. Patel, V. P. Gopinath, M. A. Lieberman, J. Phys.","D: Appl.","Phys., 33, 1323 (2000).","77.R.","Gasparik, S. Ihara, C. Yamabe, S. Satoh, Jpn.","J. Appl.","Phys., Part 1, 39, 306 (2000).","78.J.","Lei, M. Shi, J. Zhang, Eur.","Polym.","J, 36, 1277 (2000).","79.R.","Foerch, J. Izawa, N. S. Mc Intyre, J. Appl.","Polym.","Sci.",": Appl.","Polym.","Symp., 46, 415 (1990).","80.A.","E. Lefohn, N. M. Mackie, E. R. Fisher, Plasmas and Polymers, 3, 197 (1998).","81.S.","I. Kim, S. W. Lee, Y. H. Park, M. Ree, Mol.","Cryst.","Liq.","Cryst.","Sci.","Technol., Sect.","A, 349, 275 (2000).","82.J.","M. Nedelec, J. Grimblot, S. Turrell, M. Bouazaoui, J. Phys.","Chem.","B, 104, 926 (2000).","83.D.","Y. Wu; S. Li; G. W. Stanislaw, Surface treatment of polymers, U.S. Pat.","No.","5,922,161 (1999).","84.Based on the international conference on phosphorus chemistry at Duke University, Durham, N.C. (1981), Phosphorus chemistry—Proceeding of the 1981 international conference, American Chemical Society, Eds.","Library of congress CIP, Data, Vol.","171.85.R.","Neidlen, S. Buseck, Helvetica Chimica Acta, 75, 2520 (1992).","86.R.","Neidlen, P. Greulich, Helvetica Chimica Acta, 75, 2545 (1992).","87.Theilheimer, Synthetic Methods of Organic Chemistry, Finch, Theilheimer, Nutley, Tozer, Eds Karger, Interscience publishers, Basel-München-Paris-London-New-York-New Dehli-Singapore-Tokyo-Sydney, 1946-1998.88.A.","I. Vogel, Vogel's Textbook of Practical Organic Chemistry, including qualitative organic analysis, 4th ed./rev.","by the following members of the School of Chemistry, Thames (formerly Woolwich)—polytechnic, B. S. Furniss .",".",".","[et al.","], London; New York: Longman, 1978."]],"string":"[\n [\n \"Plasma surface graft process for reducing thrombogenicity\",\n \"In accordance with the present invention, there is provided a novel process for modifying the surface properties of a material that is suitable for contact with animal tissue so as to enhance its hemocompatibility and make it less thrombogenic when in use.\",\n \"This process comprises: Exposing the surface of the material to plasma treatment conditions in order to create reactive groups on said surface; activating a molecule with an activator to produce a reactive molecular species capable of forming convalent bonds with the reactive groups created on the surface of the material to form convalent bonds.\",\n \"The invention further encompasses the materials produced by this process as well as devices, such as vascular prosthesis, that are comprised of these process-modified materials.\"\n ],\n [\n \"1.A process for modifying the surface properties of a material suitable for contact with a living tissue comprising: Exposing the surface of the material to plasma treatment conditions in order to create reactive groups on said surface; Activating a molecule with an activator to produce a reactive molecular species capable of forming strong bonds with the reactive groups created on the surface of the material; and Contacting the reactive molecular species with the reactive groups created on the surface of the material to form strong bonds.\",\n \"2.A process as defined in claim 1, wherein said strong bonds are covalent bonds.\",\n \"3.A process as defined in claim 1 or 2, wherein the resulting surface of the material is compatible with living tissue.\",\n \"4.A process as defined in claim 3, wherein said tissue is blood tissue.\",\n \"5.A process as defined in claim 1, wherein: said plasma is ammonia Radio Frequency (RF) plasma and said reactive groups are amine groups; said molecule is selected from the group consisting of choline, heparin, and other molecules known to those skilled in the art for their hemocompatibility, said activator is phosphoryl chloride (POCl3) and said reactive molecular species is the oxyphosphorodichlorinated derivative of said molecule; and said strong bonds are phosphoamide-type covalent bonds.\",\n \"6.A process as defined in claim 5, wherein said plasma treatment conditions consist of the application of a RF power of between about 5 watts to about 500 watts for a time of about 10 seconds to about 30 minutes at a pressure of about 50 mtorr to about 5 torr.\",\n \"7.A process as defined in claim 6, wherein said plasma treatment conditions consist of the application of a RF power of about 20 watts for a time of about 250 seconds at a pressure of about 300 mtorr.\",\n \"8.A process as defined in claim 6, wherein said plasma treatment conditions consist of the application of a RF power of about 15 watts for a time of about 100 seconds at a pressure of about 250 mtorr.\",\n \"9.A process as defined in claim 7 or 8, wherein the third step is performed within about 2 hours of the first step.\",\n \"10.A process as defined in any one of claims 1 to 9, wherein said material suitable for contact with a living tissue is material implantable in an animal's body.\",\n \"11.A process as defined in claim 10, wherein said material implantable in an animal's body is selected from the group consisting of: Microporous expanded polytetrafluoroethylene (ePTFE), polytetrafluoroethylene (PTFE), polyvinylchloride (PVC), polyethylenes, polyesters (such as polyethylene terephtalate—PET), polypropylenes, polyurethanes, polycarbonates, silicones, PVDF and polymer-coated materials, such as metals and ceramics for example.\",\n \"12.A process as defined in claim 11, wherein said material implantable in an animal's body is ePTFE.\",\n \"13.A process as defined in claim 12, wherein said ePTFE is that of the internal surface of a vascular prosthesis.\",\n \"14.A process as defined in claim 13, wherein said vascular prosthesis has an inner diameter of about 1 to 30 mm.\",\n \"15.A material implantable in an animal's body produced by the process of any one of claims 1 to 14.16.A vascular prosthesis produced by the process of claim 13 or 14.17.A device comprising a material as defined in claim 15.18.A device as defined in claim 17, wherein said device is to be used in contact with an animal's tissue.\",\n \"19.A device as defined in claim 18, wherein said tissue is blood tissue.\",\n \"20.A device as defined in claim 18 or 19, wherein said device is selected from the group consisting of: An implant, a prosthesis, an artificial organ, a stent, a cardiac valve, an apparatus contacting blood during an extra-corporal blood circulation, an apparatus contacting blood during a dialysis treatment or any other material with surfaces coming in contact with blood.\",\n \"21.Use of a device as defined in any one of claims 17 to 20 for preventing thrombosis.\",\n \"22.A material suitable for contact with a living tissue characterized in having a surface that is resistant to neutrophil adhesion, platelet adhesion and activation but that enhances the development of fibroblasts and endothelial cells.\",\n \"23.A material as defined in claim 22 which is plasma-treated ePTFE with grafted PRC.\",\n \"24.A device comprising a material as defined in claim 22 or 23.25.A device as defined in claim 24, wherein said device is selected from the group consisting of: An implant, a prosthesis, an artificial organ, a stent, a cardiac valve, an apparatus contacting blood during an extra-corporal blood circulation, an apparatus contacting blood during a dialysis treatment or any other material with surfaces coming in contact with blood.\",\n \"26.Use of a device as defined in claim 24 or 25 for preventing thrombosis.\"\n ],\n [\n \"<SOH> BACKGROUND OF THE INVENTION <EOH>Approximately 350 000 synthetic vascular prostheses are implanted each year as arterial bypasses in Western countries and Japan.\",\n \"Made of Dacron™ (polyethyleneterephthalate) or microporous Teflon™ (expanded polytetrafluoroethylene, or ePTFE), they perform quite well in large-diameter vessels under high flow conditions but have a low patency rate when used as small-diameter bypasses (i.e., coronaries) or as medium-diameter bypasses in low-flow conditions and high-resistance locations (i.e., leg arteries).\",\n \"(The patency rate relates to a biomaterial's ability to remain pervious to blood flow when replacing an artery).\",\n \"A study on 398 ePTFE vascular prostheses implanted as medium-diameter bypasses and retrieved following complications revealed that 65% of them had to be explanted due to thrombosis.\",\n \"(1 ) Endothelial cell lining is the natural blood-compatible surface that covers the inside surface of blood vessels and heart chambers, and it is recognized to be the best possible hemocompatible surface.\",\n \"The principal techniques that have been proposed in the past to promote endothelial cell adhesion and spreading onto the luminal surfaces of vascular prostheses are: (1) endothelial cell seeding; (2) graft pre-treatment with endothelial cell mitogens; (3) increasing structural porosity; and (4) coating the surface with a protein matrix.\",\n \"While several attempts have been made, the complete endothelial cell coverage of the blood-exposed inside prosthetic surfaces has never been observed in humans.\",\n \"It is therefore evident that increasing the patency rate of arterial prostheses is a high priority in current cardiovascular research.\",\n \"It has been shown in many instances that surface modification through plasma treatment is one of the most promising techniques that may be used to improve specific hemocompatibility and general biocompatibility of polymeric materials.\",\n \"(2,3) One advantage of the plasma treatment is that it allows formation of a covalent bond between the modifier and the surface to be treated, unlike other approaches where a coating is deposited inside the prosthesis without formation of a covalent bond (4,5) , where the bond is ionic (6) or where the bioactive substance is incorporated in the biomaterial.\",\n \"(7) Following to the covalent bonding of the modifier to the surface of the material, made possible by plasma treatment, a molecule chosen for its biocompatibility properties can be covalently attached to the modifier either directly or through a spacer molecule.\",\n \"Clinically, the increased stability of a surface treatment involving plasma treatment could result in improved limb salvage rates and a possible future use in small-diameter coronary bypass operations.\",\n \"This, in turn, could minimize the incidence of re-operation for patients and lower social healthcare costs related to surgical interventions of this type.\",\n \"Plasma treatment of biomaterials is not the only process that can yield covalent bonding of molecules possessing desirable hemocompatibility and/or biocompatibility.\",\n \"However, the processes that are described in the relevant prior art frequently involve several steps.\",\n \"(8-14) To date, most of the experiments performed to improve the performance of biomaterial surfaces have been based on flat specimens of material, particularly polymeric sheets.\",\n \"Unfortunately, few experiments involving the use of plasma to activate the surface have been carried out directly on commercial prostheses with a non-planar geometry.\",\n \"Experiments done at the University of Washington, (2,15-20) which consisted mainly in treating commercial Dacron™ arterial prostheses with a plasma treatment in a tetrafluoroethylene (TFE) gas environment so as to coat the internal surfaces of the prostheses with potentially more biocompatible CF 3 groups, were a precursor to the development of the new commercial Radio Frequency Glow Discharge (RFGD)-treated vascular grafts.\",\n \"(17) Yet, despite the existence of many surface treatment systems for biomaterial surfaces, few have resulted in the treatment, as opposed to the coating, of the internal surface of tubular devices.\",\n \"(21-23) Over the last 30 years, a number of studies have been designed to test the ability of plasma to either treat or coat the surfaces of biomedical devices with a view to enhancing their biocompatibility or to modulate the interactions of polymers and tissue when prostheses are implanted in situ.\",\n \"(16,24-30) Plasma polymerizable gas has been used to coat various substrates with a thin polymeric layer, (15,17-19,24,31,32) while non-polymerizable gas has been applied to treat substrate surfaces.\",\n \"(33-38) Despite the fact that many approaches have been developed to coat or treat the surface of biomaterials, the introduction of amino groups through RFGD treatment is particularly interesting for two reasons.\",\n \"First, amino groups are known to facilitate cellular spreading on a biomaterial surface.\",\n \"(39-41) Second, amino groups react readily with other chemical functional groups, allowing for the attachment of specific molecules through interactions sufficiently strong to prevent the leaching of these molecules by the blood stream.\",\n \"(42) For example, amino groups can be used to ionically immobilize heparin, a well-known polysaccharide anticoagulant.\",\n \"(22) While many efforts have been made in the last 20 years in the field of cardiovascular prosthetic devices to develop surfaces with improved human blood compatibility, an acceptable long-term blood-compatible synthetic material has yet to be achieved.\",\n \"The complexity of the interactions between blood and synthetic materials constitutes a major interfacial problem in this respect.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>Surface treatments allow a modulation of the surface properties of a biomaterial to enhance its interfacial reaction with a biological environment.\",\n \"Low pressure plasma surface treatments are particularly advantageous in the design and development of new biocompatible materials, since they permit surface modifications without altering the bulk of the materials' properties.\",\n \"(43) In the context of the present invention, they are particularly useful for modulating different tissue/biomaterial interface properties, but their ultimate utility may reside in their significant improvement of the hemocompatibility of vascular prostheses.\",\n \"Thus, in accordance with the present invention, there is provided a novel process for modifying the surface properties of a material that is to be placed in contact with living tissues.\",\n \"This process comprises: Exposing the surface of the material to plasma treatment conditions in order to create reactive groups on the surface of the material; Activating a molecule with an activator to produce a reactive molecular species capable of forming strong bonds with the reactive groups created on the surface of the material; and Contacting the reactive molecular species with the reactive groups created on the surface of the material to form strong bonds.\",\n \"Preferably, the strong bonds formed are covalent bonds.\",\n \"In a preferred embodiment, the molecule selected for activation is compatible with living tissue, including human tissue.\",\n \"In a specific embodiment the novel process of the present invention further comprises the following features: the plasma is ammonia Radio Frequency (RF) plasma and the reactive groups are amine groups; the molecule selected for activation is chosen from the group consisting of choline, heparin, and other molecules known to those skilled in the art for their hemocompatibility, the activator is phosphoryl chloride (POCl 3 ) and the reactive molecular species is the oxyphosphorodichlorinated derivative of the molecule; and the strong bonds are phosphoamide-type covalent bonds.\",\n \"In a preferred embodiment of the specific embodiment above, the RF power is between about 5 watts to about 500 watts and is applied for a time of about 10 seconds to about 30 minutes at a pressure of about 50 mtorr to about 5 torr.\",\n \"Those skilled in the art will recognize that care has to be taken in order to avoid combinations of time and power that would deliver excessive amounts of energy to the treated surface.\",\n \"Such conditions lead to a microscopically brittle surface.\",\n \"Preferably, the RF power is about 20 watts and is applied for a time of about 250 seconds at a pressure of about 300 mtorr.\",\n \"In yet another preferred embodiment, the RF power is about 15 watts and is applied for a time of about 100 seconds at a pressure of about 250 mtorr.\",\n \"Ideally, the third step of the process is performed within about 2 hours of the first step.\",\n \"The novel process of the present invention is particularly suitable for an implantable material selected from the following group: expanded polytetrafluoroethylene (ePTFE), polytetrafluoroethylene (PTFE), polyethylenes, polyesters, polypropylenes and polyurethanes.\",\n \"In a preferred embodiment, the implantable material is ePTFE, and the treated surface is the internal surface of a vascular prosthesis.\",\n \"In a more preferred embodiment, the vascular prosthesis has an inner diameter of about 1 to 30 mm.\",\n \"The present invention further comprises the materials produced by the novel process, as well as devices comprising these materials, including any of the following: an implant, a prosthesis, an artificial organ, a stent, a cardiac valve, an apparatus contacting blood during an extra-corporal blood circulation, an apparatus contacting blood during a dialysis treatment or any other material with surfaces coming in contact with blood.\",\n \"Additionally, the present invention is meant to cover the use of any such device, particularly to prevent thrombosis in an animal, including a human being.\",\n \"By controlling the RF power, the ammonia pressure, and the treatment duration, the atomic substitution percentage may be modulated and up to 15% of the surface atoms may be substituted with nitrogen, as probed by X-ray photoelectron spectroscopy (XPS).\",\n \"On the surface, different chemical species are present after the treatment, such as amine and imine groups.\",\n \"Storage in air for up to 80 days shows a defluorination of the surface and a slow decrease of the surface nitrogen concentration during this storage.\",\n \"Ammonia RF Plasma treatment can be successfully used to uniformly treat the internal surface of an ePTFE arterial prosthesis despite the fact that care has to be taken to prevent a significant surface reorganization upon exposure to the atmosphere.\",\n \"(44,45) The ammonia RF plasma-treated internal surfaces of vascular prostheses may be reacted with a substance whose molecular structure can render the prostheses more hemocompatible and non-thrombogenic when in use.\",\n \"The molecule is preferably activated phosphorylcholine (PRC) or any other molecule with similar properties, wherein the two hydroxyl groups on the phosphate moiety have been substituted with chlorine to form the dichloro derivative of the molecule.\",\n \"This dichloro derivative readily reacts with the amino groups resulting from the ammonia RF plasma treatment of the inside surfaces of the vascular prostheses, allowing for the effective anchoring of the molecule.\",\n \"It is an object of the present invention to provide a novel process for producing a material suitable for contact with a living tissue.\",\n \"This material is characterized in having a surface that is resistant to neutrophil adhesion and thrombosis but that enhances the development of fibroblasts and endothelial cells.\",\n \"The process may be used, for example, to treat the inside surfaces of devices, such as vascular prostheses, so as to enhance their hemocompatibility and make them less thrombogenic when in use.\",\n \"A further object of the invention is therefore to provide devices treated with the novel process of the present invention.\",\n \"Other objects, advantages and features of the present invention will become more apparent upon reading of the following non restrictive description of preferred embodiments thereof, given by way of example only with reference to the accompanying drawings.\"\n ],\n [\n \"FIELD OF THE INVENTION The present invention relates to a novel process for modifying the surface properties of a material that is suitable for contact with living tissue comprising: Exposing the surface of the material to plasma treatment conditions in order to create reactive groups on said surface; activating a molecule to produce a reactive molecular species capable of forming strong bonds with the reactive groups created on the surface of the material; and contacting the reactive molecular species with the reactive groups created on the surface of the material to form strong bonds.\",\n \"The invention further encompasses the materials produced by this process as well as devices that are comprised of these process-modified materials, such as vascular prostheses.\",\n \"BACKGROUND OF THE INVENTION Approximately 350 000 synthetic vascular prostheses are implanted each year as arterial bypasses in Western countries and Japan.\",\n \"Made of Dacron™ (polyethyleneterephthalate) or microporous Teflon™ (expanded polytetrafluoroethylene, or ePTFE), they perform quite well in large-diameter vessels under high flow conditions but have a low patency rate when used as small-diameter bypasses (i.e., coronaries) or as medium-diameter bypasses in low-flow conditions and high-resistance locations (i.e., leg arteries).\",\n \"(The patency rate relates to a biomaterial's ability to remain pervious to blood flow when replacing an artery).\",\n \"A study on 398 ePTFE vascular prostheses implanted as medium-diameter bypasses and retrieved following complications revealed that 65% of them had to be explanted due to thrombosis.\",\n \"(1) Endothelial cell lining is the natural blood-compatible surface that covers the inside surface of blood vessels and heart chambers, and it is recognized to be the best possible hemocompatible surface.\",\n \"The principal techniques that have been proposed in the past to promote endothelial cell adhesion and spreading onto the luminal surfaces of vascular prostheses are: (1) endothelial cell seeding; (2) graft pre-treatment with endothelial cell mitogens; (3) increasing structural porosity; and (4) coating the surface with a protein matrix.\",\n \"While several attempts have been made, the complete endothelial cell coverage of the blood-exposed inside prosthetic surfaces has never been observed in humans.\",\n \"It is therefore evident that increasing the patency rate of arterial prostheses is a high priority in current cardiovascular research.\",\n \"It has been shown in many instances that surface modification through plasma treatment is one of the most promising techniques that may be used to improve specific hemocompatibility and general biocompatibility of polymeric materials.\",\n \"(2,3) One advantage of the plasma treatment is that it allows formation of a covalent bond between the modifier and the surface to be treated, unlike other approaches where a coating is deposited inside the prosthesis without formation of a covalent bond(4,5), where the bond is ionic(6) or where the bioactive substance is incorporated in the biomaterial.\",\n \"(7) Following to the covalent bonding of the modifier to the surface of the material, made possible by plasma treatment, a molecule chosen for its biocompatibility properties can be covalently attached to the modifier either directly or through a spacer molecule.\",\n \"Clinically, the increased stability of a surface treatment involving plasma treatment could result in improved limb salvage rates and a possible future use in small-diameter coronary bypass operations.\",\n \"This, in turn, could minimize the incidence of re-operation for patients and lower social healthcare costs related to surgical interventions of this type.\",\n \"Plasma treatment of biomaterials is not the only process that can yield covalent bonding of molecules possessing desirable hemocompatibility and/or biocompatibility.\",\n \"However, the processes that are described in the relevant prior art frequently involve several steps.\",\n \"(8-14) To date, most of the experiments performed to improve the performance of biomaterial surfaces have been based on flat specimens of material, particularly polymeric sheets.\",\n \"Unfortunately, few experiments involving the use of plasma to activate the surface have been carried out directly on commercial prostheses with a non-planar geometry.\",\n \"Experiments done at the University of Washington,(2,15-20) which consisted mainly in treating commercial Dacron™ arterial prostheses with a plasma treatment in a tetrafluoroethylene (TFE) gas environment so as to coat the internal surfaces of the prostheses with potentially more biocompatible CF3 groups, were a precursor to the development of the new commercial Radio Frequency Glow Discharge (RFGD)-treated vascular grafts.\",\n \"(17) Yet, despite the existence of many surface treatment systems for biomaterial surfaces, few have resulted in the treatment, as opposed to the coating, of the internal surface of tubular devices.\",\n \"(21-23) Over the last 30 years, a number of studies have been designed to test the ability of plasma to either treat or coat the surfaces of biomedical devices with a view to enhancing their biocompatibility or to modulate the interactions of polymers and tissue when prostheses are implanted in situ.\",\n \"(16,24-30) Plasma polymerizable gas has been used to coat various substrates with a thin polymeric layer,(15,17-19,24,31,32) while non-polymerizable gas has been applied to treat substrate surfaces.\",\n \"(33-38) Despite the fact that many approaches have been developed to coat or treat the surface of biomaterials, the introduction of amino groups through RFGD treatment is particularly interesting for two reasons.\",\n \"First, amino groups are known to facilitate cellular spreading on a biomaterial surface.\",\n \"(39-41) Second, amino groups react readily with other chemical functional groups, allowing for the attachment of specific molecules through interactions sufficiently strong to prevent the leaching of these molecules by the blood stream.\",\n \"(42) For example, amino groups can be used to ionically immobilize heparin, a well-known polysaccharide anticoagulant.\",\n \"(22) While many efforts have been made in the last 20 years in the field of cardiovascular prosthetic devices to develop surfaces with improved human blood compatibility, an acceptable long-term blood-compatible synthetic material has yet to be achieved.\",\n \"The complexity of the interactions between blood and synthetic materials constitutes a major interfacial problem in this respect.\",\n \"SUMMARY OF THE INVENTION Surface treatments allow a modulation of the surface properties of a biomaterial to enhance its interfacial reaction with a biological environment.\",\n \"Low pressure plasma surface treatments are particularly advantageous in the design and development of new biocompatible materials, since they permit surface modifications without altering the bulk of the materials' properties.\",\n \"(43) In the context of the present invention, they are particularly useful for modulating different tissue/biomaterial interface properties, but their ultimate utility may reside in their significant improvement of the hemocompatibility of vascular prostheses.\",\n \"Thus, in accordance with the present invention, there is provided a novel process for modifying the surface properties of a material that is to be placed in contact with living tissues.\",\n \"This process comprises: Exposing the surface of the material to plasma treatment conditions in order to create reactive groups on the surface of the material; Activating a molecule with an activator to produce a reactive molecular species capable of forming strong bonds with the reactive groups created on the surface of the material; and Contacting the reactive molecular species with the reactive groups created on the surface of the material to form strong bonds.\",\n \"Preferably, the strong bonds formed are covalent bonds.\",\n \"In a preferred embodiment, the molecule selected for activation is compatible with living tissue, including human tissue.\",\n \"In a specific embodiment the novel process of the present invention further comprises the following features: the plasma is ammonia Radio Frequency (RF) plasma and the reactive groups are amine groups; the molecule selected for activation is chosen from the group consisting of choline, heparin, and other molecules known to those skilled in the art for their hemocompatibility, the activator is phosphoryl chloride (POCl3) and the reactive molecular species is the oxyphosphorodichlorinated derivative of the molecule; and the strong bonds are phosphoamide-type covalent bonds.\",\n \"In a preferred embodiment of the specific embodiment above, the RF power is between about 5 watts to about 500 watts and is applied for a time of about 10 seconds to about 30 minutes at a pressure of about 50 mtorr to about 5 torr.\",\n \"Those skilled in the art will recognize that care has to be taken in order to avoid combinations of time and power that would deliver excessive amounts of energy to the treated surface.\",\n \"Such conditions lead to a microscopically brittle surface.\",\n \"Preferably, the RF power is about 20 watts and is applied for a time of about 250 seconds at a pressure of about 300 mtorr.\",\n \"In yet another preferred embodiment, the RF power is about 15 watts and is applied for a time of about 100 seconds at a pressure of about 250 mtorr.\",\n \"Ideally, the third step of the process is performed within about 2 hours of the first step.\",\n \"The novel process of the present invention is particularly suitable for an implantable material selected from the following group: expanded polytetrafluoroethylene (ePTFE), polytetrafluoroethylene (PTFE), polyethylenes, polyesters, polypropylenes and polyurethanes.\",\n \"In a preferred embodiment, the implantable material is ePTFE, and the treated surface is the internal surface of a vascular prosthesis.\",\n \"In a more preferred embodiment, the vascular prosthesis has an inner diameter of about 1 to 30 mm.\",\n \"The present invention further comprises the materials produced by the novel process, as well as devices comprising these materials, including any of the following: an implant, a prosthesis, an artificial organ, a stent, a cardiac valve, an apparatus contacting blood during an extra-corporal blood circulation, an apparatus contacting blood during a dialysis treatment or any other material with surfaces coming in contact with blood.\",\n \"Additionally, the present invention is meant to cover the use of any such device, particularly to prevent thrombosis in an animal, including a human being.\",\n \"By controlling the RF power, the ammonia pressure, and the treatment duration, the atomic substitution percentage may be modulated and up to 15% of the surface atoms may be substituted with nitrogen, as probed by X-ray photoelectron spectroscopy (XPS).\",\n \"On the surface, different chemical species are present after the treatment, such as amine and imine groups.\",\n \"Storage in air for up to 80 days shows a defluorination of the surface and a slow decrease of the surface nitrogen concentration during this storage.\",\n \"Ammonia RF Plasma treatment can be successfully used to uniformly treat the internal surface of an ePTFE arterial prosthesis despite the fact that care has to be taken to prevent a significant surface reorganization upon exposure to the atmosphere.\",\n \"(44,45) The ammonia RF plasma-treated internal surfaces of vascular prostheses may be reacted with a substance whose molecular structure can render the prostheses more hemocompatible and non-thrombogenic when in use.\",\n \"The molecule is preferably activated phosphorylcholine (PRC) or any other molecule with similar properties, wherein the two hydroxyl groups on the phosphate moiety have been substituted with chlorine to form the dichloro derivative of the molecule.\",\n \"This dichloro derivative readily reacts with the amino groups resulting from the ammonia RF plasma treatment of the inside surfaces of the vascular prostheses, allowing for the effective anchoring of the molecule.\",\n \"It is an object of the present invention to provide a novel process for producing a material suitable for contact with a living tissue.\",\n \"This material is characterized in having a surface that is resistant to neutrophil adhesion and thrombosis but that enhances the development of fibroblasts and endothelial cells.\",\n \"The process may be used, for example, to treat the inside surfaces of devices, such as vascular prostheses, so as to enhance their hemocompatibility and make them less thrombogenic when in use.\",\n \"A further object of the invention is therefore to provide devices treated with the novel process of the present invention.\",\n \"Other objects, advantages and features of the present invention will become more apparent upon reading of the following non restrictive description of preferred embodiments thereof, given by way of example only with reference to the accompanying drawings.\",\n \"BRIEF DESCRIPTION OF THE FIGURES FIG.\",\n \"1 is a schematic representation of the Radio Frequency Glow Discharge treatment system used in the process of the invention.\",\n \"FIG.\",\n \"2 shows the XPS spectrum of the inside surface of an untreated ePTFE vascular prosthesis and the XPS spectrum of the inside surface of an ammonia plasma treated ePTFE vascular prosthesis.\",\n \"FIG.\",\n \"3 shows the effect on the surface composition of treatments performed at 300 mtorr of ammonia by varying the power (20, 40 and 50 W) and treatment times (10, 120 and 275 seconds).\",\n \"FIG.\",\n \"4 shows the relationship between the pressure within the chamber and the modification of the ePTFE prosthetic surface.\",\n \"FIG.\",\n \"5 shows the percentage of nitrogen and the N/C and F/C ratios measured on the surface as a function of ammonia plasma treatment time.\",\n \"FIG.\",\n \"6 shows the derivatization reaction of a plasma treated PTFE film with chlorobenzaldehyde.\",\n \"FIG.\",\n \"7 shows the relationship between the atomic surface concentration of phosphorous grafted onto the surface (% P) and the intensity of the N+ peak at 402 eV.\",\n \"FIG.\",\n \"8 shows the cellular growth determined by the fluorescence of DNA strands using specific markers with untreated and treated prostheses as well as with “control” cell cultures.\",\n \"FIG.\",\n \"9 reproduces SEM micrographs showing the degree of accumulation of blood platelets within (a) a prosthesis treated in accordance with the process of the invention and (b) an untreated prosthesis.\",\n \"FIG.\",\n \"10 shows the quantification of the adherent blood platelets on untreated and treated prostheses.\",\n \"FIG.\",\n \"11 shows the quantification of the adherent neutrophils on untreated and treated prostheses.\",\n \"FIG.\",\n \"12 shows the influence of a treated and untreated prostheses on the clotting time.\",\n \"FIG.\",\n \"13 shows the influence of a treated and untreated prostheses on the thrombogenic index.\",\n \"FIG.\",\n \"14 displays microscopic slides of the growth of fibroblasts within a prosthesis treated in accordance with the process of the invention ((a) and (c)) and an untreated prosthesis ((b) and (d)).\",\n \"FIG.\",\n \"15 SEM images at 20× and 2000× magnification for ANBIOPA™-treated prostheses and commercially-available untreated prostheses after a one-month implantation in a canine model: a) ANBIOPA™-treated, 20×; b) untreated, 20×; c) ANBIOPA™-treated, 2000×; and d) untreated, 2000×.\",\n \"DESCRIPTION OF A PREFERRED EMBODIMENT For the present purposes, we give the following definitions of the terms “untreated” and “treated”.\",\n \"“Untreated” means a commercially available vascular prosthesis, to which the inventors did not apply any surface modification treatment.\",\n \"“Treated” means a commercially available vascular prosthesis to which the inventors applied the surface modification procedure hereafter described.\",\n \"The term “animal” as used herein is meant to signify human beings, primates, domestic animals (such as horses, cows, pigs, goats, sheep, cats, dogs, guinea pigs, mice, etc.)\",\n \"and other mammals.\",\n \"Generally, this term is used to indicate living creatures having highly-developed vascular systems.\",\n \"I. Pre-Treatment of the Inside Surface of Vascular Prostheses with Ammonia Radio Frequency (RF) Plasma Experimental Method (The Experimental Method is described with reference to FIG.\",\n \"1.)\",\n \"Materials Microporous ePTFE vascular prostheses 6 (6 and 10 mm ID) with a fibril average length of 25 microns were used, and the method is described in relation to a single prosthesis.\",\n \"A microporous ePTFE arterial prosthesis (length 3 to 10 cm) was first washed with high purity methanol for 10 minutes and vacuum-dried.\",\n \"This procedure was performed to remove any contaminants which may have been present on the surface of the “as-received” prosthesis.\",\n \"Following this washing procedure, two small holes were punctured at one end of the prosthesis, facing each other, and a polypropylene monofilament (5-0—Ethicon) 3 was inserted through both holes and a knot tied loosely above the prosthesis.\",\n \"The other end of the monofilament was rolled around a pulley 5, itself located on the horizontal shaft of an electric motor 36 to allow a vertical displacement of the sample prosthesis inside the Pyrex™ tube 2 of the RFGD treatment system.\",\n \"Gravity caused the vascular prosthesis to move down inside the Pyrex™ tube.\",\n \"For vascular grafts longer than 3 cm, it was preferable, in addition to the aforementioned knot and its attachment via the monofilament to the pulley, to attach a polypropylene monofilament 3 at the bottom of the vascular prosthesis and to attach the monofilament to the pulley by first letting the monofilament run down the tube and then up to the pulley.\",\n \"With this set-up, the motor can pull on the prosthesis when it is going down the Pyrex™ tube, and therefore friction of the prosthesis on the inside wall of the tube ceases to be a problem when moving the prosthesis down the tube.\",\n \"Prior to closing the system, a container and its cap were placed inside a bell jar 1 to collect and store the prosthesis in an argon atmosphere following the RFGD treatment, thereby minimizing any contact of the active sites on the surface of the prosthesis with air.\",\n \"Plasma Equipment As shown in FIG.\",\n \"1, the RFGD treatment system used can be generally represented by the numeral 10.This system is basically comprised of a Pyrex™ glass tube (400 mm long, 12.5 mm i.d.\",\n \"for 10 mm i.d.\",\n \"prostheses and 8 mm i.d.\",\n \"for 6 mm i.d.\",\n \"prostheses) 2 with an internal diameter 4 slightly greater than the external diameter of the prosthesis 6, a configuration which allows for RFGD generation on the internal side of the prosthesis only.\",\n \"Indeed, the path length between the external side of the prosthesis 6 and the Pyrex™ tube 4 is not long enough to allow the electron-induced cascade ionization which is necessary for plasma formation.\",\n \"(11) Therefore, the treatment of prostheses of various diameter requires the selection of a Pyrex™ tube of the appropriate diameter.\",\n \"As mentioned above, the criteria of selection is that no plasma is ignited on the outside surface of the prosthesis during its treatment.\",\n \"A space of about 1 mm all around the outside diameter of the prosthesis and the inside wall of the Pyrex™ glass tube 2 has proven to be appropriate.\",\n \"The Pyrex™ glass tube 2 has an inlet for gas injection 8 located 5 cm below its upper end.\",\n \"Three capacitively coupled copper electrodes 12a, 12b and 12c, spaced from 3 to 5 cm from each other (depending on the length of the prosthesis to treat), are attached to the middle of the tube 2.The vacuum within the chamber was created by a turbo-molecular pump 14 with a pumping speed of 60 liters/s (Model TCP015, Balzers Pfeiffer) which, in turn, is connected to a mechanical pump 16 (Model 1402, Welch Scientific Company).\",\n \"This set-up allows a pressure of close to 4×10−5 torr to be reached.\",\n \"During treatment, pressure is kept constant within the chamber by means of a capacitance pressure gauge/electromechanical valve controller 18 (type 250-1A, MKS Instruments) connected to an electromechanical valve 20 (type 248A-00200SV, MKS Instruments) and a capacitance manometer 22 (Baratron type 626A01TAE, MKS Instruments).\",\n \"Finally, gaseous molecules are excited through a radio-frequency field made by an RF generator 24 (YAESU HF transceiver FT-840, YAESU) that transmits the power, through a power meter 26, to an automatic matching network (Smartuner SG-230, SGC) 28 which then couples the RF power with an antenna made of three copper electrodes 12a-c. Electrodes 12a and 12c, located at both ends of the antenna, are connected to a ground (not shown) while the RF power is brought through the central electrode 12b.\",\n \"The treatment can be operated in a RF power range from 10 to 100 W at 13.56 MHz.\",\n \"Plasma Treatment Initially, all valves on the system are closed.\",\n \"After the introduction of the prosthesis into the bell jar 1, valve 30 is opened and the mechanical pump 16 is activated to reach a vacuum of approximately 10−3 torr.\",\n \"As soon as this pressure is attained inside the system, valve 30 is closed, valve 32 is opened and after a few seconds the turbo-molecular pump 14 is activated.\",\n \"After about one minute, valve 34 is opened and pumping inside the bell jar continued until it reaches a vacuum greater than 5×10−5 torr.\",\n \"The chamber is then isolated from the turbo pump 14 by closing valve 34 and high purity ammonia is introduced into the bell jar 1 via electromechanical valve 20 and, when the pressure is above 0.1 torr, the bell jar 1 is pumped by the mechanical pump 16 alone, through valve 30.The electromechanical valve controller 18 automatically regulates the amount of gas entering the chamber and the pressure, read by the capacitance gauge 22, settles within 1 minute to the value selected on the capacitance gauge/electromechanical valve controller.\",\n \"Typically, this configuration allows plasma generation within a pressure range of 10−3 to 1 torr.\",\n \"With the above parameters established, the plasma is ignited by turning the radio-frequency generator 24 on.\",\n \"Sufficient time is allowed for the matching network 28 to reach a standing wave ratio (SWR) signal of between 1 and 1.15, as read on the power meter 26 in order to provide optimal matching of the power input to the discharge.\",\n \"RFGD treatments are then performed using a static treatment: the prosthesis 6 is initially located just under the inlet 8 while the plasma parameters are adjusted, it is then moved from this position in the Pyrex™ glass tube 2 into the middle of the lighted area (between the three electrodes 12a, 12b and 12c) using the electric motor 36, and is maintained within the plasma for predetermined periods of time.\",\n \"Prostheses longer than 10 cm could be treated by slowly moving the prostheses across the lighted area using electric motor 36, resulting in similar grafting efficiencies.\",\n \"(45) At the end of the treatment, the Radio-Frequency generator 24 is turned off and the gas flow stopped by closing the electromechanical valve 20 via its controller 18.The mechanical pump 16 is kept in operation to remove residual gaseous ammonia from the bell-jar 1 until a pressure of approximately 50 mtorr is reached.\",\n \"It is then flushed with argon for 2 minutes by carefully opening valve 38.This is done in order to allow the excited species resulting from the plasma treatment to reach their ground state.\",\n \"After isolating the mechanical pump 16 from the bell-jar by closing valve 30, argon flow is continued inside the chamber to bring the bell jar back to atmospheric pressure.\",\n \"The prosthesis 6 is then lowered to the bottom of the Pyrex™ tube 2, the bell jar 1 is quickly opened and the prosthesis 6 is disconnected from the polypropylene microfilament 3 and put inside a capped container.\",\n \"It is then placed in a glove box purged with nitrogen where a small sample is taken for XPS studies in order to assess the success of the ammonia treatment.\",\n \"The prosthesis is then prepared for the anchoring process as described below.\",\n \"II.\",\n \"Anchoring of Molecules Mimicry of the non-thrombogenic surface of the erythrocyte has been advocated as the starting point for the development of non-thrombogenic biomaterials.\",\n \"Since phosphorylcholine (PRC) is a major component of the outer surface of the erythrocyte, materials containing it would be expected to be non-thrombogenic.\",\n \"(46-50) Moreover, it is known that PRC groups attached to polymer surfaces improve hemocompatibility.\",\n \"(51-57) In order to enhance the hemocompatibility and non-thrombogenicity of the interior surfaces of vascular prostheses, prostheses which had been pre-treated with plasma (as described above) were made to react with activated analogues of molecules such as phosphorylcholine (PRC).\",\n \"Since the ultimate goal is the implantation of such prostheses into the human body, the molecules must be solidly attached to the inside walls of the prostheses.\",\n \"Consequently, it is necessary to form strong bonds (preferably, covalent bonds) between the molecules and the amine groups created by the plasma treatment rather than to rely on the formation of ionic bonds or adsorption phenomenon to secure the molecules onto the inside surfaces of the prostheses.\",\n \"Additionally, it is desirable that any chemical treatment of this type be simple and inexpensive.\",\n \"The general structure of the molecules is represented below: Direct reaction between PRC and the amino groups present on the surface of the plasma-treated prostheses results in ionic bonds and/or surface adsorption neither of which, as previously stated, is sufficiently strong or stable for the applications contemplated.\",\n \"It is therefore necessary to enhance the reactivity of these molecules by synthesizing reactive analogues so that they may partake in the creation of phosphoamide-type covalent bonds with the amino groups on the surface of the ammonia plasma pre-treated prostheses.\",\n \"In this way, the molecules may be anchored effectively to the interior surface of the prostheses.\",\n \"In order to achieve the desired covalent reaction, the dichlorinated derivative RPO(Cl)2 of the target molecule RPO(OH)2 must first be synthesized.\",\n \"The synthesis of the dichlorinated derivatives of the molecules suitable for reaction with the amino groups on the inside surface of the pre-treated vascular prostheses may be achieved in at least two ways, as revealed diagrammatically below with PRC.\",\n \"In the first pathway, PRC, which is available commercially in the form of a calcium salt, is reacted with an acidic resin(58) to eliminate the calcium.\",\n \"The product is then made to react with phosphorus trichloride or pentachloride (or any halogenation agent such as phosgene, oxalyl chloride, thionyl chloride) to obtain the dichlorinated derivative.\",\n \"An alternative synthesis is shown in the second pathway.\",\n \"Here, the dichlorinated derivative of PRC is synthesized directly from phosphoryl chloride and choline in a single step.\",\n \"In a preferred embodiment, the preferred pathway is the second one described above which has the advantage of requiring a lesser number of steps.\",\n \"This experimental protocol takes into account such factors as reproducibility and toxicity, as well as feasibility on an industrial scale.\",\n \"Experimental Method The dichloro derivative of PRC is synthesized by the addition of phosphoryl chloride (POCl3) in chloroform solution to a chloroform solution containing choline with glass beads (3 mm diameter) at 0° C. (ice bath) in a closed glass flask, under a dry nitrogen stream to exclude the presence of water and displace the HCl resulting from the reaction.\",\n \"The mixture is then stirred at 0° C. for 1 hr, and at room temperature for 12 hours to allow the reaction to proceed, as indicated by dissolution of the choline crystals and the formation of a oily phase.\",\n \"A slight excess of choline is used to ensure that all the phosphoryl chloride is consumed during the activation reaction.\",\n \"Immediately following the synthesis of the dichloro derivative of PRC, the 3 cm prostheses are immersed at room temperature in the abovementioned glass flask and agitation is continued for an additional 2 hours.\",\n \"In the case of longer vascular prostheses (>3 cm) a simple system was designed with a 1-liter commercial glass reactor with an outlet at the bottom.\",\n \"In this case, following the 12 hours of mechanical agitation, the chloroform layer at the bottom was removed via the bottom outlet of the flask and was replaced by fresh chloroform in order to remove any remaining POCl3.A PTFE diaphragm pump is then used to circulate the activated PRC solution from the bottom of the reactor, through a section of PTFE tubing, then through a vertical glass tube in which the prosthesis is held.\",\n \"Finally, a section of PTFE tubing brings back the solution in the glass reactor for recirculation.\",\n \"When the grafting of the activated PRC has occurred, the prostheses are washed many times in water to eliminate all traces of chlorine that may remain on the phosphoamide moiety of the anchored molecule (see Step 2 of the complete reaction, below) as well as all traces of reactants and solvent.\",\n \"The prostheses are then dried under vacuum overnight before being analyzed by XPS and static SIMS to determine the level of grafting.\",\n \"Overall Reaction for ePTFE Vascular Prostheses Inner Surface Treatment The process of the present invention may be extended to bind other molecules that are highly compatible with biological tissue, such as blood tissue.\",\n \"In fact, preliminary results(63) indicate that the internal surface of commercial ePTFE arterial prostheses that were previously treated with the ammonia plasma treatment described herein can be homogeneously coated, using a grafting method different from the one presented here, with polyethylene-glycol (PEG), which is another highly biocompatible molecule.\",\n \"Characterization of Ammonia RFGD Plasma Treated Surfaces and PRC-Grafted Surfaces X-Ray Photoelectron Spectroscopy (XPS) Analyses X-ray photoelectron spectroscopy (XPS) was used to characterize the modified surfaces.\",\n \"XPS spectra were recorded using a PHI 5600-ci spectrometer (Physical Electronics).\",\n \"A monochromatic aluminum X-ray source (1486.6 eV) was used to record the survey and high resolution spectra of the prosthetic surface.\",\n \"For both type of analyses, the power source was kept at 100 W to minimize X-ray induced modifications of the specimen and the detection was performed at 45° with respect to the surface.\",\n \"The pressure within the XPS analytical chamber was kept at about 5×10−9 torr.\",\n \"Ammonia RFGD Plasma Treated ePTFE Surfaces After RFGD ammonia plasma treatments, specimens were longitudinally opened in a chamber under nitrogen positive pressure and their internal surfaces were analyzed by XPS within 30 minutes of the RFGD treatment.\",\n \"Surface atomic concentration of oxygen was always lower than 5%.\",\n \"In order to collect data of the surface modifications along the longitudinally-treated length of a specimen, the XPS analysis area was moved along the length of the prosthesis.\",\n \"To validate that the RFGD system resulted in the treatment of the internal surface only, XPS analyses of the external surface of a prosthetic specimen treated at 20 W, for 250 seconds with an ammonia pressure of 300 mtorr were conducted.\",\n \"The data (not shown) confirmed that the external surface was not modified by the glow discharge when the internal diameter of the Pyrex™ tube was chosen to be only slightly larger (1 mm) than the external diameter of the prosthesis.\",\n \"Under the pressure and power conditions used, the Debye length did not allow the formation of a stable plasma between the external surface of the prosthesis and the internal surface of the tube, because the ionization processes were insufficient to compensate for the recombination processes.\",\n \"(60) All results are discussed by considering the percentage of atomic concentration of nitrogen on the surface, and the N/C, F/C and O/C surface concentration ratios.\",\n \"It was observed that because of experimental and instrumental uncertainties, atomic concentration values above 5% are reliable to ±1% (absolute) or better.\",\n \"As gaseous ammonia was used to create the plasma, it is clear that the percentage of nitrogen would be a valid indicator of the extent of the RFGD treatment.\",\n \"Moreover, the N/C ratio probes the extent of nitrogen atom insertion onto the ePTFE surface, while the F/C ratio monitors the level of defluorination and is indicative of the amount of fluorine atoms substituted by other atoms and/or multiple bond formations within or between the chains.\",\n \"Relationship Between the Nature of the Nitrogen-Containing Species Grafted onto the Surface of the Vascular Prostheses and Treatment Parameters With a view to understanding the relationship between the nature of the nitrogen-containing species grafted onto the surface of the RFGD treated ePTFE samples, different tests were performed to investigate the effects of variable parameters such as gas pressure, RF power and treatment time.\",\n \"Prostheses measuring 3 cm in length were treated under conditions where two of the variables were kept constant while the third was varied.\",\n \"As may be seen in FIG.\",\n \"2, the RF plasma treatment allowed a surface atomic nitrogen concentration of up to 15% to be reached, with an average N/C ratio of up to 0.3, at 20 W for 250 seconds and with an ammonia pressure of 300 mtorr.\",\n \"This represents a significant improvement over previously-reported results following treatment aimed at incorporating nitrogen on ePTFE surfaces, where published data indicates a percentage of nitrogen between 1 and 3%(26,42,61) or a N/C ratio between 0.06 and 0.18(28-43,59).\",\n \"A possible explanation for the present results may reside in the cylindrical configuration of the reactor chosen maximizes the efficiency of the treatment(59).\",\n \"The three surface characterization parameters (F/C, N/C and % N) indicate that the surface modifications induced on any given prosthesis are fairly homogenous: for example, (13±1)% atomic nitrogen concentration on a 7 cm-segment of a 6 mm ID prosthesis (250 s, 300 mtorr, 15 W).\",\n \"The degree of homogeneity required for subsequent grafting of the hemocompatible molecule and obtaining of an improved performance of the vascular prostheses can only be properly determined by hemocompatibility tests.\",\n \"However, the observed relative uniformity of surface modifications following ammonia plasma treatment provides a good starting point for the subsequent grafting.\",\n \"FIG.\",\n \"3 shows the effect on the surface composition of treatments performed by keeping the pressure constant (300 mtorr) while varying the power (20, 40 and 50 W) and treatment times (10, 120 and 275 seconds).\",\n \"It appears that the nitrogen concentration reached its highest value for a RF power of 40 W. The N/C ratio measured for the experiments performed for 275 seconds was significantly higher than that measured following treatment for 120 seconds.\",\n \"On the other hand, the F/C ratio was identical for these two treatment times for all RF powers tested.\",\n \"Thus, this data suggests that, despite leading to a higher nitrogen surface concentration, longer treatment times did not promote increased fluorine substitution.\",\n \"This result could mean that different treatment times lead to the formation of different chemical species on the surface.\",\n \"As may be seen in FIG.\",\n \"4, increasing the ammonia pressure from 100 to 600 mtorr (RF power kept at 20 W and treatment time at 10 s) lead to only a slight increase of the nitrogen content: from lower to higher pressures, this content ranged from 5 to 7%, respectively and the N/C ratio which ranged from 0.14 to 0.16.However, it is clear that the defluorination of the surface is highly pressure-dependent; the F/C ratio decreased from 1.45, for a 100 mtorr pressure, to 1.1 at 600 mtorr.\",\n \"Therefore, it is evident that despite a similar nitrogen insertion, the chemical moieties formed on the surface depend upon the pressure within the chamber.\",\n \"Further experiments were carried out to determine the time required to reach an approximately constant surface concentration of nitrogen.\",\n \"The nitrogen concentration reached a plateau after a treatment time of 160 seconds, as shown by the percentage of nitrogen and the N/C ratio measured on the surface (FIG.\",\n \"5).\",\n \"Defluorination of the surface, despite showing a more complex behavior, appears to follow a similar pattern.\",\n \"The above results have shown that the pressure, the duration and the RF power of the treatment all have an effect on the chemical composition as seen from the surface characterization parameters given by the survey spectra.\",\n \"In particular, these results raise the possibility of presence of different nitrogen species as a function of the above three treatment parameters.\",\n \"As several nitrogen containing species may be formed upon the ammonia RFGD plasma treatment, we have performed surface derivatization in order to quantify the percentage of the nitrogen present as NH2 species.\",\n \"The results of the surface derivatization on model surfaces (not shown) demonstrated that chlorobenzaldehyde specifically reacts with the amine moieties as depicted in FIG.\",\n \"6.Therefore, the chlorine XPS signal of the derivatized surface allows the determination of the surface amine concentration as described by d'Agostino et al.(62).\",\n \"Using the surface derivatization technique, it was possible to show that up to 45% of all the nitrogen species grafted onto the surface during the plasma treatment are amine moieties (as shown in Table 1 by % of N0 present as NH2).\",\n \"In addition, the data presented in the Table 1 also indicate that the surface amine concentration may be controlled by changing the plasma treatment duration.\",\n \"For example, the surface concentration of NH2 on the surface goes from 3.6% for a 50 second treatment duration to 6.0% when the surface is treated for 250 seconds.\",\n \"However, too long exposures to the plasma environment should be avoided to minimise the occurrence of polytetrafluoroethylene chain scission or formation of double bonds.\",\n \"(64) TABLE 1 Quantification of amine groups on plasma-treated PTFE film Plasma treatment % of N0 Surface concentration Duration F/C N/C % N0 present as NH2 of NH2 250 s 0.499 0.269 14.3 42 6.0 100 s 0.626 0.229 11.9 42 5.0 50 s 0.865 0.249 11.6 31 3.6 One skilled in the art will appreciate that the surface amine groups can be created on the surface by methods different from the one presented here without departing from the spirit of the present invention.\",\n \"For example, amine groups can be created on the surface not only by using an ammonia plasma, but also by using plasmas of N2H4 (hydrazine),(65,66) aliphatic amines,(67) coating-forming gases such as allylamine,(68-71) to name a few non-restrictive possibilities.\",\n \"U.S. Pat.\",\n \"No.\",\n \"6,159,531,(72) for example, briefly discusses the relative merits of these gases in view of their use as precursors to surface amine groups.\",\n \"Various methods of activating the surface can also be used, other than the continuous capacitively coupled RF plasma used here.\",\n \"Without departing from the spirit of the present invention, these include, but are not limited to, pulsed plasma,(68,70,73) inductively coupled RF plasma,(74-76) corona discharge,(77,78) microwave plasma(79,80) and UV irradiation.\",\n \"(81,82) As an example, one can read U.S. Pat.\",\n \"No.\",\n \"5,922,161,(83) which teaches the use of such treatments for the partial surface oxidation of polymers.\",\n \"Furthermore, without departing from the spirit of the present invention, one skilled in the art will appreciate that surface reactive groups other than amine can successfully react with the oxyphosphorodichlorinated derivative form of phosphorylcholine or another so-activated molecule.\",\n \"Hydroxyl and thiol groups(84-87) are two non-restrictive examples of such surface groups.\",\n \"Conversely, different pathways, in addition to the one proposed here, can be followed to obtain an activated form of phosphorylcholine, as one skilled in the art knows.\",\n \"These additional activation methods include, but are not limited to, using reagents such as PCl5, PCl3, PBr5, PBr3, PI3, SOCl2, SOBr2, COCl2, COBr2, (COCl)2, (COBr)2 to directly activate phosphorylcholine through the formation of at least a reactive P-halogen bond.\",\n \"(87,88) Storage in Atmosphere of the Ammonia Plasma Treated Prostheses Surface Atomic Concentrations as a Function of the Storage Time in Air The surface atomic concentration of nitrogen on the surface of a treated prosthesis tends to slowly decrease (from 14% initially to 12% after 80 days of storage in air or a decrease of N/C from 0.3 to 0.2) and oxygen uptake is also observed (O/C increased from 0.05 to 0.3 after 20 days)(45).\",\n \"Therefore, it is preferable to perform the grafting of the molecule shortly after the plasma treatment, preferably within two hours(45).\",\n \"PRC Grafted ePTFE Surfaces The atomic surface compositions were also determined by XPS spectroscopy.\",\n \"First, the survey spectra clearly showed that there is phosphorus and chlorine atom integration in approximately similar concentrations, as expected from the molecular structure of PRC (chlorine is present as the counterion of the N(CH3)3+ moiety of PRC).\",\n \"The extent of grafting of the PRC was therefore estimated from the percentage of phosphorus detected by XPS.\",\n \"Moreover, FIG.\",\n \"7 shows that the N 1 s High Resolution XPS spectra clearly indicates that there is a good correlation between the trimethylammonium peak at 402 eV (a peak which is not present on an ammonia plasma-treated prosthesis) and the percentage of phosphorus grafted onto the prosthesis.\",\n \"Indeed, there is a linear relation between the extent of PRC grafting (characterized by % P), and the trimethylammonium peak, +N(CH3)3 (FIG.\",\n \"7).\",\n \"The C 1 s peak (not shown) for each of the three methyl groups from the trimethylammonium moiety, +N(CH3)3, followed the same trend and provided further indication of the successful grafting of PRC onto the surface.\",\n \"The PRC grafting on PTFE films was also confirmed by static SIMS (static Secondary Ion Mass Spectroscopy).\",\n \"This method identifies molecules adsorbed on a surface by recording the mass spectra of atomic or molecular particles which are emitted when a surface is bombarded by energetic primary particles.\",\n \"The secondary ions emitted from the surface result from the fragmentation of the initial surface molecules and are only detected under their ionized state (positive and negative state).\",\n \"The peaks are given in Dalton (molecular weight—gmol−1).\",\n \"Thus, from an analysis of these spectra it is possible to determine the initial structure of the molecules grafted onto the surface.\",\n \"The spectra of a PRC-grafted PTFE film (not shown) gave the following fragments and confirmed the PRC grafting.\",\n \"122.1 78.8 PO3− 58.1 +N—(CH3)3 62.8 PO2− 43 +N—(CH3)2 O—CH2—CH+ 29 +N—CH3 15 CH3+ III.\",\n \"Sterilization of Inner-Surface Modified Vascular Prostheses For the present method of surface modification to be useful, it is required that sterilization not remove the grafted molecules.\",\n \"A very widely used method for sterilization is autoclaving.\",\n \"At least two possible methods of autoclaving may be contemplated: 1) sterilization under vapor pressure: 120° C., 15 PSI, 20 min; and 2) dry sterilization: short duration and high temperature (180° C., 1 h) or for a longer duration at a lower temperature (150° C., 2 h).\",\n \"According to scientific literature, PRC derivatives are stable when subjected to the first sterilization procedure.\",\n \"In addition, ePTFE prostheses are more frequently sterilized in this manner.\",\n \"This method is recognized and used by hospitals.\",\n \"Sample PRC-grafted films and prostheses were placed in a sterilization bag and sealed.\",\n \"A sterilization marker, a piece of adhesive tape, was placed on the bag; this indicator darkens when sterilization is successful.\",\n \"Following the sterilization procedure, the sample prostheses were analyzed by XPS to determine whether their compositions and the distribution of the phosphorus atoms had been altered.\",\n \"The results are summarized in Table 2 below.\",\n \"TABLE 2 Effect of Sterilization on PRC-Grafted Prostheses Sterilization Before After After 6 months % P 5 ± 1 4 ± 1 3.4 ± 0.6* *This data was obtained from a 8 cm-segment of a 6 mm ID prosthesis (yielding 60 data points) whereas the other values were estimated from a shorter segment (1 cm, yielding 6 data points), hence the difference in the standard deviations.\",\n \"Despite the slight decrease in the percentage of phosphorus, which is taken as a measure of the amount of PRC on the surface because it is otherwise absent, PRC grafting was found to be stable when subjected to sterilization under vapor pressure and the distribution of the grafting on the surface remained homogeneous.\",\n \"The surface concentrations were stable after 6 months and the distribution remained homogeneous (the same was observed for intermediate times of 1 and 2 months for different prostheses).\",\n \"Consequently, the sterilization procedure most practiced by hospitals does not appear to adversely modify the quantity and distribution of the grafted molecules on the PRC-treated vascular prostheses.\",\n \"IV.\",\n \"In Vitro Testing of Inner-Surface Modified Vascular Prostheses A. Non-Toxicity of Ammonia RFGD Plasma Treated Prostheses To verify the non-toxicity of ammonia plasma treated prostheses, we used the diffusion test which determines whether cellular growth is inhibited by the biomaterial by using a coloring agent specific to DNA (Hoechst).\",\n \"The elution test, also known as “extract dilution”, was also used; it determines whether there is inhibition of cellular growth by looking for an extract of biomaterial.\",\n \"a) Diffusion Test Human endothelial cells derived from an umbilical cord were cultured in a 24-well plate (1×104 cells/well), taking care to pre-treat each well with gelatin before use.\",\n \"The cells were allowed to grow in Medium 199 containing 10% FBS, heparin (90 g/ml), L-Glutamine (2 mM), penicillin G (100 I.U./ml), streptomycin sulfate (100 μg/ml), amphotericin B (250 ng/ml) and ECGS (20 μg/ml).\",\n \"Circular prosthetic samples measuring 7 mm in diameter from both treated and untreated prostheses were placed in the 24 wells.\",\n \"The cultures were incubated for 3 days with 5% CO2 in a humid atmosphere.\",\n \"Subsequently, the cultures were washed, lysed, treated with Hoechst #33358 and finally transferred to another plate designed for fluorescent studies.\",\n \"Readings were taken with a Bio-Tek FL600 fluorometer, and the quantity of DNA calculated from a calibration curve.\",\n \"At the same time, cells were cultured in wells without prosthetic samples (control).\",\n \"b) Elution Test Two samples, one from a pre-treated prosthesis and the other from an untreated prosthesis, were incubated in M199 for 7 days.\",\n \"Twenty-four hours before the end, endothelial cells were cultured in a 24-well plate.\",\n \"At the seventh day, the cells were cultured in the M199 medium used with the prostheses.\",\n \"(It is important to note here that, in contrast to what is done in the Diffusion Test, only the medium is used; the sample prosthetic devices are set aside following the incubation period.)\",\n \"The endothelial cells are incubated for 3 days under the same conditions as those for the Diffusion Test and analyzed in the same way (fluorometry).\",\n \"Table 3 shows the results for the Diffusion and Elution Tests on untreated (virgin) prostheses, on ammonia plasma treated prostheses and on control samples.\",\n \"(All experiments were done in duplicate, series A and B.)\",\n \"These results demonstrate that there are no significant variations in the level of cellular proliferation.\",\n \"It may therefore be concluded that ammonia plasma treated ePTFE vascular prostheses are no more toxic than untreated ePTFE vascular prostheses.\",\n \"TABLE 3 Results of in vitro Cellular Cultures Analyzed by Fluorometry Ammonia Plasma Untreated Prostheses treated Prostheses Control Test (*104) (*104) (*104) Diffusion Series A 54 ± 1 52 ± 2 Series B 43.7 ± 0.5 43.1 ± 0.5 40 ± 1 Elution Series A 32.9 ± 0.4 33 ± 1 Series B 28 ± 1 32 ± 1 31 ± 2 Cytotoxicity of the PRC-Grafted Vascular Prostheses As discussed above, toxicity tests performed on prostheses following plasma treatment but prior to the anchoring of the molecules revealed that the plasma-treated prostheses were non-toxic, or at least no more toxic than virgin prostheses.\",\n \"The next logical step was to determine whether prostheses which had been completely treated by the process of the present invention were also non-toxic to cells, and particularly to endothelial cells which naturally line the internal surface of blood vessels.\",\n \"Endothelial cells are currently used to verify the toxicity of certain products as well as to check the efficiency of sterilization.\",\n \"In cytotoxicity testing, the prostheses are not in direct contact with endothelial cells and the incubation period is 3 days at 37° C. Cellular growth is determined by the fluorescence of DNA strands using specific markers.\",\n \"Testing was repeated several times in order to obtain the best statistical average for the cell culture results.\",\n \"To gain a better appreciation of the effect of PRC grafting on the cells, tests were also performed on untreated prostheses as well as with “control” cell cultures.\",\n \"The histogram in FIG.\",\n \"8 clearly shows that treated prostheses have no more effect on cell growth than do untreated prostheses and neither of them have a significant effect on the growth of the cells.\",\n \"Hence, the prostheses treated with the process of the invention are non-cytotoxic and in fact seem to slightly favor endothelial cell growth.\",\n \"These findings are promising given that endothelial cells must line the internal surfaces of the prostheses.\",\n \"B) Blood Platelets Adhesion Adhesion and Aggregation of Blood Platelets The adhesion and aggregation of blood platelets on prosthetic devices has a direct effect on hemocompatibility, since the platelets are involved in the coagulation cascade.\",\n \"Indeed, the adherence of platelets on the internal surfaces of prostheses may prevent the adherence of endothelial cells (the natural lining of blood vessels), and may result in thrombosis if the accumulation is too important.\",\n \"In vitro studies were performed several times in the following manner.\",\n \"Following the centrifugation of human blood, the platelet-rich plasma was extracted and placed on prostheses for one hour at 37° C. The prostheses were then treated with different solutions before analyses by SEM.\",\n \"FIG.\",\n \"9 shows SEM photographs of treated and untreated prosthetic devices.\",\n \"A comparison of the two photographs reveals that the untreated prosthesis has a significantly greater accumulation and activation of platelets than the prosthesis that had been treated with the process of the invention.\",\n \"Hence, the treated prosthesis appears to prevent much more effectively the accumulation of blood platelets.\",\n \"Quantification of Blood Platelets After centrifugation of human blood, the platelet-rich plasma (5*104 platelets/μl count by a hemocytometer) was extracted, radiolabelled with 51Cr and placed on prostheses for two hours at 37° C. The prostheses were then washed three times with sterile PBS before analyses and the amount of platelet uptake was evaluated by a gamma scintillation counter adjusted to 270-370 keV.\",\n \"This test was performed 3 times with human blood from six donors (FIG.\",\n \"10).\",\n \"In all cases, no significant difference in the adherent platelets was noticed for untreated and treated prostheses.\",\n \"C) Neutrophils Neutrophils are characteristic of the inflammatory response of an individual to a foreign material.\",\n \"After centrifugation of human blood, the leukocyte-rich suspension was extracted, radiolabelled with 111In and placed on prostheses for two hours at 37° C. The prostheses were then analyzed and the adherent neutrophils were evaluated by a gamma scintillation counter adjusted to 260-480 keV.\",\n \"This test was performed three times with blood from six different donors (FIG.\",\n \"11).\",\n \"For every individual, there were fewer adherent neutrophils on the treated prosthesis than on the untreated prosthesis.\",\n \"Thus, it appears that the inflammatory response was reduced by the PRC grafted on the prosthesis.\",\n \"D) Clotting Time Platelet adhesion to biomaterials is often used as an indication of blood compatibility, but a more clinically relevant issue is whether the adherent platelets are able to promote clot formation.\",\n \"Indeed, clotting events require the presence of other blood elements such as fibrinogen to catalyze the clotting cascade.\",\n \"Longer clotting times are taken as indicative of a better thrombogenic resistance of a prosthesis in contact with blood.\",\n \"Normal human blood was collected into a citrate anticoagulant tube and a 1-ml sample was deposited onto the surface of the prosthesis.\",\n \"The clotting time was determined visually: the blood was deemed to have clotted when there was no longer a movement of the blood in response to a tilting motion of the sample prosthesis.\",\n \"This test was performed three times with blood from six human donors, on untreated and treated (PRC-grafted) prostheses (FIG.\",\n \"12).\",\n \"As observed in FIG.\",\n \"12, clotting times were significantly longer on PRC-grafted prostheses.\",\n \"Thus, it appears that the treated prosthesis is less thrombogenic when compared to the untreated prosthesis.\",\n \"E) Thromboelastography Another important measure of the thrombogenic potential of a material is the evolution of the elastic properties of whole blood clots that form on the surface of the material.\",\n \"For a given material in contact with blood, the thrombogenicity index takes into account the coagulation time as well as the elasticity of the blood clot.\",\n \"Surfaces with good blood compatibility should exhibit low thrombogenecity indexes, as measured by a thromboelastograph.\",\n \"This technique uses a steel piston placed in a cuvette having a 1-mm clearance between the piston and the inner surface of the cuvette.\",\n \"Blood is placed inside the cuvette.\",\n \"As coagulation proceeds, the elasticity of the forming blood clot varies and these changes are detected by the torsion wire to which the piston is suspended.\",\n \"This is converted to an electric signal which is sent to a chart recorder.\",\n \"The resulting trace is called the thromboelastogram (TEG), which records the elasticity of the formed blood clot.\",\n \"This test was performed using the blood of six human donors, on samples of untreated and treated prostheses (FIG.\",\n \"13).\",\n \"Comparison of the index of thrombogenic potential for treated and untreated prostheses showed a lower thrombogenic potential for the PRC-grafted prostheses.\",\n \"Values ranged from 17 to 45% lower, except for one individual where the value was 30% higher.\",\n \"Thus, the treated prostheses improved the non-thrombogenic potential of the initial material.\",\n \"F) Fibroblast Cultures The development of an endothelium, which is the natural hemocompatible surface, on the surface of prostheses would render them more hemocompatible.\",\n \"Fibroblasts, which are cells capable of differentiation, are commonly used for in vitro hemocompatibility tests.\",\n \"By testing the adsorption of fibroblasts, one can measure the ability of a material to act as a viable substrate for the adsorption and spreading of cells.\",\n \"The adsorption and spreading of cells represent conditions that are necessary for the development of the endothelium.\",\n \"Fibroblasts were placed in direct contact with the prostheses in order to observe the ability of cells to develop on different materials.\",\n \"The cells were allowed to grow for 7 days at 37° C. The fibroblasts were then observed through a microscope using two coloring agents: the Hoëscht reagent marked the nucleus (allowing the cells to be counted) and rhodamine dyed the actin filaments in the cytoplasm (allowing the shape of the cells to be determined).\",\n \"As shown in FIG.\",\n \"14, the development of fibroblasts on the surface of the prostheses treated by the process of the present invention was greatly enhanced in comparison to the untreated prosthesis: the number of nuclei is greater, and the shape of the fibroblasts is more elongated, which shows that the cells adhere to and develop on the surface.\",\n \"G) Endothelial Cell Cultures Endothelial cells are the cells that naturally line the internal surface of blood vessels; they therefore constitute the ideal hemocompatible surface.\",\n \"Because endothelial cells are known to be more fragile and sensitive than fibroblasts, this test is more severe.\",\n \"Endothelial cells were cultured following the same protocol as that used for the fibroblasts.\",\n \"The incubation period was 3 days at 37° C. The development of endothelial cells was greatly enhanced on the prosthesis treated by the process of this invention than on the untreated one.\",\n \"FIG.\",\n \"14 shows the optical microscope image obtained after using the Hoëscht reagent to mark the nucleus of the endothelial cells.\",\n \"The number of cell nucleus is clearly more important on the treated prosthesis, as is the spreading of the cells, as observed by SEM (FIG.\",\n \"14).\",\n \"V. In Vivo Testing of Inner-Surface Modified Vascular Prostheses The in vivo response of the ANBIOPA™-treated prostheses was tested in a canine model.\",\n \"The implantation period was one month and six dogs received a prosthesis: three received an untreated Gore-Tex™ ePTFE prosthesis while the three others received an ANBIOPA™-treated prosthesis.\",\n \"Surgery (The following procedure, while described in relation to a single dog, was used on all six dogs that participated in the trial.)\",\n \"After a 24-hour fast, a conditioned mongrel dog having a weight of between 20 and 25 kg was pre-anaesthetized by intra-veinous administration of atropin sulfate (0.05 mg/kg) and acepromazin maleate (0.1 mg/kg).\",\n \"The dog's abdomen was shaved and disinfected by an application of Hibitane and Proviodine.\",\n \"The dog was then placed on a surgical table in decubitus position, anaesthetized by intra-veinous administration of Penthothal (10 mg/kg) and then ventilated with a mix of air and isofuran to maintain anaesthesia.\",\n \"After abdominal incision, the sub-renal aorta was localized and cleared of surrounding structures before the collateral arteries were tied.\",\n \"Prior to clamping, heparin (0.5 mg/kg) was injected as an anticoagulant.\",\n \"The aorta was clamped upstream and downstream of a 5 cm segment in an aorto-aortic position and this segment was replaced with a 6 mm diameter vascular prosthesis (untreated commercially available prothesis or ANBIOPA™-treated prosthesis) of the same length by termino-terminal anastomosis using a Surgilene™ monofilament.\",\n \"The dog was stitched and butorphanol tartrate (0.2 mg/kg) was subcutaneously injected as an analgesic for three days after surgery.\",\n \"Following surgery, the dog was fed with a standard diet until explantation of the prosthesis one (1) month later.\",\n \"Image Analysis SEM observation on the explanted prostheses shows clear differences between the untreated commercially available prostheses and the ANBIOPA™-treated prostheses (FIG.\",\n \"15).\",\n \"At low magnification, the surface of an ANBIOPA™-treated prosthesis remains uniform with a smooth surface and is almost deposit free (FIG.\",\n \"15a).\",\n \"Further observations at higher magnification reveal a coating consisting of several layers of red blood cells (FIG.\",\n \"15c).\",\n \"The lack of activated platelets and fibrin clot on an ANBIOPA™-treated prosthesis surface suggests an anti-thrombogenic property.\",\n \"On the other hand, an untreated commercially available prosthesis shows important deposits on the surface at low magnification (FIG.\",\n \"15b) with deposits mainly composed of a network of activated platelets and red blood cells trapped in a fibrin clot (FIG.\",\n \"15d), thus providing evidence of thrombus formation.\",\n \"Although the present invention has been described by way of preferred embodiments thereof, it can be modified without departing from the spirit and nature of the subject invention, as defined in the appended claims.\",\n \"LIST OF REFERENCES 1.R.\",\n \"Guidoin, N. Chakfé, S. Maurel, T. How, M. Batt, M. Marois and C. Gosselin, Biomaterials 14, 678 (1993).\",\n \"2.B.\",\n \"D. Ratner, A. Chilkoti and G. P. Lopez, Plasma deposition and treatment for biomedical applications, in Plasma Deposition, Treatment, and Etching of Polymers, R. D'Agotino, ed., Academic Press, New York, (1990), p. 463.3.B.\",\n \"D. Ratner, J. Biomed.\",\n \"Mat.\",\n \"Res.\",\n \"27, 837 (1993).\",\n \"4.B.\",\n \"Haimovich; A. Freeman; R. Greco, Thromboresistant surface treatment for biomaterials, U.S. Pat.\",\n \"No.\",\n \"5,578,073 (1996).\",\n \"5.P.\",\n \"D. Drumheller, Materials and method for the immobilization of bioactive species onto polymeric subtrates, U.S. Pat.\",\n \"No.\",\n \"5,874,165 (1999).\",\n \"6.J.\",\n \"R. Keogh, Ionic attachement of biomolecules with a guanidine moiety to medical devices surfaces, U.S. Pat.\",\n \"No.\",\n \"5,928,916 (1999).\",\n \"7.J.\",\n \"M. McHaney and C. E. Banas, Carbon containing vascular graft and method of making same, U.S. Pat.\",\n \"No.\",\n \"5,827,327 (1998).\",\n \"8.J.\",\n \"R. Keogh, Method for a covalent attachment of biomolecules to surfaces of medical devices, U.S. Pat.\",\n \"No.\",\n \"6,033,719 (2000).\",\n \"9.J.\",\n \"R. Keogh, Periodate oxidative method for attachment and crosslinking of biomolecules to medical device surfaces, U.S. Pat.\",\n \"No.\",\n \"6,017,741 (2000).\",\n \"10.J.\",\n \"R. Keogh, Method for attachment of biomolecules to medical devices surfaces, U.S. Pat.\",\n \"No.\",\n \"5,925,552 (1999).\",\n \"11.J.\",\n \"R. Keogh, Oxidative method for attachment of glycoproteins or glycopeptides to surfaces of medical devices, U.S. Pat.\",\n \"No.\",\n \"5,891,506 (1999).\",\n \"12.J.\",\n \"R. Keogh, Oxidative method for attachment of glycoproteins to surfaces of medical devices, U.S. Pat.\",\n \"No.\",\n \"5,728,420 (1998).\",\n \"13.P.\",\n \"T. Cahalan, M. Verhoeven, M. Hendricks, L. Cahalan, Biocompatibility of solid surfaces, U.S. Pat.\",\n \"No.\",\n \"5,702,818 (1997).\",\n \"14.P.\",\n \"E. Guire, Biocompatible device with covalently bonded biocompatible agent, U.S. Pat.\",\n \"No.\",\n \"5,263,992 (1993).\",\n \"15.D.\",\n \"G. Castner, P. Favia and B. D. Ratner, Deposition of fluorocarbon films by remote RF glow discharges, in Surface Modification of Polymeric Biomaterials, B. D. Ratner and D. G. Castner, eds., Plenum Press, New York (1997), p. 45.16.B.\",\n \"D. Ratner, D. Leach-Scampavia and D. G. Castner, Biomaterials 14, 148 (1993).\",\n \"17.A.\",\n \"S. Hoffman, A. M. Garfinkle, B. D. Ratner and S. R. Hanson, Plasma gas discharge treatment for improving the compatibility of biomaterials, U.S. Pat.\",\n \"No.\",\n \"5,034,265 (1991).\",\n \"18.T.\",\n \"A. Horbett, B. D. Ratner, J.\",\n \"A. Chinn and Y. Haque, Radio frequency plasma deposited polymers that enhance cell growth, U.S. Pat.\",\n \"No.\",\n \"4,919,659 (1990).\",\n \"19.A.\",\n \"S. Hoffman, A. Garfinkle, B. D. Ratner and S. R. Hanson, Plasma gas discharge treatment for improving the biocompatibility of biomaterials, U.S. Pat.\",\n \"No.\",\n \"4,656,083 (1987).\",\n \"20.M.\",\n \"Garfinkle, A. S. Hoffmann, B. D. Ratner, L. O. Reynolds, S. R. Hanson, Trans.\",\n \"Am.\",\n \"Soc.\",\n \"Artif.\",\n \"Intern.\",\n \"Organs 30, 432 (1984).\",\n \"21.J.\",\n \"C. Lin and S. L. Cooper, J. Appl.\",\n \"Polym.\",\n \"Sci.\",\n \": Appl.\",\n \"Polym.\",\n \"Symp.\",\n \"54, 157 (1994).\",\n \"22.P.\",\n \"V. Narayanan, J. Biomater.\",\n \"Sci.\",\n \"Polymer Edn, 6, 181 (1994).\",\n \"23.Y.\",\n \"Matsuzawa and H. Yasuda, J. Appl.\",\n \"Polym.\",\n \"Sci.\",\n \"38, 65 (1984).\",\n \"24.P.\",\n \"Favia, V. H. Perez-Luna, T. Boland, D. G. Castner and B. D. Ratner, Plasmas & Polymers 1, 299, (1996).\",\n \"25.Y.\",\n \"Babukutty, M. Kodama, H. Nomiyama, M. Kogoma and S. Okazaki, Preparation of acrylamide coated PVC tube by APG treatment and its characterization, in Advanced Biomaterials in Biomedical Engineering and Drug Delivery Systems, N. Ogata, S. W. Kim, J. Feijen and T. Okano, eds., Springer-Verlag, Tokyo (1996), p. 217.26.A.\",\n \"Dekker, K. Reitsma, T. Beugeling, A. Bantjes, J. Feijen and W. G. van Aken, Biomaterials 12, 130 (1991).\",\n \"27.T.\",\n \"A. Giroux and S. L. Cooper, J. Colloid Interface Sci.\",\n \"146, 179 (1991).\",\n \"28.N.\",\n \"Inagaki, S. Tasaka and H. Kawai, J. Adhesion Sci.\",\n \"Technol.\",\n \"3, 637 (1989).\",\n \"29.Y.\",\n \"Nakayama, T. Takahagi, F. Soeda, S. Nagaoka, J. Suzuki and A. Ishitani, J. Polym.\",\n \"Sci., Part A: Polym.\",\n \"Chem.\",\n \"26, 559 (1988).\",\n \"30.J.\",\n \"R. Hollahan, B.\",\n \"B. Stafford, R. D. Falb and S. T. Payne, J. Appl.\",\n \"Polymer Sci.\",\n \"13, 807 (1969).\",\n \"31.R.\",\n \"E. Marchant, Anticoagulant plasma polymer-modified substrate, U.S. Pat.\",\n \"No.\",\n \"5,455,040 (1995).\",\n \"32.H.\",\n \"K. Yasuda, Method of making a biocompatible prosthesis, U.S. Pat.\",\n \"No.\",\n \"4,994,298 (1991).\",\n \"33.T.\",\n \"Karwoski and Y. Matsuzawa, Glow discharge process for producing implantable devices, U.S. Pat.\",\n \"No.\",\n \"4,632,842 (1986).\",\n \"34.P.\",\n \"V. Narayanan, Radiofrequency plasma biocompatibility treatment of inside of medical tubing and the like, U.S. Pat.\",\n \"No.\",\n \"5,244,654 (1993).\",\n \"35.P.\",\n \"V. Trescony, P. Cahalan and K. Keeney, Vascular prosthesis and method, U.S. Pat.\",\n \"No.\",\n \"5,246,451 (1993).\",\n \"36.P.\",\n \"V. Narayanan, S. M. Rowland and K. D. Stanley, Treatment of metallic surfaces using radiofrequency plasma deposition and chemical attachment of bioactive agents, U.S. Pat.\",\n \"No.\",\n \"5,336,518 (1994).\",\n \"37.P.\",\n \"V. Narayanan, Radiofrequency plasma biocompatibility treatement of inside surfaces, U.S. Pat.\",\n \"No.\",\n \"5,486,357 (1996).\",\n \"38.F.\",\n \"Schué, G. Clarotti, J. Sledz, A. Mas, K. E. Geckeler, W. Göpel, A. Orsetti, Makromol.\",\n \"Chem, Macromol.\",\n \"Symp.\",\n \"73, 217 (1993).\",\n \"39.R.\",\n \"Sipehia, Biomat., Art.\",\n \"Cells & Immob.\",\n \"Biotech.\",\n \"21, 647 (1993).\",\n \"40.U.\",\n \"König, A. Augsburg, F. Simon, D. Lunkwitz, G. Hermel, C. Werner, M. Müller, H. J. Jacobash, ACS Polymer Preprints.\",\n \"38, 1079 (1997).\",\n \"41.F.\",\n \"Simon, G. Hermel, D. Lunkwitz, C. Werner, K. Eichhorn and H. J. Jacobash, Macromol.\",\n \"Symp.\",\n \"103, 243 (1996).\",\n \"42.R.\",\n \"Sipehia, G. Martucci, M. Barbarosie and C. Wu, Biomat., Art.\",\n \"Cells & Immob.\",\n \"Biotech.\",\n \"21, 455 (1993).\",\n \"43.D.\",\n \"Ratner, Biomaterials science: an interdisciplinary endeavor, in Biomaterials science: an introduction to materials in medicine, B. D. Ratner, A. S. Hoffman, F. J. Schoen, J. E. Lemons, eds., Academic Press, San Diego, (1996), p. 1.44.D.\",\n \"Mantovani, M. Castonguay, J. F. Pageau, M. Fiset and G. Laroche.\",\n \"Mater.\",\n \"Res.\",\n \"Soc.\",\n \"Preprints, 544, 21 (1998).\",\n \"45.D.\",\n \"Mantovani, M. Castonguay, J. F. Pageau, M. Fiset and G. Laroche.\",\n \"Plasmas and Polymers, 4, 207 (1999).\",\n \"46.J.\",\n \"A. Hayward and D. Chapman, Biomaterials, 5, 135 (1984).\",\n \"47.R.\",\n \"F. A. Zwall, P. Comfurius, L. M. M. van Deenen, Nature, 268, 358 (1977).\",\n \"48.R.\",\n \"F. A. Zwall, H. C. Hember, Haemostasis, 11, 12 (1982).\",\n \"49.R.\",\n \"F. A. Zwall and E. M. Bevers, Subcellular Biochemistry, 9, 163 (1983).\",\n \"50.Y.\",\n \"P. Yianni, Biocompatible surfaces based upon biomembrane mimicry, in Structural and dynamic properties of lipids and membranes, eds., P J Quinn & R J Cherry, Portland, Press Research Monograph, 1992, p. 187-216.51.A.\",\n \"A. Durrani, J.\",\n \"A. Hayward and D. Chapman, Biomaterials, 7, 121 (1986).\",\n \"52.Y.\",\n \"P. Yianni, S. R. Hanson, A.\",\n \"B. Lumsden, Journal of Surgical Research, 77, 119 (1998).\",\n \"53.K.\",\n \"Ishihara, R. Aragaki, A. Watenabe, N. Nakabayashi, J. Biomed.\",\n \"Mater.\",\n \"Res., 24, 1069 (1990).\",\n \"54.K.\",\n \"Ishihara, N. Nakabayashi, K. Fukumoto, J. Aoki, Biomaterials, 13, 145 (1992).\",\n \"55.K.\",\n \"Ishishara, H. Oshida, Y. Endo, T. Ueda, A. Watanabe, N. Nakabayashi, J. Biomed.\",\n \"Mater.\",\n \"Res., 26, 1543 (1992).\",\n \"56.K.\",\n \"Ishihara, H. Hanuyada, N. Nakabayashi, Biomaterials, 16, 873 (1995).\",\n \"57.Y.\",\n \"Iwasaki, K. Ishihara, N. Nakabayashi, G. Khang, J. H. Jeon, J. W. Lee, H. B. Lee, J Biomater Sci Polym Edn, 9, 801 (1998).\",\n \"58.A.\",\n \"E. Gal, F. J. Fash, Lipids, 12, 314 (1976).\",\n \"59.J.\",\n \"C. Lin and S. L. Cooper, J. Appl.\",\n \"Polymer Sci.:Appl.\",\n \"Polymer Symp.\",\n \"54, 157 (1994).\",\n \"60.F.\",\n \"Fracassi, Architecture of RF plasma reactors, in Plasma Processing of Polymers, R. D'Agostino, P. Favia, F. Fracassi, eds., NATO-ASI Series E: Applied Science—Vol.\",\n \"346, Kluwer Academic Publishers (1997), p. 47.61.M.\",\n \"E. Ryan and J. P. S. Badyal, Macromolecules 28, 1377 (1995).\",\n \"62.P.\",\n \"Favia, M. V. Stendardo and R. d'Agostino, Plasmas and Polymers 1, 91 (1996).\",\n \"63.S.\",\n \"F. Popescu, M. Castonguay, C. Doillon, R. C. Gaudreault, G. Laroche.\",\n \"Society for Biomaterials 25th Annual Conference, Proceeding vol.\",\n \"XXII, 574, Apr.\",\n \"28-May 2, 1999, Rhode Island, USA.\",\n \"64.P.\",\n \"Chevallier, M. Castonguay, S. Turgeon, D. Mantovani, J. C. Wittmann, G. Laroche, submitted for publication at J. Phys.\",\n \"Chem.\",\n \"65.A.\",\n \"J. Martinez, S. Manolache, V. Gonzalez, R. A.\",\n \"Young, F. Denes, J. Biomater.\",\n \"Sci., Polym.\",\n \"Ed., 11, 415 (2000).\",\n \"66.X.\",\n \"Zhang, R. A.\",\n \"Young, Mater.\",\n \"Res.\",\n \"Soc.\",\n \"Symp.\",\n \"Proc., 586, 157 (2000).\",\n \"67.J.\",\n \"G. A. Terlingen, L. M. Brenneisen, H. T. J. Super, A. P. Pijpers, A. S. Hoffman, J. Feijenand, J. Biomater.\",\n \"Sci., Polym.\",\n \"Ed., 4, 165 (1993) 68.W.\",\n \"R. Gombotz, A. S. Hoffman, J. Appl.\",\n \"Polym.\",\n \"Sci.\",\n \": Appl.\",\n \"polym.\",\n \"Symp., 42, 285 (1988).\",\n \"69.H.\",\n \"J. Griesser, Mater.\",\n \"Forum, 14, 192 (1990).\",\n \"70.C.\",\n \"L. Rinsch, X. Chen, V. Panchalingam, R. E. Eberhart, J-H. Wang, R. B. Timmons, Langmuir, 12, 2995 (1996).\",\n \"71.J.\",\n \"G. Calderon, A. Harsch, G. W. Gross, R. B. Timmons, J. Biomed.\",\n \"Mater.\",\n \"Res., 42, 597 (1998).\",\n \"72.M.\",\n \"H. Dang; P. Chiu, Coating having biological activity and medical implant having surface carrying the same and method, U.S. Pat.\",\n \"No.\",\n \"6,159,531 (2000).\",\n \"73.J.\",\n \"G. Calderon, R. B. Timmons, Macromolecules, 31, 3216 (1998).\",\n \"74.P.\",\n \"Colpo, R. Ernst, J.-P. Keradec, Plasma Sources Sci.\",\n \"Technol., 8, 587 (1999).\",\n \"75.M.\",\n \"Desilets, B. Davies, G. Soucy, P. Proulx, Can.\",\n \"J. Chem.\",\n \"Eng., 76, 707 (1998).\",\n \"76.J.\",\n \"T. Gudmundsson, A. M. Marakhtanov, K. K. Patel, V. P. Gopinath, M. A. Lieberman, J. Phys.\",\n \"D: Appl.\",\n \"Phys., 33, 1323 (2000).\",\n \"77.R.\",\n \"Gasparik, S. Ihara, C. Yamabe, S. Satoh, Jpn.\",\n \"J. Appl.\",\n \"Phys., Part 1, 39, 306 (2000).\",\n \"78.J.\",\n \"Lei, M. Shi, J. Zhang, Eur.\",\n \"Polym.\",\n \"J, 36, 1277 (2000).\",\n \"79.R.\",\n \"Foerch, J. Izawa, N. S. Mc Intyre, J. Appl.\",\n \"Polym.\",\n \"Sci.\",\n \": Appl.\",\n \"Polym.\",\n \"Symp., 46, 415 (1990).\",\n \"80.A.\",\n \"E. Lefohn, N. M. Mackie, E. R. Fisher, Plasmas and Polymers, 3, 197 (1998).\",\n \"81.S.\",\n \"I. Kim, S. W. Lee, Y. H. Park, M. Ree, Mol.\",\n \"Cryst.\",\n \"Liq.\",\n \"Cryst.\",\n \"Sci.\",\n \"Technol., Sect.\",\n \"A, 349, 275 (2000).\",\n \"82.J.\",\n \"M. Nedelec, J. Grimblot, S. Turrell, M. Bouazaoui, J. Phys.\",\n \"Chem.\",\n \"B, 104, 926 (2000).\",\n \"83.D.\",\n \"Y. Wu; S. Li; G. W. Stanislaw, Surface treatment of polymers, U.S. Pat.\",\n \"No.\",\n \"5,922,161 (1999).\",\n \"84.Based on the international conference on phosphorus chemistry at Duke University, Durham, N.C. (1981), Phosphorus chemistry—Proceeding of the 1981 international conference, American Chemical Society, Eds.\",\n \"Library of congress CIP, Data, Vol.\",\n \"171.85.R.\",\n \"Neidlen, S. Buseck, Helvetica Chimica Acta, 75, 2520 (1992).\",\n \"86.R.\",\n \"Neidlen, P. Greulich, Helvetica Chimica Acta, 75, 2545 (1992).\",\n \"87.Theilheimer, Synthetic Methods of Organic Chemistry, Finch, Theilheimer, Nutley, Tozer, Eds Karger, Interscience publishers, Basel-München-Paris-London-New-York-New Dehli-Singapore-Tokyo-Sydney, 1946-1998.88.A.\",\n \"I. Vogel, Vogel's Textbook of Practical Organic Chemistry, including qualitative organic analysis, 4th ed./rev.\",\n \"by the following members of the School of Chemistry, Thames (formerly Woolwich)—polytechnic, B. S. Furniss .\",\n \".\",\n \".\",\n \"[et al.\",\n \"], London; New York: Longman, 1978.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10468149"}}},{"rowIdx":398,"cells":{"text":{"kind":"list like","value":[["Central Fastening Element for an Axially Symmetric Gas Spring","In a central fastening element for an axially symmetric, vehicle gas spring, which includes a bellows that has central bores or cutouts in the region of its end faces, the fastening element being fixed to the vehicle body, protruding from the surroundings of the attachment point in a direction normal to it, and being encompassed by the bores or cutouts.","The fastening element includes a shaped stud or a shaped cap, the maximum outer diameter of the stud or the cap being at least less than one fifth of the maximum outer diameter of the gas-spring bellows.","A device may be provided which allows a gas spring to be installed in a simple manner and allows the spring bellows to rotate with respect to the suspension and/or the vehicle body during the initial installation and/or the initial operation."],["1-9.","(canceled) 10.A central fastening element for an axially symmetric, vehicle gas spring including a bellows having one of central bores and cutouts in a region of an end face, the fastening element configured to be fixed to a vehicle body and to protrude from surroundings of an attachment point in a normal direction and to be encompassed by the one of the bores and cutouts, comprising: at least one of a stud and a cap, one of the at least one of the stud and the cap shaped, a maximum outer diameter of the one of the stud and the cap at least less than one fifth of a maximum outer diameter of the bellows, the one of the stud and the cap including at least one necked-down portion having a diameter less than the maximum outer diameter of the one of the stud and the cap, the end face elastic in a zone of contact with the one of the stud and the cap.","11.The fastening element according to claim 10, wherein a base of the bellows axially and radially surrounds the one of the stud and the cap without a sealing joint.","12.The fastening element according to claim 10, further comprising an integrated supply line.","13.The fastening element according to claim 10, wherein the cap includes an internal thread, the stud having an external thread, the cap substantially completely surrounding the stud, the cap arranged to one of directly and indirectly contact the vehicle body.","14.The fastening element according to claim 10, wherein the stud is arranged to be screwed into a tapped hole of the vehicle body, the stud having a surface oriented in a direction normal to an axis of the stud, the surface arranged to one of directly and indirectly rest against the vehicle body.","15.The fastening element according to claim 10, wherein the stud is arranged to be screwed into a nut fastened to the vehicle body.","16.The fastening element according to claim 10, wherein a base of the bellows includes at least two superposed layers, at least one of the layers made of metal, at least one of the layers made of metal.","17.The fastening element according to claim 10, wherein a base of the bellows is arranged to rest against at least two surface sections of the one of the stud and the cap oriented in axially opposite directions.","18.The fastening element according to claim 10, wherein a base of the bellows is arranged to rest against a surface section of the one of the stud and the cap oriented in a direction of the vehicle body and against the vehicle body in a region of attachment of the one of the stud and the cap.","19.The fastening element according to claim 10, wherein the cap includes an internal thread, the stud having an external thread, the cap substantially completely surrounding the stud, the cap one of directly and indirectly contacting the vehicle body.","20.The fastening element according to claim 10, wherein the stud is screwed into a tapped hole of the vehicle body, the stud having a surface oriented in a direction normal to an axis of the stud, the surface one of directly and indirectly resting against the vehicle body.","21.The fastening element according to claim 10, wherein the stud is screwed into a nut fastened to the vehicle body.","22.The fastening element according to claim 10, wherein a base of the bellows rests against at least two surface sections of the one of the stud and the cap oriented in axially opposite directions.","23.The fastening element according to claim 10, wherein a base of the bellows rests against a surface section of the one of the stud and the cap oriented in a direction of the vehicle body and against the vehicle body in a region of attachment of the one of the stud and the cap.","24.The fastening element according to claim 10, wherein the fastening element is fixed to a vehicle body.","25.The fastening element according to claim 10, wherein the fastening element protrudes from the surroundings of the attachment point in the normal direction.","26.The fastening element according to claim 10, wherein the fastening element is encompassed by the one of the bores and cutouts.","27.A device, comprising: an axially symmetric, vehicle gas spring including a bellows having one of central bores and cutouts in a region of an end face; and a fastening element configured to be fixed to a vehicle body and to protrude from surroundings of an attachment point in a normal direction, the fastening element encompassed by the one of the bores and cutouts, the fastening element including at least one of a stud and a cap, one of the stud and the cap shaped, a maximum outer diameter of the one of the stud and the cap at least less than one fifth of a maximum outer diameter of the bellows, the one of the stud and the cap including at least one necked-down portion having a diameter less than the maximum outer diameter of the one of the stud and the cap, the end face elastic in a zone of contact with the one of the stud and the cap."],["<SOH> BACKGROUND INFORMATION <EOH>Such a device is described in European Published Patent Application No.","0 123 171.The gas spring includes, inter alia, a vertical U-bellows and two axially symmetric bodies terminating it on the upper and lower sides.","On the upper side, the gas spring is fixed to the vehicle body, e.g., to the vehicle frame, with the aid of a bolt.","This bolt is seated in a bore of the frame and is screwed to the axially symmetric upper body, a so-called plug.","The U-bellows itself is fastened to the axially symmetric, upper member with the aid of a tension band.","In this case, the diameter of the plug is as large as the inner diameter of the U-bellows.","This type of fastening requires that the bolthead on the upper side of the frame be accessible during installation.","This makes it difficult to automate the installation of the gas spring.","The gas spring tends to twist during installation, and while compressing and rebounding.","Therefore, the tightening torque of the bolt must be selected to be high enough to prevent the connection between the gas spring and the vehicle body from loosening or releasing in response to vibrations and shock.","It is an object of the present invention to provide a fastening element, which renders simple installation possible and allows the bellows to rotate with respect to the suspension and/or the vehicle frame during the initial installation and/or the initial operation."],["<SOH> SUMMARY <EOH>This object may be achieved by providing a fastening element as described herein.","To this end, the fastening element may include a shaped stud or a shaped cap, the maximum outer diameter of the stud or the cap being at least less than one fifth of the maximum outer diameter of the gas-spring bellows.","The cap or the stud has at least one necked-down portion or waist, whose outer diameter is less than the above-mentioned, maximum diameter of the cap or stud.","The end face is elastic in the zone in which it comes into contact with the stud or the cap.","The central fastening element may be attached to the vehicle body prior to the installation of the gas spring and may protrude from the vehicle body in a direction normal to it.","During installation, the gas spring is attached to, for example, the strut of the suspension and, e.g., pressed, together with it, against the shaped stud or the shaped cap.","In the following, the term, stud, also includes the shaped cap.","The elastic zone of the end face of the gas spring comes into contact with the stud in response to being slid up, and then engages with it in the manner of a snap fastener.","The stud has a necked-down portion, which is encompassed by the bore or the cutout in a form-locked or force-locked manner.","This type of fastening may allow the gas spring to be installed in an automated manner.","In this case, and during initial operation, the gas spring may rotate on the stud, which means that the twisting of the U-bellows and the increased wear caused by it are prevented.","The keyed connection between the stud and spring prevents vibrations and shock from detaching the fastening element.","The base of the gas-spring bellows surrounds the stud axially and radially.","Consequently, the gas spring is fixed in position in the axial and radial directions after installation.","For example, it may not detach when the vehicle is jacked up, or in response to a pressure drop.","The gas spring may be attached at its upper and lower ends in the same manner.","Supply lines may be run through the fastening element into the interior of the gas spring.","At least some regions of the base of the gas-spring bellows may be made of an elastic material, e.g., rubber.","This may allow the base to act as a damping layer.","The base may also be made up of multiple layers, e.g., a rubber layer and a metallic layer.","In this case, the gas-spring bellows is attached to the metallic layer.","The metallic layer is simultaneously used for increasing the strength of the gas-spring base.","Several rubber and metallic layers may also be combined.","To attach the gas spring, e.g., this rubber layer is compressed between two surface sections of the stud oriented in opposite, axial directions, or between a surface section oriented in the direction of the vehicle body, and the vehicle body.","If the base of the gas spring is made out of multiple layers, the inner layer may be, for example, a metallic layer.","The gas-spring bellows is then attached to this.","This layer may be constructed in such a manner, that it supports the rubber layer and holds the position of the gas spring on the stud.","Further details of the fastening element according to the present invention are set forth below in the subsequent description of several schematically represented, example embodiments."],["FIELD OF THE INVENTION The present invention relates to a central fastening element for an axially symmetric, vehicle gas spring, which includes a bellows that has central bores or cutouts in the region of its end faces, the fastening element being fixed to the vehicle body, protruding from the surroundings of the attachment point in a direction normal to it, and being encompassed by the bores or cutouts.","BACKGROUND INFORMATION Such a device is described in European Published Patent Application No.","0 123 171.The gas spring includes, inter alia, a vertical U-bellows and two axially symmetric bodies terminating it on the upper and lower sides.","On the upper side, the gas spring is fixed to the vehicle body, e.g., to the vehicle frame, with the aid of a bolt.","This bolt is seated in a bore of the frame and is screwed to the axially symmetric upper body, a so-called plug.","The U-bellows itself is fastened to the axially symmetric, upper member with the aid of a tension band.","In this case, the diameter of the plug is as large as the inner diameter of the U-bellows.","This type of fastening requires that the bolthead on the upper side of the frame be accessible during installation.","This makes it difficult to automate the installation of the gas spring.","The gas spring tends to twist during installation, and while compressing and rebounding.","Therefore, the tightening torque of the bolt must be selected to be high enough to prevent the connection between the gas spring and the vehicle body from loosening or releasing in response to vibrations and shock.","It is an object of the present invention to provide a fastening element, which renders simple installation possible and allows the bellows to rotate with respect to the suspension and/or the vehicle frame during the initial installation and/or the initial operation.","SUMMARY This object may be achieved by providing a fastening element as described herein.","To this end, the fastening element may include a shaped stud or a shaped cap, the maximum outer diameter of the stud or the cap being at least less than one fifth of the maximum outer diameter of the gas-spring bellows.","The cap or the stud has at least one necked-down portion or waist, whose outer diameter is less than the above-mentioned, maximum diameter of the cap or stud.","The end face is elastic in the zone in which it comes into contact with the stud or the cap.","The central fastening element may be attached to the vehicle body prior to the installation of the gas spring and may protrude from the vehicle body in a direction normal to it.","During installation, the gas spring is attached to, for example, the strut of the suspension and, e.g., pressed, together with it, against the shaped stud or the shaped cap.","In the following, the term, stud, also includes the shaped cap.","The elastic zone of the end face of the gas spring comes into contact with the stud in response to being slid up, and then engages with it in the manner of a snap fastener.","The stud has a necked-down portion, which is encompassed by the bore or the cutout in a form-locked or force-locked manner.","This type of fastening may allow the gas spring to be installed in an automated manner.","In this case, and during initial operation, the gas spring may rotate on the stud, which means that the twisting of the U-bellows and the increased wear caused by it are prevented.","The keyed connection between the stud and spring prevents vibrations and shock from detaching the fastening element.","The base of the gas-spring bellows surrounds the stud axially and radially.","Consequently, the gas spring is fixed in position in the axial and radial directions after installation.","For example, it may not detach when the vehicle is jacked up, or in response to a pressure drop.","The gas spring may be attached at its upper and lower ends in the same manner.","Supply lines may be run through the fastening element into the interior of the gas spring.","At least some regions of the base of the gas-spring bellows may be made of an elastic material, e.g., rubber.","This may allow the base to act as a damping layer.","The base may also be made up of multiple layers, e.g., a rubber layer and a metallic layer.","In this case, the gas-spring bellows is attached to the metallic layer.","The metallic layer is simultaneously used for increasing the strength of the gas-spring base.","Several rubber and metallic layers may also be combined.","To attach the gas spring, e.g., this rubber layer is compressed between two surface sections of the stud oriented in opposite, axial directions, or between a surface section oriented in the direction of the vehicle body, and the vehicle body.","If the base of the gas spring is made out of multiple layers, the inner layer may be, for example, a metallic layer.","The gas-spring bellows is then attached to this.","This layer may be constructed in such a manner, that it supports the rubber layer and holds the position of the gas spring on the stud.","Further details of the fastening element according to the present invention are set forth below in the subsequent description of several schematically represented, example embodiments.","BRIEF DESCRIPTION OF THE DRAWINGS FIG.","1 schematically illustrates the attachment of a gas spring to a vehicle body.","FIG.","2 schematically illustrates an alternative manner of attaching a gas spring to a vehicle body.","FIGS.","3 to 7 schematically illustrate alternative example embodiments of the fastening elements for the arrangements illustrated in FIGS.","1 and 2.DETAILED DESCRIPTION FIG.","1 illustrates the attachment of a gas spring 70 to a vehicle body 5, or to a strut.","In this case, gas spring 70, e.g.","an air spring, is made up of, inter alia, a U-bellows 72, which is attached to a damper plate 50.In this connection, damper plate 50 forms the base of gas-spring bellows 72.A fastening element 10 is mounted to vehicle body 5, e.g., to the vehicle frame, a body member, or a reinforced part of the body paneling.","On vehicle body 5, a bolt 15 is situated in a concave and, optionally, axial asymmetric depression 6.For example, this bolt 15 may be welded on.","Bolt 15 has a thread 16.A mushroom-shaped cap 19 is screwed onto this thread 16 via an internal thread 21.Cap 19 is axially asymmetric and has a cylindrical outer contour 23 in the upper region.","In the middle region, the contour changes, e.g., discontinuously, into a circular arc 24.This transition is referred to in the following as 35 necked-down portion 25, undercut, or waist.","In this case, the center point of circular arc 24 is, e.g., on the radius of cylindrical outer contour 23 or below it.","Curved section 24 is referred to below as a toroid.","Cap 19 changes continuously or discontinuously between toroid 24 and a conical section 28 and tapers to approximately three quarters of its upper diameter.","Instead of being semicircular, the contour of the partial cross-section of toroid 24 may also be triangular.","In the case of this contour, toroid 24 has a frustoconical lateral surface, whose imaginary apex points to the center of the U-bellows interior.","For example, the bottom surface of this frustoconical apex discontinuously joins up with necked-down portion 25 to form a barb-shaped undercut.","The frustoconical apex may also be an imaginary enveloping surface for a series of resilient barbs positioned about cap 19.After installation, these barbs sink into optionally sharp-edged groove 54 of damper plate 50, while elastically recovering.","For example, a hexagonal recess 26 is centrally situated in cap 19, cf.","FIG.","3.Damper plate 50 is positioned around cap 19.The upper side of damper plate 50 is designated as outer side 59 and the lower side is designated as inner side 58.Cylindrical damper plate 50 is made of rubber and has a central bore 52, which is smaller that the maximum outer diameter of cap 19.A recess in the form of a circular groove 54 is situated approximately halfway up bore 52.This has a contour opposite to that of toroid 24 and therefore may ensure that damper plate 50 grips cap 19 from behind.","In this connection, the region of damper plate 50 between groove 54 and outer side 59 may be compressed.","Bore 52 changes into a chamfer 57 at the upper end face of damper plate 50.Damper plate 50 is also chamferred at the lower end face.","The thickness of damper plate 50 in the middle region approximately corresponds to the length of cap 19, but the thickness in the outer region is reduced to approximately two thirds of the overall height.","In this connection, inner side 58 is approximately planar.","A metallic disk 62 and a thin rubber layer 63 are situated on the outer side 59 of damper plate 50.The two parts may be cemented on or vulcanized on.","A machined disk 64, whose central region is formed in the shape of a frustoconical shell in the direction of the center, is situated on inner side 58.The top edge of the frustoconical shell may be oriented, e.g., along groove 54 and may thus reinforce the rubber layer between bore 52 and machined disk 64.This improves, for example, the rear gripping action.","For example, this machined disk 64 may be vulcanized into damper plate 50.At least two bores 65 are situated in damper plate 50.These are cylindrical in the region of upper metallic disk 62, thin rubber layer 63 and damper plate 50.The diameter of bores.","65 in machined disk 64 is adapted to cover bolts 68.Gas-spring bellows 72 is attached to damper plate 50 by these cover bolts 68.Damper plate 50 rests against vehicle body 5 in the region of vehicle-frame depression 6, and in the region of rubber sheet 63.FIG.","2 illustrates an alternative manner of attaching gas spring 70 to vehicle body 5.As in the exemplary embodiment illustrated in FIG.","1, a bolt 15 having external thread 16 is situated in a depression 6 of vehicle body 5.A cap 19 is screwed onto a bolt 15.In this case, this cap 19 has two circumferential toroids 24 at the transition from conical part 28 to cylindrical outer contour 23, the outer diameter being greater in the region of the cap 19 of the two circumferential toroids than in the region of cylindrical outer contour 23.The spacing of the two toroids 24 is, for example, approximately as large as half the difference of the diameter of a toroid 24 and cylindrical outer contour 23.Conical part 28 of cap 19 is tapered in the downward direction.","Damper plate 50 of gas spring 70 is a cylindrical, axially symmetric rubber sheet, which has a central bore 52.Bore 52 has two circumferential grooves 54.These have a contour opposite to that of toroids 24 and therefore may ensure that damper plate 50 elastically grips cap 19 from behind.","In this connection, the region of damper plate 50 between grooves 54 is optionally compressed.","In the region of the cap, the thickness of damper plate 50 corresponds to approximately two-thirds of the length of cap 19.In this case, outer side 59 of damper plate 50 is planar.","Situated in damper plate 50 is a, e.g., metallic, machined disk 80, which may be vulcanized in.","It has a central bore 82.Near the bore 82, the thickness of this machined disk 80 is approximately twice as much as in the remainder of machined disk 80.The diameter of bore 82 is approximately one third of the overall diameter of machined disk 80.A recess 83 is situated approximately in the center of this cylindrical bore 82.Damper plate 50 engages with the former and thus allows axial, form-locked engagement.","Bore 82 of machined disk 80 surrounds damper plate 50 to provide stiffness in the region of grooves 54.Machined disk 80 has at least two countersunk bores 86 in the outer region.","Cover bolts 68 are situated in these countersunk bores 86, cf.","FIG.","1.In the exemplary embodiment illustrated in FIG.","2, metallic disks 62, 64 and/or rubber disks 63 illustrated in FIG.","1 may also be vulcanized into damper plate 50.In the two exemplary embodiments, gas-spring bellows 72 pre-mounted to damper plate 50 is installed in the designated position.","In this connection, central bore 52 of damper plate 50 is aligned with and centered on cap 19.Chamfer 57 of bore 52 is then seated on conical part 28 of cap 19.For installation purposes, damper plate 50 is pushed, optionally together with spring bellows 72, against cap 19, and pushed over toroid(s) 24, as damper plate 50 elastically expands.","Cap in this case, grooves 54 rest against toroid(s) 24.During the automatic assembly of the axle, this gas spring 70 may be snapped into place in the nonpressurized state.","Additional fastening measures, such as bonding or the application of a torque, may not be necessary for installation.","In addition, a special tool may not be required for the installation or the detachment of gas spring 70 in the exemplary embodiments.","During installation, gas spring 70 centers itself on cap 19, using chamfer 57 of damper plate 50.No torsional stress is generated in spring bellows 72 in response to it being filled with gas, since spring 70 may align itself about fastening element 10.Due to its rear engagement, gas spring 70 may not detach from its mounting in response to a drop in pressure.","If gas spring 70 is equipped, on its end faces, with one of the mountings described in the exemplary embodiments, upper or lower damper plate 50 of gas spring 70 may also be arranged to have a blind hole in place of a bore 52.In this case, the sealing of the interior of gas spring 70 may be eliminated in the region of attachment.","Damper plate 50 may also be part of spring bellows 72.In this case, the need for pre-mounting spring bellows 72 to damper plate 50 may be eliminated.","In this exemplary embodiment, the need for the upper and/or lower gaskets in the region of the cover bolts may be eliminated.","The rubber-elastic seating of damper plate 50 on vehicle body 5 allows it to acoustically decouple vehicle body 5 from the suspension.","FIGS.","3 to 7 illustrate exemplary embodiments of the form and the attachment of bolt 15, i.e., of the shaped stud, and cap 19 to vehicle frame 5.In FIG.","3, bolt 15 is welded, for example, to vehicle frame 5.Bolt 15 may also be arranged as a sleeve.","Cap 19 has a cylindrical inner bore 22.As seen from above, the final third of inner bore 22 is arranged to be a tapped hole 21.In FIG.","4, stud 15 is screwed into a thread in vehicle frame 5.To secure the connection, stud 15 is braced against vehicle frame 5 at collar 17.FIG.","5 illustrates a bolt 95, which is inserted from above, through a bore 8 of vehicle frame 5.Bolt 95 may have a special head shape.","Head 96 of bolt 95 is welded to vehicle frame 5 from the top.","Cap 19 is screwed onto bolt 15 from below.","In this case, this cap 19 has the same exemplary embodiment as in FIG.","3.Cap 19 is fastened to bolt 95 by bracing it.","In FIG.","6, a nut 93 is welded to vehicle frame 5.Stud 15 is screwed into the nut.","In this connection, stud 15 may be arranged to have or not have a collar.","When stud 15 is constructed without a collar, stud 15 is secured by bracing stud 15 against the root of the thread.","When stud 15 has a collar, stud 15 is secured by bracing the shaft collar against nut 93.FIG.","7 corresponds to FIG.","6, the difference being that a weld nut 92 is attached to the upper side of vehicle frame 5.Stud 15 is secured, for example, by tack-welding it to weld nut 92, or by cold-working the ends of the thread, e.g., using a special tool.","Other attachment variations are possible.","These may combine, for example, the elements described above."]],"string":"[\n [\n \"Central Fastening Element for an Axially Symmetric Gas Spring\",\n \"In a central fastening element for an axially symmetric, vehicle gas spring, which includes a bellows that has central bores or cutouts in the region of its end faces, the fastening element being fixed to the vehicle body, protruding from the surroundings of the attachment point in a direction normal to it, and being encompassed by the bores or cutouts.\",\n \"The fastening element includes a shaped stud or a shaped cap, the maximum outer diameter of the stud or the cap being at least less than one fifth of the maximum outer diameter of the gas-spring bellows.\",\n \"A device may be provided which allows a gas spring to be installed in a simple manner and allows the spring bellows to rotate with respect to the suspension and/or the vehicle body during the initial installation and/or the initial operation.\"\n ],\n [\n \"1-9.\",\n \"(canceled) 10.A central fastening element for an axially symmetric, vehicle gas spring including a bellows having one of central bores and cutouts in a region of an end face, the fastening element configured to be fixed to a vehicle body and to protrude from surroundings of an attachment point in a normal direction and to be encompassed by the one of the bores and cutouts, comprising: at least one of a stud and a cap, one of the at least one of the stud and the cap shaped, a maximum outer diameter of the one of the stud and the cap at least less than one fifth of a maximum outer diameter of the bellows, the one of the stud and the cap including at least one necked-down portion having a diameter less than the maximum outer diameter of the one of the stud and the cap, the end face elastic in a zone of contact with the one of the stud and the cap.\",\n \"11.The fastening element according to claim 10, wherein a base of the bellows axially and radially surrounds the one of the stud and the cap without a sealing joint.\",\n \"12.The fastening element according to claim 10, further comprising an integrated supply line.\",\n \"13.The fastening element according to claim 10, wherein the cap includes an internal thread, the stud having an external thread, the cap substantially completely surrounding the stud, the cap arranged to one of directly and indirectly contact the vehicle body.\",\n \"14.The fastening element according to claim 10, wherein the stud is arranged to be screwed into a tapped hole of the vehicle body, the stud having a surface oriented in a direction normal to an axis of the stud, the surface arranged to one of directly and indirectly rest against the vehicle body.\",\n \"15.The fastening element according to claim 10, wherein the stud is arranged to be screwed into a nut fastened to the vehicle body.\",\n \"16.The fastening element according to claim 10, wherein a base of the bellows includes at least two superposed layers, at least one of the layers made of metal, at least one of the layers made of metal.\",\n \"17.The fastening element according to claim 10, wherein a base of the bellows is arranged to rest against at least two surface sections of the one of the stud and the cap oriented in axially opposite directions.\",\n \"18.The fastening element according to claim 10, wherein a base of the bellows is arranged to rest against a surface section of the one of the stud and the cap oriented in a direction of the vehicle body and against the vehicle body in a region of attachment of the one of the stud and the cap.\",\n \"19.The fastening element according to claim 10, wherein the cap includes an internal thread, the stud having an external thread, the cap substantially completely surrounding the stud, the cap one of directly and indirectly contacting the vehicle body.\",\n \"20.The fastening element according to claim 10, wherein the stud is screwed into a tapped hole of the vehicle body, the stud having a surface oriented in a direction normal to an axis of the stud, the surface one of directly and indirectly resting against the vehicle body.\",\n \"21.The fastening element according to claim 10, wherein the stud is screwed into a nut fastened to the vehicle body.\",\n \"22.The fastening element according to claim 10, wherein a base of the bellows rests against at least two surface sections of the one of the stud and the cap oriented in axially opposite directions.\",\n \"23.The fastening element according to claim 10, wherein a base of the bellows rests against a surface section of the one of the stud and the cap oriented in a direction of the vehicle body and against the vehicle body in a region of attachment of the one of the stud and the cap.\",\n \"24.The fastening element according to claim 10, wherein the fastening element is fixed to a vehicle body.\",\n \"25.The fastening element according to claim 10, wherein the fastening element protrudes from the surroundings of the attachment point in the normal direction.\",\n \"26.The fastening element according to claim 10, wherein the fastening element is encompassed by the one of the bores and cutouts.\",\n \"27.A device, comprising: an axially symmetric, vehicle gas spring including a bellows having one of central bores and cutouts in a region of an end face; and a fastening element configured to be fixed to a vehicle body and to protrude from surroundings of an attachment point in a normal direction, the fastening element encompassed by the one of the bores and cutouts, the fastening element including at least one of a stud and a cap, one of the stud and the cap shaped, a maximum outer diameter of the one of the stud and the cap at least less than one fifth of a maximum outer diameter of the bellows, the one of the stud and the cap including at least one necked-down portion having a diameter less than the maximum outer diameter of the one of the stud and the cap, the end face elastic in a zone of contact with the one of the stud and the cap.\"\n ],\n [\n \"<SOH> BACKGROUND INFORMATION <EOH>Such a device is described in European Published Patent Application No.\",\n \"0 123 171.The gas spring includes, inter alia, a vertical U-bellows and two axially symmetric bodies terminating it on the upper and lower sides.\",\n \"On the upper side, the gas spring is fixed to the vehicle body, e.g., to the vehicle frame, with the aid of a bolt.\",\n \"This bolt is seated in a bore of the frame and is screwed to the axially symmetric upper body, a so-called plug.\",\n \"The U-bellows itself is fastened to the axially symmetric, upper member with the aid of a tension band.\",\n \"In this case, the diameter of the plug is as large as the inner diameter of the U-bellows.\",\n \"This type of fastening requires that the bolthead on the upper side of the frame be accessible during installation.\",\n \"This makes it difficult to automate the installation of the gas spring.\",\n \"The gas spring tends to twist during installation, and while compressing and rebounding.\",\n \"Therefore, the tightening torque of the bolt must be selected to be high enough to prevent the connection between the gas spring and the vehicle body from loosening or releasing in response to vibrations and shock.\",\n \"It is an object of the present invention to provide a fastening element, which renders simple installation possible and allows the bellows to rotate with respect to the suspension and/or the vehicle frame during the initial installation and/or the initial operation.\"\n ],\n [\n \"<SOH> SUMMARY <EOH>This object may be achieved by providing a fastening element as described herein.\",\n \"To this end, the fastening element may include a shaped stud or a shaped cap, the maximum outer diameter of the stud or the cap being at least less than one fifth of the maximum outer diameter of the gas-spring bellows.\",\n \"The cap or the stud has at least one necked-down portion or waist, whose outer diameter is less than the above-mentioned, maximum diameter of the cap or stud.\",\n \"The end face is elastic in the zone in which it comes into contact with the stud or the cap.\",\n \"The central fastening element may be attached to the vehicle body prior to the installation of the gas spring and may protrude from the vehicle body in a direction normal to it.\",\n \"During installation, the gas spring is attached to, for example, the strut of the suspension and, e.g., pressed, together with it, against the shaped stud or the shaped cap.\",\n \"In the following, the term, stud, also includes the shaped cap.\",\n \"The elastic zone of the end face of the gas spring comes into contact with the stud in response to being slid up, and then engages with it in the manner of a snap fastener.\",\n \"The stud has a necked-down portion, which is encompassed by the bore or the cutout in a form-locked or force-locked manner.\",\n \"This type of fastening may allow the gas spring to be installed in an automated manner.\",\n \"In this case, and during initial operation, the gas spring may rotate on the stud, which means that the twisting of the U-bellows and the increased wear caused by it are prevented.\",\n \"The keyed connection between the stud and spring prevents vibrations and shock from detaching the fastening element.\",\n \"The base of the gas-spring bellows surrounds the stud axially and radially.\",\n \"Consequently, the gas spring is fixed in position in the axial and radial directions after installation.\",\n \"For example, it may not detach when the vehicle is jacked up, or in response to a pressure drop.\",\n \"The gas spring may be attached at its upper and lower ends in the same manner.\",\n \"Supply lines may be run through the fastening element into the interior of the gas spring.\",\n \"At least some regions of the base of the gas-spring bellows may be made of an elastic material, e.g., rubber.\",\n \"This may allow the base to act as a damping layer.\",\n \"The base may also be made up of multiple layers, e.g., a rubber layer and a metallic layer.\",\n \"In this case, the gas-spring bellows is attached to the metallic layer.\",\n \"The metallic layer is simultaneously used for increasing the strength of the gas-spring base.\",\n \"Several rubber and metallic layers may also be combined.\",\n \"To attach the gas spring, e.g., this rubber layer is compressed between two surface sections of the stud oriented in opposite, axial directions, or between a surface section oriented in the direction of the vehicle body, and the vehicle body.\",\n \"If the base of the gas spring is made out of multiple layers, the inner layer may be, for example, a metallic layer.\",\n \"The gas-spring bellows is then attached to this.\",\n \"This layer may be constructed in such a manner, that it supports the rubber layer and holds the position of the gas spring on the stud.\",\n \"Further details of the fastening element according to the present invention are set forth below in the subsequent description of several schematically represented, example embodiments.\"\n ],\n [\n \"FIELD OF THE INVENTION The present invention relates to a central fastening element for an axially symmetric, vehicle gas spring, which includes a bellows that has central bores or cutouts in the region of its end faces, the fastening element being fixed to the vehicle body, protruding from the surroundings of the attachment point in a direction normal to it, and being encompassed by the bores or cutouts.\",\n \"BACKGROUND INFORMATION Such a device is described in European Published Patent Application No.\",\n \"0 123 171.The gas spring includes, inter alia, a vertical U-bellows and two axially symmetric bodies terminating it on the upper and lower sides.\",\n \"On the upper side, the gas spring is fixed to the vehicle body, e.g., to the vehicle frame, with the aid of a bolt.\",\n \"This bolt is seated in a bore of the frame and is screwed to the axially symmetric upper body, a so-called plug.\",\n \"The U-bellows itself is fastened to the axially symmetric, upper member with the aid of a tension band.\",\n \"In this case, the diameter of the plug is as large as the inner diameter of the U-bellows.\",\n \"This type of fastening requires that the bolthead on the upper side of the frame be accessible during installation.\",\n \"This makes it difficult to automate the installation of the gas spring.\",\n \"The gas spring tends to twist during installation, and while compressing and rebounding.\",\n \"Therefore, the tightening torque of the bolt must be selected to be high enough to prevent the connection between the gas spring and the vehicle body from loosening or releasing in response to vibrations and shock.\",\n \"It is an object of the present invention to provide a fastening element, which renders simple installation possible and allows the bellows to rotate with respect to the suspension and/or the vehicle frame during the initial installation and/or the initial operation.\",\n \"SUMMARY This object may be achieved by providing a fastening element as described herein.\",\n \"To this end, the fastening element may include a shaped stud or a shaped cap, the maximum outer diameter of the stud or the cap being at least less than one fifth of the maximum outer diameter of the gas-spring bellows.\",\n \"The cap or the stud has at least one necked-down portion or waist, whose outer diameter is less than the above-mentioned, maximum diameter of the cap or stud.\",\n \"The end face is elastic in the zone in which it comes into contact with the stud or the cap.\",\n \"The central fastening element may be attached to the vehicle body prior to the installation of the gas spring and may protrude from the vehicle body in a direction normal to it.\",\n \"During installation, the gas spring is attached to, for example, the strut of the suspension and, e.g., pressed, together with it, against the shaped stud or the shaped cap.\",\n \"In the following, the term, stud, also includes the shaped cap.\",\n \"The elastic zone of the end face of the gas spring comes into contact with the stud in response to being slid up, and then engages with it in the manner of a snap fastener.\",\n \"The stud has a necked-down portion, which is encompassed by the bore or the cutout in a form-locked or force-locked manner.\",\n \"This type of fastening may allow the gas spring to be installed in an automated manner.\",\n \"In this case, and during initial operation, the gas spring may rotate on the stud, which means that the twisting of the U-bellows and the increased wear caused by it are prevented.\",\n \"The keyed connection between the stud and spring prevents vibrations and shock from detaching the fastening element.\",\n \"The base of the gas-spring bellows surrounds the stud axially and radially.\",\n \"Consequently, the gas spring is fixed in position in the axial and radial directions after installation.\",\n \"For example, it may not detach when the vehicle is jacked up, or in response to a pressure drop.\",\n \"The gas spring may be attached at its upper and lower ends in the same manner.\",\n \"Supply lines may be run through the fastening element into the interior of the gas spring.\",\n \"At least some regions of the base of the gas-spring bellows may be made of an elastic material, e.g., rubber.\",\n \"This may allow the base to act as a damping layer.\",\n \"The base may also be made up of multiple layers, e.g., a rubber layer and a metallic layer.\",\n \"In this case, the gas-spring bellows is attached to the metallic layer.\",\n \"The metallic layer is simultaneously used for increasing the strength of the gas-spring base.\",\n \"Several rubber and metallic layers may also be combined.\",\n \"To attach the gas spring, e.g., this rubber layer is compressed between two surface sections of the stud oriented in opposite, axial directions, or between a surface section oriented in the direction of the vehicle body, and the vehicle body.\",\n \"If the base of the gas spring is made out of multiple layers, the inner layer may be, for example, a metallic layer.\",\n \"The gas-spring bellows is then attached to this.\",\n \"This layer may be constructed in such a manner, that it supports the rubber layer and holds the position of the gas spring on the stud.\",\n \"Further details of the fastening element according to the present invention are set forth below in the subsequent description of several schematically represented, example embodiments.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS FIG.\",\n \"1 schematically illustrates the attachment of a gas spring to a vehicle body.\",\n \"FIG.\",\n \"2 schematically illustrates an alternative manner of attaching a gas spring to a vehicle body.\",\n \"FIGS.\",\n \"3 to 7 schematically illustrate alternative example embodiments of the fastening elements for the arrangements illustrated in FIGS.\",\n \"1 and 2.DETAILED DESCRIPTION FIG.\",\n \"1 illustrates the attachment of a gas spring 70 to a vehicle body 5, or to a strut.\",\n \"In this case, gas spring 70, e.g.\",\n \"an air spring, is made up of, inter alia, a U-bellows 72, which is attached to a damper plate 50.In this connection, damper plate 50 forms the base of gas-spring bellows 72.A fastening element 10 is mounted to vehicle body 5, e.g., to the vehicle frame, a body member, or a reinforced part of the body paneling.\",\n \"On vehicle body 5, a bolt 15 is situated in a concave and, optionally, axial asymmetric depression 6.For example, this bolt 15 may be welded on.\",\n \"Bolt 15 has a thread 16.A mushroom-shaped cap 19 is screwed onto this thread 16 via an internal thread 21.Cap 19 is axially asymmetric and has a cylindrical outer contour 23 in the upper region.\",\n \"In the middle region, the contour changes, e.g., discontinuously, into a circular arc 24.This transition is referred to in the following as 35 necked-down portion 25, undercut, or waist.\",\n \"In this case, the center point of circular arc 24 is, e.g., on the radius of cylindrical outer contour 23 or below it.\",\n \"Curved section 24 is referred to below as a toroid.\",\n \"Cap 19 changes continuously or discontinuously between toroid 24 and a conical section 28 and tapers to approximately three quarters of its upper diameter.\",\n \"Instead of being semicircular, the contour of the partial cross-section of toroid 24 may also be triangular.\",\n \"In the case of this contour, toroid 24 has a frustoconical lateral surface, whose imaginary apex points to the center of the U-bellows interior.\",\n \"For example, the bottom surface of this frustoconical apex discontinuously joins up with necked-down portion 25 to form a barb-shaped undercut.\",\n \"The frustoconical apex may also be an imaginary enveloping surface for a series of resilient barbs positioned about cap 19.After installation, these barbs sink into optionally sharp-edged groove 54 of damper plate 50, while elastically recovering.\",\n \"For example, a hexagonal recess 26 is centrally situated in cap 19, cf.\",\n \"FIG.\",\n \"3.Damper plate 50 is positioned around cap 19.The upper side of damper plate 50 is designated as outer side 59 and the lower side is designated as inner side 58.Cylindrical damper plate 50 is made of rubber and has a central bore 52, which is smaller that the maximum outer diameter of cap 19.A recess in the form of a circular groove 54 is situated approximately halfway up bore 52.This has a contour opposite to that of toroid 24 and therefore may ensure that damper plate 50 grips cap 19 from behind.\",\n \"In this connection, the region of damper plate 50 between groove 54 and outer side 59 may be compressed.\",\n \"Bore 52 changes into a chamfer 57 at the upper end face of damper plate 50.Damper plate 50 is also chamferred at the lower end face.\",\n \"The thickness of damper plate 50 in the middle region approximately corresponds to the length of cap 19, but the thickness in the outer region is reduced to approximately two thirds of the overall height.\",\n \"In this connection, inner side 58 is approximately planar.\",\n \"A metallic disk 62 and a thin rubber layer 63 are situated on the outer side 59 of damper plate 50.The two parts may be cemented on or vulcanized on.\",\n \"A machined disk 64, whose central region is formed in the shape of a frustoconical shell in the direction of the center, is situated on inner side 58.The top edge of the frustoconical shell may be oriented, e.g., along groove 54 and may thus reinforce the rubber layer between bore 52 and machined disk 64.This improves, for example, the rear gripping action.\",\n \"For example, this machined disk 64 may be vulcanized into damper plate 50.At least two bores 65 are situated in damper plate 50.These are cylindrical in the region of upper metallic disk 62, thin rubber layer 63 and damper plate 50.The diameter of bores.\",\n \"65 in machined disk 64 is adapted to cover bolts 68.Gas-spring bellows 72 is attached to damper plate 50 by these cover bolts 68.Damper plate 50 rests against vehicle body 5 in the region of vehicle-frame depression 6, and in the region of rubber sheet 63.FIG.\",\n \"2 illustrates an alternative manner of attaching gas spring 70 to vehicle body 5.As in the exemplary embodiment illustrated in FIG.\",\n \"1, a bolt 15 having external thread 16 is situated in a depression 6 of vehicle body 5.A cap 19 is screwed onto a bolt 15.In this case, this cap 19 has two circumferential toroids 24 at the transition from conical part 28 to cylindrical outer contour 23, the outer diameter being greater in the region of the cap 19 of the two circumferential toroids than in the region of cylindrical outer contour 23.The spacing of the two toroids 24 is, for example, approximately as large as half the difference of the diameter of a toroid 24 and cylindrical outer contour 23.Conical part 28 of cap 19 is tapered in the downward direction.\",\n \"Damper plate 50 of gas spring 70 is a cylindrical, axially symmetric rubber sheet, which has a central bore 52.Bore 52 has two circumferential grooves 54.These have a contour opposite to that of toroids 24 and therefore may ensure that damper plate 50 elastically grips cap 19 from behind.\",\n \"In this connection, the region of damper plate 50 between grooves 54 is optionally compressed.\",\n \"In the region of the cap, the thickness of damper plate 50 corresponds to approximately two-thirds of the length of cap 19.In this case, outer side 59 of damper plate 50 is planar.\",\n \"Situated in damper plate 50 is a, e.g., metallic, machined disk 80, which may be vulcanized in.\",\n \"It has a central bore 82.Near the bore 82, the thickness of this machined disk 80 is approximately twice as much as in the remainder of machined disk 80.The diameter of bore 82 is approximately one third of the overall diameter of machined disk 80.A recess 83 is situated approximately in the center of this cylindrical bore 82.Damper plate 50 engages with the former and thus allows axial, form-locked engagement.\",\n \"Bore 82 of machined disk 80 surrounds damper plate 50 to provide stiffness in the region of grooves 54.Machined disk 80 has at least two countersunk bores 86 in the outer region.\",\n \"Cover bolts 68 are situated in these countersunk bores 86, cf.\",\n \"FIG.\",\n \"1.In the exemplary embodiment illustrated in FIG.\",\n \"2, metallic disks 62, 64 and/or rubber disks 63 illustrated in FIG.\",\n \"1 may also be vulcanized into damper plate 50.In the two exemplary embodiments, gas-spring bellows 72 pre-mounted to damper plate 50 is installed in the designated position.\",\n \"In this connection, central bore 52 of damper plate 50 is aligned with and centered on cap 19.Chamfer 57 of bore 52 is then seated on conical part 28 of cap 19.For installation purposes, damper plate 50 is pushed, optionally together with spring bellows 72, against cap 19, and pushed over toroid(s) 24, as damper plate 50 elastically expands.\",\n \"Cap in this case, grooves 54 rest against toroid(s) 24.During the automatic assembly of the axle, this gas spring 70 may be snapped into place in the nonpressurized state.\",\n \"Additional fastening measures, such as bonding or the application of a torque, may not be necessary for installation.\",\n \"In addition, a special tool may not be required for the installation or the detachment of gas spring 70 in the exemplary embodiments.\",\n \"During installation, gas spring 70 centers itself on cap 19, using chamfer 57 of damper plate 50.No torsional stress is generated in spring bellows 72 in response to it being filled with gas, since spring 70 may align itself about fastening element 10.Due to its rear engagement, gas spring 70 may not detach from its mounting in response to a drop in pressure.\",\n \"If gas spring 70 is equipped, on its end faces, with one of the mountings described in the exemplary embodiments, upper or lower damper plate 50 of gas spring 70 may also be arranged to have a blind hole in place of a bore 52.In this case, the sealing of the interior of gas spring 70 may be eliminated in the region of attachment.\",\n \"Damper plate 50 may also be part of spring bellows 72.In this case, the need for pre-mounting spring bellows 72 to damper plate 50 may be eliminated.\",\n \"In this exemplary embodiment, the need for the upper and/or lower gaskets in the region of the cover bolts may be eliminated.\",\n \"The rubber-elastic seating of damper plate 50 on vehicle body 5 allows it to acoustically decouple vehicle body 5 from the suspension.\",\n \"FIGS.\",\n \"3 to 7 illustrate exemplary embodiments of the form and the attachment of bolt 15, i.e., of the shaped stud, and cap 19 to vehicle frame 5.In FIG.\",\n \"3, bolt 15 is welded, for example, to vehicle frame 5.Bolt 15 may also be arranged as a sleeve.\",\n \"Cap 19 has a cylindrical inner bore 22.As seen from above, the final third of inner bore 22 is arranged to be a tapped hole 21.In FIG.\",\n \"4, stud 15 is screwed into a thread in vehicle frame 5.To secure the connection, stud 15 is braced against vehicle frame 5 at collar 17.FIG.\",\n \"5 illustrates a bolt 95, which is inserted from above, through a bore 8 of vehicle frame 5.Bolt 95 may have a special head shape.\",\n \"Head 96 of bolt 95 is welded to vehicle frame 5 from the top.\",\n \"Cap 19 is screwed onto bolt 15 from below.\",\n \"In this case, this cap 19 has the same exemplary embodiment as in FIG.\",\n \"3.Cap 19 is fastened to bolt 95 by bracing it.\",\n \"In FIG.\",\n \"6, a nut 93 is welded to vehicle frame 5.Stud 15 is screwed into the nut.\",\n \"In this connection, stud 15 may be arranged to have or not have a collar.\",\n \"When stud 15 is constructed without a collar, stud 15 is secured by bracing stud 15 against the root of the thread.\",\n \"When stud 15 has a collar, stud 15 is secured by bracing the shaft collar against nut 93.FIG.\",\n \"7 corresponds to FIG.\",\n \"6, the difference being that a weld nut 92 is attached to the upper side of vehicle frame 5.Stud 15 is secured, for example, by tack-welding it to weld nut 92, or by cold-working the ends of the thread, e.g., using a special tool.\",\n \"Other attachment variations are possible.\",\n \"These may combine, for example, the elements described above.\"\n ]\n]","isConversational":false},"source":{"kind":"string","value":"Patent_10468176"}}},{"rowIdx":399,"cells":{"text":{"kind":"list like","value":[["Methods and compositions for inhibiting hiv-coreceptor interactions","Novel methods and compositions are provided for inhibiting interactions between human immunodeficiency viruses (HIVs) and viral coreceptors, including CXCR4 and/or CCR5 coreceptors.","The anti-coreceptor binding agent includes a novel peptide portion of the gp120 envelope protein of HIV-1, as well as peptide analogs and mimetics of this peptide, that specifically binds to, or modulates activity of, the coreceptors(s).","The anti-coreceptor binding agent is useful as a prophylactic or therapeutic treatment to prevent or inhibit HIV binding to a susceptible cell and thereby reduces infection and/or moderates or treats related diseases.","In alternative embodiments, the peptides, analogs and mimetics are effective to inhibit direct co-receptor binding by HIV virus, coreceptor binding by HIV gp120 proteins or peptides, HIV fusion with target host cells, HIV virion entry into host cells, HIV replication, and HIV transmission between cells and hosts.","In more detailed embodiments, the anti-coreceptor binding agents of the invention are multi-tropic by exhibiting activity against HIV interactions with multiple, CXCR4 and CCR5, coreceptors."],["1.A composition comprising an effective amount of an anti-coreceptor binding agent to inhibit binding of a CXCR4 and/or CCR5 coreceptor of a subject by an HIV virus or viral protein, wherein the anti-coreceptor binding agent is a gp120 peptide, peptide analog or mimetic that specifically binds the CXCR4 and/or CCR5 coreceptor.","2.The composition of claim 1, wherein the gp120 peptide, peptide analog or mimetic is between about 12 and about 24 amino acid residues in length and comprises a conserved CXXXXXXW amino acid sequence motif, wherein X is any naturally occurring or synthetic amino acid or amino acid analog.","3.The composition of claim 1, wherein the peptide, peptide analog or mimetic is modified by addition, admixture, or conjugation of additional amino acids, peptides, proteins, chemical reagents or moieties which do not substantially alter the anti-coreceptor binding activity of the peptide.","4.The composition of claim 1, wherein the anti-coreceptor binding agent is a peptide comprising an allelic variant among native HIV gp120 peptide sequences.","5.The composition of claim 1, wherein the anti-coreceptor binding agent is formulated for delivery to subject selected from an isolated or bound coreceptor, a membrane or cell preparation comprising the coreceptor, a cell population, tissue or organ expressing the coreceptor, or a mammalian patient.","6.The composition of claim 5, wherein the anti-coreceptor binding agent is combined with a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant for administration in a prophylactic or therapeutic effective dose to a mammalian patient to prevent or inhibit HIV infection or a related disease condition or symptom in the patient.","7.The composition of claim 1, wherein the anti-coreceptor binding inhibits one or more biological activities mediated by or associated with HIV-coreceptor interactions selected from (a) direct co-receptor binding by HIV virus, (b) coreceptor binding by a HIV gp120 protein or a peptide fragment or derivative thereof, (c) HIV fusion with target host cells, (d) HIV virion entry into host cells, (e) HIV replication, and/or (f) HIV cell-cell or host-host transmission.","8.The composition of claim 1, wherein the anti-coreceptor binding agent comprises an effective formulation of an HIV-1 peptide, peptide analog or mimetic for in vivo administration to inhibit one or more biological activities selected from (a) direct co-receptor binding by HIV-1 virus, (b) coreceptor binding by a HIV-1 gp120 protein or a peptide fragment or derivative thereof, (c) HIV-1 fusion with target host cells, (d) HIV-1 virion entry into host cells, (e) HIV-1 replication, and/or (f) HIV-1 cell-cell or host-host transmission.","9.The composition of claim 1, wherein the anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic formulated for administration to a mammalian patient in a prophylactically or therapeutically effective dose to prevent or inhibit HIV-1 infection or an HIV-1-related disease condition or symptom.","10.The composition of claim 1, wherein the anti-coreceptor binding agent is a peptide that includes a conserved “CXXXXXXW” amino acid sequence motif, wherein X is any amino acid, and wherein the peptide is from about 12-17 amino acids in length and is selected from peptide 15K, comprising an amino acid sequence IRKAHCNISRAKWND (SEQ ID NO:8), or a corresponding or overlapping native peptide sequence or peptide analog that shares substantial sequence identity to the reference peptide sequence of 15K.","11.The composition of claim 10, wherein the peptide includes one or more residues occurring naturally or by substitution at a relative, aligned position corresponding to a designated position for peptide 15K, selected from: Position 1—I, M, K, S, T, L, A, V, R, P, or N; Position 2—R, G, E, K, S, T, or I; Position 3—Q, K, R, L, E, P, A, V, S, T, H, or D; Position 4—A, T, P, V, E, or S; Position 5—H, Y, F, Q, N, I, or V; Position 7—N, D, H, T, K, E, S, I, Q, V, G, or A; Position 8—I, L, V, Y, D, A; Position 9—S, N, D, T, K, Y, I, or P; Position 10—R, K, G, S, A, E, D, I, T, W, or N; Position 11—A, R, K, T, S, G, E, D, N, Q, H, V, I, or L; Position 12—K, D, R, E, K, Q, N, T, S, G, A, V, L; Position 14—N, Q, D, E, K, R, A, S, T, G, M, Y, I, H, or V; and/or Position 15—D, N, K, E, T, Q, R, S, A, I, M, or P. 12.The composition of claim 1, wherein the anti-coreceptor binding agent exhibits multi-tropic activity characterized by effective inhibition of HIV viral, or gp120 protein or peptide binding to multiple, CXCR4 and CCR5, coreceptors.","13.The composition of claim 12, wherein the multi-tropic anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic effective to inhibit one or more biological activities of both T cell tropic (lymphotropic) and macrophage tropic (m-tropic) HIV-1 viruses selected from (a) direct co-receptor binding by viruses, (b) coreceptor binding by viral gp120 proteins or peptide fragments or derivatives thereof, (c) viral fusion with target host cells, (d) virion entry into host cells, (e) viral replication, and/or (f) viral cell-cell or host-host transmission.","14.A method for inhibiting human immunodeficiency virus (HIV) interaction with a CXCR4 and/or CCR5 coreceptor comprising exposing a subject to an effective amount of an anti-coreceptor binding agent to inhibit binding of the CXCR4 and/or CCR5 coreceptor by an HIV virus or viral protein, wherein the anti-coreceptor binding agent is a gp120 peptide, peptide analog or mimetic that specifically binds the CXCR4 and/or CCR5 coreceptor.","15.The method of claim 14, wherein the gp120 peptide, peptide analog or mimetic is between about 12 and about 24 amino acid residues in length and comprises a conserved CXXXXXXW amino acid sequence motif, wherein X is any naturally occurring or synthetic amino acid or amino acid analog.","16.The method of claim 14, wherein the peptide, peptide analog or mimetic is modified by addition, admixture, or conjugation of additional amino acids, peptides, proteins, chemical reagents or moieties which do not substantially alter the anti-coreceptor binding activity of the peptide.","17.The method of claim 14, wherein the anti-coreceptor binding agent is a peptide comprising an allelic variant among native HIV gp120 peptide sequences.","18.The method of claim 14, wherein the subject is an isolated or bound CXCR4 and/or CCRS coreceptor, a membrane or cell preparation comprising the coreceptor, a cell population, tissue or organ expressing the coreceptor, or a mammalian patient.","19.The method of claim 18, wherein the subject comprises a cell population, tissue or organ selected for in vivo or ex vivo treatment or diagnostic processing.","20.The method of claim 18, wherein the subject is a mammalian patient susceptible to HIV infection and the anti-coreceptor binding agent is administered in a prophylactic or therapeutic effective dose to prevent or inhibit HIV infection or a related disease condition or symptom.","21.The method of claim 14, wherein the anti-coreceptor binding agent is administered to the subject in an amount effective to inhibit one or more biological activities mediated by or associated with HIV-coreceptor interactions selected from (a) direct co-receptor binding by HIV virus, (b) coreceptor binding by a HIV gp120 protein or a peptide fragment or derivative thereof, (c) HIV fusion with target host cells, (d) HIV virion entry into host cells, (e) HIV replication, and/or (f) HIV cell-cell or host-host transmission.","22.The method of claim 14, wherein the anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic administered to the subject in an amount effective to inhibit one or more biological activities selected from (a) direct co-receptor binding by HIV-1 virus, (b) coreceptor binding by a HIV-1 gp120 protein or a peptide fragment or derivative thereof, (c) HIV-1 fusion with target host cells, (d) HIV-1 virion entry into host cells, (e) HIV-1 replication, and/or (f) HIV-1 cell-cell or host-host transmission.","23.The method of claim 14, wherein the anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic administered to a mammalian patient in a prophylactically or therapeutically effective dose to prevent or inhibit HIV-1 infection or an HIV-1-related disease condition or symptom.","24.The method of claim 14, wherein the anti-coreceptor binding agent is a peptide that includes a conserved “CXXXXXXW” amino acid sequence motif, wherein X is any amino acid, and wherein the peptide is from about 12 to about 17 amino acids in length and is selected from peptide 15K, comprising an amino acid sequence IRKAHCNISRAKWND (SEQ ID NO:8), or a corresponding or overlapping native peptide sequence or peptide analog that shares substantial sequence identity to the peptide sequence of 15K.","25.The method of claim 24, wherein the peptide includes one or more residues occurring naturally or by substitution at a relative, aligned position corresponding to a designated position for peptide 15K, selected from: Position 1—I, M, K, S, T, L, A, V, R, P, or N; Position 2—R, G, E, K, S, T, or I; Position 3—Q, K, R, L, E, P, A, V, S, T, H, or D; Position 4—A, T, P, V, E, or S; Position 5—H, Y, F, Q, N, I or V; Position 7—N, D, H, T, K, E, S, I, Q, V, G, or A; Position 8—I, L, Y, D, A; Position 9—S, N, D, T, K, Y, I, or P; Position 10—R, K, G, S, A, E, D, I, T, W, or N; Position 11—A,R,K,T, S, G, E, D,N, Q, H, V, I, or L; Position 12—K, D, R, E, K, Q, N, T, S, G, A, V, L; Position 14—N, Q, D, E, K, R, A, S, T, G, M, Y, I, H, or V; and/or Position 15—D, N, K, E, T, Q, R, S, A, I, M, or P. 26.The method of claim 14, wherein the anti-coreceptor binding agent exhibits multi-tropic activity characterized by effective inhibition of HIV viral, or gp120 protein or peptide binding to multiple, CXCR4 and CCR5, coreceptors.","27.The method of claim 26, wherein the multi-tropic anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic administered to the subject in an amount effective to inhibit one or more biological activities of both T cell tropic (lymphotropic) and macrophage tropic (m-tropic) HIV-1 viruses selected from (a) direct co-receptor binding by viruses, (b) coreceptor binding by viral gp120 proteins or peptide fragments or derivatives thereof, (c) viral fusion with target host cells, (d) virion entry into host cells, (e) viral replication, and/or (f) viral cell-cell or host-host transmission."],["<SOH> BACKGROUND OF THE INVENTION <EOH>The envelope glycoprotein of the human immunodeficiency virus type I (HIV-1) mediates in the fusion of viral and host cell membranes necessary for virion entry (Freed et al., J. Biol.","Chem.","270:23883-23886, 1995).","The envelope glycoprotein of HIV-1 is produced by the enzymatic cleavage from a gp160 precursor protein to produce the external gp120 protein and the transmembrane gp41 protein (Capon et al., Annu.","Rev.","Immunol.","9:649-678, 1991).","Several studies have identified specific portions or domains of the gp120 protein that may elicit humoral and/or cell-mediated immune responses to HIV in susceptible host subjects, and may therefore be useful to formulate anti-HIV reagents and methods for prevention and treatment of HIV infection and related diseases.","These general HIV peptide studies describe a large, diverse assemblage of gp120 peptides that are proposed as candidates for therapeutic use, primarily in vaccine formulations to prevent and treat HIV infection and related disease.","For example, U.S. Pat.","No.","5,691,135 describes various peptides that are selected for the ability to inhibit HIV infection by stimulating VH3 and VH4 antibody responses.","The peptides are proposed for administration to a patient as an antigen in sufficient quantity to induce antibodies that exhibit superantigen binding to gp120.The disclosure considers 31 peptides obtained from the AIDS Research and Reference Program, NIH, which peptides correspond to sequences from gp120 and gp41 from different strains of HIV.","Additional gp120 peptides are described in U.S. Pat.","No.","5,939,074.In particular, this references describes peptides that are useful as “multideterminant peptide antigens” for eliciting both cell-mediated and humoral immune responses against HIV.","Additional studies have identified particular regions of the gp120 that are proposed to determine specific interactions between gp120 and CD4, which is the primary receptor on target cells mediating cellular entry by HIV-1 (Arthos et al., Cell 57:469481, 1989; Clayton et al., Nature 339:548-551, 1989; Kwong et al., Nature 393:648-659, 1998; and Landau et al., Nature 334:159-162, 1988).","In addition to CD4 binding, cellular entry by HIV-1 is thought to require additional interactions between the virus and one or more “coreceptors” on the surfaces of target cells.","Briefly, the HIV-1 particles are proposed to bind initially to the CD4 receptor, and then subsequently to a chemokine receptor present on the target cells, which is used by the virus as a “coreceptor” for mediating cellular entry.","Different strains of HIV-1 preferentially utilize different chemokine receptors that are variably expressed among HIV-1 target cells.","In particular, some strains of HIV-1, referred to as “lymphotropic” strains, bind CXCR4 chemokine receptors predominantly expressed on lymphocyte target cells, while other HIV-1 strains, termed “monocyte tropic” strains, bind CCR5 receptors predominantly expressed on cells of monocyte lineage.","Thus, the major viral coreceptors are CXCR4 (Berson et al., J. Virol.","70:6288-6295, 1996), which has a native function as a chemokine receptor for stromal derived factor-1 (SDF-1), and CCR5 (Alkhatib et al., Science 272:1955-1958, 1996; Deng et al., Nature 381:661-666, 1996; and Dragic et al., Nature 381:667-673, 1996), which functions naturally as a receptor for several chemokines, including macrophage inflammatory protein-1β (MIP-1β).","It is proposed that a conformational change occurs in gp120 following HIV binding of CD4, and that this conformation change exposes one or more binding sites on the gp120 molecule that mediate additional interactions between HIV and chemokine receptor, which are referred to in this context as “viral coreceptors” (Kwong et al., Nature 393:648-659, 1998, incorporated herein by reference).","Recent studies suggest a complicated, multi-step binding and activation model, wherein the association of gp120 with CD4 yields a CD4-gp120 complex, which subsequently associates with the viral coreceptor resulting in a structural rearrangement (e.g., conformational change) of gp120 that facilitates interaction of the gp41 envelope protein subunit with the host cell membrane, leading to viral entry (Helseth et al., J. Virol.","64:6314-6318, 1990; and Weissenhom et al., Nature 387:426-430, 1997).","Within this emergent field of investigation, numerous studies have focused on structure-function mapping of chemokine receptors, with the goals of determining binding determinants and mechanisms of action of the receptors.","Yet additional research has focused on chemokine mapping studies.","From these studies chemokine-derived peptides have been identified that reportedly comprise binding determinants of the chemokines capable of blocking native chemokine receptor-ligand interactions.","Further studies, focusing alternatively on HIV gp120 mapping, report production and testing of gp120 peptides capable of blocking HIV-coreceptor binding and HIV infectivity.","With regard to chemokine receptor studies, numerous articles report that chemokine receptors play a direct role as coreceptors for HIV cell entry.","Briefly, as noted above, it is reported that specific interactions between the HIV envelope glycoprotein gp120 and one or more chemokine receptors mediate viral entry into target cells.","More specifically, monocyte-tropic or “m-tropic” HIV strains bind to a distinct chemokine receptor, CCR5, for cell entry, while T cell lymphotropic or “T-tropic” virus use mainly CXCR4 receptors for cell entry.","More detailed, structure-function analyses have been directed toward identifying specific HIV binding determinants of chemokine receptors mediating their activity as coreceptors for HIV entry.","In conjunction with these reports, certain references focus on a concept of blocking chemokine receptor activity (e.g., receptor binding with chemokines, or with gp120) using competitive inhibitors.","Proposed inhibitors of chemokine receptor binding interactions include peptide inhibitors that mimic structures of chemokines, or of gp120, binding determinants or related structural domains.","These studies follow more basic research which shows that intact chemokines, the normal ligands of chemokine receptors, can compete with cognate chemokines, or with HIV, for binding to the target receptors.","Referring specifically to chemokine mapping studies focusing on chemokine structure-function, various publications attempt to identify and characterize receptor binding determinants of chemokines.","As noted above, certain of these references also describe chemokine-based peptides reportedly capable of blocking chemokine-receptor binding and other activities mediated by receptor-ligand (chemokine or HIV) interactions.","One such study is presented in a publication by Reckless et al., ( Biochem.","J.","340:803-811, 1999).","This study identifies a number of chemokine-derived peptides, including a peptide designated “peptide 3”, based on a human chemokine, monocyte chemotactic protein-1 (MCP-1).","Reckless and colleagues report that the peptide 3 inhibits cell migration induced by a wide range of chemokines.","Moreover, peptide 3 reportedly binds to THP-1 cells and inhibits THP-1 migration, reportedly by acting as a chemokine receptor antagonist.","On this basis, the authors propose that peptide 3 and its derivatives, including peptides ranging from 6-15 residues in length, may be useful as chemokine inhibitors.","A number of related reports focus on a distinct portion of chemokines as prospective receptor binding determinants.","In particular, the reports focus on the N-terminal region of chemokines comprising a structural element called the “N-loop”.","This distinct element follows the first two cysteine residues of a model chemokine, and is proposed to play an important role in chemokine-receptor interactions.","Other parts of chemokine molecules have also been proposed to contribute significantly as structural determinants of interactions between chemokines and their cognate receptors.","For example, Crump et al., ( EMBO J.","16:6996-7007, 1997), teach that the N-terminal eight residues of the chemokine SDF-1 form an important receptor binding site.","At this site, two residues (Lys-1 and Pro-2) were proposed to be directly involved in receptor activation.","Disruption of these residues reportedly abolished activation.","It has further been reported that SDF-1 includes a second receptor binding motif at residues 12-17 of the chemokine loop region, termed the “RFFESH motif”.","Thus, it has been widely considered that the N-terminal region and so called N-loop following the first two cysteine residues of chemokines play the most important role in mediating the interactions between chemokines and their cognate receptors (Clark-Lewis et al., J. Biol.","Chem.","266:23128-23134, 1991; Crump et al., EMBO J.","16:6996-7007, 1997; and Pakianathan et al., Biochemistry 36:9642-9648, 1997).","In one of these studies, Clark-Lewis et al.","reported that a C-terminally truncated form of IL-8 missing the α-helix and β-turn within this region manifested greatly reduced chemotactic activity.","Additional confirmation of the primary significance of this segment of chemokines for receptor binding is provided by a recent observation that the biological activity of human MIP-1β is strongly reduced by substitutions of Arg-45, and Arg-47, with Serine (Czaplewski et al., J. Biol.","Chem.","274:16077-16084, 1999, incorporated herein by reference).","Finally, a peptide corresponding to the MCP-1 sequence just preceding the C-terminal α-helix was reported to inhibit chemotaxis of THP-1 cells indicating the importance of this region for chemokine function (Reckless et al., Biochem.","J.","340:803-1121, 1999).","Additional structure-function data for the CC chemokine RANTES have been reported by Pakianathan et al., ( Biochemistry 36:9642-9648, 1997).","Notably, a number of the foregoing publications point to a complex, multi-determinant model of interactions between chemokines and their native receptors.","In this context, Pakianathan et al.","believe the data indicates that RANTES interacts with each of its receptors in a distinct and specific manner and supports a two-site model of interaction between chemokines and their receptors.","Protein mapping studies of HIV gp120 envelope have reported identification of structural determinants of gp120 responsible for mediating HIV-coreceptor interactions.","Among these studies, Rizzuto et al., Science 280:1949-1953, 1998, have described a conserved gp120 structure that is reportedly important for binding to CCR5, and have postulated generally that this structural determinant should facilitate development of pharmacologic or immunologic inhibitors of virus-receptor interactions.","Rizzuto et al., suggested “that the CCR5-binding site is likely composed of conserved gp120 elements near or within the bridging sheet and V3 loop residues.” In another report addressing gp120 structure-function analysis relating to coreceptor binding, Verrier et al., ( AIDS Res.","Hum.","Retroviruses 15:731-743, 1999), studied the effect of linear V3 peptides (21-30 amino acids in length) on infectivity of different strains of HIV-1.These studies also pointed to the V3 loop as an important determinant of coreceptor choice, whereby single amino acid substitutions in V3 were reported to dramatically alter coreceptor usage.","In conjunction with this disclosure, Verrier et al., reported that artificial, linear peptides of V3 could compete with intact gp120 for binding to CCR5 and CXCR4 and block HIV entry into cells.","More specifically, the authors reported that the most efficient peptides for blocking fusion were derived from the middle of V3, and therefore did not include sequences at the C-terminal or N-terminal ends of V3 that form the base of the V3 loop.","Also significantly, Verrier et al., (supra) pointed to a “pattern of restriction” between multiple gp120 binding determinants, whereby peptides from different HIV strains (m-tropic versus lymphotropic) discriminate in their fusion-blocking activity in a pattern that “follows the coreceptor usage of the parental envelopes from which the peptides were derived.” This indicates that the candidate peptides described by Verrier et al., (supra) would not exhibit multi-specific blocking potential against both m-tropic and lymphotropic HIV-coreceptor interactions.","In a related study, Sakaida et al., ( J. Virol.","72:9763-9670, 1998) reported that synthetic cyclized (but not linear) V3 peptides of CXCR4 and dual-tropic strains of HIV-1 (but not a CCR5 strain) can prevent binding of anti-CXCR4 antibodies, potentially by binding to the coreceptor and acting as a competitive inhibitor.","These same peptides reportedly inhibited calcium mobilization by the chemokine SDF-1 in a T cell line.","The reference proposes that V3 loop peptides can directly bind to the relevant chemokine receptor and determine coreceptor usage, and postulates that such peptides can serve as HIV-1 reagents.","Like Verrier et al., (supra) Sakaida and coworkers conclude that the coreceptor binding activities of V3 peptides are strain-dependent.","More detailed reports pertaining to the role of V3 in mediating HIV-coreceptor interactions focus on specific sites or residues within the V3 domain as reportedly critical or important residues for coreceptor binding.","For example, Tugarinov et al., ( Structure Fold Des.","8:385-395, 2000), report that a conserved, central loop sequence, GPG, within the V3 loop plays a primary role in maintaining the conformation of the loop to mediate coreceptor binding.","Kato et al.","( J. Virol.","73:5520-5526, 1999) suggest that three specific residues, confined to the central, loop portion of V3 and located distant from the C-terminal segment, are particularly important for coreceptor usage.","Xiao et al., ( Virology 240:83-92, 1998), identify a consensus motif S/GXXXGPGXXXXXXXE/D, covering the central portion of the V3 loop and excluding the C-terminal portion, as a critical motif for coreceptor binding.","Wang et al., Proc.","Natl.","Acad.","Sci.","USA 95:5740-5745, 1998), studied whether certain V3 residues conserved among HIV-1, HIV-2 and SIV determine the utilization of CCR5 as a coreceptor.","They concluded that Arg-298 (at the beginning of V3 loop) has an importance for CCR5 utilization.","The authors suggested that this residue may represent a highly conserved structural element and a useful target for developing anti-viral therapies.","In another study by Wang et al., ( Proc.","Natl.","Acad.","Sci.","USA 96: 4558-4562, 1999), additional V3 residues reported to be critical for CCR5 utilization are identified by alanine scanning mutagenesis.","One of the identified residues, A 328 , is located at the C-terminus of V3, and its substitution reportedly results in a 1,000-fold reduction of CCR5 binding activity.","However, this residue is only one of two “highly conserved” residues and six “critical” residues identified for CCR5 utilization.","Most of these residues are distinctly located at the C-terminal or central loop portion of V3, including R 298 , K 305 , I 307 , R 313 , and F 315 .","Wang and colleagues propose that “these highly invariable residues” as well as others identified in a “bridging sheet” portion of the molecule may represent “targets for antiviral designs aimed at blocking the coreceptor entry step of HIV-1 replication.” Based on the foregoing articles, there is a broadly-confirmed disclosure of V3 as a critical domain for mediating HIV-coreceptor interactions.","A large number of important or critical residues are indicated, with a substantial range of discrepancy between reports implicating different residues as being important, or critical, for coreceptor binding functions.","These references that focus on V3 uniformly teach away from excluding important “critical” or “highly conserved” V3 residues.","At the same time, these references focusing on the V3 domain uniformly fail to implicate other sequence elements that may be present in the gp120 molecule outside the V3 loop portion thereof.","Coupled with these teachings, a host of related publications point to yet additional components of gp120 that may be essential for HIV-coreceptor interactions.","Because these components appear to be complex and potentially interact to achieve coreceptor binding, their disclosure adds further complexity to understanding gp120 structure-function relationships for mediating coreceptor binding and cell entry.","In particular, Tugarinov et al., ( Structure Fold Des.","8:385-395, 2000), teach the importance of the GPG motif of V3, but also teach that “[h]igh affinity binding of gp120 to the chemokine receptors requires participation of other domains in gp120 such as the CD4i epitope.” Rizzuto et al., ( Science 280:1949-1953, 1998), suggest that the “CCR5-binding site is likely composed of conserved gp120 elements near or within the bridging sheet and V3 loop residues.” They further proposed that CD4 binding may distort the V1/V2 stem, repositioning the stem allowing the formation of the β-sheet important for CCR5 binding.","They also noted that substitution of Asp for Thr 123 , located in the V1/V2 stem and which contact CD4, significantly decreased CCR5 binding.","This report parallels others which point to a favored model of a “conformational binding site” in gp120 for mediating HIV-coreceptor interactions.","According to this model, effective binding of coreceptors by gp120 involves initial binding of the gp120 to a CD4 receptor, which brings about a “CD4 induced” conformational change in gp120 involving distant residues—that in turn leads to formation of a conformational binding determinant on gp120 to mediate HIV-coreceptor interactions.","In this context, Wu et al., ( Nature 384:179-183, 1996) suggested that HIV-1 attachment to CD4 creates a high-affinity binding site for CCR-5, leading to membrane fusion and virus entry.","Consistent with this model, recent studies have identified yet additional sites of gp120 which are reportedly important, or essential, for mediating HIV-coreceptor interactions.","These sites are generally distinct from V3, and also from the V3-flanking regions.","For example, Cho et al., ( J. Virol.","72:2509-2515, 1998), report that V1/V2 and V3 confer on HIV-1 the ability to use CXCR4 coreceptors, but that the V4 and V5 regions are also “required in conjunction with regions V1 and V3 of HIV-1DH 12 gp120 for efficient utilization of CXCR4.” Este et al., ( Mol.","Pharmacol.","52:98-104, 1997), point to structural determinants in each of the V2, V3 and C3 regions of gp120 for determining cell tropism and coreceptor utilization.","Hoffman et al., ( Mol.","Membr.","Biol.","16:57-65, 1999), concluded the V3 loop was implicated in regulating viral tropism, but, that other regions of Env, such as the V1- and V2-loops, modulated the effects of the V3-loop.","They also acknowledged that some important exceptions to this model suggested that understanding of virus tropism and Env-chemokine receptor interactions was incomplete.","Collectively, these studies point to numerous components of gp120 that may be important or essential for HIV-coreceptor interactions, and which may in fact involve multiple interactions whereby different parts of gp120 are required to work together to achieve efficient HIV-coreceptor interactions.","This complexity and the proposed requirement for multiple, distant binding determinants on gp120 to form a conformational binding determinant for coreceptor usage, may be generally considered to teach away from the utility of small peptides for effectively blocking HIV-coreceptor binding interactions, particularly to block multiple HIV strains infecting different target cell types.","Moreover, while the foregoing, separate bodies of literature individually discuss models of conserved binding elements of chemokines, or of gp120, as determinants for mediating coreceptor interactions, these reports do not teach or suggest identification of conserved binding determinants shared between gp120 and chemokines.","On the contrary, complex functional interactions and distinct binding mechanisms and determinants previously proposed for gp120 and chemokines may be considered to lead away from investigations aimed at identifying common binding determinants or mechanisms between gp120 and chemokines.","This conclusion is well supported in the literature reviewed herein above.","Based on these and other teachings, there does not appear to be a clear suggestion in the literature to use a common blocking agent for coreceptor interactions by chemokines and HIV aimed at, or patterned after, a shared binding determinant between these two distinct molecules.","The literature does not point toward any such common structural domain or binding determinant between HIV gp120 and chemokines, and the distinct structure-function reports for how HIV and chemokines are thought to interact with the subject receptors leads away from this path of inquiry.","In view of the foregoing teachings and unsolved questions in the literature, there remains an urgent need in the art for additional tools and methods to combat HIV infection and related disease.","Related to this fundamental goal, there remains a clear need for additional therapeutic agents and methods targeting HIV viral entry into host cells, preferably that will include compositions and methods to block gp120 binding to different coreceptors to inhibit viral entry and infection and ameliorate HIV-related disease.","Surprisingly, the instant invention fulfills these objects and satisfies additional objects and advantages which will become apparent from the following description."],["<SOH> SUMMARY OF THE INVENTION <EOH>The instant invention provides novel methods and compositions for inhibiting interactions between human immunodeficiency viruses (HIVs) and chemokine receptors, termed “viral coreceptors” in this context.","The methods of the invention generally comprise exposing a CXCR4 or CCR5 coreceptor of a subject to an effective amount of an anti-coreceptor binding agent of the invention to inhibit binding of the coreceptor by an HIV virus or viral protein.","Typically, the anti-coreceptor binding agent is a gp120 peptide, peptide analog or mimetic that specifically binds the coreceptor.","Within certain methods of the invention, the subject is an isolated or bound coreceptor, a membrane or cell preparation comprising the coreceptor, a cell population, tissue or organ expressing the coreceptor, or a mammalian patient.","In more detailed embodiments, the subject comprises a cell population, tissue or organ selected for in vivo or ex vivo treatment or diagnostic processing.","Alternatively, the subject may be a mammalian patient susceptible to HIV infection and the anti-coreceptor binding agent is administered in a prophylactic or therapeutically effective dose to prevent or inhibit HIV binding to a susceptible cell and thereby preventing or inhibiting infection or a related disease condition or symptom.","In typical aspects of the invention, the anti-coreceptor binding agent is administered to the subject in an amount effective to inhibit one or more biological activities mediated by or associated with HIV-coreceptor interactions selected from (a) direct co-receptor, e.g., CXCR4 and/or CCR5, binding by HIV virus, (b) coreceptor binding by a HIV gp120 protein or a peptide fragment or derivative thereof, (c) HIV fusion with target host cells, (d) HIV virion entry into host cells, (e) HIV replication, and/or (f) HIV cell-cell or host-host transmission.","In more specific embodiments, the anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic and is administered to the subject in an amount effective to inhibit one or more biological activities selected from (a) direct co-receptor, i.e., CXCR4 and/or CCR5, binding by HIV-1 virus, (b) coreceptor binding by a HIV-1 gp120 protein or a peptide fragment or derivative thereof, (c) HIV-1 fusion with target host cells, (d) HIV-1 virion entry into host cells, (e) HIV-1 replication, and/or (f) HIV-1 cell-cell or host-host transmission.","Often, the anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic administered to a mammalian patient in a prophylactically or therapeutically effective dose to prevent or inhibit HIV-1 binding infection, to a susceptible host, HIV-1 or an HIV-1-related disease condition or symptom.","In more detailed methods and compositions of the invention, the gp120 peptide, peptide analog or mimetic is between about 12 and about 24 amino acid residues in length and comprises a conserved CXXXXXXW amino acid sequence motif identified within the amino acid sequence of gp120 proteins of HIV isolates and also among diverse chemokines, wherein X is any naturally occurring or synthetic amino acid or amino acid analog.","The peptide, peptide analog or mimetic can be modified in a wide variety of ways and forms, e.g., by addition, admixture, or conjugation of additional amino acids, peptides, proteins, chemical reagents or moieties which do not substantially alter the anti-coreceptor binding activity of the peptide.","Often, the anti-coreceptor binding agent of the invention is a peptide comprising an allelic variant that is found among native HIV gp120 peptide sequences.","Within more detailed embodiments, the anti-coreceptor binding agent is a peptide of between about 12-17 amino acids in length that includes a conserved “CXXXXXXW” amino acid sequence motif, which is selected from an exemplary “reference” peptide designated 15K comprising an amino acid sequence IRKAHCNISRAKWND (SEQ ID NO:8), or is alternatively represented by a corresponding or overlapping native peptide sequence or peptide analog that shares substantial sequence identity to the reference amino acid sequence of 15K.","In various specific embodiments, the peptide includes one or more residues occurring naturally or by substitution at a relative, aligned position corresponding to a designated position for peptide 15K, selected from: Position 1—I, M, K, S, T, L, A, V, R, P, or N; Position 2—R, G, E, K, S, T, or I; Position 3—Q, K, R, L, E, P, A, V, S, T, H, or D; Position 4—A, T, P, V, E, or S; Position 5—H, Y, F, Q. N, I, or V; Position 7—N, D, H, T, K, E, S, I, Q, V, G, or A; Position 8—I, L, V, Y, D, A; Position 9—S, N, D, T, K, Y, I, or P; Position 10—R, K, G, S, A, E, D, I, T, W, or N; Position 11—A, R, K, T, S, G, E, D, N, Q, H, V, I, or L; Position 12—K, D, R, E, K, Q, N, T, S, G, A, V, L; Position 14—N, Q, D, E, K, R, A, S, T, G, M, Y, I, H, or V; and/or Position 15—D, N, K, E, T, Q, R, S, A, I, M, or P. In yet additional embodiments of the invention, the anti-coreceptor binding agent exhibits multi-tropic activity characterized by effective inhibition of HIV viral, or gp120 protein or peptide binding to multiple, CXCR4 and CCR5, coreceptors.","Often, the multi-tropic anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic administered to the subject in an amount effective to inhibit one or more biological activities of both T cell tropic (lymphotropic) and macrophage tropic (m-tropic) HIV-1 viruses selected from (a) direct co-receptor binding by viruses, (b) coreceptor binding by viral gp120 proteins or peptide fragments or derivatives thereof, (c) viral fusion with target host cells, (d) virion entry into host cells, (e) viral replication, and/or (f) viral cell-cell or host-host transmission.","Within the methods and compositions of the invention, the anti-coreceptor binding agent may be formulated in various combinations with a pharmaceutically acceptable carrier, diluent, excipient, adjuvant or other active or inactive agents, in an amount or dosage form sufficient to prevent, reduce or even alleviate HIV infection or related disease conditions or symptoms.","In yet additional aspects of the invention, the anti-coreceptor binding agent of the invention is administered according to the foregoing methods in a combinatorial formulation or coordinate treatment with one or more additional anti-HIV, antibacterial, antiviral or other therapeutically active agent(s).","Within related methods and compositions, the anti-coreceptor binding agent is admixed or co-administered, simultaneously or sequentially with one or more additional anti-HIV, antibacterial, antiviral or other therapeutically active agent(s) to prevent, reduce or even alleviate HIV infection or related conditions in a mammalian patient.","The instant invention also includes kits, packages and multicontainer units containing the anti-coreceptor binding agent, optionally with other active or inactive ingredients, and/or means for administering the same for use in the diagnosis, management and/or prevention and treatment of HIV and related conditions.","Typically, these kits include a diagnostic or pharmaceutical preparation of the anti-coreceptor binding agent, typically formulated with a biologically suitable carrier and optionally contained in a bulk dispensing container or unit or multi-unit dosage form.","Optional dispensing means can be provided, for example an intranasal spray applicator.","Packaging materials optionally include a label or instruction which indicates a desired use of the kit as described herein below.","Additional aspects of the invention include polynucleotide molecules and vector constructs encoding anti-coreceptor binding peptides and peptide analogs.","Also provided are peptide vaccines and other immunogenic compositions that elicit an immune response involving production of antibodies targeting one or more epitopes of gp120 recognized by antibodies that specifically bind an anti-coreceptor binding peptide of the invention.","In addition, the invention provides, antibodies, including monoclonal antibodies, and immunotherapeutic methods and compositions comprising such antibodies that specifically recognize anti-coreceptor binding peptides of the invention, for use as diagnostic and therapeutic reagents.","Also provided within the invention are a variety of additional diagnostic and therapeutic tools and reagents as set forth in detail in the following description."],["RELATED APPLICATION This application claims priority to provisional application Ser.","No.","60/269,534 filed on Feb. 15, 2001.BACKGROUND OF THE INVENTION The envelope glycoprotein of the human immunodeficiency virus type I (HIV-1) mediates in the fusion of viral and host cell membranes necessary for virion entry (Freed et al., J. Biol.","Chem.","270:23883-23886, 1995).","The envelope glycoprotein of HIV-1 is produced by the enzymatic cleavage from a gp160 precursor protein to produce the external gp120 protein and the transmembrane gp41 protein (Capon et al., Annu.","Rev.","Immunol.","9:649-678, 1991).","Several studies have identified specific portions or domains of the gp120 protein that may elicit humoral and/or cell-mediated immune responses to HIV in susceptible host subjects, and may therefore be useful to formulate anti-HIV reagents and methods for prevention and treatment of HIV infection and related diseases.","These general HIV peptide studies describe a large, diverse assemblage of gp120 peptides that are proposed as candidates for therapeutic use, primarily in vaccine formulations to prevent and treat HIV infection and related disease.","For example, U.S. Pat.","No.","5,691,135 describes various peptides that are selected for the ability to inhibit HIV infection by stimulating VH3 and VH4 antibody responses.","The peptides are proposed for administration to a patient as an antigen in sufficient quantity to induce antibodies that exhibit superantigen binding to gp120.The disclosure considers 31 peptides obtained from the AIDS Research and Reference Program, NIH, which peptides correspond to sequences from gp120 and gp41 from different strains of HIV.","Additional gp120 peptides are described in U.S. Pat.","No.","5,939,074.In particular, this references describes peptides that are useful as “multideterminant peptide antigens” for eliciting both cell-mediated and humoral immune responses against HIV.","Additional studies have identified particular regions of the gp120 that are proposed to determine specific interactions between gp120 and CD4, which is the primary receptor on target cells mediating cellular entry by HIV-1 (Arthos et al., Cell 57:469481, 1989; Clayton et al., Nature 339:548-551, 1989; Kwong et al., Nature 393:648-659, 1998; and Landau et al., Nature 334:159-162, 1988).","In addition to CD4 binding, cellular entry by HIV-1 is thought to require additional interactions between the virus and one or more “coreceptors” on the surfaces of target cells.","Briefly, the HIV-1 particles are proposed to bind initially to the CD4 receptor, and then subsequently to a chemokine receptor present on the target cells, which is used by the virus as a “coreceptor” for mediating cellular entry.","Different strains of HIV-1 preferentially utilize different chemokine receptors that are variably expressed among HIV-1 target cells.","In particular, some strains of HIV-1, referred to as “lymphotropic” strains, bind CXCR4 chemokine receptors predominantly expressed on lymphocyte target cells, while other HIV-1 strains, termed “monocyte tropic” strains, bind CCR5 receptors predominantly expressed on cells of monocyte lineage.","Thus, the major viral coreceptors are CXCR4 (Berson et al., J. Virol.","70:6288-6295, 1996), which has a native function as a chemokine receptor for stromal derived factor-1 (SDF-1), and CCR5 (Alkhatib et al., Science 272:1955-1958, 1996; Deng et al., Nature 381:661-666, 1996; and Dragic et al., Nature 381:667-673, 1996), which functions naturally as a receptor for several chemokines, including macrophage inflammatory protein-1β (MIP-1β).","It is proposed that a conformational change occurs in gp120 following HIV binding of CD4, and that this conformation change exposes one or more binding sites on the gp120 molecule that mediate additional interactions between HIV and chemokine receptor, which are referred to in this context as “viral coreceptors” (Kwong et al., Nature 393:648-659, 1998, incorporated herein by reference).","Recent studies suggest a complicated, multi-step binding and activation model, wherein the association of gp120 with CD4 yields a CD4-gp120 complex, which subsequently associates with the viral coreceptor resulting in a structural rearrangement (e.g., conformational change) of gp120 that facilitates interaction of the gp41 envelope protein subunit with the host cell membrane, leading to viral entry (Helseth et al., J. Virol.","64:6314-6318, 1990; and Weissenhom et al., Nature 387:426-430, 1997).","Within this emergent field of investigation, numerous studies have focused on structure-function mapping of chemokine receptors, with the goals of determining binding determinants and mechanisms of action of the receptors.","Yet additional research has focused on chemokine mapping studies.","From these studies chemokine-derived peptides have been identified that reportedly comprise binding determinants of the chemokines capable of blocking native chemokine receptor-ligand interactions.","Further studies, focusing alternatively on HIV gp120 mapping, report production and testing of gp120 peptides capable of blocking HIV-coreceptor binding and HIV infectivity.","With regard to chemokine receptor studies, numerous articles report that chemokine receptors play a direct role as coreceptors for HIV cell entry.","Briefly, as noted above, it is reported that specific interactions between the HIV envelope glycoprotein gp120 and one or more chemokine receptors mediate viral entry into target cells.","More specifically, monocyte-tropic or “m-tropic” HIV strains bind to a distinct chemokine receptor, CCR5, for cell entry, while T cell lymphotropic or “T-tropic” virus use mainly CXCR4 receptors for cell entry.","More detailed, structure-function analyses have been directed toward identifying specific HIV binding determinants of chemokine receptors mediating their activity as coreceptors for HIV entry.","In conjunction with these reports, certain references focus on a concept of blocking chemokine receptor activity (e.g., receptor binding with chemokines, or with gp120) using competitive inhibitors.","Proposed inhibitors of chemokine receptor binding interactions include peptide inhibitors that mimic structures of chemokines, or of gp120, binding determinants or related structural domains.","These studies follow more basic research which shows that intact chemokines, the normal ligands of chemokine receptors, can compete with cognate chemokines, or with HIV, for binding to the target receptors.","Referring specifically to chemokine mapping studies focusing on chemokine structure-function, various publications attempt to identify and characterize receptor binding determinants of chemokines.","As noted above, certain of these references also describe chemokine-based peptides reportedly capable of blocking chemokine-receptor binding and other activities mediated by receptor-ligand (chemokine or HIV) interactions.","One such study is presented in a publication by Reckless et al., (Biochem.","J.","340:803-811, 1999).","This study identifies a number of chemokine-derived peptides, including a peptide designated “peptide 3”, based on a human chemokine, monocyte chemotactic protein-1 (MCP-1).","Reckless and colleagues report that the peptide 3 inhibits cell migration induced by a wide range of chemokines.","Moreover, peptide 3 reportedly binds to THP-1 cells and inhibits THP-1 migration, reportedly by acting as a chemokine receptor antagonist.","On this basis, the authors propose that peptide 3 and its derivatives, including peptides ranging from 6-15 residues in length, may be useful as chemokine inhibitors.","A number of related reports focus on a distinct portion of chemokines as prospective receptor binding determinants.","In particular, the reports focus on the N-terminal region of chemokines comprising a structural element called the “N-loop”.","This distinct element follows the first two cysteine residues of a model chemokine, and is proposed to play an important role in chemokine-receptor interactions.","Other parts of chemokine molecules have also been proposed to contribute significantly as structural determinants of interactions between chemokines and their cognate receptors.","For example, Crump et al., (EMBO J.","16:6996-7007, 1997), teach that the N-terminal eight residues of the chemokine SDF-1 form an important receptor binding site.","At this site, two residues (Lys-1 and Pro-2) were proposed to be directly involved in receptor activation.","Disruption of these residues reportedly abolished activation.","It has further been reported that SDF-1 includes a second receptor binding motif at residues 12-17 of the chemokine loop region, termed the “RFFESH motif”.","Thus, it has been widely considered that the N-terminal region and so called N-loop following the first two cysteine residues of chemokines play the most important role in mediating the interactions between chemokines and their cognate receptors (Clark-Lewis et al., J. Biol.","Chem.","266:23128-23134, 1991; Crump et al., EMBO J.","16:6996-7007, 1997; and Pakianathan et al., Biochemistry 36:9642-9648, 1997).","In one of these studies, Clark-Lewis et al.","reported that a C-terminally truncated form of IL-8 missing the α-helix and β-turn within this region manifested greatly reduced chemotactic activity.","Additional confirmation of the primary significance of this segment of chemokines for receptor binding is provided by a recent observation that the biological activity of human MIP-1β is strongly reduced by substitutions of Arg-45, and Arg-47, with Serine (Czaplewski et al., J. Biol.","Chem.","274:16077-16084, 1999, incorporated herein by reference).","Finally, a peptide corresponding to the MCP-1 sequence just preceding the C-terminal α-helix was reported to inhibit chemotaxis of THP-1 cells indicating the importance of this region for chemokine function (Reckless et al., Biochem.","J.","340:803-1121, 1999).","Additional structure-function data for the CC chemokine RANTES have been reported by Pakianathan et al., (Biochemistry 36:9642-9648, 1997).","Notably, a number of the foregoing publications point to a complex, multi-determinant model of interactions between chemokines and their native receptors.","In this context, Pakianathan et al.","believe the data indicates that RANTES interacts with each of its receptors in a distinct and specific manner and supports a two-site model of interaction between chemokines and their receptors.","Protein mapping studies of HIV gp120 envelope have reported identification of structural determinants of gp120 responsible for mediating HIV-coreceptor interactions.","Among these studies, Rizzuto et al., Science 280:1949-1953, 1998, have described a conserved gp120 structure that is reportedly important for binding to CCR5, and have postulated generally that this structural determinant should facilitate development of pharmacologic or immunologic inhibitors of virus-receptor interactions.","Rizzuto et al., suggested “that the CCR5-binding site is likely composed of conserved gp120 elements near or within the bridging sheet and V3 loop residues.” In another report addressing gp120 structure-function analysis relating to coreceptor binding, Verrier et al., (AIDS Res.","Hum.","Retroviruses 15:731-743, 1999), studied the effect of linear V3 peptides (21-30 amino acids in length) on infectivity of different strains of HIV-1.These studies also pointed to the V3 loop as an important determinant of coreceptor choice, whereby single amino acid substitutions in V3 were reported to dramatically alter coreceptor usage.","In conjunction with this disclosure, Verrier et al., reported that artificial, linear peptides of V3 could compete with intact gp120 for binding to CCR5 and CXCR4 and block HIV entry into cells.","More specifically, the authors reported that the most efficient peptides for blocking fusion were derived from the middle of V3, and therefore did not include sequences at the C-terminal or N-terminal ends of V3 that form the base of the V3 loop.","Also significantly, Verrier et al., (supra) pointed to a “pattern of restriction” between multiple gp120 binding determinants, whereby peptides from different HIV strains (m-tropic versus lymphotropic) discriminate in their fusion-blocking activity in a pattern that “follows the coreceptor usage of the parental envelopes from which the peptides were derived.” This indicates that the candidate peptides described by Verrier et al., (supra) would not exhibit multi-specific blocking potential against both m-tropic and lymphotropic HIV-coreceptor interactions.","In a related study, Sakaida et al., (J. Virol.","72:9763-9670, 1998) reported that synthetic cyclized (but not linear) V3 peptides of CXCR4 and dual-tropic strains of HIV-1 (but not a CCR5 strain) can prevent binding of anti-CXCR4 antibodies, potentially by binding to the coreceptor and acting as a competitive inhibitor.","These same peptides reportedly inhibited calcium mobilization by the chemokine SDF-1 in a T cell line.","The reference proposes that V3 loop peptides can directly bind to the relevant chemokine receptor and determine coreceptor usage, and postulates that such peptides can serve as HIV-1 reagents.","Like Verrier et al., (supra) Sakaida and coworkers conclude that the coreceptor binding activities of V3 peptides are strain-dependent.","More detailed reports pertaining to the role of V3 in mediating HIV-coreceptor interactions focus on specific sites or residues within the V3 domain as reportedly critical or important residues for coreceptor binding.","For example, Tugarinov et al., (Structure Fold Des.","8:385-395, 2000), report that a conserved, central loop sequence, GPG, within the V3 loop plays a primary role in maintaining the conformation of the loop to mediate coreceptor binding.","Kato et al.","(J. Virol.","73:5520-5526, 1999) suggest that three specific residues, confined to the central, loop portion of V3 and located distant from the C-terminal segment, are particularly important for coreceptor usage.","Xiao et al., (Virology 240:83-92, 1998), identify a consensus motif S/GXXXGPGXXXXXXXE/D, covering the central portion of the V3 loop and excluding the C-terminal portion, as a critical motif for coreceptor binding.","Wang et al., Proc.","Natl.","Acad.","Sci.","USA 95:5740-5745, 1998), studied whether certain V3 residues conserved among HIV-1, HIV-2 and SIV determine the utilization of CCR5 as a coreceptor.","They concluded that Arg-298 (at the beginning of V3 loop) has an importance for CCR5 utilization.","The authors suggested that this residue may represent a highly conserved structural element and a useful target for developing anti-viral therapies.","In another study by Wang et al., (Proc.","Natl.","Acad.","Sci.","USA 96: 4558-4562, 1999), additional V3 residues reported to be critical for CCR5 utilization are identified by alanine scanning mutagenesis.","One of the identified residues, A328, is located at the C-terminus of V3, and its substitution reportedly results in a 1,000-fold reduction of CCR5 binding activity.","However, this residue is only one of two “highly conserved” residues and six “critical” residues identified for CCR5 utilization.","Most of these residues are distinctly located at the C-terminal or central loop portion of V3, including R298, K305, I307, R313, and F315.Wang and colleagues propose that “these highly invariable residues” as well as others identified in a “bridging sheet” portion of the molecule may represent “targets for antiviral designs aimed at blocking the coreceptor entry step of HIV-1 replication.” Based on the foregoing articles, there is a broadly-confirmed disclosure of V3 as a critical domain for mediating HIV-coreceptor interactions.","A large number of important or critical residues are indicated, with a substantial range of discrepancy between reports implicating different residues as being important, or critical, for coreceptor binding functions.","These references that focus on V3 uniformly teach away from excluding important “critical” or “highly conserved” V3 residues.","At the same time, these references focusing on the V3 domain uniformly fail to implicate other sequence elements that may be present in the gp120 molecule outside the V3 loop portion thereof.","Coupled with these teachings, a host of related publications point to yet additional components of gp120 that may be essential for HIV-coreceptor interactions.","Because these components appear to be complex and potentially interact to achieve coreceptor binding, their disclosure adds further complexity to understanding gp120 structure-function relationships for mediating coreceptor binding and cell entry.","In particular, Tugarinov et al., (Structure Fold Des.","8:385-395, 2000), teach the importance of the GPG motif of V3, but also teach that “[h]igh affinity binding of gp120 to the chemokine receptors requires participation of other domains in gp120 such as the CD4i epitope.” Rizzuto et al., (Science 280:1949-1953, 1998), suggest that the “CCR5-binding site is likely composed of conserved gp120 elements near or within the bridging sheet and V3 loop residues.” They further proposed that CD4 binding may distort the V1/V2 stem, repositioning the stem allowing the formation of the β-sheet important for CCR5 binding.","They also noted that substitution of Asp for Thr123, located in the V1/V2 stem and which contact CD4, significantly decreased CCR5 binding.","This report parallels others which point to a favored model of a “conformational binding site” in gp120 for mediating HIV-coreceptor interactions.","According to this model, effective binding of coreceptors by gp120 involves initial binding of the gp120 to a CD4 receptor, which brings about a “CD4 induced” conformational change in gp120 involving distant residues—that in turn leads to formation of a conformational binding determinant on gp120 to mediate HIV-coreceptor interactions.","In this context, Wu et al., (Nature 384:179-183, 1996) suggested that HIV-1 attachment to CD4 creates a high-affinity binding site for CCR-5, leading to membrane fusion and virus entry.","Consistent with this model, recent studies have identified yet additional sites of gp120 which are reportedly important, or essential, for mediating HIV-coreceptor interactions.","These sites are generally distinct from V3, and also from the V3-flanking regions.","For example, Cho et al., (J. Virol.","72:2509-2515, 1998), report that V1/V2 and V3 confer on HIV-1 the ability to use CXCR4 coreceptors, but that the V4 and V5 regions are also “required in conjunction with regions V1 and V3 of HIV-1DH12 gp120 for efficient utilization of CXCR4.” Este et al., (Mol.","Pharmacol.","52:98-104, 1997), point to structural determinants in each of the V2, V3 and C3 regions of gp120 for determining cell tropism and coreceptor utilization.","Hoffman et al., (Mol.","Membr.","Biol.","16:57-65, 1999), concluded the V3 loop was implicated in regulating viral tropism, but, that other regions of Env, such as the V1- and V2-loops, modulated the effects of the V3-loop.","They also acknowledged that some important exceptions to this model suggested that understanding of virus tropism and Env-chemokine receptor interactions was incomplete.","Collectively, these studies point to numerous components of gp120 that may be important or essential for HIV-coreceptor interactions, and which may in fact involve multiple interactions whereby different parts of gp120 are required to work together to achieve efficient HIV-coreceptor interactions.","This complexity and the proposed requirement for multiple, distant binding determinants on gp120 to form a conformational binding determinant for coreceptor usage, may be generally considered to teach away from the utility of small peptides for effectively blocking HIV-coreceptor binding interactions, particularly to block multiple HIV strains infecting different target cell types.","Moreover, while the foregoing, separate bodies of literature individually discuss models of conserved binding elements of chemokines, or of gp120, as determinants for mediating coreceptor interactions, these reports do not teach or suggest identification of conserved binding determinants shared between gp120 and chemokines.","On the contrary, complex functional interactions and distinct binding mechanisms and determinants previously proposed for gp120 and chemokines may be considered to lead away from investigations aimed at identifying common binding determinants or mechanisms between gp120 and chemokines.","This conclusion is well supported in the literature reviewed herein above.","Based on these and other teachings, there does not appear to be a clear suggestion in the literature to use a common blocking agent for coreceptor interactions by chemokines and HIV aimed at, or patterned after, a shared binding determinant between these two distinct molecules.","The literature does not point toward any such common structural domain or binding determinant between HIV gp120 and chemokines, and the distinct structure-function reports for how HIV and chemokines are thought to interact with the subject receptors leads away from this path of inquiry.","In view of the foregoing teachings and unsolved questions in the literature, there remains an urgent need in the art for additional tools and methods to combat HIV infection and related disease.","Related to this fundamental goal, there remains a clear need for additional therapeutic agents and methods targeting HIV viral entry into host cells, preferably that will include compositions and methods to block gp120 binding to different coreceptors to inhibit viral entry and infection and ameliorate HIV-related disease.","Surprisingly, the instant invention fulfills these objects and satisfies additional objects and advantages which will become apparent from the following description.","SUMMARY OF THE INVENTION The instant invention provides novel methods and compositions for inhibiting interactions between human immunodeficiency viruses (HIVs) and chemokine receptors, termed “viral coreceptors” in this context.","The methods of the invention generally comprise exposing a CXCR4 or CCR5 coreceptor of a subject to an effective amount of an anti-coreceptor binding agent of the invention to inhibit binding of the coreceptor by an HIV virus or viral protein.","Typically, the anti-coreceptor binding agent is a gp120 peptide, peptide analog or mimetic that specifically binds the coreceptor.","Within certain methods of the invention, the subject is an isolated or bound coreceptor, a membrane or cell preparation comprising the coreceptor, a cell population, tissue or organ expressing the coreceptor, or a mammalian patient.","In more detailed embodiments, the subject comprises a cell population, tissue or organ selected for in vivo or ex vivo treatment or diagnostic processing.","Alternatively, the subject may be a mammalian patient susceptible to HIV infection and the anti-coreceptor binding agent is administered in a prophylactic or therapeutically effective dose to prevent or inhibit HIV binding to a susceptible cell and thereby preventing or inhibiting infection or a related disease condition or symptom.","In typical aspects of the invention, the anti-coreceptor binding agent is administered to the subject in an amount effective to inhibit one or more biological activities mediated by or associated with HIV-coreceptor interactions selected from (a) direct co-receptor, e.g., CXCR4 and/or CCR5, binding by HIV virus, (b) coreceptor binding by a HIV gp120 protein or a peptide fragment or derivative thereof, (c) HIV fusion with target host cells, (d) HIV virion entry into host cells, (e) HIV replication, and/or (f) HIV cell-cell or host-host transmission.","In more specific embodiments, the anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic and is administered to the subject in an amount effective to inhibit one or more biological activities selected from (a) direct co-receptor, i.e., CXCR4 and/or CCR5, binding by HIV-1 virus, (b) coreceptor binding by a HIV-1 gp120 protein or a peptide fragment or derivative thereof, (c) HIV-1 fusion with target host cells, (d) HIV-1 virion entry into host cells, (e) HIV-1 replication, and/or (f) HIV-1 cell-cell or host-host transmission.","Often, the anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic administered to a mammalian patient in a prophylactically or therapeutically effective dose to prevent or inhibit HIV-1 binding infection, to a susceptible host, HIV-1 or an HIV-1-related disease condition or symptom.","In more detailed methods and compositions of the invention, the gp120 peptide, peptide analog or mimetic is between about 12 and about 24 amino acid residues in length and comprises a conserved CXXXXXXW amino acid sequence motif identified within the amino acid sequence of gp120 proteins of HIV isolates and also among diverse chemokines, wherein X is any naturally occurring or synthetic amino acid or amino acid analog.","The peptide, peptide analog or mimetic can be modified in a wide variety of ways and forms, e.g., by addition, admixture, or conjugation of additional amino acids, peptides, proteins, chemical reagents or moieties which do not substantially alter the anti-coreceptor binding activity of the peptide.","Often, the anti-coreceptor binding agent of the invention is a peptide comprising an allelic variant that is found among native HIV gp120 peptide sequences.","Within more detailed embodiments, the anti-coreceptor binding agent is a peptide of between about 12-17 amino acids in length that includes a conserved “CXXXXXXW” amino acid sequence motif, which is selected from an exemplary “reference” peptide designated 15K comprising an amino acid sequence IRKAHCNISRAKWND (SEQ ID NO:8), or is alternatively represented by a corresponding or overlapping native peptide sequence or peptide analog that shares substantial sequence identity to the reference amino acid sequence of 15K.","In various specific embodiments, the peptide includes one or more residues occurring naturally or by substitution at a relative, aligned position corresponding to a designated position for peptide 15K, selected from: Position 1—I, M, K, S, T, L, A, V, R, P, or N; Position 2—R, G, E, K, S, T, or I; Position 3—Q, K, R, L, E, P, A, V, S, T, H, or D; Position 4—A, T, P, V, E, or S; Position 5—H, Y, F, Q. N, I, or V; Position 7—N, D, H, T, K, E, S, I, Q, V, G, or A; Position 8—I, L, V, Y, D, A; Position 9—S, N, D, T, K, Y, I, or P; Position 10—R, K, G, S, A, E, D, I, T, W, or N; Position 11—A, R, K, T, S, G, E, D, N, Q, H, V, I, or L; Position 12—K, D, R, E, K, Q, N, T, S, G, A, V, L; Position 14—N, Q, D, E, K, R, A, S, T, G, M, Y, I, H, or V; and/or Position 15—D, N, K, E, T, Q, R, S, A, I, M, or P. In yet additional embodiments of the invention, the anti-coreceptor binding agent exhibits multi-tropic activity characterized by effective inhibition of HIV viral, or gp120 protein or peptide binding to multiple, CXCR4 and CCR5, coreceptors.","Often, the multi-tropic anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic administered to the subject in an amount effective to inhibit one or more biological activities of both T cell tropic (lymphotropic) and macrophage tropic (m-tropic) HIV-1 viruses selected from (a) direct co-receptor binding by viruses, (b) coreceptor binding by viral gp120 proteins or peptide fragments or derivatives thereof, (c) viral fusion with target host cells, (d) virion entry into host cells, (e) viral replication, and/or (f) viral cell-cell or host-host transmission.","Within the methods and compositions of the invention, the anti-coreceptor binding agent may be formulated in various combinations with a pharmaceutically acceptable carrier, diluent, excipient, adjuvant or other active or inactive agents, in an amount or dosage form sufficient to prevent, reduce or even alleviate HIV infection or related disease conditions or symptoms.","In yet additional aspects of the invention, the anti-coreceptor binding agent of the invention is administered according to the foregoing methods in a combinatorial formulation or coordinate treatment with one or more additional anti-HIV, antibacterial, antiviral or other therapeutically active agent(s).","Within related methods and compositions, the anti-coreceptor binding agent is admixed or co-administered, simultaneously or sequentially with one or more additional anti-HIV, antibacterial, antiviral or other therapeutically active agent(s) to prevent, reduce or even alleviate HIV infection or related conditions in a mammalian patient.","The instant invention also includes kits, packages and multicontainer units containing the anti-coreceptor binding agent, optionally with other active or inactive ingredients, and/or means for administering the same for use in the diagnosis, management and/or prevention and treatment of HIV and related conditions.","Typically, these kits include a diagnostic or pharmaceutical preparation of the anti-coreceptor binding agent, typically formulated with a biologically suitable carrier and optionally contained in a bulk dispensing container or unit or multi-unit dosage form.","Optional dispensing means can be provided, for example an intranasal spray applicator.","Packaging materials optionally include a label or instruction which indicates a desired use of the kit as described herein below.","Additional aspects of the invention include polynucleotide molecules and vector constructs encoding anti-coreceptor binding peptides and peptide analogs.","Also provided are peptide vaccines and other immunogenic compositions that elicit an immune response involving production of antibodies targeting one or more epitopes of gp120 recognized by antibodies that specifically bind an anti-coreceptor binding peptide of the invention.","In addition, the invention provides, antibodies, including monoclonal antibodies, and immunotherapeutic methods and compositions comprising such antibodies that specifically recognize anti-coreceptor binding peptides of the invention, for use as diagnostic and therapeutic reagents.","Also provided within the invention are a variety of additional diagnostic and therapeutic tools and reagents as set forth in detail in the following description.","BRIEF DESCRIPTION OF THE DRAWINGS FIG.","1 demonstrates inhibition of specific chemokine binding to CXCR4 and CCRS expressing cells by exemplary HIV gp120-derived anti-coreceptor binding peptides 15K and 15D.","FIG.","2 documents inhibition of the chemotactic response of HEK-CCR5 cells to the chemokine RANTES by the exemplary anti-coreceptor binding peptides 15K and 15D.","Migration of HEK-CCR5 cells to RANTES alone, or the combination of RANTES and designated concentrations of either 15K or 15D peptides, were determined in micro-chemotaxis chambers.","The 15K and 15D peptides did not induce detectable chemotaxis when tested at any concentration.","The results are representative of three independent experiments.","*p<0.05.FIG.","3 demonstrates inhibition of monocyte-tropic HIV-1 infection of susceptible target cells by the exemplary anti-coreceptor binding peptides 15K and l5D.","Monocyte-derived macrophages were treated with the designated concentrations of the indicated peptide for a period of 1 hr prior to addition of HIV-1JRFL.","After 2 hr, cells were washed, and viral replication was determined after 72 hr by p24 analysis.","Results are representative of 4 independent experiments.","FIG.","4 shows inhibition of T cell-tropic HIV-1 infection by the exemplary anti-coreceptor binding peptides 15K, l5D and scrambled peptide 15KS.","Peripheral blood mononuclear cells were treated with designated concentrations of peptide for a period of 1 hr prior to addition of HIV-1IIIB.","After 2 hr, cells were washed, and viral replication was determined after 48 hr by analysis of p24 levels.","Results are representative of 4 independent experiments.","FIGS.","5A through 5C demonstrate the effect of peptides 15K, 15D and scrambled peptide 15KS on the binding of anti-CXCR4 antibody 12G5 to CEMx174 cells.","FIG.","5A.","Dose dependent effect of peptide 15K in comparison with SDF-1α.","FIG.","5B.","The comparison of the effects of 15K and 15D.","FIG.","5C.","The comparison of the effects of 15K and 15KS.","Cells were preincubated at 22° C. with peptides at designated concentrations of SDF-1α for 60 min.","Then cells were incubated with FITC-labeled anti-human CXCR4 monoclonal antibodies 12G5 for 40 min at 22° C. and washed with FACS buffer prior to flow cytometry.","FIGS.","6A through 6D depict the effect of peptide pre-treatment on the mobilization of intracellular Ca2+ in THP-1 cells in response to SDF-1α.","Cells loaded with fura-2 were preincubated with peptide 15K (FIG.","6C), 15D (FIG.","6B) and 15CW (FIG.","6D) at a final concentration of 500 μM for 3 min prior to stimulation with SDF-1α (100 ng/ml).","FIGS.","7A through 7C depicts the inhibition of chemokine receptor ligand binding by peptide pre-treatment.","FIG.","7A.","Effect of pre-treatment with peptides 15K and 15D on the binding of MIP-1β to SupT1/CCR5 cells; FIG.","7B.","Effect of pre-treatment with peptides 15K and 1SD on the binding of SDF-1α to CEMx174 cells; and FIG.","7C.","Effect of pre-treatment with 15K and control peptide 15GIG on the binding of SDF-1α to CEmx174 cells.","Binding studies were performed as described in Materials and Methods.","DETAILED DESCRIPTION The present invention provides novel methods and compositions for clinical and diagnostic use in the evaluation, treatment and prevention of HIV infection and related disease conditions by reducing the ability of HIV to bind and fuse to cells.","Typically, the compositions and procedures of the invention are directed toward prophylaxis or treatment of HIV-1 infection and related clinical conditions.","The methods of the invention involve administering an effective amount of an anti-coreceptor binding agent to a subject to inhibit HIV-coreceptor interaction.","Typically, the anti-coreceptor binding agent is administered in a prophylactic or therapeutically effective dose to a mammalian patient susceptible to HIV infection.","Alternatively, the subject may comprise a susceptible cell population, tissue or organ, selected for in vivo or ex vivo treatment or diagnostic processing involving exposure of the subject to an anti-coreceptor binding agent (for example, bone marrow or other tissue or organ materials treated ex vivo before re-implantation or transplant).","The methods and compositions of the invention involve delivery or formulation of an anti-coreceptor binding agent in an amount that is effective to inhibit HIV-coreceptor interactions, and/or to inhibit one or more selected biological activities or conditions associated with, or mediated by, HIV-coreceptor interactions.","Selected biological activities include direct co-receptor binding by HIV viruses, coreceptor binding by selected viral proteins or peptides (including gp120 proteins and peptide fragments or derivatives of gp120), as well as binding by antibodies that recognize epitopes on gp120 or on a selected chemokine or HIV coreceptor.","Additional biological activities in this context include HIV infection and related activities and conditions, for example HIV fusion with target host cells, HIV virion entry into host cells, HIV propagation and related HIV infective events, as well as specific HIV-related disease conditions including the full range of clinical conditions and disease states associated with AIDS and AIDS Related Complex (ARC) (for example, Kaposi's sarcoma and opportunistic viral (e.g., herpes) and bacterial (e.g., pneumonia infections).","As used herein, the term “anti-coreceptor binding agent” is meant to include anti-coreceptor binding HIV peptides, as well as peptide analogs and peptide mimetics which exhibit comparable, or substantially the same, anti-coreceptor binding activity as a selected anti-coreceptor binding HIV peptide as described herein.","The term anti-coreceptor binding peptide includes all of the reference peptides described herein, as well as other natural or artificially selected mutant or allelic forms and derivatives of these reference peptides having the desired anti-coreceptor binding activity.","The term peptide analog refers to such artificially modified peptide analogs as chemically cleaved peptide fragments, chemically modified peptide derivatives, site directed mutant peptide variants having one or more amino acid insertions, substitutions or deletions, and the like.","The anti-coreceptor binding peptides, analogs and mimetics of the invention function to specifically inhibit HIV-1 gp120 binding interactions with chemokine receptors (HIV-1 coreceptors).","In addition, the anti-coreceptor binding agents are effective to inhibit or block HIV-1 cell fusion and virion entry, thereby impairing viral replication and transmission.","Correlated with these activities, the anti-coreceptor binding agents and methods of the invention provide for safe and effective treatment of HIV-1 infection and related diseases.","Ancillary uses are also readily implemented using these compositions and methods for diagnosing and evaluating HIV infection and related activities and disease mechanisms.","The description herein illustrates production and characterization of peptides modeled after a novel structural motif identified in the HIV-1 gp120 envelope protein.","This novel structural motif shares similarities with a corresponding structural motif identified as a conservative feature among diverse chemokines.","This conservative motif is represented in one aspect by the reference sequences identified for HIV strains HXB2, IIIB, and JRFL, which represent operable anti-coreceptor binding peptides within the invention provided as native fragments of the corresponding gp120 proteins of these strains.","As shown in Table 1, below, these sequences aligned with MIP-1β and SDF-1α embrace a novel, HIV-1 coreceptor binding motif.","TABLE AMINO ACID ALIGNMENT OF PARTIAL SEQUENCES OF GP120 FROM REPRESENTATIVE HIV STRAINS WITH PARTIAL SEQUENCES OF MIP-1β AND SDF-1α HIV strain* 326 340 HXB2 M R Q A H C N I S R A K W N N SEQ ID NO:1 326 340 III B M R Q A H C N I S R A K W N A SEQ ID NO:2 322 336 JR FL I R Q A H C N I S R A K W N D SEQ ID NO:3 Consensus I R Q A H C N V S R S E W N K SEQ ID NO:4 HIV A Consensus I R Q A H C N I S R A Q W N N SEQ ID NO:5 HIV B chemokine 47 60 MIP-1β S K Q - V C A D P S E S W V Q SEQ ID NO:6 48 61 SDF-1α N R Q - V C I D P K L K W I Q SEQ ID NO:7 *The residues numbered according to (Korber, B. and Los Alamos National Laboratory - Theoretical Biology and Biophysics Group T-10, Human retroviruses and AIDS.","1998: a compilation and analysis of nucleic acid and amino acid sequences.","Published by Theoretical Biology and Biophysics Group T-10 Los Alamos National Laboratory, Los Alamos, N.M., 1998, incorporated herein by reference).","The residues that are shared between identified regions of HIV-1 gp120 and chemokines are shown in bold.","This alignment illustrates the finding herein that the amino acid sequences of nearly all chemokines feature a Trp residue located at the beginning of a C-terminal α-helix and separated by six intervening residues from a conserved Cys residue.","Comparing this conserved motif with the gp120 molecule of HIV-1, it is further demonstrated that all HIV isolates have a similar structural motif adjacent the V3 loop.","As described in further detail below, computer modeling based on the sequence of HIV-1 strain JRFL which closely relates to a Consensus HIV-1 sequence, demonstrates that a fragment of gp120 containing a conserved “CXXXXXXW motif” can be “template forced” onto the homologous portion of the chemokine MIP-1β.","Thus, the anti-coreceptor binding peptides of the invention typically feature the above noted CXXXXXXW motif, with additional amino acids, peptides, proteins, chemical reagents or moieties combined or conjugated therewith.","These peptides include native HIV-1 peptides, such as those identified above in Table 1 and other known HIV-1 peptides that correspond to, or include, a partial or complete, homologous sequence to these exemplary peptides.","Other peptides comprising the novel structural motif that includes the above noted CXXXXXXW anti-coreceptor binding determinant can comprise allelic variants among native peptide sequences, or synthetic or mutant peptide analogs of selected native HIV-1 gp120 peptides.","Two exemplary peptides within the invention, designated 15K and 15D, include the C-terminal part of the V3 loop of gp120 and the N-terminal part of the C3 segment of the protein (Table 2).","These peptides differ from each other by the presence of a D or K (Asp or Lys) residue at the twelfth position, which difference reflects allelic sequence variation among natural HIV-1 isolates.","In addition, both of these exemplary synthetic peptides differ from corresponding, native HIV-1 sequences by a site-directed mutation comprising a substitution of K for Q (Lys for Gln) at the third residue position in the peptides, which reflects allelic sequence variation among natural HIV-1 isolates and also results in reduced susceptibility of the peptides to hydrolysis.","TABLE 2 SEQUENCES OF EXEMPLARY SYNTHETIC PEPTIDES Design- tion Sequence 15K I R K A H C N I S R A K W N D (SEQ ID NO:8) 15D I R K A H C N I S R A D W N D (SEQ ID NO:9) These exemplary synthetic peptides of the invention inhibit or block binding or “docking” interactions between the HIV-1 envelope protein gp120 and chemokine receptors (e.g., CXCR4 and CCR5) that function as “coreceptors” for HIV entry on the surface of target cells (macrophages and T lymphocytes).","The natural ligands of the coreceptors are chemokines (e.g., SDF-1α and MIP-1β), and it is further demonstrated herein that the conserved structural elements identified for the HIV gp120 protein motif that are shared with chemokines mediate critical HIV-coreceptor interactions.","Detailed functional analyses of the peptides show that both are effective in competing with chemokines for binding to CCR5- and CXCR4-expressing cells.","The peptides also inhibit monocyte chemotaxis stimulated by the chemokine RANTES (Regulated upon Activation Normal T-cell Expressed and Secreted).","In addition, the anti-coreceptor binding peptides of the invention are shown herein to be potent inhibitors of HIV replication.","In certain aspects, the peptides effect this inhibition in a multi-tropic or multi-specific manner to prevent or treat infection by both T cell tropic (lymphotropic) and macrophage tropic (m-tropic) HIV strains by blocking HIV interactions with distinct (CXCR4 and CCR5) coreceptors.","This ability to use small peptides to achieve multi-tropic (i.e., multi-receptor) blockade is a surprising and important advantage satisfied by the invention.","The anti-coreceptor binding peptides of the present invention include naturally occurring peptide variants, e.g., naturally occurring allelic variants and mutant proteins, as well as synthetic, e.g., chemically or recombinantly engineered, peptide fragments and analogs.","As used herein, anti-coreceptor binding peptide “analogs” is meant to include a modified gp120 peptide incorporating one or more amino acid substitutions, insertions, rearrangements or deletions as compared to a native amino acid sequence of an HIV-1 gp120 anti-coreceptor binding peptide domain, fragment or motif, as described herein.","Anti-coreceptor binding peptide analogs thus modified exhibit substantial anti-coreceptor binding activity comparable to that of a corresponding native peptide, which is activity that at least 50%, typically at least 75% or greater, compared to activity of the corresponding native peptide (e.g., as determined by an in vitro coreceptor binding assay or HIV-1 infection assay).","As used herein, the term “biologically active anti-coreceptor binding analogs and mimetics” refers to analogs or mimetics of native peptides which encompass the entire length, sequence or chemical structure of the corresponding native peptide but which nevertheless maintains substantial anti-coreceptor binding activity as described above in an appropriate assay system.","For purposes of the present invention, the term anti-coreceptor binding peptide “analog” thus includes derivatives or synthetic variants of a native HIV-1 gp120 anti-coreceptor binding peptide, such as amino and/or carboxyl terminal deletions and fusions, as well as intrasequence insertions, substitutions or deletions of single or multiple amino acids.","Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein.","Random insertion is also possible with suitable screening of the resulting product.","Deletional variants are characterized by removal of one or more amino acids from the sequence.","Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place.","Where a native HIV-1 gp120 anti-coreceptor binding peptide is modified by amino acid substitution, amino acids are generally replaced by other amino acids having similar, conservatively related chemical properties such as hydrophobicity, hydrophilicity, electronegativity, bulky side chains and the like.","Residue positions which are not identical to the native peptide sequence are thus replaced by amino acids having similar chemical properties, such as charge or polarity, which changes are not likely to substantially effect the properties of the peptide analog.","These and other minor alterations substantially maintain the immunoidentity (e.g., recognition by one or more monoclonal antibodies that recognize a native HIV-1 gp120 anti-coreceptor binding peptide) and other biological activities of the native peptide.","In this context, the term “conservative amino acid substitution” refers to the general interchangeability of amino acid residues having similar side chains.","For example, a group of amino acids having aliphatic side chains is alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.","Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another.","Likewise, the present invention contemplates the substitution of a polar (hydrophilic) residue such as between arginine and lysine, between glutamine and asparagine, and between threonine and serine.","Additionally, the substitution of a basic residue such as lysine, arginine or histidine for another or the substitution of an acidic residue such as aspartic acid or glutamic acid for another is also contemplated.","Exemplary conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.","Anti-coreceptor binding peptide analogs also include modified forms of a native HIV-1 gp120 anti-coreceptor binding peptide incorporating stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, or unnatural amino acids such as a, α-disubstituted amino acids, N-alkyl amino acids, lactic acid.","These and other unconventional amino acids may also be substituted or inserted within native HIV-1 gp120 anti-coreceptor binding peptides of the present invention.","Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, ω-N-methylarginine, and other similar amino acids and amino acids (e.g., 4-hydroxyproline).","For purposes of the present invention, analogs of native HIV-1 gp120 anti-coreceptor binding peptides also include single or multiple substitutions, deletions and/or additions of carbohydrate, lipid and/or proteinaceous moieties that occur naturally or artificially as structural components of gp120 peptides or are bound or otherwise associated with the peptide analog.","To facilitate production and use of anti-coreceptor binding peptide analogs within the invention, reference can be made to molecular phylogenetic studies that characterize conserved and divergent protein structural and functional elements between different HIV-1 strains, and between HIV-1 and other HIV taxa and more distantly related retroviruses.","In this regard, available studies provide detailed assessments of gp120 protein structure-function relationships on a fine molecular level.","These studies include detailed sequence comparisons identifying conserved and divergent structural elements among a large number of HIV-1 gp120 allelic variants, for example.","Each of these conserved and divergent structural elements facilitate practice of the invention by pointing to useful targets that for modifying native HIV-1 gp120 anti-coreceptor binding peptides to confer desired structural and/or functional changes.","In this context, existing sequence alignments may be analyzed, or conventional sequence alignment methods may be employed to yield sequence comparisons for analysis, to identify corresponding protein regions and amino acid positions between native HIV-1 gp120 anti-coreceptor binding peptides and related or homologous peptides bearing a structural element of interest for incorporation within an anti-coreceptor binding peptide analog.","Typically, one or more amino acid residues marking a structural element of interest in a different reference peptide sequence is incorporated within the anti-coreceptor binding peptide analog.","For example, a cDNA encoding a native HIV-1 gp120 anti-coreceptor binding peptide may be recombinantly modified at one or more corresponding amino acid position(s) (i.e., corresponding positions that match or span a similar aligned sequence element according to accepted alignment methods to residues marking the structural element of interest in a heterologous reference peptide sequence) to encode an amino acid deletion, substitution, or insertion that alters corresponding residue(s) in the native HIV-1 gp120 anti-coreceptor binding peptide to generate an operable peptide analog within the invention having an analogous structural and/or functional element as the reference peptide or protein.","Within this rational design method for constructing anti-coreceptor binding peptide analogs, the native or wild-type identity of residue(s) at amino acid positions corresponding to a structural element of interest in a heterologous reference peptide or protein may be altered to the same, or a conservatively related, residue identity as the corresponding amino acid residue(s) in the reference peptide or protein.","However, it is often possible to alter native amino acid residues non-conservatively with respect to the corresponding reference protein residue(s).","In particular, many non-conservative amino acid substitutions, particularly at divergent sites suggested to be more amenable to modification, may yield a moderate impairment or neutral effect, or even enhance a selected biological activity, compared to the function of a native HIV-1 gp120 anti-coreceptor binding peptide.","Extensive protein structure-function data are available in the literature pertaining to HIV that will serve and guide the artisan to prepare functional anti-coreceptor binding peptide analogs for use within the invention.","Structure-function relationships between HIV-1 strains and between HIV-1 and more distantly related retroviruses may be elucidated using conventional molecular phylogenetic analysis coupled with functional assays for determining anti-coreceptor binding activities described herein.","Sequence alignment and comparisons to forecast useful peptide analogs and mimetics will be further refined by analysis of crystalline structure (see, e.g., Löebermann et al., J. Mol.","Biol.","177:531-556, 1984; Huber et al., Biochemistry 28:8951-8966, 1989; Stein et al., Nature 347:99-102, 1990; Wei et al., Structural Biology 1:251-255, 1994, each incorporated herein by reference) coupled with computer modeling methods known in the art.","These analyses allow detailed structure-function mapping to identify desired structural elements and modifications for incorporation into anti-coreceptor binding peptide analogs and mimetics that will exhibit substantial anti-coreceptor binding activity for use within the methods of the invention.","Native HIV-1 gp120 anti-coreceptor binding peptides and peptide analogs within the invention are typically between about 6-35 amino acid residues in length, more typically between about 10 and 21 or 22 amino acid residues in length.","Within certain embodiments, the native peptides and analogs are between about 10-17 residues in length.","In more specific embodiments, peptides are 10, 12, 13, 15, 17, 21, or 22 amino acid residues in length.","Anti-coreceptor binding peptide analogs of the invention typically show substantial sequence identity to a corresponding native HIV-1 gp120 anti-coreceptor binding peptide sequence.","Within the foregoing length ranges, both the native HIV-1 gp120 anti-coreceptor binding peptide and the peptide analogs typically comprise the conserved “CXXXXXXW” motif, described above, which may be extended to include additional residues from the native HIV-1 gp120 anti-coreceptor binding peptide sequence or non-native residues, fusion protein members, chemical moieties and the like.","As applied to anti-coreceptor binding peptide analogs and fragments, these analogs and fragments typically exhibit substantial amino acid sequence identity to a corresponding native HIV-1 gp120 anti-coreceptor binding peptide sequence.","The term “substantial sequence identity” as used herein means that the two subject amino acid sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap penalties, share at least 80 percent sequence identity, often at least 90-95 percent or greater sequence identity.","“Percentage amino acid identity” refers to a comparison of the amino acid sequences of two peptides which, when optimally aligned, have approximately the designated percentage of the same amino acids.","Sequence comparisons are generally made to a reference sequence over a comparison window of at least 10 residue positions, frequently over a window of at least 15-20 amino acids, wherein the percentage of sequence identity is calculated by comparing a reference sequence to a second sequence, the latter of which may represent, for example, a peptide analog sequence that includes one or more deletions, substitutions or additions which total 20 percent, typically less than 5-10% of the reference sequence over the window of comparison.","The reference sequence may be a subset of a larger sequence, for example, as a segment of the HIV-1 gp120 protein.","Optimal alignment of sequences for aligning a comparison window may be conducted according to the local homology algorithm of Smith and Waterman (Adv.","Appl.","Math.","2:482, 1981), by the homology alignment algorithm of Needleman and Wunsch (J. Mol.","Biol.","48:443, 1970), by the search for similarity method of Pearson and Lipman (Proc.","Natl.","Acad.","Sci.","USA 85:2444, 1988), or by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.).","By aligning a peptide analog optimally with a corresponding native HIV-1 gp120 anti-coreceptor binding peptide, and by using appropriate assays, e.g., coreceptor binding assays, to determine a selected biological activity, one can readily identify operable peptide analogs for use within the methods and compositions of the invention.","Anti-coreceptor binding peptide analogs are typically specifically immunoreactive with antibodies raised to the corresponding native HIV-1 gp120 anti-coreceptor binding peptide.","Likewise, nucleic acids encoding functional anti-coreceptor binding peptide analogs will typically selectively hybridize to nucleic acid sequences encoding a corresponding native HIV-1 gp120 anti-coreceptor binding peptide under accepted, moderate or high stringency hybridization conditions (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989, incorporated herein by reference).","The phrase “selectively hybridizing to” refers to a selective interaction between a nucleic acid probe that hybridizes, duplexes or binds preferentially to a particular target DNA or RNA sequence, for example when the target sequence is present in a heterogenous preparation such as total cellular DNA or RNA.","Generally, nucleic acid sequences encoding functional anti-coreceptor binding peptide analogs and fragments will hybridize to nucleic acid sequences encoding native HIV-1 gp120 anti-coreceptor binding peptides under stringent conditions selected to be about 5° C. lower than the thermal melting point (Tm) for the subject sequence at a defined ionic strength and pH.","The Tm is the temperature (under defined ionic strength and pH) at which 50% of the complementary or target sequence hybridizes to a perfectly matched probe.","For discussions of nucleic acid probe design and annealing conditions, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, 1989 or Current Protocols in Molecular Biology F. Ausubel et al, ed., Greene Publishing and Wiley-Interscience, New York, 1987, each of which is incorporated herein by reference.","Typically, stringent or selective conditions will be those in which the salt concentration is at least about 0.02 molar at pH 7 and the temperature is at least about 60° C. Less stringent selective hybridization conditions can also be chosen.","As other factors may significantly affect the stringency of hybridization, including, among others, base composition and size of the complementary strands, the presence of organic solvents and the extent of base mismatching, the combination of parameters is more important than the specific measure of any one.","Anti-coreceptor binding peptide analogs of the invention typically show substantial sequence identity to a corresponding native HIV1 gp120 anti-coreceptor binding peptide sequence.","Within the foregoing length ranges, both the native HIV-1 gp120 anti-coreceptor binding peptide and the peptide analogs typically comprise the conserved “CXXXXXXW” motif, described above, which may be extended to include additional residues from the native HIV-1 gp120 anti-coreceptor binding peptide sequence, non-native residues, fusion protein members, chemical moieties, and the like.","As applied to anti-coreceptor binding peptide analogs and fragments, these analogs and fragments typically exhibit substantial amino acid sequence identity to a corresponding native HIV-1 gp120 anti-coreceptor binding peptide sequence.","Various peptide analogs are contemplated within the scope of the invention, which typically satisfy the foregoing length criteria of 10 to and include the conserved “CXXXXXXW” motif, but which incorporate selected sequence modifications at one or more positions to yield a desired structural or activity modification.","To illustrate the breadth of different peptides and peptide analogs that are useful within the invention, the following table (Table 3) provides a map of anti-coreceptor binding peptide sequence variants based on a detailed analysis of different strains of HIV-1, along with structure-function analysis directed to both gp120 and chemokine structure-function, further guided by general rules of peptide structure-function.","TABLE 3 MENU FOR CONSTRUCTING OPERABLE HIV gp120 PEPTIDES AND PEPTIDE ANALOGS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Peptide 15K I R Q A H C N I S R A K W N D SEQ ID NO:8 Peptide 15D D SEQ ID NO:9 M G K T Y D L N K R R Q N K E R P F H V D G K E D K S K L V Q T Y T S T D E E T S E S N K D K A S Q K T L T P I E A Y E G N R Q A I A V S I D E T A R V V I P I D S S S A S Q T N G T A P T V W Q A G I N H G N H V M M D A V L Y P I I H H V In accordance with the foregoing table, peptides which satisfy the above-described length criteria and include the conserved “CXXXXW” motif can also be selected from corresponding or overlapping native peptides among any known HIV isolate having a variant sequence as compared to a corresponding “reference” peptide.","As illustrated in Table 3, one reference peptide that may be used in this context is the 15K peptide, which provides an exemplary reference sequence for comparison with other peptide sequences within the invention.","Against this reference sequence, the table provides a range of amino acid residues that can be effectively substituted at the indicated residue positions (numbered in reference to the 15K peptide) which, for the substitutions designated by regular font single amino acid code, follow natural variations in the HIV gp120 available in the various HIV sequence data banks (e.g., http://hiv-web.lanl.gov/., incorporated herein by reference).","Each of these residues can be incorporated, in a native HIV gp120-derived peptide, or in a single- or multiple-substituted peptide analog of the invention, to yield effective anti-coreceptor binding agents.","Thus, peptides and peptide analogs are provided which satisfy the above-described length criteria and include the conserved “CXXXXXXW” motif, and which include any one, or any combination, of the following alternative residues occurring naturally or by substitution at the indicated positions (as designated for peptide 15K in Table 3 and determined for the subject peptide by conventional comparison and alignment against, e.g., the 15K reference sequence, as described herein): Position 1—I, M, K, S, T, L, A, V, R, P, or N; Position 2—R, G, E, K, S, T, or I; Position 3—Q, K, R, L, E, P, A, V, S, T, H, or D; Position 4—A, T, P, V, E, or S; Position 5—H, Y, F, Q, N, I, or V; Position 7—N, D, H, T, K, E, S, I, Q, V, G, or A; Position 8—I, L, V, Y, D, A; Position 9—S, N+EE, D, T, K, Y, I, or P; Position 10—R, K, G, S, A, E, D, I, T, W, or N; Position 11—A, R, K, T, S, G, E, D, N, Q, H, V, I, or L; Position 12—K, D, R, E, K, Q, N, T, S, G, A, V, L; Position 14—N, Q, D, E, K, R, A, S, T, G, M, Y, I, H, or V; Position 15—D, N, K, E, T, Q, R, S, A, I, M, or P. Certain of the foregoing residue/position alternatives, for example, the residue/position alternatives designated by underscoring in the preceding paragraph, may be selected in a greater number of peptides and analogs of the invention compared to other alternatives.","This preference may be guided based on the greater degree of conservation of the indicated residue at corresponding positions among different HIV-1 isolates, or based on a convergent or homologous occurrence of the indicated residue at corresponding positions in gp120 of a selected HIV isolate and in a selected chemokine.","Additional selections within the foregoing “menu” will be guided to favor conservative structural relationships between original and substitute residues in the preparation of anti-coreceptor binding peptide analogs, as described herein.","In accordance with these selection principals, the above-noted, alternative residues may be included in peptides and peptide analogs of the invention, singly or in any combination of 2, 3, 4 or more and up to 13 residues (e.g., as exemplified by the various combinations shown in the separate lines of Table 3), selected or substituted at the indicated positions.","Within additional aspects of the invention, peptide mimetics are provided which comprise a peptide or non-peptide molecule that mimics the tertiary binding structure and activity of the anti-coreceptor binding peptides described herein.","These peptide mimetics include recombinantly or chemically modified peptides, as well as non-peptide anti-coreceptor binding agents such as small molecule drug mimetics, as further described below.","In one aspect, peptides of the invention are modified to produce peptide mimetics by replacement of one or more naturally occurring side chains of the 20 genetically encoded amino acids (or D amino acids) with other side chains, for instance with groups such as alkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7-membered alkyl, amide, amide lower alkyl, amide di (lower alkyl), lower alkoxy, hydroxy, carboxy and the lower ester derivatives thereof, and with 4-, 5-, 6-, to 7-membered heterocyclics.","For example, proline analogs can be made in which the ring size of the proline residue is changed from 5 members to 4, 6, or 7 members.","Cyclic groups can be saturated or unsaturated, and if unsaturated, can be aromatic or non-aromatic.","Heterocyclic groups can contain one or more nitrogen, oxygen, and/or sulphur heteroatoms.","Examples of such groups include the furazanyl, furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g., morpholino), oxazolyl, piperazinyl (e.g., 1-piperazinyl), piperidyl (e.g., 1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (e.g., 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g., thiomorpholino), and triazolyl.","These heterocyclic groups can be substituted or unsubstituted.","Where a group is substituted, the substituent can be alkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl.","The peptide compounds of the invention, including peptidomimetics, can also be covalently bound to one or more of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkenes, in the manner set forth in U.S. Pat.","No.","4,640,835; U.S. Pat.","No.","4,496,689; U.S. Pat.","No.","4,301,144; U.S. Pat.","No.","4,670,417; U.S. Pat.","No.","4,791,192; or U.S. Pat.","No.","4,179,337, all which-are incorporated by reference in their entirety herein.","Other peptide analogs and mimetics within the invention include glycosylation variants, and covalent or aggregate conjugates with other chemical moieties.","Covalent derivatives can be prepared by linkage of functionalities to groups which are found in amino acid side chains or at the N- or C-termini, by means which are well known in the art.","These derivatives can include, without limitation, aliphatic esters or amides of the carboxyl terminus, or of residues containing carboxyl side chains, O-acyl derivatives of hydroxyl group-containing residues, and N-acyl derivatives of the amino terminal amino acid or amino-group containing residues, e.g., lysine or arginine.","Acyl groups are selected from the group of alkyl-moieties including C3 to C18 normal alkyl, thereby forming alkanoyl aroyl species.","Covalent attachment to carrier proteins, e.g., immunogenic moieties may also be employed.","In certain embodiments, glycosylation alterations of anti-coreceptor binding agents are included, which can be made, e.g., by modifying the glycosylation patterns of a peptide during its synthesis and processing, or in further processing steps.","Particularly preferred means for accomplishing this are by exposing the peptide to glycosylating enzymes derived from cells which normally provide such processing, e.g., mammalian glycosylation enzymes.","Deglycosylation enzymes are also contemplated.","Also embraced are versions of the same primary amino acid sequence which have other minor modifications, including phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine, or other moieties, including ribosyl groups or cross-linking reagents.","Peptidomimetics may also have amino acid residues which have been chemically modified by phosphorylation, sulfonation, biotinylation, or the addition or removal of other moieties, particularly those which have molecular shapes similar to phosphate groups.","In some embodiments, the modifications will be useful labeling reagents, or serve as purification targets, e.g., affinity ligands.","A major group of peptidomimetics within the invention are covalent conjugates of the anti-coreceptor binding peptides, or fragments thereof, with other proteins or peptides.","These derivatives can be synthesized in recombinant culture such as N- or C-terminal fusions or by the use of agents known in the art for their usefulness in cross-linking proteins through reactive side groups.","Preferred peptide and protein derivatization sites for targeting by cross-linking agents are at free amino groups, carbohydrate moieties, and cysteine residues.","Fusion polypeptides between anti-coreceptor binding peptides and other homologous or heterologous peptides and proteins are also provided.","Many growth factors and cytokines are homodimeric entities, and a repeat construct of anti-coreceptor binding peptide linked to form “cluster peptides” will yield various advantages, including lessened susceptibility to proteolytic degradation.","Various alternative multimeric constructs comprising peptides of the invention are also provided.","In one embodiment, various polypeptide fusions are provided as described in U.S. Pat.","Nos.","6,018,026 and 5,843,725, by linking one or more anti-coreceptor binding peptides of the invention with a heterologous, multimerizing polypeptide or protein, for example, immunoglobulin heavy chain constant region, or an immunoglobulin light chain constant region.","The biologically active, multimerized polypeptide fusion thus constructed can be a hetero- or homo-multimer, e.g., a heterodimer or homodimer, which may each comprise one or more distinct anti-coreceptor binding peptide(s) of the invention.","Other heterologous polypeptides may be combined with the anti-coreceptor binding agents to yield fusions comprising, e.g., a hybrid protein exhibiting heterologous (e.g., CD4) receptor binding specificity.","Likewise, heterologous fusions may be constructed exhibit a combination of properties or activities of the derivative proteins.","Other typical examples are fusions of a reporter polypeptide, e.g., CAT or luciferase, with a peptide of the invention, to facilitate localization of the fused protein (see, e.g., Dull et al., U.S. Pat.","No.","4,859,609, incorporated herein by reference).","Other gene/protein fusion partners useful in this context include bacterial beta-galactosidase, trpE, Protein A, beta-lactamase, alpha amylase, alcohol dehydrogenase, and yeast alpha mating factor (see, e.g., Godowski et al., Science 241:812-816, 1988, incorporated herein by reference).","The present invention also contemplates the use of anti-coreceptor binding agents modified by covalent or aggregative association with chemical moieties.","These derivatives generally fall into the three classes: (1) salts, (2) side chain and terminal residue covalent modifications, and (3) adsorption complexes, for example with cell membranes.","Such covalent or aggregative derivatives are useful for various purposes, for example as immunogens, as reagents in immunoassays, or in purification methods such as for affinity purification of ligands or other binding ligands.","For example, an anti-coreceptor binding agent can be immobilized by covalent bonding to a solid support such as cyanogen bromide-activated Sepharose, by methods which are well known in the art, or adsorbed onto polyolefin surfaces, with or without glutaraldehyde cross-linking, for use in the assay or purification of antibodies that specifically bind the anti-coreceptor binding agent.","The anti-coreceptor binding agent can also be labeled with a detectable group, for example radioiodinated by the chloramine T procedure, covalently bound to rare earth chelates, or conjugated to another fluorescent moiety for use in diagnostic assays.","Those of skill in the art recognize that a variety of techniques are available for constructing peptide mimetics with the same or similar desired biological activity as the corresponding peptide compound but with more favorable activity than the peptide with respect to solubility, stability, and susceptibility to hydrolysis and proteolysis (see, e.g., Morgan and Gainor, Ann.","Rep. Med.","Chem.","24:243-252, 1989, incorporated herein by reference).","The following describes methods for preparing peptide mimetics modified at the N-terminal amino group, the C-terminal carboxyl group, and/or changing ore or more of the amido linkages in the peptide to a non-amido linkage.","It being understood that two or more such modifications can be coupled in one peptide mimetic structure (e.g., modification at the C-terminal carboxyl group and inclusion of a —CH2-carbamate linkage between two amino acids in the peptide.","For N-terminal modifications, the peptides typically are synthesized as the free acid but, as noted above, can be readily prepared as the amide or ester.","One can also modify the amino and/or carboxy terminus of the peptide compounds of the invention to produce other compounds of the invention.","Amino terminus modifications include methylating (i.e., —NHCH3 or —NH(CH3)2), acetylating, adding a carbobenzoyl group, or blocking the amino terminus with any blocking group containing a carboxylate functionality defined by RCOO—, where R is selected from the group consisting of naphthyl, acridinyl, steroidyl, and similar groups.","Carboxy terminus modifications include replacing the free acid with a carboxamide group or forming a cyclic lactam at the carboxy terminus to introduce structural constraints.","Amino terminus modifications are as recited above and include alkylating, acetylating, adding a carbobenzoyl group, forming a succinimide group, and the like.","Specifically, the N-terminal amino group can then be reacted as follows: (a) to form an amide group of the formula RC(O)NH— where R is as defined above by reaction with an acid halide (e.g., RC(O)Cl) or acid anhydride.","Typically, the reaction can be conducted by contacting about equimolar or excess amounts (e.g., about 5 equivalents) of an acid halide to the peptide in an inert diluent (e.g., dichloromethane) preferably containing an excess (e.g., about 10 equivalents) of a tertiary amine, such as diisopropylethylamine, to scavenge the acid generated during reaction.","Reaction conditions are otherwise conventional (e.g., room temperature for 30 minutes).","Alkylation of the terminal amino to provide for a lower alkyl N-substitution followed by reaction with an acid halide as described above will provide for N-alkyl amide group of the formula RC(O)NR—; (b) to form a succinimide group by reaction with succinic anhydride.","As before, an approximately equimolar amount or an excess of succinic anhydride (e.g., about 5 equivalents) can be employed and the amino group is converted to the succinimide by methods well known in the art including the use of an excess (e.g., ten equivalents) of a tertiary amine such as diisopropylethylamine in a suitable inert solvent (e.g., dichloromethane) (see, for example, U.S. Pat.","No.","4,612,132, incorporated herein by reference).","It is understood that the succinic group can be substituted with, for example, C2-C6 alkyl or —SR substituents which are prepared in a conventional manner to provide for substituted succinimide at the N-terminus of the peptide.","Such alkyl substituents are prepared by reaction of a lower olefin (C2-C6) with maleic anhydride in the manner described by (U.S. Pat.","No.","4,612,132) and —SR substituents are prepared by reaction of RSH with maleic anhydride where R is as defined above; (c) to form a benzyloxycarbonyl-NH— or a substituted benzyloxycarbonyl-NH-group by reaction with approximately an equivalent amount or an excess of CBZ-Cl (i.e., benzyloxycarbonyl chloride) or a substituted CBZ-Cl in a suitable inert diluent (e.g., dichloromethane) preferably containing a tertiary amine to scavenge the acid generated during the reaction; (d) to form a sulfonamide group by reaction with an equivalent amount or an excess (e.g., 5 equivalents) of R—S(O)2Cl in a suitable inert diluent (dichloromethane) to convert the terminal amine into a sulfonamide where R is as defined above.","Preferably, the inert diluent contains excess tertiary amine (e.g., ten equivalents) such as diisopropylethylamine, to scavenge the acid generated during reaction.","Reaction conditions are otherwise conventional (e.g., room temperature for 30 minutes); (e) to form a carbamate group by reaction with an equivalent amount or an excess (e.g., 5 equivalents) of R—OC(O)Cl or R—OC(O)OC6H4-p-NO2 in a suitable inert diluent (e.g., dichloromethane) to convert the terminal amine into a carbamate where R is as defined above.","Preferably, the inert diluent contains an excess (e.g., about 10 equivalents) of a tertiary amine, such as diisopropylethylamine, to scavenge any acid generated during reaction.","Reaction conditions are otherwise conventional (e.g., room temperature for 30 minutes); and (f) to form a urea group by reaction with an equivalent amount or an excess (e.g., 5 equivalents) of R—N═C═O in a suitable inert diluent (e.g., dichloromethane) to convert the terminal amine into a urea (i.e., RNHC(O)NH—) group where R is as defined above.","Preferably, the inert diluent contains an excess (e.g., about 10 equivalents) of a tertiary amine, such as diisopropylethylamine.","Reaction conditions are otherwise conventional (e.g., room temperature for about 30 minutes).","In preparing peptide mimetics wherein the C-terminal carboxyl group is replaced by an ester (i.e., —C(O)OR where R is as defined above), resins as used to prepare peptide acids are employed, and the side chain protected peptide is cleaved with base and the appropriate alcohol, e.g., methanol.","Side chain protecting groups are then removed in the usual fashion by treatment with hydrogen fluoride to obtain the desired ester.","In preparing peptide mimetics wherein the C-terminal carboxyl group is replaced by the amide —C(O)NR3R4, where R3 and R4 independently are hydrogen or a lower alkyl, a benzhydrylamine resin is used as the solid support for peptide synthesis.","Upon completion of the synthesis, hydrogen fluoride treatment to release the peptide from the support results directly in the free peptide amide (i.e., the C-terminus is —C(O)NH2).","Alternatively, use of the chloromethylated resin during peptide synthesis coupled with reaction with ammonia to cleave the side chain protected peptide from the support yields the free peptide amide and reaction with an alkylamine or a dialkylamine yields a side chain protected alkylamide or dialkylamide (i.e., the C-terminus is —C(O)NRR1 where R and R1 independently are hydrogen or a lower alkyl).","Side chain protection is then removed in the usual fashion by treatment with hydrogen fluoride to give the free amides, alkylamides, or dialkylamides.","In another alternative embodiment, the C-terminal carboxyl group or a C-terminal ester can be induced to cyclize by internal displacement of the —OH or the ester (—OR) of the carboxyl group or ester respectively with the N-terminal amino group to form a cyclic peptide.","For example, after synthesis and cleavage to give the peptide acid, the free acid is converted to an activated ester by an appropriate carboxyl group activator such as dicyclohexylcarbodiimide (DCC) in solution, for example, in methylene chloride (CH2Cl2), dimethyl formamide (DMF) mixtures.","The cyclic peptide is then formed by internal displacement of the activated ester with the N-terminal amine.","Internal cyclization as opposed to polymerization can be enhanced by use of very dilute solutions.","Such methods are well known in the art.","One can also cyclize the anti-coreceptor binding peptides of the invention, or incorporate a desamino or descarboxy residue at the termini of the peptide, so that there is no terminal amino or carboxyl group, to decrease susceptibility to proteases or to restrict the conformation of the peptide.","C-terminal functional groups of the compounds of the present invention include amide, amide lower alkyl, amide di (lower alkyl), lower alkoxy, hydroxy, and carboxy, and the lower ester derivatives thereof, and the pharmaceutically acceptable salts thereof.","Other methods for making peptide derivatives and mimetics of the are described in Hruby et al., (Biochem J.","268:249-262, 1990, incorporated herein by reference).","According to these methods, the anti-coreceptor binding peptide compounds of the invention also serve as structural models for non-peptide mimetic compounds with similar biological activity.","Those of skill in the art recognize that a variety of techniques are available for constructing compounds with the same or similar desired biological activity as the lead peptide compound but with more favorable activity than the lead with respect to solubility, stability, and susceptibility to hydrolysis and proteolysis (see, e.g., Morgan and Gainor, Ann.","Rep. Med.","Chem.","24:243-252, 1989, incorporated herein by reference).","These techniques include replacing the peptide backbone with a backbone composed of phosphonates, amidates, carbamates, sulfonamides, secondary amines, and/or N-methylamino acids.","Peptide mimetics wherein one or more of the peptidyl linkages —C(O)NH— have been replaced by such linkages as a —CH2-carbamate linkage, a phosphonate linkage, a —CH2-sulfonamide linkage, a urea linkage, a secondary amine (—CH2NH—) linkage, and an alkylated peptidyl linkage —C(O)NR6— where R6 is lower alkyl are prepared during conventional peptide synthesis by merely substituting a suitably protected amino acid analogue for the amino acid reagent at the appropriate point during synthesis.","Suitable reagents include, for example, amino acid analogues wherein the carboxyl group of the amino acid has been replaced with a moiety suitable for forming one of the above linkages.","For example, if one desires to replace a —C(O)NR— linkage in the peptide with a —CH2-carbamate linkage (—CH2OC(O)NR—), then the carboxyl (—COOH) group of a suitably protected amino acid is first reduced to the —CH2OH group which is then converted by conventional methods to a —OC(O)Cl functionality or a para-nitrocarbonate —OC(O)O—C6H4-p-NO2 functionality.","Reaction of either of such functional groups with the free amine or an alkylated amine on the N-terminus of the partially fabricated peptide found on the solid support leads to the formation of a —CH2OC(O)NR— linkage.","For a more detailed description of the formation of such —CH2-carbamate linkages, see, e.g., Cho et al., (Science 261:1303-1305, 1993, incorporated herein by reference).","Replacement of an amido linkage in an anti-coreceptor binding peptide with a —CH2-sulfonamide linkage can be achieved by reducing the carboxyl (—COOH) group of a suitably protected amino acid to the —CH2OH group, and the hydroxyl group is then converted to a suitable leaving group such as a tosyl group by conventional methods.","Reaction of the tosylated derivative with, for example, thioacetic acid followed by hydrolysis and oxidative chlorination will provide for the —CH2—S(O)2Cl functional group which replaces the carboxyl group of the otherwise suitably protected amino acid.","Use of this suitably protected amino acid analogue in peptide synthesis provides for inclusion of an —CH2S(O)2NR— linkage which replaces the amido linkage in the peptide thereby providing a peptide mimetic.","For a more complete description on the conversion of the carboxyl group of the amino acid to a —CH2S(O)2Cl group, see, e.g., Weinstein and Boris (Chemistry & Biochemistry of Amino Acids.","Peptides and Proteins, Vol.","7, pp.","267-357, Marcel Dekker, Inc., New York, 1983, incorporated herein by reference).","Replacement of an amido linkage in an anti-coreceptor binding peptide with a urea linkage can be achieved in the manner known to the skilled artisan.","Secondary amine linkages wherein a —CH2NH— linkage replaces the amido linkage in the peptide can be prepared by employing, for example, a suitably protected dipeptide analogue wherein the carbonyl bond of the amido linkage has been reduced to a CH2 group by conventional methods.","For example, in the case of diglycine, reduction of the amide to the amine will yield after deprotection H2NCH2CH2NHCH2 COOH which is then used in N-protected form in the next coupling reaction.","The preparation of such analogues by reduction of the carbonyl group of the amido linkage in the dipeptide is well known in the art.","The anti-coreceptor binding agents of the present invention may exist in a monomeric form with no disulfide bond formed with the thiol groups of the cysteine residue(s).","Alternatively, an intermolecular disulfide bond between the thiol groups of cysteines on two or more peptides can be produced to yield a multimeric (e.g., dimeric, tetrameric or higher oligomeric) compound.","Certain of these peptides can be cyclized or dimerized via displacement of the leaving group by the sulfur of a cysteine or homocysteine residue (see, e.g., Barker et al., J. Med.","Chem.","35:2040-2048, 1992; and/or et al., J. Org.","Chem.","56:3146-3149, 1991, each incorporated herein by reference).","Thus, one or more native cysteine residues may be substituted with a homocysteine.","Intramolecular or intermolecular disulfide derivatives of anti-coreceptor binding agents provide analogs in which one of the sulfurs has been replaced by a CH2 group or other isostere for sulfur.","These analogs can be made via an intramolecular or intermolecular displacement, using methods known in the art as shown below.","One of skill in the art will readily appreciate that this displacement can also occur using other homologs of the a-amino-g-butyric acid derivative shown above and homocysteine.","All of the naturally occurring, recombinant, and synthetic peptides and peptide analogs and mimetics of the invention can be used for screening (e.g., in kits and/or screening assay methods) to identify additional compounds, including other peptides and peptide mimetics, that will function as anti-coreceptor binding agents within the methods and compositions of the invention.","Several methods of automating assays have been developed in recent years so as to permit screening of tens of thousands of compounds in a short period (see, e.g., Fodor et al., Science 251:767-773, 1991, and U.S. Pat.","Nos.","5,677,195; 5,885,837; 5,902,723; 6,027,880; 6,040,193; and 6,124,102, each incorporated herein by reference).","For example, large combinatorial libraries of the compounds can be constructed by the encoded synthetic libraries (ESL) method described in, e.g., WO 95/12608, WO 93/06121, WO 94/08051, WO 95/35503, and WO 95/30642 (each incorporated by reference).","Peptide libraries can also be generated by phage display methods (see, e.g., WO 91/18980, incorporated herein by reference).","Many other publications describing chemical diversity libraries and screening methods are also considered reflective of the state of the art pertaining to these aspects of the invention and are generally incorporated herein.","In one general screening strategy within the invention, new agonists and antagonists against HIV-coreceptor binding can be readily identified using the anti-coreceptor binding peptides of the invention incorporated within highly automated assay methods, e.g., using a purified chemokine receptor.","Of particular importance are antagonist compounds that have multi-tropic or multi-specific binding affinity or activity, i.e., which inhibit or block HIV interactions with distinct (e.g., both CXCR4 and CCR5) coreceptors and thereby inhibit or prevent infection by both T cell tropic (lymphotropic) and macrophage tropic (m-tropic) HIV strains.","One method of screening for new anti-coreceptor binding agents (e.g., small molecule drug peptide mimetics) utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant DNA molecules expressing an anti-coreceptor binding peptide.","Such cells, either in viable or fixed form, can be used for standard ligand/receptor binding assays (see, e.g., Parce et al., Science 246:243-247, 1989; and Owicki et al., Proc.","Natl.","Acad.","Sci.","USA 87:4007-4011, 1990, each incorporated herein by reference).","Competitive assays are particularly useful, where the cells are contacted and incubated with a labeled receptor or antibody having known binding affinity to the peptide ligand, and a test compound or sample whose binding affinity is being measured.","The bound and free labeled binding components are then separated to assess the degree of ligand binding.","The amount of test compound bound is inversely proportional to the amount of labeled receptor binding to the known source.","Any one of numerous techniques can be used to separate bound from free ligand to assess the degree of ligand binding.","This separation step can involve a conventional procedure such as adhesion to filters followed by washing, adhesion to plastic followed by washing, or centrifugation of the cell membranes.","Viable cells can also be used to screen for the effects of drugs on coreceptor-mediated functions and biological activities, e.g., HIV viral fusion, cell entry, replication, and the like.","Some detection methods allow for elimination of a separation step, e.g., a proximity sensitive detection system.","Another technique for drug screening within the invention involves an approach which provides high throughput screening for compounds having suitable binding affinity to a target molecule, e.g., a chemokine receptor, and is described in detail in European Patent Application 84/03564, published on Sep. 13, 1984.First, large numbers of different test compounds, e.g., small peptides, are synthesized on a solid substrate, e.g., plastic pins or some other appropriate surface, (see, e.g., Fodor et al., Science 251:767-773, 1991, and U.S. Pat.","Nos.","5,677,195; 5,885,837; 5,902,723; 6,027,880; 6,040,193; and 6,124,102, each incorporated herein by reference).","Then all of the pins are reacted with a solubilized anti-coreceptor binding agent of the invention, and washed.","The next step involves detecting bound anti-coreceptor binding agent.","Rational drug design may also be based upon structural studies of the molecular shapes of the anti-coreceptor binding agents.","Various methods are available and well known in the art for characterizing, mapping, translating, and reproducing structural features of anti-coreceptor binding agents to guide the production and selection of new anti-coreceptor binding mimetics, including for example x-ray crystallography and 2 dimensional NMR techniques.","These and other methods, for example, will allow reasoned prediction of which amino acid residues present in a selected anti-coreceptor binding peptide forms molecular contact regions necessary for peptide-coreceptor binding and specificity (see, e.g., Blundell and Johnson, Protein Crystallography, Academic Press, N.Y., 1976, incorporated herein by reference).","Operable anti-coreceptor binding analogs and mimetics within the invention retain partial, complete or enhanced activity compared to native anti-coreceptor binding peptides, for example, partial or complete activity for inhibiting HIV-coreceptor binding, HIV viral fusion, cell entry, and/or replication, or HIV-related disease occurrence or progression.","In this regard, operable anti-coreceptor binding analogs and mimetics for use within the invention will retain at least 50%, often 75%, and up to 95-100% or greater levels of one or more selected activities as compared to the same activity observed for a selected native HIV-1 gp120 anti-coreceptor binding peptide.","These biological properties of altered peptides or non-peptide mimetics can be determined according to any suitable assay disclosed or incorporated herein, for example, by determining the ability of an anti-coreceptor binding analog or mimetic to inhibit HIV viral fusion to coreceptor positive target cells.","In accordance with the description herein, the compounds of the invention are useful in vitro as unique tools for analyzing the nature and function of gp120 interactions with chemokine receptors, and will also serve as leads in various programs for designing additional peptide and non-peptide (e.g., small molecule drug) inhibitors of HIV-1.In addition, the anti-coreceptor binding peptides and peptide analogs of the invention are useful as immunogens, or components of immunogens, for eliciting an immune response in mammalian subjects, for example, to provide a protective immune response to prevent or treat HIV infection.","In this aspect, the peptides of the invention can be administered alone or in a formulation comprising the peptide and a pharmaceutically acceptable carrier or adjuvant, with or without additional active or inactive ingredients such immune modulatory agents (e.g., cytokines).","In certain embodiments, the peptides are administered as immunogens in the form of a conjugate (e.g., a multimeric peptide, or a peptide/carrier or peptide/hapten conjugate).","In one embodiment, a peptide is conjugated with a multimerizing polypeptide as described above.","Alternatively, a multimeric construct of immunogenic peptides, for example, comprising repeat peptide subunits, or containing two or more different peptides, can be employed, which contain one or multiple immunogenic epitope(s) that elicit a specific, humoral and/or cell-mediated (e.g.","CTL) immune response directed against the immunizing peptide(s).","Typically, the immune response will be marked by production of antibodies that bind the immunizing peptide(s) or peptide conjugate(s) with high affinity or avidity, but do not similarly recognize unrelated peptides.","In other embodiments, the antibodies recognize the immunizing peptide(s) or peptide conjugate(s) but fail to bind with high affinity or avidity to chemokines, or to peptides derived from chemokines.","In yet additional embodiments, antibodies generated against the anti-coreceptor binding peptide(s) or conjugate(s) bind to gp120 only when the gp120 protein is in a “bound” or “activated” state.","For example, when gp120 is bound to a CD4 receptor this may result in a conformational change or other activation event that exposes a gp120 coreceptor binding domain.","Antibodies that specifically target gp120 during this vulnerable activation period, (e.g., by recognizing one or more “masked” or “hidden” epitopes of gp120 that are not exposed for immune targeting when gp120 is in a “free” conformation or activity state, are particularly useful within the invention.","Thus, the invention also provides diagnostic and therapeutic antibodies, including monoclonal antibodies, and related compositions and methods for use in the management and treatment of HIV infection and related disease.","The antibodies specifically recognize anti-coreceptor binding peptides of the invention and are therefore useful for blocking HIV-coreceptor interactions when administered in vivo.","For example, monoclonal antibodies can be generated that specifically bind the anti-coreceptor binding peptides of the invention, which antibodies can be purified and administered to a patient to block or inhibit HIV-coreceptor interactions, including binding or “docking,” and HIV viral fusion, entry, replication and HIV-related disease occurrence or progression.","These activities are typically mediated, at least in part, by in vivo binding of the antibodies to gp120 (and therefore to intact HIV virus), at least in the protein's activated state upon binding to CD4.These immunotherapeutic reagents often include humanized antibodies, and can be combined for therapeutic use with additional active or inert ingredients as disclosed herein, e.g., in conventional pharmaceutically acceptable carriers or diluents, e.g., immunogenic adjuvants, and optionally with adjunctive or combinatorially active agents such as antiretroviral drugs.","The production of non-human monoclonal antibodies, e.g., murine or rat, can be accomplished by, for example, immunizing the animal with a preparation comprising purified peptides of the invention, or purified gp120.The immunogen, often comprising a peptide/hapten complex or other conjugate as described herein, can be obtained from a natural source, by peptides synthesis or preferably by recombinant expression.","Antibody-producing cells obtained from the immunized animals are immortalized and screened for the production of an antibody which binds to gp120 or a specific anti-coreceptor binding peptide (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual, Cold Springs Harbor Press, Cold Spring Harbor, N.Y., 1988, incorporated by reference for all purposes).","Humanized forms of mouse antibodies can be generated by linking the CDR regions of non-human antibodies to human constant regions by recombinant DNA techniques (see, e.g., Queen et al., Proc.","Natl.","Acad.","Sci.","USA 86:10029-10033, 1989 and WO 90/07861, each incorporated by reference).","Human antibodies can be obtained using phage-display methods (see, e.g., WO 91/17271; WO 92/01047, each incorporated herein by reference).","In these methods, libraries of phage are produced in which members display different antibodies on their outer surfaces.","Antibodies are usually displayed as Fv or Fab fragments.","Phage displaying antibodies with a desired specificity are selected by affinity enrichment to human cytochrome P450 or a fragment thereof.","Human antibodies are selected by competitive binding experiments, or otherwise, to have the same epitope specificity as a particular mouse antibody.","The invention further provides fragments of the intact antibodies described above.","Typically, these fragments compete with the intact antibody from which they were derived for specific binding to anti-coreceptor binding peptides and/or gp120.Antibody fragments include separate heavy chains, light chains Fab, Fab′ F(ab′)2, Fv, and single chain antibodies.","Fragments can be produced by enzymic or chemical separation of intact immunoglobulins.","For example, a F(ab′)2 fragment can be obtained from an IgG molecule by proteolytic digestion with pepsin at pH 3.0-3.5 using standard methods such as those described in Harlow and Lane, supra.","Fab fragments may be obtained from F(ab′)2 fragments by limited reduction, or from whole antibody by digestion with papain in the presence of reducing agents.","Fragments can also be produced by recombinant DNA techniques.","Segments of nucleic acids encoding selected fragments are produced by digestion of full-length coding sequences with restriction enzymes, or by de novo synthesis.","Often fragments are expressed in the form of phage-coat fusion proteins.","This manner of expression is advantageous for affinity-sharpening of antibodies.","To produce antibodies of the invention recombinantly, nucleic acids encoding light and heavy chain variable regions, optionally linked to constant regions, are inserted into expression vectors.","The light and heavy chains can be cloned in the same or different expression vectors.","The DNA segments encoding antibody chains are operably linked to control sequences in the expression vector(s) that ensure the expression of antibody chains.","Such control sequences include a signal sequence, a promoter, an enhancer, and a transcription termination sequence.","Expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosome.","E. coli is one prokaryotic host particularly useful for expressing antibodies of the present invention.","Other microbial hosts suitable for use include bacilli, such as Bacillus subtilus, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species.","In these prokaryotic hosts, one can also make expression vectors, which typically contain expression control sequences compatible with the host cell (e.g., an origin of replication) and regulatory sequences such as a lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda.","Other microbes, such as yeast, may also be used for expression.","Saccharomyces is a preferred host, with suitable vectors having expression control sequences, such as promoters, including 3-phosphoglycerate kinase or other glycolytic enzymes, and an origin of replication, termination sequences and the like as desired.","Mammalian tissue cell culture can also be used to express and produce the antibodies of the present invention (see, e.g., Winnacker, From Genes to Clones, VCH Publishers, N.Y., 1987, incorporated herein by reference).","Eukaryotic cells are preferred, because a number of suitable host cell lines capable of secreting intact antibodies have been developed.","Preferred suitable host cells for expressing nucleic acids encoding the immunoglobulins of the invention include: monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293) (Graham et al., J. Gen. Virol.","36:59, 1977, incorporated herein by reference); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary-cells-DHFR (CHO, Urlaub and Chasin, Proc.","Natl.","Acad.","Sci.","USA 77:4216, 1980, incorporated herein by reference); mouse sertoli cells (TM4, Mather, Biol.","Reprod.","23:243-251, 1980, incorporated herein by reference); monkey kidney cells (CVI ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL 1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); and, TRI cells (Mather et al., Annals N.Y. Acad.","Sci.","383:44-46, 1982, incorporated herein by reference); and baculovirus cells.","The vectors containing the polynucleotide sequences of interest (e.g., the heavy and light chain encoding sequences and expression control sequences) can be transferred into the host cell.","Calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation can be used for other cellular hosts (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989, incorporated herein by reference).","When heavy and light chains are cloned on separate expression vectors, the vectors are co-transfected to obtain expression and assembly of intact immunoglobulins.","After introduction of recombinant DNA, cell lines expressing immunoglobulin products are cell selected.","Cell lines capable of stable expression are preferred (i.e., undiminished levels of expression after fifty passages of the cell line).","Once expressed, the whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms of the present invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see, e.g., Scopes, Protein Purification, Springer-Verlag, N.Y., 1982, incorporated herein by reference).","Substantially pure immunoglobulins of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity most preferred.","The anti-coreceptor binding agents of the invention can also generally be used in drug screening compositions and procedures, as noted above, to identify additional compounds having binding affinity to chemokine receptors and/or act as agonists or antagonists to gp120-coreceptor or chemokine-chemokine receptor interactions and related biological activities.","Various screening methods and formats are available and well known in the art.","Subsequent biological assays can then be utilized to determine if the screened compound has intrinsic coreceptor binding, agonist or antagonist, or other desired activity useful within the invention.","Thus, in one example, the anti-coreceptor binding agents of the invention are useful as competitive binding agents in assays to screen for new HIV coreceptor agonists and antagonists.","In such assays, the compounds of the invention can be used without modification or can be modified in a variety of ways; for example, by labeling, such as covalently or non-covalently joining a moiety which directly or indirectly provides a detectable signal.","Direct labeling moieties include, for example, radiolabels, enzymes such as peroxidase and alkaline phosphatase (see, e.g., U.S. Pat.","No.","3,645,090; and U.S. Pat.","No.","3,940,475, each incorporated herein by reference), and fluorescent labels.","Moieties for indirect labeling include biotin and avidin, a binding pair that can be coupled to one constituent and the other to a label.","The compounds may also include spacers or linkers in cases where the compounds are to be attached to a solid support.","The anti-coreceptor binding agents of the invention can also be employed, based on their ability to bind HIV coreceptors (chemokine receptors), as reagents for detecting and/or quantifying HIV coreceptors on living cells, fixed cells, in biological fluids, in tissue homogenates, in purified, natural biological materials, and the like.","For example, by labeling such peptides, one can identify and/or quantify cells having HIV coreceptors on their surfaces.","In addition, based on their ability to bind HIV coreceptors, the peptides of the present invention can be used in in situ staining, FACS (fluorescence-activated cell sorting), Western blotting, ELISA, and the like.","Further, the peptides of the present invention can be used in receptor purification, or in purifying cells expressing HIV coreceptors.","The compounds of the present invention can also be utilized as commercial reagents for various medical research and diagnostic uses.","Such uses include but are not limited to: (1) use as a calibration standard for quantitating the activities of candidate HIV-coreceptor binding agonists and antagonists in a variety of functional assays; (2) use in structural analysis of HIV coreceptors through co-crystallization; and (3) use to investigate the mechanism of HIV coreceptor binding and activation.","The present invention also provides reagents, formulations, kits, and methods which provide significant prophylactic and therapeutic values.","In one embodiment of the invention, methods and compositions employ an anti-coreceptor binding agent for preventing and/or inhibiting HIV-1 binding to a host cell thereby ameliorating HIV, i.e., HIV-1 infection or a selected disease or condition associated therewith.","Additional methods and compositions are provided to treat, prevent or delay the occurrence or AIDS or ARC.","In yet additional embodiments, the methods and compositions of the invention can be used to treat other diseases and conditions which benefit from the compositions and methodologies disclosed herein, for example, a specific HIV-related disease or condition, such as Kaposi's sarcoma or an opportunistic viral (e.g., herpes) or bacterial (e.g., pneumonia) infection or condition.","In accordance with the various treatment methods of the invention, the anti-coreceptor binding agent is delivered to a patient or other subject in a manner consistent with conventional methodologies associated with management of the disorder for which treatment or prevention is sought.","In accordance with the disclosure herein, a prophylactically or therapeutically effective amount of an anti-coreceptor binding agent is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent and/or inhibit HIV binding to a host cell thereby preventing and/or inhibiting HIV infection and ameliorating a selected disease or condition.","The term “subject” as used herein means any mammalian patient to which the compositions of the invention may be administered.","Typical subjects intended for treatment with the compositions and methods of the present invention include humans, as well as non-human primates and other animals.","Alternate subjects for administration of anti-coreceptor binding agents of the invention (either for a diagnostic, analytic, disease management or therapeutic purpose) include cells, cell explants, tissues and organs, particularly those originating from mammalian subjects at risk of developing or presently suffering from HIV infection.","To identify subject patients for prophylaxis or treatment according to the methods of the invention, accepted screening methods are employed to determine risk factors associated with HIV infection, or to determine the status of an existing HIV infection or related condition in a subject.","These screening methods include, for example, conventional work-ups to determine sexual and drug-use related risk factors, as well as diagnostic methods such as various ELISA immunoassay methods, which are available and well known in the art to detect and/or characterize HIV infection and related disease.","These and other routine methods allow the clinician to select patients in need of therapy using the anti-coreceptor binding agents of the invention.","In accordance with these methods and principles, anti-coreceptor binding agent therapy can be implemented as an independent prophylaxis or treatment program or as a follow-up, adjunct or coordinate treatment regimen to other treatments, for example other anti-HIV treatments such as drug therapy (AZT, DDI, protease inhibitors and other anti-retroviral drugs), surgery, vaccination, immunotherapy, hormone treatment, cell, tissue, or organ transplants, and the like.","Within the compositions and methods of the invention, the anti-coreceptor binding agent is typically formulated with a pharmaceutically acceptable carrier and administered in an amount sufficient to inhibit virus binding and initiation or progression of HIV infection or a related disease or condition in the subject.","According to the methods of the invention, the anti-coreceptor binding agent can be administered to subjects by a variety of administration modes, including by intramuscular, subcutaneous, intravenous, intra-atrial, intra-articular, intraperitoneal, parenteral, oral, rectal, intranasal, intrapulmonary, transdermal or topically to the eyes, ears, skin or mucosal surfaces.","Alternatively, the anti-coreceptor binding agent may be administered ex vivo by direct exposure to cells, tissues or organs originating from a mammalian subject, for example, as a component of an ex vivo tissue or organ treatment formulation that contains the anti-coreceptor binding agent in a biologically suitable, liquid or solid carrier.","For prophylactic and treatment purposes, the anti-coreceptor binding agent can be administered to the subject in a single bolus delivery, via continuous delivery (e.g., continuous intravenous or transdermal delivery) over an extended time period, or in a repeated administration protocol (e.g., on an hourly, daily or weekly basis).","The various dosages and delivery protocols contemplated for administration of anti-coreceptor binding agents are therapeutically effective to inhibit the occurrence or alleviate one or more symptoms of HIV infection.","An “anti-HIV therapeutically effective amount” of the anti-coreceptor binding agent thus refers to an amount that is effective, at dosages and for periods of time necessary, to achieve detectable inhibition of HIV binding and/or infection (initiation or progression) or a related condition (e.g., in HIV-exposed versus unexposed, or treated versus untreated, test and control subjects).","In certain embodiments, a therapeutically effective amount of the anti-coreceptor binding agent, depending on the selected mode, frequency and duration of administration, will be effective to reduce or prevent HIV binding and infection of cells of the patient.","Alternatively or in addition to these effects, a therapeutically effective dosage of the anti-coreceptor binding agent, which can include repeated doses within an prolonged prophylaxis or treatment regimen, will alleviate one or more symptoms or detectable conditions associated with HIV infection.","Determination of effective dosages in this context is typically based on animal model studies followed up by human clinical trials and is guided by determining effective dosages and administration protocols that significantly reduce the occurrence or severity of HIV infection or related disease symptoms or conditions in the subject, which may be any of a range of accepted, e.g., murine or non-human primate, animal model subjects known in the art.","Alternatively, effective dosages can be determined using in vitro models (e.g., immunologic and histopathologic assays).","Using such models, only ordinary calculations and adjustments are typically required to determine an appropriate concentration and dose to administer an effective amount of anti-coreceptor binding agent (e.g., intranasally effective, transdermally effective, intravenously effective, or intramuscularly effective) to a subject to elicit a desired response.","In alternative embodiments, an “effective amount” or “effective dose” of the anti-coreceptor binding agent may simply inhibit one or more selected biological activity(ies) correlated with HIV-coreceptor binding, as set forth above.","The actual dosage of anti-coreceptor binding agent will of course vary according to factors such as the risk or state of infection or disease, the subject's age, and weight, as well as the established potency of the anti-coreceptor binding agent for eliciting the desired activity or biological response in the subject.","Dosage regimens can be adjusted to provide an optimum prophylactic therapeutic response.","A therapeutically effective amount is also one in which any toxic or detrimental side effects of the anti-coreceptor binding agent is outweighed by therapeutically beneficial effects.","A non-limiting range for a therapeutically effective amount of the anti-coreceptor binding agent 0.01 μg/kg-10 mg/kg, more typically between about 0.05 and 5 mg/kg, and in certain embodiments between about 0.2 and 2 mg/kg.","Dosages within this range can be achieved by single or multiple administrations, including, e.g., multiple administrations per day, daily or weekly administrations.","Per administration, it is desirable to administer at least one microgram of anti-coreceptor binding agent, more typically between about 10 μg and 5.0 mg, and in certain embodiments between about 100 μg and 1.0 or 2.0 mg to an average human subject.","It is to be further noted that for each particular subject, specific dosage regimens should be evaluated and adjusted over time according to the individual need and professional judgment of the person administering or supervising the administration of the anti-coreceptor binding agent compositions.","Dosage of the anti-coreceptor binding agent can be varied by the attending clinician to maintain a desired concentration at the target site.","For example, if an intravenous mode of delivery is selected local concentration of the anti-coreceptor binding agent in the bloodstream at a selected target tissue (e.g., circulating blood) can be between about 1-50 nanomoles of anti-coreceptor binding agent per liter, sometimes between about 1.0 nanomelia per liter and 10, 15 or 25 nanomoles per liter, depending on the subject's status and projected or measured response.","Higher or lower concentrations can be selected based on the mode of delivery, e.g., trans-epidermal delivery versus delivery to a mucosal surface.","Dosage should also be adjusted based on the release rate of the administered formulation, e.g., nasal spray versus powder, sustained release oral versus injected particle or transdermal delivery formulations, and the like.","To achieve the same serum concentration level, for example, slow-release particles with a release rate of 5 nanomolar (under standard conditions) would be administered at about twice the dosage of particles with a release rate of 10 nanomolar.","Anti-coreceptor binding agents comprising HIV-1 gp120 peptides and peptide analogs can be readily constructed using peptide synthetic techniques, such as solid phase peptide synthesis (Merrifleld synthesis), and the like, or by recombinant DNA techniques, that are well known in the art.","Techniques for making substitution mutations at predetermined sites in DNA include for example M13 mutagenesis.","Manipulation of DNA sequences to produce substitutional, insertional, or deletional variants are conveniently described elsewhere such as Sambrook et al., (Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989).","In accordance with these and related teachings, defined mutations can be introduced into a native gp120 peptide to generate analogs of interest by a variety of conventional techniques, e.g., site-directed mutagenesis of a cDNA copy of a portion of the gp120 gene encoding a selected peptide fragment, domain or motif.","This can be achieved through and intermediate of single-stranded form, such as using the MUTA-gen® kit of Bio-Rad Laboratories (Richmond, Calif.), or a method using the double-stranded plasmid directly as a template such as the Chameleon® mutagenesis kit of Strategene (La Jolla, Calif.), or by the polymerase chain reaction employing either an oligonucleotide primer or a template which contains the mutation(s) of interest.","A mutated subfragment can then be assembled into a complete anti-coreceptor binding peptide analog-encoding cDNA.","A variety of other mutagenesis techniques are known and can be routinely adapted for use in producing the mutations of interest in a anti-coreceptor binding peptide analog-encoding cDNA and corresponding peptide analog of the invention.","In accordance with the present invention, anti-coreceptor binding agents are isolated and purified before administration to a subject so that contaminants are removed.","In one method for obtaining purified anti-coreceptor binding peptides, a polynucleotide molecule, for example, a deoxyribonucleic acid (DNA) molecule, that defines a coding sequence for a selected anti-coreceptor binding peptide or peptide analog (e.g., a biologically active mutant or fragment of a peptide as disclosed herein bearing a deletion or substitution of 1, 2, 3 or more residues) is operably incorporated in a recombinant polynucleotide expression vector that direct expression of the peptide or analog in a suitable host cell.","Exemplary methods for cloning and purifying anti-coreceptor binding peptides and analogs employing these novel polynucleotides and vectors are widely known in the art.","Briefly, a polynucleotide of the invention encoding an anti-coreceptor binding peptide or peptide analog is amplified by well known methods, such as the polymerase chain reaction (PCR).","In this way the polynucleotide encoding the anti-coreceptor binding peptide, or a recombinantly modified version or portion thereof, is obtained for expression and purification according to conventional methods.","A DNA vector molecule that incorporates a DNA sequence encoding the subject peptide or analog can be operatively assembled, e.g., by linkage using appropriate restriction fragments from various plasmids which are described elsewhere.","Also contemplated by the present invention are ribonucleic acid (RNA) equivalents of the above described polynucleotides comprising a coding sequence for the subject anti-coreceptor binding peptide operatively linked in a polynucleotide expression construct for recombinant expression of the peptide or peptide analog.","Once a polynucleotide molecule encoding an anti-coreceptor binding peptide or analog is isolated and cloned, the peptide or analog can be expressed in a variety of recombinantly engineered cells.","Numerous expression systems are available for expressing a DNA encoding anti-coreceptor binding peptide.","The expression of natural or synthetic nucleic acids encoding an anti-coreceptor binding peptide is typically achieved by operably linking the DNA to a promoter (which is either constitutive or inducible) within an expression vector.","By expression vector is meant a polynucleotide molecule, linear or circular, that comprises a segment encoding the anti-coreceptor binding peptide of interest, operably linked to additional segments that provide for its transcription.","Such additional segments include promoter and terminator sequences.","An expression vector also may include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like.","Expression vectors generally are derived from plasmid or viral DNA, and can contain elements of both.","The term “operably linked” indicates that the segments are arranged so that they function in concert for their intended purposes, for example, transcription initiates in the promoter and proceeds through the coding segment to the terminator (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989, incorporated herein by reference).","Expression vectors can be constructed which contain a promoter to direct transcription, a ribosome binding site, and a transcriptional terminator.","Examples of regulatory regions suitable for this purpose in E. coli are the promoter and operator region of the E. coli tryptophan biosynthetic pathway as described by Yanofsky, (J. Bacteriol.","158:1018-1024, 1984, incorporated herein by reference) and the leftward promoter of phage lambda (Pk) as described by Herskowitz and Hagen, (Ann.","Rev.","Genet.","14:399-445, 1980, incorporated herein by reference).","The inclusion of selection markers in DNA vectors transformed in E. coli is also useful.","Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol.","Vectors used for expressing foreign genes in bacterial hosts generally will contain a selectable marker, such as a gene for antibiotic resistance, and a promoter which functions in the host cell.","Plasmids useful for transforming bacteria include pBR322 (Bolivar et al., Gene 2:95-113, 1977, incorporated herein by reference), the pUC plasmids (Messing, Meth.","Enzymol.","101:20-77, 1983; Vieira and Messing, Gene 19:259-268.1982, each incorporated herein by reference), pCQV2, and derivatives thereof Plasmids may contain both viral and bacterial elements.","A variety of prokaryotic expression systems can be used to express anti-coreceptor binding peptides and peptide analogs.","Examples include E. coli, Bacillus, Streptomyces, and the like.","Detection of the expressed peptide is achieved by methods such as radioimmunoassay, Western blotting techniques or immunoprecipitation.","For expression in eukaryotes, host cells for use in practicing the invention include mammalian, avian, plant, insect, and fungal cells.","Fungal cells, including species of yeast (e.g., Saccharomyces spp., Schizosaccharomyces spp.)","or filamentous fungi (e.g., Aspergillus spp., Neurospora spp.)","can be used as host cells within the present invention.","Strains of the yeast Saccharomyces cerevisiae can be used.","As explained briefly below, anti-coreceptor binding peptides and analogs can be expressed in these eukaryotic systems.","Suitable yeast vectors for use in the present invention include YRp7 (Struhl et al., Proc.","Natl.","Acad.","Sci.","USA 76:1035-1039, 1978, incorporated herein by reference), YEp13 (Broach et al., Gene 8:121-133, 1979, incorporated herein by reference), POT vectors (U.S. Pat.","No.","4,931,373, incorporated herein by reference), pJDB249 and pJDB219 (Beggs, Nature 275:104-108, 1978, incorporated herein by reference) and derivatives thereof.","Such vectors generally include a selectable marker, which can be one of any number of genes that exhibit a dominant phenotype for which a phenotypic assay exists to enable transformants to be selected.","Often, the selectable marker will be one that complements host cell auxotrophy, provides antibiotic resistance and/or enables a cell to utilize specific carbon sources, for example, LEU2 (Broach et al., Gene 8:121-133, 1979), URA3 (Botstein et al., Gene 8:17, 1979, incorporated herein by reference), HIS3 (Struhl et al., Proc.","Natl.","Acad.","Sci.","USA 76:1035-1039, 1978) or POT1 (U.S. Pat.","No.","4,931,373).","Another suitable selectable marker available for use within the invention is the CAT gene, which confers chloramphenicol resistance on yeast cells.","Examples of promoters for use in yeast include promoters from yeast glycolytic genes (Hitzeman et al., J. Biol.","Chem.","255:12073-12080, 1980; Alber and Kawasaki, J. Mol.","Appl.","Genet.","1:419434, 1982; U.S. Pat.","No.","4,599,311) or alcohol dehydrogenase genes (Young et al., Genetic Engineering of Microorganisms for Chemicals, Hollaender et al., eds., p. 355, Plenum, N.Y., 1982; Ammerer, Meth.","Enzymol.","101:192-201, 1983).","The TPI1 promoter (U.S. Pat.","No.","4,599,311) and the ADH2-4c promoter (Russell et al., Nature 304:652-654, 1983; and EP 284,044) also can be used.","The expression units can also include a transcriptional terminator.","An example of such a transcriptional terminator is the TPI1 terminator (Alber and Kawasaki, J. Mol.","Appl.","Genet.","1:419-434, 1982).","In addition to yeast, anti-coreceptor binding peptides and peptide analogs of the present invention can be expressed in filamentous fungi, for example, strains of the fungi Aspergillus (U.S. Pat.","No.","4,935,349, which is incorporated herein by reference).","Examples of useful promoters include those derived from Aspergillus nidulans glycolytic genes, such as the ADH3 promoter and the tpiA promoter.","An example of a suitable terminator is the ADH3 terminator (McKnight et al., EMBO J.","4: 2093-2099, 1985, incorporated herein by reference).","The expression units utilizing such components are cloned into vectors that are capable of insertion into the chromosomal DNA of Aspergillus.","Techniques for transforming fungi are well known in the literature, and have been described, for instance, by Beggs (Nature 275:104-108, 1978), Hinnen et al., (Proc.","Natl.","Acad.","Sci.","USA 75:1929-1933, 1978), Yelton et al., (Proc.","Natl.","Acad.","Sci.","USA 81:1740-1747, 1984), and Russell (Nature 301:167-169, 1983), each incorporated herein by reference.","The genotype of the host cell generally contains a genetic defect that is complemented by the selectable marker present on the expression vector.","Choice of a particular host and selectable marker is well within the level of ordinary skill in the art.","In addition to fungal cells, cultured mammalian cells can be used as host cells within the present invention.","Examples of cultured mammalian cells for use in the present invention include the COS-1 (ATCC CRL 1650), BHK, and 293 (ATCC CRL 1573; Graham et al., J. Gen. Virol.","6:59-72, 1977, incorporated herein by reference) cell lines.","An example of a BHK cell line is the BHK 570 cell line (deposited with the American Type Culture Collection under accession number CRL 10314).","In addition, a number of other mammalian cell lines can be used within the present invention, including rat Hep I (ATCC CRL 600), rat Hep II (ATCC CRL 1548), TCMK (ATCC CCL 139), human lung (ATCC CCL 75.1), human hepatoma (ATCC HTB-52), Hep G2 (ATCC HB 8065), mouse liver (ATCC CCL 29.1), NCTC 1469 (ATCC CCL 9.1) and DUKX cells (Urlaub and Chasin, Proc.","Natl.","Acad.","Sci USA 77:4216-4220, 1980, incorporated herein by reference).","Mammalian expression vectors for use in carrying out the present invention include a promoter capable of directing the transcription of a cloned cDNA.","Either viral promoters or cellular promoters can be used.","Viral promoters include the immediate early cytomegalovirus (CMV) promoter (Boshart et al., Cell 41:521-530, 1985, incorporated herein by reference) and the SV40 promoter (Subramani et al., Mol.","Cell.","Biol.","1:854-864, 1981, incorporated herein by reference).","Cellular promoters include the mouse metallothionein-1 promoter (U.S. Pat.","No.","4,579,821, incorporated herein by reference), a mouse V1 promoter (Bergman et al., Proc.","Natl.","Acad.","Sci.","USA 81:7041-7045, 1983; Grant et al., Nuc.","Acids Res.","15:5496, 1987, each incorporated herein by reference), a mouse V1 promoter (Loh et al., Cell 33:85-93, 1983, incorporated herein by reference), and the major late promoter from Adenovirus 2 (Kaufman and Sharp, Mol.","Cell.","Biol.","2:1304-13199, 1982, incorporated herein by reference).","Cloned DNA sequences can be introduced into cultured mammalian cells by, for example, calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb, Virology 52:456, 1973; each incorporated by reference herein in their entirety).","Other techniques for introducing cloned DNA sequences into mammalian cells can also be used, such as electroporation (Neumann et al., EMBO J.","1:841-845, 1982, incorporated herein by reference) or cationic lipid-mediated transfection (Hawley-Nelson et al., Focus 15:73-79, 1993, incorporated herein by reference) using, e.g., a 3:1 liposome formulation of 2,3-dioleyloxy-N-[2 (sperminecarboxyamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate and dioleoly-phosphatidylethanolamine in water Lipofectamine reagent, GIBCO-BRL).","To identify cells that have integrated the cloned DNA, a selectable marker is generally introduced into the cells along with the gene or cDNA of interest.","Examples of selectable markers for use in cultured mammalian cells include genes that confer resistance to drugs, such as neomycin, hygromycin, and methotrexate.","The selectable marker can be an amplifiable selectable marker, for example the DHFR gene.","Additional selectable markers are reviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass., which is incorporated herein by reference).","The choice of selectable markers is well within the level of ordinary skill in the art.","Selectable markers can be introduced into the cell on a separate plasmid at the same time as the polynucleotide encoding the anti-coreceptor binding peptide, or they may be introduced on the same plasmid.","If on the same plasmid, the selectable marker and the peptide-encoding polynucleotide can be under the control of different promoters or the same promoter.","Constructs of this latter type are known in the art (for example, U.S. Pat.","No.","4,713,339).","It also can be advantageous to add additional DNA, known as “carrier DNA” to the mixture which is introduced into the cells.","Transfected mammalian cells are allowed to grow for a period of time, typically 1-2 days, to begin expressing the polynucleotide sequence(s) of interest.","Drug selection is then applied to select for growth of cells that are expressing the selectable marker in a stable fashion.","For cells that have been transfected with an amplifiable selectable marker the drug concentration is increased in a stepwise manner to select for increased copy number of the cloned sequences, thereby increasing expression levels.","Host cells containing polynucleotide constructs of the present invention are then cultured to produce anti-coreceptor binding peptide.","The cells are cultured according to standard methods in a culture medium containing nutrients required for growth of the host cells.","A variety of suitable media are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins, minerals and growth factors.","The growth medium generally selects for cells containing the DNA construct by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker on the DNA construct or co-transfected with the DNA construct.","Recombinantly produced anti-coreceptor binding peptides and peptide analogs as described above can be purified by techniques well known to those of ordinary skill in the art.","For example, recombinantly produced peptides can be directly expressed or expressed as fusion proteins.","The proteins can then be purified by a combination of cell lysis (e.g., sonication) and affinity chromatography.","For fusion products, subsequent digestion of the fusion protein with an appropriate proteolytic enzyme releases the desired peptide.","The phrase “substantially purified” when referring to anti-coreceptor binding peptides or peptide analogs (including peptide fusions with other peptides and/or proteins) of the present invention, means a composition which is essentially free of other cellular components with which the peptides or analogs are associated in a non-purified, e.g., native state or environment.","Purified peptide is generally in a homogeneous state although it can be in either in a dry state or in an aqueous solution.","Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography.","Generally, substantially purified anti-coreceptor binding peptide comprises more than 80% of all macromolecular species present in a preparation prior to admixture or formulation of the peptide with a pharmaceutical carrier, excipient, buffer, absorption enhancing agent, stabilizer, preservative, adjuvant or other co-ingredient.","More typically, the peptide is purified to represent greater than 90% of all proteins present in a purified preparation.","In specific embodiments, the peptide is purified to greater than 95% purity or may be essentially homogeneous wherein other macromolecular species are not detectable by conventional techniques.","The peptides and analogs of the present invention can be purified to substantial purity by standard techniques well known in the art.","Useful purification methods include selective precipitation with such substances as ammonium sulfate; column chromatography; affinity methods, including immunopurification methods; and others.","See, for instance, R. Scopes, Protein Purification: Principles and Practice, Springer-Verlag: N.Y., 1982, incorporated herein by reference.","In general, anti-coreceptor binding peptides can be extracted from tissues or cell cultures that express the peptides and then immunoprecipitated, whereafter the peptides can be further purified by standard protein chemistry/chromatographic methods.","For therapeutic administration, anti-coreceptor binding peptides, analogs and mimetics of the invention are typically formulated with a pharmaceutically acceptable carrier.","As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption enhancing or delaying agents, and other excipients or additives that are physiologically compatible.","In specific embodiments, the carrier is suitable for intranasal, intravenous, intramuscular, subcutaneous, parenteral, oral, transmucosal or transdermal administration.","Depending on the route of administration, the active compound may be coated in a material to protect the compound from the action of acids and other natural conditions which may inactivate the compound.","In preparing pharmaceutical compositions of the present invention, it may be desirable to modify the anti-coreceptor binding agent, or combine or conjugate the peptide or mimetic compound with other agents, to alter pharmacokinetics and biodistribution of the anti-coreceptor binding agent.","A number of methods for altering pharmacokinetics and biodistribution are known to persons of ordinary skill in the art.","Examples of such methods include protection of peptides, proteins or complexes thereof in vesicles composed of other proteins, lipids, carbohydrates, or synthetic polymers.","For example, anti-coreceptor binding agents can be incorporated into liposomes in order to enhance pharmacokinetics and biodistribution characteristics.","A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al., (Ann.","Rev.","Biophys.","Bioeng.","2:467, 1980; U.S. Pat.","Nos.","4,235,871; 4,501,728 and 4,837,028, each incorporated herein by reference).","For use with liposome delivery, the anti-coreceptor binding agent is typically entrapped within the liposome, or lipid vesicle, or is bound to the outside of the vesicle.","Several strategies have been devised to increase the effectiveness of liposome-mediated delivery by targeting liposomes to specific tissues and specific cell types.","Liposome formulations, including those containing a cationic lipid, have been shown to be safe and well tolerated in human patients (Treat et al., J. Natl.","Cancer Instit.","82:1706-1710, 1990, incorporated herein by reference).","The compositions of the invention may alternatively contain as pharmaceutically acceptable carriers, substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like.","For solid compositions, conventional nontoxic pharmaceutically acceptable carriers can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.","For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95%, more typically 25% to 75% of active ingredient.","Therapeutic compositions for administering the anti-coreceptor binding agent can also be formulated as a solution, microemulsion, or other ordered structure suitable for high concentration of active ingredients.","The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.","Proper fluidity for solutions can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of a desired particle size in the case of dispersible formulations, and by the use of surfactants.","In many cases, it will be desirable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.","Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.","In certain embodiments of the invention, the anti-coreceptor binding agent is administered in a time release formulation, for example in a composition which includes a slow release polymer, or by depot injection.","The active peptide, analog or mimetic can be prepared with carriers that will protect against rapid release, for example a controlled release vehicle such as implants, transdermal patches, or microencapsulated delivery system.","Prolonged delivery of the anti-coreceptor binding agent, or a biologically active analog or mimetic thereof, in various compositions of the invention can be brought about by including in the composition agents that delay absorption, for example, aluminum monosterate hydrogels and gelatin.","When controlled release formulations of anti-coreceptor binding agents is desired, controlled release binders suitable for use in accordance with the invention include any biocompatible controlled-release material which is inert to the active ingredient and which is capable of incorporating the anti-coreceptor binding agent.","Numerous such materials are known in the art.","Useful controlled-release binders are materials which are metabolized slowly under physiological conditions following their subcutaneous or intramuscular injection in mammals (i.e., in the presence of bodily fluids which exist there).","Appropriate binders include but are not limited to biocompatible polymers and copolymers previously used in the art in sustained release formulations.","Such biocompatible compounds are non-toxic and inert to surrounding tissues, e.g., following subcutaneous or intramuscular injection, and do not trigger significant adverse effects such as immune response, inflammation, or the like.","They are metabolized into metabolic products which are also biocompatible and easily eliminated from the body.","For example, a polymeric matrix derived from copolymeric and homopolymeric polyesters having hydrolysable ester linkages may be used.","A number of these are known in the art to be biodegradable and to lead to degradation products having no or low toxicity.","Exemplary polymers include polyglycolic acids (PGA) and polylactic acids (PLA), poly(DL-lactic acid-co-glycolic acid)(DL PLGA), poly(D-lactic acid-coglycolic acid) (D PLGA) and poly(L-lactic acid-co-glycolic acid)(L PLGA).","Other useful biodegradable or bioerodable polymers include but are not limited to such polymers as poly(epsilon-caprolactone), poly(epsilon-aprolactone-CO-lactic acid), poly(epsilon.-aprolactone-CO-glycolic acid), poly(beta-hydroxy butyric acid), poly(alkyl-2-cyanoacrilate), hydrogels such as poly(hydroxyethyl methacrylate), polyamides, poly(amino acids) (i.e., L-leucine, glutamic acid, L-aspartic acid and the like), poly(ester urea), poly(2-hydroxyethyl DL-aspartamide), polyacetal polymers, polyorthoesters, polycarbonate, polymaleamides, polysaccharides and copolymers thereof.","Many methods for preparing such formulations are generally known to those skilled in the art (see, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978, incorporated herein by reference).","Useful formulations include controlled-release compositions such as are known in the art for the administration of leuprolide (trade name: Lupron.RTM.","), e.g., microcapsules (U.S. Pat.","Nos.","4,652,441 and 4,917,893, each incorporated herein by reference), injectable formulations (U.S. Pat.","No.","4,849,228, incorporated herein by reference), lactic acid-glycolic acid copolymers useful in making microcapsules or injectable formulations (U.S. Pat.","Nos.","4,677,191 and 4,728,721, each incorporated herein by reference), and sustained-release compositions for water-soluble peptides (U.S. Pat.","No.","4,675,189, incorporated herein by reference).","A long-term sustained release implant also may be used.","These can be readily constructed to deliver therapeutic levels of the anti-coreceptor binding agent for at least 10 to 20 days, often at least 30 days, up to 60 days or longer.","Long-term sustained release implants are well known to those of ordinary skill in the art and can incorporate some of the absorption delaying components described above.","Such implants can be particularly useful by placing the implant near or directly within the target tissue or cell population, thereby affecting localized, high-doses of the anti-coreceptor binding agent at one or more sites of interest.","In alternate embodiments, anti-coreceptor binding agent may be delivered to a mucosal surface, e.g., orally, intranasally, or rectally, for prophylaxis or treatment of HIV infection and related disease.","For mucosal administration, the peptide, analog or mimetic is typically combined with an inert diluent or an assimilable edible carrier.","The anti-coreceptor binding agent may be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into a subject's diet.","For oral therapeutic administration, the anti-coreceptor binding agent may be incorporated with excipients and used in the form of ingestable tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.","Of course, taste-improving substances can be added in the case of oral administration forms.","The percentage (e.g., by weight or by volume) of the anti-coreceptor binding agent in these compositions and preparations may, of course, be varied.","As noted above, the amount of anti-coreceptor binding agent in such therapeutically useful compositions is generally such that a therapeutically effective dosage will be delivered.","For oral or rectal administration, the anti-coreceptor binding agent can be worked into tablets or other solid forms by being mixed with solid, pulverulent carrier substances, such as sodium citrate, calcium carbonate or dicalcium phosphate, and binders such as polyvinyl pyrrolidone, gelatin or cellulose derivatives, possibly by adding also lubricants such as magnesium stearate, sodium lauryl sulfate, “Carbowax” or polyethylene glycol.","Solid delivery vehicles may contain a protein or peptide in a mixture with fillers, such as lactose, saccharose, mannitol, starches, such as potato starch or amylopectin, cellulose derivatives or highly dispersed silicic acids.","In soft-gelatin capsules, the active substance is dissolved or suspended in suitable liquids, such as vegetable oils or liquid polyethylene glycols.","As further forms, one can use plug capsules, e.g., of hard gelatin, as well as dosed soft-gelatin capsules comprising a softener or plasticizer, e.g., glycerin.","Alternatively, liquid dosage forms for delivering anti-coreceptor binding agents to mucosal surfaces include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules.","Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents.","Oral dosage forms optionally contain flavorants and coloring agents.","Parenteral and intravenous forms would also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.","The therapeutic compositions of the invention typically must be sterile and stable under all conditions of manufacture, storage and use.","Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.","Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.","In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.","The prevention of the action of microorganisms can be accomplished by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.","In certain embodiments of the invention, the anti-coreceptor binding agent is administered by topical delivery to a mucosal surface of the patient, for example, via intranasal or intrapulmonary delivery in the form of an aerosol spray or powder.","According to one aspect of the invention, the anti-coreceptor binding agent is delivered in an intranasally or intrapulmonarily effective amount, typically in a selected volume of administered spray or powder, to achieve a desired therapeutic result.","In related aspects of the invention, novel pharmaceutical compositions are provided for intranasal or intrapulmonary delivery that incorporate the anti-coreceptor binding agent in a powder or aqueous formulation.","Intranasal or intrapulmonary administration allows self-administration of treatment by patients, provided that sufficient safeguards are in place to control and monitor dosing and side effects.","Nasal or intrapulmonary administration also overcomes certain drawbacks of other administration forms, such as injections, that are painful and expose the patient to possible infections and may present drug bioavailability problems.","Systems for aerosol dispensing of therapeutic liquids as a spray are well known.","In one embodiment, metered doses of aerosolized anti-coreceptor binding agent are delivered by means of a specially constructed mechanical pump valve (See, for example, U.S. Pat.","No.","4,511,069, incorporated herein by reference).","This hand-held delivery device is uniquely nonvented so that sterility of the solution in the aerosol container is maintained indefinitely.","Certain nasal and intrapulmonary spray solutions within the invention comprise an anti-coreceptor binding agent in a liquid carrier that optionally includes a nonionic surfactant for enhancing absorption of the drug and one or more buffers or other additives to minimize nasal or pulmonary irritation.","In some embodiments, the nasal or pulmonary spray solution further comprises a propellant.","Thus, in certain embodiments the pharmaceutical compositions comprising an anti-coreceptor binding agent administrable in fine particulate form (e.g., between about 0.5-5.0 μm, more typically between about 1.0-2.5 μm diameter particles) comprising a surfactant and propellant as pharmaceutically acceptable carriers (see, e.g., U.S. Pat.","No.","5,902,789, incorporated herein by reference).","The surfactant must be nontoxic and soluble in the propellant.","Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride.","Mixed esters, such as mixed or natural glycerides, can be employed.","Additional carrier can be included as desired, for example lecithin for intranasal delivery.","The pH of the intranasal or intrapulmonary spray solution is typically between pH 6.8 and 7.2.Alternative means of mucosal administration for the anti-coreceptor binding agents of the invention may involve the use of powder carriers, for example ion exchange resins or adsorbent resin powders (see, e.g., U.S. Pat.","No.","5,942,242, incorporated herein by reference).","In more detailed aspects of the invention, the anti-coreceptor binding agent is stabilized to extend its effective half-life following delivery to the subject, particularly for extending metabolic persistence in an active state within the physiological environment (e.g., in the bloodstream, at a mucosal surface, or within a connective tissue compartment or fluid-filled body cavity).","For this purpose, the anti-coreceptor binding agent can be modified by chemical means, e.g., chemical conjugation, N-terminal capping, PEGylation, or recombinant means, e.g., site-directed mutagenesis or construction of fusion proteins, or formulated with various stabilizing agents or carriers.","Thus stabilized, the anti-coreceptor binding agent administered as above retains anti-coreceptor activity for an extended period (e.g., 2 to 3, up to 5 to 10 fold greater stability) under physiological conditions compared to its non-stabilized form.","Numerous reports in the literature describe the potential advantages of pegylated proteins, which include their increased resistance to proteolytic degradation, increased plasma half-life, increased solubility and decreased antigenicity and immunogenicity (Nucci et al., Advanced Drug Deliver Reviews 6:133-155, 1991; Lu et al., Int.","J. Peptide Protein Res.","43:127-138, 1994, each incorporated herein by reference).","A number of proteins, including L-asparaginase, strepto kinase, insulin, and interleukin-2 have been conjugated to a poly(ethyleneglycol) (PEG) and evaluated for their altered biochemical properties as therapeutics (see, e.g., Ho et al., Drug Metabolism and Disposition 14:349-352, 1986; Abuchowski et al., Prep.","Biochem.","9:205-211, 1979; and Rajagopaian et al., J. Clin.","Invest.","75:413-419, 1985, each incorporated herein by reference).","Although the in vitro biological activities of pegylated proteins may be decreased, this loss in activity is usually offset by the increased in vivo half-life in the bloodstream (Nucci, et al., Advanced Drug Deliver Reviews 6:133-155, 1991, incorporated herein by reference).","Several procedures have been reported for the attachment of PEG to proteins and peptides and their subsequent purification (Abuchowski et al., J. Biol.","Chem.","252:3582-3586,1977; and Beauchamp et al., Anal.","Biochem, 131:25-33, 1983, each incorporated herein by reference).","Lu et al., (Int.","J. Peptide Protein Res.","43:127-138, 1994) describe various technical considerations and compare PEGylation procedures for proteins versus peptides (see also, Katre et al., Proc.","Natl.","Acad.","Sci.","USA 84:1487-1491, 1987; Becker et al., Makromol.","Chem.","Rapid Commun.","3:217-223, 1982; Mutter et al., Makromol.","Chem.","Rapid Commun.","13:151-157, 1992; Merrifield, R. B., J.","Am.","Chem.","Soc.","85:2149-2154, 1993; Lu et al., Peptide Res.","6:142-146, 1993; Lee et al., Bioconiugate Chem.","10:973-981, 1999; Nucci et al., Adv.","Drug Deliv.","Rev.","6:133-151, 1991; Francis et al., J.","Drug Targeting 3:321-340, 1996; Zalipsky, Bioconjugate Chem.","6:150-165, 1995; Clark et al., J. Biol.","Chem.","271:21969-21977, 1996; Pettit et al., J. Biol.","Chem.","272:2312-2318, 1997; Delgado et al., Br.","J.","Cancer 73:175-182, 1996; Benhar et al., Bioconjugate Chem.","5:321-326, 1994; Benhar et al., J. Biol.","Chem.","269:13398-13404, 1994; Wang et al., Cancer Res.","53:4588-4594, 1993; Kinstler et al., Pharm.","Res.","13:996-1002, 1996, Filpula et al., Exp.","Opin.","Ther.","Patents 2:231-245, 1999; Pelegrin et al., Hum.","Gene Ther.","9:2165-2175, 1998, each incorporated herein by reference).","Following these and other teachings in the art, the conjugation of anti-coreceptor binding peptides and analogs with poly(ethyleneglycol) polymers, is readily undertaken with the expected result of prolonging circulating life and/or reducing immunogenicity while maintaining an acceptable level of activity of the PEGylated anti-coreceptor binding agent.","Amine-reactive PEG polymers for use within the invention include SC-PEG with molecular masses of 2000, 5000, 10000, 12000, and 20000; U-PEG-10000; NHS-PEG-3400-biotin; T-PEG-5000; T-PEG-12000; and TPC-PEG-5000.Chemical conjugation chemistries for these polymers have been published (see, e.g., Zalipsky, Bioconjugate Chem.","6:150-165, 1995; Greenwald et al., Bioconjugate Chem.","7:638-641, 1996; Martinez et al., Macromol.","Chem.","Phys.","198:2489-2498, 1997; Hermanson, Bioconjugate Techniques, pp.","605-618, 1996; Whitlow et al., Protein Eng.","6:989-995, 1993; Habeeb, Anal.","Biochem.","14:328-336, 1966; Zalipsky et al., Poly(ethyleneglycol) Chemistry and Biological Applications, pp.","318-341, 1997; Harlow et al., Antibodies: A Laboratory Manual pp.","553-612, Cold Spring harbor Laboratory, Plainview, N.Y., 1988; Milenic et al., Cancer Res.","51:6363-6371, 1991; Friguet et al., J. Immunol.","Methods 77:305-319, 1985, each incorporated herein by reference).","While phosphate buffers are commonly employed in these protocols, the choice of borate buffers may beneficially influence the PEGylation reaction rates and resulting products.","PEGylation of anti-coreceptor binding peptides and analogs may be achieved by modification of carboxyl sites (e.g., aspartic acid or glutamic acid groups in addition to the carboxyl terminus).","The utility of PEG-hydrazide in selective modification of carbodimide-activated protein carboxyl groups under acidic conditions has been described (Zalipsky, Bioconjugate Chem.","6:150-165, 1995; Zalipsky et al., Poly(ethyleneglycol) Chemistry and Biological Applications, pp.318-341, American Chemical Society, Washington, D.C., 1997, each incorporated herein by reference).","Alternatively, bifunctional PEG modification of anti-coreceptor binding peptides can be employed.","In some procedures, charged amino acid residues, including lysine, aspartic acid, and glutamic acid, have a marked tendency to be solvent accessible on protein surfaces.","Conjugation to carboxylic acid groups of proteins is a less frequently explored approach for production of protein bioconjugates.","However, the hydrazide/EDC chemistry described by Zalipsky et al., (Zalipsky, Bioconjugate Chem.","6:150-165, 1995; Zalipsky et al., Poly(ethyleneglycol) Chemistry and Biological Applications, pp.","318-341, American Chemical Society, Washington, D.C., 1997, each incorporated herein by reference) offers a practical method of linking PEG polymers to protein carboxylic sites.","For example, this alternate conjugation chemistry has been shown to be superior to amine linkages for PEGylation of brain-derived neurotrophic factor (BDNF) while retaining biological activity (Wu et al., Proc.","Natl.","Acad.","Sci.","U.S.A. 96:254-259, 1999, incorporated herein by reference).","Maeda and colleagues have also found carboxyl-targeted PEGylation to be the preferred approach for bilirubin oxidase conjugations (Maeda et al., Poly(ethyleneglycol) Chemistry, Biotechnical and Biomedical Applications, J. M. Harris, ed., pp.","153-169, Plenum Press, New York, 1992, incorporated herein by reference).","In addition to PEGylation, anti-coreceptor binding agents of the invention can be modified to enhance circulating half-life by shielding the peptide or analog via conjugation to other known protecting or stabilizing compounds, or by the creation of fusion proteins with the peptide or analog linked to one or more carrier proteins, such as one or more immunoglobulin chains (see, e.g., U.S. Pat.","Nos.","5,750,375; 5,843,725; 5,567,584 and 6,018,026, each incorporated herein by reference).","These modifications will decrease the degradation, sequestration or clearance of the anti-coreceptor binding agent and result in a longer half-life in a physiological environment (e.g., in the circulatory system, or at a mucosal surface).","The anti-coreceptor binding agents modified by PEGylation and other stabilizing methods are therefore useful with enhanced efficacy within the methods of the invention.","In particular, the anti-coreceptor binding agents thus modified maintain activity for greater periods at a target site of delivery compared to the unmodified peptide or analog.","Even when the anti-coreceptor binding agents are thus modified, they retain substantial biological activity for inhibiting HIV-coreceptor interactions and HIV infection and related disease.","In yet additional aspects of the invention, an anti-coreceptor binding agent is administered according to the foregoing methods in a coordinate therapy protocol with one or more additional anti-HIV agents or treatment steps.","Thus, in various embodiments an anti-coreceptor binding agent is administered coordinately with one or more adjunct or combinatorially (e.g., additively or synergistically) effective treatment agents selected from anti-retroviral drugs (e.g., nucleoside reverse transcriptase inhibitors such as AZT (zidovudine), Videx® (ddI or didanosine), or Hivid® (ddC or zalcitabine); antiviral drugs (e.g., acyclovir); antibiotics (e.g., penicillins, cephalosporins, macrolides and lincosamides such as erythromycin, gentamycin, ceftriaxone, cefixime, azithromycin, spectinomycin, ofloxacin, ciprofloxacin, cefoxitin, clindamycin, metronidazole, amoxicillin, tetracycline, doxycycline); immunomodulatory agents (e.g., interferons (IFNα, IFNβ, IFNγ), interleukins (such as IL-1 or IL-2); G-CSF, GM-CSF, levamisole) particularly interferon); or vaccine agents.","Other immune modulatory agents that are useful for coordinate administration with anti-coreceptor binding agents include corticosteroids, cytotoxic drugs, T-cell specific inhibitors, antisera, replacement immune globulins, and monoclonal antibodies.","At times, specific immunosuppressive drugs can be used in conjunction with anti-coreceptor binding agent therapy, for example, azathioprine, cyclophosphamide, or cyclosprine.","Some cytotoxic drugs may also be used for coordinate administration with anti-coreceptor binding agents, for example azathioprine, azathioprine sodium, chlorambucil, cyclophosphamide, methotrexate, methotrexate sodium, and/or clyclosprorine.","Additional combinatorial or adjunctive therapeutic agents for use within coordinate formulations and treatment methods of the invention may comprise a glucocorticoid or non-steroidal anti-inflammatory drug (NSAID).","Glucocorticoids useful within this aspect of the invention include short-acting glucocorticoids (e.g., cortisone and hydrocortisone), intermediate-acting glucocorticoids (e.g., prednisone, prednisolone, meprednisone, methylprednisolone and triamcinolone), and long-acting glucocorticoids (betamethasone, dexamethasone and paramethasone).","NSAID's useful within the invention include, aspirin, salicylates, naproxen, indomethacin, piroxicam, oxaprozin, phenylbutazone, ibuprofen, flurbiprofen, fenoprofen, and ketoprofen, and the like.","Additional therapeutic agents for use in conjunction with anti-coreceptor binding agent therapy may include the anti-malarial drug hydroxychloroquine, or sulfasalazine.","Also useful in coordinate therapy protocols with anti-coreceptor binding agents are antihistamines, including amino alkyl ethers (e.g., diphenhydramine, clemasine), ethylenediamines (e.g., pyrilamine, tripelennamine), alkylamines (e.g., brompheniramine, chlorpheniramine, dexchlorpheniramine, triprolidine), and phenothiazines (e.g., methdilazine, promethazine, trimeprazine).","Within alternate methods and compositions of the invention, the anti-coreceptor binding agent is administered as above coordinately, admixed or separately, simultaneously or sequentially, with of one or more of the foregoing “combinatorially effective or adjunct treatment agents”, in respective amounts sufficient (independently sufficient or combinatorially sufficient) to prevent or alleviate HIV infection or a disease condition or symptom associated therewith.","The instant invention also includes kits, packages and multicontainer units containing the above described pharmaceutical compositions, active ingredients, and/or means for administering the same for use in the prevention and treatment of HIV infection and related disease conditions.","Briefly, these kits include a container or formulation which contains an anti-coreceptor binding agent, typically formulated in a pharmaceutical preparation with a biologically suitable carrier.","The anti-coreceptor binding agent is optionally contained in a bulk dispensing container or unit or multi-unit dosage form.","Optional dispensing means may be provided, for example, an intranasal or intrapulmonary spray applicator.","Packaging materials optionally include a label or instruction which indicates that the pharmaceutical agent packaged therewith can be used for treating HIV and related disease conditions.","In more detailed embodiments of the invention, kits include reagents and/or devices for detecting the presence and/or status of HIV infection or related disease in a subject.","For example, an immunological or molecular probe that binds or reacts with an HIV-specific marker detectable in blood or other biological samples to be obtained from the subject.","Thus, the kits may contain ELISA probes for detecting HIV antigens, as well as additional, optional kit materials for collecting and/or processing samples for ELISA and other diagnostic assays.","The kits may also contain suitable buffers, preservatives such as protease inhibitors, direct or sandwich-type labels for labeling probes, and/or developing reagents for detecting a signal from the label.","Thus, a broad selection of therapeutic and diagnostic kits are provided within the invention based on the description herein, including kits that contain specific instructions for carrying out the prophylactic and treatment protocols and associated assays of the invention.","The invention is further illustrated by the following specific examples which are not intended in any way to limit the scope of the invention.","EXAMPLES Materials and Methods The following examples describe computer modeling, synthesis and testing of co-receptor agents that inhibit the binding of HIV-1 to CXCR4 and CCR5.Synthetic Peptides Exemplary peptides 15D, 15K and 15KS, were synthesized using known methods by Macromolecular Resources (Fort Collins, Colo.), peptide 15CW was synthesized by the Basic Research Laboratory National Cancer Institute, (Frederick, MD).","The peptides were purified by reverse-phase HPLC and the homogeneity of the peptide preparations was confirmed by mass-spectrometry.","Computer Modeling Computer-generated structural models were derived using non-hydrogen atom superimposition of homologous residues during an optimization protocol of constrained consistent valence force field (CVFF) (Dauber-Osguthorpe et al., Proteins 4:31-47, 1988; Hagler et al., J.","Am.","Chem.","Soc.","96:5319-27, 1974; and Hagler et al., Science 227:1309-15, 1985, each incorporated herein by reference), molecular dynamics sampling and conjugate gradients minimization of sampled structures.","Cells and Culture Conditions HEK-293 cells expressing human CCR5 (HEK-CCR5) and CXCR4 (HEK-CXCR4) (Howard et al., J. Biol.","Chem.","274:16228-16234, 1999, incorporated herein by reference) were cultured in Dulbecco's modified Eagle's medium (BioWhittaker, Walkersville, Md.)","containing 10% fetal bovine serum (HyClone, Logan, Utah), 2 mM glutamine, 100 units/ml penicillin and streptomycin (Quality Biologicals, Gaithersburg, Md.)","and 400 mg/ml Geneticin (Life Technologies, Inc., Rockville, Md.)","at 37° C. in a humidified 5% CO2 atmosphere.","CEMx174 and THP-1 cell lines were obtained from ATCC (Rockville, Md.).","Sup-T1 cells expressing CCR5 was the gift of James Hoxie.","Cells were cultured in RPMI-1640 medium (BioWhittaker, Walkersville, Md.)","containing 10% fetal bovine serum (FBS; HyClone, Logan, Utah), 2 mM glutamine, 100 units/ml penicillin and streptomycin (Quality Biologicals, Gaithersburg, Md.)","at 37° C. in a humidified 5% CO2 atrnosphere.","Binding Assays Binding assays were performed with HEK-CCR5 or HEK CXCR4 in triplicate by adding unlabeled competitor (anti-coreceptor binding peptide or control chemokine) and radiolabeled chemokine, 0.2 ng/ml (125I-MIP-1β or SDF-1α, specific activity 2000 Ci/mmol, NEN Life Science Products) to a cell suspension (4×105 cells in 200 μl) in RPMI 1640 supplemented with 1% bovine serum albumin and 25 mM HEPES pH 8.0 (binding medium).","Cells were then incubated at 22° C. for 40 min with continuous rotation.","After incubation cells were transferred to the tubes containing 800 μl of 10% sucrose in PBS and harvested by centrifugation.","The supernatant was aspirated and the cell-associated radioactivity was measured using a 1272 Wallac gamma counter.","Binding assays were performed with the 174xCEM and Sup-T1 cells expressing CCR5 in duplicates by adding unlabeled competitor (peptide or control chemokine) and radiolabeled chemokine, 0.2 ng/ml (125I-MIP-1β or 125I-SDF-1α, specific activity 2000 Ci/mmol, NEN life Science Products) to 300 μl cell suspension (4×106 cells/ml) in RPMI 1640 supplemented with 1% bovine serum albumin, 0.1% sodium azide and 25 mM HEPES pH 8.0 (binding medium).","Cells were then incubated at 22° C. for 30 min with continuous rotation.","After incubation cells were transferred to the tubes containing 800 μl of 10% sucrose in PBS and harvested by centrifugation, the supematant was aspirated and the cell-associated radioactivity was measured using a 1272 Wallac gamma counter.","The data were analyzed by nonlinear regression using the computer program GraphPad Prizm 3.0.Chemotaxis Assays Chemotaxis assays for HEK-293 cells transfected with CCRS was performed as previously described (Howard et al., J. Biol.","Chem.","274:16228-16234, 1999, incorporated herein by reference).","Briefly, the chemokine RANTES, diluted in binding medium to 100 ng/ml, was placed in the lower wells of a microchemotaxis chamber.","Polycarbonate membrane pretreated with rat tail collagen type 1 was placed over the chemokine solution.","The cells (1×106/ml) were suspended in binding medium along with the exemplary peptides 15D or 15K at designated concentrations and placed in the upper wells of the chamber.","After incubation for 3 hr the membrane was removed, stained using a Diff-Quik kit (Trends Scientific, Kalamazoo, Mich.) and counted.","The results are expressed as the “chemotaxis index”, which represents the ratio of the number of cells in high powered field in test versus control samples.","Flow Cytometric Analysis CEMx174 cells were pelleted and resuspended at 106 cells/ml in PBS containing 1% BSA and 0.1% sodium azide (FACS buffer).","Cells were then preincubated with designated concentrations of peptides or SDF-1-α (1 μg/ml) at 22° C. for 60 min.","FITC-labeled anti-human CXCR4 antibody (clone 12G5, BD PharMingen, San Diego, Calif.) was added to the cells per the manufactures instructions and further incubated at 22° C. for 40 minutes.","Cells were extensively washed with FACS buffer and analyzed using a FACS Calibur flowcytometer (Becton Dickinson).","HEK/CCR5 cells were suspended in Dulbecco's PBS containing 1% FCS and 0.05% NaN3 (104 cells in 100 μl) and incubated with or without 5 μg of MIP-1β (PeproTech, Rocky Hill, N.J.), 0.1 mM peptide 15D or 15K for 45 min at 22° C. Cells were then treated with FITC-conjugated anti-human CCR5 antibody (2D7; BD PharMingen, San Diego, Calif.), and incubated for 30 min at 22° C. Cells were washed twice, and analyzed using a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, Calif.).","CA2+ Mobilization Assay THP-1 cells (12×106/ml in RPMI 1640 containing 10% FSB) were loaded in the presence of 5 μM fura-2 AM (Molecular Probes, Eugene, Oreg.)","at 22° C. for 30 min in the dark.","Subsequently, cells were washed three times and resuspended (106 cells/ml) in the buffer containing 138 mM sodium chloride, 6 mM potassium chloride, 1 mM calcium chloride, 1 mM magnesium chloride, 10 mM HEPES, pH 7.4,5 mM glucose, and 0.1% bovine serum albumin.","1.93 ml of loaded cells was transferred into a quartz cuvette.","50 μl of a peptide stock solution (2×10−2 M) was added to the cells, and after 3 min of incubation 20 μl of human SDF-1α (10 μg/ml, PeproTech) was added to a stirred cuvette.","The measurements were performed using luminescence spectrophotometer LS50 B (Perkin-Elmer).","Ca2+ mobilization in the cells in response to SDF-1α was measured by analysis of the ratio of fluorescence emitted at 510 nm after sequential excitation at 340 and 380 nm.","Assays of HIV Infectivity PBMCs were obtained from whole blood of normal donors and isolated by Ficoll-Paque Plus (Pharmacia Biotech, Piscataway, N.J.) density gradient centrifugation, and plated at a cell density of 2×106 cells/ml.","Monocyte-derived macrophages (MDM) were generated from adherent human peripheral blood mononuclear cells by culture for 7 days with M-CSF (100 ng/ml).","Cultures were maintained in RPMI-1640 medium (Life Technologies, Rockville, Md.)","supplemented with 10% heat-inactivated endotoxin-free FCS (Hyclone, Logan, Utah), 10 μg/ml gentamicin, and 1 mM glutamine.","Cells were treated with designated concentrations of the exemplary peptides 15K or 15D, and after 1 hr cells were infected with HIV-1IIIB (T cell tropic) or HIV-1JRFL (monocyte tropic) at an multiplicity of infection (MOI) of 0.1.After 2 hr, cells were washed, and cultured for additional 48-72 hr, followed by analysis of HIV replication as determined by quantification of accumulated p24 in the supernatant.","The production of p24 was determined by conventional sandwich ELISA, using ELISA plates pre-coated with capture anti-p24 antibodies provided by the AIDS Vaccine Program (SAIC Frederick, NCI-FCRDC, Frederick, Md.).","The captured p24 antigen was detected using rabbit anti-HIV-1 anti-p24 antibody, and a secondary goat anti-rabbit IgG (peroxidase-labeled) antibody.","The captured p24 protein was detected using a 3,3′,5,5′-tetramethylbenzidine and hydrogen peroxide detection system (KPL Laboratories, Gaithersburg, Md.).","The reaction was read spectrophotometrically at 450 nm.","No cytotoxicity was detectable following treatment of PBMC with the peptides even at a concentration of 100 μg/ml.","Example I Structural Analysis of gp120 and Chemokines As noted above, a number of recent studies point to a role of chemokine receptors as coreceptors for HIV cell entry.","Additional studies suggest that protein fragments or peptides from chemokines or HIV corresponding to structural determinants involved in chemokine receptor (coreceptor) binding, may be useful to block HIV-coreceptor binding and therefore serve as anti-HIV reagents.","Considering that both chemokines and the HIV envelope protein gp120 are thought to directly interact with chemokine receptors, it is conceivable that this direct interaction may involve a part of the gp120 polypeptide chain that is structurally similar to (e.g., by homologous or convergent evolutionary relationship) receptor binding determinants of chemokines.","However, conventional amino acid sequence comparison between gp120 and chemokines undertaken by the present inventors did not reveal any significant amino acid identity or similarity relationship that would point to a convergent, or conservative, chemokine receptor binding domain.","Despite this apparent lack of structural homology, the present investigation focused on a putative structural element found to be conserved within the amino acid sequences of nearly all chemokines, and also shared in a corresponding structural motif postulated for the HIV-1 gp120 protein.","In particular, all chemokines studied were found to possess a Trp residue located at the beginning of a C-terminal alpha-helix.","This conserved Trp residue is separated by six residues from a 4th Cys residue, to comprise what is characterized herein as a conserved chemokine structural motif.","By comparison, in the amino acid sequences of gp120 proteins of all HIV-1 isolates there is a very similar motif in a spanning part of the conserved region 3 (C3) adjacent to the V3 loop.","Surprisingly, this motif was also found to include one or more residues within a C-terminal portion of the variable loop 3 (V3) of gp120 that are conserved between different HIV-1 strains and exhibit some homology with chemokines (Table 1).","Example II Computer Modeling of gp120 and Chemokines Computer modeling further demonstrates that novel peptide fragments of gp120 can be template forced onto a “homologous loop” of the known structure of an exemplary chemokine, MIP-1β without violation of the general rules of protein structure.","To assess the possibility that a selected region of gp120 may potentially assume a structure similar to that of chemokines, a model of the corresponding structure of gp120 was built using the structure of MIP-1β (Lodi et al., Science 263:1762-1767, 1994, incorporated herein by reference) as the template.","Based on this analysis, it was determined that a model of the three-dimensional structure of the selected gp120 segment could be generated without violation of protein stereochemistry.","In an effort to examine the functional activity of this region, peptides corresponding to the selected gp120 sequence were synthesized for further studies.","Example III Peptide Design and Synthesis Two exemplary peptides were synthesized based on a reference peptide comprising a C-terminal portion of the V3 loop and an N-terminal of the C3 domain of gp120 (see Table 4, above).","The sequence of HIV-1JRFL, which is very close to the consensus B of HIV-1 sequences (Korber and Los Alamos National Laboratory-Theoretical Biology and Biophysics Group T-10, Human Retroviruses and AIDS, 1998, A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences, Los Alamos National Laboratory, Los Alamos, NW, 1998), was used as a template for the design of anti-HIV peptides.","Synthesized peptides corresponding to a conserved structural motif of the HlV1JRFL gp120 protein, bearing the above-noted structural similarity to a corresponding, conserved motif identified in chemokines, are shown in Table 4.Because gp120 of different HIV-1 strains have some sequences variabilities even in conserved regions, peptides were designed with amino acid sequences similar to the widest range of naturally existing variants.","In many HIV strains there is a lysine (k) residue instead of glutamine (Q) in position 3 (Table 4).","Moreover, it was reasonable to change glutamine for lysine in the synthetic peptide to avoid hydrolysis and conversion to glutamic acid near the conserved positively changed arginine.","The lysine residue preceding the tryptophan (W) in the peptide designated 15K was changed to aspartic acid (D) in the peptides designated 15D because some HIV-1 isolates have aspartic acid in this position and the change provided an opportunity to explore the significance of this substitution.","For control experiments, peptides with “scrambled” sequences were also synthesized (peptide 15CW with the same amino acid composition as 15D, and 15KS with the same amino acid composition as 15K).","In some control experiments a 15 amino acid peptide, 15GIG, with an amino acid sequence unrelated to gp120 was also used (See Table 4).","TABLE 4 SEQUENCE OF SYNTHESIZED PEPTIDES SEQ Amino Acid Sequence ID 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 NO: HIVJRFL I R Q A H C N I S R A K W N D SEQ ID NO:3 15 K I R K A H C N I S R A K W N D SEQ ID NO:8 15 D I R K A H C N I S R A D W N D SEQ ID NO:9 15CW I R K A H C W I D R A D N N S SEQ ID NO:10 15KS K I N S W R A D N I H C K A R SEQ ID NO:11 15GIG G I G D P V T C L K S G A I A SEQ ID NO:12 As further detailed below, these exemplary peptides exhibited surprising anti-coreceptor binding activities, including competition with chemokines for binding to CCR5- and CXCR4-expressing cells, and inhibition of chemotaxis in chemokine-responsive cells.","Correlated with these activities, the peptides mediate potent inhibition of HIV replication in macrophages and T lymphocytes, evincing their efficacy for prophylaxis and treatment of HIV infection and related disease conditions within the compositions and methods of the invention.","This and related description herein further provides abundant structural information to enable the design and construction of a large assemblage of effective anti-coreceptor binding peptides, peptide analogs and mimetics, to serve as potent inhibitors of HIV-coreceptor interactions.","Example IV Anti-Coreceptor Binding Activity of gp120 Peptides as Demonstrated by Inhibition of Chemokine Binding to CCR5 and CXCR4 Expressing Cells Considering that the 15K and 15D peptides were designed in part based on their structural similarity to corresponding chemokine fragments, the binding activity of these peptides for CCR5 and/or CXCR4 receptors, and corresponding anti-coreceptor binding activity against chemokines, was assessed.","Experiments were carried out to determine the ability of these exemplary peptides to competitively inhibit binding of radiolabeled MIP-1β or SDF-1α to cells expressing CCR5 or CXCR4, respectively.","The results, shown in FIG.","1, demonstrate that both peptides significantly inhibit chemokine binding, although high concentrations (e.g., 100 μM) of peptide are required to mediate this effect.","Example V Anti-Coreceptor Binding Activity of gp120 Peptides as Demonstrated by Inhibition of CCR5-Mediated Chemotaxis The inhibition of binding of MIP-1β and SDF-1α to their respective receptors suggests that the exemplary peptides 15K and 15D might also inhibit chemotaxis of cells expressing cognate chemokine receptors for appropriate ligands.","To achieve this goal, the capacity of these peptides to inhibit the chemotactic response of HEK-CCR5 cells to a cognate ligand of CCR5 receptors, the chemokine RANTES, was assayed.","The results demonstrated that RANTES-directed chemotactic responses were completely blocked by the addition of either 15K or 15D (FIG.","2).","Interestingly, one of the peptides, 15D, consistently exhibited greater inhibitory activity in these experiments.","These results show that the 15K and 15D peptides interfere with chemokine receptor function.","Example VI Anti-Coreceptor Binding Activity of gp120 Peptides Mediates Inhibition of HIV Infection in Monocyte-Tropic Cells The results described in the preceding Examples indicate that the 15K and 15D peptides effectively block the ability of CCR5 to serve as a receptor to mediate chemotactic responses.","Additional experiments were thus carried out to determine the capacity of these peptides to inhibit coreceptor activity mediating cellular entry and infection by HIV-1JRFL (a monocyte-tropic HIV strain), using peripheral blood monocyte-derived macrophages.","The results, presented in FIG.","3, show that the addition of either 15K or 15D significantly reduced monocyte lineage target cell infection by HIV-1.Significant inhibition was exhibited at concentrations as low as 10 ng/ml.","Interestingly, the CCR5-selective chemokine ligand MIP-1β and the 15K and 15D peptides exhibited comparable inhibitory activities (FIG.","3).","Example VII Anti-Coreceptor Binding Activity of gp120 Peptides Mediates Inhibition of HIV Infection in Lymphocyte-Tropic Cells In view of the foregoing binding inhibition studies, and considering the homology between the 15K and 15D peptides and the CXCR4 ligand SDF-1α, it was further undertaken to determine the capacity of the synthetic peptides to inhibit CXCR4 co-receptor function.","In particular, experiments were carried out to assess the ability of 15K or 15D to alter the infection of PBMCs with the T cell-tropic HIV-1β strain.","The results, presented in FIG.","4, demonstrate that both peptides significantly reduced HIV-1 infection, in a dose-dependent manner.","Significant inhibition of HIV-1 infection was detected with concentrations of peptides as low as 10 ng/ml.","The scrambled peptide 15KS with the same amino acid composition as 15K but a randomized sequence manifested significantly less inhibition of virus infectively (FIG.","4) demonstrating the importance of the sequence of amino acids for inhibiting activity.","Example VIII Activity of gp120 Peptides in Relation to Binding of Anti-Coreceptor Antibodies To determine if peptides 15D and 15K interact directly with chemokine receptors the effect of these and control peptides on the binding of anti-chemokine receptor antibodies to CXCR4 and CCR5 was studied.","The monoclonal antibody 12G5 recognizes a conformational extracellular epitope on CXCR4.This antibody blocks the infectivity of some X4 strains of HIV-1 and HIV-2 (Endres et al., Cell 87:745-756, 1996; Hoxie et al., J. Reprod.","Immunol.","41:197-211(1998); McKnight et al., J. Virol.","74:1692-1696(1997), 19, 26).","It also inhibits the binding of SDF-1α, a natural CXCR4 ligand Schols et al., J. Exp.","Med.","186:1383-1388, 1997 (Haste et al., Mol.","Pharmacol.","60:164-173(2001), 33).","Further, the binding of 12G5 to CXCR4 receptor-expressing cells is prevented by anti-HIV compounds.","The results show that peptide 15K inhibited the binding of the 12G5 antibody to CXCR4 in a dose-dependent manner (FIG.","5A), and at a concentration of 50 μg/ml approached the efficacy of SDF-1α at 5 mg/ml.","Peptide 15D appeared to be less effective in blocking of 12G5 antibody binding to CXCR4 (FIG.","5B), which is not surprising since the binding site of CXCR4 includes several negatively charged residues Duranz et al., J. Virol.","73:2752-2761, 1999.To determine the importance of the particular amino acid sequence of the inhibitory activity of the peptides.","The effects of peptide 15K and the corresponding scrambled peptide 15KS on binding of 12G5 to CXCR4 was compared.","The scrambled peptide 15KS manifest significantly less inhibitory activity in comparison with 15K suggesting the importance of the specific peptide sequence (FIG.","5C).","Apparently there is a direct correlation between the ability of 15K to inhibit the anti-CXCR4 antibody binding and its ability to inhibit HIV-1 infectivity since the scrambled peptide 15KS with the same amino acid comparison has reduced ability to manifest both these activities.","The effect of the exemplary peptides 15K and 15D on the binding of 2D7 antibodies to CCR5 was assayed to further evaluate the mechanism of anti-coreceptor activity of the peptides.","The 2D7 monoclonal antibody binds to a conformational epitope on CCR5, and inhibits Ca2+ flux induced by RANTES.","This antibody has also been shown to inhibit HIV infection in vitro (Wu et al., J. Exp.","Med.","186:1373-81, 1997, incorporated herein by reference).","It does not, however, inhibit binding of gp120-SCD4 complexes to CCR5-expressing L1.2 murine cells (Olson et al., J. Virol.","73:4145-4155, 1999, incorporated herein by reference).","The effects of peptides 15D and 15K on 2D7 antibody binding to HEK293/CCR5 cells were analyzed by flow cytometry.","No significant effect of either of the two peptides on anti-CCR5 antibody binding was observed.","These results indicate that the peptides bind to an epitope on the CCR5 receptor that is spatially and sterically distinct from the antibody binding site.","Considering that both the peptides and the 2D7 antibodies inhibit HIV infection in vitro, these results may be interpreted as underscoring the unexpected nature and advantages of the present invention.","Example IX Effects of Peptides on Induction of Intracellular Ca2+ Concentration in CXCR4 Expressing THP-1 Cells The increase of intracellular Ca2+ concentration mediated by a chemokine receptor in response to a cognate chemokine is a reliable assay for measurement of chemokine agonist activity.","The human monocytic cell line THP-1 expresses a high level of CXCR4 and responds well to SDF-1α stimulation (FIG.","6A), so these cells were used to determine whether peptides 15D and 15K could block the activation of CXCR4.Preincubation of cells with peptide 15D (at a concentration 500 μM) for 2 min completely inhibited cellular response to SDF-1α (FIG.","6B).","Peptide 15K also inhibited Ca2+ mobilization although less efficiently than 15D (FIG.","6C).","There is a probable direct correlation between the greater ability of 15D to inhibit Ca2+ mobilization in response to SDF-1α and anti-HIV-1 activity because in some experiments higher anti-HIV-1 activity of 15D was observed in comparison with 15K (data not shown).","The scrambled peptide 15CW, with amino acid composition identical to 15D (Table 4), was less effective at inhibiting the Ca2+ mobilization response (FIG.","6D) suggesting the importance of the specific peptide sequence.","The peptide treatment alone did not induce Ca2+ mobilization.","Example X Effect of gp120 Derived Peptides 15K and 15D on Binding of Chemokines to CCRS and CXCR4 Expressing Cells Since the 15K and 15D peptides were designed based on their structural homology with a chemokine fragment, whether these peptides might exhibit binding activity for CCR5 or CXCR4 was determined.","Experiments were carried out to assess the ability of the peptides to competitively inhibit binding of radiolabeled MIP-1β or SDF-1α to cells expressing CCR4 or CXCR4, respectively.","To determine the effect of peptides exclusively on chemokine binding and to exclude the contribution of chemokine internalization, cells were incubated at room temperature in the presence of 0.1% sodium azide.","Under such conditions all cell-bound ligand could be removed by rinsing the cells with 0.1 M Gly-HCl pH 2.5, excluding the contribution of internalization to the chemokine binding.","The binding experiments demonstrated that the 15D and 15K peptides competitively inhibited chemokine binding to CCR5 (FIG.","7A) and CXCR4 (FIG.","7B).","The binding experiments did not reveal any difference in the inhibitory activity of peptides 15K and 15D in comparison with their scrambled analogs (data not shown).","The control 15-mer peptide 15GIG with a different amino acid composition manifested significantly lower competing activity (FIG.","7C).","It is possible that the particular scrambling of the peptide sequence that was performed did not change the sequences of the scrambled peptides sufficiently to significantly reduce their capacity to bind to the receptors because scrambling of these peptides may have created similar amino acid triplets in other positions of the peptides (like RAK and KAR in 15K and 15KS, Table 4).","It is possible that because of this peptide 15KS also manifested a low level of inhibitory activity (although significantly less than original peptide) on anti-CXCR4 antibody binding to the receptor (FIG.","5C).","Additional structure-functional studies are necessary to determine the minimum sequence of the peptides sufficient for inhibition of virus.","To briefly summarize the foregoing description, the interaction of HIV envelope glycoprotein gp120 with a chemokine receptor is known to be a prerequisite for viral attachment and entry into target cells (Alkhatib et al., Science 272:1955-1958, 1996; Berson et al., J. Virol.","70:6288-6295, 1996; Deng et al., Nature 381:661-666, 1996; and Dragic et al., Nature 381:667-673, 1996, each incorporated herein by reference).","Monocyte-tropic viruses (R5 strains) utilize a distinct chemokine receptor, CCR5, for cell entry, while T cell tropic viruses (X4 strains) utilize CXCR4 receptors.","Chemokine receptor binding site(s) on gp120 are thought to be formed, or exposed, after binding of gp120 to CD4 (Wu et al., Nature 384:179-183, 1996, incorporated herein by reference).","However, gp120 binding to the chemokine receptor in certain HIV strains does not require interaction with CD4 (Hoffman et al., Proc.","Natl.","Acad.","Sci.","USA 96:6359-6364, 1999; and Hoxie et al., J. Reprod.","Immunol.","41:197-211, 1998, each incorporated herein by reference).","The V3 loop of gp120 has been identified as the major determinant of cellular tropism and coreceptor specificity (Cocchi et al., Nat.","Med.","2:1244-1247, 1996; and Hwang, et al., Science 253:71-74, 1991, each incorporated herein by reference).","Nevertheless, the precise region or residues within this 35-37 amino acid V3 loop responsible for mediating these phenotypic effects has not yet been established (Hung et al., J. Virol.","73:8216-8226, 1999, incorporated herein by reference).","Synthetic cyclized peptides corresponding to the V3 loop of gp120 of X4 and dual strains of HIV-1 (but not an R5 strain) at micromolar concentrations could prevent binding of anti-CXCR4 antibodies.","Some of these peptides at micromolar concentrations inhibited the infectivity of HIV-IIIB (Sakaida et al., J. Virol.","72:9763-9770, 1998, incorporated herein by reference).","Synthetic polymer preparations including a putative V3 consensus sequence (GPGRAF, SEQ ID NO:13) of HIV-1 were reported to inhibit HIV-1 infection by an unknown mechanism.","However, it is unlikely that this inhibition was due to competition with gp120 binding to the chemokine receptor, or to CD4 (Moulard et al., J. Pept.","Res.","53:647-655, 1999, incorporated herein by reference).","Although the influence of V3 on Hw coreceptor utilization is well established, other more conservative regions of gp120 are also proposed to be involved in coreceptor usage (Rizzuto et al., Science 280:1949-1953, 1998, incorporated herein by reference).","Within the present invention, a conservative chemokine receptor binding motif of gp120 was identified which shares certain similarity in amino acid sequence that appears conserved among relevant chemokines.","More specifically, the present inventors observed that in the amino acid sequences of most chemokines there is a conserved Trp residue separated by six amino acid residues from a fourth Cys residue.","In comparison, the gp120 of all HIV isolates analyzed exhibited a comparable structural motif in the C3 region following the V3 loop.","Moreover, several residues of the C-terminal part of V3 loop also manifest some homology with a canonical chemokine sequence (Table 1).","Computer modeling as provided herein further demonstrated that the corresponding fragment of gp120 could be template forced onto a “homologous” loop of the known three dimensional structure of an exemplary chemokine MIP-1β (for specific MIP-1β structural detail, see, e.g., Lodi et al., Science 263:1762-7, 1994, incorporated herein by reference) without violation of protein stereochemistry.","Prompted in part by these discoveries, two candidate peptides were synthesized which exemplify the proposed gp120 structural motif (Table 2).","Both candidate peptides were demonstrated to compete with chemokines for binding to CCR5- and CXCR4-expressing cells.","In addition, the exemplary peptides each inhibited CCR5-directed chemotaxis.","Correlated with these anti-coreceptor activities, the peptides mediated potent inhibit replication of both monocyte-tropic HIV-1JRFL and T cell tropic HIV-1IIIB.","The X-ray structure of an HIV-1 gp120 core (not including V-loops), complexed with a two-domain fragment of CD4, and an antigen-binding fragment of neutralizing antibody, has been reported (Kwong et al., Nature 393:648-659, 1998).","Although the structure does not include variable loops of gp120 it did include the C3 portion of the fragment of gp120, which the present invention considered to be structurally similar to the chemokines and potentially involved in interaction with chemokine receptors.","In fact, the three-dimensional structure of this fragment (HIVHXB2, residues 331-340, Table 1) appeared to be similar to the corresponding MIP-1β fragment.","Both motifs are characterized by an α-helix preceded by a turn.","Both contain a Trp residue with the indole ring buried in the interior region of the turn and with the α-carbon present on an exterior turn of the helix.","The surface residues on the helices are characterized by long aliphatic side chains, terminated with polar groups, including Lys, Glu, Gln, and Asn.","Importantly, this fragment is located within the region of gp120 molecule, which was implicated in CCR5 binding (Rizzuto et al., Science 280:1949-1953, 1998).","Since the peptides 15D and 15K include only 5 residues from the C-terminal part of the V3 loop, which is more conserved than other parts of V3 loop, and 9 residues from the N-terminal part of the conservative C3 region, it was reasonable to expect that anti-HIV activity of these peptides may not be dependent on virus tropism.","The effects of the peptides on HIV-1 infectivity using R5 and X4 viruses confirmed this prediction (FIGS.","3 and 4).","The data above demonstrating that peptides 15K and 15D compete with anti-CXCR4 antibody 12G5 suggests a direct interaction of the peptide with this receptor.","Although the 15D peptide had reduced ability to block binding of the 12G5 antibody, the infectivity data indicates that the low affinity interaction of 15D was still sufficient to interfere with viral infection.","Moreover, the inhibition of the SDF-1α induced intracellular Ca2+ influx by peptides 15D and 15K also supports the conclusion that both antiviral peptides interact with chemokine receptors.","The affinity of interaction of the peptides 15D and 15K with chemokine receptors is apparently low, as evidenced by the high concentration of the peptides required to inhibit anti-CXCR4 antibody binding, mobilization of intracellular Ca2+ in response to SDF-1α, and chemokine binding.","However, even this low affinity interaction is sufficient for interference with HIV-1 infection based on the low concentration of peptides required for blocking of the viral infection of macrophages and T lymphocytes.","Indeed, it is not necessary for an efficient inhibitor of virus interaction with the coreceptor to also be a strong competitor of chemokines binding to the same receptor, because the envelope protein of HIV-1 only mimicks the highly specific binding of a chemokine with its receptor and the affinity of this interaction can be quite low (Hoffman et al., Proc.","Natl.","Acad.","Sci.","USA 99:11215-11220, 2000).","The use of low affinity anti-viral drugs interfering with HIV-1 coreceptor interaction may allow targeting multiple cellular receptors while maintaining the ability to inhibit interactions of a viral glycoprotein, which is subject to frequent mutation.","This may provide the basis for the capacity of the present peptides to inhibit infection by viruses using different coreceptors.","Moreover, the rather weak competition of peptides 15D and 15K with chemokines for receptor binding, together with potent inhibition of HIV-1 infectivity, can be therapeutically preferable to high affinity inhibitors of CXCR4 and CCR5, because 15K and 15D peptides would not compromise functions of potentially critical chemokines such as SDF-1α or the CCR5 ligands.","The present invention has identified a region of HIV-1 gp120, which is structurally similar to chemokines, and appears to be directly involved in the interaction with certain chemokine receptors.","The above findings that these peptides inhibit HIV-1 infection of human monocyte-derived macrophages and T-lymphocytes at low nanomolar concentrations suggest that these peptides, their analogs, and peptide menetics, can be used to dissect gp120 interactions with different chemokine receptors and could serve as not only leads for the design of new peptide inhibitors of HIV-1 not restricted by viral tropism, but are useful themselves as therapeutic agents to prevent binding of HIV-1 to a susceptible cell thereby reducing infection and viral replication in the treatment and prevention of HIV infection and related disease.","Moreover, it may also be possible that antibodies raised against this sequence of HIV-1 gp120 can also have anti-HIV protective and therapeutic activity by reducing or preventing HIV binding to a susceptible cell.","Although the foregoing invention has been described in detail by way of example for purposes of clarity of understanding, it will be apparent to the artisan that certain changes and modifications are comprehended by the disclosure and may be practiced without undue experimentation within the scope of the appended claims, which are presented by way of illustration not limitation.","All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes."]],"string":"[\n [\n \"Methods and compositions for inhibiting hiv-coreceptor interactions\",\n \"Novel methods and compositions are provided for inhibiting interactions between human immunodeficiency viruses (HIVs) and viral coreceptors, including CXCR4 and/or CCR5 coreceptors.\",\n \"The anti-coreceptor binding agent includes a novel peptide portion of the gp120 envelope protein of HIV-1, as well as peptide analogs and mimetics of this peptide, that specifically binds to, or modulates activity of, the coreceptors(s).\",\n \"The anti-coreceptor binding agent is useful as a prophylactic or therapeutic treatment to prevent or inhibit HIV binding to a susceptible cell and thereby reduces infection and/or moderates or treats related diseases.\",\n \"In alternative embodiments, the peptides, analogs and mimetics are effective to inhibit direct co-receptor binding by HIV virus, coreceptor binding by HIV gp120 proteins or peptides, HIV fusion with target host cells, HIV virion entry into host cells, HIV replication, and HIV transmission between cells and hosts.\",\n \"In more detailed embodiments, the anti-coreceptor binding agents of the invention are multi-tropic by exhibiting activity against HIV interactions with multiple, CXCR4 and CCR5, coreceptors.\"\n ],\n [\n \"1.A composition comprising an effective amount of an anti-coreceptor binding agent to inhibit binding of a CXCR4 and/or CCR5 coreceptor of a subject by an HIV virus or viral protein, wherein the anti-coreceptor binding agent is a gp120 peptide, peptide analog or mimetic that specifically binds the CXCR4 and/or CCR5 coreceptor.\",\n \"2.The composition of claim 1, wherein the gp120 peptide, peptide analog or mimetic is between about 12 and about 24 amino acid residues in length and comprises a conserved CXXXXXXW amino acid sequence motif, wherein X is any naturally occurring or synthetic amino acid or amino acid analog.\",\n \"3.The composition of claim 1, wherein the peptide, peptide analog or mimetic is modified by addition, admixture, or conjugation of additional amino acids, peptides, proteins, chemical reagents or moieties which do not substantially alter the anti-coreceptor binding activity of the peptide.\",\n \"4.The composition of claim 1, wherein the anti-coreceptor binding agent is a peptide comprising an allelic variant among native HIV gp120 peptide sequences.\",\n \"5.The composition of claim 1, wherein the anti-coreceptor binding agent is formulated for delivery to subject selected from an isolated or bound coreceptor, a membrane or cell preparation comprising the coreceptor, a cell population, tissue or organ expressing the coreceptor, or a mammalian patient.\",\n \"6.The composition of claim 5, wherein the anti-coreceptor binding agent is combined with a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant for administration in a prophylactic or therapeutic effective dose to a mammalian patient to prevent or inhibit HIV infection or a related disease condition or symptom in the patient.\",\n \"7.The composition of claim 1, wherein the anti-coreceptor binding inhibits one or more biological activities mediated by or associated with HIV-coreceptor interactions selected from (a) direct co-receptor binding by HIV virus, (b) coreceptor binding by a HIV gp120 protein or a peptide fragment or derivative thereof, (c) HIV fusion with target host cells, (d) HIV virion entry into host cells, (e) HIV replication, and/or (f) HIV cell-cell or host-host transmission.\",\n \"8.The composition of claim 1, wherein the anti-coreceptor binding agent comprises an effective formulation of an HIV-1 peptide, peptide analog or mimetic for in vivo administration to inhibit one or more biological activities selected from (a) direct co-receptor binding by HIV-1 virus, (b) coreceptor binding by a HIV-1 gp120 protein or a peptide fragment or derivative thereof, (c) HIV-1 fusion with target host cells, (d) HIV-1 virion entry into host cells, (e) HIV-1 replication, and/or (f) HIV-1 cell-cell or host-host transmission.\",\n \"9.The composition of claim 1, wherein the anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic formulated for administration to a mammalian patient in a prophylactically or therapeutically effective dose to prevent or inhibit HIV-1 infection or an HIV-1-related disease condition or symptom.\",\n \"10.The composition of claim 1, wherein the anti-coreceptor binding agent is a peptide that includes a conserved “CXXXXXXW” amino acid sequence motif, wherein X is any amino acid, and wherein the peptide is from about 12-17 amino acids in length and is selected from peptide 15K, comprising an amino acid sequence IRKAHCNISRAKWND (SEQ ID NO:8), or a corresponding or overlapping native peptide sequence or peptide analog that shares substantial sequence identity to the reference peptide sequence of 15K.\",\n \"11.The composition of claim 10, wherein the peptide includes one or more residues occurring naturally or by substitution at a relative, aligned position corresponding to a designated position for peptide 15K, selected from: Position 1—I, M, K, S, T, L, A, V, R, P, or N; Position 2—R, G, E, K, S, T, or I; Position 3—Q, K, R, L, E, P, A, V, S, T, H, or D; Position 4—A, T, P, V, E, or S; Position 5—H, Y, F, Q, N, I, or V; Position 7—N, D, H, T, K, E, S, I, Q, V, G, or A; Position 8—I, L, V, Y, D, A; Position 9—S, N, D, T, K, Y, I, or P; Position 10—R, K, G, S, A, E, D, I, T, W, or N; Position 11—A, R, K, T, S, G, E, D, N, Q, H, V, I, or L; Position 12—K, D, R, E, K, Q, N, T, S, G, A, V, L; Position 14—N, Q, D, E, K, R, A, S, T, G, M, Y, I, H, or V; and/or Position 15—D, N, K, E, T, Q, R, S, A, I, M, or P. 12.The composition of claim 1, wherein the anti-coreceptor binding agent exhibits multi-tropic activity characterized by effective inhibition of HIV viral, or gp120 protein or peptide binding to multiple, CXCR4 and CCR5, coreceptors.\",\n \"13.The composition of claim 12, wherein the multi-tropic anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic effective to inhibit one or more biological activities of both T cell tropic (lymphotropic) and macrophage tropic (m-tropic) HIV-1 viruses selected from (a) direct co-receptor binding by viruses, (b) coreceptor binding by viral gp120 proteins or peptide fragments or derivatives thereof, (c) viral fusion with target host cells, (d) virion entry into host cells, (e) viral replication, and/or (f) viral cell-cell or host-host transmission.\",\n \"14.A method for inhibiting human immunodeficiency virus (HIV) interaction with a CXCR4 and/or CCR5 coreceptor comprising exposing a subject to an effective amount of an anti-coreceptor binding agent to inhibit binding of the CXCR4 and/or CCR5 coreceptor by an HIV virus or viral protein, wherein the anti-coreceptor binding agent is a gp120 peptide, peptide analog or mimetic that specifically binds the CXCR4 and/or CCR5 coreceptor.\",\n \"15.The method of claim 14, wherein the gp120 peptide, peptide analog or mimetic is between about 12 and about 24 amino acid residues in length and comprises a conserved CXXXXXXW amino acid sequence motif, wherein X is any naturally occurring or synthetic amino acid or amino acid analog.\",\n \"16.The method of claim 14, wherein the peptide, peptide analog or mimetic is modified by addition, admixture, or conjugation of additional amino acids, peptides, proteins, chemical reagents or moieties which do not substantially alter the anti-coreceptor binding activity of the peptide.\",\n \"17.The method of claim 14, wherein the anti-coreceptor binding agent is a peptide comprising an allelic variant among native HIV gp120 peptide sequences.\",\n \"18.The method of claim 14, wherein the subject is an isolated or bound CXCR4 and/or CCRS coreceptor, a membrane or cell preparation comprising the coreceptor, a cell population, tissue or organ expressing the coreceptor, or a mammalian patient.\",\n \"19.The method of claim 18, wherein the subject comprises a cell population, tissue or organ selected for in vivo or ex vivo treatment or diagnostic processing.\",\n \"20.The method of claim 18, wherein the subject is a mammalian patient susceptible to HIV infection and the anti-coreceptor binding agent is administered in a prophylactic or therapeutic effective dose to prevent or inhibit HIV infection or a related disease condition or symptom.\",\n \"21.The method of claim 14, wherein the anti-coreceptor binding agent is administered to the subject in an amount effective to inhibit one or more biological activities mediated by or associated with HIV-coreceptor interactions selected from (a) direct co-receptor binding by HIV virus, (b) coreceptor binding by a HIV gp120 protein or a peptide fragment or derivative thereof, (c) HIV fusion with target host cells, (d) HIV virion entry into host cells, (e) HIV replication, and/or (f) HIV cell-cell or host-host transmission.\",\n \"22.The method of claim 14, wherein the anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic administered to the subject in an amount effective to inhibit one or more biological activities selected from (a) direct co-receptor binding by HIV-1 virus, (b) coreceptor binding by a HIV-1 gp120 protein or a peptide fragment or derivative thereof, (c) HIV-1 fusion with target host cells, (d) HIV-1 virion entry into host cells, (e) HIV-1 replication, and/or (f) HIV-1 cell-cell or host-host transmission.\",\n \"23.The method of claim 14, wherein the anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic administered to a mammalian patient in a prophylactically or therapeutically effective dose to prevent or inhibit HIV-1 infection or an HIV-1-related disease condition or symptom.\",\n \"24.The method of claim 14, wherein the anti-coreceptor binding agent is a peptide that includes a conserved “CXXXXXXW” amino acid sequence motif, wherein X is any amino acid, and wherein the peptide is from about 12 to about 17 amino acids in length and is selected from peptide 15K, comprising an amino acid sequence IRKAHCNISRAKWND (SEQ ID NO:8), or a corresponding or overlapping native peptide sequence or peptide analog that shares substantial sequence identity to the peptide sequence of 15K.\",\n \"25.The method of claim 24, wherein the peptide includes one or more residues occurring naturally or by substitution at a relative, aligned position corresponding to a designated position for peptide 15K, selected from: Position 1—I, M, K, S, T, L, A, V, R, P, or N; Position 2—R, G, E, K, S, T, or I; Position 3—Q, K, R, L, E, P, A, V, S, T, H, or D; Position 4—A, T, P, V, E, or S; Position 5—H, Y, F, Q, N, I or V; Position 7—N, D, H, T, K, E, S, I, Q, V, G, or A; Position 8—I, L, Y, D, A; Position 9—S, N, D, T, K, Y, I, or P; Position 10—R, K, G, S, A, E, D, I, T, W, or N; Position 11—A,R,K,T, S, G, E, D,N, Q, H, V, I, or L; Position 12—K, D, R, E, K, Q, N, T, S, G, A, V, L; Position 14—N, Q, D, E, K, R, A, S, T, G, M, Y, I, H, or V; and/or Position 15—D, N, K, E, T, Q, R, S, A, I, M, or P. 26.The method of claim 14, wherein the anti-coreceptor binding agent exhibits multi-tropic activity characterized by effective inhibition of HIV viral, or gp120 protein or peptide binding to multiple, CXCR4 and CCR5, coreceptors.\",\n \"27.The method of claim 26, wherein the multi-tropic anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic administered to the subject in an amount effective to inhibit one or more biological activities of both T cell tropic (lymphotropic) and macrophage tropic (m-tropic) HIV-1 viruses selected from (a) direct co-receptor binding by viruses, (b) coreceptor binding by viral gp120 proteins or peptide fragments or derivatives thereof, (c) viral fusion with target host cells, (d) virion entry into host cells, (e) viral replication, and/or (f) viral cell-cell or host-host transmission.\"\n ],\n [\n \"<SOH> BACKGROUND OF THE INVENTION <EOH>The envelope glycoprotein of the human immunodeficiency virus type I (HIV-1) mediates in the fusion of viral and host cell membranes necessary for virion entry (Freed et al., J. Biol.\",\n \"Chem.\",\n \"270:23883-23886, 1995).\",\n \"The envelope glycoprotein of HIV-1 is produced by the enzymatic cleavage from a gp160 precursor protein to produce the external gp120 protein and the transmembrane gp41 protein (Capon et al., Annu.\",\n \"Rev.\",\n \"Immunol.\",\n \"9:649-678, 1991).\",\n \"Several studies have identified specific portions or domains of the gp120 protein that may elicit humoral and/or cell-mediated immune responses to HIV in susceptible host subjects, and may therefore be useful to formulate anti-HIV reagents and methods for prevention and treatment of HIV infection and related diseases.\",\n \"These general HIV peptide studies describe a large, diverse assemblage of gp120 peptides that are proposed as candidates for therapeutic use, primarily in vaccine formulations to prevent and treat HIV infection and related disease.\",\n \"For example, U.S. Pat.\",\n \"No.\",\n \"5,691,135 describes various peptides that are selected for the ability to inhibit HIV infection by stimulating VH3 and VH4 antibody responses.\",\n \"The peptides are proposed for administration to a patient as an antigen in sufficient quantity to induce antibodies that exhibit superantigen binding to gp120.The disclosure considers 31 peptides obtained from the AIDS Research and Reference Program, NIH, which peptides correspond to sequences from gp120 and gp41 from different strains of HIV.\",\n \"Additional gp120 peptides are described in U.S. Pat.\",\n \"No.\",\n \"5,939,074.In particular, this references describes peptides that are useful as “multideterminant peptide antigens” for eliciting both cell-mediated and humoral immune responses against HIV.\",\n \"Additional studies have identified particular regions of the gp120 that are proposed to determine specific interactions between gp120 and CD4, which is the primary receptor on target cells mediating cellular entry by HIV-1 (Arthos et al., Cell 57:469481, 1989; Clayton et al., Nature 339:548-551, 1989; Kwong et al., Nature 393:648-659, 1998; and Landau et al., Nature 334:159-162, 1988).\",\n \"In addition to CD4 binding, cellular entry by HIV-1 is thought to require additional interactions between the virus and one or more “coreceptors” on the surfaces of target cells.\",\n \"Briefly, the HIV-1 particles are proposed to bind initially to the CD4 receptor, and then subsequently to a chemokine receptor present on the target cells, which is used by the virus as a “coreceptor” for mediating cellular entry.\",\n \"Different strains of HIV-1 preferentially utilize different chemokine receptors that are variably expressed among HIV-1 target cells.\",\n \"In particular, some strains of HIV-1, referred to as “lymphotropic” strains, bind CXCR4 chemokine receptors predominantly expressed on lymphocyte target cells, while other HIV-1 strains, termed “monocyte tropic” strains, bind CCR5 receptors predominantly expressed on cells of monocyte lineage.\",\n \"Thus, the major viral coreceptors are CXCR4 (Berson et al., J. Virol.\",\n \"70:6288-6295, 1996), which has a native function as a chemokine receptor for stromal derived factor-1 (SDF-1), and CCR5 (Alkhatib et al., Science 272:1955-1958, 1996; Deng et al., Nature 381:661-666, 1996; and Dragic et al., Nature 381:667-673, 1996), which functions naturally as a receptor for several chemokines, including macrophage inflammatory protein-1β (MIP-1β).\",\n \"It is proposed that a conformational change occurs in gp120 following HIV binding of CD4, and that this conformation change exposes one or more binding sites on the gp120 molecule that mediate additional interactions between HIV and chemokine receptor, which are referred to in this context as “viral coreceptors” (Kwong et al., Nature 393:648-659, 1998, incorporated herein by reference).\",\n \"Recent studies suggest a complicated, multi-step binding and activation model, wherein the association of gp120 with CD4 yields a CD4-gp120 complex, which subsequently associates with the viral coreceptor resulting in a structural rearrangement (e.g., conformational change) of gp120 that facilitates interaction of the gp41 envelope protein subunit with the host cell membrane, leading to viral entry (Helseth et al., J. Virol.\",\n \"64:6314-6318, 1990; and Weissenhom et al., Nature 387:426-430, 1997).\",\n \"Within this emergent field of investigation, numerous studies have focused on structure-function mapping of chemokine receptors, with the goals of determining binding determinants and mechanisms of action of the receptors.\",\n \"Yet additional research has focused on chemokine mapping studies.\",\n \"From these studies chemokine-derived peptides have been identified that reportedly comprise binding determinants of the chemokines capable of blocking native chemokine receptor-ligand interactions.\",\n \"Further studies, focusing alternatively on HIV gp120 mapping, report production and testing of gp120 peptides capable of blocking HIV-coreceptor binding and HIV infectivity.\",\n \"With regard to chemokine receptor studies, numerous articles report that chemokine receptors play a direct role as coreceptors for HIV cell entry.\",\n \"Briefly, as noted above, it is reported that specific interactions between the HIV envelope glycoprotein gp120 and one or more chemokine receptors mediate viral entry into target cells.\",\n \"More specifically, monocyte-tropic or “m-tropic” HIV strains bind to a distinct chemokine receptor, CCR5, for cell entry, while T cell lymphotropic or “T-tropic” virus use mainly CXCR4 receptors for cell entry.\",\n \"More detailed, structure-function analyses have been directed toward identifying specific HIV binding determinants of chemokine receptors mediating their activity as coreceptors for HIV entry.\",\n \"In conjunction with these reports, certain references focus on a concept of blocking chemokine receptor activity (e.g., receptor binding with chemokines, or with gp120) using competitive inhibitors.\",\n \"Proposed inhibitors of chemokine receptor binding interactions include peptide inhibitors that mimic structures of chemokines, or of gp120, binding determinants or related structural domains.\",\n \"These studies follow more basic research which shows that intact chemokines, the normal ligands of chemokine receptors, can compete with cognate chemokines, or with HIV, for binding to the target receptors.\",\n \"Referring specifically to chemokine mapping studies focusing on chemokine structure-function, various publications attempt to identify and characterize receptor binding determinants of chemokines.\",\n \"As noted above, certain of these references also describe chemokine-based peptides reportedly capable of blocking chemokine-receptor binding and other activities mediated by receptor-ligand (chemokine or HIV) interactions.\",\n \"One such study is presented in a publication by Reckless et al., ( Biochem.\",\n \"J.\",\n \"340:803-811, 1999).\",\n \"This study identifies a number of chemokine-derived peptides, including a peptide designated “peptide 3”, based on a human chemokine, monocyte chemotactic protein-1 (MCP-1).\",\n \"Reckless and colleagues report that the peptide 3 inhibits cell migration induced by a wide range of chemokines.\",\n \"Moreover, peptide 3 reportedly binds to THP-1 cells and inhibits THP-1 migration, reportedly by acting as a chemokine receptor antagonist.\",\n \"On this basis, the authors propose that peptide 3 and its derivatives, including peptides ranging from 6-15 residues in length, may be useful as chemokine inhibitors.\",\n \"A number of related reports focus on a distinct portion of chemokines as prospective receptor binding determinants.\",\n \"In particular, the reports focus on the N-terminal region of chemokines comprising a structural element called the “N-loop”.\",\n \"This distinct element follows the first two cysteine residues of a model chemokine, and is proposed to play an important role in chemokine-receptor interactions.\",\n \"Other parts of chemokine molecules have also been proposed to contribute significantly as structural determinants of interactions between chemokines and their cognate receptors.\",\n \"For example, Crump et al., ( EMBO J.\",\n \"16:6996-7007, 1997), teach that the N-terminal eight residues of the chemokine SDF-1 form an important receptor binding site.\",\n \"At this site, two residues (Lys-1 and Pro-2) were proposed to be directly involved in receptor activation.\",\n \"Disruption of these residues reportedly abolished activation.\",\n \"It has further been reported that SDF-1 includes a second receptor binding motif at residues 12-17 of the chemokine loop region, termed the “RFFESH motif”.\",\n \"Thus, it has been widely considered that the N-terminal region and so called N-loop following the first two cysteine residues of chemokines play the most important role in mediating the interactions between chemokines and their cognate receptors (Clark-Lewis et al., J. Biol.\",\n \"Chem.\",\n \"266:23128-23134, 1991; Crump et al., EMBO J.\",\n \"16:6996-7007, 1997; and Pakianathan et al., Biochemistry 36:9642-9648, 1997).\",\n \"In one of these studies, Clark-Lewis et al.\",\n \"reported that a C-terminally truncated form of IL-8 missing the α-helix and β-turn within this region manifested greatly reduced chemotactic activity.\",\n \"Additional confirmation of the primary significance of this segment of chemokines for receptor binding is provided by a recent observation that the biological activity of human MIP-1β is strongly reduced by substitutions of Arg-45, and Arg-47, with Serine (Czaplewski et al., J. Biol.\",\n \"Chem.\",\n \"274:16077-16084, 1999, incorporated herein by reference).\",\n \"Finally, a peptide corresponding to the MCP-1 sequence just preceding the C-terminal α-helix was reported to inhibit chemotaxis of THP-1 cells indicating the importance of this region for chemokine function (Reckless et al., Biochem.\",\n \"J.\",\n \"340:803-1121, 1999).\",\n \"Additional structure-function data for the CC chemokine RANTES have been reported by Pakianathan et al., ( Biochemistry 36:9642-9648, 1997).\",\n \"Notably, a number of the foregoing publications point to a complex, multi-determinant model of interactions between chemokines and their native receptors.\",\n \"In this context, Pakianathan et al.\",\n \"believe the data indicates that RANTES interacts with each of its receptors in a distinct and specific manner and supports a two-site model of interaction between chemokines and their receptors.\",\n \"Protein mapping studies of HIV gp120 envelope have reported identification of structural determinants of gp120 responsible for mediating HIV-coreceptor interactions.\",\n \"Among these studies, Rizzuto et al., Science 280:1949-1953, 1998, have described a conserved gp120 structure that is reportedly important for binding to CCR5, and have postulated generally that this structural determinant should facilitate development of pharmacologic or immunologic inhibitors of virus-receptor interactions.\",\n \"Rizzuto et al., suggested “that the CCR5-binding site is likely composed of conserved gp120 elements near or within the bridging sheet and V3 loop residues.” In another report addressing gp120 structure-function analysis relating to coreceptor binding, Verrier et al., ( AIDS Res.\",\n \"Hum.\",\n \"Retroviruses 15:731-743, 1999), studied the effect of linear V3 peptides (21-30 amino acids in length) on infectivity of different strains of HIV-1.These studies also pointed to the V3 loop as an important determinant of coreceptor choice, whereby single amino acid substitutions in V3 were reported to dramatically alter coreceptor usage.\",\n \"In conjunction with this disclosure, Verrier et al., reported that artificial, linear peptides of V3 could compete with intact gp120 for binding to CCR5 and CXCR4 and block HIV entry into cells.\",\n \"More specifically, the authors reported that the most efficient peptides for blocking fusion were derived from the middle of V3, and therefore did not include sequences at the C-terminal or N-terminal ends of V3 that form the base of the V3 loop.\",\n \"Also significantly, Verrier et al., (supra) pointed to a “pattern of restriction” between multiple gp120 binding determinants, whereby peptides from different HIV strains (m-tropic versus lymphotropic) discriminate in their fusion-blocking activity in a pattern that “follows the coreceptor usage of the parental envelopes from which the peptides were derived.” This indicates that the candidate peptides described by Verrier et al., (supra) would not exhibit multi-specific blocking potential against both m-tropic and lymphotropic HIV-coreceptor interactions.\",\n \"In a related study, Sakaida et al., ( J. Virol.\",\n \"72:9763-9670, 1998) reported that synthetic cyclized (but not linear) V3 peptides of CXCR4 and dual-tropic strains of HIV-1 (but not a CCR5 strain) can prevent binding of anti-CXCR4 antibodies, potentially by binding to the coreceptor and acting as a competitive inhibitor.\",\n \"These same peptides reportedly inhibited calcium mobilization by the chemokine SDF-1 in a T cell line.\",\n \"The reference proposes that V3 loop peptides can directly bind to the relevant chemokine receptor and determine coreceptor usage, and postulates that such peptides can serve as HIV-1 reagents.\",\n \"Like Verrier et al., (supra) Sakaida and coworkers conclude that the coreceptor binding activities of V3 peptides are strain-dependent.\",\n \"More detailed reports pertaining to the role of V3 in mediating HIV-coreceptor interactions focus on specific sites or residues within the V3 domain as reportedly critical or important residues for coreceptor binding.\",\n \"For example, Tugarinov et al., ( Structure Fold Des.\",\n \"8:385-395, 2000), report that a conserved, central loop sequence, GPG, within the V3 loop plays a primary role in maintaining the conformation of the loop to mediate coreceptor binding.\",\n \"Kato et al.\",\n \"( J. Virol.\",\n \"73:5520-5526, 1999) suggest that three specific residues, confined to the central, loop portion of V3 and located distant from the C-terminal segment, are particularly important for coreceptor usage.\",\n \"Xiao et al., ( Virology 240:83-92, 1998), identify a consensus motif S/GXXXGPGXXXXXXXE/D, covering the central portion of the V3 loop and excluding the C-terminal portion, as a critical motif for coreceptor binding.\",\n \"Wang et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 95:5740-5745, 1998), studied whether certain V3 residues conserved among HIV-1, HIV-2 and SIV determine the utilization of CCR5 as a coreceptor.\",\n \"They concluded that Arg-298 (at the beginning of V3 loop) has an importance for CCR5 utilization.\",\n \"The authors suggested that this residue may represent a highly conserved structural element and a useful target for developing anti-viral therapies.\",\n \"In another study by Wang et al., ( Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 96: 4558-4562, 1999), additional V3 residues reported to be critical for CCR5 utilization are identified by alanine scanning mutagenesis.\",\n \"One of the identified residues, A 328 , is located at the C-terminus of V3, and its substitution reportedly results in a 1,000-fold reduction of CCR5 binding activity.\",\n \"However, this residue is only one of two “highly conserved” residues and six “critical” residues identified for CCR5 utilization.\",\n \"Most of these residues are distinctly located at the C-terminal or central loop portion of V3, including R 298 , K 305 , I 307 , R 313 , and F 315 .\",\n \"Wang and colleagues propose that “these highly invariable residues” as well as others identified in a “bridging sheet” portion of the molecule may represent “targets for antiviral designs aimed at blocking the coreceptor entry step of HIV-1 replication.” Based on the foregoing articles, there is a broadly-confirmed disclosure of V3 as a critical domain for mediating HIV-coreceptor interactions.\",\n \"A large number of important or critical residues are indicated, with a substantial range of discrepancy between reports implicating different residues as being important, or critical, for coreceptor binding functions.\",\n \"These references that focus on V3 uniformly teach away from excluding important “critical” or “highly conserved” V3 residues.\",\n \"At the same time, these references focusing on the V3 domain uniformly fail to implicate other sequence elements that may be present in the gp120 molecule outside the V3 loop portion thereof.\",\n \"Coupled with these teachings, a host of related publications point to yet additional components of gp120 that may be essential for HIV-coreceptor interactions.\",\n \"Because these components appear to be complex and potentially interact to achieve coreceptor binding, their disclosure adds further complexity to understanding gp120 structure-function relationships for mediating coreceptor binding and cell entry.\",\n \"In particular, Tugarinov et al., ( Structure Fold Des.\",\n \"8:385-395, 2000), teach the importance of the GPG motif of V3, but also teach that “[h]igh affinity binding of gp120 to the chemokine receptors requires participation of other domains in gp120 such as the CD4i epitope.” Rizzuto et al., ( Science 280:1949-1953, 1998), suggest that the “CCR5-binding site is likely composed of conserved gp120 elements near or within the bridging sheet and V3 loop residues.” They further proposed that CD4 binding may distort the V1/V2 stem, repositioning the stem allowing the formation of the β-sheet important for CCR5 binding.\",\n \"They also noted that substitution of Asp for Thr 123 , located in the V1/V2 stem and which contact CD4, significantly decreased CCR5 binding.\",\n \"This report parallels others which point to a favored model of a “conformational binding site” in gp120 for mediating HIV-coreceptor interactions.\",\n \"According to this model, effective binding of coreceptors by gp120 involves initial binding of the gp120 to a CD4 receptor, which brings about a “CD4 induced” conformational change in gp120 involving distant residues—that in turn leads to formation of a conformational binding determinant on gp120 to mediate HIV-coreceptor interactions.\",\n \"In this context, Wu et al., ( Nature 384:179-183, 1996) suggested that HIV-1 attachment to CD4 creates a high-affinity binding site for CCR-5, leading to membrane fusion and virus entry.\",\n \"Consistent with this model, recent studies have identified yet additional sites of gp120 which are reportedly important, or essential, for mediating HIV-coreceptor interactions.\",\n \"These sites are generally distinct from V3, and also from the V3-flanking regions.\",\n \"For example, Cho et al., ( J. Virol.\",\n \"72:2509-2515, 1998), report that V1/V2 and V3 confer on HIV-1 the ability to use CXCR4 coreceptors, but that the V4 and V5 regions are also “required in conjunction with regions V1 and V3 of HIV-1DH 12 gp120 for efficient utilization of CXCR4.” Este et al., ( Mol.\",\n \"Pharmacol.\",\n \"52:98-104, 1997), point to structural determinants in each of the V2, V3 and C3 regions of gp120 for determining cell tropism and coreceptor utilization.\",\n \"Hoffman et al., ( Mol.\",\n \"Membr.\",\n \"Biol.\",\n \"16:57-65, 1999), concluded the V3 loop was implicated in regulating viral tropism, but, that other regions of Env, such as the V1- and V2-loops, modulated the effects of the V3-loop.\",\n \"They also acknowledged that some important exceptions to this model suggested that understanding of virus tropism and Env-chemokine receptor interactions was incomplete.\",\n \"Collectively, these studies point to numerous components of gp120 that may be important or essential for HIV-coreceptor interactions, and which may in fact involve multiple interactions whereby different parts of gp120 are required to work together to achieve efficient HIV-coreceptor interactions.\",\n \"This complexity and the proposed requirement for multiple, distant binding determinants on gp120 to form a conformational binding determinant for coreceptor usage, may be generally considered to teach away from the utility of small peptides for effectively blocking HIV-coreceptor binding interactions, particularly to block multiple HIV strains infecting different target cell types.\",\n \"Moreover, while the foregoing, separate bodies of literature individually discuss models of conserved binding elements of chemokines, or of gp120, as determinants for mediating coreceptor interactions, these reports do not teach or suggest identification of conserved binding determinants shared between gp120 and chemokines.\",\n \"On the contrary, complex functional interactions and distinct binding mechanisms and determinants previously proposed for gp120 and chemokines may be considered to lead away from investigations aimed at identifying common binding determinants or mechanisms between gp120 and chemokines.\",\n \"This conclusion is well supported in the literature reviewed herein above.\",\n \"Based on these and other teachings, there does not appear to be a clear suggestion in the literature to use a common blocking agent for coreceptor interactions by chemokines and HIV aimed at, or patterned after, a shared binding determinant between these two distinct molecules.\",\n \"The literature does not point toward any such common structural domain or binding determinant between HIV gp120 and chemokines, and the distinct structure-function reports for how HIV and chemokines are thought to interact with the subject receptors leads away from this path of inquiry.\",\n \"In view of the foregoing teachings and unsolved questions in the literature, there remains an urgent need in the art for additional tools and methods to combat HIV infection and related disease.\",\n \"Related to this fundamental goal, there remains a clear need for additional therapeutic agents and methods targeting HIV viral entry into host cells, preferably that will include compositions and methods to block gp120 binding to different coreceptors to inhibit viral entry and infection and ameliorate HIV-related disease.\",\n \"Surprisingly, the instant invention fulfills these objects and satisfies additional objects and advantages which will become apparent from the following description.\"\n ],\n [\n \"<SOH> SUMMARY OF THE INVENTION <EOH>The instant invention provides novel methods and compositions for inhibiting interactions between human immunodeficiency viruses (HIVs) and chemokine receptors, termed “viral coreceptors” in this context.\",\n \"The methods of the invention generally comprise exposing a CXCR4 or CCR5 coreceptor of a subject to an effective amount of an anti-coreceptor binding agent of the invention to inhibit binding of the coreceptor by an HIV virus or viral protein.\",\n \"Typically, the anti-coreceptor binding agent is a gp120 peptide, peptide analog or mimetic that specifically binds the coreceptor.\",\n \"Within certain methods of the invention, the subject is an isolated or bound coreceptor, a membrane or cell preparation comprising the coreceptor, a cell population, tissue or organ expressing the coreceptor, or a mammalian patient.\",\n \"In more detailed embodiments, the subject comprises a cell population, tissue or organ selected for in vivo or ex vivo treatment or diagnostic processing.\",\n \"Alternatively, the subject may be a mammalian patient susceptible to HIV infection and the anti-coreceptor binding agent is administered in a prophylactic or therapeutically effective dose to prevent or inhibit HIV binding to a susceptible cell and thereby preventing or inhibiting infection or a related disease condition or symptom.\",\n \"In typical aspects of the invention, the anti-coreceptor binding agent is administered to the subject in an amount effective to inhibit one or more biological activities mediated by or associated with HIV-coreceptor interactions selected from (a) direct co-receptor, e.g., CXCR4 and/or CCR5, binding by HIV virus, (b) coreceptor binding by a HIV gp120 protein or a peptide fragment or derivative thereof, (c) HIV fusion with target host cells, (d) HIV virion entry into host cells, (e) HIV replication, and/or (f) HIV cell-cell or host-host transmission.\",\n \"In more specific embodiments, the anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic and is administered to the subject in an amount effective to inhibit one or more biological activities selected from (a) direct co-receptor, i.e., CXCR4 and/or CCR5, binding by HIV-1 virus, (b) coreceptor binding by a HIV-1 gp120 protein or a peptide fragment or derivative thereof, (c) HIV-1 fusion with target host cells, (d) HIV-1 virion entry into host cells, (e) HIV-1 replication, and/or (f) HIV-1 cell-cell or host-host transmission.\",\n \"Often, the anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic administered to a mammalian patient in a prophylactically or therapeutically effective dose to prevent or inhibit HIV-1 binding infection, to a susceptible host, HIV-1 or an HIV-1-related disease condition or symptom.\",\n \"In more detailed methods and compositions of the invention, the gp120 peptide, peptide analog or mimetic is between about 12 and about 24 amino acid residues in length and comprises a conserved CXXXXXXW amino acid sequence motif identified within the amino acid sequence of gp120 proteins of HIV isolates and also among diverse chemokines, wherein X is any naturally occurring or synthetic amino acid or amino acid analog.\",\n \"The peptide, peptide analog or mimetic can be modified in a wide variety of ways and forms, e.g., by addition, admixture, or conjugation of additional amino acids, peptides, proteins, chemical reagents or moieties which do not substantially alter the anti-coreceptor binding activity of the peptide.\",\n \"Often, the anti-coreceptor binding agent of the invention is a peptide comprising an allelic variant that is found among native HIV gp120 peptide sequences.\",\n \"Within more detailed embodiments, the anti-coreceptor binding agent is a peptide of between about 12-17 amino acids in length that includes a conserved “CXXXXXXW” amino acid sequence motif, which is selected from an exemplary “reference” peptide designated 15K comprising an amino acid sequence IRKAHCNISRAKWND (SEQ ID NO:8), or is alternatively represented by a corresponding or overlapping native peptide sequence or peptide analog that shares substantial sequence identity to the reference amino acid sequence of 15K.\",\n \"In various specific embodiments, the peptide includes one or more residues occurring naturally or by substitution at a relative, aligned position corresponding to a designated position for peptide 15K, selected from: Position 1—I, M, K, S, T, L, A, V, R, P, or N; Position 2—R, G, E, K, S, T, or I; Position 3—Q, K, R, L, E, P, A, V, S, T, H, or D; Position 4—A, T, P, V, E, or S; Position 5—H, Y, F, Q. N, I, or V; Position 7—N, D, H, T, K, E, S, I, Q, V, G, or A; Position 8—I, L, V, Y, D, A; Position 9—S, N, D, T, K, Y, I, or P; Position 10—R, K, G, S, A, E, D, I, T, W, or N; Position 11—A, R, K, T, S, G, E, D, N, Q, H, V, I, or L; Position 12—K, D, R, E, K, Q, N, T, S, G, A, V, L; Position 14—N, Q, D, E, K, R, A, S, T, G, M, Y, I, H, or V; and/or Position 15—D, N, K, E, T, Q, R, S, A, I, M, or P. In yet additional embodiments of the invention, the anti-coreceptor binding agent exhibits multi-tropic activity characterized by effective inhibition of HIV viral, or gp120 protein or peptide binding to multiple, CXCR4 and CCR5, coreceptors.\",\n \"Often, the multi-tropic anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic administered to the subject in an amount effective to inhibit one or more biological activities of both T cell tropic (lymphotropic) and macrophage tropic (m-tropic) HIV-1 viruses selected from (a) direct co-receptor binding by viruses, (b) coreceptor binding by viral gp120 proteins or peptide fragments or derivatives thereof, (c) viral fusion with target host cells, (d) virion entry into host cells, (e) viral replication, and/or (f) viral cell-cell or host-host transmission.\",\n \"Within the methods and compositions of the invention, the anti-coreceptor binding agent may be formulated in various combinations with a pharmaceutically acceptable carrier, diluent, excipient, adjuvant or other active or inactive agents, in an amount or dosage form sufficient to prevent, reduce or even alleviate HIV infection or related disease conditions or symptoms.\",\n \"In yet additional aspects of the invention, the anti-coreceptor binding agent of the invention is administered according to the foregoing methods in a combinatorial formulation or coordinate treatment with one or more additional anti-HIV, antibacterial, antiviral or other therapeutically active agent(s).\",\n \"Within related methods and compositions, the anti-coreceptor binding agent is admixed or co-administered, simultaneously or sequentially with one or more additional anti-HIV, antibacterial, antiviral or other therapeutically active agent(s) to prevent, reduce or even alleviate HIV infection or related conditions in a mammalian patient.\",\n \"The instant invention also includes kits, packages and multicontainer units containing the anti-coreceptor binding agent, optionally with other active or inactive ingredients, and/or means for administering the same for use in the diagnosis, management and/or prevention and treatment of HIV and related conditions.\",\n \"Typically, these kits include a diagnostic or pharmaceutical preparation of the anti-coreceptor binding agent, typically formulated with a biologically suitable carrier and optionally contained in a bulk dispensing container or unit or multi-unit dosage form.\",\n \"Optional dispensing means can be provided, for example an intranasal spray applicator.\",\n \"Packaging materials optionally include a label or instruction which indicates a desired use of the kit as described herein below.\",\n \"Additional aspects of the invention include polynucleotide molecules and vector constructs encoding anti-coreceptor binding peptides and peptide analogs.\",\n \"Also provided are peptide vaccines and other immunogenic compositions that elicit an immune response involving production of antibodies targeting one or more epitopes of gp120 recognized by antibodies that specifically bind an anti-coreceptor binding peptide of the invention.\",\n \"In addition, the invention provides, antibodies, including monoclonal antibodies, and immunotherapeutic methods and compositions comprising such antibodies that specifically recognize anti-coreceptor binding peptides of the invention, for use as diagnostic and therapeutic reagents.\",\n \"Also provided within the invention are a variety of additional diagnostic and therapeutic tools and reagents as set forth in detail in the following description.\"\n ],\n [\n \"RELATED APPLICATION This application claims priority to provisional application Ser.\",\n \"No.\",\n \"60/269,534 filed on Feb. 15, 2001.BACKGROUND OF THE INVENTION The envelope glycoprotein of the human immunodeficiency virus type I (HIV-1) mediates in the fusion of viral and host cell membranes necessary for virion entry (Freed et al., J. Biol.\",\n \"Chem.\",\n \"270:23883-23886, 1995).\",\n \"The envelope glycoprotein of HIV-1 is produced by the enzymatic cleavage from a gp160 precursor protein to produce the external gp120 protein and the transmembrane gp41 protein (Capon et al., Annu.\",\n \"Rev.\",\n \"Immunol.\",\n \"9:649-678, 1991).\",\n \"Several studies have identified specific portions or domains of the gp120 protein that may elicit humoral and/or cell-mediated immune responses to HIV in susceptible host subjects, and may therefore be useful to formulate anti-HIV reagents and methods for prevention and treatment of HIV infection and related diseases.\",\n \"These general HIV peptide studies describe a large, diverse assemblage of gp120 peptides that are proposed as candidates for therapeutic use, primarily in vaccine formulations to prevent and treat HIV infection and related disease.\",\n \"For example, U.S. Pat.\",\n \"No.\",\n \"5,691,135 describes various peptides that are selected for the ability to inhibit HIV infection by stimulating VH3 and VH4 antibody responses.\",\n \"The peptides are proposed for administration to a patient as an antigen in sufficient quantity to induce antibodies that exhibit superantigen binding to gp120.The disclosure considers 31 peptides obtained from the AIDS Research and Reference Program, NIH, which peptides correspond to sequences from gp120 and gp41 from different strains of HIV.\",\n \"Additional gp120 peptides are described in U.S. Pat.\",\n \"No.\",\n \"5,939,074.In particular, this references describes peptides that are useful as “multideterminant peptide antigens” for eliciting both cell-mediated and humoral immune responses against HIV.\",\n \"Additional studies have identified particular regions of the gp120 that are proposed to determine specific interactions between gp120 and CD4, which is the primary receptor on target cells mediating cellular entry by HIV-1 (Arthos et al., Cell 57:469481, 1989; Clayton et al., Nature 339:548-551, 1989; Kwong et al., Nature 393:648-659, 1998; and Landau et al., Nature 334:159-162, 1988).\",\n \"In addition to CD4 binding, cellular entry by HIV-1 is thought to require additional interactions between the virus and one or more “coreceptors” on the surfaces of target cells.\",\n \"Briefly, the HIV-1 particles are proposed to bind initially to the CD4 receptor, and then subsequently to a chemokine receptor present on the target cells, which is used by the virus as a “coreceptor” for mediating cellular entry.\",\n \"Different strains of HIV-1 preferentially utilize different chemokine receptors that are variably expressed among HIV-1 target cells.\",\n \"In particular, some strains of HIV-1, referred to as “lymphotropic” strains, bind CXCR4 chemokine receptors predominantly expressed on lymphocyte target cells, while other HIV-1 strains, termed “monocyte tropic” strains, bind CCR5 receptors predominantly expressed on cells of monocyte lineage.\",\n \"Thus, the major viral coreceptors are CXCR4 (Berson et al., J. Virol.\",\n \"70:6288-6295, 1996), which has a native function as a chemokine receptor for stromal derived factor-1 (SDF-1), and CCR5 (Alkhatib et al., Science 272:1955-1958, 1996; Deng et al., Nature 381:661-666, 1996; and Dragic et al., Nature 381:667-673, 1996), which functions naturally as a receptor for several chemokines, including macrophage inflammatory protein-1β (MIP-1β).\",\n \"It is proposed that a conformational change occurs in gp120 following HIV binding of CD4, and that this conformation change exposes one or more binding sites on the gp120 molecule that mediate additional interactions between HIV and chemokine receptor, which are referred to in this context as “viral coreceptors” (Kwong et al., Nature 393:648-659, 1998, incorporated herein by reference).\",\n \"Recent studies suggest a complicated, multi-step binding and activation model, wherein the association of gp120 with CD4 yields a CD4-gp120 complex, which subsequently associates with the viral coreceptor resulting in a structural rearrangement (e.g., conformational change) of gp120 that facilitates interaction of the gp41 envelope protein subunit with the host cell membrane, leading to viral entry (Helseth et al., J. Virol.\",\n \"64:6314-6318, 1990; and Weissenhom et al., Nature 387:426-430, 1997).\",\n \"Within this emergent field of investigation, numerous studies have focused on structure-function mapping of chemokine receptors, with the goals of determining binding determinants and mechanisms of action of the receptors.\",\n \"Yet additional research has focused on chemokine mapping studies.\",\n \"From these studies chemokine-derived peptides have been identified that reportedly comprise binding determinants of the chemokines capable of blocking native chemokine receptor-ligand interactions.\",\n \"Further studies, focusing alternatively on HIV gp120 mapping, report production and testing of gp120 peptides capable of blocking HIV-coreceptor binding and HIV infectivity.\",\n \"With regard to chemokine receptor studies, numerous articles report that chemokine receptors play a direct role as coreceptors for HIV cell entry.\",\n \"Briefly, as noted above, it is reported that specific interactions between the HIV envelope glycoprotein gp120 and one or more chemokine receptors mediate viral entry into target cells.\",\n \"More specifically, monocyte-tropic or “m-tropic” HIV strains bind to a distinct chemokine receptor, CCR5, for cell entry, while T cell lymphotropic or “T-tropic” virus use mainly CXCR4 receptors for cell entry.\",\n \"More detailed, structure-function analyses have been directed toward identifying specific HIV binding determinants of chemokine receptors mediating their activity as coreceptors for HIV entry.\",\n \"In conjunction with these reports, certain references focus on a concept of blocking chemokine receptor activity (e.g., receptor binding with chemokines, or with gp120) using competitive inhibitors.\",\n \"Proposed inhibitors of chemokine receptor binding interactions include peptide inhibitors that mimic structures of chemokines, or of gp120, binding determinants or related structural domains.\",\n \"These studies follow more basic research which shows that intact chemokines, the normal ligands of chemokine receptors, can compete with cognate chemokines, or with HIV, for binding to the target receptors.\",\n \"Referring specifically to chemokine mapping studies focusing on chemokine structure-function, various publications attempt to identify and characterize receptor binding determinants of chemokines.\",\n \"As noted above, certain of these references also describe chemokine-based peptides reportedly capable of blocking chemokine-receptor binding and other activities mediated by receptor-ligand (chemokine or HIV) interactions.\",\n \"One such study is presented in a publication by Reckless et al., (Biochem.\",\n \"J.\",\n \"340:803-811, 1999).\",\n \"This study identifies a number of chemokine-derived peptides, including a peptide designated “peptide 3”, based on a human chemokine, monocyte chemotactic protein-1 (MCP-1).\",\n \"Reckless and colleagues report that the peptide 3 inhibits cell migration induced by a wide range of chemokines.\",\n \"Moreover, peptide 3 reportedly binds to THP-1 cells and inhibits THP-1 migration, reportedly by acting as a chemokine receptor antagonist.\",\n \"On this basis, the authors propose that peptide 3 and its derivatives, including peptides ranging from 6-15 residues in length, may be useful as chemokine inhibitors.\",\n \"A number of related reports focus on a distinct portion of chemokines as prospective receptor binding determinants.\",\n \"In particular, the reports focus on the N-terminal region of chemokines comprising a structural element called the “N-loop”.\",\n \"This distinct element follows the first two cysteine residues of a model chemokine, and is proposed to play an important role in chemokine-receptor interactions.\",\n \"Other parts of chemokine molecules have also been proposed to contribute significantly as structural determinants of interactions between chemokines and their cognate receptors.\",\n \"For example, Crump et al., (EMBO J.\",\n \"16:6996-7007, 1997), teach that the N-terminal eight residues of the chemokine SDF-1 form an important receptor binding site.\",\n \"At this site, two residues (Lys-1 and Pro-2) were proposed to be directly involved in receptor activation.\",\n \"Disruption of these residues reportedly abolished activation.\",\n \"It has further been reported that SDF-1 includes a second receptor binding motif at residues 12-17 of the chemokine loop region, termed the “RFFESH motif”.\",\n \"Thus, it has been widely considered that the N-terminal region and so called N-loop following the first two cysteine residues of chemokines play the most important role in mediating the interactions between chemokines and their cognate receptors (Clark-Lewis et al., J. Biol.\",\n \"Chem.\",\n \"266:23128-23134, 1991; Crump et al., EMBO J.\",\n \"16:6996-7007, 1997; and Pakianathan et al., Biochemistry 36:9642-9648, 1997).\",\n \"In one of these studies, Clark-Lewis et al.\",\n \"reported that a C-terminally truncated form of IL-8 missing the α-helix and β-turn within this region manifested greatly reduced chemotactic activity.\",\n \"Additional confirmation of the primary significance of this segment of chemokines for receptor binding is provided by a recent observation that the biological activity of human MIP-1β is strongly reduced by substitutions of Arg-45, and Arg-47, with Serine (Czaplewski et al., J. Biol.\",\n \"Chem.\",\n \"274:16077-16084, 1999, incorporated herein by reference).\",\n \"Finally, a peptide corresponding to the MCP-1 sequence just preceding the C-terminal α-helix was reported to inhibit chemotaxis of THP-1 cells indicating the importance of this region for chemokine function (Reckless et al., Biochem.\",\n \"J.\",\n \"340:803-1121, 1999).\",\n \"Additional structure-function data for the CC chemokine RANTES have been reported by Pakianathan et al., (Biochemistry 36:9642-9648, 1997).\",\n \"Notably, a number of the foregoing publications point to a complex, multi-determinant model of interactions between chemokines and their native receptors.\",\n \"In this context, Pakianathan et al.\",\n \"believe the data indicates that RANTES interacts with each of its receptors in a distinct and specific manner and supports a two-site model of interaction between chemokines and their receptors.\",\n \"Protein mapping studies of HIV gp120 envelope have reported identification of structural determinants of gp120 responsible for mediating HIV-coreceptor interactions.\",\n \"Among these studies, Rizzuto et al., Science 280:1949-1953, 1998, have described a conserved gp120 structure that is reportedly important for binding to CCR5, and have postulated generally that this structural determinant should facilitate development of pharmacologic or immunologic inhibitors of virus-receptor interactions.\",\n \"Rizzuto et al., suggested “that the CCR5-binding site is likely composed of conserved gp120 elements near or within the bridging sheet and V3 loop residues.” In another report addressing gp120 structure-function analysis relating to coreceptor binding, Verrier et al., (AIDS Res.\",\n \"Hum.\",\n \"Retroviruses 15:731-743, 1999), studied the effect of linear V3 peptides (21-30 amino acids in length) on infectivity of different strains of HIV-1.These studies also pointed to the V3 loop as an important determinant of coreceptor choice, whereby single amino acid substitutions in V3 were reported to dramatically alter coreceptor usage.\",\n \"In conjunction with this disclosure, Verrier et al., reported that artificial, linear peptides of V3 could compete with intact gp120 for binding to CCR5 and CXCR4 and block HIV entry into cells.\",\n \"More specifically, the authors reported that the most efficient peptides for blocking fusion were derived from the middle of V3, and therefore did not include sequences at the C-terminal or N-terminal ends of V3 that form the base of the V3 loop.\",\n \"Also significantly, Verrier et al., (supra) pointed to a “pattern of restriction” between multiple gp120 binding determinants, whereby peptides from different HIV strains (m-tropic versus lymphotropic) discriminate in their fusion-blocking activity in a pattern that “follows the coreceptor usage of the parental envelopes from which the peptides were derived.” This indicates that the candidate peptides described by Verrier et al., (supra) would not exhibit multi-specific blocking potential against both m-tropic and lymphotropic HIV-coreceptor interactions.\",\n \"In a related study, Sakaida et al., (J. Virol.\",\n \"72:9763-9670, 1998) reported that synthetic cyclized (but not linear) V3 peptides of CXCR4 and dual-tropic strains of HIV-1 (but not a CCR5 strain) can prevent binding of anti-CXCR4 antibodies, potentially by binding to the coreceptor and acting as a competitive inhibitor.\",\n \"These same peptides reportedly inhibited calcium mobilization by the chemokine SDF-1 in a T cell line.\",\n \"The reference proposes that V3 loop peptides can directly bind to the relevant chemokine receptor and determine coreceptor usage, and postulates that such peptides can serve as HIV-1 reagents.\",\n \"Like Verrier et al., (supra) Sakaida and coworkers conclude that the coreceptor binding activities of V3 peptides are strain-dependent.\",\n \"More detailed reports pertaining to the role of V3 in mediating HIV-coreceptor interactions focus on specific sites or residues within the V3 domain as reportedly critical or important residues for coreceptor binding.\",\n \"For example, Tugarinov et al., (Structure Fold Des.\",\n \"8:385-395, 2000), report that a conserved, central loop sequence, GPG, within the V3 loop plays a primary role in maintaining the conformation of the loop to mediate coreceptor binding.\",\n \"Kato et al.\",\n \"(J. Virol.\",\n \"73:5520-5526, 1999) suggest that three specific residues, confined to the central, loop portion of V3 and located distant from the C-terminal segment, are particularly important for coreceptor usage.\",\n \"Xiao et al., (Virology 240:83-92, 1998), identify a consensus motif S/GXXXGPGXXXXXXXE/D, covering the central portion of the V3 loop and excluding the C-terminal portion, as a critical motif for coreceptor binding.\",\n \"Wang et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 95:5740-5745, 1998), studied whether certain V3 residues conserved among HIV-1, HIV-2 and SIV determine the utilization of CCR5 as a coreceptor.\",\n \"They concluded that Arg-298 (at the beginning of V3 loop) has an importance for CCR5 utilization.\",\n \"The authors suggested that this residue may represent a highly conserved structural element and a useful target for developing anti-viral therapies.\",\n \"In another study by Wang et al., (Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 96: 4558-4562, 1999), additional V3 residues reported to be critical for CCR5 utilization are identified by alanine scanning mutagenesis.\",\n \"One of the identified residues, A328, is located at the C-terminus of V3, and its substitution reportedly results in a 1,000-fold reduction of CCR5 binding activity.\",\n \"However, this residue is only one of two “highly conserved” residues and six “critical” residues identified for CCR5 utilization.\",\n \"Most of these residues are distinctly located at the C-terminal or central loop portion of V3, including R298, K305, I307, R313, and F315.Wang and colleagues propose that “these highly invariable residues” as well as others identified in a “bridging sheet” portion of the molecule may represent “targets for antiviral designs aimed at blocking the coreceptor entry step of HIV-1 replication.” Based on the foregoing articles, there is a broadly-confirmed disclosure of V3 as a critical domain for mediating HIV-coreceptor interactions.\",\n \"A large number of important or critical residues are indicated, with a substantial range of discrepancy between reports implicating different residues as being important, or critical, for coreceptor binding functions.\",\n \"These references that focus on V3 uniformly teach away from excluding important “critical” or “highly conserved” V3 residues.\",\n \"At the same time, these references focusing on the V3 domain uniformly fail to implicate other sequence elements that may be present in the gp120 molecule outside the V3 loop portion thereof.\",\n \"Coupled with these teachings, a host of related publications point to yet additional components of gp120 that may be essential for HIV-coreceptor interactions.\",\n \"Because these components appear to be complex and potentially interact to achieve coreceptor binding, their disclosure adds further complexity to understanding gp120 structure-function relationships for mediating coreceptor binding and cell entry.\",\n \"In particular, Tugarinov et al., (Structure Fold Des.\",\n \"8:385-395, 2000), teach the importance of the GPG motif of V3, but also teach that “[h]igh affinity binding of gp120 to the chemokine receptors requires participation of other domains in gp120 such as the CD4i epitope.” Rizzuto et al., (Science 280:1949-1953, 1998), suggest that the “CCR5-binding site is likely composed of conserved gp120 elements near or within the bridging sheet and V3 loop residues.” They further proposed that CD4 binding may distort the V1/V2 stem, repositioning the stem allowing the formation of the β-sheet important for CCR5 binding.\",\n \"They also noted that substitution of Asp for Thr123, located in the V1/V2 stem and which contact CD4, significantly decreased CCR5 binding.\",\n \"This report parallels others which point to a favored model of a “conformational binding site” in gp120 for mediating HIV-coreceptor interactions.\",\n \"According to this model, effective binding of coreceptors by gp120 involves initial binding of the gp120 to a CD4 receptor, which brings about a “CD4 induced” conformational change in gp120 involving distant residues—that in turn leads to formation of a conformational binding determinant on gp120 to mediate HIV-coreceptor interactions.\",\n \"In this context, Wu et al., (Nature 384:179-183, 1996) suggested that HIV-1 attachment to CD4 creates a high-affinity binding site for CCR-5, leading to membrane fusion and virus entry.\",\n \"Consistent with this model, recent studies have identified yet additional sites of gp120 which are reportedly important, or essential, for mediating HIV-coreceptor interactions.\",\n \"These sites are generally distinct from V3, and also from the V3-flanking regions.\",\n \"For example, Cho et al., (J. Virol.\",\n \"72:2509-2515, 1998), report that V1/V2 and V3 confer on HIV-1 the ability to use CXCR4 coreceptors, but that the V4 and V5 regions are also “required in conjunction with regions V1 and V3 of HIV-1DH12 gp120 for efficient utilization of CXCR4.” Este et al., (Mol.\",\n \"Pharmacol.\",\n \"52:98-104, 1997), point to structural determinants in each of the V2, V3 and C3 regions of gp120 for determining cell tropism and coreceptor utilization.\",\n \"Hoffman et al., (Mol.\",\n \"Membr.\",\n \"Biol.\",\n \"16:57-65, 1999), concluded the V3 loop was implicated in regulating viral tropism, but, that other regions of Env, such as the V1- and V2-loops, modulated the effects of the V3-loop.\",\n \"They also acknowledged that some important exceptions to this model suggested that understanding of virus tropism and Env-chemokine receptor interactions was incomplete.\",\n \"Collectively, these studies point to numerous components of gp120 that may be important or essential for HIV-coreceptor interactions, and which may in fact involve multiple interactions whereby different parts of gp120 are required to work together to achieve efficient HIV-coreceptor interactions.\",\n \"This complexity and the proposed requirement for multiple, distant binding determinants on gp120 to form a conformational binding determinant for coreceptor usage, may be generally considered to teach away from the utility of small peptides for effectively blocking HIV-coreceptor binding interactions, particularly to block multiple HIV strains infecting different target cell types.\",\n \"Moreover, while the foregoing, separate bodies of literature individually discuss models of conserved binding elements of chemokines, or of gp120, as determinants for mediating coreceptor interactions, these reports do not teach or suggest identification of conserved binding determinants shared between gp120 and chemokines.\",\n \"On the contrary, complex functional interactions and distinct binding mechanisms and determinants previously proposed for gp120 and chemokines may be considered to lead away from investigations aimed at identifying common binding determinants or mechanisms between gp120 and chemokines.\",\n \"This conclusion is well supported in the literature reviewed herein above.\",\n \"Based on these and other teachings, there does not appear to be a clear suggestion in the literature to use a common blocking agent for coreceptor interactions by chemokines and HIV aimed at, or patterned after, a shared binding determinant between these two distinct molecules.\",\n \"The literature does not point toward any such common structural domain or binding determinant between HIV gp120 and chemokines, and the distinct structure-function reports for how HIV and chemokines are thought to interact with the subject receptors leads away from this path of inquiry.\",\n \"In view of the foregoing teachings and unsolved questions in the literature, there remains an urgent need in the art for additional tools and methods to combat HIV infection and related disease.\",\n \"Related to this fundamental goal, there remains a clear need for additional therapeutic agents and methods targeting HIV viral entry into host cells, preferably that will include compositions and methods to block gp120 binding to different coreceptors to inhibit viral entry and infection and ameliorate HIV-related disease.\",\n \"Surprisingly, the instant invention fulfills these objects and satisfies additional objects and advantages which will become apparent from the following description.\",\n \"SUMMARY OF THE INVENTION The instant invention provides novel methods and compositions for inhibiting interactions between human immunodeficiency viruses (HIVs) and chemokine receptors, termed “viral coreceptors” in this context.\",\n \"The methods of the invention generally comprise exposing a CXCR4 or CCR5 coreceptor of a subject to an effective amount of an anti-coreceptor binding agent of the invention to inhibit binding of the coreceptor by an HIV virus or viral protein.\",\n \"Typically, the anti-coreceptor binding agent is a gp120 peptide, peptide analog or mimetic that specifically binds the coreceptor.\",\n \"Within certain methods of the invention, the subject is an isolated or bound coreceptor, a membrane or cell preparation comprising the coreceptor, a cell population, tissue or organ expressing the coreceptor, or a mammalian patient.\",\n \"In more detailed embodiments, the subject comprises a cell population, tissue or organ selected for in vivo or ex vivo treatment or diagnostic processing.\",\n \"Alternatively, the subject may be a mammalian patient susceptible to HIV infection and the anti-coreceptor binding agent is administered in a prophylactic or therapeutically effective dose to prevent or inhibit HIV binding to a susceptible cell and thereby preventing or inhibiting infection or a related disease condition or symptom.\",\n \"In typical aspects of the invention, the anti-coreceptor binding agent is administered to the subject in an amount effective to inhibit one or more biological activities mediated by or associated with HIV-coreceptor interactions selected from (a) direct co-receptor, e.g., CXCR4 and/or CCR5, binding by HIV virus, (b) coreceptor binding by a HIV gp120 protein or a peptide fragment or derivative thereof, (c) HIV fusion with target host cells, (d) HIV virion entry into host cells, (e) HIV replication, and/or (f) HIV cell-cell or host-host transmission.\",\n \"In more specific embodiments, the anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic and is administered to the subject in an amount effective to inhibit one or more biological activities selected from (a) direct co-receptor, i.e., CXCR4 and/or CCR5, binding by HIV-1 virus, (b) coreceptor binding by a HIV-1 gp120 protein or a peptide fragment or derivative thereof, (c) HIV-1 fusion with target host cells, (d) HIV-1 virion entry into host cells, (e) HIV-1 replication, and/or (f) HIV-1 cell-cell or host-host transmission.\",\n \"Often, the anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic administered to a mammalian patient in a prophylactically or therapeutically effective dose to prevent or inhibit HIV-1 binding infection, to a susceptible host, HIV-1 or an HIV-1-related disease condition or symptom.\",\n \"In more detailed methods and compositions of the invention, the gp120 peptide, peptide analog or mimetic is between about 12 and about 24 amino acid residues in length and comprises a conserved CXXXXXXW amino acid sequence motif identified within the amino acid sequence of gp120 proteins of HIV isolates and also among diverse chemokines, wherein X is any naturally occurring or synthetic amino acid or amino acid analog.\",\n \"The peptide, peptide analog or mimetic can be modified in a wide variety of ways and forms, e.g., by addition, admixture, or conjugation of additional amino acids, peptides, proteins, chemical reagents or moieties which do not substantially alter the anti-coreceptor binding activity of the peptide.\",\n \"Often, the anti-coreceptor binding agent of the invention is a peptide comprising an allelic variant that is found among native HIV gp120 peptide sequences.\",\n \"Within more detailed embodiments, the anti-coreceptor binding agent is a peptide of between about 12-17 amino acids in length that includes a conserved “CXXXXXXW” amino acid sequence motif, which is selected from an exemplary “reference” peptide designated 15K comprising an amino acid sequence IRKAHCNISRAKWND (SEQ ID NO:8), or is alternatively represented by a corresponding or overlapping native peptide sequence or peptide analog that shares substantial sequence identity to the reference amino acid sequence of 15K.\",\n \"In various specific embodiments, the peptide includes one or more residues occurring naturally or by substitution at a relative, aligned position corresponding to a designated position for peptide 15K, selected from: Position 1—I, M, K, S, T, L, A, V, R, P, or N; Position 2—R, G, E, K, S, T, or I; Position 3—Q, K, R, L, E, P, A, V, S, T, H, or D; Position 4—A, T, P, V, E, or S; Position 5—H, Y, F, Q. N, I, or V; Position 7—N, D, H, T, K, E, S, I, Q, V, G, or A; Position 8—I, L, V, Y, D, A; Position 9—S, N, D, T, K, Y, I, or P; Position 10—R, K, G, S, A, E, D, I, T, W, or N; Position 11—A, R, K, T, S, G, E, D, N, Q, H, V, I, or L; Position 12—K, D, R, E, K, Q, N, T, S, G, A, V, L; Position 14—N, Q, D, E, K, R, A, S, T, G, M, Y, I, H, or V; and/or Position 15—D, N, K, E, T, Q, R, S, A, I, M, or P. In yet additional embodiments of the invention, the anti-coreceptor binding agent exhibits multi-tropic activity characterized by effective inhibition of HIV viral, or gp120 protein or peptide binding to multiple, CXCR4 and CCR5, coreceptors.\",\n \"Often, the multi-tropic anti-coreceptor binding agent is an HIV-1 peptide, peptide analog or mimetic administered to the subject in an amount effective to inhibit one or more biological activities of both T cell tropic (lymphotropic) and macrophage tropic (m-tropic) HIV-1 viruses selected from (a) direct co-receptor binding by viruses, (b) coreceptor binding by viral gp120 proteins or peptide fragments or derivatives thereof, (c) viral fusion with target host cells, (d) virion entry into host cells, (e) viral replication, and/or (f) viral cell-cell or host-host transmission.\",\n \"Within the methods and compositions of the invention, the anti-coreceptor binding agent may be formulated in various combinations with a pharmaceutically acceptable carrier, diluent, excipient, adjuvant or other active or inactive agents, in an amount or dosage form sufficient to prevent, reduce or even alleviate HIV infection or related disease conditions or symptoms.\",\n \"In yet additional aspects of the invention, the anti-coreceptor binding agent of the invention is administered according to the foregoing methods in a combinatorial formulation or coordinate treatment with one or more additional anti-HIV, antibacterial, antiviral or other therapeutically active agent(s).\",\n \"Within related methods and compositions, the anti-coreceptor binding agent is admixed or co-administered, simultaneously or sequentially with one or more additional anti-HIV, antibacterial, antiviral or other therapeutically active agent(s) to prevent, reduce or even alleviate HIV infection or related conditions in a mammalian patient.\",\n \"The instant invention also includes kits, packages and multicontainer units containing the anti-coreceptor binding agent, optionally with other active or inactive ingredients, and/or means for administering the same for use in the diagnosis, management and/or prevention and treatment of HIV and related conditions.\",\n \"Typically, these kits include a diagnostic or pharmaceutical preparation of the anti-coreceptor binding agent, typically formulated with a biologically suitable carrier and optionally contained in a bulk dispensing container or unit or multi-unit dosage form.\",\n \"Optional dispensing means can be provided, for example an intranasal spray applicator.\",\n \"Packaging materials optionally include a label or instruction which indicates a desired use of the kit as described herein below.\",\n \"Additional aspects of the invention include polynucleotide molecules and vector constructs encoding anti-coreceptor binding peptides and peptide analogs.\",\n \"Also provided are peptide vaccines and other immunogenic compositions that elicit an immune response involving production of antibodies targeting one or more epitopes of gp120 recognized by antibodies that specifically bind an anti-coreceptor binding peptide of the invention.\",\n \"In addition, the invention provides, antibodies, including monoclonal antibodies, and immunotherapeutic methods and compositions comprising such antibodies that specifically recognize anti-coreceptor binding peptides of the invention, for use as diagnostic and therapeutic reagents.\",\n \"Also provided within the invention are a variety of additional diagnostic and therapeutic tools and reagents as set forth in detail in the following description.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS FIG.\",\n \"1 demonstrates inhibition of specific chemokine binding to CXCR4 and CCRS expressing cells by exemplary HIV gp120-derived anti-coreceptor binding peptides 15K and 15D.\",\n \"FIG.\",\n \"2 documents inhibition of the chemotactic response of HEK-CCR5 cells to the chemokine RANTES by the exemplary anti-coreceptor binding peptides 15K and 15D.\",\n \"Migration of HEK-CCR5 cells to RANTES alone, or the combination of RANTES and designated concentrations of either 15K or 15D peptides, were determined in micro-chemotaxis chambers.\",\n \"The 15K and 15D peptides did not induce detectable chemotaxis when tested at any concentration.\",\n \"The results are representative of three independent experiments.\",\n \"*p<0.05.FIG.\",\n \"3 demonstrates inhibition of monocyte-tropic HIV-1 infection of susceptible target cells by the exemplary anti-coreceptor binding peptides 15K and l5D.\",\n \"Monocyte-derived macrophages were treated with the designated concentrations of the indicated peptide for a period of 1 hr prior to addition of HIV-1JRFL.\",\n \"After 2 hr, cells were washed, and viral replication was determined after 72 hr by p24 analysis.\",\n \"Results are representative of 4 independent experiments.\",\n \"FIG.\",\n \"4 shows inhibition of T cell-tropic HIV-1 infection by the exemplary anti-coreceptor binding peptides 15K, l5D and scrambled peptide 15KS.\",\n \"Peripheral blood mononuclear cells were treated with designated concentrations of peptide for a period of 1 hr prior to addition of HIV-1IIIB.\",\n \"After 2 hr, cells were washed, and viral replication was determined after 48 hr by analysis of p24 levels.\",\n \"Results are representative of 4 independent experiments.\",\n \"FIGS.\",\n \"5A through 5C demonstrate the effect of peptides 15K, 15D and scrambled peptide 15KS on the binding of anti-CXCR4 antibody 12G5 to CEMx174 cells.\",\n \"FIG.\",\n \"5A.\",\n \"Dose dependent effect of peptide 15K in comparison with SDF-1α.\",\n \"FIG.\",\n \"5B.\",\n \"The comparison of the effects of 15K and 15D.\",\n \"FIG.\",\n \"5C.\",\n \"The comparison of the effects of 15K and 15KS.\",\n \"Cells were preincubated at 22° C. with peptides at designated concentrations of SDF-1α for 60 min.\",\n \"Then cells were incubated with FITC-labeled anti-human CXCR4 monoclonal antibodies 12G5 for 40 min at 22° C. and washed with FACS buffer prior to flow cytometry.\",\n \"FIGS.\",\n \"6A through 6D depict the effect of peptide pre-treatment on the mobilization of intracellular Ca2+ in THP-1 cells in response to SDF-1α.\",\n \"Cells loaded with fura-2 were preincubated with peptide 15K (FIG.\",\n \"6C), 15D (FIG.\",\n \"6B) and 15CW (FIG.\",\n \"6D) at a final concentration of 500 μM for 3 min prior to stimulation with SDF-1α (100 ng/ml).\",\n \"FIGS.\",\n \"7A through 7C depicts the inhibition of chemokine receptor ligand binding by peptide pre-treatment.\",\n \"FIG.\",\n \"7A.\",\n \"Effect of pre-treatment with peptides 15K and 15D on the binding of MIP-1β to SupT1/CCR5 cells; FIG.\",\n \"7B.\",\n \"Effect of pre-treatment with peptides 15K and 1SD on the binding of SDF-1α to CEMx174 cells; and FIG.\",\n \"7C.\",\n \"Effect of pre-treatment with 15K and control peptide 15GIG on the binding of SDF-1α to CEmx174 cells.\",\n \"Binding studies were performed as described in Materials and Methods.\",\n \"DETAILED DESCRIPTION The present invention provides novel methods and compositions for clinical and diagnostic use in the evaluation, treatment and prevention of HIV infection and related disease conditions by reducing the ability of HIV to bind and fuse to cells.\",\n \"Typically, the compositions and procedures of the invention are directed toward prophylaxis or treatment of HIV-1 infection and related clinical conditions.\",\n \"The methods of the invention involve administering an effective amount of an anti-coreceptor binding agent to a subject to inhibit HIV-coreceptor interaction.\",\n \"Typically, the anti-coreceptor binding agent is administered in a prophylactic or therapeutically effective dose to a mammalian patient susceptible to HIV infection.\",\n \"Alternatively, the subject may comprise a susceptible cell population, tissue or organ, selected for in vivo or ex vivo treatment or diagnostic processing involving exposure of the subject to an anti-coreceptor binding agent (for example, bone marrow or other tissue or organ materials treated ex vivo before re-implantation or transplant).\",\n \"The methods and compositions of the invention involve delivery or formulation of an anti-coreceptor binding agent in an amount that is effective to inhibit HIV-coreceptor interactions, and/or to inhibit one or more selected biological activities or conditions associated with, or mediated by, HIV-coreceptor interactions.\",\n \"Selected biological activities include direct co-receptor binding by HIV viruses, coreceptor binding by selected viral proteins or peptides (including gp120 proteins and peptide fragments or derivatives of gp120), as well as binding by antibodies that recognize epitopes on gp120 or on a selected chemokine or HIV coreceptor.\",\n \"Additional biological activities in this context include HIV infection and related activities and conditions, for example HIV fusion with target host cells, HIV virion entry into host cells, HIV propagation and related HIV infective events, as well as specific HIV-related disease conditions including the full range of clinical conditions and disease states associated with AIDS and AIDS Related Complex (ARC) (for example, Kaposi's sarcoma and opportunistic viral (e.g., herpes) and bacterial (e.g., pneumonia infections).\",\n \"As used herein, the term “anti-coreceptor binding agent” is meant to include anti-coreceptor binding HIV peptides, as well as peptide analogs and peptide mimetics which exhibit comparable, or substantially the same, anti-coreceptor binding activity as a selected anti-coreceptor binding HIV peptide as described herein.\",\n \"The term anti-coreceptor binding peptide includes all of the reference peptides described herein, as well as other natural or artificially selected mutant or allelic forms and derivatives of these reference peptides having the desired anti-coreceptor binding activity.\",\n \"The term peptide analog refers to such artificially modified peptide analogs as chemically cleaved peptide fragments, chemically modified peptide derivatives, site directed mutant peptide variants having one or more amino acid insertions, substitutions or deletions, and the like.\",\n \"The anti-coreceptor binding peptides, analogs and mimetics of the invention function to specifically inhibit HIV-1 gp120 binding interactions with chemokine receptors (HIV-1 coreceptors).\",\n \"In addition, the anti-coreceptor binding agents are effective to inhibit or block HIV-1 cell fusion and virion entry, thereby impairing viral replication and transmission.\",\n \"Correlated with these activities, the anti-coreceptor binding agents and methods of the invention provide for safe and effective treatment of HIV-1 infection and related diseases.\",\n \"Ancillary uses are also readily implemented using these compositions and methods for diagnosing and evaluating HIV infection and related activities and disease mechanisms.\",\n \"The description herein illustrates production and characterization of peptides modeled after a novel structural motif identified in the HIV-1 gp120 envelope protein.\",\n \"This novel structural motif shares similarities with a corresponding structural motif identified as a conservative feature among diverse chemokines.\",\n \"This conservative motif is represented in one aspect by the reference sequences identified for HIV strains HXB2, IIIB, and JRFL, which represent operable anti-coreceptor binding peptides within the invention provided as native fragments of the corresponding gp120 proteins of these strains.\",\n \"As shown in Table 1, below, these sequences aligned with MIP-1β and SDF-1α embrace a novel, HIV-1 coreceptor binding motif.\",\n \"TABLE AMINO ACID ALIGNMENT OF PARTIAL SEQUENCES OF GP120 FROM REPRESENTATIVE HIV STRAINS WITH PARTIAL SEQUENCES OF MIP-1β AND SDF-1α HIV strain* 326 340 HXB2 M R Q A H C N I S R A K W N N SEQ ID NO:1 326 340 III B M R Q A H C N I S R A K W N A SEQ ID NO:2 322 336 JR FL I R Q A H C N I S R A K W N D SEQ ID NO:3 Consensus I R Q A H C N V S R S E W N K SEQ ID NO:4 HIV A Consensus I R Q A H C N I S R A Q W N N SEQ ID NO:5 HIV B chemokine 47 60 MIP-1β S K Q - V C A D P S E S W V Q SEQ ID NO:6 48 61 SDF-1α N R Q - V C I D P K L K W I Q SEQ ID NO:7 *The residues numbered according to (Korber, B. and Los Alamos National Laboratory - Theoretical Biology and Biophysics Group T-10, Human retroviruses and AIDS.\",\n \"1998: a compilation and analysis of nucleic acid and amino acid sequences.\",\n \"Published by Theoretical Biology and Biophysics Group T-10 Los Alamos National Laboratory, Los Alamos, N.M., 1998, incorporated herein by reference).\",\n \"The residues that are shared between identified regions of HIV-1 gp120 and chemokines are shown in bold.\",\n \"This alignment illustrates the finding herein that the amino acid sequences of nearly all chemokines feature a Trp residue located at the beginning of a C-terminal α-helix and separated by six intervening residues from a conserved Cys residue.\",\n \"Comparing this conserved motif with the gp120 molecule of HIV-1, it is further demonstrated that all HIV isolates have a similar structural motif adjacent the V3 loop.\",\n \"As described in further detail below, computer modeling based on the sequence of HIV-1 strain JRFL which closely relates to a Consensus HIV-1 sequence, demonstrates that a fragment of gp120 containing a conserved “CXXXXXXW motif” can be “template forced” onto the homologous portion of the chemokine MIP-1β.\",\n \"Thus, the anti-coreceptor binding peptides of the invention typically feature the above noted CXXXXXXW motif, with additional amino acids, peptides, proteins, chemical reagents or moieties combined or conjugated therewith.\",\n \"These peptides include native HIV-1 peptides, such as those identified above in Table 1 and other known HIV-1 peptides that correspond to, or include, a partial or complete, homologous sequence to these exemplary peptides.\",\n \"Other peptides comprising the novel structural motif that includes the above noted CXXXXXXW anti-coreceptor binding determinant can comprise allelic variants among native peptide sequences, or synthetic or mutant peptide analogs of selected native HIV-1 gp120 peptides.\",\n \"Two exemplary peptides within the invention, designated 15K and 15D, include the C-terminal part of the V3 loop of gp120 and the N-terminal part of the C3 segment of the protein (Table 2).\",\n \"These peptides differ from each other by the presence of a D or K (Asp or Lys) residue at the twelfth position, which difference reflects allelic sequence variation among natural HIV-1 isolates.\",\n \"In addition, both of these exemplary synthetic peptides differ from corresponding, native HIV-1 sequences by a site-directed mutation comprising a substitution of K for Q (Lys for Gln) at the third residue position in the peptides, which reflects allelic sequence variation among natural HIV-1 isolates and also results in reduced susceptibility of the peptides to hydrolysis.\",\n \"TABLE 2 SEQUENCES OF EXEMPLARY SYNTHETIC PEPTIDES Design- tion Sequence 15K I R K A H C N I S R A K W N D (SEQ ID NO:8) 15D I R K A H C N I S R A D W N D (SEQ ID NO:9) These exemplary synthetic peptides of the invention inhibit or block binding or “docking” interactions between the HIV-1 envelope protein gp120 and chemokine receptors (e.g., CXCR4 and CCR5) that function as “coreceptors” for HIV entry on the surface of target cells (macrophages and T lymphocytes).\",\n \"The natural ligands of the coreceptors are chemokines (e.g., SDF-1α and MIP-1β), and it is further demonstrated herein that the conserved structural elements identified for the HIV gp120 protein motif that are shared with chemokines mediate critical HIV-coreceptor interactions.\",\n \"Detailed functional analyses of the peptides show that both are effective in competing with chemokines for binding to CCR5- and CXCR4-expressing cells.\",\n \"The peptides also inhibit monocyte chemotaxis stimulated by the chemokine RANTES (Regulated upon Activation Normal T-cell Expressed and Secreted).\",\n \"In addition, the anti-coreceptor binding peptides of the invention are shown herein to be potent inhibitors of HIV replication.\",\n \"In certain aspects, the peptides effect this inhibition in a multi-tropic or multi-specific manner to prevent or treat infection by both T cell tropic (lymphotropic) and macrophage tropic (m-tropic) HIV strains by blocking HIV interactions with distinct (CXCR4 and CCR5) coreceptors.\",\n \"This ability to use small peptides to achieve multi-tropic (i.e., multi-receptor) blockade is a surprising and important advantage satisfied by the invention.\",\n \"The anti-coreceptor binding peptides of the present invention include naturally occurring peptide variants, e.g., naturally occurring allelic variants and mutant proteins, as well as synthetic, e.g., chemically or recombinantly engineered, peptide fragments and analogs.\",\n \"As used herein, anti-coreceptor binding peptide “analogs” is meant to include a modified gp120 peptide incorporating one or more amino acid substitutions, insertions, rearrangements or deletions as compared to a native amino acid sequence of an HIV-1 gp120 anti-coreceptor binding peptide domain, fragment or motif, as described herein.\",\n \"Anti-coreceptor binding peptide analogs thus modified exhibit substantial anti-coreceptor binding activity comparable to that of a corresponding native peptide, which is activity that at least 50%, typically at least 75% or greater, compared to activity of the corresponding native peptide (e.g., as determined by an in vitro coreceptor binding assay or HIV-1 infection assay).\",\n \"As used herein, the term “biologically active anti-coreceptor binding analogs and mimetics” refers to analogs or mimetics of native peptides which encompass the entire length, sequence or chemical structure of the corresponding native peptide but which nevertheless maintains substantial anti-coreceptor binding activity as described above in an appropriate assay system.\",\n \"For purposes of the present invention, the term anti-coreceptor binding peptide “analog” thus includes derivatives or synthetic variants of a native HIV-1 gp120 anti-coreceptor binding peptide, such as amino and/or carboxyl terminal deletions and fusions, as well as intrasequence insertions, substitutions or deletions of single or multiple amino acids.\",\n \"Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein.\",\n \"Random insertion is also possible with suitable screening of the resulting product.\",\n \"Deletional variants are characterized by removal of one or more amino acids from the sequence.\",\n \"Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place.\",\n \"Where a native HIV-1 gp120 anti-coreceptor binding peptide is modified by amino acid substitution, amino acids are generally replaced by other amino acids having similar, conservatively related chemical properties such as hydrophobicity, hydrophilicity, electronegativity, bulky side chains and the like.\",\n \"Residue positions which are not identical to the native peptide sequence are thus replaced by amino acids having similar chemical properties, such as charge or polarity, which changes are not likely to substantially effect the properties of the peptide analog.\",\n \"These and other minor alterations substantially maintain the immunoidentity (e.g., recognition by one or more monoclonal antibodies that recognize a native HIV-1 gp120 anti-coreceptor binding peptide) and other biological activities of the native peptide.\",\n \"In this context, the term “conservative amino acid substitution” refers to the general interchangeability of amino acid residues having similar side chains.\",\n \"For example, a group of amino acids having aliphatic side chains is alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.\",\n \"Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another.\",\n \"Likewise, the present invention contemplates the substitution of a polar (hydrophilic) residue such as between arginine and lysine, between glutamine and asparagine, and between threonine and serine.\",\n \"Additionally, the substitution of a basic residue such as lysine, arginine or histidine for another or the substitution of an acidic residue such as aspartic acid or glutamic acid for another is also contemplated.\",\n \"Exemplary conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.\",\n \"Anti-coreceptor binding peptide analogs also include modified forms of a native HIV-1 gp120 anti-coreceptor binding peptide incorporating stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, or unnatural amino acids such as a, α-disubstituted amino acids, N-alkyl amino acids, lactic acid.\",\n \"These and other unconventional amino acids may also be substituted or inserted within native HIV-1 gp120 anti-coreceptor binding peptides of the present invention.\",\n \"Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, ω-N-methylarginine, and other similar amino acids and amino acids (e.g., 4-hydroxyproline).\",\n \"For purposes of the present invention, analogs of native HIV-1 gp120 anti-coreceptor binding peptides also include single or multiple substitutions, deletions and/or additions of carbohydrate, lipid and/or proteinaceous moieties that occur naturally or artificially as structural components of gp120 peptides or are bound or otherwise associated with the peptide analog.\",\n \"To facilitate production and use of anti-coreceptor binding peptide analogs within the invention, reference can be made to molecular phylogenetic studies that characterize conserved and divergent protein structural and functional elements between different HIV-1 strains, and between HIV-1 and other HIV taxa and more distantly related retroviruses.\",\n \"In this regard, available studies provide detailed assessments of gp120 protein structure-function relationships on a fine molecular level.\",\n \"These studies include detailed sequence comparisons identifying conserved and divergent structural elements among a large number of HIV-1 gp120 allelic variants, for example.\",\n \"Each of these conserved and divergent structural elements facilitate practice of the invention by pointing to useful targets that for modifying native HIV-1 gp120 anti-coreceptor binding peptides to confer desired structural and/or functional changes.\",\n \"In this context, existing sequence alignments may be analyzed, or conventional sequence alignment methods may be employed to yield sequence comparisons for analysis, to identify corresponding protein regions and amino acid positions between native HIV-1 gp120 anti-coreceptor binding peptides and related or homologous peptides bearing a structural element of interest for incorporation within an anti-coreceptor binding peptide analog.\",\n \"Typically, one or more amino acid residues marking a structural element of interest in a different reference peptide sequence is incorporated within the anti-coreceptor binding peptide analog.\",\n \"For example, a cDNA encoding a native HIV-1 gp120 anti-coreceptor binding peptide may be recombinantly modified at one or more corresponding amino acid position(s) (i.e., corresponding positions that match or span a similar aligned sequence element according to accepted alignment methods to residues marking the structural element of interest in a heterologous reference peptide sequence) to encode an amino acid deletion, substitution, or insertion that alters corresponding residue(s) in the native HIV-1 gp120 anti-coreceptor binding peptide to generate an operable peptide analog within the invention having an analogous structural and/or functional element as the reference peptide or protein.\",\n \"Within this rational design method for constructing anti-coreceptor binding peptide analogs, the native or wild-type identity of residue(s) at amino acid positions corresponding to a structural element of interest in a heterologous reference peptide or protein may be altered to the same, or a conservatively related, residue identity as the corresponding amino acid residue(s) in the reference peptide or protein.\",\n \"However, it is often possible to alter native amino acid residues non-conservatively with respect to the corresponding reference protein residue(s).\",\n \"In particular, many non-conservative amino acid substitutions, particularly at divergent sites suggested to be more amenable to modification, may yield a moderate impairment or neutral effect, or even enhance a selected biological activity, compared to the function of a native HIV-1 gp120 anti-coreceptor binding peptide.\",\n \"Extensive protein structure-function data are available in the literature pertaining to HIV that will serve and guide the artisan to prepare functional anti-coreceptor binding peptide analogs for use within the invention.\",\n \"Structure-function relationships between HIV-1 strains and between HIV-1 and more distantly related retroviruses may be elucidated using conventional molecular phylogenetic analysis coupled with functional assays for determining anti-coreceptor binding activities described herein.\",\n \"Sequence alignment and comparisons to forecast useful peptide analogs and mimetics will be further refined by analysis of crystalline structure (see, e.g., Löebermann et al., J. Mol.\",\n \"Biol.\",\n \"177:531-556, 1984; Huber et al., Biochemistry 28:8951-8966, 1989; Stein et al., Nature 347:99-102, 1990; Wei et al., Structural Biology 1:251-255, 1994, each incorporated herein by reference) coupled with computer modeling methods known in the art.\",\n \"These analyses allow detailed structure-function mapping to identify desired structural elements and modifications for incorporation into anti-coreceptor binding peptide analogs and mimetics that will exhibit substantial anti-coreceptor binding activity for use within the methods of the invention.\",\n \"Native HIV-1 gp120 anti-coreceptor binding peptides and peptide analogs within the invention are typically between about 6-35 amino acid residues in length, more typically between about 10 and 21 or 22 amino acid residues in length.\",\n \"Within certain embodiments, the native peptides and analogs are between about 10-17 residues in length.\",\n \"In more specific embodiments, peptides are 10, 12, 13, 15, 17, 21, or 22 amino acid residues in length.\",\n \"Anti-coreceptor binding peptide analogs of the invention typically show substantial sequence identity to a corresponding native HIV-1 gp120 anti-coreceptor binding peptide sequence.\",\n \"Within the foregoing length ranges, both the native HIV-1 gp120 anti-coreceptor binding peptide and the peptide analogs typically comprise the conserved “CXXXXXXW” motif, described above, which may be extended to include additional residues from the native HIV-1 gp120 anti-coreceptor binding peptide sequence or non-native residues, fusion protein members, chemical moieties and the like.\",\n \"As applied to anti-coreceptor binding peptide analogs and fragments, these analogs and fragments typically exhibit substantial amino acid sequence identity to a corresponding native HIV-1 gp120 anti-coreceptor binding peptide sequence.\",\n \"The term “substantial sequence identity” as used herein means that the two subject amino acid sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap penalties, share at least 80 percent sequence identity, often at least 90-95 percent or greater sequence identity.\",\n \"“Percentage amino acid identity” refers to a comparison of the amino acid sequences of two peptides which, when optimally aligned, have approximately the designated percentage of the same amino acids.\",\n \"Sequence comparisons are generally made to a reference sequence over a comparison window of at least 10 residue positions, frequently over a window of at least 15-20 amino acids, wherein the percentage of sequence identity is calculated by comparing a reference sequence to a second sequence, the latter of which may represent, for example, a peptide analog sequence that includes one or more deletions, substitutions or additions which total 20 percent, typically less than 5-10% of the reference sequence over the window of comparison.\",\n \"The reference sequence may be a subset of a larger sequence, for example, as a segment of the HIV-1 gp120 protein.\",\n \"Optimal alignment of sequences for aligning a comparison window may be conducted according to the local homology algorithm of Smith and Waterman (Adv.\",\n \"Appl.\",\n \"Math.\",\n \"2:482, 1981), by the homology alignment algorithm of Needleman and Wunsch (J. Mol.\",\n \"Biol.\",\n \"48:443, 1970), by the search for similarity method of Pearson and Lipman (Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 85:2444, 1988), or by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.).\",\n \"By aligning a peptide analog optimally with a corresponding native HIV-1 gp120 anti-coreceptor binding peptide, and by using appropriate assays, e.g., coreceptor binding assays, to determine a selected biological activity, one can readily identify operable peptide analogs for use within the methods and compositions of the invention.\",\n \"Anti-coreceptor binding peptide analogs are typically specifically immunoreactive with antibodies raised to the corresponding native HIV-1 gp120 anti-coreceptor binding peptide.\",\n \"Likewise, nucleic acids encoding functional anti-coreceptor binding peptide analogs will typically selectively hybridize to nucleic acid sequences encoding a corresponding native HIV-1 gp120 anti-coreceptor binding peptide under accepted, moderate or high stringency hybridization conditions (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989, incorporated herein by reference).\",\n \"The phrase “selectively hybridizing to” refers to a selective interaction between a nucleic acid probe that hybridizes, duplexes or binds preferentially to a particular target DNA or RNA sequence, for example when the target sequence is present in a heterogenous preparation such as total cellular DNA or RNA.\",\n \"Generally, nucleic acid sequences encoding functional anti-coreceptor binding peptide analogs and fragments will hybridize to nucleic acid sequences encoding native HIV-1 gp120 anti-coreceptor binding peptides under stringent conditions selected to be about 5° C. lower than the thermal melting point (Tm) for the subject sequence at a defined ionic strength and pH.\",\n \"The Tm is the temperature (under defined ionic strength and pH) at which 50% of the complementary or target sequence hybridizes to a perfectly matched probe.\",\n \"For discussions of nucleic acid probe design and annealing conditions, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, 1989 or Current Protocols in Molecular Biology F. Ausubel et al, ed., Greene Publishing and Wiley-Interscience, New York, 1987, each of which is incorporated herein by reference.\",\n \"Typically, stringent or selective conditions will be those in which the salt concentration is at least about 0.02 molar at pH 7 and the temperature is at least about 60° C. Less stringent selective hybridization conditions can also be chosen.\",\n \"As other factors may significantly affect the stringency of hybridization, including, among others, base composition and size of the complementary strands, the presence of organic solvents and the extent of base mismatching, the combination of parameters is more important than the specific measure of any one.\",\n \"Anti-coreceptor binding peptide analogs of the invention typically show substantial sequence identity to a corresponding native HIV1 gp120 anti-coreceptor binding peptide sequence.\",\n \"Within the foregoing length ranges, both the native HIV-1 gp120 anti-coreceptor binding peptide and the peptide analogs typically comprise the conserved “CXXXXXXW” motif, described above, which may be extended to include additional residues from the native HIV-1 gp120 anti-coreceptor binding peptide sequence, non-native residues, fusion protein members, chemical moieties, and the like.\",\n \"As applied to anti-coreceptor binding peptide analogs and fragments, these analogs and fragments typically exhibit substantial amino acid sequence identity to a corresponding native HIV-1 gp120 anti-coreceptor binding peptide sequence.\",\n \"Various peptide analogs are contemplated within the scope of the invention, which typically satisfy the foregoing length criteria of 10 to and include the conserved “CXXXXXXW” motif, but which incorporate selected sequence modifications at one or more positions to yield a desired structural or activity modification.\",\n \"To illustrate the breadth of different peptides and peptide analogs that are useful within the invention, the following table (Table 3) provides a map of anti-coreceptor binding peptide sequence variants based on a detailed analysis of different strains of HIV-1, along with structure-function analysis directed to both gp120 and chemokine structure-function, further guided by general rules of peptide structure-function.\",\n \"TABLE 3 MENU FOR CONSTRUCTING OPERABLE HIV gp120 PEPTIDES AND PEPTIDE ANALOGS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Peptide 15K I R Q A H C N I S R A K W N D SEQ ID NO:8 Peptide 15D D SEQ ID NO:9 M G K T Y D L N K R R Q N K E R P F H V D G K E D K S K L V Q T Y T S T D E E T S E S N K D K A S Q K T L T P I E A Y E G N R Q A I A V S I D E T A R V V I P I D S S S A S Q T N G T A P T V W Q A G I N H G N H V M M D A V L Y P I I H H V In accordance with the foregoing table, peptides which satisfy the above-described length criteria and include the conserved “CXXXXW” motif can also be selected from corresponding or overlapping native peptides among any known HIV isolate having a variant sequence as compared to a corresponding “reference” peptide.\",\n \"As illustrated in Table 3, one reference peptide that may be used in this context is the 15K peptide, which provides an exemplary reference sequence for comparison with other peptide sequences within the invention.\",\n \"Against this reference sequence, the table provides a range of amino acid residues that can be effectively substituted at the indicated residue positions (numbered in reference to the 15K peptide) which, for the substitutions designated by regular font single amino acid code, follow natural variations in the HIV gp120 available in the various HIV sequence data banks (e.g., http://hiv-web.lanl.gov/., incorporated herein by reference).\",\n \"Each of these residues can be incorporated, in a native HIV gp120-derived peptide, or in a single- or multiple-substituted peptide analog of the invention, to yield effective anti-coreceptor binding agents.\",\n \"Thus, peptides and peptide analogs are provided which satisfy the above-described length criteria and include the conserved “CXXXXXXW” motif, and which include any one, or any combination, of the following alternative residues occurring naturally or by substitution at the indicated positions (as designated for peptide 15K in Table 3 and determined for the subject peptide by conventional comparison and alignment against, e.g., the 15K reference sequence, as described herein): Position 1—I, M, K, S, T, L, A, V, R, P, or N; Position 2—R, G, E, K, S, T, or I; Position 3—Q, K, R, L, E, P, A, V, S, T, H, or D; Position 4—A, T, P, V, E, or S; Position 5—H, Y, F, Q, N, I, or V; Position 7—N, D, H, T, K, E, S, I, Q, V, G, or A; Position 8—I, L, V, Y, D, A; Position 9—S, N+EE, D, T, K, Y, I, or P; Position 10—R, K, G, S, A, E, D, I, T, W, or N; Position 11—A, R, K, T, S, G, E, D, N, Q, H, V, I, or L; Position 12—K, D, R, E, K, Q, N, T, S, G, A, V, L; Position 14—N, Q, D, E, K, R, A, S, T, G, M, Y, I, H, or V; Position 15—D, N, K, E, T, Q, R, S, A, I, M, or P. Certain of the foregoing residue/position alternatives, for example, the residue/position alternatives designated by underscoring in the preceding paragraph, may be selected in a greater number of peptides and analogs of the invention compared to other alternatives.\",\n \"This preference may be guided based on the greater degree of conservation of the indicated residue at corresponding positions among different HIV-1 isolates, or based on a convergent or homologous occurrence of the indicated residue at corresponding positions in gp120 of a selected HIV isolate and in a selected chemokine.\",\n \"Additional selections within the foregoing “menu” will be guided to favor conservative structural relationships between original and substitute residues in the preparation of anti-coreceptor binding peptide analogs, as described herein.\",\n \"In accordance with these selection principals, the above-noted, alternative residues may be included in peptides and peptide analogs of the invention, singly or in any combination of 2, 3, 4 or more and up to 13 residues (e.g., as exemplified by the various combinations shown in the separate lines of Table 3), selected or substituted at the indicated positions.\",\n \"Within additional aspects of the invention, peptide mimetics are provided which comprise a peptide or non-peptide molecule that mimics the tertiary binding structure and activity of the anti-coreceptor binding peptides described herein.\",\n \"These peptide mimetics include recombinantly or chemically modified peptides, as well as non-peptide anti-coreceptor binding agents such as small molecule drug mimetics, as further described below.\",\n \"In one aspect, peptides of the invention are modified to produce peptide mimetics by replacement of one or more naturally occurring side chains of the 20 genetically encoded amino acids (or D amino acids) with other side chains, for instance with groups such as alkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7-membered alkyl, amide, amide lower alkyl, amide di (lower alkyl), lower alkoxy, hydroxy, carboxy and the lower ester derivatives thereof, and with 4-, 5-, 6-, to 7-membered heterocyclics.\",\n \"For example, proline analogs can be made in which the ring size of the proline residue is changed from 5 members to 4, 6, or 7 members.\",\n \"Cyclic groups can be saturated or unsaturated, and if unsaturated, can be aromatic or non-aromatic.\",\n \"Heterocyclic groups can contain one or more nitrogen, oxygen, and/or sulphur heteroatoms.\",\n \"Examples of such groups include the furazanyl, furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g., morpholino), oxazolyl, piperazinyl (e.g., 1-piperazinyl), piperidyl (e.g., 1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (e.g., 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g., thiomorpholino), and triazolyl.\",\n \"These heterocyclic groups can be substituted or unsubstituted.\",\n \"Where a group is substituted, the substituent can be alkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl.\",\n \"The peptide compounds of the invention, including peptidomimetics, can also be covalently bound to one or more of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkenes, in the manner set forth in U.S. Pat.\",\n \"No.\",\n \"4,640,835; U.S. Pat.\",\n \"No.\",\n \"4,496,689; U.S. Pat.\",\n \"No.\",\n \"4,301,144; U.S. Pat.\",\n \"No.\",\n \"4,670,417; U.S. Pat.\",\n \"No.\",\n \"4,791,192; or U.S. Pat.\",\n \"No.\",\n \"4,179,337, all which-are incorporated by reference in their entirety herein.\",\n \"Other peptide analogs and mimetics within the invention include glycosylation variants, and covalent or aggregate conjugates with other chemical moieties.\",\n \"Covalent derivatives can be prepared by linkage of functionalities to groups which are found in amino acid side chains or at the N- or C-termini, by means which are well known in the art.\",\n \"These derivatives can include, without limitation, aliphatic esters or amides of the carboxyl terminus, or of residues containing carboxyl side chains, O-acyl derivatives of hydroxyl group-containing residues, and N-acyl derivatives of the amino terminal amino acid or amino-group containing residues, e.g., lysine or arginine.\",\n \"Acyl groups are selected from the group of alkyl-moieties including C3 to C18 normal alkyl, thereby forming alkanoyl aroyl species.\",\n \"Covalent attachment to carrier proteins, e.g., immunogenic moieties may also be employed.\",\n \"In certain embodiments, glycosylation alterations of anti-coreceptor binding agents are included, which can be made, e.g., by modifying the glycosylation patterns of a peptide during its synthesis and processing, or in further processing steps.\",\n \"Particularly preferred means for accomplishing this are by exposing the peptide to glycosylating enzymes derived from cells which normally provide such processing, e.g., mammalian glycosylation enzymes.\",\n \"Deglycosylation enzymes are also contemplated.\",\n \"Also embraced are versions of the same primary amino acid sequence which have other minor modifications, including phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine, or other moieties, including ribosyl groups or cross-linking reagents.\",\n \"Peptidomimetics may also have amino acid residues which have been chemically modified by phosphorylation, sulfonation, biotinylation, or the addition or removal of other moieties, particularly those which have molecular shapes similar to phosphate groups.\",\n \"In some embodiments, the modifications will be useful labeling reagents, or serve as purification targets, e.g., affinity ligands.\",\n \"A major group of peptidomimetics within the invention are covalent conjugates of the anti-coreceptor binding peptides, or fragments thereof, with other proteins or peptides.\",\n \"These derivatives can be synthesized in recombinant culture such as N- or C-terminal fusions or by the use of agents known in the art for their usefulness in cross-linking proteins through reactive side groups.\",\n \"Preferred peptide and protein derivatization sites for targeting by cross-linking agents are at free amino groups, carbohydrate moieties, and cysteine residues.\",\n \"Fusion polypeptides between anti-coreceptor binding peptides and other homologous or heterologous peptides and proteins are also provided.\",\n \"Many growth factors and cytokines are homodimeric entities, and a repeat construct of anti-coreceptor binding peptide linked to form “cluster peptides” will yield various advantages, including lessened susceptibility to proteolytic degradation.\",\n \"Various alternative multimeric constructs comprising peptides of the invention are also provided.\",\n \"In one embodiment, various polypeptide fusions are provided as described in U.S. Pat.\",\n \"Nos.\",\n \"6,018,026 and 5,843,725, by linking one or more anti-coreceptor binding peptides of the invention with a heterologous, multimerizing polypeptide or protein, for example, immunoglobulin heavy chain constant region, or an immunoglobulin light chain constant region.\",\n \"The biologically active, multimerized polypeptide fusion thus constructed can be a hetero- or homo-multimer, e.g., a heterodimer or homodimer, which may each comprise one or more distinct anti-coreceptor binding peptide(s) of the invention.\",\n \"Other heterologous polypeptides may be combined with the anti-coreceptor binding agents to yield fusions comprising, e.g., a hybrid protein exhibiting heterologous (e.g., CD4) receptor binding specificity.\",\n \"Likewise, heterologous fusions may be constructed exhibit a combination of properties or activities of the derivative proteins.\",\n \"Other typical examples are fusions of a reporter polypeptide, e.g., CAT or luciferase, with a peptide of the invention, to facilitate localization of the fused protein (see, e.g., Dull et al., U.S. Pat.\",\n \"No.\",\n \"4,859,609, incorporated herein by reference).\",\n \"Other gene/protein fusion partners useful in this context include bacterial beta-galactosidase, trpE, Protein A, beta-lactamase, alpha amylase, alcohol dehydrogenase, and yeast alpha mating factor (see, e.g., Godowski et al., Science 241:812-816, 1988, incorporated herein by reference).\",\n \"The present invention also contemplates the use of anti-coreceptor binding agents modified by covalent or aggregative association with chemical moieties.\",\n \"These derivatives generally fall into the three classes: (1) salts, (2) side chain and terminal residue covalent modifications, and (3) adsorption complexes, for example with cell membranes.\",\n \"Such covalent or aggregative derivatives are useful for various purposes, for example as immunogens, as reagents in immunoassays, or in purification methods such as for affinity purification of ligands or other binding ligands.\",\n \"For example, an anti-coreceptor binding agent can be immobilized by covalent bonding to a solid support such as cyanogen bromide-activated Sepharose, by methods which are well known in the art, or adsorbed onto polyolefin surfaces, with or without glutaraldehyde cross-linking, for use in the assay or purification of antibodies that specifically bind the anti-coreceptor binding agent.\",\n \"The anti-coreceptor binding agent can also be labeled with a detectable group, for example radioiodinated by the chloramine T procedure, covalently bound to rare earth chelates, or conjugated to another fluorescent moiety for use in diagnostic assays.\",\n \"Those of skill in the art recognize that a variety of techniques are available for constructing peptide mimetics with the same or similar desired biological activity as the corresponding peptide compound but with more favorable activity than the peptide with respect to solubility, stability, and susceptibility to hydrolysis and proteolysis (see, e.g., Morgan and Gainor, Ann.\",\n \"Rep. Med.\",\n \"Chem.\",\n \"24:243-252, 1989, incorporated herein by reference).\",\n \"The following describes methods for preparing peptide mimetics modified at the N-terminal amino group, the C-terminal carboxyl group, and/or changing ore or more of the amido linkages in the peptide to a non-amido linkage.\",\n \"It being understood that two or more such modifications can be coupled in one peptide mimetic structure (e.g., modification at the C-terminal carboxyl group and inclusion of a —CH2-carbamate linkage between two amino acids in the peptide.\",\n \"For N-terminal modifications, the peptides typically are synthesized as the free acid but, as noted above, can be readily prepared as the amide or ester.\",\n \"One can also modify the amino and/or carboxy terminus of the peptide compounds of the invention to produce other compounds of the invention.\",\n \"Amino terminus modifications include methylating (i.e., —NHCH3 or —NH(CH3)2), acetylating, adding a carbobenzoyl group, or blocking the amino terminus with any blocking group containing a carboxylate functionality defined by RCOO—, where R is selected from the group consisting of naphthyl, acridinyl, steroidyl, and similar groups.\",\n \"Carboxy terminus modifications include replacing the free acid with a carboxamide group or forming a cyclic lactam at the carboxy terminus to introduce structural constraints.\",\n \"Amino terminus modifications are as recited above and include alkylating, acetylating, adding a carbobenzoyl group, forming a succinimide group, and the like.\",\n \"Specifically, the N-terminal amino group can then be reacted as follows: (a) to form an amide group of the formula RC(O)NH— where R is as defined above by reaction with an acid halide (e.g., RC(O)Cl) or acid anhydride.\",\n \"Typically, the reaction can be conducted by contacting about equimolar or excess amounts (e.g., about 5 equivalents) of an acid halide to the peptide in an inert diluent (e.g., dichloromethane) preferably containing an excess (e.g., about 10 equivalents) of a tertiary amine, such as diisopropylethylamine, to scavenge the acid generated during reaction.\",\n \"Reaction conditions are otherwise conventional (e.g., room temperature for 30 minutes).\",\n \"Alkylation of the terminal amino to provide for a lower alkyl N-substitution followed by reaction with an acid halide as described above will provide for N-alkyl amide group of the formula RC(O)NR—; (b) to form a succinimide group by reaction with succinic anhydride.\",\n \"As before, an approximately equimolar amount or an excess of succinic anhydride (e.g., about 5 equivalents) can be employed and the amino group is converted to the succinimide by methods well known in the art including the use of an excess (e.g., ten equivalents) of a tertiary amine such as diisopropylethylamine in a suitable inert solvent (e.g., dichloromethane) (see, for example, U.S. Pat.\",\n \"No.\",\n \"4,612,132, incorporated herein by reference).\",\n \"It is understood that the succinic group can be substituted with, for example, C2-C6 alkyl or —SR substituents which are prepared in a conventional manner to provide for substituted succinimide at the N-terminus of the peptide.\",\n \"Such alkyl substituents are prepared by reaction of a lower olefin (C2-C6) with maleic anhydride in the manner described by (U.S. Pat.\",\n \"No.\",\n \"4,612,132) and —SR substituents are prepared by reaction of RSH with maleic anhydride where R is as defined above; (c) to form a benzyloxycarbonyl-NH— or a substituted benzyloxycarbonyl-NH-group by reaction with approximately an equivalent amount or an excess of CBZ-Cl (i.e., benzyloxycarbonyl chloride) or a substituted CBZ-Cl in a suitable inert diluent (e.g., dichloromethane) preferably containing a tertiary amine to scavenge the acid generated during the reaction; (d) to form a sulfonamide group by reaction with an equivalent amount or an excess (e.g., 5 equivalents) of R—S(O)2Cl in a suitable inert diluent (dichloromethane) to convert the terminal amine into a sulfonamide where R is as defined above.\",\n \"Preferably, the inert diluent contains excess tertiary amine (e.g., ten equivalents) such as diisopropylethylamine, to scavenge the acid generated during reaction.\",\n \"Reaction conditions are otherwise conventional (e.g., room temperature for 30 minutes); (e) to form a carbamate group by reaction with an equivalent amount or an excess (e.g., 5 equivalents) of R—OC(O)Cl or R—OC(O)OC6H4-p-NO2 in a suitable inert diluent (e.g., dichloromethane) to convert the terminal amine into a carbamate where R is as defined above.\",\n \"Preferably, the inert diluent contains an excess (e.g., about 10 equivalents) of a tertiary amine, such as diisopropylethylamine, to scavenge any acid generated during reaction.\",\n \"Reaction conditions are otherwise conventional (e.g., room temperature for 30 minutes); and (f) to form a urea group by reaction with an equivalent amount or an excess (e.g., 5 equivalents) of R—N═C═O in a suitable inert diluent (e.g., dichloromethane) to convert the terminal amine into a urea (i.e., RNHC(O)NH—) group where R is as defined above.\",\n \"Preferably, the inert diluent contains an excess (e.g., about 10 equivalents) of a tertiary amine, such as diisopropylethylamine.\",\n \"Reaction conditions are otherwise conventional (e.g., room temperature for about 30 minutes).\",\n \"In preparing peptide mimetics wherein the C-terminal carboxyl group is replaced by an ester (i.e., —C(O)OR where R is as defined above), resins as used to prepare peptide acids are employed, and the side chain protected peptide is cleaved with base and the appropriate alcohol, e.g., methanol.\",\n \"Side chain protecting groups are then removed in the usual fashion by treatment with hydrogen fluoride to obtain the desired ester.\",\n \"In preparing peptide mimetics wherein the C-terminal carboxyl group is replaced by the amide —C(O)NR3R4, where R3 and R4 independently are hydrogen or a lower alkyl, a benzhydrylamine resin is used as the solid support for peptide synthesis.\",\n \"Upon completion of the synthesis, hydrogen fluoride treatment to release the peptide from the support results directly in the free peptide amide (i.e., the C-terminus is —C(O)NH2).\",\n \"Alternatively, use of the chloromethylated resin during peptide synthesis coupled with reaction with ammonia to cleave the side chain protected peptide from the support yields the free peptide amide and reaction with an alkylamine or a dialkylamine yields a side chain protected alkylamide or dialkylamide (i.e., the C-terminus is —C(O)NRR1 where R and R1 independently are hydrogen or a lower alkyl).\",\n \"Side chain protection is then removed in the usual fashion by treatment with hydrogen fluoride to give the free amides, alkylamides, or dialkylamides.\",\n \"In another alternative embodiment, the C-terminal carboxyl group or a C-terminal ester can be induced to cyclize by internal displacement of the —OH or the ester (—OR) of the carboxyl group or ester respectively with the N-terminal amino group to form a cyclic peptide.\",\n \"For example, after synthesis and cleavage to give the peptide acid, the free acid is converted to an activated ester by an appropriate carboxyl group activator such as dicyclohexylcarbodiimide (DCC) in solution, for example, in methylene chloride (CH2Cl2), dimethyl formamide (DMF) mixtures.\",\n \"The cyclic peptide is then formed by internal displacement of the activated ester with the N-terminal amine.\",\n \"Internal cyclization as opposed to polymerization can be enhanced by use of very dilute solutions.\",\n \"Such methods are well known in the art.\",\n \"One can also cyclize the anti-coreceptor binding peptides of the invention, or incorporate a desamino or descarboxy residue at the termini of the peptide, so that there is no terminal amino or carboxyl group, to decrease susceptibility to proteases or to restrict the conformation of the peptide.\",\n \"C-terminal functional groups of the compounds of the present invention include amide, amide lower alkyl, amide di (lower alkyl), lower alkoxy, hydroxy, and carboxy, and the lower ester derivatives thereof, and the pharmaceutically acceptable salts thereof.\",\n \"Other methods for making peptide derivatives and mimetics of the are described in Hruby et al., (Biochem J.\",\n \"268:249-262, 1990, incorporated herein by reference).\",\n \"According to these methods, the anti-coreceptor binding peptide compounds of the invention also serve as structural models for non-peptide mimetic compounds with similar biological activity.\",\n \"Those of skill in the art recognize that a variety of techniques are available for constructing compounds with the same or similar desired biological activity as the lead peptide compound but with more favorable activity than the lead with respect to solubility, stability, and susceptibility to hydrolysis and proteolysis (see, e.g., Morgan and Gainor, Ann.\",\n \"Rep. Med.\",\n \"Chem.\",\n \"24:243-252, 1989, incorporated herein by reference).\",\n \"These techniques include replacing the peptide backbone with a backbone composed of phosphonates, amidates, carbamates, sulfonamides, secondary amines, and/or N-methylamino acids.\",\n \"Peptide mimetics wherein one or more of the peptidyl linkages —C(O)NH— have been replaced by such linkages as a —CH2-carbamate linkage, a phosphonate linkage, a —CH2-sulfonamide linkage, a urea linkage, a secondary amine (—CH2NH—) linkage, and an alkylated peptidyl linkage —C(O)NR6— where R6 is lower alkyl are prepared during conventional peptide synthesis by merely substituting a suitably protected amino acid analogue for the amino acid reagent at the appropriate point during synthesis.\",\n \"Suitable reagents include, for example, amino acid analogues wherein the carboxyl group of the amino acid has been replaced with a moiety suitable for forming one of the above linkages.\",\n \"For example, if one desires to replace a —C(O)NR— linkage in the peptide with a —CH2-carbamate linkage (—CH2OC(O)NR—), then the carboxyl (—COOH) group of a suitably protected amino acid is first reduced to the —CH2OH group which is then converted by conventional methods to a —OC(O)Cl functionality or a para-nitrocarbonate —OC(O)O—C6H4-p-NO2 functionality.\",\n \"Reaction of either of such functional groups with the free amine or an alkylated amine on the N-terminus of the partially fabricated peptide found on the solid support leads to the formation of a —CH2OC(O)NR— linkage.\",\n \"For a more detailed description of the formation of such —CH2-carbamate linkages, see, e.g., Cho et al., (Science 261:1303-1305, 1993, incorporated herein by reference).\",\n \"Replacement of an amido linkage in an anti-coreceptor binding peptide with a —CH2-sulfonamide linkage can be achieved by reducing the carboxyl (—COOH) group of a suitably protected amino acid to the —CH2OH group, and the hydroxyl group is then converted to a suitable leaving group such as a tosyl group by conventional methods.\",\n \"Reaction of the tosylated derivative with, for example, thioacetic acid followed by hydrolysis and oxidative chlorination will provide for the —CH2—S(O)2Cl functional group which replaces the carboxyl group of the otherwise suitably protected amino acid.\",\n \"Use of this suitably protected amino acid analogue in peptide synthesis provides for inclusion of an —CH2S(O)2NR— linkage which replaces the amido linkage in the peptide thereby providing a peptide mimetic.\",\n \"For a more complete description on the conversion of the carboxyl group of the amino acid to a —CH2S(O)2Cl group, see, e.g., Weinstein and Boris (Chemistry & Biochemistry of Amino Acids.\",\n \"Peptides and Proteins, Vol.\",\n \"7, pp.\",\n \"267-357, Marcel Dekker, Inc., New York, 1983, incorporated herein by reference).\",\n \"Replacement of an amido linkage in an anti-coreceptor binding peptide with a urea linkage can be achieved in the manner known to the skilled artisan.\",\n \"Secondary amine linkages wherein a —CH2NH— linkage replaces the amido linkage in the peptide can be prepared by employing, for example, a suitably protected dipeptide analogue wherein the carbonyl bond of the amido linkage has been reduced to a CH2 group by conventional methods.\",\n \"For example, in the case of diglycine, reduction of the amide to the amine will yield after deprotection H2NCH2CH2NHCH2 COOH which is then used in N-protected form in the next coupling reaction.\",\n \"The preparation of such analogues by reduction of the carbonyl group of the amido linkage in the dipeptide is well known in the art.\",\n \"The anti-coreceptor binding agents of the present invention may exist in a monomeric form with no disulfide bond formed with the thiol groups of the cysteine residue(s).\",\n \"Alternatively, an intermolecular disulfide bond between the thiol groups of cysteines on two or more peptides can be produced to yield a multimeric (e.g., dimeric, tetrameric or higher oligomeric) compound.\",\n \"Certain of these peptides can be cyclized or dimerized via displacement of the leaving group by the sulfur of a cysteine or homocysteine residue (see, e.g., Barker et al., J. Med.\",\n \"Chem.\",\n \"35:2040-2048, 1992; and/or et al., J. Org.\",\n \"Chem.\",\n \"56:3146-3149, 1991, each incorporated herein by reference).\",\n \"Thus, one or more native cysteine residues may be substituted with a homocysteine.\",\n \"Intramolecular or intermolecular disulfide derivatives of anti-coreceptor binding agents provide analogs in which one of the sulfurs has been replaced by a CH2 group or other isostere for sulfur.\",\n \"These analogs can be made via an intramolecular or intermolecular displacement, using methods known in the art as shown below.\",\n \"One of skill in the art will readily appreciate that this displacement can also occur using other homologs of the a-amino-g-butyric acid derivative shown above and homocysteine.\",\n \"All of the naturally occurring, recombinant, and synthetic peptides and peptide analogs and mimetics of the invention can be used for screening (e.g., in kits and/or screening assay methods) to identify additional compounds, including other peptides and peptide mimetics, that will function as anti-coreceptor binding agents within the methods and compositions of the invention.\",\n \"Several methods of automating assays have been developed in recent years so as to permit screening of tens of thousands of compounds in a short period (see, e.g., Fodor et al., Science 251:767-773, 1991, and U.S. Pat.\",\n \"Nos.\",\n \"5,677,195; 5,885,837; 5,902,723; 6,027,880; 6,040,193; and 6,124,102, each incorporated herein by reference).\",\n \"For example, large combinatorial libraries of the compounds can be constructed by the encoded synthetic libraries (ESL) method described in, e.g., WO 95/12608, WO 93/06121, WO 94/08051, WO 95/35503, and WO 95/30642 (each incorporated by reference).\",\n \"Peptide libraries can also be generated by phage display methods (see, e.g., WO 91/18980, incorporated herein by reference).\",\n \"Many other publications describing chemical diversity libraries and screening methods are also considered reflective of the state of the art pertaining to these aspects of the invention and are generally incorporated herein.\",\n \"In one general screening strategy within the invention, new agonists and antagonists against HIV-coreceptor binding can be readily identified using the anti-coreceptor binding peptides of the invention incorporated within highly automated assay methods, e.g., using a purified chemokine receptor.\",\n \"Of particular importance are antagonist compounds that have multi-tropic or multi-specific binding affinity or activity, i.e., which inhibit or block HIV interactions with distinct (e.g., both CXCR4 and CCR5) coreceptors and thereby inhibit or prevent infection by both T cell tropic (lymphotropic) and macrophage tropic (m-tropic) HIV strains.\",\n \"One method of screening for new anti-coreceptor binding agents (e.g., small molecule drug peptide mimetics) utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant DNA molecules expressing an anti-coreceptor binding peptide.\",\n \"Such cells, either in viable or fixed form, can be used for standard ligand/receptor binding assays (see, e.g., Parce et al., Science 246:243-247, 1989; and Owicki et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 87:4007-4011, 1990, each incorporated herein by reference).\",\n \"Competitive assays are particularly useful, where the cells are contacted and incubated with a labeled receptor or antibody having known binding affinity to the peptide ligand, and a test compound or sample whose binding affinity is being measured.\",\n \"The bound and free labeled binding components are then separated to assess the degree of ligand binding.\",\n \"The amount of test compound bound is inversely proportional to the amount of labeled receptor binding to the known source.\",\n \"Any one of numerous techniques can be used to separate bound from free ligand to assess the degree of ligand binding.\",\n \"This separation step can involve a conventional procedure such as adhesion to filters followed by washing, adhesion to plastic followed by washing, or centrifugation of the cell membranes.\",\n \"Viable cells can also be used to screen for the effects of drugs on coreceptor-mediated functions and biological activities, e.g., HIV viral fusion, cell entry, replication, and the like.\",\n \"Some detection methods allow for elimination of a separation step, e.g., a proximity sensitive detection system.\",\n \"Another technique for drug screening within the invention involves an approach which provides high throughput screening for compounds having suitable binding affinity to a target molecule, e.g., a chemokine receptor, and is described in detail in European Patent Application 84/03564, published on Sep. 13, 1984.First, large numbers of different test compounds, e.g., small peptides, are synthesized on a solid substrate, e.g., plastic pins or some other appropriate surface, (see, e.g., Fodor et al., Science 251:767-773, 1991, and U.S. Pat.\",\n \"Nos.\",\n \"5,677,195; 5,885,837; 5,902,723; 6,027,880; 6,040,193; and 6,124,102, each incorporated herein by reference).\",\n \"Then all of the pins are reacted with a solubilized anti-coreceptor binding agent of the invention, and washed.\",\n \"The next step involves detecting bound anti-coreceptor binding agent.\",\n \"Rational drug design may also be based upon structural studies of the molecular shapes of the anti-coreceptor binding agents.\",\n \"Various methods are available and well known in the art for characterizing, mapping, translating, and reproducing structural features of anti-coreceptor binding agents to guide the production and selection of new anti-coreceptor binding mimetics, including for example x-ray crystallography and 2 dimensional NMR techniques.\",\n \"These and other methods, for example, will allow reasoned prediction of which amino acid residues present in a selected anti-coreceptor binding peptide forms molecular contact regions necessary for peptide-coreceptor binding and specificity (see, e.g., Blundell and Johnson, Protein Crystallography, Academic Press, N.Y., 1976, incorporated herein by reference).\",\n \"Operable anti-coreceptor binding analogs and mimetics within the invention retain partial, complete or enhanced activity compared to native anti-coreceptor binding peptides, for example, partial or complete activity for inhibiting HIV-coreceptor binding, HIV viral fusion, cell entry, and/or replication, or HIV-related disease occurrence or progression.\",\n \"In this regard, operable anti-coreceptor binding analogs and mimetics for use within the invention will retain at least 50%, often 75%, and up to 95-100% or greater levels of one or more selected activities as compared to the same activity observed for a selected native HIV-1 gp120 anti-coreceptor binding peptide.\",\n \"These biological properties of altered peptides or non-peptide mimetics can be determined according to any suitable assay disclosed or incorporated herein, for example, by determining the ability of an anti-coreceptor binding analog or mimetic to inhibit HIV viral fusion to coreceptor positive target cells.\",\n \"In accordance with the description herein, the compounds of the invention are useful in vitro as unique tools for analyzing the nature and function of gp120 interactions with chemokine receptors, and will also serve as leads in various programs for designing additional peptide and non-peptide (e.g., small molecule drug) inhibitors of HIV-1.In addition, the anti-coreceptor binding peptides and peptide analogs of the invention are useful as immunogens, or components of immunogens, for eliciting an immune response in mammalian subjects, for example, to provide a protective immune response to prevent or treat HIV infection.\",\n \"In this aspect, the peptides of the invention can be administered alone or in a formulation comprising the peptide and a pharmaceutically acceptable carrier or adjuvant, with or without additional active or inactive ingredients such immune modulatory agents (e.g., cytokines).\",\n \"In certain embodiments, the peptides are administered as immunogens in the form of a conjugate (e.g., a multimeric peptide, or a peptide/carrier or peptide/hapten conjugate).\",\n \"In one embodiment, a peptide is conjugated with a multimerizing polypeptide as described above.\",\n \"Alternatively, a multimeric construct of immunogenic peptides, for example, comprising repeat peptide subunits, or containing two or more different peptides, can be employed, which contain one or multiple immunogenic epitope(s) that elicit a specific, humoral and/or cell-mediated (e.g.\",\n \"CTL) immune response directed against the immunizing peptide(s).\",\n \"Typically, the immune response will be marked by production of antibodies that bind the immunizing peptide(s) or peptide conjugate(s) with high affinity or avidity, but do not similarly recognize unrelated peptides.\",\n \"In other embodiments, the antibodies recognize the immunizing peptide(s) or peptide conjugate(s) but fail to bind with high affinity or avidity to chemokines, or to peptides derived from chemokines.\",\n \"In yet additional embodiments, antibodies generated against the anti-coreceptor binding peptide(s) or conjugate(s) bind to gp120 only when the gp120 protein is in a “bound” or “activated” state.\",\n \"For example, when gp120 is bound to a CD4 receptor this may result in a conformational change or other activation event that exposes a gp120 coreceptor binding domain.\",\n \"Antibodies that specifically target gp120 during this vulnerable activation period, (e.g., by recognizing one or more “masked” or “hidden” epitopes of gp120 that are not exposed for immune targeting when gp120 is in a “free” conformation or activity state, are particularly useful within the invention.\",\n \"Thus, the invention also provides diagnostic and therapeutic antibodies, including monoclonal antibodies, and related compositions and methods for use in the management and treatment of HIV infection and related disease.\",\n \"The antibodies specifically recognize anti-coreceptor binding peptides of the invention and are therefore useful for blocking HIV-coreceptor interactions when administered in vivo.\",\n \"For example, monoclonal antibodies can be generated that specifically bind the anti-coreceptor binding peptides of the invention, which antibodies can be purified and administered to a patient to block or inhibit HIV-coreceptor interactions, including binding or “docking,” and HIV viral fusion, entry, replication and HIV-related disease occurrence or progression.\",\n \"These activities are typically mediated, at least in part, by in vivo binding of the antibodies to gp120 (and therefore to intact HIV virus), at least in the protein's activated state upon binding to CD4.These immunotherapeutic reagents often include humanized antibodies, and can be combined for therapeutic use with additional active or inert ingredients as disclosed herein, e.g., in conventional pharmaceutically acceptable carriers or diluents, e.g., immunogenic adjuvants, and optionally with adjunctive or combinatorially active agents such as antiretroviral drugs.\",\n \"The production of non-human monoclonal antibodies, e.g., murine or rat, can be accomplished by, for example, immunizing the animal with a preparation comprising purified peptides of the invention, or purified gp120.The immunogen, often comprising a peptide/hapten complex or other conjugate as described herein, can be obtained from a natural source, by peptides synthesis or preferably by recombinant expression.\",\n \"Antibody-producing cells obtained from the immunized animals are immortalized and screened for the production of an antibody which binds to gp120 or a specific anti-coreceptor binding peptide (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual, Cold Springs Harbor Press, Cold Spring Harbor, N.Y., 1988, incorporated by reference for all purposes).\",\n \"Humanized forms of mouse antibodies can be generated by linking the CDR regions of non-human antibodies to human constant regions by recombinant DNA techniques (see, e.g., Queen et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 86:10029-10033, 1989 and WO 90/07861, each incorporated by reference).\",\n \"Human antibodies can be obtained using phage-display methods (see, e.g., WO 91/17271; WO 92/01047, each incorporated herein by reference).\",\n \"In these methods, libraries of phage are produced in which members display different antibodies on their outer surfaces.\",\n \"Antibodies are usually displayed as Fv or Fab fragments.\",\n \"Phage displaying antibodies with a desired specificity are selected by affinity enrichment to human cytochrome P450 or a fragment thereof.\",\n \"Human antibodies are selected by competitive binding experiments, or otherwise, to have the same epitope specificity as a particular mouse antibody.\",\n \"The invention further provides fragments of the intact antibodies described above.\",\n \"Typically, these fragments compete with the intact antibody from which they were derived for specific binding to anti-coreceptor binding peptides and/or gp120.Antibody fragments include separate heavy chains, light chains Fab, Fab′ F(ab′)2, Fv, and single chain antibodies.\",\n \"Fragments can be produced by enzymic or chemical separation of intact immunoglobulins.\",\n \"For example, a F(ab′)2 fragment can be obtained from an IgG molecule by proteolytic digestion with pepsin at pH 3.0-3.5 using standard methods such as those described in Harlow and Lane, supra.\",\n \"Fab fragments may be obtained from F(ab′)2 fragments by limited reduction, or from whole antibody by digestion with papain in the presence of reducing agents.\",\n \"Fragments can also be produced by recombinant DNA techniques.\",\n \"Segments of nucleic acids encoding selected fragments are produced by digestion of full-length coding sequences with restriction enzymes, or by de novo synthesis.\",\n \"Often fragments are expressed in the form of phage-coat fusion proteins.\",\n \"This manner of expression is advantageous for affinity-sharpening of antibodies.\",\n \"To produce antibodies of the invention recombinantly, nucleic acids encoding light and heavy chain variable regions, optionally linked to constant regions, are inserted into expression vectors.\",\n \"The light and heavy chains can be cloned in the same or different expression vectors.\",\n \"The DNA segments encoding antibody chains are operably linked to control sequences in the expression vector(s) that ensure the expression of antibody chains.\",\n \"Such control sequences include a signal sequence, a promoter, an enhancer, and a transcription termination sequence.\",\n \"Expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosome.\",\n \"E. coli is one prokaryotic host particularly useful for expressing antibodies of the present invention.\",\n \"Other microbial hosts suitable for use include bacilli, such as Bacillus subtilus, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species.\",\n \"In these prokaryotic hosts, one can also make expression vectors, which typically contain expression control sequences compatible with the host cell (e.g., an origin of replication) and regulatory sequences such as a lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda.\",\n \"Other microbes, such as yeast, may also be used for expression.\",\n \"Saccharomyces is a preferred host, with suitable vectors having expression control sequences, such as promoters, including 3-phosphoglycerate kinase or other glycolytic enzymes, and an origin of replication, termination sequences and the like as desired.\",\n \"Mammalian tissue cell culture can also be used to express and produce the antibodies of the present invention (see, e.g., Winnacker, From Genes to Clones, VCH Publishers, N.Y., 1987, incorporated herein by reference).\",\n \"Eukaryotic cells are preferred, because a number of suitable host cell lines capable of secreting intact antibodies have been developed.\",\n \"Preferred suitable host cells for expressing nucleic acids encoding the immunoglobulins of the invention include: monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293) (Graham et al., J. Gen. Virol.\",\n \"36:59, 1977, incorporated herein by reference); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary-cells-DHFR (CHO, Urlaub and Chasin, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 77:4216, 1980, incorporated herein by reference); mouse sertoli cells (TM4, Mather, Biol.\",\n \"Reprod.\",\n \"23:243-251, 1980, incorporated herein by reference); monkey kidney cells (CVI ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL 1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); and, TRI cells (Mather et al., Annals N.Y. Acad.\",\n \"Sci.\",\n \"383:44-46, 1982, incorporated herein by reference); and baculovirus cells.\",\n \"The vectors containing the polynucleotide sequences of interest (e.g., the heavy and light chain encoding sequences and expression control sequences) can be transferred into the host cell.\",\n \"Calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation can be used for other cellular hosts (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989, incorporated herein by reference).\",\n \"When heavy and light chains are cloned on separate expression vectors, the vectors are co-transfected to obtain expression and assembly of intact immunoglobulins.\",\n \"After introduction of recombinant DNA, cell lines expressing immunoglobulin products are cell selected.\",\n \"Cell lines capable of stable expression are preferred (i.e., undiminished levels of expression after fifty passages of the cell line).\",\n \"Once expressed, the whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms of the present invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see, e.g., Scopes, Protein Purification, Springer-Verlag, N.Y., 1982, incorporated herein by reference).\",\n \"Substantially pure immunoglobulins of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity most preferred.\",\n \"The anti-coreceptor binding agents of the invention can also generally be used in drug screening compositions and procedures, as noted above, to identify additional compounds having binding affinity to chemokine receptors and/or act as agonists or antagonists to gp120-coreceptor or chemokine-chemokine receptor interactions and related biological activities.\",\n \"Various screening methods and formats are available and well known in the art.\",\n \"Subsequent biological assays can then be utilized to determine if the screened compound has intrinsic coreceptor binding, agonist or antagonist, or other desired activity useful within the invention.\",\n \"Thus, in one example, the anti-coreceptor binding agents of the invention are useful as competitive binding agents in assays to screen for new HIV coreceptor agonists and antagonists.\",\n \"In such assays, the compounds of the invention can be used without modification or can be modified in a variety of ways; for example, by labeling, such as covalently or non-covalently joining a moiety which directly or indirectly provides a detectable signal.\",\n \"Direct labeling moieties include, for example, radiolabels, enzymes such as peroxidase and alkaline phosphatase (see, e.g., U.S. Pat.\",\n \"No.\",\n \"3,645,090; and U.S. Pat.\",\n \"No.\",\n \"3,940,475, each incorporated herein by reference), and fluorescent labels.\",\n \"Moieties for indirect labeling include biotin and avidin, a binding pair that can be coupled to one constituent and the other to a label.\",\n \"The compounds may also include spacers or linkers in cases where the compounds are to be attached to a solid support.\",\n \"The anti-coreceptor binding agents of the invention can also be employed, based on their ability to bind HIV coreceptors (chemokine receptors), as reagents for detecting and/or quantifying HIV coreceptors on living cells, fixed cells, in biological fluids, in tissue homogenates, in purified, natural biological materials, and the like.\",\n \"For example, by labeling such peptides, one can identify and/or quantify cells having HIV coreceptors on their surfaces.\",\n \"In addition, based on their ability to bind HIV coreceptors, the peptides of the present invention can be used in in situ staining, FACS (fluorescence-activated cell sorting), Western blotting, ELISA, and the like.\",\n \"Further, the peptides of the present invention can be used in receptor purification, or in purifying cells expressing HIV coreceptors.\",\n \"The compounds of the present invention can also be utilized as commercial reagents for various medical research and diagnostic uses.\",\n \"Such uses include but are not limited to: (1) use as a calibration standard for quantitating the activities of candidate HIV-coreceptor binding agonists and antagonists in a variety of functional assays; (2) use in structural analysis of HIV coreceptors through co-crystallization; and (3) use to investigate the mechanism of HIV coreceptor binding and activation.\",\n \"The present invention also provides reagents, formulations, kits, and methods which provide significant prophylactic and therapeutic values.\",\n \"In one embodiment of the invention, methods and compositions employ an anti-coreceptor binding agent for preventing and/or inhibiting HIV-1 binding to a host cell thereby ameliorating HIV, i.e., HIV-1 infection or a selected disease or condition associated therewith.\",\n \"Additional methods and compositions are provided to treat, prevent or delay the occurrence or AIDS or ARC.\",\n \"In yet additional embodiments, the methods and compositions of the invention can be used to treat other diseases and conditions which benefit from the compositions and methodologies disclosed herein, for example, a specific HIV-related disease or condition, such as Kaposi's sarcoma or an opportunistic viral (e.g., herpes) or bacterial (e.g., pneumonia) infection or condition.\",\n \"In accordance with the various treatment methods of the invention, the anti-coreceptor binding agent is delivered to a patient or other subject in a manner consistent with conventional methodologies associated with management of the disorder for which treatment or prevention is sought.\",\n \"In accordance with the disclosure herein, a prophylactically or therapeutically effective amount of an anti-coreceptor binding agent is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent and/or inhibit HIV binding to a host cell thereby preventing and/or inhibiting HIV infection and ameliorating a selected disease or condition.\",\n \"The term “subject” as used herein means any mammalian patient to which the compositions of the invention may be administered.\",\n \"Typical subjects intended for treatment with the compositions and methods of the present invention include humans, as well as non-human primates and other animals.\",\n \"Alternate subjects for administration of anti-coreceptor binding agents of the invention (either for a diagnostic, analytic, disease management or therapeutic purpose) include cells, cell explants, tissues and organs, particularly those originating from mammalian subjects at risk of developing or presently suffering from HIV infection.\",\n \"To identify subject patients for prophylaxis or treatment according to the methods of the invention, accepted screening methods are employed to determine risk factors associated with HIV infection, or to determine the status of an existing HIV infection or related condition in a subject.\",\n \"These screening methods include, for example, conventional work-ups to determine sexual and drug-use related risk factors, as well as diagnostic methods such as various ELISA immunoassay methods, which are available and well known in the art to detect and/or characterize HIV infection and related disease.\",\n \"These and other routine methods allow the clinician to select patients in need of therapy using the anti-coreceptor binding agents of the invention.\",\n \"In accordance with these methods and principles, anti-coreceptor binding agent therapy can be implemented as an independent prophylaxis or treatment program or as a follow-up, adjunct or coordinate treatment regimen to other treatments, for example other anti-HIV treatments such as drug therapy (AZT, DDI, protease inhibitors and other anti-retroviral drugs), surgery, vaccination, immunotherapy, hormone treatment, cell, tissue, or organ transplants, and the like.\",\n \"Within the compositions and methods of the invention, the anti-coreceptor binding agent is typically formulated with a pharmaceutically acceptable carrier and administered in an amount sufficient to inhibit virus binding and initiation or progression of HIV infection or a related disease or condition in the subject.\",\n \"According to the methods of the invention, the anti-coreceptor binding agent can be administered to subjects by a variety of administration modes, including by intramuscular, subcutaneous, intravenous, intra-atrial, intra-articular, intraperitoneal, parenteral, oral, rectal, intranasal, intrapulmonary, transdermal or topically to the eyes, ears, skin or mucosal surfaces.\",\n \"Alternatively, the anti-coreceptor binding agent may be administered ex vivo by direct exposure to cells, tissues or organs originating from a mammalian subject, for example, as a component of an ex vivo tissue or organ treatment formulation that contains the anti-coreceptor binding agent in a biologically suitable, liquid or solid carrier.\",\n \"For prophylactic and treatment purposes, the anti-coreceptor binding agent can be administered to the subject in a single bolus delivery, via continuous delivery (e.g., continuous intravenous or transdermal delivery) over an extended time period, or in a repeated administration protocol (e.g., on an hourly, daily or weekly basis).\",\n \"The various dosages and delivery protocols contemplated for administration of anti-coreceptor binding agents are therapeutically effective to inhibit the occurrence or alleviate one or more symptoms of HIV infection.\",\n \"An “anti-HIV therapeutically effective amount” of the anti-coreceptor binding agent thus refers to an amount that is effective, at dosages and for periods of time necessary, to achieve detectable inhibition of HIV binding and/or infection (initiation or progression) or a related condition (e.g., in HIV-exposed versus unexposed, or treated versus untreated, test and control subjects).\",\n \"In certain embodiments, a therapeutically effective amount of the anti-coreceptor binding agent, depending on the selected mode, frequency and duration of administration, will be effective to reduce or prevent HIV binding and infection of cells of the patient.\",\n \"Alternatively or in addition to these effects, a therapeutically effective dosage of the anti-coreceptor binding agent, which can include repeated doses within an prolonged prophylaxis or treatment regimen, will alleviate one or more symptoms or detectable conditions associated with HIV infection.\",\n \"Determination of effective dosages in this context is typically based on animal model studies followed up by human clinical trials and is guided by determining effective dosages and administration protocols that significantly reduce the occurrence or severity of HIV infection or related disease symptoms or conditions in the subject, which may be any of a range of accepted, e.g., murine or non-human primate, animal model subjects known in the art.\",\n \"Alternatively, effective dosages can be determined using in vitro models (e.g., immunologic and histopathologic assays).\",\n \"Using such models, only ordinary calculations and adjustments are typically required to determine an appropriate concentration and dose to administer an effective amount of anti-coreceptor binding agent (e.g., intranasally effective, transdermally effective, intravenously effective, or intramuscularly effective) to a subject to elicit a desired response.\",\n \"In alternative embodiments, an “effective amount” or “effective dose” of the anti-coreceptor binding agent may simply inhibit one or more selected biological activity(ies) correlated with HIV-coreceptor binding, as set forth above.\",\n \"The actual dosage of anti-coreceptor binding agent will of course vary according to factors such as the risk or state of infection or disease, the subject's age, and weight, as well as the established potency of the anti-coreceptor binding agent for eliciting the desired activity or biological response in the subject.\",\n \"Dosage regimens can be adjusted to provide an optimum prophylactic therapeutic response.\",\n \"A therapeutically effective amount is also one in which any toxic or detrimental side effects of the anti-coreceptor binding agent is outweighed by therapeutically beneficial effects.\",\n \"A non-limiting range for a therapeutically effective amount of the anti-coreceptor binding agent 0.01 μg/kg-10 mg/kg, more typically between about 0.05 and 5 mg/kg, and in certain embodiments between about 0.2 and 2 mg/kg.\",\n \"Dosages within this range can be achieved by single or multiple administrations, including, e.g., multiple administrations per day, daily or weekly administrations.\",\n \"Per administration, it is desirable to administer at least one microgram of anti-coreceptor binding agent, more typically between about 10 μg and 5.0 mg, and in certain embodiments between about 100 μg and 1.0 or 2.0 mg to an average human subject.\",\n \"It is to be further noted that for each particular subject, specific dosage regimens should be evaluated and adjusted over time according to the individual need and professional judgment of the person administering or supervising the administration of the anti-coreceptor binding agent compositions.\",\n \"Dosage of the anti-coreceptor binding agent can be varied by the attending clinician to maintain a desired concentration at the target site.\",\n \"For example, if an intravenous mode of delivery is selected local concentration of the anti-coreceptor binding agent in the bloodstream at a selected target tissue (e.g., circulating blood) can be between about 1-50 nanomoles of anti-coreceptor binding agent per liter, sometimes between about 1.0 nanomelia per liter and 10, 15 or 25 nanomoles per liter, depending on the subject's status and projected or measured response.\",\n \"Higher or lower concentrations can be selected based on the mode of delivery, e.g., trans-epidermal delivery versus delivery to a mucosal surface.\",\n \"Dosage should also be adjusted based on the release rate of the administered formulation, e.g., nasal spray versus powder, sustained release oral versus injected particle or transdermal delivery formulations, and the like.\",\n \"To achieve the same serum concentration level, for example, slow-release particles with a release rate of 5 nanomolar (under standard conditions) would be administered at about twice the dosage of particles with a release rate of 10 nanomolar.\",\n \"Anti-coreceptor binding agents comprising HIV-1 gp120 peptides and peptide analogs can be readily constructed using peptide synthetic techniques, such as solid phase peptide synthesis (Merrifleld synthesis), and the like, or by recombinant DNA techniques, that are well known in the art.\",\n \"Techniques for making substitution mutations at predetermined sites in DNA include for example M13 mutagenesis.\",\n \"Manipulation of DNA sequences to produce substitutional, insertional, or deletional variants are conveniently described elsewhere such as Sambrook et al., (Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989).\",\n \"In accordance with these and related teachings, defined mutations can be introduced into a native gp120 peptide to generate analogs of interest by a variety of conventional techniques, e.g., site-directed mutagenesis of a cDNA copy of a portion of the gp120 gene encoding a selected peptide fragment, domain or motif.\",\n \"This can be achieved through and intermediate of single-stranded form, such as using the MUTA-gen® kit of Bio-Rad Laboratories (Richmond, Calif.), or a method using the double-stranded plasmid directly as a template such as the Chameleon® mutagenesis kit of Strategene (La Jolla, Calif.), or by the polymerase chain reaction employing either an oligonucleotide primer or a template which contains the mutation(s) of interest.\",\n \"A mutated subfragment can then be assembled into a complete anti-coreceptor binding peptide analog-encoding cDNA.\",\n \"A variety of other mutagenesis techniques are known and can be routinely adapted for use in producing the mutations of interest in a anti-coreceptor binding peptide analog-encoding cDNA and corresponding peptide analog of the invention.\",\n \"In accordance with the present invention, anti-coreceptor binding agents are isolated and purified before administration to a subject so that contaminants are removed.\",\n \"In one method for obtaining purified anti-coreceptor binding peptides, a polynucleotide molecule, for example, a deoxyribonucleic acid (DNA) molecule, that defines a coding sequence for a selected anti-coreceptor binding peptide or peptide analog (e.g., a biologically active mutant or fragment of a peptide as disclosed herein bearing a deletion or substitution of 1, 2, 3 or more residues) is operably incorporated in a recombinant polynucleotide expression vector that direct expression of the peptide or analog in a suitable host cell.\",\n \"Exemplary methods for cloning and purifying anti-coreceptor binding peptides and analogs employing these novel polynucleotides and vectors are widely known in the art.\",\n \"Briefly, a polynucleotide of the invention encoding an anti-coreceptor binding peptide or peptide analog is amplified by well known methods, such as the polymerase chain reaction (PCR).\",\n \"In this way the polynucleotide encoding the anti-coreceptor binding peptide, or a recombinantly modified version or portion thereof, is obtained for expression and purification according to conventional methods.\",\n \"A DNA vector molecule that incorporates a DNA sequence encoding the subject peptide or analog can be operatively assembled, e.g., by linkage using appropriate restriction fragments from various plasmids which are described elsewhere.\",\n \"Also contemplated by the present invention are ribonucleic acid (RNA) equivalents of the above described polynucleotides comprising a coding sequence for the subject anti-coreceptor binding peptide operatively linked in a polynucleotide expression construct for recombinant expression of the peptide or peptide analog.\",\n \"Once a polynucleotide molecule encoding an anti-coreceptor binding peptide or analog is isolated and cloned, the peptide or analog can be expressed in a variety of recombinantly engineered cells.\",\n \"Numerous expression systems are available for expressing a DNA encoding anti-coreceptor binding peptide.\",\n \"The expression of natural or synthetic nucleic acids encoding an anti-coreceptor binding peptide is typically achieved by operably linking the DNA to a promoter (which is either constitutive or inducible) within an expression vector.\",\n \"By expression vector is meant a polynucleotide molecule, linear or circular, that comprises a segment encoding the anti-coreceptor binding peptide of interest, operably linked to additional segments that provide for its transcription.\",\n \"Such additional segments include promoter and terminator sequences.\",\n \"An expression vector also may include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like.\",\n \"Expression vectors generally are derived from plasmid or viral DNA, and can contain elements of both.\",\n \"The term “operably linked” indicates that the segments are arranged so that they function in concert for their intended purposes, for example, transcription initiates in the promoter and proceeds through the coding segment to the terminator (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989, incorporated herein by reference).\",\n \"Expression vectors can be constructed which contain a promoter to direct transcription, a ribosome binding site, and a transcriptional terminator.\",\n \"Examples of regulatory regions suitable for this purpose in E. coli are the promoter and operator region of the E. coli tryptophan biosynthetic pathway as described by Yanofsky, (J. Bacteriol.\",\n \"158:1018-1024, 1984, incorporated herein by reference) and the leftward promoter of phage lambda (Pk) as described by Herskowitz and Hagen, (Ann.\",\n \"Rev.\",\n \"Genet.\",\n \"14:399-445, 1980, incorporated herein by reference).\",\n \"The inclusion of selection markers in DNA vectors transformed in E. coli is also useful.\",\n \"Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol.\",\n \"Vectors used for expressing foreign genes in bacterial hosts generally will contain a selectable marker, such as a gene for antibiotic resistance, and a promoter which functions in the host cell.\",\n \"Plasmids useful for transforming bacteria include pBR322 (Bolivar et al., Gene 2:95-113, 1977, incorporated herein by reference), the pUC plasmids (Messing, Meth.\",\n \"Enzymol.\",\n \"101:20-77, 1983; Vieira and Messing, Gene 19:259-268.1982, each incorporated herein by reference), pCQV2, and derivatives thereof Plasmids may contain both viral and bacterial elements.\",\n \"A variety of prokaryotic expression systems can be used to express anti-coreceptor binding peptides and peptide analogs.\",\n \"Examples include E. coli, Bacillus, Streptomyces, and the like.\",\n \"Detection of the expressed peptide is achieved by methods such as radioimmunoassay, Western blotting techniques or immunoprecipitation.\",\n \"For expression in eukaryotes, host cells for use in practicing the invention include mammalian, avian, plant, insect, and fungal cells.\",\n \"Fungal cells, including species of yeast (e.g., Saccharomyces spp., Schizosaccharomyces spp.)\",\n \"or filamentous fungi (e.g., Aspergillus spp., Neurospora spp.)\",\n \"can be used as host cells within the present invention.\",\n \"Strains of the yeast Saccharomyces cerevisiae can be used.\",\n \"As explained briefly below, anti-coreceptor binding peptides and analogs can be expressed in these eukaryotic systems.\",\n \"Suitable yeast vectors for use in the present invention include YRp7 (Struhl et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 76:1035-1039, 1978, incorporated herein by reference), YEp13 (Broach et al., Gene 8:121-133, 1979, incorporated herein by reference), POT vectors (U.S. Pat.\",\n \"No.\",\n \"4,931,373, incorporated herein by reference), pJDB249 and pJDB219 (Beggs, Nature 275:104-108, 1978, incorporated herein by reference) and derivatives thereof.\",\n \"Such vectors generally include a selectable marker, which can be one of any number of genes that exhibit a dominant phenotype for which a phenotypic assay exists to enable transformants to be selected.\",\n \"Often, the selectable marker will be one that complements host cell auxotrophy, provides antibiotic resistance and/or enables a cell to utilize specific carbon sources, for example, LEU2 (Broach et al., Gene 8:121-133, 1979), URA3 (Botstein et al., Gene 8:17, 1979, incorporated herein by reference), HIS3 (Struhl et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 76:1035-1039, 1978) or POT1 (U.S. Pat.\",\n \"No.\",\n \"4,931,373).\",\n \"Another suitable selectable marker available for use within the invention is the CAT gene, which confers chloramphenicol resistance on yeast cells.\",\n \"Examples of promoters for use in yeast include promoters from yeast glycolytic genes (Hitzeman et al., J. Biol.\",\n \"Chem.\",\n \"255:12073-12080, 1980; Alber and Kawasaki, J. Mol.\",\n \"Appl.\",\n \"Genet.\",\n \"1:419434, 1982; U.S. Pat.\",\n \"No.\",\n \"4,599,311) or alcohol dehydrogenase genes (Young et al., Genetic Engineering of Microorganisms for Chemicals, Hollaender et al., eds., p. 355, Plenum, N.Y., 1982; Ammerer, Meth.\",\n \"Enzymol.\",\n \"101:192-201, 1983).\",\n \"The TPI1 promoter (U.S. Pat.\",\n \"No.\",\n \"4,599,311) and the ADH2-4c promoter (Russell et al., Nature 304:652-654, 1983; and EP 284,044) also can be used.\",\n \"The expression units can also include a transcriptional terminator.\",\n \"An example of such a transcriptional terminator is the TPI1 terminator (Alber and Kawasaki, J. Mol.\",\n \"Appl.\",\n \"Genet.\",\n \"1:419-434, 1982).\",\n \"In addition to yeast, anti-coreceptor binding peptides and peptide analogs of the present invention can be expressed in filamentous fungi, for example, strains of the fungi Aspergillus (U.S. Pat.\",\n \"No.\",\n \"4,935,349, which is incorporated herein by reference).\",\n \"Examples of useful promoters include those derived from Aspergillus nidulans glycolytic genes, such as the ADH3 promoter and the tpiA promoter.\",\n \"An example of a suitable terminator is the ADH3 terminator (McKnight et al., EMBO J.\",\n \"4: 2093-2099, 1985, incorporated herein by reference).\",\n \"The expression units utilizing such components are cloned into vectors that are capable of insertion into the chromosomal DNA of Aspergillus.\",\n \"Techniques for transforming fungi are well known in the literature, and have been described, for instance, by Beggs (Nature 275:104-108, 1978), Hinnen et al., (Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 75:1929-1933, 1978), Yelton et al., (Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 81:1740-1747, 1984), and Russell (Nature 301:167-169, 1983), each incorporated herein by reference.\",\n \"The genotype of the host cell generally contains a genetic defect that is complemented by the selectable marker present on the expression vector.\",\n \"Choice of a particular host and selectable marker is well within the level of ordinary skill in the art.\",\n \"In addition to fungal cells, cultured mammalian cells can be used as host cells within the present invention.\",\n \"Examples of cultured mammalian cells for use in the present invention include the COS-1 (ATCC CRL 1650), BHK, and 293 (ATCC CRL 1573; Graham et al., J. Gen. Virol.\",\n \"6:59-72, 1977, incorporated herein by reference) cell lines.\",\n \"An example of a BHK cell line is the BHK 570 cell line (deposited with the American Type Culture Collection under accession number CRL 10314).\",\n \"In addition, a number of other mammalian cell lines can be used within the present invention, including rat Hep I (ATCC CRL 600), rat Hep II (ATCC CRL 1548), TCMK (ATCC CCL 139), human lung (ATCC CCL 75.1), human hepatoma (ATCC HTB-52), Hep G2 (ATCC HB 8065), mouse liver (ATCC CCL 29.1), NCTC 1469 (ATCC CCL 9.1) and DUKX cells (Urlaub and Chasin, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci USA 77:4216-4220, 1980, incorporated herein by reference).\",\n \"Mammalian expression vectors for use in carrying out the present invention include a promoter capable of directing the transcription of a cloned cDNA.\",\n \"Either viral promoters or cellular promoters can be used.\",\n \"Viral promoters include the immediate early cytomegalovirus (CMV) promoter (Boshart et al., Cell 41:521-530, 1985, incorporated herein by reference) and the SV40 promoter (Subramani et al., Mol.\",\n \"Cell.\",\n \"Biol.\",\n \"1:854-864, 1981, incorporated herein by reference).\",\n \"Cellular promoters include the mouse metallothionein-1 promoter (U.S. Pat.\",\n \"No.\",\n \"4,579,821, incorporated herein by reference), a mouse V1 promoter (Bergman et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 81:7041-7045, 1983; Grant et al., Nuc.\",\n \"Acids Res.\",\n \"15:5496, 1987, each incorporated herein by reference), a mouse V1 promoter (Loh et al., Cell 33:85-93, 1983, incorporated herein by reference), and the major late promoter from Adenovirus 2 (Kaufman and Sharp, Mol.\",\n \"Cell.\",\n \"Biol.\",\n \"2:1304-13199, 1982, incorporated herein by reference).\",\n \"Cloned DNA sequences can be introduced into cultured mammalian cells by, for example, calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb, Virology 52:456, 1973; each incorporated by reference herein in their entirety).\",\n \"Other techniques for introducing cloned DNA sequences into mammalian cells can also be used, such as electroporation (Neumann et al., EMBO J.\",\n \"1:841-845, 1982, incorporated herein by reference) or cationic lipid-mediated transfection (Hawley-Nelson et al., Focus 15:73-79, 1993, incorporated herein by reference) using, e.g., a 3:1 liposome formulation of 2,3-dioleyloxy-N-[2 (sperminecarboxyamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate and dioleoly-phosphatidylethanolamine in water Lipofectamine reagent, GIBCO-BRL).\",\n \"To identify cells that have integrated the cloned DNA, a selectable marker is generally introduced into the cells along with the gene or cDNA of interest.\",\n \"Examples of selectable markers for use in cultured mammalian cells include genes that confer resistance to drugs, such as neomycin, hygromycin, and methotrexate.\",\n \"The selectable marker can be an amplifiable selectable marker, for example the DHFR gene.\",\n \"Additional selectable markers are reviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass., which is incorporated herein by reference).\",\n \"The choice of selectable markers is well within the level of ordinary skill in the art.\",\n \"Selectable markers can be introduced into the cell on a separate plasmid at the same time as the polynucleotide encoding the anti-coreceptor binding peptide, or they may be introduced on the same plasmid.\",\n \"If on the same plasmid, the selectable marker and the peptide-encoding polynucleotide can be under the control of different promoters or the same promoter.\",\n \"Constructs of this latter type are known in the art (for example, U.S. Pat.\",\n \"No.\",\n \"4,713,339).\",\n \"It also can be advantageous to add additional DNA, known as “carrier DNA” to the mixture which is introduced into the cells.\",\n \"Transfected mammalian cells are allowed to grow for a period of time, typically 1-2 days, to begin expressing the polynucleotide sequence(s) of interest.\",\n \"Drug selection is then applied to select for growth of cells that are expressing the selectable marker in a stable fashion.\",\n \"For cells that have been transfected with an amplifiable selectable marker the drug concentration is increased in a stepwise manner to select for increased copy number of the cloned sequences, thereby increasing expression levels.\",\n \"Host cells containing polynucleotide constructs of the present invention are then cultured to produce anti-coreceptor binding peptide.\",\n \"The cells are cultured according to standard methods in a culture medium containing nutrients required for growth of the host cells.\",\n \"A variety of suitable media are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins, minerals and growth factors.\",\n \"The growth medium generally selects for cells containing the DNA construct by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker on the DNA construct or co-transfected with the DNA construct.\",\n \"Recombinantly produced anti-coreceptor binding peptides and peptide analogs as described above can be purified by techniques well known to those of ordinary skill in the art.\",\n \"For example, recombinantly produced peptides can be directly expressed or expressed as fusion proteins.\",\n \"The proteins can then be purified by a combination of cell lysis (e.g., sonication) and affinity chromatography.\",\n \"For fusion products, subsequent digestion of the fusion protein with an appropriate proteolytic enzyme releases the desired peptide.\",\n \"The phrase “substantially purified” when referring to anti-coreceptor binding peptides or peptide analogs (including peptide fusions with other peptides and/or proteins) of the present invention, means a composition which is essentially free of other cellular components with which the peptides or analogs are associated in a non-purified, e.g., native state or environment.\",\n \"Purified peptide is generally in a homogeneous state although it can be in either in a dry state or in an aqueous solution.\",\n \"Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography.\",\n \"Generally, substantially purified anti-coreceptor binding peptide comprises more than 80% of all macromolecular species present in a preparation prior to admixture or formulation of the peptide with a pharmaceutical carrier, excipient, buffer, absorption enhancing agent, stabilizer, preservative, adjuvant or other co-ingredient.\",\n \"More typically, the peptide is purified to represent greater than 90% of all proteins present in a purified preparation.\",\n \"In specific embodiments, the peptide is purified to greater than 95% purity or may be essentially homogeneous wherein other macromolecular species are not detectable by conventional techniques.\",\n \"The peptides and analogs of the present invention can be purified to substantial purity by standard techniques well known in the art.\",\n \"Useful purification methods include selective precipitation with such substances as ammonium sulfate; column chromatography; affinity methods, including immunopurification methods; and others.\",\n \"See, for instance, R. Scopes, Protein Purification: Principles and Practice, Springer-Verlag: N.Y., 1982, incorporated herein by reference.\",\n \"In general, anti-coreceptor binding peptides can be extracted from tissues or cell cultures that express the peptides and then immunoprecipitated, whereafter the peptides can be further purified by standard protein chemistry/chromatographic methods.\",\n \"For therapeutic administration, anti-coreceptor binding peptides, analogs and mimetics of the invention are typically formulated with a pharmaceutically acceptable carrier.\",\n \"As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption enhancing or delaying agents, and other excipients or additives that are physiologically compatible.\",\n \"In specific embodiments, the carrier is suitable for intranasal, intravenous, intramuscular, subcutaneous, parenteral, oral, transmucosal or transdermal administration.\",\n \"Depending on the route of administration, the active compound may be coated in a material to protect the compound from the action of acids and other natural conditions which may inactivate the compound.\",\n \"In preparing pharmaceutical compositions of the present invention, it may be desirable to modify the anti-coreceptor binding agent, or combine or conjugate the peptide or mimetic compound with other agents, to alter pharmacokinetics and biodistribution of the anti-coreceptor binding agent.\",\n \"A number of methods for altering pharmacokinetics and biodistribution are known to persons of ordinary skill in the art.\",\n \"Examples of such methods include protection of peptides, proteins or complexes thereof in vesicles composed of other proteins, lipids, carbohydrates, or synthetic polymers.\",\n \"For example, anti-coreceptor binding agents can be incorporated into liposomes in order to enhance pharmacokinetics and biodistribution characteristics.\",\n \"A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al., (Ann.\",\n \"Rev.\",\n \"Biophys.\",\n \"Bioeng.\",\n \"2:467, 1980; U.S. Pat.\",\n \"Nos.\",\n \"4,235,871; 4,501,728 and 4,837,028, each incorporated herein by reference).\",\n \"For use with liposome delivery, the anti-coreceptor binding agent is typically entrapped within the liposome, or lipid vesicle, or is bound to the outside of the vesicle.\",\n \"Several strategies have been devised to increase the effectiveness of liposome-mediated delivery by targeting liposomes to specific tissues and specific cell types.\",\n \"Liposome formulations, including those containing a cationic lipid, have been shown to be safe and well tolerated in human patients (Treat et al., J. Natl.\",\n \"Cancer Instit.\",\n \"82:1706-1710, 1990, incorporated herein by reference).\",\n \"The compositions of the invention may alternatively contain as pharmaceutically acceptable carriers, substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like.\",\n \"For solid compositions, conventional nontoxic pharmaceutically acceptable carriers can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.\",\n \"For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95%, more typically 25% to 75% of active ingredient.\",\n \"Therapeutic compositions for administering the anti-coreceptor binding agent can also be formulated as a solution, microemulsion, or other ordered structure suitable for high concentration of active ingredients.\",\n \"The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.\",\n \"Proper fluidity for solutions can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of a desired particle size in the case of dispersible formulations, and by the use of surfactants.\",\n \"In many cases, it will be desirable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.\",\n \"Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.\",\n \"In certain embodiments of the invention, the anti-coreceptor binding agent is administered in a time release formulation, for example in a composition which includes a slow release polymer, or by depot injection.\",\n \"The active peptide, analog or mimetic can be prepared with carriers that will protect against rapid release, for example a controlled release vehicle such as implants, transdermal patches, or microencapsulated delivery system.\",\n \"Prolonged delivery of the anti-coreceptor binding agent, or a biologically active analog or mimetic thereof, in various compositions of the invention can be brought about by including in the composition agents that delay absorption, for example, aluminum monosterate hydrogels and gelatin.\",\n \"When controlled release formulations of anti-coreceptor binding agents is desired, controlled release binders suitable for use in accordance with the invention include any biocompatible controlled-release material which is inert to the active ingredient and which is capable of incorporating the anti-coreceptor binding agent.\",\n \"Numerous such materials are known in the art.\",\n \"Useful controlled-release binders are materials which are metabolized slowly under physiological conditions following their subcutaneous or intramuscular injection in mammals (i.e., in the presence of bodily fluids which exist there).\",\n \"Appropriate binders include but are not limited to biocompatible polymers and copolymers previously used in the art in sustained release formulations.\",\n \"Such biocompatible compounds are non-toxic and inert to surrounding tissues, e.g., following subcutaneous or intramuscular injection, and do not trigger significant adverse effects such as immune response, inflammation, or the like.\",\n \"They are metabolized into metabolic products which are also biocompatible and easily eliminated from the body.\",\n \"For example, a polymeric matrix derived from copolymeric and homopolymeric polyesters having hydrolysable ester linkages may be used.\",\n \"A number of these are known in the art to be biodegradable and to lead to degradation products having no or low toxicity.\",\n \"Exemplary polymers include polyglycolic acids (PGA) and polylactic acids (PLA), poly(DL-lactic acid-co-glycolic acid)(DL PLGA), poly(D-lactic acid-coglycolic acid) (D PLGA) and poly(L-lactic acid-co-glycolic acid)(L PLGA).\",\n \"Other useful biodegradable or bioerodable polymers include but are not limited to such polymers as poly(epsilon-caprolactone), poly(epsilon-aprolactone-CO-lactic acid), poly(epsilon.-aprolactone-CO-glycolic acid), poly(beta-hydroxy butyric acid), poly(alkyl-2-cyanoacrilate), hydrogels such as poly(hydroxyethyl methacrylate), polyamides, poly(amino acids) (i.e., L-leucine, glutamic acid, L-aspartic acid and the like), poly(ester urea), poly(2-hydroxyethyl DL-aspartamide), polyacetal polymers, polyorthoesters, polycarbonate, polymaleamides, polysaccharides and copolymers thereof.\",\n \"Many methods for preparing such formulations are generally known to those skilled in the art (see, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978, incorporated herein by reference).\",\n \"Useful formulations include controlled-release compositions such as are known in the art for the administration of leuprolide (trade name: Lupron.RTM.\",\n \"), e.g., microcapsules (U.S. Pat.\",\n \"Nos.\",\n \"4,652,441 and 4,917,893, each incorporated herein by reference), injectable formulations (U.S. Pat.\",\n \"No.\",\n \"4,849,228, incorporated herein by reference), lactic acid-glycolic acid copolymers useful in making microcapsules or injectable formulations (U.S. Pat.\",\n \"Nos.\",\n \"4,677,191 and 4,728,721, each incorporated herein by reference), and sustained-release compositions for water-soluble peptides (U.S. Pat.\",\n \"No.\",\n \"4,675,189, incorporated herein by reference).\",\n \"A long-term sustained release implant also may be used.\",\n \"These can be readily constructed to deliver therapeutic levels of the anti-coreceptor binding agent for at least 10 to 20 days, often at least 30 days, up to 60 days or longer.\",\n \"Long-term sustained release implants are well known to those of ordinary skill in the art and can incorporate some of the absorption delaying components described above.\",\n \"Such implants can be particularly useful by placing the implant near or directly within the target tissue or cell population, thereby affecting localized, high-doses of the anti-coreceptor binding agent at one or more sites of interest.\",\n \"In alternate embodiments, anti-coreceptor binding agent may be delivered to a mucosal surface, e.g., orally, intranasally, or rectally, for prophylaxis or treatment of HIV infection and related disease.\",\n \"For mucosal administration, the peptide, analog or mimetic is typically combined with an inert diluent or an assimilable edible carrier.\",\n \"The anti-coreceptor binding agent may be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into a subject's diet.\",\n \"For oral therapeutic administration, the anti-coreceptor binding agent may be incorporated with excipients and used in the form of ingestable tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.\",\n \"Of course, taste-improving substances can be added in the case of oral administration forms.\",\n \"The percentage (e.g., by weight or by volume) of the anti-coreceptor binding agent in these compositions and preparations may, of course, be varied.\",\n \"As noted above, the amount of anti-coreceptor binding agent in such therapeutically useful compositions is generally such that a therapeutically effective dosage will be delivered.\",\n \"For oral or rectal administration, the anti-coreceptor binding agent can be worked into tablets or other solid forms by being mixed with solid, pulverulent carrier substances, such as sodium citrate, calcium carbonate or dicalcium phosphate, and binders such as polyvinyl pyrrolidone, gelatin or cellulose derivatives, possibly by adding also lubricants such as magnesium stearate, sodium lauryl sulfate, “Carbowax” or polyethylene glycol.\",\n \"Solid delivery vehicles may contain a protein or peptide in a mixture with fillers, such as lactose, saccharose, mannitol, starches, such as potato starch or amylopectin, cellulose derivatives or highly dispersed silicic acids.\",\n \"In soft-gelatin capsules, the active substance is dissolved or suspended in suitable liquids, such as vegetable oils or liquid polyethylene glycols.\",\n \"As further forms, one can use plug capsules, e.g., of hard gelatin, as well as dosed soft-gelatin capsules comprising a softener or plasticizer, e.g., glycerin.\",\n \"Alternatively, liquid dosage forms for delivering anti-coreceptor binding agents to mucosal surfaces include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules.\",\n \"Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents.\",\n \"Oral dosage forms optionally contain flavorants and coloring agents.\",\n \"Parenteral and intravenous forms would also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.\",\n \"The therapeutic compositions of the invention typically must be sterile and stable under all conditions of manufacture, storage and use.\",\n \"Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.\",\n \"Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.\",\n \"In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.\",\n \"The prevention of the action of microorganisms can be accomplished by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.\",\n \"In certain embodiments of the invention, the anti-coreceptor binding agent is administered by topical delivery to a mucosal surface of the patient, for example, via intranasal or intrapulmonary delivery in the form of an aerosol spray or powder.\",\n \"According to one aspect of the invention, the anti-coreceptor binding agent is delivered in an intranasally or intrapulmonarily effective amount, typically in a selected volume of administered spray or powder, to achieve a desired therapeutic result.\",\n \"In related aspects of the invention, novel pharmaceutical compositions are provided for intranasal or intrapulmonary delivery that incorporate the anti-coreceptor binding agent in a powder or aqueous formulation.\",\n \"Intranasal or intrapulmonary administration allows self-administration of treatment by patients, provided that sufficient safeguards are in place to control and monitor dosing and side effects.\",\n \"Nasal or intrapulmonary administration also overcomes certain drawbacks of other administration forms, such as injections, that are painful and expose the patient to possible infections and may present drug bioavailability problems.\",\n \"Systems for aerosol dispensing of therapeutic liquids as a spray are well known.\",\n \"In one embodiment, metered doses of aerosolized anti-coreceptor binding agent are delivered by means of a specially constructed mechanical pump valve (See, for example, U.S. Pat.\",\n \"No.\",\n \"4,511,069, incorporated herein by reference).\",\n \"This hand-held delivery device is uniquely nonvented so that sterility of the solution in the aerosol container is maintained indefinitely.\",\n \"Certain nasal and intrapulmonary spray solutions within the invention comprise an anti-coreceptor binding agent in a liquid carrier that optionally includes a nonionic surfactant for enhancing absorption of the drug and one or more buffers or other additives to minimize nasal or pulmonary irritation.\",\n \"In some embodiments, the nasal or pulmonary spray solution further comprises a propellant.\",\n \"Thus, in certain embodiments the pharmaceutical compositions comprising an anti-coreceptor binding agent administrable in fine particulate form (e.g., between about 0.5-5.0 μm, more typically between about 1.0-2.5 μm diameter particles) comprising a surfactant and propellant as pharmaceutically acceptable carriers (see, e.g., U.S. Pat.\",\n \"No.\",\n \"5,902,789, incorporated herein by reference).\",\n \"The surfactant must be nontoxic and soluble in the propellant.\",\n \"Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride.\",\n \"Mixed esters, such as mixed or natural glycerides, can be employed.\",\n \"Additional carrier can be included as desired, for example lecithin for intranasal delivery.\",\n \"The pH of the intranasal or intrapulmonary spray solution is typically between pH 6.8 and 7.2.Alternative means of mucosal administration for the anti-coreceptor binding agents of the invention may involve the use of powder carriers, for example ion exchange resins or adsorbent resin powders (see, e.g., U.S. Pat.\",\n \"No.\",\n \"5,942,242, incorporated herein by reference).\",\n \"In more detailed aspects of the invention, the anti-coreceptor binding agent is stabilized to extend its effective half-life following delivery to the subject, particularly for extending metabolic persistence in an active state within the physiological environment (e.g., in the bloodstream, at a mucosal surface, or within a connective tissue compartment or fluid-filled body cavity).\",\n \"For this purpose, the anti-coreceptor binding agent can be modified by chemical means, e.g., chemical conjugation, N-terminal capping, PEGylation, or recombinant means, e.g., site-directed mutagenesis or construction of fusion proteins, or formulated with various stabilizing agents or carriers.\",\n \"Thus stabilized, the anti-coreceptor binding agent administered as above retains anti-coreceptor activity for an extended period (e.g., 2 to 3, up to 5 to 10 fold greater stability) under physiological conditions compared to its non-stabilized form.\",\n \"Numerous reports in the literature describe the potential advantages of pegylated proteins, which include their increased resistance to proteolytic degradation, increased plasma half-life, increased solubility and decreased antigenicity and immunogenicity (Nucci et al., Advanced Drug Deliver Reviews 6:133-155, 1991; Lu et al., Int.\",\n \"J. Peptide Protein Res.\",\n \"43:127-138, 1994, each incorporated herein by reference).\",\n \"A number of proteins, including L-asparaginase, strepto kinase, insulin, and interleukin-2 have been conjugated to a poly(ethyleneglycol) (PEG) and evaluated for their altered biochemical properties as therapeutics (see, e.g., Ho et al., Drug Metabolism and Disposition 14:349-352, 1986; Abuchowski et al., Prep.\",\n \"Biochem.\",\n \"9:205-211, 1979; and Rajagopaian et al., J. Clin.\",\n \"Invest.\",\n \"75:413-419, 1985, each incorporated herein by reference).\",\n \"Although the in vitro biological activities of pegylated proteins may be decreased, this loss in activity is usually offset by the increased in vivo half-life in the bloodstream (Nucci, et al., Advanced Drug Deliver Reviews 6:133-155, 1991, incorporated herein by reference).\",\n \"Several procedures have been reported for the attachment of PEG to proteins and peptides and their subsequent purification (Abuchowski et al., J. Biol.\",\n \"Chem.\",\n \"252:3582-3586,1977; and Beauchamp et al., Anal.\",\n \"Biochem, 131:25-33, 1983, each incorporated herein by reference).\",\n \"Lu et al., (Int.\",\n \"J. Peptide Protein Res.\",\n \"43:127-138, 1994) describe various technical considerations and compare PEGylation procedures for proteins versus peptides (see also, Katre et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 84:1487-1491, 1987; Becker et al., Makromol.\",\n \"Chem.\",\n \"Rapid Commun.\",\n \"3:217-223, 1982; Mutter et al., Makromol.\",\n \"Chem.\",\n \"Rapid Commun.\",\n \"13:151-157, 1992; Merrifield, R. B., J.\",\n \"Am.\",\n \"Chem.\",\n \"Soc.\",\n \"85:2149-2154, 1993; Lu et al., Peptide Res.\",\n \"6:142-146, 1993; Lee et al., Bioconiugate Chem.\",\n \"10:973-981, 1999; Nucci et al., Adv.\",\n \"Drug Deliv.\",\n \"Rev.\",\n \"6:133-151, 1991; Francis et al., J.\",\n \"Drug Targeting 3:321-340, 1996; Zalipsky, Bioconjugate Chem.\",\n \"6:150-165, 1995; Clark et al., J. Biol.\",\n \"Chem.\",\n \"271:21969-21977, 1996; Pettit et al., J. Biol.\",\n \"Chem.\",\n \"272:2312-2318, 1997; Delgado et al., Br.\",\n \"J.\",\n \"Cancer 73:175-182, 1996; Benhar et al., Bioconjugate Chem.\",\n \"5:321-326, 1994; Benhar et al., J. Biol.\",\n \"Chem.\",\n \"269:13398-13404, 1994; Wang et al., Cancer Res.\",\n \"53:4588-4594, 1993; Kinstler et al., Pharm.\",\n \"Res.\",\n \"13:996-1002, 1996, Filpula et al., Exp.\",\n \"Opin.\",\n \"Ther.\",\n \"Patents 2:231-245, 1999; Pelegrin et al., Hum.\",\n \"Gene Ther.\",\n \"9:2165-2175, 1998, each incorporated herein by reference).\",\n \"Following these and other teachings in the art, the conjugation of anti-coreceptor binding peptides and analogs with poly(ethyleneglycol) polymers, is readily undertaken with the expected result of prolonging circulating life and/or reducing immunogenicity while maintaining an acceptable level of activity of the PEGylated anti-coreceptor binding agent.\",\n \"Amine-reactive PEG polymers for use within the invention include SC-PEG with molecular masses of 2000, 5000, 10000, 12000, and 20000; U-PEG-10000; NHS-PEG-3400-biotin; T-PEG-5000; T-PEG-12000; and TPC-PEG-5000.Chemical conjugation chemistries for these polymers have been published (see, e.g., Zalipsky, Bioconjugate Chem.\",\n \"6:150-165, 1995; Greenwald et al., Bioconjugate Chem.\",\n \"7:638-641, 1996; Martinez et al., Macromol.\",\n \"Chem.\",\n \"Phys.\",\n \"198:2489-2498, 1997; Hermanson, Bioconjugate Techniques, pp.\",\n \"605-618, 1996; Whitlow et al., Protein Eng.\",\n \"6:989-995, 1993; Habeeb, Anal.\",\n \"Biochem.\",\n \"14:328-336, 1966; Zalipsky et al., Poly(ethyleneglycol) Chemistry and Biological Applications, pp.\",\n \"318-341, 1997; Harlow et al., Antibodies: A Laboratory Manual pp.\",\n \"553-612, Cold Spring harbor Laboratory, Plainview, N.Y., 1988; Milenic et al., Cancer Res.\",\n \"51:6363-6371, 1991; Friguet et al., J. Immunol.\",\n \"Methods 77:305-319, 1985, each incorporated herein by reference).\",\n \"While phosphate buffers are commonly employed in these protocols, the choice of borate buffers may beneficially influence the PEGylation reaction rates and resulting products.\",\n \"PEGylation of anti-coreceptor binding peptides and analogs may be achieved by modification of carboxyl sites (e.g., aspartic acid or glutamic acid groups in addition to the carboxyl terminus).\",\n \"The utility of PEG-hydrazide in selective modification of carbodimide-activated protein carboxyl groups under acidic conditions has been described (Zalipsky, Bioconjugate Chem.\",\n \"6:150-165, 1995; Zalipsky et al., Poly(ethyleneglycol) Chemistry and Biological Applications, pp.318-341, American Chemical Society, Washington, D.C., 1997, each incorporated herein by reference).\",\n \"Alternatively, bifunctional PEG modification of anti-coreceptor binding peptides can be employed.\",\n \"In some procedures, charged amino acid residues, including lysine, aspartic acid, and glutamic acid, have a marked tendency to be solvent accessible on protein surfaces.\",\n \"Conjugation to carboxylic acid groups of proteins is a less frequently explored approach for production of protein bioconjugates.\",\n \"However, the hydrazide/EDC chemistry described by Zalipsky et al., (Zalipsky, Bioconjugate Chem.\",\n \"6:150-165, 1995; Zalipsky et al., Poly(ethyleneglycol) Chemistry and Biological Applications, pp.\",\n \"318-341, American Chemical Society, Washington, D.C., 1997, each incorporated herein by reference) offers a practical method of linking PEG polymers to protein carboxylic sites.\",\n \"For example, this alternate conjugation chemistry has been shown to be superior to amine linkages for PEGylation of brain-derived neurotrophic factor (BDNF) while retaining biological activity (Wu et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"U.S.A. 96:254-259, 1999, incorporated herein by reference).\",\n \"Maeda and colleagues have also found carboxyl-targeted PEGylation to be the preferred approach for bilirubin oxidase conjugations (Maeda et al., Poly(ethyleneglycol) Chemistry, Biotechnical and Biomedical Applications, J. M. Harris, ed., pp.\",\n \"153-169, Plenum Press, New York, 1992, incorporated herein by reference).\",\n \"In addition to PEGylation, anti-coreceptor binding agents of the invention can be modified to enhance circulating half-life by shielding the peptide or analog via conjugation to other known protecting or stabilizing compounds, or by the creation of fusion proteins with the peptide or analog linked to one or more carrier proteins, such as one or more immunoglobulin chains (see, e.g., U.S. Pat.\",\n \"Nos.\",\n \"5,750,375; 5,843,725; 5,567,584 and 6,018,026, each incorporated herein by reference).\",\n \"These modifications will decrease the degradation, sequestration or clearance of the anti-coreceptor binding agent and result in a longer half-life in a physiological environment (e.g., in the circulatory system, or at a mucosal surface).\",\n \"The anti-coreceptor binding agents modified by PEGylation and other stabilizing methods are therefore useful with enhanced efficacy within the methods of the invention.\",\n \"In particular, the anti-coreceptor binding agents thus modified maintain activity for greater periods at a target site of delivery compared to the unmodified peptide or analog.\",\n \"Even when the anti-coreceptor binding agents are thus modified, they retain substantial biological activity for inhibiting HIV-coreceptor interactions and HIV infection and related disease.\",\n \"In yet additional aspects of the invention, an anti-coreceptor binding agent is administered according to the foregoing methods in a coordinate therapy protocol with one or more additional anti-HIV agents or treatment steps.\",\n \"Thus, in various embodiments an anti-coreceptor binding agent is administered coordinately with one or more adjunct or combinatorially (e.g., additively or synergistically) effective treatment agents selected from anti-retroviral drugs (e.g., nucleoside reverse transcriptase inhibitors such as AZT (zidovudine), Videx® (ddI or didanosine), or Hivid® (ddC or zalcitabine); antiviral drugs (e.g., acyclovir); antibiotics (e.g., penicillins, cephalosporins, macrolides and lincosamides such as erythromycin, gentamycin, ceftriaxone, cefixime, azithromycin, spectinomycin, ofloxacin, ciprofloxacin, cefoxitin, clindamycin, metronidazole, amoxicillin, tetracycline, doxycycline); immunomodulatory agents (e.g., interferons (IFNα, IFNβ, IFNγ), interleukins (such as IL-1 or IL-2); G-CSF, GM-CSF, levamisole) particularly interferon); or vaccine agents.\",\n \"Other immune modulatory agents that are useful for coordinate administration with anti-coreceptor binding agents include corticosteroids, cytotoxic drugs, T-cell specific inhibitors, antisera, replacement immune globulins, and monoclonal antibodies.\",\n \"At times, specific immunosuppressive drugs can be used in conjunction with anti-coreceptor binding agent therapy, for example, azathioprine, cyclophosphamide, or cyclosprine.\",\n \"Some cytotoxic drugs may also be used for coordinate administration with anti-coreceptor binding agents, for example azathioprine, azathioprine sodium, chlorambucil, cyclophosphamide, methotrexate, methotrexate sodium, and/or clyclosprorine.\",\n \"Additional combinatorial or adjunctive therapeutic agents for use within coordinate formulations and treatment methods of the invention may comprise a glucocorticoid or non-steroidal anti-inflammatory drug (NSAID).\",\n \"Glucocorticoids useful within this aspect of the invention include short-acting glucocorticoids (e.g., cortisone and hydrocortisone), intermediate-acting glucocorticoids (e.g., prednisone, prednisolone, meprednisone, methylprednisolone and triamcinolone), and long-acting glucocorticoids (betamethasone, dexamethasone and paramethasone).\",\n \"NSAID's useful within the invention include, aspirin, salicylates, naproxen, indomethacin, piroxicam, oxaprozin, phenylbutazone, ibuprofen, flurbiprofen, fenoprofen, and ketoprofen, and the like.\",\n \"Additional therapeutic agents for use in conjunction with anti-coreceptor binding agent therapy may include the anti-malarial drug hydroxychloroquine, or sulfasalazine.\",\n \"Also useful in coordinate therapy protocols with anti-coreceptor binding agents are antihistamines, including amino alkyl ethers (e.g., diphenhydramine, clemasine), ethylenediamines (e.g., pyrilamine, tripelennamine), alkylamines (e.g., brompheniramine, chlorpheniramine, dexchlorpheniramine, triprolidine), and phenothiazines (e.g., methdilazine, promethazine, trimeprazine).\",\n \"Within alternate methods and compositions of the invention, the anti-coreceptor binding agent is administered as above coordinately, admixed or separately, simultaneously or sequentially, with of one or more of the foregoing “combinatorially effective or adjunct treatment agents”, in respective amounts sufficient (independently sufficient or combinatorially sufficient) to prevent or alleviate HIV infection or a disease condition or symptom associated therewith.\",\n \"The instant invention also includes kits, packages and multicontainer units containing the above described pharmaceutical compositions, active ingredients, and/or means for administering the same for use in the prevention and treatment of HIV infection and related disease conditions.\",\n \"Briefly, these kits include a container or formulation which contains an anti-coreceptor binding agent, typically formulated in a pharmaceutical preparation with a biologically suitable carrier.\",\n \"The anti-coreceptor binding agent is optionally contained in a bulk dispensing container or unit or multi-unit dosage form.\",\n \"Optional dispensing means may be provided, for example, an intranasal or intrapulmonary spray applicator.\",\n \"Packaging materials optionally include a label or instruction which indicates that the pharmaceutical agent packaged therewith can be used for treating HIV and related disease conditions.\",\n \"In more detailed embodiments of the invention, kits include reagents and/or devices for detecting the presence and/or status of HIV infection or related disease in a subject.\",\n \"For example, an immunological or molecular probe that binds or reacts with an HIV-specific marker detectable in blood or other biological samples to be obtained from the subject.\",\n \"Thus, the kits may contain ELISA probes for detecting HIV antigens, as well as additional, optional kit materials for collecting and/or processing samples for ELISA and other diagnostic assays.\",\n \"The kits may also contain suitable buffers, preservatives such as protease inhibitors, direct or sandwich-type labels for labeling probes, and/or developing reagents for detecting a signal from the label.\",\n \"Thus, a broad selection of therapeutic and diagnostic kits are provided within the invention based on the description herein, including kits that contain specific instructions for carrying out the prophylactic and treatment protocols and associated assays of the invention.\",\n \"The invention is further illustrated by the following specific examples which are not intended in any way to limit the scope of the invention.\",\n \"EXAMPLES Materials and Methods The following examples describe computer modeling, synthesis and testing of co-receptor agents that inhibit the binding of HIV-1 to CXCR4 and CCR5.Synthetic Peptides Exemplary peptides 15D, 15K and 15KS, were synthesized using known methods by Macromolecular Resources (Fort Collins, Colo.), peptide 15CW was synthesized by the Basic Research Laboratory National Cancer Institute, (Frederick, MD).\",\n \"The peptides were purified by reverse-phase HPLC and the homogeneity of the peptide preparations was confirmed by mass-spectrometry.\",\n \"Computer Modeling Computer-generated structural models were derived using non-hydrogen atom superimposition of homologous residues during an optimization protocol of constrained consistent valence force field (CVFF) (Dauber-Osguthorpe et al., Proteins 4:31-47, 1988; Hagler et al., J.\",\n \"Am.\",\n \"Chem.\",\n \"Soc.\",\n \"96:5319-27, 1974; and Hagler et al., Science 227:1309-15, 1985, each incorporated herein by reference), molecular dynamics sampling and conjugate gradients minimization of sampled structures.\",\n \"Cells and Culture Conditions HEK-293 cells expressing human CCR5 (HEK-CCR5) and CXCR4 (HEK-CXCR4) (Howard et al., J. Biol.\",\n \"Chem.\",\n \"274:16228-16234, 1999, incorporated herein by reference) were cultured in Dulbecco's modified Eagle's medium (BioWhittaker, Walkersville, Md.)\",\n \"containing 10% fetal bovine serum (HyClone, Logan, Utah), 2 mM glutamine, 100 units/ml penicillin and streptomycin (Quality Biologicals, Gaithersburg, Md.)\",\n \"and 400 mg/ml Geneticin (Life Technologies, Inc., Rockville, Md.)\",\n \"at 37° C. in a humidified 5% CO2 atmosphere.\",\n \"CEMx174 and THP-1 cell lines were obtained from ATCC (Rockville, Md.).\",\n \"Sup-T1 cells expressing CCR5 was the gift of James Hoxie.\",\n \"Cells were cultured in RPMI-1640 medium (BioWhittaker, Walkersville, Md.)\",\n \"containing 10% fetal bovine serum (FBS; HyClone, Logan, Utah), 2 mM glutamine, 100 units/ml penicillin and streptomycin (Quality Biologicals, Gaithersburg, Md.)\",\n \"at 37° C. in a humidified 5% CO2 atrnosphere.\",\n \"Binding Assays Binding assays were performed with HEK-CCR5 or HEK CXCR4 in triplicate by adding unlabeled competitor (anti-coreceptor binding peptide or control chemokine) and radiolabeled chemokine, 0.2 ng/ml (125I-MIP-1β or SDF-1α, specific activity 2000 Ci/mmol, NEN Life Science Products) to a cell suspension (4×105 cells in 200 μl) in RPMI 1640 supplemented with 1% bovine serum albumin and 25 mM HEPES pH 8.0 (binding medium).\",\n \"Cells were then incubated at 22° C. for 40 min with continuous rotation.\",\n \"After incubation cells were transferred to the tubes containing 800 μl of 10% sucrose in PBS and harvested by centrifugation.\",\n \"The supernatant was aspirated and the cell-associated radioactivity was measured using a 1272 Wallac gamma counter.\",\n \"Binding assays were performed with the 174xCEM and Sup-T1 cells expressing CCR5 in duplicates by adding unlabeled competitor (peptide or control chemokine) and radiolabeled chemokine, 0.2 ng/ml (125I-MIP-1β or 125I-SDF-1α, specific activity 2000 Ci/mmol, NEN life Science Products) to 300 μl cell suspension (4×106 cells/ml) in RPMI 1640 supplemented with 1% bovine serum albumin, 0.1% sodium azide and 25 mM HEPES pH 8.0 (binding medium).\",\n \"Cells were then incubated at 22° C. for 30 min with continuous rotation.\",\n \"After incubation cells were transferred to the tubes containing 800 μl of 10% sucrose in PBS and harvested by centrifugation, the supematant was aspirated and the cell-associated radioactivity was measured using a 1272 Wallac gamma counter.\",\n \"The data were analyzed by nonlinear regression using the computer program GraphPad Prizm 3.0.Chemotaxis Assays Chemotaxis assays for HEK-293 cells transfected with CCRS was performed as previously described (Howard et al., J. Biol.\",\n \"Chem.\",\n \"274:16228-16234, 1999, incorporated herein by reference).\",\n \"Briefly, the chemokine RANTES, diluted in binding medium to 100 ng/ml, was placed in the lower wells of a microchemotaxis chamber.\",\n \"Polycarbonate membrane pretreated with rat tail collagen type 1 was placed over the chemokine solution.\",\n \"The cells (1×106/ml) were suspended in binding medium along with the exemplary peptides 15D or 15K at designated concentrations and placed in the upper wells of the chamber.\",\n \"After incubation for 3 hr the membrane was removed, stained using a Diff-Quik kit (Trends Scientific, Kalamazoo, Mich.) and counted.\",\n \"The results are expressed as the “chemotaxis index”, which represents the ratio of the number of cells in high powered field in test versus control samples.\",\n \"Flow Cytometric Analysis CEMx174 cells were pelleted and resuspended at 106 cells/ml in PBS containing 1% BSA and 0.1% sodium azide (FACS buffer).\",\n \"Cells were then preincubated with designated concentrations of peptides or SDF-1-α (1 μg/ml) at 22° C. for 60 min.\",\n \"FITC-labeled anti-human CXCR4 antibody (clone 12G5, BD PharMingen, San Diego, Calif.) was added to the cells per the manufactures instructions and further incubated at 22° C. for 40 minutes.\",\n \"Cells were extensively washed with FACS buffer and analyzed using a FACS Calibur flowcytometer (Becton Dickinson).\",\n \"HEK/CCR5 cells were suspended in Dulbecco's PBS containing 1% FCS and 0.05% NaN3 (104 cells in 100 μl) and incubated with or without 5 μg of MIP-1β (PeproTech, Rocky Hill, N.J.), 0.1 mM peptide 15D or 15K for 45 min at 22° C. Cells were then treated with FITC-conjugated anti-human CCR5 antibody (2D7; BD PharMingen, San Diego, Calif.), and incubated for 30 min at 22° C. Cells were washed twice, and analyzed using a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, Calif.).\",\n \"CA2+ Mobilization Assay THP-1 cells (12×106/ml in RPMI 1640 containing 10% FSB) were loaded in the presence of 5 μM fura-2 AM (Molecular Probes, Eugene, Oreg.)\",\n \"at 22° C. for 30 min in the dark.\",\n \"Subsequently, cells were washed three times and resuspended (106 cells/ml) in the buffer containing 138 mM sodium chloride, 6 mM potassium chloride, 1 mM calcium chloride, 1 mM magnesium chloride, 10 mM HEPES, pH 7.4,5 mM glucose, and 0.1% bovine serum albumin.\",\n \"1.93 ml of loaded cells was transferred into a quartz cuvette.\",\n \"50 μl of a peptide stock solution (2×10−2 M) was added to the cells, and after 3 min of incubation 20 μl of human SDF-1α (10 μg/ml, PeproTech) was added to a stirred cuvette.\",\n \"The measurements were performed using luminescence spectrophotometer LS50 B (Perkin-Elmer).\",\n \"Ca2+ mobilization in the cells in response to SDF-1α was measured by analysis of the ratio of fluorescence emitted at 510 nm after sequential excitation at 340 and 380 nm.\",\n \"Assays of HIV Infectivity PBMCs were obtained from whole blood of normal donors and isolated by Ficoll-Paque Plus (Pharmacia Biotech, Piscataway, N.J.) density gradient centrifugation, and plated at a cell density of 2×106 cells/ml.\",\n \"Monocyte-derived macrophages (MDM) were generated from adherent human peripheral blood mononuclear cells by culture for 7 days with M-CSF (100 ng/ml).\",\n \"Cultures were maintained in RPMI-1640 medium (Life Technologies, Rockville, Md.)\",\n \"supplemented with 10% heat-inactivated endotoxin-free FCS (Hyclone, Logan, Utah), 10 μg/ml gentamicin, and 1 mM glutamine.\",\n \"Cells were treated with designated concentrations of the exemplary peptides 15K or 15D, and after 1 hr cells were infected with HIV-1IIIB (T cell tropic) or HIV-1JRFL (monocyte tropic) at an multiplicity of infection (MOI) of 0.1.After 2 hr, cells were washed, and cultured for additional 48-72 hr, followed by analysis of HIV replication as determined by quantification of accumulated p24 in the supernatant.\",\n \"The production of p24 was determined by conventional sandwich ELISA, using ELISA plates pre-coated with capture anti-p24 antibodies provided by the AIDS Vaccine Program (SAIC Frederick, NCI-FCRDC, Frederick, Md.).\",\n \"The captured p24 antigen was detected using rabbit anti-HIV-1 anti-p24 antibody, and a secondary goat anti-rabbit IgG (peroxidase-labeled) antibody.\",\n \"The captured p24 protein was detected using a 3,3′,5,5′-tetramethylbenzidine and hydrogen peroxide detection system (KPL Laboratories, Gaithersburg, Md.).\",\n \"The reaction was read spectrophotometrically at 450 nm.\",\n \"No cytotoxicity was detectable following treatment of PBMC with the peptides even at a concentration of 100 μg/ml.\",\n \"Example I Structural Analysis of gp120 and Chemokines As noted above, a number of recent studies point to a role of chemokine receptors as coreceptors for HIV cell entry.\",\n \"Additional studies suggest that protein fragments or peptides from chemokines or HIV corresponding to structural determinants involved in chemokine receptor (coreceptor) binding, may be useful to block HIV-coreceptor binding and therefore serve as anti-HIV reagents.\",\n \"Considering that both chemokines and the HIV envelope protein gp120 are thought to directly interact with chemokine receptors, it is conceivable that this direct interaction may involve a part of the gp120 polypeptide chain that is structurally similar to (e.g., by homologous or convergent evolutionary relationship) receptor binding determinants of chemokines.\",\n \"However, conventional amino acid sequence comparison between gp120 and chemokines undertaken by the present inventors did not reveal any significant amino acid identity or similarity relationship that would point to a convergent, or conservative, chemokine receptor binding domain.\",\n \"Despite this apparent lack of structural homology, the present investigation focused on a putative structural element found to be conserved within the amino acid sequences of nearly all chemokines, and also shared in a corresponding structural motif postulated for the HIV-1 gp120 protein.\",\n \"In particular, all chemokines studied were found to possess a Trp residue located at the beginning of a C-terminal alpha-helix.\",\n \"This conserved Trp residue is separated by six residues from a 4th Cys residue, to comprise what is characterized herein as a conserved chemokine structural motif.\",\n \"By comparison, in the amino acid sequences of gp120 proteins of all HIV-1 isolates there is a very similar motif in a spanning part of the conserved region 3 (C3) adjacent to the V3 loop.\",\n \"Surprisingly, this motif was also found to include one or more residues within a C-terminal portion of the variable loop 3 (V3) of gp120 that are conserved between different HIV-1 strains and exhibit some homology with chemokines (Table 1).\",\n \"Example II Computer Modeling of gp120 and Chemokines Computer modeling further demonstrates that novel peptide fragments of gp120 can be template forced onto a “homologous loop” of the known structure of an exemplary chemokine, MIP-1β without violation of the general rules of protein structure.\",\n \"To assess the possibility that a selected region of gp120 may potentially assume a structure similar to that of chemokines, a model of the corresponding structure of gp120 was built using the structure of MIP-1β (Lodi et al., Science 263:1762-1767, 1994, incorporated herein by reference) as the template.\",\n \"Based on this analysis, it was determined that a model of the three-dimensional structure of the selected gp120 segment could be generated without violation of protein stereochemistry.\",\n \"In an effort to examine the functional activity of this region, peptides corresponding to the selected gp120 sequence were synthesized for further studies.\",\n \"Example III Peptide Design and Synthesis Two exemplary peptides were synthesized based on a reference peptide comprising a C-terminal portion of the V3 loop and an N-terminal of the C3 domain of gp120 (see Table 4, above).\",\n \"The sequence of HIV-1JRFL, which is very close to the consensus B of HIV-1 sequences (Korber and Los Alamos National Laboratory-Theoretical Biology and Biophysics Group T-10, Human Retroviruses and AIDS, 1998, A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences, Los Alamos National Laboratory, Los Alamos, NW, 1998), was used as a template for the design of anti-HIV peptides.\",\n \"Synthesized peptides corresponding to a conserved structural motif of the HlV1JRFL gp120 protein, bearing the above-noted structural similarity to a corresponding, conserved motif identified in chemokines, are shown in Table 4.Because gp120 of different HIV-1 strains have some sequences variabilities even in conserved regions, peptides were designed with amino acid sequences similar to the widest range of naturally existing variants.\",\n \"In many HIV strains there is a lysine (k) residue instead of glutamine (Q) in position 3 (Table 4).\",\n \"Moreover, it was reasonable to change glutamine for lysine in the synthetic peptide to avoid hydrolysis and conversion to glutamic acid near the conserved positively changed arginine.\",\n \"The lysine residue preceding the tryptophan (W) in the peptide designated 15K was changed to aspartic acid (D) in the peptides designated 15D because some HIV-1 isolates have aspartic acid in this position and the change provided an opportunity to explore the significance of this substitution.\",\n \"For control experiments, peptides with “scrambled” sequences were also synthesized (peptide 15CW with the same amino acid composition as 15D, and 15KS with the same amino acid composition as 15K).\",\n \"In some control experiments a 15 amino acid peptide, 15GIG, with an amino acid sequence unrelated to gp120 was also used (See Table 4).\",\n \"TABLE 4 SEQUENCE OF SYNTHESIZED PEPTIDES SEQ Amino Acid Sequence ID 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 NO: HIVJRFL I R Q A H C N I S R A K W N D SEQ ID NO:3 15 K I R K A H C N I S R A K W N D SEQ ID NO:8 15 D I R K A H C N I S R A D W N D SEQ ID NO:9 15CW I R K A H C W I D R A D N N S SEQ ID NO:10 15KS K I N S W R A D N I H C K A R SEQ ID NO:11 15GIG G I G D P V T C L K S G A I A SEQ ID NO:12 As further detailed below, these exemplary peptides exhibited surprising anti-coreceptor binding activities, including competition with chemokines for binding to CCR5- and CXCR4-expressing cells, and inhibition of chemotaxis in chemokine-responsive cells.\",\n \"Correlated with these activities, the peptides mediate potent inhibition of HIV replication in macrophages and T lymphocytes, evincing their efficacy for prophylaxis and treatment of HIV infection and related disease conditions within the compositions and methods of the invention.\",\n \"This and related description herein further provides abundant structural information to enable the design and construction of a large assemblage of effective anti-coreceptor binding peptides, peptide analogs and mimetics, to serve as potent inhibitors of HIV-coreceptor interactions.\",\n \"Example IV Anti-Coreceptor Binding Activity of gp120 Peptides as Demonstrated by Inhibition of Chemokine Binding to CCR5 and CXCR4 Expressing Cells Considering that the 15K and 15D peptides were designed in part based on their structural similarity to corresponding chemokine fragments, the binding activity of these peptides for CCR5 and/or CXCR4 receptors, and corresponding anti-coreceptor binding activity against chemokines, was assessed.\",\n \"Experiments were carried out to determine the ability of these exemplary peptides to competitively inhibit binding of radiolabeled MIP-1β or SDF-1α to cells expressing CCR5 or CXCR4, respectively.\",\n \"The results, shown in FIG.\",\n \"1, demonstrate that both peptides significantly inhibit chemokine binding, although high concentrations (e.g., 100 μM) of peptide are required to mediate this effect.\",\n \"Example V Anti-Coreceptor Binding Activity of gp120 Peptides as Demonstrated by Inhibition of CCR5-Mediated Chemotaxis The inhibition of binding of MIP-1β and SDF-1α to their respective receptors suggests that the exemplary peptides 15K and 15D might also inhibit chemotaxis of cells expressing cognate chemokine receptors for appropriate ligands.\",\n \"To achieve this goal, the capacity of these peptides to inhibit the chemotactic response of HEK-CCR5 cells to a cognate ligand of CCR5 receptors, the chemokine RANTES, was assayed.\",\n \"The results demonstrated that RANTES-directed chemotactic responses were completely blocked by the addition of either 15K or 15D (FIG.\",\n \"2).\",\n \"Interestingly, one of the peptides, 15D, consistently exhibited greater inhibitory activity in these experiments.\",\n \"These results show that the 15K and 15D peptides interfere with chemokine receptor function.\",\n \"Example VI Anti-Coreceptor Binding Activity of gp120 Peptides Mediates Inhibition of HIV Infection in Monocyte-Tropic Cells The results described in the preceding Examples indicate that the 15K and 15D peptides effectively block the ability of CCR5 to serve as a receptor to mediate chemotactic responses.\",\n \"Additional experiments were thus carried out to determine the capacity of these peptides to inhibit coreceptor activity mediating cellular entry and infection by HIV-1JRFL (a monocyte-tropic HIV strain), using peripheral blood monocyte-derived macrophages.\",\n \"The results, presented in FIG.\",\n \"3, show that the addition of either 15K or 15D significantly reduced monocyte lineage target cell infection by HIV-1.Significant inhibition was exhibited at concentrations as low as 10 ng/ml.\",\n \"Interestingly, the CCR5-selective chemokine ligand MIP-1β and the 15K and 15D peptides exhibited comparable inhibitory activities (FIG.\",\n \"3).\",\n \"Example VII Anti-Coreceptor Binding Activity of gp120 Peptides Mediates Inhibition of HIV Infection in Lymphocyte-Tropic Cells In view of the foregoing binding inhibition studies, and considering the homology between the 15K and 15D peptides and the CXCR4 ligand SDF-1α, it was further undertaken to determine the capacity of the synthetic peptides to inhibit CXCR4 co-receptor function.\",\n \"In particular, experiments were carried out to assess the ability of 15K or 15D to alter the infection of PBMCs with the T cell-tropic HIV-1β strain.\",\n \"The results, presented in FIG.\",\n \"4, demonstrate that both peptides significantly reduced HIV-1 infection, in a dose-dependent manner.\",\n \"Significant inhibition of HIV-1 infection was detected with concentrations of peptides as low as 10 ng/ml.\",\n \"The scrambled peptide 15KS with the same amino acid composition as 15K but a randomized sequence manifested significantly less inhibition of virus infectively (FIG.\",\n \"4) demonstrating the importance of the sequence of amino acids for inhibiting activity.\",\n \"Example VIII Activity of gp120 Peptides in Relation to Binding of Anti-Coreceptor Antibodies To determine if peptides 15D and 15K interact directly with chemokine receptors the effect of these and control peptides on the binding of anti-chemokine receptor antibodies to CXCR4 and CCR5 was studied.\",\n \"The monoclonal antibody 12G5 recognizes a conformational extracellular epitope on CXCR4.This antibody blocks the infectivity of some X4 strains of HIV-1 and HIV-2 (Endres et al., Cell 87:745-756, 1996; Hoxie et al., J. Reprod.\",\n \"Immunol.\",\n \"41:197-211(1998); McKnight et al., J. Virol.\",\n \"74:1692-1696(1997), 19, 26).\",\n \"It also inhibits the binding of SDF-1α, a natural CXCR4 ligand Schols et al., J. Exp.\",\n \"Med.\",\n \"186:1383-1388, 1997 (Haste et al., Mol.\",\n \"Pharmacol.\",\n \"60:164-173(2001), 33).\",\n \"Further, the binding of 12G5 to CXCR4 receptor-expressing cells is prevented by anti-HIV compounds.\",\n \"The results show that peptide 15K inhibited the binding of the 12G5 antibody to CXCR4 in a dose-dependent manner (FIG.\",\n \"5A), and at a concentration of 50 μg/ml approached the efficacy of SDF-1α at 5 mg/ml.\",\n \"Peptide 15D appeared to be less effective in blocking of 12G5 antibody binding to CXCR4 (FIG.\",\n \"5B), which is not surprising since the binding site of CXCR4 includes several negatively charged residues Duranz et al., J. Virol.\",\n \"73:2752-2761, 1999.To determine the importance of the particular amino acid sequence of the inhibitory activity of the peptides.\",\n \"The effects of peptide 15K and the corresponding scrambled peptide 15KS on binding of 12G5 to CXCR4 was compared.\",\n \"The scrambled peptide 15KS manifest significantly less inhibitory activity in comparison with 15K suggesting the importance of the specific peptide sequence (FIG.\",\n \"5C).\",\n \"Apparently there is a direct correlation between the ability of 15K to inhibit the anti-CXCR4 antibody binding and its ability to inhibit HIV-1 infectivity since the scrambled peptide 15KS with the same amino acid comparison has reduced ability to manifest both these activities.\",\n \"The effect of the exemplary peptides 15K and 15D on the binding of 2D7 antibodies to CCR5 was assayed to further evaluate the mechanism of anti-coreceptor activity of the peptides.\",\n \"The 2D7 monoclonal antibody binds to a conformational epitope on CCR5, and inhibits Ca2+ flux induced by RANTES.\",\n \"This antibody has also been shown to inhibit HIV infection in vitro (Wu et al., J. Exp.\",\n \"Med.\",\n \"186:1373-81, 1997, incorporated herein by reference).\",\n \"It does not, however, inhibit binding of gp120-SCD4 complexes to CCR5-expressing L1.2 murine cells (Olson et al., J. Virol.\",\n \"73:4145-4155, 1999, incorporated herein by reference).\",\n \"The effects of peptides 15D and 15K on 2D7 antibody binding to HEK293/CCR5 cells were analyzed by flow cytometry.\",\n \"No significant effect of either of the two peptides on anti-CCR5 antibody binding was observed.\",\n \"These results indicate that the peptides bind to an epitope on the CCR5 receptor that is spatially and sterically distinct from the antibody binding site.\",\n \"Considering that both the peptides and the 2D7 antibodies inhibit HIV infection in vitro, these results may be interpreted as underscoring the unexpected nature and advantages of the present invention.\",\n \"Example IX Effects of Peptides on Induction of Intracellular Ca2+ Concentration in CXCR4 Expressing THP-1 Cells The increase of intracellular Ca2+ concentration mediated by a chemokine receptor in response to a cognate chemokine is a reliable assay for measurement of chemokine agonist activity.\",\n \"The human monocytic cell line THP-1 expresses a high level of CXCR4 and responds well to SDF-1α stimulation (FIG.\",\n \"6A), so these cells were used to determine whether peptides 15D and 15K could block the activation of CXCR4.Preincubation of cells with peptide 15D (at a concentration 500 μM) for 2 min completely inhibited cellular response to SDF-1α (FIG.\",\n \"6B).\",\n \"Peptide 15K also inhibited Ca2+ mobilization although less efficiently than 15D (FIG.\",\n \"6C).\",\n \"There is a probable direct correlation between the greater ability of 15D to inhibit Ca2+ mobilization in response to SDF-1α and anti-HIV-1 activity because in some experiments higher anti-HIV-1 activity of 15D was observed in comparison with 15K (data not shown).\",\n \"The scrambled peptide 15CW, with amino acid composition identical to 15D (Table 4), was less effective at inhibiting the Ca2+ mobilization response (FIG.\",\n \"6D) suggesting the importance of the specific peptide sequence.\",\n \"The peptide treatment alone did not induce Ca2+ mobilization.\",\n \"Example X Effect of gp120 Derived Peptides 15K and 15D on Binding of Chemokines to CCRS and CXCR4 Expressing Cells Since the 15K and 15D peptides were designed based on their structural homology with a chemokine fragment, whether these peptides might exhibit binding activity for CCR5 or CXCR4 was determined.\",\n \"Experiments were carried out to assess the ability of the peptides to competitively inhibit binding of radiolabeled MIP-1β or SDF-1α to cells expressing CCR4 or CXCR4, respectively.\",\n \"To determine the effect of peptides exclusively on chemokine binding and to exclude the contribution of chemokine internalization, cells were incubated at room temperature in the presence of 0.1% sodium azide.\",\n \"Under such conditions all cell-bound ligand could be removed by rinsing the cells with 0.1 M Gly-HCl pH 2.5, excluding the contribution of internalization to the chemokine binding.\",\n \"The binding experiments demonstrated that the 15D and 15K peptides competitively inhibited chemokine binding to CCR5 (FIG.\",\n \"7A) and CXCR4 (FIG.\",\n \"7B).\",\n \"The binding experiments did not reveal any difference in the inhibitory activity of peptides 15K and 15D in comparison with their scrambled analogs (data not shown).\",\n \"The control 15-mer peptide 15GIG with a different amino acid composition manifested significantly lower competing activity (FIG.\",\n \"7C).\",\n \"It is possible that the particular scrambling of the peptide sequence that was performed did not change the sequences of the scrambled peptides sufficiently to significantly reduce their capacity to bind to the receptors because scrambling of these peptides may have created similar amino acid triplets in other positions of the peptides (like RAK and KAR in 15K and 15KS, Table 4).\",\n \"It is possible that because of this peptide 15KS also manifested a low level of inhibitory activity (although significantly less than original peptide) on anti-CXCR4 antibody binding to the receptor (FIG.\",\n \"5C).\",\n \"Additional structure-functional studies are necessary to determine the minimum sequence of the peptides sufficient for inhibition of virus.\",\n \"To briefly summarize the foregoing description, the interaction of HIV envelope glycoprotein gp120 with a chemokine receptor is known to be a prerequisite for viral attachment and entry into target cells (Alkhatib et al., Science 272:1955-1958, 1996; Berson et al., J. Virol.\",\n \"70:6288-6295, 1996; Deng et al., Nature 381:661-666, 1996; and Dragic et al., Nature 381:667-673, 1996, each incorporated herein by reference).\",\n \"Monocyte-tropic viruses (R5 strains) utilize a distinct chemokine receptor, CCR5, for cell entry, while T cell tropic viruses (X4 strains) utilize CXCR4 receptors.\",\n \"Chemokine receptor binding site(s) on gp120 are thought to be formed, or exposed, after binding of gp120 to CD4 (Wu et al., Nature 384:179-183, 1996, incorporated herein by reference).\",\n \"However, gp120 binding to the chemokine receptor in certain HIV strains does not require interaction with CD4 (Hoffman et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 96:6359-6364, 1999; and Hoxie et al., J. Reprod.\",\n \"Immunol.\",\n \"41:197-211, 1998, each incorporated herein by reference).\",\n \"The V3 loop of gp120 has been identified as the major determinant of cellular tropism and coreceptor specificity (Cocchi et al., Nat.\",\n \"Med.\",\n \"2:1244-1247, 1996; and Hwang, et al., Science 253:71-74, 1991, each incorporated herein by reference).\",\n \"Nevertheless, the precise region or residues within this 35-37 amino acid V3 loop responsible for mediating these phenotypic effects has not yet been established (Hung et al., J. Virol.\",\n \"73:8216-8226, 1999, incorporated herein by reference).\",\n \"Synthetic cyclized peptides corresponding to the V3 loop of gp120 of X4 and dual strains of HIV-1 (but not an R5 strain) at micromolar concentrations could prevent binding of anti-CXCR4 antibodies.\",\n \"Some of these peptides at micromolar concentrations inhibited the infectivity of HIV-IIIB (Sakaida et al., J. Virol.\",\n \"72:9763-9770, 1998, incorporated herein by reference).\",\n \"Synthetic polymer preparations including a putative V3 consensus sequence (GPGRAF, SEQ ID NO:13) of HIV-1 were reported to inhibit HIV-1 infection by an unknown mechanism.\",\n \"However, it is unlikely that this inhibition was due to competition with gp120 binding to the chemokine receptor, or to CD4 (Moulard et al., J. Pept.\",\n \"Res.\",\n \"53:647-655, 1999, incorporated herein by reference).\",\n \"Although the influence of V3 on Hw coreceptor utilization is well established, other more conservative regions of gp120 are also proposed to be involved in coreceptor usage (Rizzuto et al., Science 280:1949-1953, 1998, incorporated herein by reference).\",\n \"Within the present invention, a conservative chemokine receptor binding motif of gp120 was identified which shares certain similarity in amino acid sequence that appears conserved among relevant chemokines.\",\n \"More specifically, the present inventors observed that in the amino acid sequences of most chemokines there is a conserved Trp residue separated by six amino acid residues from a fourth Cys residue.\",\n \"In comparison, the gp120 of all HIV isolates analyzed exhibited a comparable structural motif in the C3 region following the V3 loop.\",\n \"Moreover, several residues of the C-terminal part of V3 loop also manifest some homology with a canonical chemokine sequence (Table 1).\",\n \"Computer modeling as provided herein further demonstrated that the corresponding fragment of gp120 could be template forced onto a “homologous” loop of the known three dimensional structure of an exemplary chemokine MIP-1β (for specific MIP-1β structural detail, see, e.g., Lodi et al., Science 263:1762-7, 1994, incorporated herein by reference) without violation of protein stereochemistry.\",\n \"Prompted in part by these discoveries, two candidate peptides were synthesized which exemplify the proposed gp120 structural motif (Table 2).\",\n \"Both candidate peptides were demonstrated to compete with chemokines for binding to CCR5- and CXCR4-expressing cells.\",\n \"In addition, the exemplary peptides each inhibited CCR5-directed chemotaxis.\",\n \"Correlated with these anti-coreceptor activities, the peptides mediated potent inhibit replication of both monocyte-tropic HIV-1JRFL and T cell tropic HIV-1IIIB.\",\n \"The X-ray structure of an HIV-1 gp120 core (not including V-loops), complexed with a two-domain fragment of CD4, and an antigen-binding fragment of neutralizing antibody, has been reported (Kwong et al., Nature 393:648-659, 1998).\",\n \"Although the structure does not include variable loops of gp120 it did include the C3 portion of the fragment of gp120, which the present invention considered to be structurally similar to the chemokines and potentially involved in interaction with chemokine receptors.\",\n \"In fact, the three-dimensional structure of this fragment (HIVHXB2, residues 331-340, Table 1) appeared to be similar to the corresponding MIP-1β fragment.\",\n \"Both motifs are characterized by an α-helix preceded by a turn.\",\n \"Both contain a Trp residue with the indole ring buried in the interior region of the turn and with the α-carbon present on an exterior turn of the helix.\",\n \"The surface residues on the helices are characterized by long aliphatic side chains, terminated with polar groups, including Lys, Glu, Gln, and Asn.\",\n \"Importantly, this fragment is located within the region of gp120 molecule, which was implicated in CCR5 binding (Rizzuto et al., Science 280:1949-1953, 1998).\",\n \"Since the peptides 15D and 15K include only 5 residues from the C-terminal part of the V3 loop, which is more conserved than other parts of V3 loop, and 9 residues from the N-terminal part of the conservative C3 region, it was reasonable to expect that anti-HIV activity of these peptides may not be dependent on virus tropism.\",\n \"The effects of the peptides on HIV-1 infectivity using R5 and X4 viruses confirmed this prediction (FIGS.\",\n \"3 and 4).\",\n \"The data above demonstrating that peptides 15K and 15D compete with anti-CXCR4 antibody 12G5 suggests a direct interaction of the peptide with this receptor.\",\n \"Although the 15D peptide had reduced ability to block binding of the 12G5 antibody, the infectivity data indicates that the low affinity interaction of 15D was still sufficient to interfere with viral infection.\",\n \"Moreover, the inhibition of the SDF-1α induced intracellular Ca2+ influx by peptides 15D and 15K also supports the conclusion that both antiviral peptides interact with chemokine receptors.\",\n \"The affinity of interaction of the peptides 15D and 15K with chemokine receptors is apparently low, as evidenced by the high concentration of the peptides required to inhibit anti-CXCR4 antibody binding, mobilization of intracellular Ca2+ in response to SDF-1α, and chemokine binding.\",\n \"However, even this low affinity interaction is sufficient for interference with HIV-1 infection based on the low concentration of peptides required for blocking of the viral infection of macrophages and T lymphocytes.\",\n \"Indeed, it is not necessary for an efficient inhibitor of virus interaction with the coreceptor to also be a strong competitor of chemokines binding to the same receptor, because the envelope protein of HIV-1 only mimicks the highly specific binding of a chemokine with its receptor and the affinity of this interaction can be quite low (Hoffman et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 99:11215-11220, 2000).\",\n \"The use of low affinity anti-viral drugs interfering with HIV-1 coreceptor interaction may allow targeting multiple cellular receptors while maintaining the ability to inhibit interactions of a viral glycoprotein, which is subject to frequent mutation.\",\n \"This may provide the basis for the capacity of the present peptides to inhibit infection by viruses using different coreceptors.\",\n \"Moreover, the rather weak competition of peptides 15D and 15K with chemokines for receptor binding, together with potent inhibition of HIV-1 infectivity, can be therapeutically preferable to high affinity inhibitors of CXCR4 and CCR5, because 15K and 15D peptides would not compromise functions of potentially critical chemokines such as SDF-1α or the CCR5 ligands.\",\n \"The present invention has identified a region of HIV-1 gp120, which is structurally similar to chemokines, and appears to be directly involved in the interaction with certain chemokine receptors.\",\n \"The above findings that these peptides inhibit HIV-1 infection of human monocyte-derived macrophages and T-lymphocytes at low nanomolar concentrations suggest that these peptides, their analogs, and peptide menetics, can be used to dissect gp120 interactions with different chemokine receptors and could serve as not only leads for the design of new peptide inhibitors of HIV-1 not restricted by viral tropism, but are useful themselves as therapeutic agents to prevent binding of HIV-1 to a susceptible cell thereby reducing infection and viral replication in the treatment and prevention of HIV infection and related disease.\",\n \"Moreover, it may also be possible that antibodies raised against this sequence of HIV-1 gp120 can also have anti-HIV protective and therapeutic activity by reducing or preventing HIV binding to a susceptible cell.\",\n \"Although the foregoing invention has been described in detail by way of example for purposes of clarity of understanding, it will be apparent to the artisan that certain changes and modifications are comprehended by the disclosure and may be practiced without undue experimentation within the scope of the appended claims, which are presented by way of illustration not limitation.\",\n \"All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.\"\n 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"Reformation of soft soil and system therefor",
"A system and a method for draining soft soil A bordered with soil B are disclosed.",
"The system includes airtight sheet means 10 which covers the soft soil A to assist in vacuuming the soft soil A.",
"The system further includes water gathering pip... | Patent_10451309 |
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"Method and apparatus for background segmentation based on motion localization",
"A system (1000) and method of detecting static background on a video sequence of images with moving foreground objects is described.",
"The method includes localizing moving objects in each frame and training a backgroun... | Patent_10451517 |
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"Gasket for a filtration element and module integrating a filtration element fitted with such a gasket",
"The invention provides a sealing gasket for mounting in a passage (5) of a support plate (4) to surround the end of a tubular-shaped filter element (3) provided with at least one fluid flow channel (3... | Patent_10451532 |
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"PACKAGED INTEGRATED CIRCUITS AND METHODS OF PRODUCING THEREOF",
"A packaged integrated circuit and method for producing thereof, including an integrated circuit substrate lying in a substrate plane and having electrical circuitry formed thereon, a package enclosing the integrated circuit substrate and de... | Patent_10451564 |
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"Conveyor lift",
"A conveyor lift having a device for engaging a product to transfer the product from a conveyor at one level to a conveyor at another level wherein the lift is driven by at least one drive comprising a motor, and a coupling to couple the motor to the lift wherein the coupling comprises a ... | Patent_10451571 |
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"Phage diplay libraries of human vh fragments",
"Phage display libraries are taught in which the recombinant phage population displays a plurality of potential binding fragments having preferred characteristics of solubility and/or intermolecular interaction.",
"Also taught are methods of biasing disp... | Patent_10451585 |
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"Solder foil semiconductor device and electronic device",
"A solder foil formed from a material comprising particles of Cu, etc.",
"as metal particles and Sn particles as solder particles by rolling is suitable for solder bonding at a high temperature side in temperature-hierarchical bonding, and semi... | Patent_10451610 |
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"Surface-functionalised carrier material, method for the production thereof and solid phase synthesis method",
"The invention concerns a novel surface-functionalized carrier material with a polymeric surface and at least one linker compound according to the general formula (I), which is covalently bound t... | Patent_10451654 |
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"Interconnecting module for the base of electronic equipment casing",
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"Dispensing Apparatus",
"A dispensing apparatus (1) including a combination of a tube (2), cap (4) and plunger (3).",
"The tube is arranged for carrying a collapsible sausage (6) and includes longitudinally extending ribs (8) projecting inwardly of the tube and defining longitudinal grooves (7) thereb... | Patent_10451671 |
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"Compositions for oral use",
"An oral composition comprising trifluoromethionine as an effective component is provided as a useful oral composition for preventing and/or treating intraoral diseases such as bad breath, periodontal disease, alveolar pyorrhea, etc., which is safe even if daily used."
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... | Patent_10451678 |
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"Sulphur-containing amphiphilic agents for the transfer of biologically active molecules into cells",
"The present invention relates to amphiphilic, cationic sulpho-substituted phosphatidyl ethanolamine analogs and their salts, which are capable of complexing biopolymers such as DNA, RNA, oligonucleotides... | Patent_10451687 |
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"Optical system having a holographic optical element",
"An optical laser system wherein a holographic optical element (HOE) replaces a bulky feedback system comprising a large number of optical element.",
"The feedback system is adjusted so that the laser device and the feedback system cooperate to se... | Patent_10451724 |
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"Device for combustion of a carbon containing fuel in a nitrogen free atmosphere and a method for operating said device",
"The present invention relates to a device for combustion of a carbon containing fuel in a nitrogen free atmosphere, and a method for operating said device.",
"The device may be in... | Patent_10451729 |
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"Antiglare,anticlouding element",
"An anti-glare and anti-fogging element 1 consisting of an anti-glare element 5 having an electrochromic cell 4 between a front transparent base plate 2 and a back transparent base plate 3 and an anti-fogging element 8 formed on the front transparent base plate 2, which i... | Patent_10451978 |
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"Method and apparatus for unified storage of data for storage area network systems and network attached storage systems",
"A unified data storage apparatus for storing data for both storage area network and network attached storage system architectures on the same storage medium.",
"The storage medium... | Patent_10465631 |
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"Apparatus and method for preparing cerium oxide nanoparticles",
"This invention provides a method for preparing cerium oxide nanoparticles with a narrow size distribution.",
"The cerium oxide nanoparticles obtained by the method of the invention are nearly all crystalline.",
"The method comprises... | Patent_10465942 |
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"Circuit board router apparatus and method thereof",
"A circuit board router (10) and method thereof.",
"De-paneling of printed circuit boards (62) off a panel 860) is efficiently increased by a router (40) which is positioned at a location above the panel (60).",
"A fixture positions the panel 86... | Patent_10465957 |
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"Interface materials and methods of production and use thereof",
"Layered interlace materials described herein comprise at least one crosslinkable thermal interface component and at least one compliant fibrous interface component coupled to the thermal interface component.",
"A method of forming layer... | Patent_10465968 |
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"Casing",
"The invention relates to casing for portable communication devices and light guides for such devices.",
"The casing (2) has a light guide for over-laying a display located in the display region (4) to deliver front lighting to the display and extending across the input region (5), the light... | Patent_10465983 |
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"Device and method for analyzing ion channels in membranes",
"The present invention relates to devices and methods for analyzing ion channels in membranes.",
"The invention is characterized by a biochip comprising a substrate in which openings are provided in the form of an M×N matrix for receiving th... | Patent_10466018 |
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"Mammalian tribbles signaling pathways and methods and reagents related thereto",
"The invention provides methods and reagents for modulating mitogen activated protein kinase pathways using mammalian tribbles homologs (htrb)."
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[
"1.An isolated htrb-1 encoding nucleic acid comprising a nucleotid... | Patent_10466020 |
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"Micromechanical flow sensor with tensile coating",
"A sensor integrated on a semiconductor device (1), in particular a flow sensor, comprises a measuring element (2) on a membrane (5).",
"In order to prevent a buckling of the membrane (5) a tensile coating (9) is applied.",
"The coating covers th... | Patent_10466026 |
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"Graft copolymers and impact-resistant flame-retardant resin compositions containing the same",
"The present invention provides a polyorganosiloxane-containing graft copolymer which is obtainable by polymerizing 0.5 to 10 parts by weight of a vinyl monomer (B) comprising 100 to 50% by weight of a polyfunc... | Patent_10466130 |
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"Stens with drug-containing amphiphilic polymer coating",
"A vascular stent having an amphiphilic polymer coating is loaded with a restenosis inhibiting agent which is sparingly soluble in water, whereby delayed release of the agent takes place after implantation of the stent."
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[
"1.Intravascul... | Patent_10466144 |
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"Modulating insulin receptor signaling",
"Human ISM genes are identified as modulators of INR signaling and thus are therapeutic targets for disorders associated with defective INR signaling.",
"Methods for identifying modulators of ISM, comprising screening for agents that modulate the activity of IS... | Patent_10466162 |
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"Power converter control for automatic maximum power point tracking",
"The invention concerns a method and a circuit for maximum power point tracking of a variable power source from a comparison of an image of the power (P) supplied by the power source, the circuit comprising two elements (14, 31) providi... | Patent_10466303 |
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"High shrink polyethylene films",
"A homogeneous blend of a low density polyethylene with a metallocene-catalysed polyethylene having a density of from 0.906 g/cm3 and a Dow Rheology Index of at least 5/MI2, MI2 being the melt index measured according to ASTMD-1238 condition 190° C./2.16 kg and the Dow Rh... | Patent_10466307 |
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"Extraction bedplate with laser or water jet cut apertures",
"The present invention relates to extraction bedplates (10), (110), (210), (310), (410), (510), (610) for use in apparatus (5) for defiberizing paper making stock and methods for making such bedplates.",
"Preferred methods for making such be... | Patent_10466308 |
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"Former and headbox for said former",
"A former for a machine for the production of a fibrous web, particularly a paper or cardboard web including a headbox equipped with at least one stock suspension feed, one turbulence block equipped with several channels and/or a tube generator equipped with several c... | Patent_10466354 |
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"Gmg-3 gmg-4 and gmg-6 polynucleotides and polypeptides and uses thereof",
"The present invention relates to the field of metabolic research.",
"Metabolic disorders, such as obesity, are a public health problem that is serious and widespread.",
"GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypepti... | Patent_10466375 |
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"Metabolic gene polynucleotides and polypeptides and uses thereof",
"The present invention relates to the field of metabolic research.",
"Metabolic disorders, such as obesity, are a public health problem that is serious and widespread.",
"GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides hav... | Patent_10466376 |
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"Watermarked paper",
"Watermarked paper, preferably shadow-watermarked paper is produced in conventional manner and then size-press dyed using a pigment dyestuff composition.",
"This results in unusual and attractive decorative effects as a result of preferential dye take-up in parts of the watermarke... | Patent_10466416 |
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"Device for metering a urea soulution",
"A device for metering urea solutions permitting a reliable reduction of nitrogen oxides in the exhaust gas of an internal combustion engine is provided.",
"This is achieved by the fact that the device for metering the urea solution includes a sensor unit for mo... | Patent_10466505 |
[
[
"Surface passivation organic polymers and elastomers",
"Surface treatment of organic polymer materials and material surfaces with oligo- or polysaccharide as passivation agent for deliberate alteration of the sorption properties, the diffusion barrier function, lubricative wetting and/or biocompatibility ... | Patent_10466507 |
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[
"Method of enhancing the efficiency of data flow in communication systems",
"A method of improving the flow of data in a communication system having at least two links, comprising inserting into one of said links at least one enhancer, the operation of which is to a) communicate with a the unit upstream o... | Patent_10466598 |
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"Image processing method and program for processing image",
"The invention relates to error distribution processing used for two-valued or multi-valued reproduction on a system recording or displaying a gradation image with several levels.",
"The texture by error distribution processing is suppressed ... | Patent_10466603 |
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[
"Promoting cell regeneration and/or cell differentiation with non-metabolizable sugar and a polymeric absorbent",
"A composition comprising at least one absorbent and at least one non-metabolizable sugar, and a method for using the composition, for promoting cell regeneration and/or cell differentiation."... | Patent_10466619 |
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"Implant",
"An artefact which is suitable for use as an implant is provided.",
"The artefact includes a body having at least an outer surface layer of a calcium phosphate-based material.",
"The outer surface layer has a surface area of at least 1,5m2/g.",
"A plurality of micropores are provide... | Patent_10466659 |
[
[
"Myoelectrically activated respiratory leak sealing",
"The method and system are for sealing/unsealing (regulating) airway leaks occuring between the ventilator circuit and respiratory airways during lung ventilatory support in response to myoelectrical activity of diaphgram.",
"Myolectrical activity ... | Patent_10466699 |
[
[
"Photodynamic stimulation device and methods",
"A treatment device which uses a light radiation of multiple wavelengths and pulse-shaped electromagnetic fields for the photodynamic stimulation of cells, especially cells of human tissue, and also for the activation and stimulation of light sensitive substa... | Patent_10466734 |
[
[
"Miniature precision bearings for minisystems or microsystems and method for assembling such systems",
"The aim of the invention is to obtain a cost-effective solution for providing a microsystem with bearings, which have sufficiently high precision and long-term stress resistance.",
"The invention pr... | Patent_10466792 |
[
[
"Vectors capable of imparting herbicide resistance and viral cross protection and methods",
"A nucleic acid vector for concurrently imparting herbicide resistance to a plant and cross protecting the plant.",
"The vector includes sufficient potyvirus nucleic acid sequence to permit viral replication an... | Patent_10466794 |
[
[
"Virus causing respiratory tract illness in susceptible mammals",
"The invention relates to the field of virology.",
"The invention provides an isolated essentially mammalian negative-sense single stranded RNA virus (MPV) within the sub-family Pneumovirinae of the family Paramyxoviridae and identifiab... | Patent_10466811 |
[
[
"Use of the slug gene, or of the transcription or expression products thereof in the detection and/or treatment of cancerous cells",
"Method for the detection of cancerous cells, in particular, mesenchymal tumoral cells, in a biological sample suspected of containing said malignant cells.",
"The metho... | Patent_10466817 |
[
[
"Audit system and method",
"The invention provides a system and method for reducing and potentially eliminating the review of source documents by auditors to determine whether there is compliance of an audited subject area with a predetermined set of rules.",
"The inventive system and method provides ... | Patent_10466831 |
[
[
"Method and apparatus for solid or solution phase reaction under ambient or inert conditions",
"The present invention generally provides a novel, automation-compatible solid or solution phase reaction vessel, as well as methods for using such a vessel.",
"Generally, the reaction vessel comprises a mic... | Patent_10466867 |
[
[
"Setting real time clocks in communications netwroks",
"A request requesting a real time clock (RTC) value in a communications network is sent from a network element to a management system via a data communications network.",
"The time taken from the sending of the request to the receipt of the RTC va... | Patent_10466880 |
[
[
"Sample carrier",
"The invention relates to a sample carrier which is used to receive chemical and/or biological samples, comprising a receiving part (30) having recesses (32) for receiving the samples.",
"The recesses (32) are sealed by means of a base part (38).",
"The base part (38) is glued to... | Patent_10466883 |
[
[
"Medical image segmentation apparatus and method thereof",
"An apparatus and method for segmenting a medical image are provided.",
"The apparatus includes a user interface unit, which provides interface with a user, a volume data input unit, which inputs medical image information; a two-dimensional im... | Patent_10466884 |
[
[
"Method and circuit for forming an atm cell",
"A pressure pulse generator for use in transmitting pressure signals to surface in a fluid-based drilling system having a housing having an inlet to admit drilling fluid to the interior of housing, and an outlet to discharge fluid from the interior of the hous... | Patent_10466984 |
[
[
"Human hyperpolarization-activated cyclic nucleotide-gated cation channel hcn1",
"The present invention is directed to novel human DNA sequences encoding human HCN1 proteins, the protein encoded by the DNA sequences, vectors comprising the DNA sequences, host cells containing the vectors, and methods of i... | Patent_10466992 |
[
[
"Transient eutectic phase process for ceramic-metal bonding metallization and compositing",
"A method for directly joining ceramics (10) and metals (12).",
"The method involves forming a structure having a ceramic component (10), a more refractory metallic component and a less refractory metallic-mate... | Patent_10467006 |
[
[
"Mutant sh-3 binding protein compositions and methods",
"The invention provides mutant SH3-binding protein (SH3BP2) nucleic acids, polypeptides, and agents which selectively bind to the mutant SH3BP2 molecules and which do not bind to the wild type SH3BP2 molecules.",
"Methods for selecting agents whi... | Patent_10467033 |
[
[
"Network selection in a mobile telecommunications system",
"A mobile telephone (1) in a cellular telecommunications system uses data stored in a SIM cards (10) select a network (4) for registration and to perform optimise call routing to a call destination.",
"If data relevant to networks which are cu... | Patent_10467055 |
[
[
"Network conduit for providing access to data services",
"A web service conduit (3) receives data access requests from browsers (11) from hyperlinks on pages generated by web sites (4), converts the data access requests to web service access requests, and invokes the corresponding web services (40) with t... | Patent_10467100 |
[
[
"Chemical vapor deposition devices and methods",
"Apparatus is described for rapidly coating a large area, or for rapidly producing a powder.",
"In one embodiment, a liquid having a coating chemical is pumped from a liquid reservoir to a distribution manifold.",
"From the distribution manifold, th... | Patent_10467139 |
[
[
"Method of using a cyclooxygenase-2 inhibitor and sex steroids as a combination therapy for the treatment and prevention of dismenorrhea",
"The present invention provides methods for the treatment and prevention of dysmenorrhea in a woman using a combination of a cyclooxygenase-2 inhibitor and sex steroid... | Patent_10467146 |
[
[
"Gel emulsions in the form of o/w emulsions having a hydrocolloid content",
"The invention relates to cosmetic preparations in the form of gel emulsions on preparations of the O/W emulsion type having a hydrocolloid content.",
"Said hydrocolloids are selected from the group of preparations based on (i... | Patent_10467176 |
[
[
"Antisense modulation of wrn espression",
"Antisense compounds, compositions and methods are provided for modulating the expression of WRN.",
"The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding WRN.",
"Methods of using these c... | Patent_10467182 |
[
[
"Method and Device for Carrying Out the Functional Check and Functional Checking of a Technical Unit",
"The invention relates to a method for carrying out the functional check and a functional checking of a technical unit, comprising the following steps, creation of a check program, by selection and stora... | Patent_10467332 |
[
[
"Androgenic 7-substituted 11-halogen steroids",
"The invention relates to 11β-halogen steroids with general formula (I), whereby R11 is halogen, X—Y-Z represents a group with one of the two structures CH═C—C or CH2—C═C and the other radicals have the meaning that is indicated in the claims, also the produ... | Patent_10467352 |
[
[
"Normalizing circuit with reduced error voltage",
"A circuit for calibrating a voltage signal.",
"The circuit includes a first voltage-to-current converter receiving the signal to be calibrated and a reference voltage, and providing a current proportional to the voltage difference between the voltage ... | Patent_10467378 |
[
[
"Process for the polymerization of olefins",
"The present invention relates to a process for homopolymerization of ethylene or copolymerization of ethylene with alpha-olefins by contacting ethylene or ethylene and alpha-olefin with a catalyst composition comprising: (a) a solid catalyst precursor comprisi... | Patent_10467389 |
[
[
"Modulating apparatus and method, and dsv control bit producing method",
"The present invention relates to a modulation apparatus and method and a DSV-control-bit generating method for suppressing an increase in circuit size of the modulation apparatus.",
"When an input data stream is supplied to a DS... | Patent_10467399 |
[
[
"Systems and methods for automatic carburetor enrichment during cold start",
"An automatic carburetor (20) enrichment system that controls the air-fuel mixture during cold start of an engine (10) having a carburetor including a fuel bowl (22) and an induction passage (23), includes a sensor (50) that prov... | Patent_10467451 |
[
[
"Sequence of the photorhabdus luminescens strain tt01 genome and uses",
"The present invention relates to the genomic sequence and to nucleotide sequences encoding polypeptides of Photorhabdus luminescens.",
"The present invention further relates to polypeptides involved in operons involved in the bio... | Patent_10467478 |
[
[
"Benzo'fisoindole derivatives with affinity to the ep4 receptor",
"The present invention relates to a compound of formula (I): corresponding pharmaceutical compositions, preparation processes, and/or methods of using the aforementioned compounds and/or compositions."
],
[
"1.A compound of formula ... | Patent_10467487 |
[
[
"Protein-coupled receptor",
"The present application provides a gene encoding a G protein-coupled receptor termed nGPCR-14; constructs and recombinant host cells incorporating the genes; the nGPCR-14 polypeptides encoded by the gene; antibodies to the nGPCR-x polypeptides; and methods of making and using ... | Patent_10467492 |
[
[
"Pharmaceutical formulation",
"The present invention provides a pharmaceutical formulation which comprises a core comprising a first active ingredient, a coating comprising a second active ingredient which is incompatible with the first active ingredient and a barrier between the core and the coating whic... | Patent_10467504 |
[
[
"Method of education and scholastic management for cyber education system utilizing internet",
"This invention relates to the method of cyber education through internet capable to check the true attendance of a cyber class by a fingerprint and calculate the accumulative learning time of each registered le... | Patent_10467520 |
[
[
"Fuel cell arrangement and method for operating a fuel cell arrangement",
"A fuel cell arrangement (10) and its operation are described.",
"This fuel cell arrangement (10) comprises one or more fuel cells (1) to which a combustible gas or a cathode gas is fed and which supply an electric power Pext to... | Patent_10467525 |
[
[
"Recombinant proteinase k",
"The invention concerns recombinant proteinase K. Furthermore a method for producing recombinant proteinase K is disclosed, which is characterized in that a) a host cell is transformed with a recombinant nucleic acid which codes for the zymogenic precursor of proteinase K, b) t... | Patent_10467532 |
[
[
"Antibody complexes and methods for immunolabeling",
"The present invention provides labeling reagents and methods for labeling primary antibodies and for detecting a target in a sample using an immuno-labeled complex that comprises a target-binding antibody and one or more labeling reagents.",
"The l... | Patent_10467550 |
[
[
"Gssp4 polynucleotides and polypeptides and uses thereof",
"The present invention relates to the field of metabolic research.",
"Metabolic disorders, such as obesity, are a public health problem that is serious and widespread.",
"GSSP4 polypeptides have been identified that are believed to be bene... | Patent_10467554 |
[
[
"Method, module, device and server for voice recognition",
"The invention concerns a voice recognition method implemented in at least a terminal, the voice recognition method using a language model, comprising the following steps: detecting at least a non-recognised expression in one of the terminals; rec... | Patent_10467586 |
[
[
"Tissue transglutaminase",
"The present invention relates to tissue transglutaminase (tTG), and more particularly to DNA coding for and polypeptides containing, the primary structural conformaion of one or more epitopes of tTG and to the use thereof in the detection of autoantibodies and/or lymphocytes pr... | Patent_10467587 |
[
[
"Dispensing device",
"The invention relates to a dispensing device for dispensing particle suspensions (12), especially cell suspensions, comprising a reservoir (10) for receiving the particle suspension (12).",
"A suspending agent (26) is provided in the reservoir (10) that influences the particle su... | Patent_10467596 |
[
[
"Information processing apparatus and method",
"When reproducing content, a client reads attribute information defined in the header of content data corresponding to a content ID specified by a user.",
"When the read attribute information meets an attribute condition defined in a license stored in a s... | Patent_10467603 |
[
[
"Gonococcal proteins and nucleic acids",
"The invention provides proteins from gonococcus (Neisseria gonorrhoeae), including amino acid sequences, the corresponding nucleotide sequences, expression data, and serological data.",
"The proteins are useful antigens for vaccines, immunogenic compositions, ... | Patent_10467657 |
[
[
"Dry shaver for variable heights of cut",
"A dry shaver (11) has a single cutter blade (15) and a single cooperating shear blade (17).",
"The shear blade (17) is an elongate flexible foil, the thickness of which is different at different regions (21, 22) along the elongate extent of the foil (17).",
... | Patent_10467689 |
[
[
"Extracts derived from chenopodium plants and uses thereof",
"The present invention relates to pesticides.",
"More particularly, the present invention relates to botanical pesticides.",
"In particular, the present invention relates to compositions and methods for controlling plant-infesting pests ... | Patent_10467696 |
[
[
"Method and apparatus for treating renal disease with hemodialysis utilizing pulsatile pump",
"A method of removing toxins from blood from a patient in need of such toxin removal includes: providing a countercurrent dialysis filter (20) having a blood compartment and a dialysate compartment separated from... | Patent_10467755 |
[
[
"Dynamic management of access rights lists in a portable electronic object",
"Access rights lists, such as capacity or access control lists, are dynamically managed in a data processing element such as a smart card from an administrator server.",
"To access an access rights list from the server, the l... | Patent_10467763 |
[
[
"Use of type 4 phosphodiesterase inhibitors in myocardial diseases",
"The invention relates to the use of type 4 phosphodiesterase inhibitors to treat myocardial diseases."
],
[
"1.Use of a) compounds of formula I disclosed in EP 0763534 in which B is an aromatic heterocycle having 1 to 4 N, O and... | Patent_10467793 |
[
[
"Substrate processing method and substrate processing apparatus",
"In a substrate processing apparatus, a control electrode (131) separates a process space (11C) including a substrate to be processed and a plasma formation space (11B) not including the substrate.",
"The control electrode includes a co... | Patent_10467820 |
[
[
"Immunogenic compositions comprising an antigen and a purified m protein from respiratory syncytial virus",
"Methods and compositions for enhancing an immune response to an antigen in a host are provided.",
"Immunogenic composition comprising an antigen and an amount of purified M protein from respira... | Patent_10467828 |
[
[
"Sequences involved in phenomena of tumour suppression, tumour reversion, apoptosis and/or virus resistance and their use as medicines",
"This invention is directed to sequences that are involved in the molecular pathways of tumor suppression, tumor revision, apoptosis and/or virus resistance.",
"The ... | Patent_10467851 |
[
[
"System and method for an auction of multiple types of items",
"An improved system and method for a computer-implemented auction in which multiple types of items are auctioned together without imposing a particular division of supply or demand among the individual types of items.",
"In some embodiment... | Patent_10467868 |
[
[
"Packet data recording method and system",
"The present invention provides for a data recording system including data recording means having a plurality of network interface cards in which a plurality of network interface cards can be employed within a single recording mean and each card can be provided w... | Patent_10467899 |
[
[
"Method and system for multiple hosts anycast routing",
"Conventional anycast networks provide a service which allows a sender to access the nearest of a group of receivers sharing a common anycast address.",
"In contrast thereto, multicast networks establish data communications between a sender and a... | Patent_10467910 |
[
[
"Enhanced data storage and retrieval devices and systems and methods for utilizing the same",
"The invention relates generally to store information on magnetic or optical storage media by using one or more novel approaches alone or in combination.",
"These novel approaches are capable of using at leas... | Patent_10467912 |
[
[
"Apparatus and method for splicing sliver of yarn during yarn formation and processing",
"An apparatus (20) and methods are provided for splicing selected portions of sliver (S) such as during yarn formation and processing.",
"The apparatus (20) preferably includes a needle carrying member (35) having... | Patent_10467924 |
[
[
"Production of nanoparticles from methyl vinyl ether and maleic anhydride for the administration of hydrophilic pharmaceuticals, more particularly of puric and pyrimidinic bases",
"Manufacture of nanoparticles on the basis of methyl vinyl ether and maleic acid for the administration of pharmaceuticals of ... | Patent_10467983 |
[
[
"Method for the administration of a subscriber card for mobile telephony equipment of the type with auxiliary reader and embedded system for the implementation of the method",
"The invention concerns a method for the administration of a subscriber SIM card 3 inserted in the main smartcard reader of a mobi... | Patent_10468033 |
[
[
"Estimating signal strength measurements in a telecommunications system",
"The number of measurements being performed in a telecommunications system can be reduced by performing a predetermined measurement in Cell A and in co-located Cell B, step 1, for example a received signal code power (RSCP) measurem... | Patent_10468046 |
[
[
"Device with flexible roller shutter",
"This invention concerns a shutter system comprising, firstly, drive systems allowing the movement of the shutter (1) along a path between a closed and an open position, said shutter (1) being intended to close an opening (3) or other aperture and, secondly, detectio... | Patent_10468129 |
[
[
"Plasma surface graft process for reducing thrombogenicity",
"In accordance with the present invention, there is provided a novel process for modifying the surface properties of a material that is suitable for contact with animal tissue so as to enhance its hemocompatibility and make it less thrombogenic ... | Patent_10468149 |
[
[
"Central Fastening Element for an Axially Symmetric Gas Spring",
"In a central fastening element for an axially symmetric, vehicle gas spring, which includes a bellows that has central bores or cutouts in the region of its end faces, the fastening element being fixed to the vehicle body, protruding from t... | Patent_10468176 |
[
[
"Methods and compositions for inhibiting hiv-coreceptor interactions",
"Novel methods and compositions are provided for inhibiting interactions between human immunodeficiency viruses (HIVs) and viral coreceptors, including CXCR4 and/or CCR5 coreceptors.",
"The anti-coreceptor binding agent includes a ... | Patent_10468182 |